TY - JOUR A1 - Kittler, Katrin A1 - Hurtaud-Pessel, D. A1 - Maul, Ronald A1 - Kolrep, F. A1 - Fessard, V. T1 - In vitro metabolism of the cyanotoxin cylindrospermopsin in HepaRG cells and liver tissue fractions JF - Toxicon N2 - No evidence for phase I metabolites of the cyanotoxin cylindrospermopsin (CYN) was given using HepaRG cells and different liver tissue fractions when studying metabolic conversion. Although the application of ketoconazole, a CYP3A4 inhibitor, led to a decreased cytotoxicity of CYN, no metabolites were detected applying high resolution mass spectrometry. Quantification of non-modified CYN led to recovery rates of almost 100%. Consequently, reduction of CYN toxicity in the presence of metabolism inhibiting agents must be attributed to alternative pathways. KW - Cylindrospermopsin KW - Metabolism KW - Liver tissue fractions KW - HepaRG cells KW - LC-HRMS KW - Quantification KW - Cyanotoxin PY - 2016 DO - https://doi.org/10.1016/j.toxicon.2015.11.007 SN - 0041-0101 SN - 1879-3150 VL - 110 SP - 47 EP - 50 PB - Elsevier CY - Oxford [u.a.] AN - OPUS4-35222 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Mekonnen, Tessema Fenta A1 - Panne, Ulrich A1 - Koch, Matthias T1 - Electrochemistry coupled online to liquid chromatography-mass spectrometry for fast simulation of biotransformation reactions of the insecticide chlorpyrifos JF - Analytical and Bioanalytical Chemistry (ABC) N2 - An automated method is presented for fast simulation of (bio)transformation products (TPs) of the organophosphate insecticide chlorpyrifos CPF)based on electrochemistry coupled online to liquid chromatography-mass spectrometry (EC-LC-MS). Oxidative TPs were produced by a boron doped diamond (BDD) electrode, separated by reversed phase HPLC and online detected by electrospray ionization-mass spectrometry (ESI-MS). Furthermore, EC oxidative TPs were investigated by HPLC-tandem mass spectrometry (LC-MS/MS) and FT-ICR high resolution mass spectrometry (HRMS) and compared to in-vitro assay metabolites (rat and human liver microsomes). Main phase I metabolites of CPF: chlorpyrifos oxon (CPF oxon), trichloropyridinol (TCP), diethylthiophosphate (DETP), diethylphosphate (DEP), desethyl chlorpyrifos (De-CPF), and desethyl chlorpyrifos oxon (De-CPF oxon), were successfully identified by the developed EC-LC-MS method. The EC-LC-MS method showed similar metabolites compared to the in-vitro assay with possibilities of determining reactive species. Our results reveal that online EC-(LC)-MS brings an advantage on time of analysis by eliminating sample preparation steps and Matrix complexity compared to conventional in-vivo or in-vitro methods. KW - Organophosphate agrochemicals KW - Electrochemical oxidation KW - Metabolism KW - In vitro KW - EC-LC-MS KW - LC-MS/MS PY - 2017 DO - https://doi.org/10.1007/s00216-017-0277-y SN - 1618-2642 SN - 1618-2650 VL - 409 IS - 13 SP - 3359 EP - 3368 PB - Springer CY - Berlin Heidelberg AN - OPUS4-40179 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Mekonnen, Tessema Fenta A1 - Panne, Ulrich A1 - Koch, Matthias T1 - Prediction of biotransformation products of the fungicide fluopyram by electrochemistry coupled online to liquid chromatography-mass spectrometry and comparison with in vitro microsomal assays JF - Analytical and Bioanalytical Chemistry N2 - Biotransformation processes of fluopyram (FLP), a new succinate dehydrogenase inhibitor (SDHI) fungicide, were investigated by electrochemistry (EC) coupled online to liquid chromatography (LC) and electrospray mass spectrometry (ESI-MS). Oxidative phase I metabolite production was achieved using an electrochemical flow-through cell equipped with a boron doped diamond (BDD) electrode. Structural elucidation and prediction of oxidative metabolism pathways were assured by retention time, isotopic patterns, fragmentation, and accurate mass measurements using EC/LC/MS, LC-MS/MS, and/or high resolution mass spectrometry (HRMS). The results obtained by EC were compared with conventional in vitro studies by incubating FLP with rat and human liver microsomes (RLM, HLM). Known phase I metabolites of FLP (benzamide, benzoic acid, 7-hydroxyl, 8-hydroxyl, 7,8-dihydroxyl FLP, lactam FLP, pyridyl acetic acid, and Z/E-olefin FLP) were successfully simulated by EC/LC/MS. New metabolites including an imide, hydroxyl lactam, and 7-hydroxyl pyridyl acetic acid oxidative metabolites were predicted for the first time in our study using EC/LC/MS and liver microsomes. We found oxidation by dechlorination to be one of the major metabolism mechanisms of FLP. Thus, our results revealed that EC/LC/MS-based metabolic elucidation was more advantageous on time and cost of analysis and enabled matrix-free detection with valuable information about the mechanisms and intermediates of metabolism processes. KW - Metabolism KW - EC/LC/MS KW - Electrochemical oxidation KW - Biotransformation KW - SDHI-fungicide PY - 2018 UR - https://link.springer.com/article/10.1007%2Fs00216-018-0933-x#citeas DO - https://doi.org/10.1007/s00216-018-0933-x SN - 1618-2650 SN - 1618-2642 VL - 410 IS - 10 SP - 2607 EP - 2617 PB - Springer CY - Heidelberg AN - OPUS4-44491 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Nikolic, Nela A1 - Dal Co, Alma A1 - Kiviet, Daniel J. A1 - Bergmiller, Tobias A1 - Littmann, Sten A1 - Kuypers, Marcel M. M. A1 - Ackermann, Martin A1 - Schreiber, Frank T1 - Cell-to-cell variation and specialization in sugar metabolism in clonal bacterial populations JF - PLoS Genetics N2 - While we have good understanding of bacterial metabolism at the population level, we know little about the metabolic behavior of individual cells: do single cells in clonal populations sometimes specialize on different metabolic pathways? Such metabolic specialization could be driven by stochastic gene expression and could provide individual cells with growth benefits of specialization. We measured the degree of phenotypic specialization in two parallel metabolic pathways, the assimilation of glucose and arabinose. We grew Escherichia coli in chemostats, and used isotope-labeled sugars in combination with nanometer-scale secondary ion mass spectrometry and mathematical modeling to quantify sugar assimilation at the single-cell level. We found large variation in metabolic activities between single cells, both in absolute assimilation and in the degree to which individual cells specialize in the assimilation of different sugars. Analysis of transcriptional reporters indicated that this variation was at least partially based on cell-to-cell variation in gene expression. Metabolic differences between cells in clonal populations could potentially reduce metabolic incompatibilities between different pathways, and increase the rate at which parallel reactions can be performed. KW - Metabolism KW - Escherichia coli KW - Phenotypic diversity KW - Phenotypic heterogeneity PY - 2017 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-438873 UR - http://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1007122 DO - https://doi.org/10.1371/journal.pgen.1007122 SN - 1553-7404 VL - 13 IS - 12 SP - e1007122, 1 EP - e1007122, 24 PB - Public Library of Science CY - Cambridge, United Kingdom AN - OPUS4-43887 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Pech, S. A1 - Rehberg, M. A1 - Janke, R. A1 - Benndorf, D. A1 - Genzel, Y. A1 - Muth, Thilo A1 - Sickmann, A. A1 - Rapp, E. A1 - Reichl, U. T1 - Tracking changes in adaptation to suspension growth for MDCK cells: cell growth correlates with levels of metabolites, enzymes and proteins JF - Applied Microbiology and Biotechnology N2 - Adaptations of animal cells to growth in suspension culture concern in particular viral vaccine production, where very specific aspects of virus-host cell interaction need to be taken into account to achieve high cell specific yields and overall process productivity. So far, the complexity of alterations on the metabolism, enzyme, and proteome level required for adaptation is only poorly understood. In this study, for the first time, we combined several complex analytical approaches with the aim to track cellular changes on different levels and to unravel interconnections and correlations. Therefore, a Madin-Darby canine kidney (MDCK) suspension cell line, adapted earlier to growth in suspension, was cultivated in a 1-L bioreactor. Cell concentrations and cell volumes, extracellular metabolite concentrations, and intracellular enzyme activities were determined. The experimental data set was used as the input for a segregated growth model that was already applied to describe the growth dynamics of the parental adherent cell line. In addition, the cellular proteome was analyzed by liquid chromatography coupled to tandem mass spectrometry using a label-free protein quantification method to unravel altered cellular processes for the suspension and the adherent cell line. Four regulatory mechanisms were identified as a response of the adaptation of adherent MDCK cells to growth in suspension. These regulatory mechanisms were linked to the proteins caveolin, cadherin-1, and pirin. Combining cell, metabolite, enzyme, and protein measurements with mathematical modeling generated a more holistic view on cellular processes involved in the adaptation of an adherent cell line to suspension growth. KW - MDCK cell KW - Proteome KW - Metabolism KW - Enzyme activity KW - Suspension growth PY - 2021 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-522070 DO - https://doi.org/10.1007/s00253-021-11150-z VL - 105 IS - 5 SP - 1861 EP - 1874 PB - Springer AN - OPUS4-52207 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Schebb, N.H. A1 - Franze, B. A1 - Maul, Ronald A1 - Ranganathan, A. A1 - Hammock, B.D. T1 - In vitro glucuronidation of the antibacterial triclocarban and its oxidative metabolites JF - Drug metabolism and disposition N2 - Triclocarban (3,4,4'-trichlorocarbanilide; TCC) is widely used as an antibacterial in bar soaps. During use of these soaps, a significant portion of TCC is absorbed by humans. For the elimination from the body, glucuronidation plays a key role in both biliary and renal clearance. To investigate this metabolic pathway, we performed microsomal incubations of TCC and its hydroxylated metabolites 2'-OH-TCC, 3'-OH-TCC, and 6-OH-TCC. Using a new liquid chromatography-UV-mass spectrometry method, we could show a rapid glucuronidation for all OH-TCCs by the uridine-5'-diphosphate-glucuronosyltransferases (UGT) present in liver microsomes of humans (HLM), cynomolgus monkeys (CLM), rats (RLM), and mice (MLM). Among the tested human UGT isoforms, UGT1A7, UGT1A8, and UGT1A9 showed the highest activity for the conjugation of hydroxylated TCC metabolites followed by UGT1A1, UGT1A3, and UGT1A10. Due to this broad pattern of active UGTs, OH-TCCs can be efficiently glucuronidated in various tissues, as shown for microsomes from human kidney (HKM) and intestine (HIM). The major renal metabolites in humans, TCC-N-glucuronide and TCC-N'-glucuronide, were formed at very low conversion rates (<1%) by microsomal incubations. Low amounts of N-glucuronides were generated by HLM, HIM, and HKM, as well as by MLM and CLM, but not by RLM, according to the observed species specificity of this metabolic pathway. Among the human UGT isoforms, only UGT1A9 had activity for the N-glucuronidation of TCC. These results present an anomaly where in vivo the predominant urinary metabolites of TCC are N and N'-glucuronides, but these compounds are slowly produced in vitro. KW - 3,4,4'-trichlorocarbanilide KW - Metabolism KW - Glucuronidation PY - 2012 DO - https://doi.org/10.1124/dmd.111.042283 SN - 0090-9556 SN - 1521-009X VL - 40 IS - 1 SP - 25 EP - 31 PB - ASPET CY - Bethesda, Md., USA AN - OPUS4-25731 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Schreiber, Frank A1 - Ackermann, M. T1 - Environmental drivers of metabolic heterogeneity in clonal microbial populations JF - Current Opinion in Biotechnology N2 - Microorganisms perform multiple metabolic functions that shape the global cycling of elements, health and disease of their host organisms, and biotechnological processes. The rates, at which different metabolic activities are performed by individual cells, can vary between genetically identical cells within clonal populations. While the molecular mechanisms that result in such metabolic heterogeneity have attracted considerable interest, the environmental conditions that shape heterogeneity and its consequences have received attention only in recent years. Here, we review the environmental drivers that lead to metabolic heterogeneity with a focus on nutrient limitation, temporal fluctuations and spatial structure, and the functional consequences of such heterogeneity. We highlight studies using single-cell methods that allow direct investigation of metabolic heterogeneity and discuss the relevance of metabolic heterogeneity in complex microbial communities. KW - Phenotypic variation KW - NanoSIMS KW - Diversity KW - Metabolism PY - 2019 UR - https://www.sciencedirect.com/science/article/abs/pii/S095816691930134X?via%3Dihub DO - https://doi.org/10.1016/j.copbio.2019.11.018 VL - 62 SP - 202 EP - 211 PB - Elsevier Ltd. AN - OPUS4-50194 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Vocks, M. A1 - Adam, Christian A1 - Lesjean, B. A1 - Gnirrs, R. A1 - Kraume, M. T1 - Enhanced post-denitrification without addition of an external carbon source in membrane bioreactors JF - Water research N2 - This study investigates a post-denitrification process without the addition of an external carbon source combined with an enhanced biological phosphorus removal (EBPR) in a membrane bioreactor (MBR). Three trial plants, with two different process configurations, were operated on two different sites, and a variety of accompanying batch tests were conducted. It was shown that even without dosing of an external carbon source, denitrification rates (DNR) much above endogenous rates could be obtained in post-denitrification systems. Furthermore, the anaerobic reactor located ahead of the process had a positive impact on the DNR. Given these surprising results, the project team decided to identify the carbon source used by the microorganisms in the post-denitrification process. Batch tests could demonstrate that lysis products do not play a major role as a C-source for post-denitrification. The following hypothesis was proposed to explain the observations: the glycogen, internally stored by the substrate accumulating bacteria, if anaerobic conditions are followed by aerobic conditions could act as carbon source for denitrification in post-denitrification system. First exploratory batch tests, where the glycogen evolution was monitored, corroborate this assumption. KW - Post-denitrification KW - EBPR KW - Glycogen KW - MBR KW - Metabolism KW - Internal substrate KW - Denitrification rate PY - 2005 DO - https://doi.org/10.1016/j.watres.2005.05.049 SN - 0043-1354 VL - 39 IS - 14 SP - 3360 EP - 3368 PB - Elsevier CY - Amsterdam AN - OPUS4-10760 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -