Persistent organic pollutants : aberrant DNA methylation underlying potential health effects
Dungen, M.W. van den - \ 2016
Wageningen University. Promotor(en): Tinka Murk; Ellen Kampman, co-promotor(en): Wilma Steegenga; Dieuwertje van Gils-Kok. - Wageningen : Wageningen University - ISBN 9789462577893 - 207
persistent organic pollutants - dna methylation - molecular genetics - epigenetics - health hazards - toxic substances - endocrine disruptors - eels - fish consumption - toxicology - persistente organische verontreinigende stoffen - dna-methylering - moleculaire genetica - epigenetica - gezondheidsgevaren - toxische stoffen - hormoonverstoorders - palingen - visconsumptie - toxicologie
Wild caught fish, especially marine fish, can contain high levels of persistent organic pollutants (POPs). In the Netherlands, especially eel from the main rivers have high POP levels. This led to a ban in 2011 on eel fishing due to health concerns. Many of the marine POPs have been related to adverse health effects such as endocrine disruption, neurodevelopmental problems, immune suppression and cancer. Although some mechanisms of action of POPs are clear, like dioxins binding to the aryl hydrocarbon receptor and OH-PCBs binding to thyroid transport proteins, not all adverse health effects can be explained by these mechanisms of action. Epigenetic phenomena, such as DNA methylation, have been proposed as a possible molecular mechanism underlying adverse health effects. DNA methylation is a heritable modification, which refers to the addition of a methyl group to cytosine in a CpG dinucleotide. Observational studies have indeed shown that POPs can affect global DNA methylation, although results are inconsistent. Some animal studies as well as in vitro experiments suggest that POPs can affect gene-specific DNA methylation, however, the biological significance and relevance for humans is not clear. Therefore, this thesis aimed to 1) study the accumulation of POPs in men consuming eel from high-polluted areas 2) elucidate whether seafood-related POPs can induce aberrant DNA methylation and 3) to determine whether DNA methylation is related to functional endpoints and gene expression in vitro.
For this purpose eight POPs that are abundantly present in seafood were chosen, namely 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorobiphenyl (PCB) 126 and 153, perfluorooctanesulfonic acid (PFOS), hexabromocyclododecane (HBCD), 2,2′,4,4′- tetrabromodiphenyl ether (BDE-47), tributyltin (TBT), and methylmercury (MeHg). Chapter 2 describes the in vitro effects of these POPs and mixtures thereof in H295R adrenocortical carcinoma cells. Relative responses for 13 steroid hormones and 7 genes involved in the steroidogenic pathway, and CYP1A1, were analysed. PFOS induced the most pronounced effects on steroid hormone levels by significantly affecting 9 out of 13 hormone levels measured, with the largest increases found for 17β-estradiol, corticosterone, and cortisol. Furthermore, TCDD, both PCBs, and TBT significantly altered steroidogenesis. Increased steroid hormone levels were accompanied by related increased gene expression levels. The differently expressed genes were MC2R, CYP11B1, CYP11B2, and CYP19A1 and changes in gene expression levels were more sensitive than changes in hormone levels. The POP mixtures tested showed mostly additive effects, especially for DHEA and 17β-estradiol levels. This study shows that some seafood POPs are capable of altering steroidogenesis in H295R cells at concentrations that mixtures might reach in human blood, suggesting that adverse health effects cannot be excluded. DNA methylation was not measured in this study due to the short exposure time, which was expected not to be sufficient for long-term epigenetic marks. Therefore, in chapters 3A and 3B a differentiation experiment was performed enabling long-term exposure to POPs. Human mesenchymal stem cells (hMSCs) were differentiated into mature adipocytes over a time-course of 10 days. The transcriptional regulatory cascade involved in adipocyte differentiation has been extensively studied, however the mechanisms driving the transcription are poorly understood. In chapter 3A we therefore first explored the involvement of DNA methylation in transcriptional regulation during adipocyte differentiation. Genome-wide changes in DNA methylation were measured as well as the expression of adipogenic genes. The majority of these genes showed significant expression changes during the differentiation process. There were, however, only a couple of these differentially expressed genes that were differentially methylated. Genome-wide DNA methylation changes were most often located in intergenic regions, and underrepresented close to the transcription start site. This suggested that changes in DNA methylation are not the underlying mechanism regulating gene expression during adipocyte differentiation. Nevertheless, we explored DNA methylation differences after continuous exposure to POPs to investigate whether this could be an underlying mechanism by which POPs affect adipocyte differentiation. TCDD and PFOS decreased lipid accumulation, while TBT increased lipid accumulation. TCDD and TBT induced opposite gene expression profiles, whereas after PFOS exposure gene expression remained relatively stable. Genome-wide DNA methylation analysis showed that all three POPs affected DNA methylation patterns in adipogenic and other genes, but without concomitant gene expression changes. Differential methylation was again predominantly detected in intergenic regions, where the biological relevance of alterations in DNA methylation is unclear. This study demonstrated that POPs, at environmentally relevant levels, are able to induce differential DNA methylation in differentiating adipocytes. However, the biological relevance of this aberrant DNA methylation remains unclear.
The in vitro results showed a proof of principle that POPs could be capable of altering DNA methylation. To this date, no human studies were performed investigating the relationship between POP levels and genome-wide DNA methylation. In order to investigate this, we first measured POP levels in eel consumers from the high-polluted areas (areas with a ban on eel fishing) and compared these levels to men consuming eel from low-polluted areas or aquaculture (chapter 4). We aimed to investigate the accumulation of these POPs and determine whether the predictions made in an earlier risk assessment were valid. This was indeed the case as levels of dioxins and dioxin-like compounds were on average 2.5 times higher in men consuming eel from high-polluted areas. Furthermore, PCBs with their hydroxylated metabolites, and perfluoroalkyl substances (PFASs) were, up to ten times, higher in these consumers. Especially the high levels of dioxins and dioxin-like compounds as well as the OH-PCBs are expected to be of health concern. We continued this research in chapter 5 by associating all the measured POPs to clinical parameters related to e.g. thyroid hormones and liver enzymes, but found no relationship. Subsequently, we investigated the association between dioxins and dioxin-like compounds, the sum of seven indicator PCBs, and PFOS with genome-wide DNA methylation. We detected a number of differentially methylated regions (DMRs) related to genes involved in carcinogenesis (e.g. BRCA1, MAGEE2, HOXA5), the immune system (e.g. RNF39, HLA-DQB1), in retinol homeostasis (DHRS4L2), or in metabolism (CYP1A1). In contrast to the in vitro data, most significant effects were detected in CpG islands and were annotated close to the promoter region. This suggests that the differential methylation might be related to differential expression and possibly induce adverse health effects. The hypermethylation of some of these gene related to cancer could be an explanation of the carcinogenic effects that are observed with POP exposure.
Based on the results of this thesis we can conclude that the consumption of eel from high-polluted areas lead to accumulation of POPs above safe levels and that POP levels are associated with gene-specific DNA methylation in vitro as well as in environmentally exposed men. More research, however, is needed to fully elucidate the biological implications of this aberrant DNA methylation. A first step can be to measure histone modifications, as these two epigenetic marks together are likely better in predicting gene expression. The second step can be to investigate the potential health effects related to these epigenetic marks and to determine whether there is a causal relationship. Although at this point there is a lack of knowledge with regard to health effects caused by DNA methylation, the consumption of eel from these high-polluted areas is ill- advised, because adverse health effects cannot be excluded based on our results and can even be expected based on literature.
Proteomics as a tool to gain more insight into sub-lethal toxicological effects
Miller, Ingrid - \ 2016
Wageningen University. Promotor(en): Tinka Murk, co-promotor(en): A.C. Gutleb; T. Serchi. - Wageningen : Wageningen University - ISBN 9789462578210 - 182
proteomics - laboratory methods - sublethal effects - toxic substances - endocrine disruptors - food consumption - toxicology - animal experiments - eiwitexpressieanalyse - laboratoriummethoden - subletale effecten - toxische stoffen - hormoonverstoorders - voedselconsumptie - toxicologie - dierproeven
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.
Thyroid in a jar: towards an integrated in vitro testing strategy for thyroid-active compounds
Jomaa, B. - \ 2015
Wageningen University. Promotor(en): Ivonne Rietjens, co-promotor(en): Jac Aarts; Ad Peijnenburg. - Wageningen : Wageningen University - ISBN 9789090290089 - 187
schildklierziekten - schildklierhormonen - hormoonverstoorders - in vitro - assays - celgroei - thyroid diseases - thyroid hormones - endocrine disruptors - in vitro - assays - cell growth
Jomaa, B. (2015). Thyroid in a Jar: Towards an Integrated In Vitro Testing Strategy for Thyroid-Active Compounds. PhD thesis, Wageningen University, the Netherlands
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.
Liver DNA methylation analysis in adult female C57BL/6JxFVB mice following perinatal exposure to bisphenol A
Esterik, J.C. van; Vitins, A.P. ; Hodemaekers, H.M. ; Kamstra, J.H. ; Legler, J. ; Pennings, J.L.A. ; Steegenga, W.T. ; Lute, C. ; Jelinek, J. ; Issa, J.P. ; Dollé, M.E.T. ; Ven, L.T.M. van der - \ 2015
Toxicology Letters 232 (2015)1. - ISSN 0378-4274 - p. 293 - 300.
endocrine disruptors - disease - bpa - xenoestrogens - association - epigenetics - expression - nutrition - evolution - chemicals
Bisphenol A (BPA) is a compound released from plastics and other consumer products used in everyday life. BPA exposure early in fetal development is proposed to contribute to programming of chronic diseases like obesity and diabetes, by affecting DNA methylation levels. Previously, we showed that in utero and lactational exposure of C57BL/6JxFVB hybrid mice via maternal feed using a dose range of 0–3000 µg/kg body weight/day resulted in a sex-dependent altered metabolic phenotype in offspring at 23 weeks of age. The most univocal effects were observed in females, with reduced body weights and related metabolic effects associated with perinatal BPA exposure. To identify whether the effects of BPA in females are associated with changes in DNA methylation, this was analyzed in liver, which is important in energy homeostasis. Measurement of global DNA methylation did not show any changes. Genome-wide DNA methylation analysis at specific CpG sites in control and 3000 µg/kg body weight/day females with the digital restriction enzyme analysis of methylation (DREAM) assay revealed potential differences, that could, however, not be confirmed by bisulfite pyrosequencing. Overall, we demonstrated that the observed altered metabolic phenotype in female offspring after maternal exposure to BPA was not detectably associated with liver DNA methylation changes. Still, other tissues may be more informative.
Towards an integrated in vitro strategy for estrogenicity testing
Wang, S. ; Aarts, H.J.M. ; Haan, L.H.J. de; Argyriou, D. ; Peijnenburg, A.A.C.M. ; Rietjens, I.M.C.M. ; Bovee, T.F.H. - \ 2014
Journal of Applied Toxicology 34 (2014)9. - ISSN 0260-437X - p. 1031 - 1040.
coregulator binding assay - reporter gene assay - ovarian cell-line - receptor-beta - endocrine disruptors - chemicals - alpha - cancer - vivo - expression
In order to define an in vitro integrated testing strategy (ITS) for estrogenicity, a set of 23 reference compounds representing diverse chemical classes were tested in a series of in vitro assays including proliferation and reporter gene assays. Outcomes of these assays were combined with published results for estrogen receptor (ER) binding assays and the OECD validated BG1Luc ER transcriptional activation (TA) assay and compared with the outcomes of the in vivo uterotrophic assay to investigate which assays most accurately predict the in vivo uterotrophic effect and to identify discrepancies between the in vitro assays and the in vivo uterotrophic assay. All in vitro assays used revealed a reasonable to good correlation (R2¿=¿0.62–0.87) with the in vivo uterotrophic assay but the combination of the yeast estrogen bioassay with the U2OS ERa-CALUX assay seems most promising for an ITS for in vitro estrogenicity testing. The main outliers identified when correlating data from the different in vitro assays and the in vivo uterotrophic assay were 4-hydroxytamoxifen, testosterone and to a lesser extent apigenin, tamoxifen and kepone. Based on the modes of action possibly underlying these discrepancies it becomes evident that to further improve the ITS and ultimately replace animal testing for (anti-)estrogenic effects, the selected bioassays have to be combined with other types of in vitro assays, including for instance in vitro models for digestion, bioavailability and metabolism of the compounds under investigation
Development and validation of in vitro bioassays for thyroid hormone receptor mediated endocrine disruption
Freitas, J. de - \ 2012
Wageningen University. Promotor(en): Tinka Murk; Ivonne Rietjens, co-promotor(en): J.D. Furlow. - [S.l.] : s.n. - ISBN 9789461734372 - 192
hormoonverstoorders - biotesten - in vitro - schildklierhormonen - endocrine disruptors - bioassays - in vitro - thyroid hormones
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.
Optimization and prevalidation of the in vitro ER alpha CALUX method to test estrogenic and antiestrogenic activity of compounds
Burg, B. van der; Winter, R. ; Weimer, M. ; Berckmans, P. ; Suzuki, G. ; Gijsbers, L. ; Jonas, A. ; Linden, S. van der; Witters, H. ; Aarts, J.M.M.J.G. ; Legler, J. ; Kopp-Schneider, A. ; Bremer, S. - \ 2010
Reproductive Toxicology 30 (2010)1. - ISSN 0890-6238 - p. 73 - 80.
cell-line - transactivation assay - endocrine disruptors - bioassays - androgen - chemicals - panel - beta
Estrogenicity of chemicals has received significant attention and is linked to endocrine-disrupting activities. However, there is a paucity of validated methods to assess estrogenicity in vitro. We have established a robust method to test estrogenic and antiestrogenic activity of compounds in vitro, as an alternative to using animal models such as the uterotrophic assay. To this end we optimized protocols to be used in combination with CALUX reporter gene assays and carried out an in house prevalidation, followed by two rounds of tests to establish transferability. Problems in the initial test with transferability were solved by isolation of a novel cell clone of the ER alpha CALUX line with greatly improved stability and luciferase levels. This cell line proved to be a very suitable and reliable predictor of estrogenicity of chemicals and was able to readily rank a range of chemicals on the basis of their EC50 values.
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)
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
De som der delen : rationale risioschatting van milieucontaminanten
Murk, A.J. - \ 2008
Wageningen : Wageningen Universiteit - ISBN 9789085852681 - 47
toxicologie - risicoschatting - gezondheidsgevaren - milieueffect - ecotoxicologie - hormoonverstoorders - openbare redes - toxicology - risk assessment - health hazards - environmental impact - ecotoxicology - endocrine disruptors - public speeches
Markers of endocrine disruption in fish
Bogers, R. - \ 2008
Wageningen University. Promotor(en): Ivonne Rietjens; Tinka Murk, co-promotor(en): J. Legler. - S.l. : S.n. - ISBN 9789085852025 - 133
oestrogene eigenschappen - pimephales promelas - screenen - oppervlaktewater - hormoonverstoorders - biomarkers - ecotoxicologie - oestrogenic properties - pimephales promelas - screening - surface water - endocrine disruptors - biomarkers - ecotoxicology
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
Nieuwe verontreinigingen in de bodem : een verkennende literatuurstudie naar de mogelijke risico's van hormoonverstoorders en diergeneesmiddelen
Lahr, J. - \ 2007
Wageningen : Alterra (Alterra-rapport 1619) - 80
bodemverontreiniging - hormoonverstoorders - geneesmiddelen - geneesmiddelenresiduen - verontreinigende stoffen - risicoschatting - voedselkwaliteit - literatuuroverzichten - veterinaire producten - bodemkwaliteit - soil pollution - endocrine disruptors - drugs - drug residues - pollutants - risk assessment - food quality - literature reviews - veterinary products - soil quality
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
Novel in vitro, ex vivo and in vivo assays elucidating the effects of endocrine disrupting compounds (EDCs) on thyroid hormone action
Schriks, M. - \ 2006
Wageningen University. Promotor(en): Ivonne Rietjens, co-promotor(en): Tinka Murk; J.D. Furlow. - Wageningen : S.n. - ISBN 9789085044840 - 159
in vitro kweek - schildklierhormonen - xenopus laevis - hormoonverstoorders - in vivo experimenten - in vitro culture - thyroid hormones - xenopus laevis - endocrine disruptors - in vivo experimentation
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 .
Impact of triphenyltin acetate in microcosms simulating floodplain lakes; II comparison of species sensitivity distributions between laboratory and semi-field
Roessink, I. ; Belgers, J.D.M. ; Crum, S.J.H. ; Brink, P.J. van den; Brock, T.C.M. - \ 2006
Ecotoxicology 15 (2006)5. - ISSN 0963-9292 - p. 411 - 424.
fresh-water microcosms - prosobranch snails mollusca - organotin compounds - fungicide carbendazim - aquatic invertebrates - endocrine disruptors - lambda-cyhalothrin - primary producers - daphnia-magna - tributyltin
The study objectives were to shed light on the types of freshwater organism that are sensitive to triphenyltin acetate (TPT) and to compare the laboratory and microcosm sensitivities of the invertebrate community. The responses of a wide array of freshwater taxa (including invertebrates, phytoplankton and macrophytes) from acute laboratory Single Species Tests (SST) were compared with the concentration¿response relationships of aquatic populations in two types of freshwater microcosms. Representatives of several taxonomic groups of invertebrates, and several phytoplankton and vascular plant species proved to be sensitive to TPT, illustrating its diverse modes of toxic action. Statistically calculated ecological risk thresholds (HC5 values) based on 96 h laboratory EC50 values for invertebrates were 1.3 ¿g/l, while these values on the basis of microcosm-Species Sensitivity Distributions (SSD) for invertebrates in sampling weeks 2¿8 after TPT treatment ranged from 0.2 to 0.6 ¿g/l based on nominal peak concentrations. Responses observed in the microcosms did not differ between system types and sampling dates, indicating that ecological threshold levels are not affected by different community structures including taxa sensitive to TPT. The laboratory-derived invertebrate SSD curve was less sensitive than the curves from the microcosms. Possible explanations for the more sensitive field response are delayed effects and/or additional chronic exposure via the food chain in the microcosms
Detecting the effects of environmentally relevant concentrations of thyroid hormone disrupting compounds on amphibian development
Gutleb, A.C. - \ 2006
Wageningen University. Promotor(en): Ivonne Rietjens, co-promotor(en): Tinka Murk. - Wageningen : s.n. - ISBN 9789085043546 - 168
schildklierhormonen - amphibia - embryonale ontwikkeling - foetale ontwikkeling - xenopus laevis - hormoonverstoorders - thyroid hormones - amphibia - embryonic development - fetal development - xenopus laevis - endocrine disruptors
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.
|Introduction of ethoxy-resorufin-o-deethylase activity by halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons in primary hepatocytes of the green frog (Rana esculenta)
Rouhani Rankouhi, T. ; Koomen, B. ; Sanderson, J.T. ; Bosveld, A.T.C. ; Seinen, W. ; Berg, M. van den - \ 2005
Environmental Toxicology and Chemistry 24 (2005)6. - ISSN 0730-7268 - p. 1428 - 1435.
polychlorinated-biphenyls pcbs - cell-line - in-vitro - cytochrome-p4501a induction - endocrine disruptors - population declines - xenopus-laevis - fish - cultures - liver
In this study, we measured the ethoxy-resorufin-O-deethylase (EROD) activity in primary hepatocytes of the common green frog Rana esculenta as a biomarker for cytochrome P4501A induction. We exposed hepatocytes derived from male and female frogs to several halogenated aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran (PCDF), polychlorinated biphenyls (PCB-126, PCB-118). and polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP), chrysene, anthracene, and pyrene. Exposure to PCB-118, anthracene, and pyrene, up to 1 μ M, did not induce EROD activity, whereas TCDD and PCDF induced EROD activity maximally. In our primary frog hepatocytes, exposure to chrysene and BaP resulted in median effective concentration values (EC50) in the high nM range (82-1035 nM). Exposure to TCDD, PCDF and PCB-126 resulted in EC50 values of 0.4 to 8, 0.07 to 0.7, and 3 to 133 nM, respectively, which is in the same range as EC50 values found in primary hepatocytes of birds. Compared to our frog hepatocytes, primary rat hepatocytes seem to be more sensitive to TCDD, chrysene. and BaP.
Effects of environmental and natural estrogens on vitellogenin production in hepatocytes of the brown frog (Rana temporaria)
Rouhani Rankouhi, T. ; Sanderson, J.T. ; Holsteijn, I. van; Kooten, P. van; Bosveld, A.T.C. ; Berg, M. van den - \ 2005
Aquatic Toxicology 71 (2005)1. - ISSN 0166-445X - p. 97 - 101.
endocrine disruptors - population declines - ethynyl estradiol - in-vitro - amphibians - chemicals - exposure - assay - model - vivo
The objective of this study was to investigate the ability of the natural estrogens and synthetic estrogens as well as the estrogen mimics to induce estrogen-receptor mediated vitellogenesis in primary hepatocytes of the brown frog (Rana temporaria). Based on EC50 values the following order was determined for the potency of the estrogens: 17beta-estradiol (EC50: 19-43 nM) approximate to ethynylestradiol (EC50: 13-80 nM) > estrone (EC50: 218-241 nM) > DES (EC50: 338-3537 nM). Exposure to bisphenol A and methoxychlor concentrations up to 100 muM did not have any effect on in vitro vitellogenesis. (C) 2004 Elsevier B.V. All rights reserved.
An integrated assessment of estrogenic contamination and biological effects in the aquatic environment of the Netherlands
Vethaak, A.D. ; Lahr, J. ; Schrap, S.M. ; Belfroid, A.C. ; Rijs, G.B.J. ; Gerritsen, A. ; Boer, J. de; Bulder, A.S. ; Grinwis, G.C.M. ; Kuiper, R.V. ; Legler, J. ; Murk, A.J. ; Peijnenburg, W. ; Verkaar, H.J.M. ; Voogt, P. de - \ 2005
Chemosphere 59 (2005)4. - ISSN 0045-6535 - p. 511 - 524.
waterverontreiniging - hormonen - aquatisch milieu - monitoring - nederland - aquatische ecosystemen - hormoonverstoorders - water pollution - hormones - aquatic environment - monitoring - netherlands - aquatic ecosystems - endocrine disruptors - sewage-treatment plants - flounder platichthys-flesus - reporter gene assays - e-screen assay - waste-water - surface-water - alkylphenol polyethoxylates - degradation-products - sexual disruption
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 strougly 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 17a-ethynylestradiol) and possibly also nonylphenol(ethoxylate)s are primarily responsible for these effects.
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
Toxicological profiling of sediments with in vitro mechanisms-based bioassays for endocrine disruption
Houtman, C.J. ; Cenijn, P.H. ; Hamers, T. ; Lamoree, M.H. ; Legler, J. ; Murk, A.J. ; Brouwer, A. - \ 2004
Environmental Toxicology and Chemistry 23 (2004)1. - ISSN 0730-7268 - p. 32 - 40.
biotesten - sediment - toxiciteit - hormonen - estuaria - rivieren - nederland - hormoonverstoorders - waterbodems - ecotoxicologie - rijn - maas - bioassays - sediment - toxicity - hormones - estuaries - rivers - netherlands - endocrine disruptors - water bottoms - ecotoxicology - river rhine - river meuse - reporter gene assays - estrogenic activity - aromatic-hydrocarbons - human transthyretin - expression assays - toxic potency - extracts - chemicals - exposure - wildlife
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.