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.
Toxicogenomics-based in vitro alternatives for estrogenicity testing
Wang, S. - \ 2013
Wageningen University. Promotor(en): Ivonne Rietjens, co-promotor(en): Toine Bovee; Jac Aarts. - S.l. : s.n. - ISBN 9789461735812 - 193
oestrogene eigenschappen - oestrogenen - in vitro - toxicogenomica - testprocedure - alternatieve methoden - methodologie - oestrogenic properties - oestrogens - in vitro - toxicogenomics - test procedure - alternative methods - methodology
Testing chemicals for their endocrine-disrupting potential, including interference with estrogen receptor signaling, is an important aspect to assess the safety of currently used and newly developed chemicals. The standard test for disruption of normal estrogen function is the in vivo uterotrophic assay in immature or ovariectomised rodents with uterus weight as a crucial read-out parameter. Due to the high costs, ethical objections and labour intensiveness of the in vivo uterotrophic assay, the development of an in vitro test battery for in vivo estrogenicity has high priority. The aim of the present thesis was to develop an integrated testing strategy (ITS), based on existing and newly developed in vitro assays for estrogenicity testing, allowing easy high-throughput screening and prioritization of chemicals. An ITS preferentially based on in vitro assays would be a crucial step towards refinement, reduction, and ultimately replacement of current animal testing for estrogenic and other endocrine disrupting effects.
To reach this aim, several presently available and newly developed in vitro bioassays were selected and evaluated for optimal representation of the estrogenic effects occurring in the uterus/endometrium in vivo. Results show that the yeast estrogen bioassay revealed the best correlation with the in vivo uterotrophic assay(R2=0.87). The estrogenic potencies predicted by the peptide microarray also correlated very well with the uterotrophic assay and 30 coactivators on the peptide microarray resulted in correlation coefficient values higher than 0.85. The present thesis thus provides proof-of-principle that combining in vitro assays measuring different steps in the estrogen receptor signaling pathway enables accurate prediction of the estrogenic effects in vivo. By including the androgen reporter gene assays as well as the H295R steroidogenesis assay, the extended testing panel even goes beyond estrogenicity testing, as it can detect possible (anti)androgenic effects and effects on steroidogenesis that are not covered by the in vivo uterotrophic assay. The integrated in vitro testing strategy presented in this thesis may therefore allow easy high-throughput screening and prioritization of chemicals, thereby contributing to refinement, reduction and to some extent even replacement of current animal testing for estrogenic effects.
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.
Hormoonverstoring in oppervlaktewater; waargenomen en veronderstelde effecten in de natuur
Lahr, J. ; Lange, H.J. de - \ 2009
Utrecht : Stowa (Rapport / STOWA 2009 38) - ISBN 9789057734588 - 27
hormonen - waterverontreiniging - oppervlaktewater - aquatisch milieu - waterorganismen - effecten - nadelige gevolgen - oestrogenen - fauna - toxicologie - hormoonverstoorders - aquatische ecosystemen - ecotoxicologie - hormones - water pollution - surface water - aquatic environment - aquatic organisms - effects - adverse effects - oestrogens - fauna - toxicology - endocrine disruptors - aquatic ecosystems - ecotoxicology
In laboratoria wordt het nodige onderzoek verricht naar de hormonale, of hormoonverstorende werking van een groot aantal stoffen. Van een aantal van deze stoffen is inmiddels aangetoond dat ze in risicovolle concentraties voorkomen in het watermilieu. Maar het is vaak niet bekend wat de daadwerkelijke, waarneembare effecten van deze hormoonverstorende stoffen zijn op (aquatische) organismen. Bij een aantal diersoorten is aangetoond dat de verhouding tussen het aantal mannetjes en vrouwtjes niet meer gelijk is en tevens dat er soms geslachtsverandering optreedt. Dit rapport vat samen wat er op dit ogenblik bekend is over de hormoonverstorende effecten van stoffen op aquatische organismen. Onderzoekers onderscheidden: schelpen en slakken, Kreeftachtigen en insecten, Vissen; Amfibieën; Vogels en zoogdieren (otters)
Fate of the estrogen nonylphenol in river sediment: availability, mass transfer and biodegradation
Weert, J.P.A. de - \ 2009
Wageningen University. Promotor(en): Huub Rijnaarts, co-promotor(en): Alette Langenhoff; Tim Grotenhuis. - [S.l. : S.n. - ISBN 9789085854661 - 143
verontreinigde sedimenten - rivieren - bodemverontreiniging - verontreinigende stoffen - oestrogenen - waterorganismen - biodegradatie - aquatische toxicologie - bodemsanering - contaminated sediments - rivers - soil pollution - pollutants - oestrogens - aquatic organisms - biodegradation - aquatic toxicology - soil remediation
Veel riviersedimenten zijn in het verleden verontreinigd geraakt met estrogene verbindingen, die toxische effecten kunnen veroorzaken op aquatische organismen, zoals de vervrouwlijking van mannelijke vissen. Een van deze estrogene verbindingen is nonylfenol (NP). Nonylfenol is een organische verbinding die bestaat uit een fenolgroep met een lineaire of een vertakte keten van negen koolstofatomen. Voornamelijk mengsels van vertakte NP-isomeren komen voor als verontreiniging in het milieu. Sedimenten die verontreinigd zijn met NP kunnen functioneren als secundaire bron van verontreiniging van het rivierwater, waar het toxische effecten kan veroorzaken op aquatische organismen. Het risico van toxische effecten door NP, dat aanwezig is in het sediment, wordt bepaald door de beschikbaarheid van NP in het sediment, het massatransport vanuit het sediment naar het rivierwater en de mogelijkheid voor biologische afbraak van NP in het sediment of het rivierwater.
Xeno-estrogenic compounds in precipitation
Peters, R.J.B. ; Beeltje, H. ; Delft, R.J. - \ 2008
Journal of Environmental Monitoring 10 (2008). - ISSN 1464-0325 - p. 760 - 769.
neerslag - regen - oestrogenen - hormonen - verontreiniging - geurstoffen - hormoonverstoorders - precipitation - rain - oestrogens - hormones - pollution - odours - endocrine disruptors - polychlorinated-biphenyls - musk fragrances - german bight - great-lakes - air - urban - atmosphere - exposure - samples - norway
The exposure to some chemicals can lead to hormone disrupting effects. Presently, much attention is focused on so-called xeno-estrogens, synthetic compounds that interact with hormone receptors causing a number of reactions that eventually lead to effects related to reproduction and development. The current study was initiated to investigate the presence of a number of such compounds in precipitation as a follow-up on a previous study in which pesticide concentrations in air and precipitation were determined. Rainwater samples were collected at about 50 locations in The Netherlands in a four week period. The samples were analysed for bisphenol-A, alkylphenols and alkylphenol ethoxylates, phthalates, flame retardants and synthetic musk compounds. The results clearly indicated the presence of these compounds in precipitation. The concentrations ranged from the low ng l-1 range for flame retardants to several thousands of ng l-1 for the phthalates. Bisphenol-A was found in 30% of the samples in concentrations up to 130 ng l-1, while alkylphenols and alkylphenol ethoxylates were found in virtually all locations in concentrations up to 920 ng l-1 for the individual compounds. Phthalates were by far the most abundant xeno-estrogens in the precipitation samples and were found in every sample. Di-isodecyl phthalate was found in a surprisingly high concentration of almost 100000 ng l-1. Polybrominated flame retardants were found in the low ng l-1 range and generally in less than 20% of the samples. Noticeable was the finding of hexabromocyclododecane, a replacement for the polybrominted diphenyl ethers at one location in a concentration of almost 2000 ng l-1. Finally, as expected, synthetic musk compounds were detected in almost all samples. This is especially true for the polycyclic musks HHCB and AHTN. Nitro musks were found, but only on a few locations. Kriging techniques were used to calculate precipitation concentrations in between actual sampling locations to produce contour plots for a number of compounds. These plots clearly show located emission sources for a number of compounds such as bisphenol-A, nonylphenol ethoxylate, phthalates and AHTN. On the contrary, the results for HHCB and some phthalates indicated diffuse emission patterns, probably as the result of the use of consumer products containing these compounds
Anaerobic biodegradation of estrogens-hard to digest
Mes, T.Z.D. de; Kujawa, K. ; Zeeman, G. ; Lettinga, G. - \ 2008
Water Science and Technology 57 (2008)8. - ISSN 0273-1223 - p. 1177 - 1182.
geactiveerd slib - varkensmest - oestrogenen - afvalwaterbehandeling - anaërobe behandeling - biodegradatie - korrelslib - activated sludge - pig manure - oestrogens - waste water treatment - anaerobic treatment - biodegradation - granular sludge - waste-water treatment - removal - sewage - combination - behavior - sludge - plant
Although many publications are available on the fate of estrone (E1), 17b-estradiol (E2) and 17a-ethynylestradiol (EE2) during aerobic wastewater treatment, little is published on their fate under strictly anaerobic conditions. Present research investigated the digestibility of E1 and EE2, using digested pig manure, granular UASB sludge, UASB-septic tank sludge and activated sludge as inocula. Besides, actual concentrations were measured in a UASB septic tank treating black water. Under anaerobic conditions E1 is reduced to E2 but the extent of this reduction depends on type of inoculum. No significant loss of the sum of E1 and E2 and of EE2 was observed. Adsorption was responsible for a 32¿35% loss of E1 and E2 from the liquid phase in the UASB septic tank and the effluent still contained considerable concentrations of respectively 4.02 mg/l and 18.79 mg/l for E1 and E2 with a large fraction present in conjugated form. No EE2 was detected in the UASB effluent
Fate of estrogens in biological treatment of concentrated black water
Mes, T.Z.D. de - \ 2007
Wageningen University. Promotor(en): Gatze Lettinga, co-promotor(en): Grietje Zeeman; Katarzyna Kujawa. - [S.l.] : S.n. - ISBN 9789085047537 - 154
oestrogenen - oestrogene eigenschappen - rioolslib - waterverontreiniging - afvalwaterbehandeling - hormonen - biologische behandeling - riolering - oestrogens - oestrogenic properties - sewage sludge - water pollution - waste water treatment - hormones - biological treatment - sewerage
Feminisation of male fish is for a large part due to compounds entering surface waters via wastewater. For domestic wastewater, two natural estrogens, estrone and 17-estradiol and the synthetic estrogen, constituent of the contraceptive pill, are mainly responsible for this effect. These compounds are excreted by humans and in conventional treatment systems sometimes insufficiently removed. A solution can be found in the implementation of innovative sanitation concepts like source separated collection and treatment of black water (toilet), grey water (shower, kitchen, laundry) and rain. As the three compounds will be mainly present in black water, contamination by storm water overflows is completely excluded and the concentrated character of the wastewater allows for more energy efficient treatment systems. Present research showed the first step, anaerobic treatment where energy is as well produced as conserved, is unfavourable to satisfactory remove these compounds and the aerobic post-treatment only partly. As the volume of the stream is remarkably smaller compared to conventional systems (7 liter black water per person per day against 200 liter wastewater per person per day), a necessary tertiary treatment, for which ozonation is promising, will be more compact. Besides, current research shed light on several parameters influencing the degradation of estrogens in biological systems
Development, validation and routine application of the in vitro REA and DR-CALUX reporter gene bioassays for the screening of estrogenic compounds and dioxins in food and feed
Bovee, T.F.H. - \ 2006
Wageningen University. Promotor(en): Ivonne Rietjens; Ron Hoogenboom; Michel Nielen. - [S.l.] : S.n. - ISBN 9789085043935 - 133
biotesten - dioxinen - oestrogenen - bioassays - dioxins - oestrogens
A dedicated cell-line was developed by the Department of Toxicology of Wageningen University in a joined project with the University of California in Davis and the RIKILT-WUR - Institute of Food Safety in Wageningen. This DR-CALUX ® bioassay was tested, optimised and validated for its use to determine low elevated levels of dioxins in bovine milk around the existing limits. It was shown that this mammalian cell based test is very sensitive for 2,3,7,8-substituted dioxins and related PCBs, thereby reflecting the relative potencies (TEF) of these compounds as set by the World Health Organisation (WHO). These toxic equivalency factors (WHO-TEFs) express the toxicity of a compound in comparison to the most toxic compoundcongener 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, TEF=1). The response obtained with a mixture of dioxins was additive, in accordance with the TEQ-principle. Milk fat was isolated by centrifugation followed by clean-up of the fat with n-pentane, removal of the fat on a 33% H2SO4silica column, and determination of Ah receptor agonist activity with the DR-CALUX ® bioassay. To investigate the performance of this 33% H2SO4silica method, milk fat was cleaned with activated carbon and spiked with a mixture of 17 different 2,3,7,8-substituted PCDD and PCDF congeners at 1, 3, 6, 9, 12 and 15 pg WHO-TEQ per g fat, as confirmed by GC/MS. In this concentration range, the method showed a recovery of TEQs around 67% (58-87%). The reproducibility showed a coefficient of variation (CV) varying between 4% and 54%, with the exception of the sample spiked at 1 pg WHO-TEQ/g (CV 97%). The repeatability determined with the sample spiked at 6 pg WHO-TEQ per g showed a CV of 10% These results clearly demonstrate that the reproducibility of the silica-CALUX procedure with samples containing more than 1 pg WHO-TEQ per g fat is relatively good, in particular regarding the fact that no internal standards could be used in the assay for correction of data for varying recoveries. The fact that the CV was much higher for the sample spiked at the lowest level, confirmed the calculated limit of quantification of 1 pg WHO-TEQ per g fat. The current tolerance limit for bovine milk in the EU is 3 pg WHO-TEQ per g fat, with an action limit of 2 pg WHO-TEQ per g fat. Therefore, the DR-CALUX ® bioassay can be a useful pre-screening tool for selecting milk samples that may contain dioxin levels exceeding this tolerance limit. This was supported by the results obtained with 22 field samples, since all five samples exceeding the 2 pg WHO-TEQ per g fat concentration gave a higher response in the DR-CALUX ® bioassay.The DR-CALUX ® bioassay in combination with the 33% H2SO4clean-up procedure results in a specific test for the determination of dioxins and dioxin-like PCBs, allowing the screening of relatively large sets of samples for the presence of unacceptable high levels of these compounds. This results in a reduction of costs involved in the analysis of food for the presence of these compounds, enabling more intense monitoring programs.Following the successful optimisation and validation of the test for milk fat, the bioassay was first used at RIKILT in the food and feed area during the 1998 Brazilian citrus pulp incident. The test procedure was subsequently optimised and validated for animal feed. During the German bakery waste incident in 2003, animal feed was contaminated with dioxins due to the use of waste wood for drying of the material. Besides Germany, the material was also shipped to the Netherlands. Levels up to 12 ng WHO-TEQ/kg were detected, being about 15 times over the current limit of 0.75 ng WHO-TEQ/kg. A combined strategy of screening with the DRCALUX -bioassay and the HRGC/HRMS confirmatory method was used in the Netherlands to rapidly control the incident. Pigs were contaminated by the incident but only to a very limited extent. Despite the rather low limits for pig meat (1 pg WHO-TEQ per g fat), the DR-CALUX ® bioassay, in combination with an extra acid pre-treatment of the fat samples, showed excellent performance, confirming once again the value of this bioassay. Shown during the recent incidents with kaolinic clay (2004) and the contaminated HCl used for gelatine production (2006), the assay is still the best availablescreening test for dioxins and dioxin-like PCBs.The second aim of the research in this thesis was to develop, validate and apply a new recombinant yeast screen to detect chemicals with an estrogenic mode of action in animal feed, urine and illegal preparations. A recombinant yeast cell that stably expresses the human estrogen receptor α (hERα) and yeast enhanced green fluorescent protein (yEGFP) as a reporter protein in response to estrogens was developed at the RIKILT. The EC50 revealed by the RIKILT yeast Estrogen bioAssay (REA) was 0.5 nM for 17b-estradiol and was comparable with reported EC50 values for yeast estrogen bioassays that contain β-galactosidase as a reporter. However, the yEGFP assay can be performed completely in 96 well plates within 4 hours and does not need require cell wall disruption, nor does it need the addition of a substrate. This makes the test sensitive, rapid and convenient with high reproducibility and small variation. The robustness and ease of the yeast cells in combination with the qualities ofyEGFP,ensure that the assay will be suited to be used as a high through put system.The properties of the RIKILT yeast Estrogen bioAssay expressing thehERα(REA) were further studied by testing a series of estrogenic compounds. In addition, a similar assay was developed based on the stable expression of human estrogen receptor β (hERβ). When exposed to 17b-estradiol, the maximum transcriptional activity of the hERb cytosensor was only about 40% of the activity observed with hERa, but the concentration where half-maximal activation is reached (EC50), was about 5 times lower. The relative estrogenic potencies (REP), defined as the ratio between the EC50 of 17b-estradiol and the EC50 of the compound, of the synthetic hormones dienestrol, hexestrol and especially mestranol were higher with ERa than with ERβ, while DES was slightly more potent with ERb. The gestagens progesterone and medroxyprogesterone-acetate showed no response, whereas the androgen testosterone showed a very weak response and only at high concentrations. The isoflavones genistein, genistin, daidzein and daidzin, the coumestran coumestrol and the flavonoid naringenin were relatively more potent with ERb than with ERa. Coumestrol and genistein were by far the most potent of these compounds with ERb. However, 8-prenylnaringenin, a phytoestrogen present in hops, was relatively more potent with ERa than with ERb and was actually the most potent phytoestrogen with ERa. The data demonstrate that the REA shows clear dose-response curves when exposed to estrogenic compounds. Since good dose-response curves can be obtained after only 4 h of exposure, the often questioned permeability of the yeast cell wall does not seem to be an obstacle in our yeast estrogen bioassays.The RIKILT yeast Estrogen bioAssay stably expressing human estrogen receptor α (REA) was validated as a qualitative screening method for the determination of estrogenic activity in calf urine and animal feed. These validations were performed according to EC Decision 2002/657, which prescribes the determination of the detection capability (CCb), the specificity and the stability. To determine these performance characteristics, twenty blank urine samples of 19 week old calves were collected and spiked with 17b-estradiol (E2b) at 1 ng/ml-1, diethylstilbestrol (DES) at 1 ng/ml-1, 17a-ethynylestradiol (EE2) at 1 ngml-1a-zearalanol at 50 ngml-1 or mestranol at 10 ng/ml-1. Following enzymatic deconjugation and solid phase extraction, 100 ml equivalents of these blank and spiked urine samples were screened for estrogenic activity in a 96 well plate using the REA. All of these blank and low estrogen spiked feed samples fulfilled the CCα and CCβ criterions, meaning that all 20 blank urine samples gave a signal below the determined decision limit CCα and were thus classified as compliant and at least 19 out of the 20 spiked samples gave a signal above this CCα (β=5%) and were thus classified as suspect. The specificity of the method was determined with blank urine samples spiked with a high dose of testosterone or progesterone (1000 ng/ml-1). No response to these substances was detected in the REA. There was also no interference of a high dose of testosterone or progesterone on the response of a low dose of the estrogens. Stability of urine samples was checked with spiked urine samples that were kept frozen for up to 90 days, showing that urine samples could be stored at -20°C for up to 60 days without changing the screening result of the assay. The assay was validated for animal feed in a simalar way, using twenty blank animal feed samples, including milk replacers and wet and dry feed samples.As all the performance characteristics met the criteria that were put forward in EC Decision 2002/657 for validation of a qualitative screening method, the described clean-up/yeast estrogen bioassay procedures were proven to be valid for the determination of estrogenic activity in calf urine and animal feed. The clean-up procedures for urine and feed samples are relatively simple and the yeast estrogen bioassay, using yEGFP as a reporter protein, is sensitive, rapid, convenient and reproducible. Due to the good sensitivity of the bioassay, only 2 ml of urine or 1 gram of feed were enough to be processed. Combined this resulted in a low cost bioassay that is suited to be used as a high through-put system for the screening of estrogenic activity in calf urine and animal feed. Like the DR CALUX ® assay, Tthe method acquired an ISO 17025 accreditation status in the Netherlands for both of these matrices. The examples of the MPA-incident with wet pig feed and the fishfeed,described in Chapter 7 of the thesis, demonstrate the applicability of the bioassay method as an early warning system for pharmaceutical waste and hormone use respectively. This is the first successful example of a developed, validated and applied bioassay for the screening of hormonal substances in feed. At present this method has been in routine use at RIKILT for more than two years.Overall the work presented in this thesis shows that bioassays are valuable tools for rapid and high throughput screening of samples for both known and unknown compounds. As such they may contribute to an earlier detection of new emerging risks and prevent the use of illegal growth-promoting agents with thus far unknown identity.
Oestrogene effecten in vissen in regionale wateren nabij rwzi's
Rijs, G. ; Gerritsen, A. ; Lahr, J. ; Bulder, A. - \ 2004
H2O : tijdschrift voor watervoorziening en afvalwaterbehandeling 37 (2004)5. - ISSN 0166-8439 - p. 15 - 18.
vissen - abramis brama - oestrogenen - hormonen - aquatisch milieu - oppervlaktewater - waterverontreiniging - rioolafvalwater - nederland - aquatische ecosystemen - zuiveringsinstallaties - fishes - abramis brama - oestrogens - hormones - aquatic environment - surface water - water pollution - sewage effluent - netherlands - aquatic ecosystems - purification plants
Het Landelijk Onderzoek oEstrogene Stoffen (LOES) heeft laten zien dat hormoonontregelende stoffen bijna overal in lage concentraties in het Nederlandse watermilieu voorkomen. Vissen in regionale wateren blijken evenwel een groter risico te lopen op nadelige effecten, zoals vervrouwelijking, dan de vissen in de wat grotere wateren. De oorzaak hiervoor lijkt te liggen in het feit dat wanneer regionale wateren onder directe invloed van lozingen met hormoonontregelende stoffen staan in kleinere wateren relatief weinig verdunning optreedt. Eén van de emissiebronnen die uitgebreid onderzocht is, is het effluent van een rioolwaterzuivering
Verwijdering van hormoonverstorende stoffen in rwzi's
Loeffen, P. ; Lahr, J. ; Derksen, A. ; Uijterlinde, C. ; Roeleveld, P. - \ 2004
H2O : tijdschrift voor watervoorziening en afvalwaterbehandeling 37 (2004)5. - ISSN 0166-8439 - p. 19 - 21.
afvalwaterbehandeling - rioolafvalwater - hormonen - oestrogenen - waterzuivering - oppervlaktewater - waterverontreiniging - zuiveringsinstallaties - hormoonverstoorders - waste water treatment - sewage effluent - hormones - oestrogens - water treatment - surface water - water pollution - purification plants - endocrine disruptors
Door de STOWA is eind vorig jaar een literatuurstudie naar de verwijdering van hormoonverstorende stoffen (ook wel endocrine disrupting chemicals of EDC's genoemd) in rioolwaterzuiveringsinstallaties verricht. Hieruit blijkt dat, ondanks een redelijke verwijdering in de rwzi, de concentraties van bepaalde EDC's in het effluent nog steeds kunnen leiden tot biologische effecten. De grootste risico's geven de oestrogene hormonen 17a-ethinyloestradiol ('de pil'), 17beta-oestradiol en oestron en de industriële detergenten nonylfenol en nonylfenolethoxylaten. Het is onduidelijk hoe de huidige rioolwaterzuiveringsinstallaties geoptimaliseerd kunnen worden om hormoonverstorende stoffen te verwijderen. Geavanceerde technieken lijken de beste resultaten op te leveren
In vitro biomonitoring in polar extracts of solid phase matrices reveals the presence of unknown compounds with estrogenic activity
Legler, J. ; Leonards, P.E.G. ; Spenkelink, A. ; Murk, A.J. - \ 2003
Ecotoxicology 12 (2003). - ISSN 0963-9292 - p. 239 - 249.
oppervlaktewater - waterorganismen - sediment - vissen - oestrogenen - hormonen - aquatisch milieu - biologische monitoring - ecotoxicologie - surface water - aquatic organisms - sediment - fishes - oestrogens - hormones - aquatic environment - biomonitoring - ecotoxicology - endocrine disruption - canada
Determination of estrogenic activity has so far mainly concentrated on the assessment of compounds in surface water and effluent. This study is one of the first to biomonitor (xeno-)estrogens in sediment, suspended particulate matter and aquatic organisms. The relatively polar acetone extracts from these solid phase matrices do not contain the well-known estrogenic compounds such as hormones, alkylphenols and phthalates. An in vitro ‘estrogen receptor-mediated chemical activated luciferase gene expression’ (ER-CALUX) assay was applied to samples from various locations in the Netherlands
Determination of estrogenic activity has so far mainly concentrated on the assessment of compounds in surface water and effluent. This study is one of the first to biomonitor (xeno-)estrogens in sediment, suspended particulate matter and aquatic organisms. The relatively polar acetone extracts from these solid phase matrices do not contain the well-known estrogenic compounds such as hormones, alkylphenols and phthalates. An in vitro,estrogen receptor-mediated chemical activated luciferase gene expression' (ER-CALUX) assay was applied to samples from various locations in the Netherlands. Estrogenic activity measured in polar fractions of particulate matter and sediment extracts ranged from below detection limit to up to 4.5 pmol estradiol equivalents (EEQ)/g dry weight. Estrogenic activity in freshwater river sediments was up to five times higher compared to sediments from large lakes and coastal locations. Tissue extracts EEQs were determined in bream (Abramis brama), flounder (Platichthys flesus), freshwater mussels (Dreissena polymorpha) and marine mussels (Mytilus edulis). The highest biota EEQ levels were found in the freshwater zebra mussel (30 pmol EEQ/g lipid). One sample site showed greatly elevated EEQs in sediment and biota, which correlated with effects found in the wild populations of bream. The EEQ activity of the unknown compounds in the polar fraction mostly was much higher than the calculated EEQ levels based on known estrogens in the non-polar fraction (previously published data).
Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays
Legler, J. ; Dennekamp, M. ; Vethaak, A.D. ; Brouwer, A. ; Koeman, J.H. ; Burg, B. van der; Murk, A.J. - \ 2002
Science of the Total Environment 293 (2002). - ISSN 0048-9697 - p. 69 - 83.
sediment - oestrogenen - toxische stoffen - afvalwater - organisch bodemmateriaal - hormonen - waterbodems - xenobiotica - waddenzee - sediment - oestrogens - hormones - toxic substances - waste water - soil organic matter - water bottoms - xenobiotics - wadden sea - in-vitro - saccharomyces-cerevisiae - polychlorinated-biphenyls - environmental estrogens - receptor - activation - metabolism - mechanism - system - transcription
Sediments may be the ultimate sink for persistent (xeno-) estrogenic compounds released into the aquatic environment. Sediment-associated estrogenic potency was measured with an estrogen receptor-mediated luciferase reporter gene (ER-CALUX) assay and compared with a recombinant yeast screen. The ER-CALUX assay was more sensitive to 17beta-estradiol (E2) than the recombinant yeast screen, with an EC50 of 6 pM E2 compared to 100 pM in the yeast screen. Yeast cells were unable to distinguish the anti-estrogens ICI 182 780 and (4-hydroxy)tamoxifen, which were agonistic in the yeast. Acetone-soluble fractions of hexane/acetone extracts of sediments showed higher estrogenic potency than hexane-soluble extracts in the ER-CALUX assay. Sediments obtained from industrialized areas such as the Port of Rotterdam showed the highest estrogenic potency of the 12 marine sediments tested (up to 40 pmol estradiol equivalents per gram sediment). The estrogenic activity of individual chemicals that can be found in sediments including: alkylphenol ethoxylates and carboxylates; phthalates; and pesticides, was tested. Increasing sidechain length of various nonylphenol ethoxylates resulted in decreased estrogenic activity. Of the phthalates tested, butylbenzylphthalate was the most estrogenic, though with a potency approximately 100 000 times less than E2. The organochlorine herbicides atrazine and simazine failed to induce reporter gene activity. As metabolic activation may be required to induce estrogenic activity, a metabolic transformation step was added to the ER-CALUX assay using incubation of compounds with liver microsomes obtained from PCB-treated rats. Results indicate that metabolites of E2, NP and bisphenol A were less active than the parent compounds, while metabolites of methoxychlor were more estrogenic following microsomal incubations. (C) 2002 Elsevier Science B.V. All rights reserved.
Detection of estrogenic potency in wastewater and surface water with three in vitro bioassays
Murk, A.J. ; Legler, J. ; Lipzig, M.M.H. van; Meerman, J.H.N. ; Belfroid, A.C. ; Spenkelink, A. ; Burg, B. van der; Rijs, G.B.J. ; Vethaak, D. - \ 2002
Environmental Toxicology and Chemistry 21 (2002)1. - ISSN 0730-7268 - p. 16 - 23.
oppervlaktewater - afvalwater - oestrogenen - biotesten - hormonen - surface water - waste water - oestrogens - hormones - bioassays
A study was performed to optimize sample preparation and application of three in vitro assays for measuring estrogenic potency in environmental extracts. The three assays applied were an estrogen receptor (ER)-binding assay and two reporter gene effect assays: a yeast estrogen screen (YES) and the ER-mediated chemically activated luciferase gene expression (ER-CALUX) assay. All assays were able to detect estrogenicity, but the amounts of material needed for the assays differed greatly between the three assays (ER-binding assay ≫ YES > ER-CALUX). In addition, in the ER-binding assay, both agonists and antagonists give an estrogenic response, resulting in higher estradiol equivalency (EEQ) levels than both the ER-CALUX and the YES assay for the same samples. The EEQs found in wastewater treatment plants (WTPs) with the ER-CALUX assay were in the range of 4 to 440 and 0.11 to 59 pmol/L for influent and effluent, respectively. Water extracts from four large rivers had levels ranging from 0.25 to 1.72 pmol/L. Extracts from suspended matter and sludge contained estrogenic potency of 0.26 to 2.49 and 1.6 to 41 pmol EEQ/g dry weight, respectively. In WTPs, the average reduction of estrogenic potency in effluent compared to influent was 90 to 95% in municipal WTPs and about 50% in industrial WTPs. In influent, 30% of the ER-CALUX activity could not be explained by the calculated potencies based on chemical analysis of a number of known (xeno)estrogens; in effluent the unexplained fraction was 80%. These first results of analyzing estrogenic potency in WTP water and surface water in The Netherlands indicate that further studies are warranted to investigate the actual risks for aquatic systems.
|The effects of nonylphenol on Rana temporaria tadpole survival, development and longitudinal growth
Poorte, J. de; Naber, A.B. ; Stumpel, A.H.P. ; Bosveld, A.T.C. - \ 2000
In: Endocrine-disrupting compounds: wildlife and human health risks : proceedings of a symposium 27 October 1998, The Hague / Vethaak, A.D., Rijksinstituut voor Kust en Zee - ISBN 9789036934046 - p. 137 - 141.
amphibia - rana temporaria - oestrogenen - fenolen - overleving - amfibieën - ecotoxicologie - endocrinologie - fauna - herpetologie - kikkers - milieuverontreiniging - amphibia - rana temporaria - oestrogens - phenols - survival - fauna
|Mogelijkheden voor isolatie van fyto-oestrogenen
Swallow, K. ; Langelaan, B. ; Zondervan, C. ; Jonge, H.D. ; Wichers, H. ; Soler Rivas, C. - \ 2000
Voedingsmiddelentechnologie 33 (2000)24. - ISSN 0042-7934 - p. 11 - 13.
geslachtshormonen - oestrogenen - plantenoestrogenen - steroïdhormonen - diethylstilbestrol - voedselhygiëne - voedingsmiddelen - ziektepreventie - voedselindustrie - voedseltechnologie - agro-industriële sector - sex hormones - oestrogens - plant oestrogens - steroid hormones - diethylstilbestrol - food hygiene - foods - disease prevention - food industry - food technology - agroindustrial sector
Samenwerking tussen het Food Processing Development Centre in Leduc (Canada) en het Agrotechnologisch Onderzoeksinstituut ATO in Wageningen leidde tot de ontwikkeling van een snelle detectiemethode voor bepaling van fyto-oestrogenen in planten. Deze spelen een belangrijke rol in onze gezondheid en bij het voorkomen van ziekten
|Application of 3 in vitro bioassays for oestrogenicity in waste water treatment plants and large rivers
Murk, A.J. ; Belfroid, A.C. ; Meerman, J.H.N. ; Legler, J. ; Burg, B. van den; Schafer, A.J. ; Rijs, G.B.J. ; Vethaak, A.D. - \ 2000
In: Endocrine disrupting compounds : Wildlife and human health risks, The Hague 1998 The Hague : - p. 38 - 43.
afvalwaterbehandeling - biotesten - oestrogenen - oppervlaktewater - rivieren - hormonen - zuiveringsinstallaties - waste water treatment - surface water - rivers - bioassays - oestrogens - hormones - purification plants
Hormoonontregelaars in water opsporen met biologische effectmetingen
Murk, A.J. ; Belfroid, A. ; Vethaak, D. - \ 2000
H2O : tijdschrift voor watervoorziening en afvalwaterbehandeling 33 (2000)1. - ISSN 0166-8439 - p. 20 - 23.
toxische stoffen - hormonen - geslachtshormonen - oestrogenen - synthetische oestrogenen - oestrogene eigenschappen - waterverontreiniging - waterkwaliteit - oppervlaktewater - afvalwater - rioolwater - samenstelling - eigenschappen - chemische eigenschappen - chemische analyse - tests - testen - assays - in vitro - Nederland - xenobiotica - toxic substances - hormones - sex hormones - oestrogens - synthetic oestrogens - oestrogenic properties - water pollution - water quality - surface water - waste water - sewage - composition - properties - chemical properties - chemical analysis - tests - testing - assays - in vitro - Netherlands - xenobiotics
Uitleg over de pricipes van verschillende in-vitro testen gebruikt voor het bepalen van de oestrogene potentie van monsters oppervlaktewater en afvalwater, en enige resultaten voor verschillende locaties in Nederland
Natuurlijke androgenen, oestrogenen en progestagenen in bloed, urine en faeces van jonge runderen : een literatuurstudie
Berende, P.L.M. ; Ginkel, L.A. van; Schilt, R. ; Arts, C.J.M. ; Stephany, R.W. ; Hartog, J.M.P. de - \ 1988
Wageningen : RIKILT (Rapport / RIKILT 88.29) - 26
rundveehouderij - kalveren - androgenen - oestrogenen - progestogenen - geslachtshormonen - urine-analyse - bloedanalyse - fecesonderzoek - diergezondheid - literatuuroverzichten - cattle husbandry - calves - androgens - oestrogens - progestogens - sex hormones - urine analysis - blood analysis - faecal examination - animal health - literature reviews
Een literatuurstudie is verricht betreffende de gehalten aan androgenen, oestrogenen en progestagenen in bloed, urine en faeces bij vleeskalveren. Omdat er niet voldoende gegevens beschikbaar waren voor genoemde hormonen in de 3 genoemde matrices bij vleeskalveren zijn ook literatuurgegevens van mannelijke en vrouwelijke fokkalveren in de leeftijdscategorie van ca. 7 tot ca. 11 maanden verwerkt. De meeste analyses zijn uitgevoerd met een radioimmunochemische methode (RIA).
Naturally occurring androgens, estrogens and progestogens in the blood, urine and feces of young cattle : a literature survey
Berende, P.L.M. ; Ginkel, L.A. van; Schilt, R. ; Arts, C.J.M. ; Stephany, R.W. ; Hartog, J.M.P. den - \ 1988
Wageningen : RIKILT (Report / RIKILT 88.60) - 38
kalveren - hormonen - androgenen - oestrogenen - progestogenen - bloed - urine - feces - calves - hormones - androgens - oestrogens - progestogens - blood - urine - faeces
The sources discussed in this literature study give the level of androgens, estrogens, and progestogens found in the blood, urine and feces of veal calves. Since there were not eneough data available in these three matrices for veal valves, the literature data for male and female breeding calves in the age group of 7-11 months has also been added.