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The research described in this thesis investigated the amount and potential effects of EDCs present in human milk using samples derived from the Norwegian HUMIS cohort. This work focused on three main actors of the endocrine system, the estrogens, androgens, and thyroid hormones. The first aim of the thesis was to develop a suitable method to properly extract EDCs from human milk samples, focusing on both polar and apolar endocrine active contaminants. The second aim was to perform a pilot study on a limited number of breast milk samples, using the existing ERα and AR CALUX bioassays, in agonistic and antagonistic mode. This phase was also used to evaluate the nature and origin of the observed EDC activity, as well as the contribution of endogenous hormones to the measurements. The third goal was to measure the EDC activity in a larger set of human milk samples to evaluate the potential association between anti-androgenic EDCs and an androgen-dependent deformity: cryptorchidism. The last objective was the development and application of novel thyroid hormone-based bioassays, TRβ CALUX and TTR-TRβ CALUX assays. These bioassays were used to evaluate the impact of breast milk contaminants, including well-known PFAS (perfluorooctanoic acid or PFOA; perfluorooctane sulfonic acid or PFOS), on the thyroid system. The thesis is divided in two distinct parts: Chapter Two and Chapter Three focusing on sex steroid disruption, and Chapter Four and Chapter Five covering thyroid system interferences, respectively. In Chapter Two, the method developed to extract EDCs from breast milk samples, used throughout the thesis, is described. In this chapter, estrogenic, anti-estrogenic, androgenic, and anti-androgenic activities derived from ten human milk extracts were analyzed on the (anti-)ERα and (anti-)AR CALUX bioassays. To rule out the impact of endogenous hormones in the measured activity, a pooled breast milk sample presenting anti-androgenic activity was fractionated and screened for natural hormones and metabolites by means of non-target screening using time-of-flight mass spectrometry UHPLC-Q-TOF-MS/MS. Chapter Three describes a larger-scale study involving 199 participants and presents results of anti-androgenic EDC activity in breast milk samples. In this chapter, we investigated the potential association between anti-androgenic activity in mothers’ milk and the occurrence of cryptorchidism, an androgen-dependent deformity, in the offspring. Moreover, we estimated the overall extent of exposure of a nursing child to anti-androgenic EDCs via breastfeeding. In Chapter Four, the TRβ CALUX bioassay, allowing screening of TRβ disrupting activity, and the TTR-TRβ CALUX assay, designed to detect competing properties towards T4 for TTR binding, were evaluated using well-known reference compounds. The performance of the TTR-TRβ CALUX assay was also evaluated during a short pilot study involving water samples. In Chapter Five the newly validated TRβ and TTR-TRβ CALUX bioassays were further used to assess a set of thirteen PFAS, known to affect the TH system (Chang et al. 2008; Thibodeaux et al. 2003) for thyroid-disrupting activities. Subsequently, ten breast milk extracts, with known PFOS and PFOA concentrations, were analyzed on the same assays. In this chapter, the impact of PFOS and PFOA levels was weighed in respect to thyroid-disrupting activity in breast milk. Furthermore, the average exposure to thyroid-disrupting EDCs during the first year of life of a nursing infant through breastfeeding was estimated. Finally, Chapter Six reviews and discusses the most important outcomes of the studies regarding hormonal-disruption derived from the presence of EDCs in Norwegian breast milk samples. General conclusions and an outlook on future perspective are presented.

Parabenen zijn stoffen die als conserveermiddel in verschillende consumentenproducten kunnen worden gebruikt, zoals persoonlijke verzorgingsproducten, voedsel en medicijnen. Ze gaan de groei van schimmels en bacteriën tegen. Parabenen worden er van verdacht dat ze een hormoonverstorende werking hebben. Hormoonverstorende stoffen kunnen de hormoonhuishouding in de war brengen. Het RIVM heeft in een literatuurstudie voor de drie meest gebruikte parabenen (methyl-, ethyl- en propylparabeen) onderzocht of deze als een hormoonverstorende stof beschouwd kunnen worden. De beschikbare gegevens uit dierstudies die in de literatuur zijn beschreven, leveren echter onvoldoende informatie om hierover een conclusie te trekken. In de studie is ook bekeken of de mogelijke hormoonverstorende effecten zijn meegenomen in de wettelijke beoordelingskaders die van toepassing zijn. Omdat er onvoldoende gegevens zijn, is dat niet het geval bij de huidige risicobeoordeling. De blootstelling door persoonlijke verzorgingsproducten is behoorlijk goed onderzocht en lijkt in het algemeen het meest aan de totale blootstelling bij te dragen. De blootstelling vanuit voedsel blijkt verwaarloosbaar. Voor een acceptabele, eerste schatting van de blootstelling vanuit medicijnen is te weinig informatie beschikbaar. Uit de studie blijkt verder dat de mate waarin mensen aan de afzonderlijke parabenen worden blootgesteld naar schatting lager lijkt te zijn dan de hoeveelheid waarbij een gezondheidseffect kan worden verwacht. Voor deze blootstellingsschattingen zijn veiligheidshalve zeer ongunstige aannames gebruikt. In de praktijk worden mensen echter aan een combinatie van verschillende stoffen blootgesteld. Het is nog onduidelijk of en hoe deze gecombineerde blootstelling aan de verschillende parabenen meegenomen kan worden in de risicobeoordeling. Om hiaten in de kennis te vullen adviseert het RIVM om aanvullend onderzoek te doen naar de mogelijk hormoonverstorende werking van de parabenen en de blootstellingsschatting te verfijnen. Hiervoor worden aanbevelingen aangereikt.

Parabenen zijn stoffen die als conserveermiddel in verschillende consumentenproducten kunnen worden gebruikt, zoals persoonlijke verzorgingsproducten, voedsel en medicijnen. Ze gaan de groei van schimmels en bacteriën tegen. Parabenen worden er van verdacht dat ze een hormoonverstorende werking hebben. Hormoonverstorende stoffen kunnen de hormoonhuishouding in de war brengen. Het RIVM heeft in een literatuurstudie voor de drie meest gebruikte parabenen (methyl-, ethyl- en propylparabeen) onderzocht of deze als een hormoonverstorende stof beschouwd kunnen worden. De beschikbare gegevens uit dierstudies die in de literatuur zijn beschreven, leveren echter onvoldoende informatie om hierover een conclusie te trekken. In de studie is ook bekeken of de mogelijke hormoonverstorende effecten zijn meegenomen in de wettelijke beoordelingskaders die van toepassing zijn. Omdat er onvoldoende gegevens zijn, is dat niet het geval bij de huidige risicobeoordeling. De blootstelling door persoonlijke verzorgingsproducten is behoorlijk goed onderzocht en lijkt in het algemeen het meest aan de totale blootstelling bij te dragen. De blootstelling vanuit voedsel blijkt verwaarloosbaar. Voor een acceptabele, eerste schatting van de blootstelling vanuit medicijnen is te weinig informatie beschikbaar. Uit de studie blijkt verder dat de mate waarin mensen aan de afzonderlijke parabenen worden blootgesteld naar schatting lager lijkt te zijn dan de hoeveelheid waarbij een gezondheidseffect kan worden verwacht. Voor deze blootstellingsschattingen zijn veiligheidshalve zeer ongunstige aannames gebruikt. In de praktijk worden mensen echter aan een combinatie van verschillende stoffen blootgesteld. Het is nog onduidelijk of en hoe deze gecombineerde blootstelling aan de verschillende parabenen meegenomen kan worden in de risicobeoordeling. Om hiaten in de kennis te vullen adviseert het RIVM om aanvullend onderzoek te doen naar de mogelijk hormoonverstorende werking van de parabenen en de blootstellingsschatting te verfijnen. Hiervoor worden aanbevelingen aangereikt.

alleen digitaal verschenen

alleen digitaal verschenen

This thesis focuses on a modern analytical method, proteomics, to investigate its use in the field of toxicological research. Proteomics is a high resolution method which separates all proteins present in a sample at a clearly defined state and compares this pattern to another one, under slightly different conditions (e.g. after exposure to a chemical). Protein changes may give rise to or reflect disease/harm of the individual and can be attributed to alterations in body functions/regulation systems. Analysis conditions and different varieties of proteomic methods are explained, and a brief introduction given where proteomics is already applied in toxicology. A specific investigation has been performed with the flame retardant HBCD (i.e. hexabromocyclododecane). It is a compound that accumulates in lipid tissue from where it is only slowly removed. Its mechanism of action is not yet completely understood and sometimes seems to be contradictory. Rats were exposed to HBCD in very low doses for just one week and liver proteins were compared to those of unexposed animals. As HBCD is suggested to disturb the thyroid system, both healthy and hypothyroid rats were investigated, of both genders. In female rats, not in males, some specific liver protein changes were seen in glucose/carbohydrate and lipid metabolism, and also in some stress related proteins. Changes were not dependent on the thyroid function of the females. These results are in line with previous findings that female rats were more susceptible to HBCD than males. In a further step, protein patterns of unexposed animals of both genders were compared, revealing gender-dependent differences that exceeded the effects seen in any of the other comparisons, mainly in the pathways that were also affected by HBCD in females. A previous proteomic study on serum proteins has also shown clear gender-dependent concentration differences in rats. This underlines the importance of performing studies both in female and male individuals. The detection of considerable gender-dependent protein alterations confirms that proteomics is a biochemical tool with high sensitivity and large potential also in toxicological research.

An extensive study was carried out in the Netherlands on the occurrence of a number of estrogenic compounds in surface water, sediment, biota, wastewater, rainwater and on the associated effects in fish. Compounds investigated included natural and synthetic hormones, phthalates, alkylphenol(ethoxylate)s and bisphenol-A. The results showed that almost all selected (xeno-)estrogens were present at low concentrations in the aquatic environment. Locally, they were found at higher levels. Hormones and nonylphenol(ethoxylate)s were present in concentrations that are reportedly high enough to cause estrogenic effects in fish. Field surveys did not disclose significant estrogenic effects in male flounder (Platichthys flesus) in the open sea and in Dutch estuaries. Minor to moderate estrogenic effects were observed in bream (Abramis brama) in major inland surface waters such as lowland rivers and a harbor area. The prevalence of feminizing effects in male fish is largest in small regional surface waters that are strongly influenced by sources of potential hormone-disrupting compounds. High concentrations of plasma vitellogenin and an increased prevalence of ovotestes occurred in wild male bream in a small river receiving a considerable load of effluent from a large sewage treatment plant. After employing in vitro and in vivo bioassays, both in situ and in the laboratory, we conclude that in this case hormones (especially 17α-ethynylestradiol) and possibly also nonylphenol(ethoxylate)s are primarily responsible for these effects. © 2005 Elsevier Ltd. All rights reserved.

In vitro bioassays are valuable tools for screening environmental samples for the presence of bioactive (e.g., endocrine-disrupting) compounds. They can be used to direct chemical analysis of active compounds in toxicity identification and evaluation (TIE) approaches. In the present study, five in vitro bioassays were used to profile toxic potencies in sediments, with emphasis on endocrine disruption. Nonpolar total and acid-treated stable extracts of sediments from 15 locations in the Rhine Meuse estuary area in The Netherlands were assessed. Dioxin-like and estrogenic activities (using dioxin-responsive chemical-activated luciferase gene expression [DR-CALUX] and estrogen-responsive chemical-activated luciferase gene expression [ER-CALUX] assays) as well as genotoxicity (UMU test) and nonspecific toxic potency (Vibrio fischeri assay) were observed in sediment extracts. For the first time, to our knowledge, in vitro displacement of thyroid hormone thyroxine (T4) from the thyroid hormone transport protein thransthyretin by sediment extracts was observed, indicating the presence of compounds potentially able to disrupt T4 plasma transport processes. Antiestrogenic activity was also observed in sediment. The present study showed the occurrence of endocrine-disrupting potencies in sediments from the Dutch delta and the suitability of the ER- and DR-CALUX bioassays to direct endocrine-disruption TIE studies.

Jomaa, B. (2015). Thyroid in a Jar: Towards an Integrated In Vitro Testing Strategy for Thyroid-Active Compounds. PhD thesis, Wageningen University, the Netherlands Abstract The aim of this thesis was to find in vitro and toxicogenomics-based alternatives to in vivo thyroid hormone disruption tests. In vitro alternatives can help reduce the amount of animal testing required under the European Union regulation for the registration, evaluation, authorization and restriction of chemicals (REACH). Moreover, with the use of human cell lines and human-identical synthetic proteins, interspecies differences can be reduced and in some cases eliminated. This thesis has shed light on the relevance of current in vitro assays for thyroid and pituitary cell proliferation, has led to the development of the TSH screen, a luminol-based thyroid peroxidase inhibition assay and the zebrafish-based general development score (GDS) for the detection of developmental toxicants, including those that act through the thyroid hormone system. Moreover, the microarray assay for real-time coregulator-nuclear receptor interaction (MARCoNI) assay was used to reveal the modulating effects of thyroid-active compounds on TRα and TRβ interactions with a peptide array representing 66 different coregulators. These developments along with an in-depth analysis of the thyroid hormone system and the presentation of the state of the art in thyroid disruption testing have highlighted the progress made and at the same time have underlined the challenges that lay ahead.

Jomaa, B. (2015). Thyroid in a Jar: Towards an Integrated In Vitro Testing Strategy for Thyroid-Active Compounds. PhD thesis, Wageningen University, the Netherlands Abstract The aim of this thesis was to find in vitro and toxicogenomics-based alternatives to in vivo thyroid hormone disruption tests. In vitro alternatives can help reduce the amount of animal testing required under the European Union regulation for the registration, evaluation, authorization and restriction of chemicals (REACH). Moreover, with the use of human cell lines and human-identical synthetic proteins, interspecies differences can be reduced and in some cases eliminated. This thesis has shed light on the relevance of current in vitro assays for thyroid and pituitary cell proliferation, has led to the development of the TSH screen, a luminol-based thyroid peroxidase inhibition assay and the zebrafish-based general development score (GDS) for the detection of developmental toxicants, including those that act through the thyroid hormone system. Moreover, the microarray assay for real-time coregulator-nuclear receptor interaction (MARCoNI) assay was used to reveal the modulating effects of thyroid-active compounds on TRα and TRβ interactions with a peptide array representing 66 different coregulators. These developments along with an in-depth analysis of the thyroid hormone system and the presentation of the state of the art in thyroid disruption testing have highlighted the progress made and at the same time have underlined the challenges that lay ahead.

De onderzoeksgroep Analytische en milieuchemie (ANCH) op de Vrije Universiteit Brussel onderzocht het binnenhuisstof van 12 kleuterscholen op hormoonverstorende activiteit in opdracht van de partij Groen. Auteur: T. Vandermarken, Vrije Universiteit Brussel

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 het kader van het project 'endocriene verstoring humaan' worden onder meer actuele ontwikkelingen gerapporteerd over de relatie tussen endocriene verstoring en humane effecten. Dit rapport geeft een overzicht van de presentaties die over dit onderwerp gegeven werden tijdens een workshop, gehouden in mei 2000 in Kopenhagen, die werd georganiserd door prof. Niels Skakkebaek. De workshop bracht een niet onaanzienlijke groep expert wetenschappers op dit terrein bijeen. Zowel actuele overzichtsverhalen als nieuwe resultaten van lopende researchprojecten werden gepresenteerd. De workshop concludeerde dat endocriene verstoring een onderwerp van zorg blijft, hoewel causale relaties tussen blootstelling aan endocriene stoffen via het milieu en gezondheidseffecten bij de mens tot op heden niet zijn aangetoond. Er zijn met name meer humane blootstellingsgegevens nodig als basis voor een actuele risicoschatting voor de mens met betrekking tot endocriene verstoring.

In het kader van het project 'endocriene verstoring humaan' worden onder meer actuele ontwikkelingen gerapporteerd over de relatie tussen endocriene verstoring en humane effecten. Dit rapport geeft een overzicht van de presentaties die over dit onderwerp gegeven werden tijdens een workshop, gehouden in mei 2000 in Kopenhagen, die werd georganiserd door prof. Niels Skakkebaek. De workshop bracht een niet onaanzienlijke groep expert wetenschappers op dit terrein bijeen. Zowel actuele overzichtsverhalen als nieuwe resultaten van lopende researchprojecten werden gepresenteerd. De workshop concludeerde dat endocriene verstoring een onderwerp van zorg blijft, hoewel causale relaties tussen blootstelling aan endocriene stoffen via het milieu en gezondheidseffecten bij de mens tot op heden niet zijn aangetoond. Er zijn met name meer humane blootstellingsgegevens nodig als basis voor een actuele risicoschatting voor de mens met betrekking tot endocriene verstoring.

Thyroid hormones regulate crucial processes in vertebrates such as reproduction, development and energy metabolism. Endocrine disruption via the thyroid hormone system is gaining more attention both from scientists and regulators, because of the increasing incidence of hormone-related cancers and developmental defects, and the requirement that newly marketed compounds are tested for thyroid hormone disruption. To reduce the number of experimental animals used and to increase the insight into the mechanisms of toxic interference with the thyroid hormone receptor function, we developed and validated functional in vitro bioassays for thyroid hormone receptor-mediated toxicity. These assays enable quick identification and quantification of specific thyroid hormone receptor disrupting potency of compounds and contribute to the further establishment of a battery of in vitro tests for hazard identification of thyroid active compounds.

Thyroid hormones regulate crucial processes in vertebrates such as reproduction, development and energy metabolism. Endocrine disruption via the thyroid hormone system is gaining more attention both from scientists and regulators, because of the increasing incidence of hormone-related cancers and developmental defects, and the requirement that newly marketed compounds are tested for thyroid hormone disruption. To reduce the number of experimental animals used and to increase the insight into the mechanisms of toxic interference with the thyroid hormone receptor function, we developed and validated functional in vitro bioassays for thyroid hormone receptor-mediated toxicity. These assays enable quick identification and quantification of specific thyroid hormone receptor disrupting potency of compounds and contribute to the further establishment of a battery of in vitro tests for hazard identification of thyroid active compounds.

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)

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)

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

Within the project on endocrine disruption and human health one aim is to keep track with the state of the art as regards endocrine disrupter research relating to human health effects. This report summarizes the presentations given at the Workshop on the subject held in May 2000 in Copenhagen, which was organised by professor Niels Skakkebaek. The workshop brought together a considerable group of key scientists in the field. Both timely overviews as well as current research results were presented. The workshop concluded that endocrine disruption remains a matter of concern, although causal relationships between exposure to environmental endocrine disrupters and human health effects have not been proven to date. Particularly, human exposure data are needed to found a solid basis for actual human risk assessment in the field of endocrine disruption.

een verkennende studie van de wetenschappelijke literatuur naar het voorkomen en de mogelijke risico’s van hormoonverstorende stoffen (inclusief hormonen) en diergeneesmiddelen in het bodemmilieu. De belangrijkste conclusie is dat er nog veel onbekend is over het voorkomen van deze stoffen in de bodem. Dit komt vooral doordat er zeer weinig metingen zijn verricht. Omdat er geen inzicht bestaat in de concentratieniveaus en ook nauwelijks in de effecten, zijn de risico’s van deze stoffen voor bodemgezondheid, voedselkwaliteit en de gezondheid van landbouwhuisdieren moeilijk in te schatten. Verder is de omvang van de verontreiniging met de meeste van deze stoffen onbekend en kan dus de schaal van de problematiek moeilijk worden vastgesteld

een verkennende studie van de wetenschappelijke literatuur naar het voorkomen en de mogelijke risico’s van hormoonverstorende stoffen (inclusief hormonen) en diergeneesmiddelen in het bodemmilieu. De belangrijkste conclusie is dat er nog veel onbekend is over het voorkomen van deze stoffen in de bodem. Dit komt vooral doordat er zeer weinig metingen zijn verricht. Omdat er geen inzicht bestaat in de concentratieniveaus en ook nauwelijks in de effecten, zijn de risico’s van deze stoffen voor bodemgezondheid, voedselkwaliteit en de gezondheid van landbouwhuisdieren moeilijk in te schatten. Verder is de omvang van de verontreiniging met de meeste van deze stoffen onbekend en kan dus de schaal van de problematiek moeilijk worden vastgesteld

Persistent organic pollutants such as PCBs have been hypothesized to contribute to the observed global decline of amphibian populations. Thyroid hormone (TH) disruption is one of the possible mechanisms for effects of xenobiotics on amphibian development. In addition to the important functions shared with other vertebrates, TH also has an important function in amphibians. The metamorphosis of amphibian larvae to froglet needs a TH surge shortly before the onset of metamorphosis to proceed. To study the potential disruption during two specific life-stages a bioassay with exposure during very early developmental stages and one for exposure of tadpoles just before the onset of TH dependent metamorphosis were developed. The assays were optimized, validated with PCBs as standards and applied for testing of realistic dosages of polluted sediment extracts. In addition, an in vitro assay using the TH dependent growth of a cell-line was established as a screening tool.The studies presented in this thesis reveal that the currently used early life stage test (FETAX) does not detect effects of the tested PCBs and apolar sediment extracts during the 96-hour test period whereas the newly developed prolonged-FETAX showed significant delayed effects of very low exposure concentrations on body weight, and on the period until- and percentage of animals finishing successful metamorphosis.In the so-called Synchronized Amphibian Metamorphosis Assay thiourea was used to synchronize tadpole development in NF stage 54 thereby starting experiments with a very homogenous group of animals. Significant effects of oral exposure to PCBs and apolar sediment extracts were found on the period until metamorphosis, and the distribution of the developmental stages of tadpoles that did not finish metamorphosis.The approach with exposing embryos (prolonged-FETAX) and tadpoles (Synchronized Amphibian Metamorphosis Assay) may finally better reflect environmental risks of apolar compounds than limiting the exposure to solely a single life-stage or water-exposure. The effects of the highly diluted apolar sediment extracts suggest the presence of TH disrupting compounds in the aquatic environment and possible effects of such compounds on development of amphibians and possibly other aquatic vertebrate species in the wild. The in vitro mode! showed its suitability and importance to study specific aspects of endocrine disrupting potency of toxic compounds.

The last years, both scientific and public concern about the possible threat of compounds in the environment that may affect endocrine functions has increased. Thus far, the majority of endocrine disruptor research has focused on the interference of compounds with the sex hormone homeostasis. Less attention has been paid to disruption of the hypothalamus-pituitary-thyroid (HPT) axis, despite the fact that several lines of evidence suggest that this system is also susceptible to disruption by compounds present in the environment. Disturbances in thyroid hormone (TH) homeostasis may lead to mosaic effects on development, growth patterns and metabolism in vertebrates including mammals and amphibians. Recent studies now indicate that the Thyroid hormone Receptors (TRs) also are targets of industrial compounds, but the effects and mechanisms are difficult to establish since suitable test assays are very limited. In part, this may be because research has generally focused on the ability of compounds to affect TH transport, TH metabolism and TH blood plasma levels but not on disruption of TR mediated TH-action.The research presented in this thesis aims to enhance insight into the mechanisms underlying the effects of endocrine disrupting compounds (EDCs) on TR-mediated TH-action. To this end, newly developed and validated in vitro and in vivo assays are applied in addition to an ex vivo assay using isolated tail tissue. This ex vivo model more resembles the natural situation for cells than in vitro assays, but excludes the feedback mechanisms from the TH axis orextracellularmetabolism which may obscure TR-mediated responses. In the assays exposure is performed in combination with TH in order to closer approach the exposure of cells under physiological conditions, where TH is present during important vertebrate fetal developmental periods. The in vitro , ex vivo and in vivo assay responses also are compared to study to what degree the in vitro and ex vivo assays can predict the in vivo thyroid hormone disrupting potency of compounds. Apart from the development of newtestsystemsfor thyroid hormone disruption, the studies presented in this thesis also provide indications for the mechanism ofEDCson the level of the TR. Furthermore, the results illustrate thatthe in vitro results are highly predictive for the effects as observed in vivo . Finally, it can be concluded that testing of compounds for potential TH disrupting effects, should be carried out in presence and absence of TH at its EC 50 .

Persistent organic pollutants such as PCBs have been hypothesized to contribute to the observed global decline of amphibian populations. Thyroid hormone (TH) disruption is one of the possible mechanisms for effects of xenobiotics on amphibian development. In addition to the important functions shared with other vertebrates, TH also has an important function in amphibians. The metamorphosis of amphibian larvae to froglet needs a TH surge shortly before the onset of metamorphosis to proceed. To study the potential disruption during two specific life-stages a bioassay with exposure during very early developmental stages and one for exposure of tadpoles just before the onset of TH dependent metamorphosis were developed. The assays were optimized, validated with PCBs as standards and applied for testing of realistic dosages of polluted sediment extracts. In addition, an in vitro assay using the TH dependent growth of a cell-line was established as a screening tool.The studies presented in this thesis reveal that the currently used early life stage test (FETAX) does not detect effects of the tested PCBs and apolar sediment extracts during the 96-hour test period whereas the newly developed prolonged-FETAX showed significant delayed effects of very low exposure concentrations on body weight, and on the period until- and percentage of animals finishing successful metamorphosis.In the so-called Synchronized Amphibian Metamorphosis Assay thiourea was used to synchronize tadpole development in NF stage 54 thereby starting experiments with a very homogenous group of animals. Significant effects of oral exposure to PCBs and apolar sediment extracts were found on the period until metamorphosis, and the distribution of the developmental stages of tadpoles that did not finish metamorphosis.The approach with exposing embryos (prolonged-FETAX) and tadpoles (Synchronized Amphibian Metamorphosis Assay) may finally better reflect environmental risks of apolar compounds than limiting the exposure to solely a single life-stage or water-exposure. The effects of the highly diluted apolar sediment extracts suggest the presence of TH disrupting compounds in the aquatic environment and possible effects of such compounds on development of amphibians and possibly other aquatic vertebrate species in the wild. The in vitro mode! showed its suitability and importance to study specific aspects of endocrine disrupting potency of toxic compounds.

The last years, both scientific and public concern about the possible threat of compounds in the environment that may affect endocrine functions has increased. Thus far, the majority of endocrine disruptor research has focused on the interference of compounds with the sex hormone homeostasis. Less attention has been paid to disruption of the hypothalamus-pituitary-thyroid (HPT) axis, despite the fact that several lines of evidence suggest that this system is also susceptible to disruption by compounds present in the environment. Disturbances in thyroid hormone (TH) homeostasis may lead to mosaic effects on development, growth patterns and metabolism in vertebrates including mammals and amphibians. Recent studies now indicate that the Thyroid hormone Receptors (TRs) also are targets of industrial compounds, but the effects and mechanisms are difficult to establish since suitable test assays are very limited. In part, this may be because research has generally focused on the ability of compounds to affect TH transport, TH metabolism and TH blood plasma levels but not on disruption of TR mediated TH-action.The research presented in this thesis aims to enhance insight into the mechanisms underlying the effects of endocrine disrupting compounds (EDCs) on TR-mediated TH-action. To this end, newly developed and validated in vitro and in vivo assays are applied in addition to an ex vivo assay using isolated tail tissue. This ex vivo model more resembles the natural situation for cells than in vitro assays, but excludes the feedback mechanisms from the TH axis orextracellularmetabolism which may obscure TR-mediated responses. In the assays exposure is performed in combination with TH in order to closer approach the exposure of cells under physiological conditions, where TH is present during important vertebrate fetal developmental periods. The in vitro , ex vivo and in vivo assay responses also are compared to study to what degree the in vitro and ex vivo assays can predict the in vivo thyroid hormone disrupting potency of compounds. Apart from the development of newtestsystemsfor thyroid hormone disruption, the studies presented in this thesis also provide indications for the mechanism ofEDCson the level of the TR. Furthermore, the results illustrate thatthe in vitro results are highly predictive for the effects as observed in vivo . Finally, it can be concluded that testing of compounds for potential TH disrupting effects, should be carried out in presence and absence of TH at its EC 50 .

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

Omdat de ontwikkeling van amfibieën sterk hormoon-gereguleerd is, is onderzocht in hoeverre de kikker een geschikte indicator is voor hormoonverstorende stoffen in het milieu. Hiertoe zijn in het laboratorium blootstellingstudies uitgevoerd met kikkerlarven en volwassen Rana kikkers, welke algemeen in Nederland voorkomen. Onderzocht is in hoeverre oestradiol (E2) en tetrachloordibenzo-p-dioxine (TCDD) de ontwikkeling, overleving, lichaams- en orgaangewichten, geslachtsorganen en -hormonen, en specifieke cytochroom P450 enzymen beïnvloeden. Ook is de onderlinge samenhang van deze ettectparameters bestudeerd. In een in-situ veldstudie zijn de overleving en ontwikkeling van larven van Rana Kikkers, die zijn uitgezet in de sterk verontreinigde Volgermeerpolder en een niet-verontreinigde referentielocatie, beschouwd. Naast dit alles zijn juveniele Rana kikkers gevangen uit beide locaties, waarin interne concentraties dioxine-achtige stoffen en activiteiten van specifieke cytochroom P450 enzymen zijn bepaald.

Omdat de ontwikkeling van amfibieën sterk hormoon-gereguleerd is, is onderzocht in hoeverre de kikker een geschikte indicator is voor hormoonverstorende stoffen in het milieu. Hiertoe zijn in het laboratorium blootstellingstudies uitgevoerd met kikkerlarven en volwassen Rana kikkers, welke algemeen in Nederland voorkomen. Onderzocht is in hoeverre oestradiol (E2) en tetrachloordibenzo-p-dioxine (TCDD) de ontwikkeling, overleving, lichaams- en orgaangewichten, geslachtsorganen en -hormonen, en specifieke cytochroom P450 enzymen beïnvloeden. Ook is de onderlinge samenhang van deze ettectparameters bestudeerd. In een in-situ veldstudie zijn de overleving en ontwikkeling van larven van Rana Kikkers, die zijn uitgezet in de sterk verontreinigde Volgermeerpolder en een niet-verontreinigde referentielocatie, beschouwd. Naast dit alles zijn juveniele Rana kikkers gevangen uit beide locaties, waarin interne concentraties dioxine-achtige stoffen en activiteiten van specifieke cytochroom P450 enzymen zijn bepaald.

The research described in this thesis was aimed at developing an in vitro cell-based reporter gene system applicable to the detection of the illegal use of androgenic growth promoters in cattle, and the presence of potential endocrine disrupters present in surface waters and interfering with androgenic action. The system is based on a luciferase reporter gene placed under transcriptional control of an authenticated androgen-responsive element (ARE) and an endogenously expressed androgen receptor. This system allows for the integration of the effects of certain modulators of androgenic signal transduction. A second important goal of the research was to gain insight into the mechanisms underlying enhanced growth promotion by mixtures of androgenic and estrogenic compounds. The use of such mixtures, which results in activation and subsequent interaction of multiple steroid receptors, is occasionally observed in illegal hormonal treatments of cattle.When applied to the screening of calf urine samples for anabolic androgens, the developed AR-LUX assay was able to identify androgen-treated animals with similar results as obtained by standard GC-MS analysis. However, both techniques should be regarded as complementary rather than interchangeable screening tools. Liquid samples confiscated at cattle farms outside theNetherlandswere found to generate a very strong response in the AR-LUX assay despite the fact that GC-MS analysis did not detect the presence of any anabolic compounds. Possibly, the samples contained a mixture of conventional androgenic compounds, each at undetectably low amounts and/or (novel) unknown compounds not tested for by GC-MS. These results emphasize the additional value of the developed AR-LUX assay.Also, the AR-LUX assay was used to determine the androgenic activity of a number of aquatic environmental samples. A number of these samples were found to contain androgenic activity at varying concentrations. Interestingly, in 2 samples containing androgens, enhancing interactive mixture effects were observed, which were probably due to interactions by estrogenic compounds and estrogen receptor activation.Our research furthermore indicates that certain established progestagens are able to activate ARE-mediated luciferase expression via progesterone receptors; we hypothesise preferentially through the progesterone receptor-α isoform. This indicates that androgen reporter assays based on the activation of the androgen receptor alone rather than on activation of a response element might produce results quite different from those observed in assay systems featuring multiple steroid receptors. This further emphasizes the notion that the AR-LUX assay is not merely detecting activation of the androgen receptor by androgens, but also allows for the detection of other androgenic substances that regulate gene expression via alternative pathways leading to activation of an established androgen response element.

The research described in this thesis was aimed at developing an in vitro cell-based reporter gene system applicable to the detection of the illegal use of androgenic growth promoters in cattle, and the presence of potential endocrine disrupters present in surface waters and interfering with androgenic action. The system is based on a luciferase reporter gene placed under transcriptional control of an authenticated androgen-responsive element (ARE) and an endogenously expressed androgen receptor. This system allows for the integration of the effects of certain modulators of androgenic signal transduction. A second important goal of the research was to gain insight into the mechanisms underlying enhanced growth promotion by mixtures of androgenic and estrogenic compounds. The use of such mixtures, which results in activation and subsequent interaction of multiple steroid receptors, is occasionally observed in illegal hormonal treatments of cattle.When applied to the screening of calf urine samples for anabolic androgens, the developed AR-LUX assay was able to identify androgen-treated animals with similar results as obtained by standard GC-MS analysis. However, both techniques should be regarded as complementary rather than interchangeable screening tools. Liquid samples confiscated at cattle farms outside theNetherlandswere found to generate a very strong response in the AR-LUX assay despite the fact that GC-MS analysis did not detect the presence of any anabolic compounds. Possibly, the samples contained a mixture of conventional androgenic compounds, each at undetectably low amounts and/or (novel) unknown compounds not tested for by GC-MS. These results emphasize the additional value of the developed AR-LUX assay.Also, the AR-LUX assay was used to determine the androgenic activity of a number of aquatic environmental samples. A number of these samples were found to contain androgenic activity at varying concentrations. Interestingly, in 2 samples containing androgens, enhancing interactive mixture effects were observed, which were probably due to interactions by estrogenic compounds and estrogen receptor activation.Our research furthermore indicates that certain established progestagens are able to activate ARE-mediated luciferase expression via progesterone receptors; we hypothesise preferentially through the progesterone receptor-α isoform. This indicates that androgen reporter assays based on the activation of the androgen receptor alone rather than on activation of a response element might produce results quite different from those observed in assay systems featuring multiple steroid receptors. This further emphasizes the notion that the AR-LUX assay is not merely detecting activation of the androgen receptor by androgens, but also allows for the detection of other androgenic substances that regulate gene expression via alternative pathways leading to activation of an established androgen response element.