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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.
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.
Hydrogen-powered road vehicles : the health benfits and drawbacks of a new fuel
Passchier, W.F. ; Erisman, J.W. ; Hazel, P.J. van den; Heederik, D.J.J. ; Leemans, R. ; Legler, J. ; Sluijs, J.P. ; Dogger, J.W. - \ 2009
Den Haag : Health Council of the Netherlands (Horizon Scanning Report 2008/016E)
voertuigen - motorvoertuigen - autorijden - brandstoffen - specifiek brandstofverbruik - waterstof - effecten - onbedoelde effecten - nadelige gevolgen - volksgezondheid - nederland - vehicles - motor cars - motoring - fuels - specific fuel consumption - hydrogen - effects - nontarget effects - adverse effects - public health - netherlands
Because of the political, social and environmental problems associated with dependency on fossil fuels, there is considerable interest in alternative energy sources. Hydrogen is regarded as a promising option, particularly as a fuel for road vehicles. The Dutch Energy Research Centre (ECN) recently published a vision of the future, in which it suggested that by 2050 more than half of all cars in the Netherlands could be running on hydrogen. A switch to using hydrogen as the primary energy source for road vehicles would have far-reaching social consequences. As with all technological developments, opportunities would be created, but drawbacks would inevitably be encountered as well. Some of the disadvantages associated with hydrogen are already known, and are to some degree manageable. It is likely, however, that other drawbacks would come to light only once hydrogen-powered cars were actually in use. With that thought in mind, and in view of the social significance of a possible transition to hydrogen, it was decided that the Health Council should assess the positive and negative effects that hydrogen use could have on public health. It is particularly important to make such an assessment at the present early stage in the development of hydrogen technologies, so that gaps in existing scientific knowledge may be identified and appropriate strategies may be developed for addressing such gaps. This report has been produced by the Health and Environment Surveillance Committee, which has special responsibility for the identification of important correlations between environmental factors and public health.
Markers of endocrine disruption in fish
Bogers, R. - \ 2008
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 - screening - surface water - endocrine disruptors - 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
An in vitro/in vovo screening assay as a sensitive tool to assess endocrine disruptive activity in surface water
Bogers, R. ; Buitenweg, S. ; Geuijen, I. ; Waart, B. van de; Kuiper, R. ; Linden, S. van der; Puijker, L. ; Murk, A.J. ; Burg, B. van der; Legler, J. - \ 2007
Environment International 33 (2007)3. - ISSN 0160-4120 - p. 292 - 301.
minnow pimephales-promelas - vitellogenin messenger-rna - secondary sex characteristics - treated sewage effluent - medaka oryzias-latipes - fathead minnow - cyprinodon-variegatus - estrogenic activity - synthetic estrogen - rainbow-trout
Adult male fathead minnow were exposed for 14 or 28-days under flow-through conditions to undiluted filtered water samples from the rivers Meuse and Rhine in the Netherlands. The experiment included two vessels per treatment each containing 10 fish and samples of five fish were taken after 14 and 28 days. Additional groups were exposed to 17¿-ethinylestradiol (EE2) as a reference and untreated drinking water as a negative control. Major endpoints examined included induction of vitellogenin (VTG) synthesis, VTG mRNA activity, hepato- and gonadosomatic indices (HSI and GSI) and gonadal histology. No significant difference was recorded in body weight or mean GSI values between the various treatments. Only exposure to Meuse water resulted in significantly higher HSI means after 14 days. Histological examination showed no apparent effects on gonadal tissue except for eosinophilic blood plasma in fish exposed to Meuse water or EE2. After 14 and 28 days, elevated VTG and VTG mRNA levels were measured in most livers of the fish exposed to Meuse water, but not in the fish exposed to Rhine water. This was confirmed by measuring estrogenic responses in the in vitro ER CALUX® assay. Induction of VTG synthesis proved to be the most sensitive endpoint in the Non Spawning Male Fish Assay for in vivo detection of bio-available estrogenic activity supplementary to a sensitive in vitro assay. The other endpoints examined varied too much and required a higher number of fish or replicates to achieve sufficient power for statistical testing making them less animal friendly.
|The in vivo transgenic zebrafish reporter gene assay for bioanalysis of exposure and effects of estrogenic chemicals in the aquatic environment
Legler, J. ; Zee, M. van der; Ven, L.T.M. van der; Jonkers, C.C.H. ; Vethaak, A.D. ; Burg, B. van der; Murk, A.J. - \ 2006
In: Estrogens and xenoestrogens in the aquatic environment: An integrated approach for field monitoring and effect assessment / Vethaak, D., Schrap, M, de Voogt, P., Pensacola : SETAC-scientific book (SETAC Technical Publications Series ) - ISBN 9781880611852 - 512 p.
|Bioanalysis of estrogenic and dioxin-like activity in field samples
Legler, J. ; Bulder, A.S. ; Voogt, P. de; Schrap, S.M. ; Murk, A.J. - \ 2006
In: Estrogens and xeno-estrogens in the aquatic environment: An integrated approach for field monitoring and effect assessment / Voogt P. de Vethaak D., Schrap M., SETAC - ISBN 9781880611852 - 512 p.
Biolonical validation of a sample preparation method for ER-CALUX bioanalysis of estrogenic activity in sediment using mixtures of xeno-estrogens
Houtman, C.J. ; Houten, Y.K. Van; Leonards, P.E.G. ; Brouwer, A. ; Lamoree, M.H. ; Legler, J. - \ 2006
Environmental Science and Technology 40 (2006)7. - ISSN 0013-936X - p. 2455 - 2461.
in-vitro bioassays - cell-proliferation - xenoestrogens - chemicals - assay
The combined estrogenic effects of mixtures of environmental pollutants in the in vitro ER-CALUX (chemical activated luciferase gene expression) bioassay were examined to biologically validate a sample preparation method for the analysis of estrogenic compounds in sediment. The method used accelerated solvent extraction (ASE) and gel permeation chromatography (GPC) and was validated with respect to recovery of biological response taking mixture effects into account. Four mixtures of three to six xeno-estrogenic compounds (bisphenol A, 4-nonylphenol, (4,4`-dichlorodiphenyl)trichloroethane, (2,4`-dichlorodiphenyl)trichloroethane, dieldrin, 4-n-octylphenol, ¿-chlordane, dibutylphthalate, (4,4`-dichlorodiphenyl)dichloroethylene, and 2,4,5-trichlorobiphenyl) were prepared. Experimentally determined mixture effects were well described by the concept of concentration addition (CA), as expected for similarly acting compounds. Observed estradiol equivalence factors of the mixtures (on average 1.2 ± 0.3) agreed very well with the value predicted according to CA. The sample preparation method was then applied to pure mixtures of standards and to sediment spiked with one of the mixtures. Recoveries of estrogenic compounds were estimated by determination of their mixture potencies in ER-CALUX and compared to the mixture effects predicted by CA. Recoveries of estrogenic activity were between 80 and 129%, indicating that the additive behavior of mixtures of xeno-estrogens is well conserved during sample preparation. Together with an average repeatability of 18.3%, low average limit of detection (2.6 ± 1.8 pg of EEQ/g), and coefficient of variance (3.5 ± 3.3%), this demonstrated the suitability of the sample preparation method for the analysis of mixtures of (xeno-)estrogenic compounds in sediment with the ER-CALUX assay
Development of chronic tests for endocrine active chemicals. Part 2. An extended fish earli-life stage test with an androgenic chemical in the fathead minnow (Pimephales promelas)
Bogers, R. ; Vries-Buitenweg, S. de; Gils, H.A.M. van; Baltussen, E. ; Hargreaves, A. ; Waart, B. van de; Roode, D.F. de; Legler, J. ; Murk, A.J. - \ 2006
Aquatic Toxicology 80 (2006)2. - ISSN 0166-445X - p. 119 - 130.
medaka oryzias-latipes - zebrafish danio-rerio - secondary sex characteristics - mill effluent - in-vivo - nonaromatizable androgens - receptor agonist - methyltestosterone - disruption - growth
The Endocrine Modulators Study Group (EMSG) of the European Chemical Industry has proposed an extended fish early-life stage (ELS) test based on OECD test guideline 210 in combination with a fish pair-breeding reproduction study as a possible alternative for fish full life cycle testing. In this paper the androgen methyldihydrotestosterone (MDHT) was tested in an extended ELS test with fathead minnow supplementary to such a test with the weak estrogen 4-tert-pentylphenol (4TPP). Main endpoints were secondary sexual characteristics (SSC), plasma vitellogenin (VTG) induction and gonadal development. Early blastula embryos were exposed to 0, 0.10, 0.32 and 1.0 ¿g MDHT l-1 for up to 114 days post-hatch (dph). A batch of fish exposed to 1.0 ¿g l-1 was transferred to clean water after 30 or 63 dph for the remainder of the study. Ethinylestradiol (EE2) was included as estrogenic reference substance at 0.01 ¿g l-1. Exposure to MDHT had no significant effect on hatching success or survival, but significantly increased the condition factor of fish exposed for 63 and 114 dph (up to 150% of the control). At 63 dph MDHT exposure induced appearance of tubercles on the snout (a male SSC) of more than 80% of fish. Compared to the controls, plasma VTG was not detectable or significantly lower in fish exposed to MDHT at 0.10 ¿g/l, but not significantly affected at higher MDHT concentrations. Both lower levels of MDHT significantly inhibited the development of female gonads as of 30 dph. Fish exposed to MDHT at 0.32 and 1.0 ¿g l-1 showed higher incidences of mixed sex gonads (10-25%) and smaller testes or dysplasia of gonadal tissue. Dysplasia was present in 80% of the fish continuously exposed to 1.0 ¿g l-1 up to 114 dph, but reversible when fish were transferred to dilution water. Results indicate that suppression of ovarian development was the most sensitive endpoint for MDHT exposure after 30 dph. Other endpoints (e.g., growth and SSC) required exposure during at least up to 63 dph to yield a significant effect. Androgenic effects on VTG production required even longer exposure, i.e., until sufficient number of females had matured
In vitro profiling of the endocrine-disrupting potency of brominated flame retardants
Hamers, T.H.M. ; Kamstra, J.H. ; Sonneveld, E. ; Murk, A.J. ; Kester, M.H.A. ; Andersson, P.L. ; Legler, J. ; Brouwer, A. - \ 2006
Toxicological sciences 92 (2006)1. - ISSN 1096-6080 - p. 157 - 173.
polybrominated diphenyl ethers - receptor active compounds - bisphenol-a - estrogen sulfotransferase - developmental exposure - tetrabromobisphenol-a - human transthyretin - brain-development - gene-expression - cell-line
Over the last years, increasing evidence has become available that some brominated flame retardants (BFRs) may have endocrine disrupting (ED) potencies. The goal of the current study was to perform a systematic in vitro screening of the ED potencies of BFRs (1) to elucidate possible modes of action of BFRs in man and wildlife, and (2) to classify BFRs with similar profiles of ED potencies. A test set of twenty-seven individual BFRs was selected, consisting of nineteen polybrominated diphenylethers (PBDE) congeners, tetrabromobisphenol-A (TBBPA), hexabromocyclododecane (HBCD), 2,4,6-tribromophenol (246-TBP), ortho-hydroxylated BDE-47 (6OH-BDE-47), and TBBPA-bis(2,3)dibromopropylether (TBBPA-DBPE). All BFRs were tested for their potency to interact with the arylhydrocarbon receptor (AhR), androgen receptor (AR), progesterone receptor (PR), and estrogen receptor (ER). In addition, all BFRs were tested for their potency to inhibit estradiol (E2) sulfation by E2-sulfotransferase (E2SULT), to interfere with thyroid hormone 3,3`,5-triiodothyronine (T3) mediated cell proliferation, and to compete with T3-precursor thyroxine (T4) for binding to the plasma transport protein transthyretin (TTR). The results of the in vitro screening indicated that BFRs have ED potencies, some of which had not or only marginally been described before (AR-antagonism, PR-antagonism, E2SULT inhibition, and potentiation of T3-mediated effects). For some BFRs, the potency to induce AR-antagonism, E2SULT inhibition and TTR competition was higher than for natural ligands or clinical drugs used as positive controls. Based on their similarity in ED profiles, BFRs were classified into five different clusters. These findings support further investigation of the potential endocrine disrupting effects of these environmentally relevant BFRs in man and wildlife.
Estrogenic endpoints in fish early life-stage tests: luciferase and vitellogenin induction in estrogen-responsive transgenic zebrafish
Bogers, R. ; Mutsaerds, E. ; Druke, J. ; Roode, D.F. de; Murk, A.J. ; Burg, B. van der; Legler, J. - \ 2006
Environmental Toxicology and Chemistry 25 (2006)1. - ISSN 0730-7268 - p. 241 - 247.
in-vivo - pimephales-promelas - fathead minnow - danio-rerio - sewage effluent - rainbow-trout - stw effluent - chemicals - exposure - vitro
This study incorporated specific endpoints for estrogenic activity in the early life-stage (ELS) test, as described in Guideline 210 of the Organization for Economic Cooperation and Development and traditionally used for toxicity screening of chemicals. A transgenic zebrafish model expressing an estrogen receptor-mediated luciferase reporter gene was exposed to ethinylestradiol (EE2), and luciferase activity as well as vitellogenin (VTG) was measured. Concentrations of EE2 were tested at 1, 3, or 10 ng/L for 30 d from fertilization or during only the last 4 d with dimethylsulfoxide (DMSO) as presolvent (0.01%). Exposure to EE2 induced no toxic effects. Mean body weights were significantly higher in groups exposed for 30 d in the presence of DMSO, but condition factors were not affected. Significant luciferase and VTG induction occurred following 30-d exposure (3 and 10 ng EE2/L), while only VTG levels were affected in the 4-d exposure (10 ng EE2/L). This study demonstrated the usefulness of incorporating estrogenic endpoints in the OECD ELS test, fitting the requirements for screening estrogenic activity of chemicals. Quantitative measurement of both VTG and luciferase activity proved to be rapid and sensitive. Additional value of using transgenic zebrafish lies in combining VTG measurement with the more mechanistic approach of luciferase induction in one experiment.
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 - 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.
Intra- and interlaboratory calibration of the DR Calux bioassay for the analysis of dioxins and dioxin-like chemicals in sediments
Besselink, H.T. ; Schipper, C.A. ; Klamer, H. ; Leonards, P.E.G. ; Verhaar, H. ; Felzel, E. ; Murk, A.J. ; Thain, J. ; Hosoe, K. ; Schoeters, G. ; Legler, J. ; Brouwer, A. - \ 2004
Environmental Toxicology and Chemistry 23 (2004)12. - ISSN 0730-7268 - p. 2781 - 2789.
dioxinen - toxiciteit - biotesten - waterbodems - ecotoxicologie - dioxins - toxicity - bioassays - water bottoms - ecotoxicology - equivalency factors tefs - in-vitro bioassay - round-robin - pcbs - pcdds - pcdfs - support - water - assay
In the Fourth National Policy Document on Water Management in the Netherlands, it is defined that in 2003, in addition to the assessment of chemical substances, special guidelines for the assessment of dredged material should be recorded. The assessment of dredged material is based on integrated chemical and biological effect measurements. Among others, the DR CALUX® (dioxin responsive–chemically activated luciferase expression) bioassay has tentatively been recommended for inclusion in the dredged material assessment. To ensure the reliability of this bioassay, an intra- and interlaboratory validation study, or ring test, was performed, organized by the Dutch National Institute for Coastal and Marine Management (RIKZ) in cooperation with BioDetection Systems BV (BDS)
In the Fourth National Policy Document on Water Management in the Netherlands , it is defined that in 2003, in addition to the assessment of chemical substances, special guidelines for the assessment of dredged material should be recorded. The assessment of dredged material is based on integrated chemical and biological effect measurements. Among others, the DR CALUX(R) (dioxin responsive-chemically activated luciferase expression) bioassay has tentatively been recommended for inclusion in the dredged material assessment. To ensure the reliability of this bioassay, an intra- and interlaboratory validation study, or ring test, was performed, organized by the Dutch National Institute for Coastal and Marine Management (RIKZ) in cooperation with BioDetection Systems BV (BDS). The intralaboratory repeatability and reproducibility and the limit of detection (LOD) and quantification (LOQ) of the DR CALUX bioassay were determined by analyzing sediment extracts and dimethyl sulfoxide (DMSO) blanks. The highest observed repeatability was found to be 24.1%, whereas the highest observed reproducibility was calculated to be 19.9%. Based on the obtained results, the LOD and LOQ to be applied for the bioassay are 0.3 and 1.0 pM, respectively. The interlaboratory calibration study was divided into three phases, starting with analyzing pure chemicals. During the second phase, sediment extracts were analyzed, whereas in the third phase, whole sediments had to be extracted, cleaned, and analyzed. The average interlaboratory repeatability increased from 14.6% for the analysis of pure compound to 26.1% for the analysis of whole matrix. A similar increase in reproducibility with increasing complexity of handlings was observed with the interlaboratory reproducibility of 6.5% for pure compound and 27.9% for whole matrix. The results of this study are intended as a starting point for implementing the integrated chemical-biological assessment strategy and for systematic monitoring of dredged materials and related materials in the coming years.
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 - 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.
Estrogenic and esterase-inhibiting potency in rainwater in relation to pesticide concentrations, sampling season and location
Hamers, T.H.M. ; Brink, P.J. van den; Mos, L. ; Linden, S.C. van der; Legler, J. ; Koeman, J.H. ; Murk, A.J. - \ 2003
Environmental Pollution 123 (2003). - ISSN 0269-7491 - p. 47 - 65.
reporter gene assays - organochlorine pesticides - pyrethroid insecticides - atmospheric transport - cell-line - in-vivo - receptor - trout - organophosphate - xenobiotics
In a year-round monitoring program (1998), pesticide composition and toxic potency of the mix of pollutants present in rainwater were measured. The goal of the study was to relate atmospheric deposition of toxic potency and pesticide composition to each other and to sampling period and local agricultural activity. Rainwater was collected in 26 consecutive periods of 14 days in a background location (BACK) and in two locations representative for different agricultural practices, i.e. intensive greenhouse horticulture (HORT) and flower bulb culture (BULB). Samples were chemically analyzed for carbamate (CARB), organophosphate (OP) and organochlorine (OC) pesticides and metabolites. Esterase inhibiting potency of rainwater extracts was measured in a specially developed bio-assay with honeybee esterases and was expressed as an equivalent concentration of the model inhibitor dichlorvos. Estrogenic potency of the extracts was measured in the ER-CALUX reporter gene assay and was expressed as an equivalent concentration of estradiol. Multivariate principal component analysis (PCA) techniques proved to be valuable tools to analyze the numerous pesticide concentrations in relation to toxic potency, sampling location, and sampling season. Pesticide composition in rainwater depended much more on sampling season than on sampling location, but differences between SPRING and SUMMER were mainly attributed to local differences in agricultural practice. On average, the esterase inhibiting potency exceeded the maximum permissible concentration set for dichlorvos in The Netherlands, and was significantly higher in HORT than in BACK and BULB. Esterase inhibition correlated significantly with OP and GARB concentrations, as expected given the working mechanism of these insecticides. The estrogenic potency incidentally exceeded NOEC levels reported for aquatic organisms and was highest in SPRING. Although estrogenic potency of rainwater correlated with OC concentrations, the ER-CALUX responses could not be attributed to any particular pesticides. Besides, the contribution of non-analyzed xeno-estrogens as alkylphenol(-ethoxylates) and bisphenol-A to the estrogenic potency of rainwater could not be excluded. Further research should focus on the chemical identification of estrogenic compounds in rainwater. In addition, more attention should be given to the ecological consequences of atmospheric deposition of individual pesticides and of total toxic potencies that regularly exceed environmental criteria for Dutch surface waters and/or toxic threshold values for aquatic organisms. (C) 2003 Elsevier Science Ltd. All rights reserved.
In vitro biomonitoring in polar extracts of solid phase matrices reveals the presence of unknown compounds with estrogenic activity
Legler, J. ; Leonards, P.E.G. ; Spenkelink, A. ; Murk, A.J. - \ 2003
Ecotoxicology 12 (2003). - ISSN 0963-9292 - p. 239 - 249.
oppervlaktewater - waterorganismen - sediment - vissen - oestrogenen - hormonen - aquatisch milieu - biologische monitoring - ecotoxicologie - surface water - aquatic organisms - fishes - oestrogens - hormones - aquatic environment - biomonitoring - ecotoxicology - endocrine disruption - canada
Determination of estrogenic activity has so far mainly concentrated on the assessment of compounds in surface water and effluent. This study is one of the first to biomonitor (xeno-)estrogens in sediment, suspended particulate matter and aquatic organisms. The relatively polar acetone extracts from these solid phase matrices do not contain the well-known estrogenic compounds such as hormones, alkylphenols and phthalates. An in vitro ‘estrogen receptor-mediated chemical activated luciferase gene expression’ (ER-CALUX) assay was applied to samples from various locations in the Netherlands
Determination of estrogenic activity has so far mainly concentrated on the assessment of compounds in surface water and effluent. This study is one of the first to biomonitor (xeno-)estrogens in sediment, suspended particulate matter and aquatic organisms. The relatively polar acetone extracts from these solid phase matrices do not contain the well-known estrogenic compounds such as hormones, alkylphenols and phthalates. An in vitro,estrogen receptor-mediated chemical activated luciferase gene expression' (ER-CALUX) assay was applied to samples from various locations in the Netherlands. Estrogenic activity measured in polar fractions of particulate matter and sediment extracts ranged from below detection limit to up to 4.5 pmol estradiol equivalents (EEQ)/g dry weight. Estrogenic activity in freshwater river sediments was up to five times higher compared to sediments from large lakes and coastal locations. Tissue extracts EEQs were determined in bream (Abramis brama), flounder (Platichthys flesus), freshwater mussels (Dreissena polymorpha) and marine mussels (Mytilus edulis). The highest biota EEQ levels were found in the freshwater zebra mussel (30 pmol EEQ/g lipid). One sample site showed greatly elevated EEQs in sediment and biota, which correlated with effects found in the wild populations of bream. The EEQ activity of the unknown compounds in the polar fraction mostly was much higher than the calculated EEQ levels based on known estrogens in the non-polar fraction (previously published data).
Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays
Legler, J. ; Dennekamp, M. ; Vethaak, A.D. ; Brouwer, A. ; Koeman, J.H. ; Burg, B. van der; Murk, A.J. - \ 2002
Science of the Total Environment 293 (2002). - ISSN 0048-9697 - p. 69 - 83.
sediment - oestrogenen - toxische stoffen - afvalwater - organisch bodemmateriaal - hormonen - waterbodems - xenobiotica - waddenzee - oestrogens - hormones - toxic substances - waste water - soil organic matter - water bottoms - xenobiotics - wadden sea - in-vitro - saccharomyces-cerevisiae - polychlorinated-biphenyls - environmental estrogens - receptor - activation - metabolism - mechanism - system - transcription
Sediments may be the ultimate sink for persistent (xeno-) estrogenic compounds released into the aquatic environment. Sediment-associated estrogenic potency was measured with an estrogen receptor-mediated luciferase reporter gene (ER-CALUX) assay and compared with a recombinant yeast screen. The ER-CALUX assay was more sensitive to 17beta-estradiol (E2) than the recombinant yeast screen, with an EC50 of 6 pM E2 compared to 100 pM in the yeast screen. Yeast cells were unable to distinguish the anti-estrogens ICI 182 780 and (4-hydroxy)tamoxifen, which were agonistic in the yeast. Acetone-soluble fractions of hexane/acetone extracts of sediments showed higher estrogenic potency than hexane-soluble extracts in the ER-CALUX assay. Sediments obtained from industrialized areas such as the Port of Rotterdam showed the highest estrogenic potency of the 12 marine sediments tested (up to 40 pmol estradiol equivalents per gram sediment). The estrogenic activity of individual chemicals that can be found in sediments including: alkylphenol ethoxylates and carboxylates; phthalates; and pesticides, was tested. Increasing sidechain length of various nonylphenol ethoxylates resulted in decreased estrogenic activity. Of the phthalates tested, butylbenzylphthalate was the most estrogenic, though with a potency approximately 100 000 times less than E2. The organochlorine herbicides atrazine and simazine failed to induce reporter gene activity. As metabolic activation may be required to induce estrogenic activity, a metabolic transformation step was added to the ER-CALUX assay using incubation of compounds with liver microsomes obtained from PCB-treated rats. Results indicate that metabolites of E2, NP and bisphenol A were less active than the parent compounds, while metabolites of methoxychlor were more estrogenic following microsomal incubations. (C) 2002 Elsevier Science B.V. All rights reserved.
Comparison of in vivo and in vitro reporter gene assays for short-term screening of estrogenic activity
Legler, J. ; Zeinstra, L.M. ; Schuitemaker, F. ; Lanser, P.H. ; Bogerd, J. ; Brouwer, A. ; Vethaak, A.D. ; Voogt, P. de; Murk, A.J. ; Burg, B. van der - \ 2002
Environmental Science and Technology 36 (2002). - ISSN 0013-936X - p. 4410 - 4415.
trout oncorhynchus-mykiss - medaka oryzias-latipes - rainbow-trout - alkylphenol ethoxylates - in-vitro - chemicals - receptor - nonylphenol - xenobiotics - disruption
Functional in vitro and in vivo reporter gene assays have recently been developed for the rapid determination of exposure to (xeno)estrogens. The in vitro estrogen receptor (ER)-mediated chemically activated luciferase gene expression (ER-CALUX) assay uses T47D human breast cancer cells stably transfected with an ER-mediated luciferase gene construct. In the in vivo assay, transgenic zebrafish are used in which the same luciferase construct has been stably introduced. In both assays, luciferase reporter gene activity can be easily quantified following short-term exposure to chemicals activating endogenous estrogen receptors. The objective of this study was to compare responses by known (xeno)estrogenic compounds in both assays. Exposure to the (xeno)estrogens estradiol (E2), estrone, ethynylestradiol (EE2), o,p'-DDT, nonylphenol (NP), and di(2-ethylhexyl)phthalate (DEHP) revealed that EE2 was the most potent (xeno)estrogen tested and was 100 times more potent than E2 in the transgenic zebrafish assay, whereas in the in vitro ER-CALUX assay, EE2 and E2 were equipotent. Although the xenoestrogens o,p'-DDT and NP were full estrogen agonists in the in vitro ER-CALUX assay, only o,p'-DDT demonstrated weak dose-related estrogenic activity in vivo. To determine if differences in reporter gene activity maybe explained by differential affinity of (xeno)estrogens to human and zebrafish ERs, full-length sequences of the zebrafish ER subtypes alpha, beta, and gamma were cloned, and transactivation by (xeno)estrogens was compared to human ERalpha and ERbeta. Using transiently transfected recombinant ER and reporter gene constructs, EE2 also showed relatively potent activation of zebrafish ERalpha and ERbeta compared to human ERalpha and ERbeta. Zebrafish ERbeta and ERgamma showed higher transactivation by (xeno)estrogens relative to E2 than human ERbeta.
Biological measurement of estrogenic activity in urine and bile conjugates with the in vitro ER-Calus reporter gene assay
Legler, J. ; Jonas, A. ; Lahr, J. ; Vethaak, A.D. ; Brouwer, A. ; Murk, A.J. - \ 2002
Environmental Toxicology and Chemistry 21 (2002)3. - ISSN 0730-7268 - p. 473 - 479.
Although estrogens are excreted as biologically inactive conjugates, they can be reconverted to an active form, possibly by bacteria. A simple method was developed to deconjugate estrogen metabolites present in human urine and fish bile back to active estrogens by enzymatic hydrolysis with b-glucuronidase or live Escherichia coli cells. Deconjugated extracts were tested for estrogenic activity in the in vitro stable estrogen receptor–mediated chemical-activated luciferase gene expression (ER-CALUX) assay. Estrogen glucuronides in urine obtained from human males and females were effectively converted to active forms after incubation with b-glucuronidase or E. coli. The highest estrogenic activity was found in deconjugated metabolites from urine of a pregnant woman, in which levels up to 3,000 nmol estradiol equivalents per liter of urine were found after overnight incubation of urine with E. coli. Bile sampled from male bream and flounder from various freshwater and marine locations was also deconjugated and a good correlation was found between high biliary estrogenic activity and elevated levels of xenoestrogenic activity in surface water as well as in plasma vitellogenin. Therefore, the measurement of deconjugated bile could form a useful (indirect) biomarker for internal dose of xenoestrogens in male fish
Detection of estrogenic potency in wastewater and surface water with three in vitro bioassays
Murk, A.J. ; Legler, J. ; Lipzig, M.M.H. van; Meerman, J.H.N. ; Belfroid, A.C. ; Spenkelink, A. ; Burg, B. van der; Rijs, G.B.J. ; Vethaak, D. - \ 2002
Environmental Toxicology and Chemistry 21 (2002)1. - ISSN 0730-7268 - p. 16 - 23.
oppervlaktewater - afvalwater - oestrogenen - biotesten - hormonen - surface water - waste water - oestrogens - hormones - bioassays
A study was performed to optimize sample preparation and application of three in vitro assays for measuring estrogenic potency in environmental extracts. The three assays applied were an estrogen receptor (ER)-binding assay and two reporter gene effect assays: a yeast estrogen screen (YES) and the ER-mediated chemically activated luciferase gene expression (ER-CALUX) assay. All assays were able to detect estrogenicity, but the amounts of material needed for the assays differed greatly between the three assays (ER-binding assay ≫ YES > ER-CALUX). In addition, in the ER-binding assay, both agonists and antagonists give an estrogenic response, resulting in higher estradiol equivalency (EEQ) levels than both the ER-CALUX and the YES assay for the same samples. The EEQs found in wastewater treatment plants (WTPs) with the ER-CALUX assay were in the range of 4 to 440 and 0.11 to 59 pmol/L for influent and effluent, respectively. Water extracts from four large rivers had levels ranging from 0.25 to 1.72 pmol/L. Extracts from suspended matter and sludge contained estrogenic potency of 0.26 to 2.49 and 1.6 to 41 pmol EEQ/g dry weight, respectively. In WTPs, the average reduction of estrogenic potency in effluent compared to influent was 90 to 95% in municipal WTPs and about 50% in industrial WTPs. In influent, 30% of the ER-CALUX activity could not be explained by the calculated potencies based on chemical analysis of a number of known (xeno)estrogens; in effluent the unexplained fraction was 80%. These first results of analyzing estrogenic potency in WTP water and surface water in The Netherlands indicate that further studies are warranted to investigate the actual risks for aquatic systems.