The influence of phase II conjugation on the biological activity of flavonoids
Beekmann, K. - \ 2016
Wageningen University. Promotor(en): Ivonne Rietjens; Peter van Bladeren, co-promotor(en): L. Actis-Goretta. - Wageningen : Wageningen University - ISBN 9789462577640 - 171
flavonoids - biological activity - in vitro - biosynthesis - peroxisomes - microarrays - daidzein - genistein - oestrogen receptors - isoflavones - quercetin - kaempferol - serine proteinases - threonine - flavonoïden - biologische activiteit - in vitro - biosynthese - peroxisomen - microarrays - daidzin - genisteïne - oestrogeenreceptoren - isoflavonen - quercetine - kaempferol - serine proteïnasen - threonine
Flavonoid consumption is often correlated with a wide range of health effects, such as the prevention of cardiovascular diseases, neurodegenerative diseases, and diabetes. These effects are usually ascribed to the activity of the parent flavonoid aglycones, even though these forms of the flavonoids generally have a low systemic bioavailability. During uptake, flavonoids undergo phase II metabolism and are present in the systemic circulation nearly exclusively as conjugated metabolites. The aim of this thesis was to study the effect of conjugation on the biological activity of selected flavonoids towards different endpoints relevant for human health. To this end, conjugation with glucuronic acid was taken as the model type of conjugation because this modification is generally observed to be the most important metabolic conjugation reaction for flavonoids in man.
A review of scientific literature published until early 2012 reveals that metabolic conjugation can affect the biological activity of flavonoids in different ways. Conjugation can increase, decrease, inverse or not affect the biological activity, depending on the flavonoid, the type and position of conjugation, the endpoint studied, and the assay system used. Based on the literature reviewed it is concluded that the effect of conjugation has to be studied on a case-by-case basis.
As the research on the biological activity of biologically relevant flavonoid conjugates is often hampered by the generally low commercial availability and high prices of these conjugates, a simple and versatile method for the biosynthesis of metabolically relevant flavonoid conjugates is described. Using this method, relevant conjugates can be prepared from different flavonoid substrates in sufficient quantities for in vitro bioassays. Further, an efficient strategy for the identification of these flavonoid conjugates by LC-MS and 1H-NMR using MetIDB (Metabolite Identification Database), a publicly accessible database of predicted and experimental 1H-NMR spectra of flavonoids, is presented.
To study the effect of conjugation on the biological activities of flavonoids, several different assay systems and endpoints were used to study the activity of different flavonoids and their conjugates. The effects of quercetin, kaempferol, and their main plasma conjugates quercetin-3-O-glucuronide and kaempferol-3-O-glucuronide (K-3G) on different endpoints related to peroxisome proliferator-activated receptor (PPAR)-γ were studied. PPAR-γ activation is reported to have positive health effects related to adipogenesis, insulin resistance and inflammation. The presented results show that the flavonoid aglycones increased PPAR-γ mediated gene expression in a stably transfected reporter gene cell line, and that glucuronidation diminished this effect. These observed increases in reporter gene expression were accompanied by increased PPAR-γ receptor-mRNA expression upon exposure to kaempferol, an effect that was also reduced by glucuronidation. Using the cell-free Microarray Assay for Real-time Coregulator-Nuclear receptor Interaction (MARCoNI) it was demonstrated that, unlike the known PPAR-γ agonist rosiglitazone, neither the flavonoid aglycones nor the conjugates are agonistic ligands of the PPAR-γ receptor. Supporting the hypothesis that the tested compounds have a different mode of action from normal LBD agonism, quercetin appeared to synergistically increase the effect of rosiglitazone in the reporter gene assay. The modes of action behind the observed effects remain to be elucidated and might include effects on protein kinase activities affecting expression of the PPAR-γ receptor, or posttranscriptional modifications of PPAR-γ.
Another type of nuclear receptor known to be targeted by certain flavonoids are the estrogen receptor (ER)α- and ERβ. ERs are the main targets of estrogenic compounds, and upon their activation different transcriptional responses with opposite effects on cell proliferation and apoptosis are elicited; ERα activation stimulates cell proliferation, while ERβ activation causes apoptosis and reduces ERα mediated induction of cell proliferation. Using the MARCoNI assay, the intrinsic estrogenic effects of the two main dietary isoflavones daidzein and genistein, and their plasma conjugates daidzein-7-O-glucuronide and genistein-7-O-glucuronide on the ligand induced coregulator binding of ERα- and ERβ-LBD were studied and compared to the effect of the positive control 17β-estradiol (E2). The results show that the tested isoflavone compounds are less potent agonists of ERα- and ERβ-LBD than E2, although they modulate the LBD-coregulator interactions in a manner similar to E2. Genistein is shown to be a more potent agonist than daidzein for both receptor subtypes. While in the MARCoNI assay genistein had a strong preference for ERβ-LBD activation over ERα-LBD activation, daidzein had a slight preference for ERα-LBD activation over ERβ-LBD activation. Glucuronidation reduced the intrinsic agonistic activities of both daidzein and genistein to induce ERα-LBD and ERβ-LBD - coregulator interactions and increased their average half maximal effective concentrations (EC50s) by 8 to 4,400 times. The results presented further show that glucuronidation changed the preferential activation of genistein from ERβ-LBD to ERα-LBD and increased the preferential activation of daidzein for ERα-LBD; this is of special interest given that ERβ activation, which is counteracting the possible adverse effects of ERα activation, is considered one of the supposedly beneficial modes of action of isoflavones.
Many flavonoids are reported to be inhibitors of protein kinases. To study the effect of conjugation on the inhibition of serine/threonine protein kinases by flavonoids, kaempferol and its main plasma conjugate K-3G were selected as model compounds. Protein kinases are involved in a wide range of physiological processes by controlling signaling cascades and regulating protein functions; modulation of their activities can have a wide range of biological effects. The inhibitory effects of kaempferol, K-3G, and the broad-specificity protein kinase inhibitor staurosporine on the phosphorylation activity of recombinant protein kinase A (PKA) and of a lysate prepared from the hepatocellular carcinoma cell line HepG2 were studied using a microarray platform that determines the phosphorylation of 141 putative serine/threonine phosphorylation sites derived from human proteins. The results reveal that glucuronidation reduces the intrinsic potency of kaempferol to inhibit the phosphorylation activity of PKA and HepG2 lysate on average about 16 and 3.5 times, respectively. It is shown that the inhibitory activity of K-3G in the experiments conducted was not caused by deconjugation to the aglycone. Furthermore, the results show that kaempferol and K-3G, unlike the broad-specificity protein kinase inhibitor staurosporine, did not appear to inhibit all protein kinases present in the HepG2 lysate to a similar extent, indicating that kaempferol selectively targets protein kinases, a characteristic that appeared not to be affected by glucuronidation. The fact that K-3G appeared to be only a few times less potent than kaempferol implies that K-3G does not necessarily need to be deconjugated to the aglycone to exert potential inhibitory effects on protein kinases.
The results obtained in the present thesis support the conclusion that glucuronidation of flavonoids does not necessarily abolish their activity and that flavonoid glucuronides may be biologically active themselves, albeit at higher concentrations than the parent aglycones. In line with the conclusions from the earlier literature review, an updated literature review on the effect of conjugation on the biological activity of flavonoids concludes that that the effect of conjugation on the biological activity of flavonoids depends on the type and position of conjugation, the endpoint studied and the assay system used. Based on the results described and the literature reviewed in this thesis, several recommendations and perspectives for future research are formulated. Several methodological considerations are formulated that need to be taken into account when studying the biological activity of flavonoids and their conjugates to avoid confounding results. Further, the relevance of the gut microbiome for flavonoid bioactivity is highlighted, and considerations regarding the pharmacokinetics and pharmacodynamics of flavonoids in vivo are formulated. Altogether, it can be concluded that circulating flavonoid conjugates may exert biological activities themselves, and that understanding these is a prerequisite to successfully elucidate the mechanisms of action behind the biological activities linked to flavonoid consumption.
Estrogenicity and metabolism of prenylated flavonoids and isoflavonoids
Schans, M.G.M. van de - \ 2015
Wageningen University. Promotor(en): Harry Gruppen, co-promotor(en): Jean-Paul Vincken; Toine Bovee. - Wageningen : Wageningen University - ISBN 9789462574748 - 180
flavonoïden - isoflavonoïden - glycine max - sojabonen - oestrogeenreceptoren - zoethout - glycyrrhiza glabra - in vitro - flavonoids - isoflavonoids - glycine max - soyabeans - oestrogen receptors - liquorice - glycyrrhiza glabra - in vitro
Binding of (prenylated) flavonoids and isoflavonoids to the human estrogen receptors (hERs) might result in beneficial health effects in vivo. To understand structure-activity relationships of prenylated (iso)flavonoids towards the hERs, prenylated (iso)flavonoids were purified from extracts of licorice roots and elicited soybean seedlings. It was observed that prenylation can modulate estrogenicity. Unprenylated, chain and δ-position pyran prenylated (iso)flavonoids show an agonistic mode of action, whereas α/β-position pyran, α/β-position furan and double chain prenylated (iso)flavonoids show an antagonistic mode of action towards hERα in the yeast bioassay. The mode of estrogenic action of prenylated (iso)flavonoids could be related to structural features of the hER. In particular, the increase in length of α/β-position pyran prenylated compounds was related to indirect antagonism. It was also shown that heat and acid affected the stability of 6a-hydroxy-pterocarpans, converting them into their respective 6a,11a-pterocarpenes and consequently modulate their estrogenicity. Six prenylated isoflavonoids acted as SERMs and eight prenylated isoflavonoids showed ER subtype-selective behavior. The kind of prenylation (chain, furan or pyran) was most important for determining SERM activity, whereas additionally the backbone structure, i.e. the presence of an additional D-ring, was of importance for determining ER subtype-selectivity. To determine structure-metabolism relationships, in vitro conversion of purified prenylated (iso)flavonoids by liver enzymes was studied. These compounds can be extensively metabolized by phase I and II enzymes. A glucuronidation yield between 70-80% was observed. It was also shown that pyran and chain prenylation gave more complex hydroxylation patterns with 4 or more than 6 hydroxyl isomers, respectively, compared to unprenylated compounds (only 1 hydroxyl isomer).
Factors influencing ER subtype-mediated cell proliferation and apoptosis
Evers, N.M. - \ 2014
Wageningen University. Promotor(en): John Groten; Ivonne Rietjens, co-promotor(en): A.G.H. Ederveen. - Wageningen : Wageningen University - ISBN 9789461739469 - 243
oestrogenen - oestrogeenreceptoren - oestrogene eigenschappen - weefselproliferatie - apoptose - oestrogens - oestrogen receptors - oestrogenic properties - tissue proliferation - apoptosis
The aim of the current thesis is to elucidate the role of estrogen receptor (ER)αand ERβin cell proliferation and apoptosis induced by estrogenic compounds. Special attention is paid to the importance of the receptor preference of the estrogenic compounds, the cellular ERα/ERβratio, the role of coregulators, and ER-mediated induction of protein expression. In chapter 1 estrogenic compounds and their interaction with estrogen receptors are described and the two different estrogen receptors, ERαand ERβ, are introduced. It is described how estrogenic compounds eventually exert biological effects through coregulator recruitment upon ER binding, transcription initiation, and
Chapter 2 describes under which conditions T47D-ERβbreast cancer cells with tetracycline-dependent ERβexpression and constant ERαexpression best mimic ERα/ERβratios in breast and other estrogen-sensitive tissues in vivo in rat as well as in human. At protein and mRNA level, ERαand ERβlevels and ratios are determined in T47D-ERβcells exposed to a range of tetracycline concentrations and in rat and human breast, prostate, and uterus or endometrium. The ERα/ERβratio in rat mammary gland and in human breast tissue can be mimicked by exposing the T47D-ERβcells to >150 ng/ml tetracycline, but the ERα/ERβratio of other estrogen-sensitive rat and human tissues can also be mimicked. The ERα/ERβratios in MCF-7 and native T47D cells are high due to a lack of ERβexpression
and therefore do not reflect ratios in rat and human tissues. It is demonstrated how these different tissues might vary in their proliferative response towards 17β-estradiol (E2) by exposing T47D-ERβcells to E2 under defined tetracycline concentrations.
In chapter 3 the modulation of the interaction of ERαand ERβwith coregulators in the ligand-dependent responses induced by estrogenic compounds is investigated. To this end, selective ERαand ERβagonists are characterized for intrinsic relative potency reflected by EC50 and maximal efficacy towards ERαand ERβin ER-selective reporter gene assays, and subsequently tested for stimulation of cell proliferation in T47D-ERβcells with variable ERα/ERβratio and for ligand-dependent modulation of the interaction of ERαand ERβwith coregulators using the Microarray Assay for Real-time Coregulator – Nuclear receptor Interaction (MARCoNI) with 154 unique nuclear receptor coregulator peptides derived from 66 different coregulators. Results obtained reveal an important influence of the ERα/ERβratio and receptor selectivity of the compounds on stimulation of cell proliferation. ERαagonists activate cell proliferation whereas ERβseems to suppress ERα-mediated cell proliferation. The responses in the MARCoNI assay reveal that the modulation of the interaction of ERαor ERβwith coregulators by a specific agonist are very similar indicating only a limited number of differences upon ERαor ERβactivation by a specific ligand. Differences in the modulation of the interaction of the ERs with coregulators between the different agonists are more substantial and can be used to classify the different agonists by hierarchical clustering. The results obtained corroborate that the ultimate effect of the model compounds on proliferation of estrogen-responsive cells depends on the intrinsic relative potency of the agonist towards ERαand ERβand the cellular ERα/ERβratio whereas differences in the modulation of the interaction of the different ERs with coregulators for a given ligand might also contribute to the compound-specific pharmacology. Based on ligand-dependent differences in the modulation of the interaction of the ERs with coregulators, the MARCoNI assay is able to classify the different ERαand ERβagonists discriminating between different agonists for the same receptor, a characteristic not defined by the ER-selective reporter gene or proliferation assays. It is concluded that differences in the modulation of the interaction of ERαand ERβwith coregulators contribute to the ligand-dependent responses but do not fully explain the differences in pharmacology between ER-mediated responses by the different estrogenic compounds.
To investigate if this is also the case for ER antagonists, chapter 4 handles the modulation of the interaction of ERαand ERβwith coregulators in the ligand-dependent responses induced by the ER antagonistic compounds 4-hydroxytamoxifen (4OHT) and fulvestrant. Comparison of these results to ligand-dependent interaction of ERαand
ERβwith coregulators expressed in modulation index (MI) profiles for the ER agonist E2 elucidates whether differences in the (ant)agonist-dependent interaction of ERαand ERβwith coregulators contribute to the differences in (ant)agonist responses. To this end, the selected ER antagonistic compounds are first characterized for intrinsic relative potency reflected by IC50 and maximal efficacy towards ERαand ERβusing ER-selective U2OS reporter gene assays, and subsequently tested for ligand-dependent modulation of the interaction of ERαand ERβwith coregulators using the MARCoNI assay. Results obtained with the U2OS reporter gene assays indicate a preference of 4OHT to bind ERβand find fulvestrant to be less ER-specific. The responses in the MARCoNI assay reveal that ERα-
and ERβ-mediated interaction with coregulators expressed in MI profiles are similar for 4OHT and fulvestrant and generally opposite to the MI profile of the ER agonist E2. Hierarchical clustering with Euclidian distance as the cluster distance metric, based on the MI profiles, is able to clearly discriminate the two compounds with ER antagonistic
properties from the ER agonist E2. Taken together the data reveal that modulation of the interaction of ERs with coregulators discriminates ER agonists from antagonists but does not discriminate between the preferential ERβantagonistic compound 4OHT and the less specific ER antagonist fulvestrant. It is concluded that differences in modulation of the interaction of ERαand ERβwith coregulators contribute to the differences in ligand-dependent responses induced by ER agonists and ER antagonists, but the importance of the subtle differences in modulation of the interaction of ERs with coregulators between the ER antagonistic compounds 4OHT and fulvestrant for the ultimate biological effect remains to be established.
To further investigate whether 4OHT, the active metabolite of the breast cancer drug tamoxifen, exerts ERα/ERβratio-dependent effects on cell proliferation and apoptosis, in chapter 5 the results of a quantitative proteomics study are described. This is of importance given that the ERα/ERβratio usually increases in tumorous tissue compared to normal tissue due to decreased ERβexpression. ERα/ERβratio-dependent effects of 4OHT on cell proliferation and apoptosis of the T47D-ERβhuman breast cancer cell line with tetracycline-dependent ERβexpression are detected. In the cells expressing only ERαdecreased cell proliferation and increased apoptosis is induced by 4OHT, which is opposite to the effects detected in cells expressing ERαand ERβ, where increased cell
proliferation and decreased apoptosis upon 4OHT exposure is found. Post-translational modifications like acetylation, methylation, and phosphorylation of several ribosomal and mitochondrial protein groups are induced by 4OHT, mostly in T47D-ERβcells with both ERαand ERβexpressed. Altogether the results suggest that effects of 4OHT on major biological functions like cell proliferation and apoptosis in the T47D-ERβcells are affected by the ERα/ERβratio. 4OHT may have differential cellular effects, being more effective in reducing cell proliferation and increasing apoptosis if ERαdominates and ERβexpression levels are low since 4OHT then antagonizes ERα.
Chapter 6 presents a discussion on the implications of the mechanisms of action of several estrogenic compounds discussed in this thesis. Altogether the results of the present thesis have elucidated the action of different estrogenic compounds, their interaction with the two ER subtypes, and the subsequent recruitment or rejection of coregulators, as well as the resulting effects on cell proliferation and apoptosis, and these results emphasize the importance of the ERα/ERβratio for the ultimate effects of estrogenic compounds on cell proliferation and apoptosis.
Unravelling the mechanism of differential biological responses induced by food-borne xeno- and phyto-estrogenic compounds
Sotoca Covaleda, A.M. - \ 2010
Wageningen University. Promotor(en): Ivonne Rietjens; Tinka Murk, co-promotor(en): Jacques Vervoort. - [S.l. : S.n. - ISBN 9789085857075 - 186
oestrogenen - plantenoestrogenen - synthetische oestrogenen - oestrogeenreceptoren - genexpressie - eiwitexpressieanalyse - transcriptomics - oestrogens - plant oestrogens - synthetic oestrogens - oestrogen receptors - gene expression - proteomics - transcriptomics
The multiple actions of estradiol and other estrogenic compounds in mammalian physiology are brought about, on a molecular level, as a result of complex signalling pathways, and mediated by at least two receptors namely estrogen receptor (ER) α and ERβ.
The aim of the work presented in this thesis was to obtain insight in the role of ERα, ERβ and the ratio of ERα/ERβ present within a cell, in the cellular response to estrogen-like compounds. To this end, this thesis addressed the transcriptional activity at both the gene and protein level and effects on cell proliferation under the influence of specifically-acting estrogen-like molecules when varying the ratio of ERα/ERβ present in the cells under study. The ultimate aim was to link the data on cell proliferation as the biological end-point to the transcriptomics and proteomics data.
|Development of an in vivo assay for oestrogenic potency using transgenic zebrafish
Legler, J. ; Broekhof, J. ; Brouwer, A. ; Murk, A.J. ; Saag, P. van der; Vethaak, A.D. ; Burg, B. van der - \ 2000
In: Endocrine Disrupting Compounds : Wildlife and human health risks, The Hague 1998 The Hague : - p. 109 - 112.
transgene dieren - vissen - danio rerio - biotesten - oestrogeenreceptoren - reporter-genen - waterdieren - ecotoxicologie - transgenic animals - aquatic animals - fishes - bioassays - oestrogen receptors - reporter genes - ecotoxicology