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Environmental chemicals can induce thyroid disruption through a number of mechanisms including altered thyroid hormone biosynthesis and transport, as well as activation and inhibition of the thyroid receptor. In the current study six in vitro bioassays indicative of different mechanisms of thyroid disruption and one whole animal in vivo assay were applied to 9 model compounds and 4 different water samples (treated wastewater, surface water, drinking water and ultra-pure lab water; both unspiked and spiked with model compounds) to determine their ability to detect thyroid active compounds. Most assays correctly identified and quantified the model compounds as agonists or antagonists, with the reporter gene assays being the most sensitive. However, the reporter gene assays did not detect significant thyroid activity in any of the water samples, suggesting that activation or inhibition of the thyroid hormone receptor is not a relevant mode of action for thyroid endocrine disruptors in water. The thyroperoxidase (TPO) inhibition assay and transthyretin (TTR) displacement assay (FITC) detected activity in the surface water and treated wastewater samples, but more work is required to assess if this activity is a true measure of thyroid activity or matrix interference. The whole animal Xenopus Embryonic Thyroid Assay (XETA) detected some activity in the unspiked surface water and treated wastewater extracts, but not in unspiked drinking water, and appears to be a suitable assay to detect thyroid activity in environmental waters., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Amphibian metamorphosis assays are used to evaluate potential effects of endocrine disrupting compounds on the thyroid hormone axis. In this study, Xenopus laevis tadpoles are kept in a solution of 0.2% thiourea (TU) to arrest and synchronise them in their development. The advantage of this synchronized amphibian metamorphosis assays is that synchronised tadpoles are available at any time to start metamorphosis experiments, and experimental groups are much more homogenous at the start of experimental exposure compared with groups selected from an untreated pool of animals. The water volume per animal was kept constant throughout the experimental period to overcome the influence of declining numbers of animals per aquarium due to metamorphosis and mortality on the density dependent development of the remaining tadpoles. Clophen A50 (a technical PCB mixture), the single congener 3,3',4,4'-tetrachlorobiphenyl (PCB 77) and apolar sediment extracts that were previously tested positive in the T-Screen, an in vitro proliferation assay for thyroid hormone disruption, were tested in the Synchronized Amphibian Metamorphosis Assay. Endpoints studied were mortality, malformations, body weight, and percentage of metamorphosed froglets at the end of the 60-day experimental period, percentage of tadpoles in different developmental stages, and developmental stage-dependent awarded penalty points. Dietary exposure to Clophen A50 (0.2-50mg/kg food) resulted in a significant increased percentage of tadpoles that did not pass metamorphosis at concentrations higher than 2mg/kg food. Time until metamorphosis in those animals that were able to metamorphose after the 60-days experimental period was significantly decreased. Dietary exposure to PCB 77, a congener that can be readily metabolised, did not result in significant effects in any exposure group (2-500 microg/kg food). Apolar sediment extracts from two of the three sites that are contaminated with a wide variety of chemicals significantly decreased the percentage of metamorphosed animals and significantly increased the number of tadpoles that remained in early and late metamorphic stages. These effects already occurred when the extracts where diluted more than 1000 times (on an organic carbon base) compared to environmental concentrations. The rank of potency was comparable to results obtained with the T-screen. This suggests the presence of thyroid hormone disrupting compounds in the aquatic environment and possible effects of such compounds on animal development in the wild.

Thyroid hormone regulates amphibian metamorphosis, including the transformation of a tadpole into a froglet and regression of the tail. Xenopus laevis tadpole tail tips in organ culture (ex vivo) undergo regression when exposed to 3,3',5-triiodo-l-thyronine (T(3)) and interference by chemicals with this process was utilized as a bioassay to detect thyroid hormone disruption. In the present study the bioassay was further validated by investigating its response to compound induced T(3)-antagonism and - potentiation. Tadpole tail tips were exposed to two brominated flame retardants (BFRs) in presence or absence of T(3) at its EC(50) (20 nM). T(3)-induced tail tip regression was antagonized by 2,2',3,3',4,4',5,5',6-nona brominated diphenyl ether (BDE206) and potentiated by hexabromocyclododecane (HBCD) in a concentration dependent manner, which was consistent with results obtained with a in vitro T(3)-dependent proliferation bioassay termed the T-screen. Neither compound induced any effect in the absence of T(3). The results indicate that studying possible hormone disrupting effects of agonistic, antagonistic or potentiating compounds should include combined exposure with the natural hormone at around its EC(50) concentration. The results obtained with the tail tip exposures were in accordance with the T-screen predictions, and occurred at BFR-concentrations that were only 5-50 times those of T(3). The bioassay proved to be suitable not only for detecting T(3)-agonism, but also for antagonism and potentiation.

The present study investigates chemical thyroid hormone disruption at the level of thyroid hormone receptor (TR) functioning. To this end the (ant)agonistic action of a series of xenobiotics was tested in the newly developed T-screen. This assay makes use of a GH3 rat pituitary cell line, that specifically proliferates when exposed to 3,3',5-triiodo-L-thyronine (T3). The growth stimulatory effect is mediated via T3-receptors. (Ant)agonistic and potentiating action of compounds was studied in absence and presence of T3 at its EC50 level (0.25 nM). The compounds tested included the specific TR-antagonist amiodarone, as well as a series of brominated diphenyl ethers (BDEs), including specifically synthesized BDEs with a structural resemblance to 3,5-diiodo-L-thyronine (T2), T3 and T4 (3,3',5,5'-tetraiodo-L-thyronine). The results obtained reveal that only BDE206 and amiodarone are specific antagonists. Interestingly some compounds which did not respond in the T-screen in absence of T3, potentiated effects when tested in combination with T3. This points at possibilities for disruption at the TR in vivo, where exposure generally occurs in presence of T3. Altogether the results of the present study show that the newly developed T-screen can be used as a valuable tool for identification and quantification of compounds active in disturbing thyroid hormone homeostasis at the level of TR-functioning.

Some compounds, including brominated diphenyl ethers (BDEs), can interfere with thyroid hormone (TH) receptor (TR)-mediated TH-signalling. In this study, the TR isoform selectivity of some TH disrupting compounds was investigated with TRα/β specific reporter gene assays. For this purpose, the effects of compounds on 3,3′,5-triiodothyronine (T3)-induced TRα-or TRβ-activation were tested in green monkey kidney fibroblast (CV-1) cells transiently transfected with Xenopus TRs and a luciferase reporter gene. The T3-like BDE-OH and diiodobiphenyl (DIB) increased T3-induced TRα-activation, but not T3-induced TRβ-activation. BDE28 (100nM) did not act via TRα, but almost tripled T3-induced TRβ-activation relative to T3 at its EC50. BDE206 (100nM) was antagonistic on both TRs with a maximum repression −54% relative to T3 at its EC50. Contrary to previous results obtained with the T-screen, HBCD was inactive. The present study illustrates the importance of testing potential TH disrupting compounds in model systems that enable independent characterization of effects on both T3-induced TRs. [Copyright &y& Elsevier]

In the prolonged-FETAX (prolonged-Frog Embryo Teratogenic Assay-Xenopus) tadpoles are allowed to develop until metamorphosis after an initial 4 day early life-stage exposure (FETAX). PCB 77 (3,4,3′,4′-tetrachlorobiphenyl) and sediment extracts were used in the presented experiments. Concentrations of PCB 77 (0.03 nM=8.55 ng/L; 3 nM=855 ng/L; 300 nM=85.5 μg/L, 30,000 nM=8.55 mg/L) were derived on the basis of dioxin-like toxic equivalency concentrations known to be present in pore-water. The results were compared to those obtained with the standard FETAX. In the prolonged-FETAX the percentage of animals passing metamorphosis within 115 days was significantly decreased in the groups exposed to 300 or 30,000 nM PCB 77. Significantly increased percentages of tadpoles were halted in thyroid hormone dependent early metamorphic NF stages ≤55 (3 nM PCB 77) and metamorphic stages 56–60 tadpoles (300 nM PCB 77 and 30,000 nM PCB 77). Non-polar sediment extracts from two contaminated Dutch sites significantly decreased, and one significantly increased the percentage of animals that passed metamorphosis. These results were in accordance with earlier in vitro results in a thyroid hormone dependent assay and strongly suggest the presence of thyroid hormone disrupting compounds in the aquatic environment. In the classical FETAX neither PCB 77 nor non-polar sediment extracts induced any effects. The results of the present study indicate that the prolonged-FETAX is an important in vivo tool in addition to the in vitro T-screen to assess long-term effects of early life-stage exposure to environmentally relevant concentrations of (mixtures of) compounds and that affect thyroid hormone dependent physiology. [Copyright &y& Elsevier]

Abstract: The T-Screen represents an in vitro bioassay based on thyroid hormone dependent cell proliferation of a rat pituitary tumour cell line (GH3) in serum-free medium. It can be used to study interference of compounds with thyroid hormone at the cellular level, thus bridging the gap between limitations of assays using either isolated molecules (enzymes, transport proteins) or complex in vivo experiments with all the complex feedback mechanisms present. Compounds are tested both in the absence and presence of thyroid hormone (EC50 concentration of T3) to test for both agonistic and antagonistic potency. Thyroid hormones (3,3′-5-triiodothyronine: T3 and 3,3′,5,5′-tetraiodothyroxine: T4) and compounds resembling the structure of thyroid hormones (3,3′-5-triiodothyroacetic acid: Triac; 3,3′,5,5′-tetraiodothyroacetic acid: Tetrac) induced cell growth, with the rank order Triac > T3 > Tetrac > T4 (relative potency = 1.35 > 1 > 0.29 > 0.07), which is identical to published affinities of these compounds for nuclear thyroid hormone receptors. Exposure to 5,5′-diphenylhydantoin (DPH) in the presence of 0.25nM T3 resulted in up to 60% decreased cell growth at 200μM DPH. No effect of DPH on basal metabolic activity of GH3 cells was observed at this concentration. Fentinchloride (IC50 = 21nM) decreased cell growth induced by 0.25nM T3, whereas parallel exposure to these concentrations in the absence of T3 did not alter basal metabolic activities of GH3 cells. Apolar sediment extracts from the Dommel (34%) and Terneuzen (14%) decreased cell growth in the presence of 0.25nM T3, whereas the extract from Hoogeveen increased cell growth (26%) and the extract from North Sea Channel had no effect. The T-Screen proved to be a fast and functional assay for assessing thyroid hormone receptor active potencies of pure chemicals or environmental mixtures. [Copyright &y& Elsevier]