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Chubanov V.,Walther Straub Institute of Pharmacology and Toxicology | Ferioli S.,Walther Straub Institute of Pharmacology and Toxicology | Gudermann T.,Walther Straub Institute of Pharmacology and Toxicology
Cell Calcium | Year: 2017

Transient receptor potential cation channel subfamily M member 7 (TRPM7) is a plasma membrane ion channel linked to a cytosolic protein kinase domain. Genetic inactivation of this bi-functional protein revealed its crucial role in Ca2+ signalling, Mg2+ metabolism, immune responses, cell motility, proliferation and differentiation. Malfunctions of TRPM7 are associated with anoxic neuronal death, cardiac fibrosis, tumour progression and macrothrombocytopenia. Recently, several groups have identified small organic compounds acting as inhibitors or activators of the TRPM7 channel. In follow-up studies, the identified TRPM7 modulators were successfully used to uncover new cellular functions of TRPM7 in situ including a crucial role of TRPM7 in Ca2+ signaling and Ca2+ dependent cellular processes. Hence, TRPM7 has been defined as a promising drug target. Here, we summarize the progress in this quickly developing field. © 2017 Elsevier Ltd.


Schulz S.D.,Albert Ludwigs University of Freiburg | Ruppell C.,Albert Ludwigs University of Freiburg | Tomakidi P.,Albert Ludwigs University of Freiburg | Steinberg T.,Albert Ludwigs University of Freiburg | And 4 more authors.
Dental Materials | Year: 2015

Objectives The aim of this study was the detection of putative gene expression-related effects of dental composites in conventional and interactive gingival cell systems. Methods Conventional monoculture (MC) and interactive cell systems (ICS) comprising human gingival fibroblast (HGF) and immortalized human gingival keratinocytes (IHGK) were exposed for 24 h and 7 days according to ISO10993-12:2012 manufactured eluates of different composites (Ceram X®, Filtek™ Supreme XT, Filtek™ Silorane, Fusio™ Liquid Dentin, and Vertise™ Flow). qRT-PCR-based mRNA analysis for biomarkers indicating cell proliferation, differentiation, apoptosis, inflammation, and adhesion was performed. Apoptotic cells were quantified by annexin-V labeling. Results Due to low RNA amounts, qPCR could not be performed for Vertise™ Flow and Fusio™ Liquid Dentin at day 7. At 24 h, flowables yielded increased transcription for biomarkers of inflammation and apoptosis in IHGK, irrespective of the cell system. HGF cultures displayed lower transcription for cell adhesion markers in both cell systems. Filtek™ Supreme XT showed increased differentiation by elevated filaggrin gene expression in both cell systems for IHGK at day 7, while Filtek™ Silorane and Ceram X® yielded elevation of inflammation biomarkers in both cell types. Annexin-V labeling revealed high apoptosis rates for both flowables and Filtek™ Supreme XT for IHGK, while low rates were detected for Filtek™ Silorane and Ceram X®. Significance Among the composites evaluated, exposition of IHGK and HGF in conventional and interactive cell systems demonstrated most pronounced gene expression alterations in response to flowables, coinciding with elevated levels of apoptosis. © 2015 Academy of Dental Materials.


Mangerich A.,University of Konstanz | Debiak M.,University of Konstanz | Birtel M.,University of Konstanz | Ponath V.,University of Konstanz | And 13 more authors.
Toxicology Letters | Year: 2016

Mustard agents are potent DNA alkylating agents with mutagenic, cytotoxic and vesicant properties. They include bi-functional agents, such as sulfur mustard (SM) or nitrogen mustard (mustine, HN2), as well as mono-functional agents, such as "half mustard" (CEES). Whereas SM has been used as a chemical warfare agent, several nitrogen mustard derivatives, such as chlorambucil and cyclophosphamide, are being used as established chemotherapeutics. Upon induction of specific forms of genotoxic stimuli, several poly(ADP-ribose) polymerases (PARPs) synthesize the nucleic acid-like biopolymer poly(ADP-ribose) (PAR) by using NAD+ as a substrate. Previously, it was shown that SM triggers cellular poly(ADP-ribosyl) ation (PARylation), but so far this phenomenon is poorly characterized. In view of the protective effects of PARP inhibitors, the latter have been proposed as a treatment option of SM-exposed victims. In an accompanying article (Debiak et al., 2016), we have provided an optimized protocol for the analysis of the CEES-induced PARylation response in HaCaT keratinocytes, which forms an experimental basis to further analyze mustard-induced PARylation and its functional consequences, in general. Thus, in the present study, we performed a comprehensive characterization of the PARylation response in HaCaT cells after treatment with four different mustard agents, i.e., SM, CEES, HN2, and chlorambucil, on a qualitative, quantitative and functional level. In particular, we recorded substance-specific as well as dose- and time-dependent PARylation responses using independent bioanalytical methods based on single-cell immuno-fluorescence microscopy and quantitative isotope dilution mass spectrometry. Furthermore, we analyzed if and how PARylation contributes to mustard-induced toxicity by treating HaCaT cells with CEES, SM, and HN2 in combination with the clinically relevant PARP inhibitor ABT888. As evaluated by a novel immunofluorescence-based protocol for the detection of N7-ETE-guanine DNA adducts, the excision rate of CEES-induced DNA adducts was not affected by PARP inhibition. Furthermore, while CEES induced moderate changes in cellular NAD+ levels, annexin V/PI flow cytometry analysis revealed that these changes did not affect CEES-induced short-term cytotoxicity 24 h after treatment. In contrast, PARP inhibition impaired cell proliferation and clonogenic survival, and potentiated micronuclei formation of HaCaT cells upon CEES treatment. Similarly, PARP inhibition affected clonogenic survival of cells treated with bi-functional mustards such as SM and HN2. In conclusion, we demonstrate that PARylation plays a functional role in mustard-induced cellular stress response with substance-specific differences. Since PARP inhibitors exhibit therapeutic potential to treat SM-related pathologies and to sensitize cancer cells for mustard-based chemotherapy, potential long-term effects of PARP inhibition on genomic stability and carcinogenesis should be carefully considered when pursuing such a strategy. © 2015.


Durner J.,Walther Straub Institute of Pharmacology and Toxicology | Durner J.,Ludwig Maximilians University of Munich | Obermaier J.,Ludwig Maximilians University of Munich | Draenert M.,Ludwig Maximilians University of Munich | Ilie N.,Ludwig Maximilians University of Munich
Dental Materials | Year: 2012

Objectives: This study's purpose was to measure and compare the degree of conversion (DC) and the amount of elutable substances from modern resin-based composites (RBCs) as function of polymerization time. One nano-hybrid RBC based on tricyclodecane-(TCD)-urethane (Venus ® Diamond) and two conventionally formulated RBCs (TetricEvo Ceram ®, Filtek™ Supreme XTE) were considered. Method: DC (n = 5) was investigated in real time for 5 min by Fourier transform infrared spectroscopy (ATR-FTIR) in a filling depth of 2 mm at varied irradiation times (5, 10, 20, 40 s). After storing the specimens in ethanol/water for 7 d at 37 °C the eluates were analyzed by gas chromatography/mass spectrometry. Results were statistically analyzed using a multivariate analysis (α = 0.05) an independent t-test (p < 0.05) and a Pearson correlation analysis. Results: In all groups increasing curing time resulted in a significant increase in DC. For TetricEvo Ceram ® a high significant inverse correlation was found between DC and the amount of eluted camphorquinone (CQ, Pearson correlation coefficient = -0.88), ethylene glycol dimethacrylate (EGDMA, -0.73), 4-N,N-dimethylaminobenzoic acid ethylester (DMABEE, -0.87), triethylene glycol dimethacrylate (TEGDMA, -0.68), Tinuvin P (-0.71) and bisphenol-A-polyetheylene glycol dimethacrylate (BisEMA, -0.84). Unexpectedly DC and the amount of eluted methyl acrylate (MAA) correlated directly (0.72). In the specimens of Venus ® Diamond a significant inverse correlation was found between DC and the amount of eluted CQ (-0.69) and TEGDMA (-0.50), whereas in the specimen of Filtek™ Supreme XTE DC correlated with CQ (-0.96), EGDMA (-0.70), DMABEE (-0.87), TEGDMA (-0.92) and MAA (-0.92). Significance: This study demonstrated a strong inverse correlation between DC and elutable substances in RBCs. Both evaluation methods emphasis the importance of an adequate polymerization (20, 40 s), since short curing-times (5, 10 s) resulted in lower DC and higher amount of eluted substances with toxic potential. © 2012 Academy of Dental Materials.


Ilie N.,Ludwig Maximilians University of Munich | Kessler A.,Ludwig Maximilians University of Munich | Durner J.,Ludwig Maximilians University of Munich | Durner J.,Walther Straub Institute of Pharmacology and Toxicology
Journal of Dentistry | Year: 2013

Objectives To assess the effect of irradiation time and distance of the light tip on the micro-mechanical properties and polymerisation kinetics of two bulk-fill resin-based composites at simulated clinically relevant filling depth. Methods Micro-mechanical properties (Vickers hardness (HV), depth of cure (DOC) and indentation modulus (E)) and polymerisation kinetics (real-time increase of degree of cure (DC)) of two bulk-fill resin-based composites (Tetric EvoCeram® Bulk Fill, Ivoclar Vivadent and x-tra base, Voco) were assessed at varying depth (0.1-6 mm in 100 μm steps for E and HV and 0.1, 2, 4 and 6 mm for DC), irradiation time (10, 20 or 40 s, Elipar Freelight2) and distances from the light tip (0 and 7 mm). Curing unit's irradiance was monitored in 1 mm steps at distances up to 10 mm away from the light tip on a laboratory-grade spectrometer. Results Multivariate analysis (α = 0.05), Student's t-test and Pearson correlation analysis were considered. The influence of material on the measured mechanical properties was significant (η2 = 0.080 for E and 0.256 for HV), while the parameters irradiation time, distance from the light tip and depth emphasise a stronger influence on Tetric EvoCeram® Bulk Fill. The polymerisation kinetics could be described by an exponential sum function, distinguishing between the gel and the glass phase. The above mentioned parameters strongly influenced the start of polymerisation (gel phase), and were of less importance for the glass phase. Conclusions Both materials enable at least 4 mm thick increments to be cured in one step under clinically relevant curing conditions. Clinical significance The susceptibility to variation in irradiance was material dependent, thus properties measured under clinically simulated curing conditions might vary to a different extent from those measured under ideal curing conditions. © 2013 Elsevier Ltd.


Debiak M.,University of Konstanz | Lex K.,University of Konstanz | Ponath V.,University of Konstanz | Burckhardt-Boer W.,University of Konstanz | And 6 more authors.
Toxicology Letters | Year: 2016

Sulfur mustard (SM) is a bifunctional alkylating agent with a long history of use as a chemical weapon. Although its last military use is dated for the eighties of the last century, a potential use in terroristic attacks against civilians remains a significant threat. Thus, improving medical therapy of mustard exposed individuals is still of particular interest. PARP inhibitors were recently brought into the focus as a potential countermeasure for mustard-induced pathologies, supported by the availability of efficient compounds successfully tested in cancer therapy.PARP activation after SM treatment was reported in several cell types and tissues under various conditions; however, a detailed characterization of this phenomenon is still missing. This study provides the basis for such studies by developing and optimizing experimental conditions to investigate poly(ADP-ribosyl)ation (PARylation) in HaCaT keratinocytes upon treatment with the monofunctional alkylating agent 2-chloroethyl ethyl sulfide ("half mustard", CEES). By using an immunofluorescence-based approach, we show that optimization of experimental conditions with regards to the type of solvent, dilution factors and treatment procedure is essential to obtain a homogenous PAR staining in HaCaT cell cultures. Furthermore, we demonstrate that different CEES treatment protocols significantly influence the cytotoxicity profiles of treated cells. Using an optimized treatment protocol, our data reveals that CEES induces a dose- and time-dependent dynamic PARylation response in HaCaT cells that could be completely blocked by treating cells with the clinically relevant pharmacological PARP inhibitor ABT888 (also known as veliparib). Finally, siRNA experiments show that CEES-induced PAR formation is predominantly due to the activation of PARP1. In conclusion, this study provides a detailed analysis of the CEES-induced PARylation response in HaCaT keratinocytes, which forms an experimental basis to study the molecular mechanism of PARP1 activation and its functional consequences after mustard treatment in general. Such a study is presented in an accompanying article (. Mangerich et al., 2016). © 2015.


Ilie N.,Ludwig Maximilians University of Munich | Obermaier J.,Ludwig Maximilians University of Munich | Durner J.,Ludwig Maximilians University of Munich | Durner J.,Walther Straub Institute of Pharmacology and Toxicology
Clinical Oral Investigations | Year: 2014

Objectives: The aim of our study was to analyse whether the irradiation time and/or the modulation of irradiation time influence the degree of conversion (DC) and the amount of elutable substances from modern nano-hybrid resin-based composites (RBCs). Materials and methods: The DC was recorded in real time for 5 min by means of attenuated total reflectance-Fourier transform infrared spectroscopy (n = 5) on the lower surface of 2-mm-thick samples irradiated with continuous and modulated irradiation times for 20 s and 40 s. The modulated times comprise a short polymerisation (2 s or 5 s) followed by a rest period of 1 min and an additional polymerisation to complete 20 s and 40 s of polymerisation (2 s + 18 s, 5 s + 15 s, 2 s + 38 s and 5 s + 35 s). After storing the specimens in ethanol/water for 7 days at 37 °C, the eluates were analysed by gas chromatography-mass spectrometry. Results were statistically analyzed using a one-way ANOVA analysis (α = 0.05). Results: The effect of irradiation time on DC is similar in all three analyzed materials, showing a significant increase in DC by increasing irradiation time from 20 s to 40 s, while the DC is not influenced within one irradiation time (20 s or 40 s) by the modulation of time. Conclusions: The type and amount of eluates are strongly dependent from the material and the irradiation protocol. Clinical relevance: An interrupt irradiation of RBCs is clinically feasible, reducing in general the amount of elutable substances at similar DC as the corresponding continuous polymerisation. © 2013 Springer-Verlag Berlin Heidelberg.


Durner J.,Walther Straub Institute of Pharmacology and Toxicology
Dental materials : official publication of the Academy of Dental Materials | Year: 2012

This study's purpose was to measure and compare the degree of conversion (DC) and the amount of elutable substances from modern resin-based composites (RBCs) as function of polymerization time. One nano-hybrid RBC based on tricyclodecane-(TCD)-urethane (Venus(®) Diamond) and two conventionally formulated RBCs (TetricEvo Ceram(®), Filtek™ Supreme XTE) were considered. DC (n=5) was investigated in real time for 5min by Fourier transform infrared spectroscopy (ATR-FTIR) in a filling depth of 2mm at varied irradiation times (5, 10, 20, 40s). After storing the specimens in ethanol/water for 7 d at 37°C the eluates were analyzed by gas chromatography/mass spectrometry. Results were statistically analyzed using a multivariate analysis (α=0.05) an independent t-test (p<0.05) and a Pearson correlation analysis. In all groups increasing curing time resulted in a significant increase in DC. For TetricEvo Ceram(®) a high significant inverse correlation was found between DC and the amount of eluted camphorquinone (CQ, Pearson correlation coefficient=-0.88), ethylene glycol dimethacrylate (EGDMA, -0.73), 4-N,N-dimethylaminobenzoic acid ethylester (DMABEE, -0.87), triethylene glycol dimethacrylate (TEGDMA, -0.68), Tinuvin P (-0.71) and bisphenol-A-polyetheylene glycol dimethacrylate (BisEMA, -0.84). Unexpectedly DC and the amount of eluted methyl acrylate (MAA) correlated directly (0.72). In the specimens of Venus(®) Diamond a significant inverse correlation was found between DC and the amount of eluted CQ (-0.69) and TEGDMA (-0.50), whereas in the specimen of Filtek™ Supreme XTE DC correlated with CQ (-0.96), EGDMA (-0.70), DMABEE (-0.87), TEGDMA (-0.92) and MAA (-0.92). This study demonstrated a strong inverse correlation between DC and elutable substances in RBCs. Both evaluation methods emphasis the importance of an adequate polymerization (20, 40s), since short curing-times (5, 10s) resulted in lower DC and higher amount of eluted substances with toxic potential. Copyright © 2012 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.


Sevkusic M.,Walther Straub Institute of Pharmacology and Toxicology | Schuster L.,Walther Straub Institute of Pharmacology and Toxicology | Rothmund L.,Walther Straub Institute of Pharmacology and Toxicology | Dettinger K.,Walther Straub Institute of Pharmacology and Toxicology | And 7 more authors.
Dental Materials | Year: 2014

Objectives Constituents of dental composites can be released from dental fillings after polymerization. The aim of this study was to examine the time-related elution and breakdown of separable constituents of polymerized composites using deuterated solvents. Method Elution and breakdown of constituents were investigated with deuterated solvents methanol and water by gas chromatography/mass spectrometry of following composites for 180 days: Filtek™ Supreme XT, Filtek™ Supreme XT Flow, Tetric Ceram®, Tetric Flow®, Grandio®, Grandio® Flow. Results Within 180 days no compounds were formed as the products of breakdown. 19 compounds were identified as elution products: Bis-EMA, TEGDMA, DDDMA, EGDMA, MAA, BPA, CQ, HQME, DMABEE, CSA, BL, TEG, BHT, TINP, TPP, TPSB, DEDHTP, DCHP, ß-PHEA. The highest concentration of Bis-EMA was measured for Tetric Flow® in deuterated methanol on day 90 at 36.993 mmol/l and in deuterated water also on day 90 at 0.031 mmol/l. The highest TEGDMA concentrations were measured for Grandio® Flow in deuterated methanol on day 60 at 1.322 mmol/l and for Filtek™ Supreme XT Flow in deuterated water on day 3 at 0.689 mmol/l. The highest BPA concentration was measured for Tetric Flow® in deuterated methanol on day 90 at 1.469 mmol/l. The highest BPA concentration was measured for Grandio® in deuterated water on day 180 at 0.007 mmol/l. Significance Examination of time-related elution indicates that various elution products (e.g. Bis-EMA, BPA) were only released in small quantities during the first 90 days, but in high quantities between day 90 and day 180. © 2014 Academy of Dental Materials.


PubMed | Walther Straub Institute of Pharmacology and Toxicology
Type: Comparative Study | Journal: Dental materials : official publication of the Academy of Dental Materials | Year: 2012

This studys purpose was to measure and compare the degree of conversion (DC) and the amount of elutable substances from modern resin-based composites (RBCs) as function of polymerization time. One nano-hybrid RBC based on tricyclodecane-(TCD)-urethane (Venus() Diamond) and two conventionally formulated RBCs (TetricEvo Ceram(), Filtek Supreme XTE) were considered.DC (n=5) was investigated in real time for 5min by Fourier transform infrared spectroscopy (ATR-FTIR) in a filling depth of 2mm at varied irradiation times (5, 10, 20, 40s). After storing the specimens in ethanol/water for 7 d at 37C the eluates were analyzed by gas chromatography/mass spectrometry. Results were statistically analyzed using a multivariate analysis (=0.05) an independent t-test (p<0.05) and a Pearson correlation analysis.In all groups increasing curing time resulted in a significant increase in DC. For TetricEvo Ceram() a high significant inverse correlation was found between DC and the amount of eluted camphorquinone (CQ, Pearson correlation coefficient=-0.88), ethylene glycol dimethacrylate (EGDMA, -0.73), 4-N,N-dimethylaminobenzoic acid ethylester (DMABEE, -0.87), triethylene glycol dimethacrylate (TEGDMA, -0.68), Tinuvin P (-0.71) and bisphenol-A-polyetheylene glycol dimethacrylate (BisEMA, -0.84). Unexpectedly DC and the amount of eluted methyl acrylate (MAA) correlated directly (0.72). In the specimens of Venus() Diamond a significant inverse correlation was found between DC and the amount of eluted CQ (-0.69) and TEGDMA (-0.50), whereas in the specimen of Filtek Supreme XTE DC correlated with CQ (-0.96), EGDMA (-0.70), DMABEE (-0.87), TEGDMA (-0.92) and MAA (-0.92).This study demonstrated a strong inverse correlation between DC and elutable substances in RBCs. Both evaluation methods emphasis the importance of an adequate polymerization (20, 40s), since short curing-times (5, 10s) resulted in lower DC and higher amount of eluted substances with toxic potential.

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