Publications by authors named "Sebastian Zellmer"

26 Publications

  • Page 1 of 1

Novel indirect co-culture of immortalised hepatocytes with monocyte derived macrophages is characterised by pro-inflammatory cytokine networks.

Toxicol In Vitro 2021 Mar 1:105134. Epub 2021 Mar 1.

German Federal Institute for Risk Assessment (BfR), Department of Chemical and Product Safety, Max-Dohrn Straße 8-10, 10589 Berlin, Germany.

The liver is composed of different cell populations. Interactions of different cell populations can be investigated by a newly established indirect co-culture system consisting of immortalised primary human hepatocytes and human monocyte derived macrophages (MDMs). Using the time-dependent cytokine secretion of the co-cultures and single cultures, correlation networks (including the cytokines G-CSF, CCL3, MCP-1, CCL20, FGF, TGF-β1, GM-CSF, IL-8 IL-6, IL-1β, and IL-18) were generated and the correlations were validated by application of IL-8 and TNF-α-neutralising antibodies. The data reveal that IL-8 is crucial for the interaction between hepatocytes and macrophages in vitro. In addition, transcriptome analyses showed that a change in the ratio between macrophages and hepatocytes may trigger pro-inflammatory signalling pathways of the acute phase response and the complement system (release of, e.g., certain cyto- and chemokines). Using diclofenac and LPS showed that the release of cytokines is increasing with higher ratios of MDMs. Altogether we could demonstrate that the current co-culture system is better suited to mirror the in vivo situation when compared to previously established co-culture systems composed of HepG2 and differentiated THP-1 cells. Further, our data reveal that the cytokine IL-8 is crucial for the interaction between hepatocytes and macrophages in vitro.
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http://dx.doi.org/10.1016/j.tiv.2021.105134DOI Listing
March 2021

Indirect co-cultivation of HepG2 with differentiated THP-1 cells induces AHR signalling and release of pro-inflammatory cytokines.

Toxicol In Vitro 2020 Oct 30;68:104957. Epub 2020 Jul 30.

German Federal Institute for Risk Assessment (BfR), Department of Chemical and Product Safety, Max-Dohrn Strasse 8-10, 10589 Berlin, Germany.

HepG2 and THP-1 cells, the latter differentiated by phorbol 12-myristate 13-acetate (PMA), were co-cultured and characterized for typical liver-specific functions, such as xenobiotic detoxification, lipid and cholesterol metabolism. Furthermore, liver injury-associated pathways, such as inflammation, were studied. In general, the co-cultivation of these cells produced a pro-inflammatory system, as indicated by increased levels of cytokines (IL-8, TGF-α, IL-6, GM-CSF, G-CSF, TGF-β, and hFGF) in the respective supernatant. Increased expression levels of target genes of the aryl hydrocarbon receptor (AHR), e.g., CYP1A1, CYP1A2 and CYP1B1, were detected, accompanied by the increased enzyme activity of CYP1A1. Moreover, transcriptome analyses indicated a significant upregulation of cholesterol biosynthesis, which could be reduced to baseline levels by lovastatin. In contrast, total de novo lipid synthesis was reduced in co-cultured HepG2 cells. Key events of the adverse outcome pathway (AOP) for fibrosis were activated by the co-cultivation, however, no increase in the concentration of extracellular collagen was detected. This indicates, that AOP should be used with care. In summary, the indirect co-culture of HepG2/THP-1 cells results in an increased release of pro-inflammatory cytokines, an activation of the AHR pathway and an increased enzymatic CYP1A activity.
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http://dx.doi.org/10.1016/j.tiv.2020.104957DOI Listing
October 2020

MCCP: are medium-chain chlorinated paraffins of concern for humans?

Arch Toxicol 2020 03 26;94(3):955-957. Epub 2020 Feb 26.

Chemical and Product Safety, German Federal Institute for Risk Assessment, Berlin, Germany.

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http://dx.doi.org/10.1007/s00204-020-02681-xDOI Listing
March 2020

Aggregated aluminium exposure: risk assessment for the general population.

Arch Toxicol 2019 12 28;93(12):3503-3521. Epub 2019 Oct 28.

German Federal Institute for Risk Assessment (BfR), Max-Dohrn-Strasse 8-10, 10589, Berlin, Germany.

Aluminium is one of the most abundant elements in earth's crust and its manifold uses result in an exposure of the population from many sources. Developmental toxicity, effects on the urinary tract and neurotoxicity are known effects of aluminium and its compounds. Here, we assessed the health risks resulting from total consumer exposure towards aluminium and various aluminium compounds, including contributions from foodstuffs, food additives, food contact materials (FCM), and cosmetic products. For the estimation of aluminium contents in foodstuff, data from the German "Pilot-Total-Diet-Study" were used, which was conducted as part of the European TDS-Exposure project. These were combined with consumption data from the German National Consumption Survey II to yield aluminium exposure via food for adults. It was found that the average weekly aluminium exposure resulting from food intake amounts to approx. 50% of the tolerable weekly intake (TWI) of 1 mg/kg body weight (bw)/week, derived by the European Food Safety Authority (EFSA). For children, data from the French "Infant Total Diet Study" and the "Second French Total Diet Study" were used to estimate aluminium exposure via food. As a result, the TWI can be exhausted or slightly exceeded-particularly for infants who are not exclusively breastfed and young children relying on specially adapted diets (e.g. soy-based, lactose free, hypoallergenic). When taking into account the overall aluminium exposure from foods, cosmetic products (cosmetics), pharmaceuticals and FCM from uncoated aluminium, a significant exceedance of the EFSA-derived TWI and even the PTWI of 2 mg/kg bw/week, derived by the Joint FAO/WHO Expert Committee on Food Additives, may occur. Specifically, high exposure levels were found for adolescents aged 11-14 years. Although exposure data were collected with special regard to the German population, it is also representative for European and comparable to international consumers. From a toxicological point of view, regular exceedance of the lifetime tolerable aluminium intake (TWI/PTWI) is undesirable, since this results in an increased risk for health impairments. Consequently, recommendations on how to reduce overall aluminium exposure are given.
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http://dx.doi.org/10.1007/s00204-019-02599-zDOI Listing
December 2019

Hepatic co-cultures in vitro reveal suitable to detect Nrf2-mediated oxidative stress responses on the bladder carcinogen o-anisidine.

Toxicol In Vitro 2017 Apr 12;40:153-160. Epub 2017 Jan 12.

Department of Chemical and Product Safety, German Federal Institute for Risk Assessment (BfR), Max-Dohrn-Strasse 8-10, 10589 Berlin, Germany.

The azo dye o-anisidine is known as an industrial and environmental pollutant. Metabolites of o-anisidine remain in the liver for >24h. However, the toxicological impact of o-anisidine on the liver and its individual cell types, e.g., hepatocytes and immune cells, is currently poorly understood. A novel co-culture system, composed of HepG2 or Huh-7 cells, and differentiated THP-1 cells was used to study the metabolic capacity towards o-anisidine, and compared to primary murine hepatocytes which express high enzyme activities. As model compounds the carcinogenic arylamine o-anisidine and its non-carcinogenic isomer, p-anisidine, as well as caffeine were used. Global proteome analysis revealed an activation of eIF2 and Nrf2-mediated oxidative stress response pathways only in co-cultures after treatment with o-anisidine. This was confirmed via detection of reactive oxygen species. In addition, the mitochondrial membrane potential decreased already after 3h treatment of cells, which correlated with a decrease of ATP levels (R>0.92). In the supernatant of co-cultured, but not single-cultured HepG2 and Huh-7 cells, o-anisidine caused increases of damage-associated proteins, such as HMGB1 (high mobility group box-1) protein. In summary, only co-cultures of HepG2 and THP-1 cells predict o-anisidine induced stress responsive pathways, since the system has a higher sensitivity compared to single cultured cells.
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http://dx.doi.org/10.1016/j.tiv.2017.01.006DOI Listing
April 2017

Hedgehog signaling is a potent regulator of liver lipid metabolism and reveals a GLI-code associated with steatosis.

Elife 2016 05 17;5. Epub 2016 May 17.

Institute of Biochemistry, Faculty of Medicine, Leipzig University, Leipzig, Germany.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in industrialized countries and is increasing in prevalence. The pathomechanisms, however, are poorly understood. This study assessed the unexpected role of the Hedgehog pathway in adult liver lipid metabolism. Using transgenic mice with conditional hepatocyte-specific deletion of Smoothened in adult mice, we showed that hepatocellular inhibition of Hedgehog signaling leads to steatosis by altering the abundance of the transcription factors GLI1 and GLI3. This steatotic 'Gli-code' caused the modulation of a complex network of lipogenic transcription factors and enzymes, including SREBP1 and PNPLA3, as demonstrated by microarray analysis and siRNA experiments and could be confirmed in other steatotic mouse models as well as in steatotic human livers. Conversely, activation of the Hedgehog pathway reversed the "Gli-code" and mitigated hepatic steatosis. Collectively, our results reveal that dysfunctions in the Hedgehog pathway play an important role in hepatic steatosis and beyond.
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http://dx.doi.org/10.7554/eLife.13308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4869931PMC
May 2016

Characterization of chemical-induced sterile inflammation in vitro: application of the model compound ketoconazole in a human hepatic co-culture system.

Arch Toxicol 2017 Feb 10;91(2):799-810. Epub 2016 Mar 10.

Department of Chemical and Product Safety, German Federal Institute for Risk Assessment (BfR), Max-Dohrn-Strasse 8-10, 10589, Berlin, Germany.

Liver injury as a result of a sterile inflammation is closely linked to the activation of immune cells, including macrophages, by damaged hepatocytes. This interaction between immune cells and hepatocytes is as yet not considered in any of the in vitro test systems applied during the generation of new drugs. Here, we established and characterized a novel in vitro co-culture model with two human cell lines, HepG2 and differentiated THP-1. Ketoconazole, an antifungal drug known for its hepatotoxicity, was used as a model compound in the testing of the co-culture. Single cultures of HepG2 and THP-1 cells were studied as controls. Different metabolism patterns of ketoconazole were observed for the single and co-culture incubations as well as for the different cell types. The main metabolite N-deacetyl ketoconazole was found in cell pellets, but not in supernatants of cell cultures. Global proteome analysis showed that the NRF2-mediated stress response and the CXCL8 (IL-8) pathway were induced by ketoconazole treatment under co-culture conditions. The upregulation and ketoconazole-induced secretion of several pro-inflammatory cytokines, including CXCL8, TNF-α and CCL3, was observed in the co-culture system only, but not in single cell cultures. Taking together, we provide evidence that the co-culture model applied might be suitable to serve as tool for the prediction of chemical-induced sterile inflammation in liver tissue in vivo.
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http://dx.doi.org/10.1007/s00204-016-1686-yDOI Listing
February 2017

Model-guided identification of a therapeutic strategy to reduce hyperammonemia in liver diseases.

J Hepatol 2016 Apr 27;64(4):860-71. Epub 2015 Nov 27.

Leibniz Research Centre for Working Environment and Human Factors at the Technical University Dortmund, Dortmund, Germany. Electronic address:

Background & Aims: Recently, spatial-temporal/metabolic mathematical models have been established that allow the simulation of metabolic processes in tissues. We applied these models to decipher ammonia detoxification mechanisms in the liver.

Methods: An integrated metabolic-spatial-temporal model was used to generate hypotheses of ammonia metabolism. Predicted mechanisms were validated using time-resolved analyses of nitrogen metabolism, activity analyses, immunostaining and gene expression after induction of liver damage in mice. Moreover, blood from the portal vein, liver vein and mixed venous blood was analyzed in a time dependent manner.

Results: Modeling revealed an underestimation of ammonia consumption after liver damage when only the currently established mechanisms of ammonia detoxification were simulated. By iterative cycles of modeling and experiments, the reductive amidation of alpha-ketoglutarate (α-KG) via glutamate dehydrogenase (GDH) was identified as the lacking component. GDH is released from damaged hepatocytes into the blood where it consumes ammonia to generate glutamate, thereby providing systemic protection against hyperammonemia. This mechanism was exploited therapeutically in a mouse model of hyperammonemia by injecting GDH together with optimized doses of cofactors. Intravenous injection of GDH (720 U/kg), α-KG (280 mg/kg) and NADPH (180 mg/kg) reduced the elevated blood ammonia concentrations (>200 μM) to levels close to normal within only 15 min.

Conclusion: If successfully translated to patients the GDH-based therapy might provide a less aggressive therapeutic alternative for patients with severe hyperammonemia.
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http://dx.doi.org/10.1016/j.jhep.2015.11.018DOI Listing
April 2016

Optimality in the zonation of ammonia detoxification in rodent liver.

Arch Toxicol 2015 Nov 5;89(11):2069-78. Epub 2015 Oct 5.

Department of Simulation and Optimal Processes, Ilmenau University of Technology, Helmholtzplatz 5, 98693, Ilmenau, Germany.

The rodent liver eliminates toxic ammonia. In mammals, three enzymes (or enzyme systems) are involved in this process: glutaminase, glutamine synthetase and the urea cycle enzymes, represented by carbamoyl phosphate synthetase. The distribution of these enzymes for optimal ammonia detoxification was determined by numerical optimization. This in silico approach predicted that the enzymes have to be zonated in order to achieve maximal removal of toxic ammonia and minimal changes in glutamine concentration. Using 13 compartments, representing hepatocytes, the following predictions were generated: glutamine synthetase is active only within a narrow pericentral zone. Glutaminase and carbamoyl phosphate synthetase are located in the periportal zone in a non-homogeneous distribution. This correlates well with the paradoxical observation that in a first step glutamine-bound ammonia is released (by glutaminase) although one of the functions of the liver is detoxification by ammonia fixation. The in silico approach correctly predicted the in vivo enzyme distributions also for non-physiological conditions (e.g. starvation) and during regeneration after tetrachloromethane (CCl4) intoxication. Metabolite concentrations of glutamine, ammonia and urea in each compartment, representing individual hepatocytes, were predicted. Finally, a sensitivity analysis showed a striking robustness of the results. These bioinformatics predictions were validated experimentally by immunohistochemistry and are supported by the literature. In summary, optimization approaches like the one applied can provide valuable explanations and high-quality predictions for in vivo enzyme and metabolite distributions in tissues and can reveal unknown metabolic functions.
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http://dx.doi.org/10.1007/s00204-015-1596-4DOI Listing
November 2015

Regulatory toxicology in the twenty-first century: challenges, perspectives and possible solutions.

Arch Toxicol 2015 Jun 28;89(6):823-50. Epub 2015 Mar 28.

German Federal Institute for Risk Assessment (BfR), Max-Dohrn-Strasse 8-10, 10589, Berlin, Germany.

The advent of new testing systems and "omics"-technologies has left regulatory toxicology facing one of the biggest challenges for decades. That is the question whether and how these methods can be used for regulatory purposes. The new methods undoubtedly enable regulators to address important open questions of toxicology such as species-specific toxicity, mixture toxicity, low-dose effects, endocrine effects or nanotoxicology, while promising faster and more efficient toxicity testing with the use of less animals. Consequently, the respective assays, methods and testing strategies are subject of several research programs worldwide. On the other hand, the practical application of such tests for regulatory purposes is a matter of ongoing debate. This document summarizes key aspects of this debate in the light of the European "regulatory status quo", while elucidating new perspectives for regulatory toxicity testing.
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http://dx.doi.org/10.1007/s00204-015-1510-0DOI Listing
June 2015

A systematic evaluation of the use of physiologically based pharmacokinetic modeling for cross-species extrapolation.

J Pharm Sci 2015 Jan 12;104(1):191-206. Epub 2014 Nov 12.

Computational Systems Biology, Bayer Technology Services GmbH, Leverkusen, Germany; Institute of Applied Microbiology, RWTH Aachen, Aachen, Germany.

Transfer of knowledge along the different phases of drug development is a fundamental process in pharmaceutical research. In particular, cross-species extrapolation between different laboratory animals and further on to first-in-human trials is challenging because of the uncertain comparability of physiological processes. Physiologically based pharmacokinetic (PBPK) modeling allows translation of mechanistic knowledge from one species to another by specifically considering physiological and biochemical differences in between. We here evaluated different knowledge-driven approaches for cross-species extrapolation by systematically incorporating specific model parameter domains of a target species into the PBPK model of a reference species. Altogether, 15 knowledge-driven approaches were applied to murine and human PBPK models of 10 exemplary drugs resulting in 300 different extrapolations. Statistical analysis of the quality of the different extrapolations revealed not only species-specific physiology as the key determinant in cross-species extrapolation but also identified a synergistic effect when considering both kinetic rate constants and gene expression profiles of relevant enzymes and transporters. Moreover, we show that considering species-specific physiology, plasma protein binding, enzyme and transport kinetics, as well as tissue-specific gene expression profiles in PBPK modeling increases accuracy of cross-species extrapolations and thus supports first-in-human trials based on prior preclinical knowledge.
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http://dx.doi.org/10.1002/jps.24214DOI Listing
January 2015

Integrated metabolic spatial-temporal model for the prediction of ammonia detoxification during liver damage and regeneration.

Hepatology 2014 Dec 12;60(6):2040-51. Epub 2014 May 12.

Clinic for Gastroenterology, Hepatology, and Infectiology, Heinrich Heine University, Düsseldorf, Germany.

Unlabelled: The impairment of hepatic metabolism due to liver injury has high systemic relevance. However, it is difficult to calculate the impairment of metabolic capacity from a specific pattern of liver damage with conventional techniques. We established an integrated metabolic spatial-temporal model (IM) using hepatic ammonia detoxification as a paradigm. First, a metabolic model (MM) based on mass balancing and mouse liver perfusion data was established to describe ammonia detoxification and its zonation. Next, the MM was combined with a spatial-temporal model simulating liver tissue damage and regeneration after CCl4 intoxication. The resulting IM simulated and visualized whether, where, and to what extent liver damage compromised ammonia detoxification. It allowed us to enter the extent and spatial patterns of liver damage and then calculate the outflow concentrations of ammonia, glutamine, and urea in the hepatic vein. The model was validated through comparisons with (1) published data for isolated, perfused livers with and without CCl4 intoxication and (2) a set of in vivo experiments. Using the experimentally determined portal concentrations of ammonia, the model adequately predicted metabolite concentrations over time in the hepatic vein during toxin-induced liver damage and regeneration in rodents. Further simulations, especially in combination with a simplified model of blood circulation with three ammonia-detoxifying compartments, indicated a yet unidentified process of ammonia consumption during liver regeneration and revealed unexpected concomitant changes in amino acid metabolism in the liver and at extrahepatic sites.

Conclusion: The IM of hepatic ammonia detoxification considerably improves our understanding of the metabolic impact of liver disease and highlights the importance of integrated modeling approaches on the way toward virtual organisms.
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http://dx.doi.org/10.1002/hep.27136DOI Listing
December 2014

The extended TILAR approach: a novel tool for dynamic modeling of the transcription factor network regulating the adaption to in vitro cultivation of murine hepatocytes.

BMC Syst Biol 2012 Nov 29;6:147. Epub 2012 Nov 29.

Leibniz Institute for Natural Product Research and Infection Biology - Hans-Knöll-Institute, Beutenbergstr. 11a, D-07745 Jena, Germany.

Background: Network inference is an important tool to reveal the underlying interactions of biological systems. In the liver, a complex system of transcription factors is active to distribute signals and induce the cellular response following extracellular stimuli. Plenty of information is available about single transcription factors important for the different functions of the liver, but little is known about their causal relations to each other.

Results: Given a DNA microarray time series dataset of collagen monolayers cultured murine hepatocytes, we identified 22 differentially expressed genes for which the corresponding protein is known to exhibit transcription factor activity. We developed the Extended TILAR (ExTILAR) network inference algorithm based on the modeling concept of the previously published TILAR algorithm. Using ExTILAR, we inferred a transcription factor network based on gene expression data which puts these important genes into a functional context. This way, we identified a previously unknown relationship between Tgif1 and Atf3 which we validated experimentally. Beside its known role in metabolic processes, this extends the knowledge about Tgif1 in hepatocytes towards a possible influence of processes such as proliferation and cell cycle. Moreover, two positive (i.e. double negative) regulatory loops were predicted that could give rise to bistable behavior. We further evaluated the performance of ExTILAR by systematic inference of an in silico network.

Conclusions: We present the ExTILAR algorithm, which combines the advantages of the regression based inference algorithm TILAR, like large network sizes processable and low computational costs, with the advantages of dynamic network models based on ordinary differential equation (i.e. in silico knock-down simulations). Like TILAR, ExTILAR makes use of various prior-knowledge types such as transcription factor binding site information and gene interaction knowledge to infer biologically meaningful gene regulatory networks. Therefore, ExTILAR is especially useful when a large number of genes is modeled using a small number of experimental data points.
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http://dx.doi.org/10.1186/1752-0509-6-147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3573979PMC
November 2012

Modulation of glutamine metabolism by the PI(3)K-PKB-FOXO network regulates autophagy.

Nat Cell Biol 2012 Aug 22;14(8):829-37. Epub 2012 Jul 22.

Department of Immunology, University Medical Center Utrecht, Utrecht 3584CX, The Netherlands.

The PI(3)K-PKB-FOXO signalling network provides a major intracellular hub for the regulation of cell proliferation, survival and stress resistance. Here we report an unexpected role for FOXO transcription factors in regulating autophagy by modulating intracellular glutamine levels. To identify transcriptional targets of this network, we performed global transcriptional analyses after conditional activation of the key components PI(3)K, PKB/Akt, FOXO3 and FOXO4. Using this pathway approach, we identified glutamine synthetase as being transcriptionally regulated by PI(3)K-PKB-FOXO signalling. Conditional activation of FOXO also led to an increased level of glutamine production. FOXO activation resulted in mTOR inhibition by preventing the translocation of mTOR to lysosomal membranes in a glutamine-synthetase-dependent manner. This resulted in an increased level of autophagy as measured by LC3 lipidation, p62 degradation and fluorescent imaging of multiple autophagosomal markers. Inhibition of FOXO3-mediated autophagy increased the level of apoptosis, suggesting that the induction of autophagy by FOXO3-mediated glutamine synthetase expression is important for cellular survival. These findings reveal a growth-factor-responsive network that can directly modulate autophagy through the regulation of glutamine metabolism.
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http://dx.doi.org/10.1038/ncb2536DOI Listing
August 2012

Phosphatidylcholine dimers can be easily misinterpreted as cardiolipins in complex lipid mixtures: a matrix-assisted laser desorption/ionization time-of-flight mass spectrometric study of lipids from hepatocytes.

Rapid Commun Mass Spectrom 2011 Sep;25(18):2619-26

University of Leipzig, Faculty of Medicine, Institute of Medical Physics and Biophysics, Härtelstrasse 16-18, D-04107 Leipzig, Germany.

The liver is an important organ that is particularly involved in the lipid metabolism of the organism. Thus, high interest is nowadays focused on the lipid composition of the liver and particularly the liver parenchymal cells, the hepatocytes. Hepatocytes contain common phospholipids (PL) such as phosphatidylcholines, -ethanolamines and -inositols, for instance, that can be easily analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) even without previous separation of the PL mixture. However, in addition to common PL, hepatocytes possess also significant amounts of cardiolipin (CLP). The MS analysis of this PL is quite challenging because it (a) has a higher mass than common lipids and (b) possesses a higher negative charge. We will show here that caution is required if CLP is analyzed directly from the total lipid extract because PC dimers may be interpreted as cardiolipins if the positive ion MALDI mass spectra are analyzed.
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http://dx.doi.org/10.1002/rcm.5161DOI Listing
September 2011

Transcription factors ETF, E2F, and SP-1 are involved in cytokine-independent proliferation of murine hepatocytes.

Hepatology 2010 Dec 26;52(6):2127-36. Epub 2010 Oct 26.

Institute of Biochemistry, Medical Faculty, University of Leipzig, Leipzig, Germany.

Unlabelled: The cellular basis of liver regeneration has been intensely investigated for many years. However, the mechanisms initiating hepatocyte "plasticity" and priming for proliferation are not yet fully clear. We investigated alterations in gene expression patterns during the first 72 hours of C57BL/6N mouse hepatocyte culture on collagen monolayers (CM), which display a high basal frequency of proliferation in the absence of cytokines. Although many metabolic genes were down-regulated, genes related to mitogen-activated protein kinase (MAPK) signaling and cell cycle were up-regulated. The latter genes showed an overrepresentation of transcription factor binding sites (TFBS) for ETF (TEA domain family member 2), E2F1 (E2F transcription factor 1), and SP-1 (Sp1 transcription factor) (P < 0.001), all depending on MAPK signaling. Time-dependent increase of ERK1/2 phosphorylation occurred during the first 48 hours (and beyond) in the absence of cytokines, accompanied by an enhanced bromodeoxyuridine labeling index of 20%. The MEK inhibitor PD98059 blunted these effects indicating MAPK signaling as major trigger for this cytokine-independent proliferative response. In line with these in vitro findings, liver tissue of mice challenged with CCl(4) displayed hepatocytes with intense p-ERK1/2 staining and nuclear SP-1 and E2F1 expression. Furthermore, differentially expressed genes in mice after partial hepatectomy contained overrepresented TFBS for ETF, E2F1, and SP-1 and displayed increased expression of E2F1.

Conclusion: Cultivation of murine hepatocytes on CM primes cells for proliferation through cytokine-independent activation of MAPK signaling. The transcription factors ETF, E2F1, and SP-1 seem to play a pronounced role in mediating proliferation-dependent differential gene expression. Similar events, but on a shorter time-scale, occur very early after liver damage in vivo.
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http://dx.doi.org/10.1002/hep.23930DOI Listing
December 2010

Prediction and validation of cell alignment along microvessels as order principle to restore tissue architecture in liver regeneration.

Proc Natl Acad Sci U S A 2010 Jun 19;107(23):10371-6. Epub 2010 May 19.

Interdisciplinary Centre for Bioinformatics (IZBI), University of Leipzig, Härtelstrasse 16-18, D-04107 Leipzig, Germany.

Only little is known about how cells coordinately behave to establish functional tissue structure and restore microarchitecture during regeneration. Research in this field is hampered by a lack of techniques that allow quantification of tissue architecture and its development. To bridge this gap, we have established a procedure based on confocal laser scans, image processing, and three-dimensional tissue reconstruction, as well as quantitative mathematical modeling. As a proof of principle, we reconstructed and modeled liver regeneration in mice after damage by CCl(4), a prototypical inducer of pericentral liver damage. We have chosen the regenerating liver as an example because of the tight link between liver architecture and function: the complex microarchitecture formed by hepatocytes and microvessels, i.e. sinusoids, ensures optimal exchange of metabolites between blood and hepatocytes. Our model captures all hepatocytes and sinusoids of a liver lobule during a 16 days regeneration process. The model unambiguously predicted a so-far unrecognized mechanism as essential for liver regeneration, whereby daughter hepatocytes align along the orientation of the closest sinusoid, a process which we named "hepatocyte-sinusoid alignment" (HSA). The simulated tissue architecture was only in agreement with the experimentally obtained data when HSA was included into the model and, moreover, no other likely mechanism could replace it. In order to experimentally validate the model of prediction of HSA, we analyzed the three-dimensional orientation of daughter hepatocytes in relation to the sinusoids. The results of this analysis clearly confirmed the model prediction. We believe our procedure is widely applicable in the systems biology of tissues.
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http://dx.doi.org/10.1073/pnas.0909374107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2890786PMC
June 2010

Heterogeneous expression of suppressor of cytokine signalling 2 (SOCS-2) in liver tissue.

J Anat 2009 Aug 14;215(2):176-83. Epub 2009 May 14.

Institute of Biochemistry, Medical Faculty, University of Leipzig, Leipzig, Germany.

Suppressor of cytokine signalling 2 (SOCS-2), a dual effector of growth hormone signalling, was found to be heterogeneously expressed in murine liver parenchyma. Data from Affymetrix gene arrays, confirmed by quantitative RT-PCR using preparations of periportal and pericentral hepatocyte subpopulations as well as immunohistochemical detection, showed a preferential expression of SOCS-2 in pericentral hepatocytes. Stimulation of cultured periportal and pericentral hepatocyte subpopulations by different concentrations of growth hormone for 1 h resulted at 100 ng mL(-1) in a 1.6-fold and 4.3-fold increase of SOCS-2 mRNA, respectively. Likewise, insulin-like growth factor-1, another physiological target of growth hormone, was stimulated preferentially in pericentral hepatocytes. As growth hormone receptor was found to be homogeneously expressed in mouse liver parenchyma, our data indicate that growth hormone signalling downstream of growth hormone receptor is more sensitive and/or effective in pericentral than in periportal hepatocytes. Presumably, the heterogeneous distribution of SOCS-2 may contribute to the pericentral preference of growth hormone action via differential feedback.
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http://dx.doi.org/10.1111/j.1469-7580.2009.01085.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2740965PMC
August 2009

4-Aminoethylamino-emodin--a novel potent inhibitor of GSK-3beta--acts as an insulin-sensitizer avoiding downstream effects of activated beta-catenin.

J Cell Mol Med 2010 Jun 3;14(6A):1276-93. Epub 2009 Oct 3.

Institute of Biochemistry, Medical Faculty, University of Leipzig, Leipzig, Germany.

Glycogen synthase kinase-3beta (GSK-3beta) is a key target and effector of downstream insulin signalling. Using comparative protein kinase assays and molecular docking studies we characterize the emodin-derivative 4-[N-2-(aminoethyl)-amino]-emodin (L4) as a sensitive and potent inhibitor of GSK-3beta with peculiar features. Compound L4 shows a low cytotoxic potential compared to other GSK-3beta inhibitors determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assay and cellular ATP levels. Physiologically, L4 acts as an insulin-sensitizing agent that is able to enhance hepatocellular glycogen and fatty acid biosynthesis. These functions are particularly stimulated in the presence of elevated concentrations of glucose and in synergy with the hormone action at moderate but not high insulin levels. In contrast to other low molecular weight GSK-3beta inhibitors (SB216763 and LiCl) or Wnt-3alpha-conditioned medium, however, L4 does not induce reporter and target genes of activated beta-catenin such as TOPflash, Axin2 and glutamine synthetase. Moreover, when present together with SB216763 or LiCl, L4 counteracts expression of TOPflash or induction of glutamine synthetase by these inhibitors. Because L4 slightly activates beta-catenin on its own, these results suggest that a downstream molecular step essential for activation of gene transcription by beta-catenin is also inhibited by L4. It is concluded that L4 represents a potent insulin-sensitizing agent favouring physiological effects of insulin mediated by GSK-3beta inhibition but avoiding hazardous effects such as activation of beta-catenin-dependent gene expression which may lead to aberrant induction of cell proliferation and cancer.
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http://dx.doi.org/10.1111/j.1582-4934.2009.00701.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3828845PMC
June 2010

Keratinocytes as depository of ammonium-inducible glutamine synthetase: age- and anatomy-dependent distribution in human and rat skin.

PLoS One 2009 10;4(2):e4416. Epub 2009 Feb 10.

Department of Clinical Pharmacology, University Hospital of Tübingen, Tübingen, Germany.

In inner organs, glutamine contributes to proliferation, detoxification and establishment of a mechanical barrier, i.e., functions essential for skin, as well. However, the age-dependent and regional peculiarities of distribution of glutamine synthetase (GS), an enzyme responsible for generation of glutamine, and factors regulating its enzymatic activity in mammalian skin remain undisclosed. To explore this, GS localization was investigated using immunohistochemistry and double-labeling of young and adult human and rat skin sections as well as skin cells in culture. In human and rat skin GS was almost completely co-localized with astrocyte-specific proteins (e.g. GFAP). While GS staining was pronounced in all layers of the epidermis of young human skin, staining was reduced and more differentiated among different layers with age. In stratum basale and in stratum spinosum GS was co-localized with the adherens junction component beta-catenin. Inhibition of, glycogen synthase kinase 3beta in cultured keratinocytes and HaCaT cells, however, did not support a direct role of beta-catenin in regulation of GS. Enzymatic and reverse transcriptase polymerase chain reaction studies revealed an unusual mode of regulation of this enzyme in keratinocytes, i.e., GS activity, but not expression, was enhanced about 8-10 fold when the cells were exposed to ammonium ions. Prominent posttranscriptional up-regulation of GS activity in keratinocytes by ammonium ions in conjunction with widespread distribution of GS immunoreactivity throughout the epidermis allows considering the skin as a large reservoir of latent GS. Such a depository of glutamine-generating enzyme seems essential for continuous renewal of epidermal permeability barrier and during pathological processes accompanied by hyperammonemia.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0004416PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2637544PMC
March 2009

Cadmium, cobalt and lead cause stress response, cell cycle deregulation and increased steroid as well as xenobiotic metabolism in primary normal human bronchial epithelial cells which is coordinated by at least nine transcription factors.

Arch Toxicol 2008 Aug 25;82(8):513-24. Epub 2008 Jul 25.

Institute of Environmental Toxicology, Martin-Luther-University, Halle-Wittenberg, Halle/Saale, Germany.

Workers occupationally exposed to cadmium, cobalt and lead have been reported to have increased levels of DNA damage. To analyze whether in vivo relevant concentrations of heavy metals cause systematic alterations in RNA expression patterns, we performed a gene array study using primary normal human bronchial epithelial cells. Cells were incubated with 15 microg/l Cd(II), 25 microg/l Co(II) and 550 microg/l Pb(II) either with individual substances or in combination. Differentially expressed genes were filtered out and used to identify enriched GO categories as well as KEGG pathways and to identify transcription factors whose binding sites are enriched in a given set of promoters. Interestingly, combined exposure to Cd(II), Co(II) and Pb(II) caused a coordinated response of at least seven stress response-related transcription factors, namely Oct-1, HIC1, TGIF, CREB, ATF4, SRF and YY1. A stress response was further corroborated by up regulation of genes involved in glutathione metabolism. A second major response to heavy metal exposure was deregulation of the cell cycle as evidenced by down regulation of the transcription factors ELK-1 and the Ets transcription factor GABP, as well as deregulation of genes involved in purine and pyrimidine metabolism. A third and surprising response was up regulation of genes involved in steroid metabolism, whereby promoter analysis identified up regulation of SRY that is known to play a role in sex determination. A forth response was up regulation of xenobiotic metabolising enzymes, particularly of dihydrodiol dehydrogenases 1 and 2 (AKR1C1, AKR1C2). Incubations with individual heavy metals showed that the response of AKR1C1 and AKR1C2 was predominantly caused by lead. In conclusion, we have shown that in vivo relevant concentrations of Cd(II), Co(II) and Pb(II) cause a complex and coordinated response in normal human bronchial epithelial cells. This study gives an overview of the most responsive genes.
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http://dx.doi.org/10.1007/s00204-008-0331-9DOI Listing
August 2008

Improved procedure for the separation of major stratum corneum lipids by means of automated multiple development thin-layer chromatography.

J Chromatogr B Analyt Technol Biomed Life Sci 2002 Nov;780(2):443-50

Institute of Pharmaceutics and Biopharmaceutics, Martin Luther University, W-Langenbeck-Str 4, 06120 Halle (S), Germany.

The separation of the major stratum corneum lipids, i.e., ceramides, fatty acids, cholesterol and its esters by means of high-performance thin-layer chromatography is hereby presented. The used automated multiple development technique allows the reproducible development of a 17-step solvent gradient also capable of separating seven ceramide classes in the same run. Reliable quantification has been performed after visualisation and densitometric scanning. The present approach is less time and solvent-consuming than previously described procedures. The application to samples obtained by in vivo skin surface extraction with hexane-ethanol (2:1) demonstrates that the method can be routinely used for diagnostic purposes.
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http://dx.doi.org/10.1016/s1570-0232(02)00654-2DOI Listing
November 2002

Evidence for the hydrolysis of topical applied liposomal lipids in human stratum corneum.

Cell Mol Biol Lett 2002 ;7(3):885-8

Labsoft Diagnostics AG, Robert-Franz-Ring 21, 06108 Halle, Germany.

Liposomes were incubated with an extract of human plantar stratum corneum. The liposomal lipids were hydrolysed, if composed of soy-bean phosphatidylcholine or phosphatidylglycerol. Rigid lipids were not degraded. The temperature optimum of the hydrolysis was between 30-35degC. CaCl enhanced, while EDTA reduced the rate of hydrolysis, indicating that the hydrolysis is due to a phospholipase A(2).
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May 2003

Evidence for the hydrolysis of topical applied liposomal lipids in human stratum corneum.

Cell Mol Biol Lett 2002 ;7(2):272

Labsoft Diagnostics AG, Robert-Franz-Ring 21, 06108 Halle, Germany.

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October 2002

Isolation, cultivation, and differentiation of normal human epidermal keratinocytes in serum-free medium.

Methods Mol Biol 2002 ;188:179-84

Institute of Biochemistry, Medical Faculty, University of Leipzig, Leipzig, Germany.

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http://dx.doi.org/10.1385/1-59259-185-X:179DOI Listing
November 2002

Thermolabile Liposomes with a High Fusion Efficacy at 42°C can be Made of Dipalmitoylphosphatidylcholine/Fatty Alcohol Mixtures in the Molar Ratio of 1/2.

J Liposome Res 1994 ;4(3):1091-1113

a Medizinische Biophysik (Urologische Klinik und Polyklinik), Technische Universität Miinchen, Klinikum r.d.L, Ismaningerstraβe 22, D-81675 Miinchen, EU, Germany.

Liposomes made of dipalmitoylphosphatidylcholine (DPPC), dipalmitoyl-phosphatidylglycerol (DPPG), and different long-chain fatty alcohols were investigated with respect to their colloidal stability, chain-melting phase transition temperature, and temperature dependent inter-vesicle fusion. In particular, the practical usefulness of the stoichiometric 1/2 (mol/mol) mixtures of the phospholipids and fatty alcohols, mainly elaidoyl alcohol (EL-OH) were studied. The mole fraction of DPPG in the bilayers of such vesicles affects crucially the colloidal stability of the resulting lipid suspensions; at least 15 mol-% of DPPG (relative to DPPC) must be incorporated into the bilayers in order to make the liposome suspension colloidally sufficiently stable at room temperature. The corresponding DPPC/DPPG/EL-OH (0.85/0.15/2) mixed lipid vesicles undergo a lamellar-gel to inverted hexagonal (H) phase transition at 52.7°C, however, and then fuse and aggregate massively. The related phase transition temperature of the DPPC/DPPG/palmitelaidoyl alcohol (0.85/0.15/2) mixture is 48.4°C. This indicates that the chain-melting phase transition temperature of the investigated lipid mixtures is rather sensitive to the alcohol chain-length. This transition temperature is independent, however, of the bulk proton concentration in the pH region between 4.9 and 7.2. Stoichiometric 1/2 mixtures of phospholipids and EL-OH have a high propensity for the inter-vesicle fusion at 42°C and neutral pH. The reason for such fusion 10°C below the lamellar-to-nonlamellar phase transition temperature are the defects that are generated during the chain-melting of the (partly segregated) phospholipid component at 42°C; the proximity of the lamellar to non-lamellar phase transition temperature of the phospholipid/fatty alcohol (1/2) complex at 52°C also plays an important role.
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http://dx.doi.org/10.3109/08982109409018624DOI Listing
January 1994