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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 protein expression. 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.

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 protein expression. 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.

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

Lignanen zijn een belangrijke bron van fyto-oestrogenen in onze voeding. De hormoonachtige werking van deze stoffen zou het lichaam kunnen beschermen tegen kanker en hart- en vaatziekten. In Nederland is onderzoek gedaan naar de inname van lignanen en de relatie met deze chronische ziekten. De verbanden die in deze studies gevonden werden, waren niet eenduidig. Mogelijk beschermen lignanen tegen darmkanker in een vroeg stadium van de ziekte, maar hebben ze later weinig effect. In sommige groepen mensen werd echter juist een verhoogd risico gevonden

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.

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.

The aim of this study was to establish a relation between zearalenone contamination of crops in the Polish province of Wielkopolska and its occurrence in aquatic ecosystems close by the crop fields. Water samples were collected from water bodies such as drainage ditches, wells, or watercourses located in four agricultural areas. Moreover, control water samples were collected from the Bogdanka river, which was located outside the agricultural areas and near an urban area. Cereal samples were collected in the harvest season from each agricultural area close to tested water bodies. Zearalenone (ZEA) was found in all water and cereal samples. The highest concentrations were recorded in the postharvest season (September to October) and the lowest in the winter and spring. Mean ZEA concentrations in water ranged between 1.0 ng L-1 and 80.6 ng L-1, and in cereals from 3.72 ng g-1 to 28.97 ng g-1. Our results confi rm that mycotoxins are transported to aquatic systems by rain water through soil., Cilj ovog istraživanja bio je pojasniti učestalost pojave mikotoksina u vodenim ekosustavima i njihove korelacije sa stupnjem zaraze žitarica (uzgajanih u blizini vodospremnika), čija su zrna onečišćena (kontaminirana) mikotoksinima te problem prolaska mikotoksina kroz tlo u vodeni okoliš (onečišćenje podzemnih voda mikotoksinima). Uzorci vode prikupljeni su u regiji Wielkopolska iz vodenih tijela poput odvodnih jaraka i zdenaca, odnosno vodotoka smještenih u područjima koja se rabe za poljoprivredu. Dio uzoraka vode prikupljen je iz rijeke Bogdanka, u rubnom području grada Poznańa. U sezoni žetve sa svake poljoprivredne površine smještene u neposrednoj blizini testiranih vodenih tijela prikupljeni su uzorci žitarica. U svim analiziranim uzorcima vode i žitarica potvrđena je prisutnost zearalenona (ZEA). Najviše koncentracije mikotoksina u uzorcima sa svih poljoprivrednih površina zabilježene su u jesen nakon sezone žetve (rujan-listopad), dok su najniže vrijednosti izmjerene zimi i u proljeće. Srednje koncentracije zearalenona u vodi bile su u rasponu od 1,0 ng L-1 do 80,6 ng L-1. U žitarica je prosječna razina zearalenona iznosila 3,72 ng g-1 do 28,97 ng g-1, što govori u prilog vjerodostojnosti naše polazišne hipoteze o prijenosu mikotoksina kroz tlo nakon njihova ispiranja s površine u jarke za odvodnju.

Prenylatedisoflavonoids are found in large amountsin soya bean (Glycine max) germinated under stress and in licorice (Glycyrrhiza glabra). Prenylation of isoflavonoids has been associated with modification of theirestrogenic activity. The aims of this thesis were (1) to provide a structural characterisation of isoflavonoids, in particular the prenylated isoflavonoids occurring in soya and licorice, (2) to increase the estrogenic activity of soya beans by a malting treatment in the presence of a food-grade fungus, and (3) to correlate the in vitroagonistic/antagonistic estrogenicity with the presence of prenylated isoflavonoids. A reversed-phase UHPLC-DAD-MSn-based screening method was developed that allowed the tentative identification of all prenylated flavonoids in licorice extracts using neutral losses diagnosticfor prenylation. Moreover, both the chain and pyran ring can be discriminated. This method was also employed on extracts from soya seedlings, which were challenged with the food-grade fungus Rhizopusspp. Besides known prenylated pterocarpans, also novel prenylated isoflavones and coumestans were found in soya. Subsequently, a licorice root extract was fractionated and the fractions were screened for estrogenic activityusing an in vitro yeast ER bioassay.Several fractions were considered estrogenically active on one or both ER-subtypes (α and β). The estrogenic activity of some fractions was associated with the presence of glabrene, a prenylated isoflavene. The predominant phytoestrogen of licorice root, glabridin, did not show any agonistic activity, but was found to bea potent ERα antagonist. Large-scale Rhizopus-challenging of soya seedlings resulted in a 10 to 12 fold increase of the isoflavonoid content, in a diversification of isoflavonoid composition, andin an increase of estrogenic activity. The measured estrogenicity of the extracts on both ERs was lower than the theoretically calculated estrogenicity, indicating the presence of antagonists. This antagonistic activity was hypothesized to be associated with the presence of prenylated isoflavonoids.

The aim of the present thesis was to study the in vivo relevance of in vitro detected estrogenic effects of food associated compounds, with emphasis on prenatal exposure. The estrogenic potency of 21 food packaging-associated compounds was studied in ERα or ERβ transfected U2-OS (human osteoblasts devoid of endogenous estrogen receptors) cell lines. Six plasticizers and three anti-oxidants were slightly estrogenic in the ERα cells. BPA, NP, tris (2-ethylhexyl) trimellitate (TEHTM), propyl gallate and butylated hydroxy anisole (BHA) were estrogenic both in ERα and ERβ cells. These compounds appeared to be more estrogenic relative to estradiol (E2) in ERβ than in ERα cells. To study the in vivo relevance of these effects, in vivo biomarker-responses of BPA, NP, DEHA, DEHP, DIHP, p,p’-DDE and quercetin were studied in ER-Luc male mice. Of these seven compounds, BPA, DEHP and quercetin induced estrogenic effects after a single oral dosage at exposure levels 10-104 times higher than the established Tolerable Daily Intakes (TDI’s). It remains to be seen whether these margins are sufficiently high to allow the conclusion that these compounds are unlikely to represent a human health risk. As exposure to estrogenic compounds during developmental stages could be an important risk factor in developing hormone dependent cancers later in life, these seven compounds were studied in pregnant ER-Luc mice as well. After oral exposure of the mother animal the compounds were unable to significantly induce luc-activity in any of the tissues including fetuses. Unexpectedly however, NP, BPA and DIHP significantly lowered the placental luc-activity. The results indicate that at the current levels of exposure to food associated estrogenic compounds, direct estrogenic effects in the fetus are not expected. The mechanism and consequences of the significant luc-reduction in the placenta should be investigated to a further extent to elucidate its possible significance. Because of the absence of significant luc-induction in the fetuses, the fate and distribution of radioactively labeled E2, NP and p,p’-DDE were studied in pregnant C57black mice in order to investigate whether the compounds can actually reach the fetuses. E2 did not reach the fetus at a level above the detection limit, NP and p,p’-DDE levels were above the detection limit in fetuses exposed via the mother from GD8-16. Levels of E2 and NP detected in the placenta were significantly higher than those in the fetuses, up to five fold for E2 and three fold for NP, possibly due to prevention of transport of the compounds to the fetuses by placental binding proteins. Based on the findings of the present thesis it was concluded that in spite of the in vitro estrogenicity of various food-born estrogens, in vivo estrogenic effects are not likely expected as estrogenicity in vivo is shown at levels 10-104 fold higher than the TDI’s for humans and placental binding proteins may reduce fetal exposure. However, the estrogenic compounds were given as a single acute dose, whereas TDI values and also risks associated with dietary exposure can be expected to result from chronic combined exposure. Therefore, it remains to be seen whether the estimated margins are sufficiently high. Future experiments are therefore needed that focus on long term and combined exposure. Another factor that remains to be solved relates to the fact that only in in vitro assays the relative ability of compounds to differentially activate ERα or ERβ can be quantified. All together the results of the present thesis reveal that the extent at which in vitro estrogenic effects may result in in vivo estrogenicity may vary with the compound under study and should be evaluated on a case by case basis taking differences in absorption, distribution, metabolism and excretion, the intrinsic estrogenic potency of chemicals for either the ERα or ERβ and the differential levels of expression of these receptors in different tissues in vivo into account.

Het doel van het onderzoek in dit proefschrift was het bepalen van de gevoeligheid en bruikbaarheid van verschillende ‘markers’ voor onderzoek naar de hormoonverstorende potentie van chemische stoffen in toxiciteitstesten met de vis als testorganisme

Het doel van het onderzoek in dit proefschrift was het bepalen van de gevoeligheid en bruikbaarheid van verschillende ‘markers’ voor onderzoek naar de hormoonverstorende potentie van chemische stoffen in toxiciteitstesten met de vis als testorganisme

Lignanen zijn een belangrijke bron van fyto-oestrogenen in onze voeding. De hormoonachtige werking van deze stoffen zou het lichaam kunnen beschermen tegen kanker en hart- en vaatziekten. In Nederland is onderzoek gedaan naar de inname van lignanen en de relatie met deze chronische ziekten. De verbanden die in deze studies gevonden werden, waren niet eenduidig. Mogelijk beschermen lignanen tegen darmkanker in een vroeg stadium van de ziekte, maar hebben ze later weinig effect. In sommige groepen mensen werd echter juist een verhoogd risico gevonden

The aim of the present thesis was to study the in vivo relevance of in vitro detected estrogenic effects of food associated compounds, with emphasis on prenatal exposure. The estrogenic potency of 21 food packaging-associated compounds was studied in ERα or ERβ transfected U2-OS (human osteoblasts devoid of endogenous estrogen receptors) cell lines. Six plasticizers and three anti-oxidants were slightly estrogenic in the ERα cells. BPA, NP, tris (2-ethylhexyl) trimellitate (TEHTM), propyl gallate and butylated hydroxy anisole (BHA) were estrogenic both in ERα and ERβ cells. These compounds appeared to be more estrogenic relative to estradiol (E2) in ERβ than in ERα cells. To study the in vivo relevance of these effects, in vivo biomarker-responses of BPA, NP, DEHA, DEHP, DIHP, p,p’-DDE and quercetin were studied in ER-Luc male mice. Of these seven compounds, BPA, DEHP and quercetin induced estrogenic effects after a single oral dosage at exposure levels 10-104 times higher than the established Tolerable Daily Intakes (TDI’s). It remains to be seen whether these margins are sufficiently high to allow the conclusion that these compounds are unlikely to represent a human health risk. As exposure to estrogenic compounds during developmental stages could be an important risk factor in developing hormone dependent cancers later in life, these seven compounds were studied in pregnant ER-Luc mice as well. After oral exposure of the mother animal the compounds were unable to significantly induce luc-activity in any of the tissues including fetuses. Unexpectedly however, NP, BPA and DIHP significantly lowered the placental luc-activity. The results indicate that at the current levels of exposure to food associated estrogenic compounds, direct estrogenic effects in the fetus are not expected. The mechanism and consequences of the significant luc-reduction in the placenta should be investigated to a further extent to elucidate its possible significance. Because of the absence of significant luc-induction in the fetuses, the fate and distribution of radioactively labeled E2, NP and p,p’-DDE were studied in pregnant C57black mice in order to investigate whether the compounds can actually reach the fetuses. E2 did not reach the fetus at a level above the detection limit, NP and p,p’-DDE levels were above the detection limit in fetuses exposed via the mother from GD8-16. Levels of E2 and NP detected in the placenta were significantly higher than those in the fetuses, up to five fold for E2 and three fold for NP, possibly due to prevention of transport of the compounds to the fetuses by placental binding proteins. Based on the findings of the present thesis it was concluded that in spite of the in vitro estrogenicity of various food-born estrogens, in vivo estrogenic effects are not likely expected as estrogenicity in vivo is shown at levels 10-104 fold higher than the TDI’s for humans and placental binding proteins may reduce fetal exposure. However, the estrogenic compounds were given as a single acute dose, whereas TDI values and also risks associated with dietary exposure can be expected to result from chronic combined exposure. Therefore, it remains to be seen whether the estimated margins are sufficiently high. Future experiments are therefore needed that focus on long term and combined exposure. Another factor that remains to be solved relates to the fact that only in in vitro assays the relative ability of compounds to differentially activate ERα or ERβ can be quantified. All together the results of the present thesis reveal that the extent at which in vitro estrogenic effects may result in in vivo estrogenicity may vary with the compound under study and should be evaluated on a case by case basis taking differences in absorption, distribution, metabolism and excretion, the intrinsic estrogenic potency of chemicals for either the ERα or ERβ and the differential levels of expression of these receptors in different tissues in vivo into account.

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 De Nederlandse anaerobe zuiveringsinstallaties kunnen onvoldoende oestrogenen afbreken, ontdekte De Mes. Daarnaast ontdekte de promovenda dat het probleem nog een graadje serieuzer was dan ze aanvankelijk had gedacht. Enkele tientallen procenten van de vrouwelijke hormonen verlaten het lichaam als een metaboliet zonder hormonale werking. Het lichaam heeft er chemische groepen aan geplakt waardoor de hormonen niet meer actief zijn. Het zuiveringsproces schroeft die groepen er echter weer af. Het proces kan de ontwaakte oestrogenen afbreken, maar heeft daarvoor dan nog meer tijd nodig. ‘We zullen onze manier van zuiveren radicaal moeten veranderen’, concludeert De Mes. ‘Dat is alleen betaalbaar als we het afvalwater van onze toiletten apart gaan zuiveren. Alleen op die manier kun je intensiever en langer zuiveren.’

An ecotoxicological study of produced formation water (PFW) from the Berge Helene FPSO at the Chinguetti oil field, operated by Woodside in Mauritania was performed. The study was requested by MRAG Ltd as a part of Woodside’s Environmental Monitoring Plan.

An ecotoxicological study of produced formation water (PFW) from the Berge Helene FPSO at the Chinguetti oil field, operated by Woodside in Mauritania was performed. The study was requested by MRAG Ltd as a part of Woodside’s Environmental Monitoring Plan.

De hoeveelheden phytoestrogenen in proteïnerijke groenten en de mogelijke effecten op de groei van zeugen worden onderzocht

De hoeveelheden phytoestrogenen in proteïnerijke groenten en de mogelijke effecten op de groei van zeugen worden onderzocht

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