Transcriptomic signatures of peroxisome proliferator-activated receptor a (PPARa) in different mouse liver models identify novel aspects of its biology
Szalowska, E. ; Tesfay, H.A. ; Hijum, S.A.F.T. van; Kersten, A.H. - \ 2014
BMC Genomics 15 (2014). - ISSN 1471-2164 - 14 p.
fatty-acid transport - kupffer cells - gene-expression - microarray experiments - primary hepatocytes - insulin-resistance - rat-liver - inflammation - secretion - agonists
Background The peroxisome proliferator-activated receptor alpha (PPARa) is a ligand-activated transcription factor that regulates lipid catabolism and inflammation and is hepatocarcinogenic in rodents. It is presumed that the functions of PPARa in liver depend on cross-talk between parenchymal (hepatocytes) and non-parenchymal (Kupffer and endothelial cells) fractions as well as inter-organ interactions. In order to determine how cellular composition and inter-organ interactions influence gene expression upon pharmacological activation of PPARa, we performed a meta-analysis of transcriptomics data obtained from mouse hepatocytes (containing only the parenchymal fraction), mouse liver slices (containing both fractions), and mouse livers exposed to a PPARa agonist. The aim was to obtain a comprehensive view of common and model-specific PPARa-dependent genes and biological processes to understand the impact of cross-talk between parenchymal and non-parenchymal fractions as well as the effect of inter-organ interactions on the hepatic PPARa transcriptome. To this end we analyzed microarray data of experiments performed in mouse primary hepatocytes treated with the PPARa agonist Wy14643 for 6 or 24 h (in vitro), mouse precision cut liver slices treated with Wy14643 for 24 h (ex vivo), and livers of wild type and Ppara knockout mice treated with Wy14643 for 6 h or 5 days (in vivo). Results In all models, activation of PPARa significantly altered processes related to various aspects of lipid metabolism. In ex vivo and in vivo models, PPARa activation significantly regulated processes involved in inflammation; these processes were unaffected in hepatocytes. Only in vivo models showed significant regulation of genes involved in coagulation, carcinogenesis, as well as vesicular trafficking and extracellular matrix. Conclusions PPARa-dependent regulation of genes/processes involved in lipid metabolism is mostly independent of the presence of non-parenchymal cells or systemic factors, as it was observed in all liver models. PPARa-dependent regulation of inflammatory genes requires the presence of non-parenchymal cells, as it was observed only ex vivo and in vivo. However, the full spectrum of PPARa biology at the level of lipid metabolism, immunity, carcinogenesis, as well as novel aspects of PPARa signaling such as coagulation, vesicular trafficking and the extracellular matrix, seems to require systemic factors, as it was observed exclusively in vivo.
Model Steatogenic Compounds (Amiodarone, Valproic Acid, and Tetracycline) Alter Lipid Metabolism by Different Mechanisms in Mouse Liver Slices
Szalowska, E. ; Burg, B. van der; Man, H.Y. ; Hendriksen, P.J.M. ; Peijnenburg, A.A.C.M. - \ 2014
PLoS ONE 9 (2014)1. - ISSN 1932-6203 - 15 p.
gene-expression - rat-liver - hepatic steatosis - heparg cells - mitochondrial dysfunction - therapeutic targets - induced cholestasis - oxidative stress - fatty-acids - drug
Although drug induced steatosis represents a mild type of hepatotoxicity it can progress into more severe non-alcoholic steatohepatitis. Current models used for safety assessment in drug development and chemical risk assessment do not accurately predict steatosis in humans. Therefore, new models need to be developed to screen compounds for steatogenic properties. We have studied the usefulness of mouse precision-cut liver slices (PCLS) as an alternative to animal testing to gain more insight into the mechanisms involved in the steatogenesis. To this end, PCLS were incubated 24 h with the model steatogenic compounds: amiodarone (AMI), valproic acid (VA), and tetracycline (TET). Transcriptome analysis using DNA microarrays was used to identify genes and processes affected by these compounds. AMI and VA upregulated lipid metabolism, whereas processes associated with extracellular matrix remodelling and inflammation were downregulated. TET downregulated mitochondrial functions, lipid metabolism, and fibrosis. Furthermore, on the basis of the transcriptomics data it was hypothesized that all three compounds affect peroxisome proliferator activated-receptor (PPAR) signaling. Application of PPAR reporter assays classified AMI and VA as PPAR¿ and triple PPARa/(ß/d)/¿ agonist, respectively, whereas TET had no effect on any of the PPARs. Some of the differentially expressed genes were considered as potential candidate biomarkers to identify PPAR agonists (i.e. AMI and VA) or compounds impairing mitochondrial functions (i.e. TET). Finally, comparison of our findings with publicly available transcriptomics data showed that a number of processes altered in the mouse PCLS was also affected in mouse livers and human primary hepatocytes exposed to known PPAR agonists. Thus mouse PCLS are a valuable model to identify early mechanisms of action of compounds altering lipid metabolism
Effect of reduced food intake on toxicokinetics of halogenated organic contaminants in herring gull (Larus argentatus) chicks
Routti, H. ; Helgason, L.B. ; Arukwe, A. ; Wolkers, J. ; Heimstad, E.S. ; Harju, M. ; Berg, V. ; Gabrielsen, G.W. - \ 2013
Environmental Toxicology and Chemistry 32 (2013)1. - ISSN 0730-7268 - p. 156 - 164.
arctic seabirds - polychlorinated-biphenyls - organochlorine contaminants - o-dealkylation - rat-liver - biotransformation - blood - bioaccumulation - metabolites - pcbs
The aim of the present study was to investigate how contaminant exposure and reduced food intake affect tissue distribution and biotransformation of halogenated organic contaminants (HOCs) in Arctic seabirds using herring gull (Larus argentatus) as a model species. Herring gull chicks were exposed for 44 d to cod liver oil containing a typical mixture of contaminants. Following exposure, food intake was reduced for a one-week period in a subgroup of the chicks. Polyclorinated biphenyls, organochlorine pesticides, and brominated flame retardants, as well as a wide range of hydroxy, methyl sulfone, and methoxy compounds were measured in liver, brain, and plasma samples. Additionally, phase I biotransformation enzyme activities and phase I and II messenger ribonucleic acid (mRNA) expression were investigated in the liver, brain, or both. Both contaminant exposure and reduced food intake had an increasing effect on the concentrations of HOCs and their metabolites. The HOC exposure and reduced food intake also led to increased 7-ethoxyresorufin-O-deethylation (EROD) activity, whereas mRNA expression of the biotransformation enzymes increased only following the reduced food intake. Tissue distribution of HOCs and their metabolites was not affected by either contaminant exposure or reduced food intake. In conclusion, the results indicate that biotransformation capacity and formation of HOC metabolites increase during reduced food intake. This finding supports the hypothesis that reduced food intake increases the susceptibility of Arctic animals to the effects of lipophilic HOCs. Environ. Toxicol. Chem. 2013;32:156164. (c) 2012 SETAC
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.
A role for the peroxisomal 3-ketoacyl-CoA thiolase B enzyme in the control of PPAR alpha-mediated upregulation of SREBP-2 target genes in the liver
Fidaleo, M. ; Arnauld, S. ; Clemencet, M.C. ; Chevillard, G. ; Royer, M.C. ; Bruycker, M. de; Wanders, R.J.A. ; Athias, A. ; Gresti, J. ; Clouet, P. ; Degrace, P. ; Kersten, A.H. ; Espeel, M. ; Latruffe, N. ; Nicolas-Frances, V. ; Mandard, S. - \ 2011
Biochimie 93 (2011)5. - ISSN 0300-9084 - p. 876 - 891.
acyl-coa oxidase - proliferator-activated receptors - element-binding proteins - fatty-acid oxidation - beta-oxidation - rat-liver - cholesterol-synthesis - 3-oxoacyl-coa thiolase - neuronal migration - zellweger-syndrome
Peroxisomal 3-ketoacyl-CoA thiolase B (Thb) catalyzes the final step in the peroxisomal beta-oxidation of straight-chain acyl-CoAs and is under the transcription control of the nuclear hormone receptor PPAR alpha. PPARa binds to and is activated by the synthetic compound Wy14,643 (Wy). Here, we show that the magnitude of Wy-mediated induction of peroxisomal beta-oxidation of radiolabeled (1-(14)C) palmitate was significantly reduced in mice deficient for Thb. In contrast, mitochondrial beta-oxidation was unaltered in Thb(-1-) mice. Given that Wy-treatment induced Acox1 and MFP-1/-2 activity at a similar level in both genotypes, we concluded that the thiolase step alone was responsible for the reduced peroxisomal beta-oxidation of fatty acids. Electron microscopic analysis and cytochemical localization of catalase indicated that peroxisome proliferation in the liver after Wy-treatment was normal in Thb(-/-) mice. Intriguingly, micro-array analysis revealed that mRNA levels of genes encoding cholesterol biosynthesis enzymes were upregulated by Wy in Wild-Type (WT) mice but not in Thb(-1-) mice, which was confirmed at the protein level for the selected genes. The non-induction of genes encoding cholesterol biosynthesis enzymes by Wy in Thb(-1-) mice appeared to be unrelated to defective SREBP-2 or PPARa signaling. No difference was observed in the plasma lathosterol/cholesterol ratio (a marker for de novo cholesterol biosynthesis) between Wy-treated WT and Thb(-1-) mice, suggesting functional compensation. Overall, we conclude that ThA and SCPx/SCP2 thiolases cannot fully compensate for the absence of ThB. In addition, our data indicate that ThB is involved in the regulation of genes encoding cholesterol biosynthesis enzymes in the liver, suggesting that the peroxisome could be a promising candidate for the correction of cholesterol imbalance in dyslipidemia.
Application of toxicogenomics in hepatic systems toxicology for risk assessment: Acetaminophen as a case study
Kienhuis, A.S. ; Bessems, J.G.M. ; Pennings, J.L.A. ; Driessen, M. ; Luijten, M. ; Delft, J.H.M. van; Ven, L.T.M. van der - \ 2011
Toxicology and Applied Pharmacology 250 (2011). - ISSN 0041-008X - p. 96 - 107.
gene-expression - rat-liver - induced hepatotoxicity - end-points - toxicity - profiles - hepatocytes - mechanisms - ontology - reveals
Hepatic systems toxicology is the integrative analysis of toxicogenomic technologies, e.g., transcriptomics, proteomics, and metabolomics, in combination with traditional toxicology measures to improve the understanding of mechanisms of hepatotoxic action. Hepatic toxicology studies that have employed toxicogenomic technologies to date have already provided a proof of principle for the value of hepatic systems toxicology in hazard identification. In the present review, acetaminophen is used as a model compound to discuss the application of toxicogenomics in hepatic systems toxicology for its potential role in the risk assessment process, to progress from hazard identification towards hazard characterization. The toxicogenomics-based parallelogram is used to identify current achievements and limitations of acetaminophen toxicogenomic in vivo and in vitro studies for in vitro-to-in vivo and interspecies comparisons, with the ultimate aim to extrapolate animal studies to humans in vivo. This article provides a model for comparison of more species and more in vitro models enhancing the robustness of common toxicogenomic responses and their relevance to human risk assessment. To progress to quantitative dose–response analysis needed for hazard characterization, in hepatic systems toxicology studies, generation of toxicogenomic data of multiple doses/concentrations and time points is required. Newly developed bioinformatics tools for quantitative analysis of toxicogenomic data can aid in the elucidation of dose-responsive effects. The challenge herein is to assess which toxicogenomic responses are relevant for induction of the apical effect and whether perturbations are sufficient for the induction of downstream events, eventually causing toxicity.
Stabilization and immobilization of Trypanosoma brucei ornithine decarboxylase for the biobased production of 1,4-diaminobutane
Könst, P.M. ; Franssen, M.C.R. ; Scott, E.L. ; Sanders, J.P.M. - \ 2011
Green Chemistry 13 (2011)5. - ISSN 1463-9262 - p. 1167 - 1174.
nitrogen-containing chemicals - rat-liver - ralstonia-eutropha - pseudomonas-putida - escherichia-coli - amino-acids - covalent immobilization - cyanophycin synthetase - recombinant strains - thiol compounds
Using the biorefinery concept, L-arginine could become widely available from biomass waste streams via the nitrogen storage polypeptide cyanophycin. In our pursuit to develop a route from biobased L-arginine to 1,4-diaminobutane, one of the monomers in nylon-4,6, we were previously successful in the stabilization and immobilization of Bacillus subtilis arginase. In the present study, we investigated the stabilization and immobilization of Trypanosoma brucei ornithine decarboxylase (EC 18.104.22.168) (TbODC) for its application in the decarboxylation of L-ornithine, the final step in the envisioned route towards 1,4-diaminobutane. The stability observed for TbODC in vitro was substantially improved upon addition of dithiothreitol (DTT), which not only has a stabilizing, but also an activating effect. For optimal TbODC performance, the pH should be controlled at pH 8 and the ionic strength should be kept to a minimum. The temperature for optimal productivity is 40 °C. Immobilization of TbODC on Sepabeads EC-HFA was most successful, leading to an almost three-fold improvement in operational stability as compared to the soluble enzyme. Overall, we demonstrated that by optimization of reaction conditions and covalent immobilization the productivity of TbODC was vastly improved, opening up possibilities for its application in the biobased production of 1,4-diaminobutane
Flavonoids and alkenylbenzenes; new concepts in bioactivation studies
Rietjens, I.M.C.M. ; Al-Husainy, W.A.A.M. ; Boersma, M.G. - \ 2011
Chemico-Biological Interactions 192 (2011)1-2. - ISSN 0009-2797 - p. 87 - 95.
mediated gene-expression - quercetin quinone methide - dna-adducts - human liver - rat-liver - estragole bioactivation - intestinal-absorption - anticancer properties - metabolic stability - safety assessment
The present paper focuses on the biological reactive intermediates formed from two categories of botanical ingredients: flavonoids and alkenylbenzenes. The paper especially presents an overview of three concepts in bioactivation studies on flavonoids and alkenylbenzenes elucidated by our recent studies. These new concepts include (i) the fact that reactive electrophilic quinone/quinone methide type metabolites of flavonoids may be the intermediates required for the induction of the beneficial gene expression through electrophile responsive element (EpRE)-mediated pathways, pointing at a possible beneficial effect of a reactive intermediate, (ii) the development of physiologically based kinetic (PBK) and physiologically based dynamic (PBD) models providing a new way to obtain insight in levels of formation of biologically reactive and unstable intermediates in vivo at high but also more realistic low dose levels, and (iii) the concept of the matrix effect that should be taken into account when studying the bioactivation of food-borne genotoxic carcinogens including the alkenylbenzenes, the bioactivation of which was shown to be inhibited by flavonoids. Together the results presented reveal that by studying the mode of action (MOA) new concepts in bioactivation studies of importance for future risk assessment and/or risk-benefit assessment of the flavonoids and alkenylbenzenes are obtained.
Absorption, distribution and biliary excretion of cafestol, a potent cholesterol elevating compound in unfiltered coffees in mice
Cruchten, S.T.J. van; Waart, D.R. de; Kunne, C. ; Hooiveld, G.J.E.J. ; Boekschoten, M.V. ; Katan, M.B. ; Oude Elferink, R.P.J. ; Witkamp, R.F. - \ 2010
Drug Metabolism and Disposition 38 (2010)4. - ISSN 0090-9556 - p. 635 - 640.
chemopreventive components kahweol - raising factor - liver aminotransferases - diterpenes cafestol - serum-lipids - rat-liver - metabolism - 4-ipomeanol - enzymes - beans
Cafestol is a diterpene present in unfiltered coffees. It is the most potent cholesterol-elevating compound present in the human diet. However, the precise mechanisms underlying this effect are still unclear. In contrast, cafestol is also known as a hepatoprotective compound which is likely to be related to the induction of glutathione biosynthesis and conjugation. In the present study we investigated whole body distribution, biliary excretion and portal bioavailability of cafestol in mice. First, dissection was used to study distribution. Five hours after an oral dose with 3H labeled cafestol, most activity was found in small intestine, liver and bile. These results were confirmed by quantitative whole body autoradiography in a time course study which also showed elimination of all radioactivity within 48 hours after administration. Next, radiolabeled cafestol was dosed i.v. to bile duct cannulated mice. Five hours post dose 20% of the radioactivity was found in bile. Bile contained several metabolites but no parent compound. After intestinal administration of radioactive cafestol to portal vein cannulated mice, cafestol was shown to be rapidly absorbed into the portal vein as parent compound, a glucuronide and an unidentified metabolite. From the presence of a glucuronide in bile that can be deconjugated by a bacterial enzyme and the prolonged absorption of parent compound from the GI tract we hypothesize that cafestol undergoes enterohepatic cycling. Together with our earlier observation that epoxidation of the furan ring occurs in liver these findings merit further research on the process of accumulation of this coffee ingredient in liver and intestinal tract
The role of epoxidation and electrophile-responsive element-regulated gene transcription in the potentially beneficial and harmful effects of the coffee components cafestol and kahweol
Cruchten, S.T.J. van; Haan, L.H.J. de; Mulder, P.P.J. ; Kunne, C. ; Boekschoten, M.V. ; Katan, M.B. ; Aarts, J.M.M.J.G. ; Witkamp, R.F. - \ 2010
Journal of Nutritional Biochemistry 21 (2010)8. - ISSN 0955-2863 - p. 757 - 763.
cholesterol-raising factor - nf-kappa-b - rat-liver - glutathione transferase - normolipidemic subjects - diterpenes cafestol - boiled coffee - serum-lipids - cancer-risk - metabolism
Cafestol and kahweol are diterpene compounds present in unfiltered coffees. Cafestol is known as the most potent cholesterol-raising agent that may be present in the human diet. Remarkably, the mechanisms behind this effect have only been partly resolved so far. Even less is known about the metabolic fate of cafestol and kahweol. From the structure of cafestol, carrying a furan moiety, we hypothesized that epoxidation may not only be an important biotransformation route but that this also plays a role in its effects found. In bile duct-cannulated mice, dosed with cafestol, we were able to demonstrate the presence of epoxy-glutathione (GSH) conjugates, GSH conjugates and glucuronide conjugates. In addition, it was shown that cafestol was able to induce an electrophile-responsive element (EpRE). Using a murine hepatoma cell line with a luciferase reporter gene under control of an EpRE from the human NQO1 regulatory region, we also found that metabolic activation by CYP450 enzymes is needed for EpRE induction. Furthermore, raising intracellular GSH resulted in a decrease in EpRE-mediated gene induction, whereas lowering intracellular GSH levels increased EpRE-mediated gene induction. In conclusion, evidence suggests that cafestol induces EpRE, apparently via a bioactivation process that possibly involves epoxidation of the furan ring. The epoxides themselves appear subject to conjugation with GSH. The effects on EpRE can also explain the induction of GSH which seems to be involved in the reported beneficial effects of cafestol, for example, when administered with aflatoxin B1 or other toxic or carcinogenic compounds
Short communication: Genome-wide scan for bovine milk-fat composition. II. Quantitative trait loci for long-chain fatty acids
Schennink, A. ; Stoop, W.M. ; Visker, M.H.P.W. ; Poel, J.J. van der; Bovenhuis, H. ; Arendonk, J.A.M. van - \ 2009
Journal of Dairy Science 92 (2009)9. - ISSN 0022-0302 - p. 4676 - 4682.
dairy-cattle - genetic-parameters - f-2 population - rat-liver - fish-oil - dgat1 - polymorphism - trans - cows - qtl
We present the results of a genome-wide scan to identify quantitative trait loci (QTL) that contribute to genetic variation in long-chain milk fatty acids. Milk-fat composition phenotypes were available on 1,905 Dutch Holstein-Friesian cows. A total of 849 cows and their 7 sires were genotyped for 1,341 single nucleotide polymorphisms across all Bos taurus autosomes (BTA). We detected significant QTL on BTA14, BTA15, and BTA16: for C18:1 cis-9, C18:1 cis-12, C18:2 cis-9,12, CLA cis-9,trans-11, C18:3 cis-9,12,15, the C18 index, the total index, total saturated fatty acids, total unsaturated fatty acids (UFA), and the ratio of saturated fatty acids:unsaturated fatty acids on BTA14; for C18:1 trans fatty acids on BTA15; and for the C18 and CLA indices on BTA16. The QTL explained 3 to 19% of the phenotypic variance. Suggestive QTL were found on 16 other chromosomes. The diacylglycerol acyltransferase 1 (DGAT1) K232A polymorphism on BTA14, which is known to influence fatty acid composition, most likely explains the QTL that was detected on BTA14
Influence of TCDD and natural Ah receptor agonists on benzo[a]pyrene-DNA adduct formation in the Caco-2 human colon cell line
Waard, W.J. de; Kok, T.M.C.M. de; Maas, L.M. ; Peijnenburg, A.A.C.M. ; Hoogenboom, L.A.P. ; Aarts, H.J.M. ; Schooten, F.J. van - \ 2008
Mutagenesis 23 (2008)1. - ISSN 0267-8357 - p. 67 - 73.
aryl-hydrocarbon receptor - dna-adducts - cancer-risk - rat-liver - vegetables - cytochrome-p450 - activation - indole-3-carbinol - detoxication - carcinogen
Several compounds originating from cruciferous vegetables and citrus fruits bind to and activate the aryl hydrocarbon receptor (AhR). This receptor plays an important role in the toxicity of the known tumour promoter and potent AhR-agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, vegetables and fruits are generally considered as healthy. Therefore, besides the AhR activation, the natural AhR agonists (NAhRAs) are assumed to show other health-concerning effects. AhR activation induces several cytochrome P450 phase I enzymes involved, e.g. in the bioactivation of carcinogenic polycyclic aromatic hydrocarbons, like benzo[a]pyrene (BaP), and may as such stimulate DNA adduct formation of those compounds. Therefore, the influence of TCDD, indolo[3,2-b]carbazole (ICZ, an NAhRA originating from cruciferous vegetables) and an NAhRA-containing extract of grapefruit juice (GJE) on BaP¿DNA adduct formation in the human Caco-2 cell line was studied. Also, we investigated if different effects of TCDD, ICZ and GJE on adduct formation could be related to the modulation of transcription of biotransformation- and DNA-repair enzymes. Co-exposure to high AhR-activating concentrations of both TCDD and ICZ significantly reduced the amount of BaP¿DNA adducts at 0.1 µM BaP, while at higher concentrations of BaP no influence was observed. In contrast, exposure to 0.1 µM BaP combined with GJE showed a significant increase in BaP¿DNA adducts, and a significant decrease at 0.3 and 1 µM BaP. These differences could not be related to transcription of the phase I and II enzymes CYP1A1, CYP1B1, NQO1, GSTP1 and UGT1A6 or to transcription of the nucleotide excision repair enzymes ERCC1, XPA, XPC, XPF and XPG. We conclude that ICZ showed a similar effect on BaP¿DNA adduct formation than TCDD, while GJE influenced the adduct formation in a different way. The difference in the influence on adduct formation may be due to effects at the level of enzyme activity, rather than gene expression
Human glutathione S-transferase-mediated glutathione conjugation of curcumin and efflux of these conjugates in Caco-2 cells
Usta, M. ; Wortelboer, H.M. ; Vervoort, J.J.M. ; Boersma, M.G. ; Rietjens, I.M.C.M. ; Bladeren, P.J. van; Cnubben, N.H.P. - \ 2007
Chemical Research in Toxicology 20 (2007)12. - ISSN 0893-228X - p. 1895 - 1902.
cancer cells - chemopreventive agent - antioxidant mechanism - drug transporters - dietary curcumin - ethacrynic-acid - rat-liver - apoptosis - enzymes - proliferation
Curcumin, an alpha,beta-unsaturated carbonyl compound, reacts with glutathione, leading to the formation of two monoglutathionyl curcumin conjugates. In the present study, the structures of both glutathione conjugates of curcumin were identified by LC-MS and one- and two-dimensional H-1 NMR analysis, and their formation in incubations with human intestinal and liver cytosol and purified human glutathione S-transferases and also in human Caco-2 cells was characterized. The results obtained demonstrate the site for glutathione conjugation to be the C1 atom, leading to two diastereoisomeric monoglutathionyl curcumin conjugates (CURSG-1 and CURSG-2). The formation of both glutathionyl conjugates appeared to be reversible. The monoglutathionyl curcumin conjugates decompose with a t(1/2) of about 4 h to curcumin and other unidentified degradation products. Both human intestinal and liver cytosol catalyzed curcumin glutathione conjugation. At saturating substrate concentrations, human GSTM1a-1a and GSTA1-1 are shown to be especially active in the formation of CURSG-1, whereas GSTP1-1 and GSTA2-2 have no preference for the formation of CURSG-1 or CURSG-2. GSTT1-1 hardly catalyzes the glutathione conjugation of curcumin. In the Caco-2 human intestinal monolayer transwell model, CURSG-1 and CURSG-2 were formed at a ratio of about 2:1 followed by their excretion, which appeared to be three times higher to the apical (lumen) side than to the basolateral (blood) side. Given that GSTM1a-1a and GSTP1-1 are present in the intestinal epithelial cells, it can be concluded that efficient glutathione conjugation of curcumin may already occur in the enterocytes, followed by an efficient excretion of these glutathione conjugates to the lumen, thereby reducing the bioavailability of (unconjugated) curcumin. In conclusion, the present study identifies the nature of the diastereoisomeric monoglutathionyl curcumin conjugates, CURSG-1 and CURSG-2 formed in biological systems, and reveals that conjugate formation is catalyzed by GSTM1a-1a, GSTA1-1, and/or GSTP1-1 with different stereoselective preference. The formation of glutathione conjugates can already occur during intestinal transport, after which the monoglutathionyl conjugates are efficiently excreted to the intestinal lumen, thereby influencing the bioavailability of curcumin and, as a result, its beneficial biological effects.
An antibody produced in tobacco expressing a hybrid ß-1,4-galactosyltransferase is essentially devoid of plant carbohydrate epitopes
Bakker, H. ; Rouwendal, G.J.A. ; Karnoup, A.S. ; Florack, D.E.A. ; Stoopen, G.M. ; Helsper, J.P.F.G. ; Ree, R. van; Die, I. van; Bosch, H.J. - \ 2006
Proceedings of the National Academy of Sciences of the United States of America 103 (2006)20. - ISSN 0027-8424 - p. 7577 - 7582.
desorption/ionization mass-spectrometry - asparagine-linked oligosaccharides - rat-liver - immunoglobulin-g - n-glycans - substrate-specificity - endoplasmic-reticulum - developmental-stage - transgenic plants - mannosidase-ii
N-glycosylation of a mAb may have a major impact on its therapeutic merits. Here, we demonstrate that expression of a hybrid enzyme (called xylGalT), consisting of the N-terminal domain of Arabidopsis thaliana xylosyltransferase and the catalytic domain of human -1,4-galactosyltransferase I (GalT), in tobacco causes a sharp reduction of N-glycans with potentially immunogenic core-bound xylose (Xyl) and fucose (Fuc) residues as shown by Western blot and MALDI-TOF MS analysis. A radioallergosorbent test inhibition assay with proteins purified from leaves of WT and these transgenic tobacco plants using sera from allergic patients suggests a significant reduction of potential immunogenicity of xylGalT proteins. A mAb purified from leaves of plants expressing xylGalT displayed an N-glycan profile that featured high levels of galactose, undetectable xylose, and a trace of fucose. Hence, a transgenic plant expressing the hybrid GalT might yield more effective and safer monoclonals for therapeutic purposes than WT plants and even transgenic plants expressing the unchanged GalT.
Choline supplemented as phosphatidylcholine decreases fasting and postmethionine-loading plasma homocysteine concentrations in healthy men
Olthof, M.R. ; Brink, E.J. ; Katan, M.B. ; Verhoef, P. - \ 2005
American Journal of Clinical Nutrition 82 (2005)1. - ISSN 0002-9165 - p. 111 - 117.
phosphatidylethanolamine n-methyltransferase - methotrexate treatment - rat-liver - parenteral-nutrition - vascular-disease - dna methylation - folic-acid - deficiency - betaine - folate
Background: A high homocysteine concentration is a potential risk factor for cardiovascular disease that can be reduced through betaine supplementation. Choline is the precursor for betaine, but the effects of choline supplementation on plasma total homocysteine (tHcy) concentrations in healthy humans are unknown. Objective: The objective was to investigate whether supplementation with phosphatidylcholine, the form in which choline occurs in foods, reduces fasting and postmethionine-loading concentrations of plasma tHcy in healthy men with mildly elevated plasma tHcy concentrations. Design: In a crossover study, 26 men ingested 2.6 g choline/d (as phosphatidylcholine) or a placebo oil mixture for 2 wk in random order. Fatty acid composition and fat content were similar for both treatments. A methionine-loading test was performed on the first and last days of each supplementation period. Results: Phosphatidylcholine supplementation for 2 wk decreased mean fasting plasma tHcy by 18% (¿3.0 µmol/L; 95% CI: ¿3.9, ¿2.1 µmol/L). On the first day of supplementation, a single dose of phosphatidylcholine containing 1.5 g choline reduced the postmethionine-loading increase in tHcy by 15% (¿4.8 µmol/L; 95% CI: ¿6.8, ¿2.8 µmol/L). Phosphatidylcholine supplementation for 2 wk reduced the postmethionine-loading increase in tHcy by 29% (¿9.2 µmol/L; 95% CI: ¿11.3, ¿7.2 µmol/L). All changes were relative to placebo. Conclusions: A high daily dose of choline, supplemented as phosphatidylcholine, lowers fasting as well as postmethionine-loading plasma tHcy concentrations in healthy men with mildly elevated tHcy concentrations. If high homocysteine concentrations indeed cause cardiovascular disease, choline intake may reduce cardiovascular disease risk in humans
Alkoxyresorufin-O-deethylase activities and polychlorinated biphenyl patterns in shrews as biomarkers in environmental risk assessments: sensitivity and specificity
Brink, N.W. van den; Bosveld, A.T.C. - \ 2005
Environmental Science and Technology 39 (2005)18. - ISSN 0013-936X - p. 7337 - 7343.
rat-liver - cytochrome-p450 induction - small mammals - exposure - metabolism - organochlorine - netherlands - earthworms - habitats - mink
Alkoxyresorufin-O-deethylase (AROD) biomarkers are useful indicators of the exposure of organisms to dioxin-like compounds. In the current study, an in vivo validation of the use of such biomarkers in shrews was conducted. Furthermore, the use of changes in polychlorinated biphenyl (PCB) patterns as an animal-friendly alternative to AROD biomarkers was evaluated. Two experiments and a field study were conducted in which dose-response relations were established between levels of Sigma-PCBs in shrews on one hand and their AROD activities and changes in PCB patterns on the other. We demonstrate that the changes in PCB patterns are as sensitive as the classic AROD biomarkers. The experiments also showed a substrate-specific induction of AROD biomarkers and a related PCB congener-specific metabolism. This implies that congener-specific analysis of PCBs can reveal activities of specific AROD biomarkers. Gender-specific induction of AROD activities in shrews was shown in the field study, whereas the relationship between exposure and changes in PCB patterns did not differ between genders. It is concluded that (i) AROD biomarkers are useful biomarkers to assess exposure of shrews to specific organochlorines and that (ii) changes in PCB patterns can be used as an animal-friendly alternative to these AROD biomarkers.
Studies on the DT-diaphorase-catalysed reaction employing quinones as substrates: evidence for a covalent modification of DT-diaphorase by tetrachloro-p-benzoquinone
Osman, A.M. ; Boeren, J.A. - \ 2004
Chemico-Biological Interactions 147 (2004)1. - ISSN 0009-2797 - p. 99 - 108.
one-electron - rat-liver - acceptor oxidoreductase - mechanism - reductase - nitrobenzimidazoles - cytotoxicity - conversion - protein - nad(p)h
In this study, the kinetic parameters, Vmax and Km, of rat liver DT-diaphorase were determined for a series of p-benzoquinones, with methyl, methoxy, cyano, hydroxy and halo substituents. The results show that there is no correlation between the experimentally determined rates of p-benzoquinone reduction by DT-diaphorase and the calculated chemical reactivity of the examined substrates as expressed by the energy of the lowest unoccupied molecular orbital, E(LUMO). However, a reasonable correlation was found between the natural logarithm of Vmax/Km and the partition coefficient of the p-benzoquinones (r=0.81). Furthermore, tetrachloro-p-benzoquinone, one of the tested quinones is shown to be an inhibitor of rat DT-diaphorase. The presence of bovine serum albumin (BSA) in the incubation mixture protects DT-diaphorase against the inactivation by tetrachloro-p-benzoquinone, probably by interacting with the quinone. Maldi-Tof analysis of the incubation mixture of the purified DT-diaphorase and tetrachloro-p-benquinone showed that every subunit of the enzyme shifted about +414 amu, whereas the dimer shifted about +849 amu relative to control values. This indicates a covalent modification of the rat liver DT-diaphorase by tetrachloro-p-benzoquinone.
A new hypotensive polyunsaturated fatty acid dietary combination regulates oleic acid accumulation by suppression of stearoyl CoA desaturase 1 gene expression in the SHR model of genetic hypertension
Bellenger, J. ; Bellenger, S. ; Clement, L. ; Mandard, S.J. ; Diot, C. ; Poisson, J.P. ; Narce, M. - \ 2004
FASEB Journal 18 (2004)2. - ISSN 0892-6638 - p. 773 - +.
element-binding protein-1 - liver-microsomes - rat-liver - delta-6-desaturase activity - peroxidative mechanism - docosahexaenoic acid - delta-6 desaturase - lipid-composition - synthase - mice
Polyunsaturated fatty acids (PUFA) are known to repress SCD-1 gene expression, key enzyme of monounsaturated fatty acid biosynthesis. Alterations of the monounsaturated/saturated fatty acids ratio have been implicated in various diseases related to the metabolic syndrome, including hypertension. We previously evidenced that lipogenesis end-products accumulated in spontaneously hypertensive rats (SHR), and that a dietary combination of n-6/n-3 PUFA had hypotensive effects. Our present objective was to test the hypothesis that these SHR liver lipid disorders might be modulated, in response to this hypotensive combination, by changes in SCD-1 expression and activity. So we studied, in hepatocytes, SCD-1 transcription by Northern blotting, as well as plasma and liver fatty acid composition by gas-liquid chromatography. Liver SCD-1 gene expression was suppressed by 50%, and in different lipid classes, relative abundance of stearic and oleic acids decreased. Consequently, the Delta9 desaturation index, calculated from the ratio of oleic vs. stearic acids, decreased. In addition, the level of circulating saturated fatty acids decreased when one of oleic acids increased. These data provided evidence that the tested hypotensive PUFA combination reverses the high monounsaturated/saturated fatty acids ratio associated to hypertension in SHR, via a regulation monounsaturated fatty acid relative abundance by repression of SCD-1 gene.
Bromobenzene-induced hepatotoxicity at the transcriptome level
Heijne, W.H.M. ; Slitt, A.L. ; Bladeren, P.J. van; Groten, J.P. ; Klaassen, C.D. ; Stierum, R.H. ; Ommen, B. van - \ 2004
Toxicological sciences 79 (2004)2. - ISSN 1096-6080 - p. 411 - 422.
glyceraldehyde-3-phosphate dehydrogenase gene - acute-phase response - rat-liver - glutathione - element - identification - expression - induction - binding - acetaminophen
Rats were exposed to three levels of bromobenzene, sampled at 6, 24, and 48 h, and liver gene expression profiles were determined to identify dose and time-related changes. Expression of many genes changed transiently, and dependent on the dose. Few changes were identified after 6 h, but many genes were differentially expressed after 24 h, while after 48 h, only the high dose elicited large effects. Differentially expressed genes were involved in drug metabolism (upregulated GSTs, mEH, NQO1, Mrps, downregulated CYPs, sulfotransferases), oxidative stress (induced HO-1, peroxiredoxin, ferritin), GSH depletion (induced GCS-l, GSTA, GSTM) the acute phase response, and in processes like cholesterol, fatty acid and protein metabolism, and intracellular signaling. Trancriptional regulation via the electrophile and sterol response elements seemed to mediate part of the response to bromobenzene. Recovery of the liver was suggested in response to BB by the altered expression of genes involved in protein synthesis and cytoskeleton rearrangement. Furthermore, after 48 h, rats in the mid dose group showed no toxicity, and gene expression patterns resembled the normal situation. For certain genes (e.g., CYP4A, metallothioneins), intraday variation in expression levels was found, regardless of the treatment. Selected cDNA microarray measurements were confirmed using the specific and sensitive branched DNA signal amplification assay.
A progressive familial intrahepatic cholestasis type 2 mutation causes an unstable, temperature-sensitive bile salt export pump
Plass, J.R.M. ; Mol, O. ; Heegsma, J. ; Geuken, M. ; Elling, G. ; Müller, M.R. - \ 2004
Journal of Hepatology 40 (2004)1. - ISSN 0168-8278 - p. 24 - 30.
transmembrane conductance regulator - canalicular membrane-vesicles - p-glycoprotein - rat-liver - substrate-specificity - dependent transport - atp - expression - mrp1 - taurocholate
Background Aims: Progressive familial intrahepatic cholestasis type 2 (PFIC-2) patients have a defect in the hepatocanalicular bile salt secretion. The disease is caused by mutations in the bile salt export pump (BSEP). Ten different missense mutations have been described. In this study, we analysed the effect of the D482G PFIC-2 mutation on BSEP function. Methods: Adenosine triphosphatase (ATPase) and taurocholate transport assays were performed with full-length mouse Bsep (mBsep) with and without the D482G mutation. The effect on expression and subcellular sorting was studied in HepG2 cells, stably expressing enhanced green fluorescent protein (EGFP)-tagged mBsep proteins. Results: The D482G mutation did not significantly affect the taurocholate transport activity of mBsep, even though the bile salt-inducible ATPase activity of the mutant protein was slightly reduced. Protein expression and canalicular sorting were strongly affected by the D482G mutation. Mutant EGFP-mBsep protein was only partly glycosylated and detected in both the canalicular membrane and the cytoplasm. At 30degreesC, the mutant mRNA and protein levels were strongly increased, and the protein was predominantly glycosylated and efficiently targeted to the canalicular membrane. Conclusions: These data suggest that PFIC-2 patients with the D482G mutation express a functional, but highly unstable, temperature-sensitive bile salt export pump. (C) 2003 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.