Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Aflatoxin B1 Conversion by Black Soldier Fly (Hermetia illucens) Larval Enzyme Extracts
Meijer, Nathan ; Stoopen, Geert ; Fels-Klerx, H.J. van der; Loon, Joop J.A. van; Carney, John ; Bosch, Guido - \ 2019
Toxins 11 (2019)9. - ISSN 2072-6651
aflatoxin - black soldier fly - BSFL - cytochrome P450 - enzyme induction - Hermetia illucens - metabolic conversion - mycotoxin - S9 fraction

The larvae of the black soldier fly (Hermetia illucens L., BSFL) have received increased industrial interest as a novel protein source for food and feed. Previous research has found that insects, including BSFL, are capable of metabolically converting aflatoxin B1 (AFB1), but recovery of total AFB1 is less than 20% when accounting for its conversion to most known metabolites. The aim of this study was to examine the conversion of AFB1 by S9 extracts of BSFL reared on substrates with or without AFB1. Liver S9 of Aroclor-induced rats was used as a reference. To investigate whether cytochrome P450 enzymes are involved in the conversion of AFB1, the inhibitor piperonyl butoxide (PBO) was tested in a number of treatments. The results showed that approximately 60% of AFB1 was converted to aflatoxicol and aflatoxin P1. The remaining 40% of AFB1 was not converted. Cytochrome P450s were indeed responsible for metabolic conversion of AFB1 into AFP1, and a cytoplasmic reductase was most likely responsible for conversion of AFB1 into aflatoxicol.

Determination of genotoxic potencies of pyrrolizidine alkaloids in HepaRG cells using the γH2AX assay
Louisse, Jochem ; Rijkers, Deborah ; Stoopen, Geert ; Holleboom, Wendy Jansen ; Delagrange, Mona ; Molthof, Elise ; Mulder, Patrick P.J. ; Hoogenboom, Ron L.A.P. ; Audebert, Marc ; Peijnenburg, Ad A.C.M. - \ 2019
Food and Chemical Toxicology 131 (2019). - ISSN 0278-6915
Genotoxicity - HepaRG - Pyrrolizidine alkaloids (PAs) - Relative potency factor (RPF) - γH2AX assay

Pyrrolizidine alkaloids (PAs) are secondary metabolites from plants that have been found in substantial amounts in herbal supplements, infusions and teas. Several PAs cause cancer in animal bioassays, mediated via a genotoxic mode of action, but for the majority of the PAs, carcinogenicity data are lacking. It is assumed in the risk assessment that all PAs have the same potency as riddelliine, which is considered to be one of the most potent carcinogenic PAs in rats. This may overestimate the risks, since many PAs are expected to have lower potencies. In this study we determined the concentration-dependent genotoxicity of 37 PAs representing different chemical classes using the γH2AX in cell western assay in HepaRG human liver cells. Based on these in vitro data, PAs were grouped into different potency classes. The group with the highest potency consists particularly of open diester PAs and cyclic diester PAs (including riddelliine). The group of the least potent or non-active PAs includes the monoester PAs, non-esterified necine bases, PA N-oxides, and the unsaturated PA trachelanthamine. This study reveals differences in in vitro genotoxic potencies of PAs, supporting that the assumption that all PAs have a similar potency as riddelliine is rather conservative.

Adverse Outcome Pathway-Driven Analysis of Liver Steatosis in Vitro : A Case Study with Cyproconazole
Luckert, Claudia ; Braeuning, Albert ; Sousa, Georges De; Durinck, Sigrid ; Katsanou, Efrosini S. ; Konstantinidou, Parthena ; Machera, Kyriaki ; Milani, Emanuela S. ; Peijnenburg, Ad A.C.M. ; Rahmani, Roger ; Rajkovic, Andreja ; Rijkers, Deborah ; Spyropoulou, Anastasia ; Stamou, Marianna ; Stoopen, Geert ; Sturla, Shana ; Wollscheid, Bernd ; Zucchini-Pascal, Nathalie ; Lampen, Alfonso - \ 2018
Chemical Research in Toxicology 31 (2018)8. - ISSN 0893-228X - p. 784 - 798.

Adverse outcome pathways (AOPs) describe causal relationships between molecular perturbation and adverse cellular effects and are being increasingly adopted for linking in vitro mechanistic toxicology to in vivo data from regulatory toxicity studies. In this work, a case study was performed by developing a bioassay toolbox to assess key events in the recently proposed AOP for chemically induced liver steatosis. The toolbox is comprised of in vitro assays to measure nuclear receptor activation, gene and protein expression, lipid accumulation, mitochondrial respiration, and formation of fatty liver cells. Assay evaluation was performed in human HepaRG hepatocarcinoma cells exposed to the model compound cyproconazole, a fungicide inducing steatosis in rodents. Cyproconazole dose-dependently activated RARα and PXR, two molecular initiating events in the steatosis AOP. Moreover, cyproconazole provoked a disruption of mitochondrial functions and induced triglyceride accumulation and the formation of fatty liver cells as described in the AOP. Gene and protein expression analysis, however, showed expression changes different from those proposed in the AOP, thus suggesting that the current version of the AOP might not fully reflect the complex mechanisms linking nuclear receptor activation and liver steatosis. Our study shows that cyproconazole induces steatosis in human liver cells in vitro and demonstrates the utility of systems-based approaches in the mechanistic assessment of molecular and cellular key events in an AOP. AOP-driven in vitro testing as demonstrated can further improve existing AOPs, provide insight regarding molecular mechanisms of toxicity, and inform predictive risk assessment.

Dose-Dependent Increase of Triglyceride Levels in Human HepaRG Liver Cells Exposed to the Fungicide Cyproconazole
Peijnenburg, A.A.C.M. ; Rijkers, Deborah ; Stoopen, G.M. - \ 2017
Cyproconazole is a triazole antifungal which has been associated with
the development of hepatic steatosis, also referred to as fatty liver
disease, in rodents. An Adverse Outcome Pathway (AOP) for steatosis has
recently been proposed and one important Key Event (KE) in this AOP
concerns the accumulation of triglycerides. The present work is part of
a larger effort that aims to explore the usefulness of the human liver cell
line HepaRG as an in vitro system to address various KEs in the steatosis
AOP and to test foodborne chemicals and chemical mixtures for steatogenic
properties. In this study, HepaRG cells were exposed for 24 and
72 hours to increasing, non-cytotoxic, concentrations of cyproconazole.
Upon exposure, cells were lysed by sonication and extracted with a
mixture of iso-octane and ethylacetate (75:25) to specifically extract
triglycerides. For further analysis of the fatty acid composition of the
triglycerides, extracts were treated with sodium methoxide and BF3 to
generate fatty acid methyl esters (FAMEs). Triglycerides and FAMEs were
analysed using Gas Chromatography with Flame Ionization Detection
(GC-FID). The results indicated that cyproconazole treatment of HepaRG
cells results in a dose-dependent accumulation of triglycerides. The
effect was strongest after 72 hours of exposure. Accumulation was most
pronounced for C50, C52, and C54 triglycerides containing mainly C16
and C18 fatty acids. This outcome underlines the value of the HepaRG
cell line as a model system for studying toxicant-induced liver steatosis.
Acknowledgement: This work has been funded by the EU project EuroMix
(Grant Agreement 633172; and the Dutch Ministry
of Economic Affairs.
Assessment of the genotoxic potential of pyrrolizidine alkaloids using human cell lines and the gammaH2AX In Cell Western technique
Stoopen, G.M. ; Audebert, Marc ; Peijnenburg, A.A.C.M. - \ 2017
- 1 p.
Effects of digested onion extracts on intestinal gene expression using rat intestine slices
Wit, N.J.W. de; Hulst, M.M. ; Govers, C.C.F.M. ; Meulen, J. van der; Hoef, A.M.A. van; Stoopen, G.M. ; Hamers, A.R.M. ; Hoekman, A.J.W. ; Vos, C.H. de; Bovee, T.F.H. ; Smits, M.A. ; Mes, J.J. ; Hendriksen, P.J.M. - \ 2016
Rattus norvegicus - GSE84179 - PRJNA328225
Rat small intestine precision cut slices were exposed for 6 hours to in vitro digested yellow (YOd) and white onion extracts (WOd) that was followed by transcriptomics analysis. The digestion was performed to mimic the digestion that in vivo takes place in the stomach and small intestine. The transcriptomics response of the rat small intestine precision cut slices was compared to that of human Caco-2 cells and the pig in-situ small intestinal segment perfusion. The microarray data for the human Caco-2 cells (GSE83893) and the pig in-situ small intestinal segment perfusion (GSE83908) have been submitted separately from the current data on rat intestine. The goal was to obtain more insight into to which extent mode of actions depend on the experimental model. A main outcome was that each of the three models pointed to the same mode of action: induction of oxidative stress and particularly the Keap1-Nrf2 pathway.
Exposure of the Human HepaRG Liver Cell Line to Pyrrolizidine Alkaloids Results in a Gene Expression Profile Characteristic for Genotoxic Carcinogens
Peijnenburg, A.A.C.M. ; Stoopen, G.M. ; Polman, T.H.G. ; Hendriksen, P.J.M. - \ 2016
- 1 p.
Pyrrolizidine alkaloids (PAs) are secondary metabolites found in many
plant species and can be present as contaminants in food, herbal teas,
plant food supplements, and animal feed. A large number of PAs are toxic
not only to livestock and wildlife but also to humans. Hepatotoxicity,
genotoxicity, and carcinogenicity are the main toxicities observed in
experimental animals treated with PAs. However, there is little direct
evidence for mutagenicity and carcinogenicity of PAs in humans and epidemiological data are lacking. The present study aimed to contribute to
a better evaluation of the genotoxic and carcinogenic risks of PAs for humans.
To that end, the human hepatoma cell line HepaRG was exposed
in vitro for 72 hours to six PAs (riddelliine, retrorsine, echimidine, monocrotaline, lasiocarpine, senkirkine), and the genotoxic carcinogen benzo[
a]pyrene, the non-genotoxic carcinogen bis(2-ethylhexyl) phthalate,
and the non-carcinogen mannitol as controls. Upon exposure, RNA was
isolated and effects on whole genome mRNA expression were analysed
using DNA microarrays. Pathway analysis showed that several processes
involved in genotoxicity, including DNA damage response, p53 pathway
and cell cycle checkpoints, were modulated by the PAs. Comparison of
the array data with transcriptome data obtained by others upon exposure
of HepaRG to various model hepatocarcinogens showed that each
of the PAs used in the present study generated a gene expression profile
that is characteristic for genotoxic carcinogens.
Arabidopsis myrosinases link the glucosinolate-myrosinase system and the cuticle
Ahuja, Ishita ; Vos, Ric C.H. de; Rohloff, Jens ; Stoopen, Geert M. ; Halle, Kari K. ; Ahmad, Samina Jam Nazeer ; Hoang, Linh ; Hall, Robert D. ; Bones, Atle M. - \ 2016
Scientific Reports 6 (2016). - ISSN 2045-2322

Both physical barriers and reactive phytochemicals represent two important components of a plant's defence system against environmental stress. However, these two defence systems have generally been studied independently. Here, we have taken an exclusive opportunity to investigate the connection between a chemical-based plant defence system, represented by the glucosinolate-myrosinase system, and a physical barrier, represented by the cuticle, using Arabidopsis myrosinase (thioglucosidase; TGG) mutants. The tgg1, single and tgg1 tgg2 double mutants showed morphological changes compared to wild-type plants visible as changes in pavement cells, stomatal cells and the ultrastructure of the cuticle. Extensive metabolite analyses of leaves from tgg mutants and wild-type Arabidopsis plants showed altered levels of cuticular fatty acids, fatty acid phytyl esters, glucosinolates, and indole compounds in tgg single and double mutants as compared to wild-type plants. These results point to a close and novel association between chemical defence systems and physical defence barriers.

Transfer of pyrrolizidine alkaloids from various herbs to eggs and meat in laying hens
Mulder, Patrick P.J. ; Witte, Susannah L. de; Stoopen, Geert M. ; Meulen, Jan van der; Wikselaar, Piet G. van; Gruys, Erik ; Groot, Maria J. ; Hoogenboom, Ron L.A.P. - \ 2016
Food Additives & Contaminants. Pt. A, Chemistry, Analysis, Control, Exposure & Risk Assessment 33 (2016)12. - ISSN 1944-0049 - p. 1826 - 1893.
eggs - laying hens - liver - meat - Pyrrolizidine alkaloids - transfer

To investigate the potential transfer of pyrrolizidine alkaloids (PAs), laying hens were fed for 14 days with diets containing 0.5% of dried common ragwort, common groundsel, narrow-leaved ragwort or viper’s bugloss, or 0.1% of common heliotrope. This resulted in total PA levels in feed of respectively 5.5, 11.1, 53.1, 5.9 and 21.7 mg kg 1, with varying composition. PAs were transferred to eggs, in particular yolk, with steady-state levels of respectively 12, 21, 216, 2 and 36 µg kg 1. Overall transfer rates for the sum of PAs were estimated between 0.02% and 0.23%, depending on the type of PAs in the feed. In animals slaughtered shortly after the last exposure, levels in meat were slightly lower than those in eggs, levels in livers somewhat higher. When switched to clean feed, levels in eggs gradually decreased, but after 14 days were still above detection limits in the hens exposed to higher PA levels. Similar was the case for meat and especially kidneys and livers. It is concluded that the intake of PA containing herbs by laying hens may result in levels in eggs and meat that could be of concern for consumers, and as such should be avoided.

Effects of digested onion extracts on intestinal gene expression: an interspecies comparison using different intestine models
Wit, N.J.W. de; Hulst, M.M. ; Govers, C.C.F.M. ; Meulen, J. van der; Hoef, A.M.A. van; Stoopen, G.M. ; Hamers, A.R.M. ; Hoekman, A.J.W. ; Vos, C.H. de; Bovee, T.F.H. ; Smits, M.A. ; Mes, J.J. ; Hendriksen, P.J.M. - \ 2016
PLoS ONE 11 (2016)9. - ISSN 1932-6203 - 18 p.
Human intestinal tissue samples are barely accessible to study potential health benefits of nutritional compounds. Numbers of animals used in animal trials, however, need to be minimalized. Therefore, we explored the applicability of in vitro (human Caco-2 cells) and ex vivo intestine models (rat precision cut intestine slices and the pig in-situ small intestinal segment perfusion (SISP) technique) to study the effect of food compounds. In vitro digested yellow (YOd) and white onion extracts (WOd) were used as model food compounds and transcriptomics was applied to obtain more insight into which extent mode of actions depend on the model. The three intestine models shared 9,140 genes which were used to compare the responses to digested onions between the models. Unsupervised clustering analysis showed that genes up- or down-regulated by WOd in human Caco-2 cells and rat intestine slices were similarly regulated by YOd, indicating comparable modes of action for the two onion species. Highly variable responses to onion were found in the pig SISP model. By focussing only on genes with significant differential expression, in combination with a fold change > 1.5, 15 genes showed similar onion-induced expression in human Caco-2 cells and rat intestine slices and 2 overlapping genes were found between the human Caco-2 and pig SISP model. Pathway analyses revealed that mainly processes related to oxidative stress, and especially the Keap1-Nrf2 pathway, were affected by onions in all three models. Our data fit with previous in vivo studies showing that the beneficial effects of onions are mostly linked to their antioxidant properties. Taken together, our data indicate that each of the in vitro and ex vivo intestine models used in this study, taking into account their limitations, can be used to determine modes of action of nutritional compounds and can thereby reduce the number of animals used in conventional nutritional intervention studies.
The impact of PPARα activation on whole genome gene expression in human precision-cut liver slices
Janssen, Aafke ; Bretzel, Bark ; Stoopen, Geert ; Berends, Frits J. ; Janssen, Ignace M. ; Peijnenburg, Ad ; Kersten, Sander - \ 2015
Homo sapiens - GSE71731 - PRJNA291929
Background: Studies in mice have shown that PPARα is an important regulator of lipid metabolism in liver and a key transcription factor involved in the adaptive response to fasting. However, much less is known about the role of PPARα in human liver. Here we set out to study the function of PPARα in human liver via analysis of whole genome gene regulation in human liver slices treated with the PPARα agonist Wy14643.
Prenylation and Backbone Structure of Flavonoids and Isoflavonoids from Licorice and Hop Influence Their Phase i and II Metabolism
De Schans, Milou G.M. Van; Bovee, Toine F.H. ; Stoopen, Geert M. ; Lorist, Marlies ; Gruppen, Harry ; Vincken, Jean Paul - \ 2015
Journal of Agricultural and Food Chemistry 63 (2015)49. - ISSN 0021-8561 - p. 10628 - 10640.
glucuronidation - hydroxylation - phytoestrogens - prenylated flavonoids - prenylated isoflavonoids - sulfation

In vitro liver metabolism of 11 prenylated flavonoids and isoflavonoids was investigated by determining their phase I glucuronyl and sulfate metabolites using pork liver preparations. One hundred metabolites were annotated using RP-UHPLC-ESI-MSn. A mass spectrometry-based data interpretation guideline was proposed for the tentative annotation of the position of hydroxyl groups, considering its relevance for estrogenic activity. To relate structure to metabolism, compounds were classified on the basis of three criteria: backbone structure (isoflavene, isoflavan, or flavanone), number of prenyl groups (0, 1, or 2), and prenyl configuration (chain or pyran). Glucuronidation was most extensive for isoflavenes and for unprenylated compounds (yield of 90-100%). Pyran and chain prenylation gave more complex hydroxylation patterns with 4 or more than 6 hydroxyl isomers, respectively, as compared to unprenylated compounds (only 1 hydroxyl isomer). Moreover, the number of hydroxyl isomers also increased with the number of prenyl groups.

The impact of PPARa activation on whole genome gene expression in human precision cut liver slices
Janssen, A.W.F. ; Stoopen, G.M. ; Peijnenburg, A.A.C.M. ; Kersten, A.H. - \ 2015
BMC Genomics 16 (2015). - ISSN 1471-2164 - 13 p.
Background: Studies in mice have shown that PPARα is an important regulator of lipid metabolism in liver and key transcription factor involved in the adaptive response to fasting. However, much less is known about the role of PPARα in human liver. Methods: Here we set out to study the function of PPARα in human liver via analysis of whole genome gene regulation in human liver slices treated with the PPARα agonist Wy14643. Results: Quantitative PCR indicated that PPARα is well expressed in human liver and human liver slices and that the classical PPARα targets PLIN2, VLDLR, ANGPTL4, CPT1A and PDK4 are robustly induced by PPARα activation. Transcriptomics analysis indicated that 617 genes were upregulated and 665 genes were downregulated by PPARα activation (q value < 0.05). Many genes induced by PPARα activation were involved in lipid metabolism (ACSL5, AGPAT9, FADS1, SLC27A4), xenobiotic metabolism (POR, ABCC2, CYP3A5) or the unfolded protein response, whereas most of the downregulated genes were involved in immune-related pathways. Among the most highly repressed genes upon PPARα activation were several chemokines (e.g. CXCL9-11, CCL8, CX3CL1, CXCL6), interferon γ-induced genes (e.g. IFITM1, IFIT1, IFIT2, IFIT3) and numerous other immune-related genes (e.g. TLR3, NOS2, and LCN2). Comparative analysis of gene regulation by Wy14643 between human liver slices and primary human hepatocytes showed that down-regulation of gene expression by PPARα is much better captured by liver slices as compared to primary hepatocytes. In particular, PPARα activation markedly suppressed immunity/inflammation-related genes in human liver slices but not in primary hepatocytes. Finally, several putative new target genes of PPARα were identified that were commonly induced by PPARα activation in the two human liver model systems, including TSKU, RHOF, CA12 and VSIG10L. Conclusion: Our paper demonstrates the suitability and superiority of human liver slices over primary hepatocytes for studying the functional role of PPARα in human liver. Our data underscore the major role of PPARα in regulation of hepatic lipid and xenobiotic metabolism in human liver and reveal a marked immuno-suppressive/anti-inflammatory effect of PPARα in human liver slices that may be therapeutically relevant for non-alcoholic fatty liver disease.
Analysis of effects of Herbabolus on milk quality
Groot, M.J. ; Alewijn, M. ; Driessen-van Lankveld, W.D.M. ; Lommen, A. ; Stoopen, G.M. ; Venema, D.P. ; Pikkemaat, M.G. ; Rijk, T.C. de - \ 2015
Wageningen : RIKILT Wageningen UR (RIKILT report 2015.007)
melkproductie - melkkwaliteit - melkveehouderij - melkkoeien - vergelijkend onderzoek - geneeskrachtige kruiden - milk production - milk quality - dairy farming - dairy cows - comparative research - herbal drugs
The Herbabolus is a mix of plant components for cows to improve their health during the transition period. The bolus contains a mixture of herbs including garlic (Garlicin), oregano and yucca. The effects of the bolus on milk quality is investigated. The results are discussed in this report.
In vitro safety assessment of herbal preparations: a toxicogenomics approach
Stoopen, G.M. ; Muller, R. ; Hendriksen, P.J.M. ; Mulder, P.P.J. ; Peijnenburg, A.A.C.M. - \ 2014
In: Book of abstracts the The 18th International Congress on In Vitro Toxicology - Advanced in vitro methods. - - p. 43 - 43.
In vitro safety assessment of herbal preparations: a toxicogenomics approach
Stoopen, G.M. ; Muller, R. ; Hendriksen, P.J.M. ; Mulder, P.P.J. ; Peijnenburg, A.A.C.M. - \ 2014
Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemolsynthase activity
Yang, T. ; Gao, L. ; Hu, H. ; Stoopen, G.M. ; Wang, C. ; Jongsma, M.A. - \ 2014
Journal of Biological Chemistry 289 (2014). - ISSN 0021-9258 - p. 36325 - 36335.
dimethylallyl diphosphate - squalene synthase - monoterpene synthase - terpene synthases - biosynthesis - expression - arabidopsis - metabolism - cloning - leaves
Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme inthe biosynthesis of pyrethrins, the most widely used plant-derivedpesticide.CDScatalyzes c1’-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP). Three proteinsare known to catalyzethis cyclopropanation reactionof terpene precursors.Two of them, phytoene and squalenesynthase, arebifunctional enzymes with both prenyltransferase and terpenesynthase activity. CDS, the other member,was reported to perform only the prenyltransferase step.Here, we show thatthe NDXXD catalytic motif of CDS,under lowersubstrate conditions prevalent inplants,also catalyzesthe next step converting CPP into chrysanthemolby hydrolyzing the diphosphatemoiety.The enzymatic hydrolysis reactionfollowed conventional Michaelis-Menten kinetics, withaKM value for CPP of 196 µM.For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemolwas ~100 µM, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemolat arate of 0.12 –0.16 µg•h-1•g-1FW. We propose that CDS should be renamed a chrysanthemol synthase(CHS)utilizingDMAPP as substrate.
Valencene synthase from the heartwood of Nootka cypress (Callitropsis nootkatensis) for biotechnological production of valencene
Beekwilder, M.J. ; Houwelingen, A.M.M.L. van; Cankar, K. ; Dijk, A.D.J. van; Jong, R. de; Stoopen, G.M. ; Bouwmeester, H.J. ; Achkar, J. ; Sonke, Th. ; Bosch, H.J. - \ 2014
Plant Biotechnology Journal 12 (2014)2. - ISSN 1467-7644 - p. 174 - 182.
antimalarial agent artemisinin - functional-characterization - phytophthora-ramorum - ixodes-scapularis - terpene synthases - essential oil - biosynthesis - yeast - metabolism - expression
Nootkatone is one of the major terpenes in the heartwood of the Nootka cypress Callitropsis nootkatensis. It is an oxidized sesquiterpene, which has been postulated to be derived from valencene. Both valencene and nootkatone are used for flavouring citrus beverages and are considered among the most valuable terpenes used at commercial scale. Functional evaluation of putative terpene synthase genes sourced by large-scale EST sequencing from Nootka cypress wood revealed a valencene synthase gene (CnVS). CnVS expression in different tissues from the tree correlates well with nootkatone content, suggesting that CnVS represents the first dedicated gene in the nootkatone biosynthetic pathway in C. nootkatensis The gene belongs to the gymnosperm-specific TPS-d subfamily of terpenes synthases and its protein sequence has low similarity to known citrus valencene synthases. In vitro, CnVS displays high robustness under different pH and temperature regimes, potentially beneficial properties for application in different host and physiological conditions. Biotechnological production of sesquiterpenes has been shown to be feasible, but productivity of microbial strains expressing valencene synthase from Citrus is low, indicating that optimization of valencene synthase activity is needed. Indeed, expression of CnVS in Saccharomyces cerevisiae indicated potential for higher yields. In an optimized Rhodobacter sphaeroides strain, expression of CnVS increased valencene yields 14-fold to 352 mg/L, bringing production to levels with industrial potential
Treatment of mouse liver slices with cholestatic hepatotoxicants results in down-regulation of Fxr and its target genes
Szalowska, E. ; Stoopen, G.M. ; Groot, M.J. ; Hendriksen, P.J.M. ; Peijnenburg, A.A.C.M. - \ 2013
BMC Medical Genomics 6 (2013). - ISSN 1755-8794 - 18 p.
farnesoid-x receptor - cyclosporine-a - bile-acid - rat-liver - intrahepatic cholestasis - expression analysis - primary hepatocytes - oxidative stress - drug - metabolism
Background Unexpected cholestasis substantially contributes to drug failure in clinical trials. Current models used for safety assessment in drug development do not accurately predict cholestasis in humans. Therefore, it is of relevance to develop new screening models that allow identifying drugs with cholestatic properties. Methods We employed mouse precision cut liver slices (PCLS), which were incubated 24 h with two model cholestatic compounds: cyclosporin A (CsA) and chlorpromazine (CPZ). Subsequently, transcriptome analysis using DNA microarrays and q-PCR were performed to identify relevant biological processes and biomarkers. Additionally, histology was carried out and levels of triglycerides (TG) and bile acids (BA) were measured. To verify the ex vivo mouse data, these were compared with publically available human data relevant for cholestasis. Results Whole genome gene expression analysis showed that CsA up-regulated pathways related to NF-¿B, ER stress and inflammation. Both CsA and CPZ down-regulated processes related to extracellular matrix (ECM) remodelling, BA homeostasis, Fxr signalling, and energy metabolism. The differential expression of a number of characteristic genes (e.g. Abcg5, Abcg8, Klf15, and Baat) could be confirmed by q-PCR. Histology revealed that CsA but not CPZ induced “ballooning” of hepatocytes. No effects on TG and BA levels were observed after incubation of PCLS with CsA and CPZ. A substantial number of processes altered in CsA- and CPZ-treated mouse PCLS ex vivo was also found to be affected in liver biopsies of cholestatic patients. Conclusion The present study demonstrated that mouse PCLS can be used as a tool to identify mechanisms of action of cholestatic model compounds. The induction of general stress responses and down-regulated Fxr signalling could play a role in the development of drug induced cholestasis. Importantly, comparative data analysis showed that the ex vivo mouse findings are also relevant for human pathology. Moreover, this work provides a set of genes that are potentially useful to assess drugs for cholestatic properties.
Effect of oxygen concentration and selected protocol factors on viability and gene expression of mouse liver slices
Szalowska, E. ; Stoopen, G.M. ; Rijk, J.C.W. ; Wang, S. ; Hendriksen, P.J.M. ; Groot, M.J. ; Ossenkoppele, J.S. ; Peijnenburg, A.A.C.M. - \ 2013
Toxicology in Vitro 27 (2013)5. - ISSN 0887-2333 - p. 1513 - 1524.
precision-cut liver - toxicity - metabolism - culture - glucose - tension - systems - cells - toxicogenomics - activation
Precision cut liver slices (PCLSs) are widely used as a model to study hepatotoxicity. For culturing of PCLS diverse protocols are used which could affect slices viability and results. We aimed to identify the most optimal culture protocol for mouse PCLS. Slices were cultured for 24 h under different concentrations of serum, glucose, insulin, and oxygen. Thereafter, slices viability was assessed by biochemical methods. Transcriptome analysis was performed to identify changes introduced by culture at different oxygen concentrations (20%, 40%, 60%, and 80% of oxygen). Medium composition did not affect the slices viability. Although metabolic competence was unaffected by oxygen concentrations, culturing at 80% of oxygen yielded slices with the best biochemical characteristics. The comparison of uncultured vs. cultured slices revealed 2524 genes to be differentially expressed. Genes involved in drug metabolism, peroxisomal and mitochondrial functions were down-regulated while several adaptive/stress response processes were up-regulated. Moreover, 80% of oxygen was the most favorable condition with respect to maintenance of expression of genes involved in drug and energy metabolism. The outcome of this study indicates that mouse PCLS are a valuable tool in research on hepatic functions and toxicity, particularly if they are cultured under a controlled oxygen concentration of 80%.
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