Product Application Laboratory
Product Application Laboratory
Riebeling C.,German Federal Institute for Risk Assessment |
Pirow R.,German Federal Institute for Risk Assessment |
Becker K.,Bayer AG |
Buesen R.,German Federal Institute for Risk Assessment |
And 14 more authors.
Toxicological Sciences | Year: 2011
Teratogenicity can be predicted in vitro using the embryonic stem cell test (EST). The EST, which is based on the morphometric measurement of cardiomyocyte differentiation and cytotoxicity parameters, represents a scientifically validated method for the detection and classification of chemicals according to their teratogenic potency. Furthermore, an abbreviated protocol applying flow cytometry of intracellular marker proteins to determine differentiation into the cardiomyocyte lineage is available. Although valproic acid (VPA) is in worldwide clinical use as antiepileptic drug, it exhibits two severe side effects, i.e., teratogenicity and hepatotoxicity. These limitations have led to extensive research into derivatives of VPA. Here we chose VPA as model compound to test the applicability domain and to further evaluate the reliability of the EST. To this end, we study six closely related congeners of VPA and demonstrate that both the standard and the molecular flow cytometry-based EST are well suited to indicate differences in the teratogenic potency among VPA analogs that differ only in chirality or side chain length. Our data show that identical results can be obtained by using the standard EST or a shortened protocol based on flow cytometry of intracellular marker proteins. Both in vitro protocols enable to reliably determine differentiation of murine stem cells toward the cardiomyocyte lineage and to assess its chemical-mediated inhibition. © The Author 2011. Published by Oxford University Press on behalf of the Society of Toxicology.
Ni J.,DuPont Company |
Ouyang H.,DuPont Company |
Seto C.,Product Application Laboratory |
Sakuma T.,Product Application Laboratory |
And 5 more authors.
Bioanalysis | Year: 2010
Background: The objective of this study was to evaluate the sensitivity requirement for LC-MS/MS as an analytical tool to characterize metabolites in plasma and urine at microdoses in rats and to investigate proportionality of metabolite exposure from a microdose of 1.67 μg/kg to a high dose of 5000 μg/kg for atorvastatin, ofloxacin, omeprazole and tamoxifen. Results: Only the glucuronide metabolite of ofloxacin, the hydroxylation metabolite of omeprazole and the hydration metabolite of tamoxifen were characterized in rat plasma at microdose by LC-MS/MS. The exposure of detected metabolites of omeprazole and tamoxifen appeared to increase in a nonproportional manner with increasing doses. Exposure of ortho- and para-hydroxyatorvastatin, but not atorvastatin and lactone, increased proportionally with increasing doses. Conclusion: LC-MS/MS has demonstrated its usefulness for detecting and characterizing the major metabolites in plasma and urine at microdosing levels in rats. The exposure of metabolites at microdose could not simply be used to predict their exposure at higher doses. © 2010 Future Science Ltd.
Irarrazabal C.E.,U.S. National Institutes of Health |
Irarrazabal C.E.,University of Los Andes, Chile |
Gallazzini M.,U.S. National Institutes of Health |
Schnetz M.P.,U.S. National Institutes of Health |
And 8 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2010
High NaCl elevates activity of the osmoprotective transcription factor TonEBP/OREBP by increasing its phosphorylation, transactivating activity, and localization to the nucleus. We investigated the possible role in this activation of phospholipase C-γ1 (PLC-γ1), which has a predicted binding site at TonEBP/OREBP-phospho-Y143. We find the following. (i) Activation of TonEBP/ OREBP transcriptional activity by high NaCl is reduced in PLC-γ1 null cells and in HEK293 cells in which PLC-γ1 is knocked down by a specific siRNA. (ii) High NaCl increases phosphorylation of TonEBP/ OREBP at Y143. (iii) Wild-type PLC-γ1 coimmunoprecipitates with wild-type TonEBP/OREBP but not TonEBP/OREBP-Y143A, and the coimmunoprecipitation is increased by high NaCl. (iv) PLC-γ1 is part of the protein complex that associates with TonEBP/OREBP at its DNA binding site. (v) Knockdown of PLC-γ1 or overexpression of a PLC-γ1-SH3 deletion mutant reduces high NaCl-dependent TonEBP/OREBP transactivating activity. (vi) Nuclear localization of PLC-γ1 is increased by high NaCl. (vii) High NaCl-induced nuclear localization of TonEBP/OREBP is reduced if cells lack PLC-γ1, if PLC-γ1 mutated in its SH2C domain is overexpressed, or if Y143 in TonEBP/OREBP is mutated to alanine. (viii) Expression of recombinant PLC-γ1 restores nuclear localization of wild-type TonEBP/ OREBP in PLC-γ1 null cells but not of TonEBP/OREBP-Y143A. (ix) The PLC-γ1 phospholipase inhibitor U72133 inhibits nuclear localization of TonEBP/OREBP but not the increase of its transactivating activity. We conclude that, when NaCl is elevated, TonEBP/OREBP becomes phosphorylated at Y143, resulting in binding of PLC-γ1 to that site, which contributes to TonEBP/OREBP transcriptional activity, transactivating activity, and nuclear localization.
Bouschen W.,Justus Liebig University |
Schulz O.,Justus Liebig University |
Eikely D.,Justus Liebig University |
Eikely D.,Product Application Laboratory |
Spengler B.,Justus Liebig University
Rapid Communications in Mass Spectrometry | Year: 2010
Matrix preparation techniques such as air spraying or vapor deposition were investigated with respect to lateral migration, integration of analyte into matrix crystals and achievable lateral resolution for the purpose of high-resolution biological imaging. The accessible mass range was found to be beyond 5000 u with sufficient analytical sensitivity. Gas-assisted spraying methods (using oxygen-free gases) provide a good compromise between crystal integration of analyte and analyte migration within the sample. Controlling preparational parameters with this method, however, is difficult. Separation of the preparation procedure into two steps, instead, leads to an improved control of migration and incorporation. The first step is a dry vapor deposition of matrix onto the investigated sample. In a second step, incorporation of analyte into the matrix crystal is enhanced by a controlled recrystallization of matrix in a saturated water atmosphere. With this latter method an effective analytical resolution of 2μm in the x and y direction was achieved for scanning microprobe matrix-assisted laser desorption/ionization imaging mass spectrometry (SMALDI-MS). Cultured A-498 cells of human renal carcinoma were successfully investigated by high-resolution MALDI imaging using the new preparation techniques. © 2010 John Wiley & Sons, Ltd.