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Zebrafish Eleutheroembryos Provide a Suitable Vertebrate Model for Screening Chemicals that Impair Thyroid Hormone Synthesis Benedicte Thienpont,† Angele Tingaud-Sequeira,‡ Eva Prats,§ Carlos Barata,† Patrick J. Babin,‡ and Demetrio Raldua†,* †
Institute of Environmental Assessment and Water Research (IDÆA-CSIC), Jordi Girona 18, 08034, Barcelona, Spain Maladies Rares: Genetique et Metabolisme (MRGM), University of Bordeaux, EA 4576, F-33400 Talence, France § Molecular Biology Institute of Barcelona (IBMB-CSIC), Jordi Girona 18, 08034, Barcelona, Spain ‡
bS Supporting Information ABSTRACT: Thyroxine-immunofluorescence quantitative disruption test (TIQDT) was designed to provide a simple, rapid, alternative bioassay for assessing the potential of chemical pollutants and drugs to disrupt thyroid gland function. This study demonstrated that zebrafish eleutheroembryos provided a suitable vertebrate model, not only for screening the potential thyroid disrupting effect of molecules, but also for estimating the potential hazards associated with exposure to chemicals directly impairing thyroxine (T4) synthesis. Amitrole, potassium perchlorate, potassium thiocyanate, methimazole (MMI), phloroglucinol, 6-propyl-2-thiouracil, ethylenethiourea, benzophenone-2, resorcinol, pyrazole, sulfamethoxazole, sodium bromide, mancozeb, and genistein were classified as thyroid gland function disruptors. Concordance between TIQDT on zebrafish and mammalian published data was very high and the physiological relevance of T4-intrafollicular content was clearly higher than regulation at the transcriptional level of tg or slc5a5. Moreover, concentrationresponse analysis provided information about the thyroid disrupting potency and hazard of selected positive compounds. Finally, the effect of perchlorate, but not MMI, was completely rescued by lowmicromolar amounts of iodide. TIQDT performed on zebrafish eleutheroembryos is an alternative whole-organism screening assay that provides relevant information for environmental and human risk assessments.
’ INTRODUCTION Thyroid hormones (THs) play essential roles in neural proliferation, differentiation, migration, synaptogenesis, and myelination during human nervous system development.1 The fetal thyroid begins to grow around the end of the first trimester, but does not begin producing its own thyroid hormones until the second trimester. The hypothalamic-pituitary-thyroid (HPT) axis is not mature until the last trimester.2 Therefore, the only source of TH available to the fetus during the first trimester of pregnancy is the mother. Several studies have drawn attention to the impact of the TH status of the mother on the future neuropsychological development of the child. On the one hand, observation of human populations with severe iodine deficiency (ID) revealed that maternal hypothyroxinemia early in pregnancy was not only the cause of the birth of neurological cretins, but also of less severe mental deficits affecting a large proportion of the apparently “normal” population in the same areas. These deficits, as well as the cretin births, are irreversible consequences of iodine deficits, preventable by an adequate supply of iodine during the first months of gestation. On the other hand, several studies carried out in areas without severe ID suggested that maternal T4 played an important role in the neuropsychological development of their progeny.3 Thus, children born to women with thyroxine (T4) levels in the lowest 10th percentile of the normal range had a higher risk of low intelligence quotient (five r 2011 American Chemical Society
to six points below average) and attention deficits, as well as difficulty with motor coordination, balance, and other psychomotor problems.4,5 Taken together, these studies present strong evidence that maternal TH plays a role in fetal brain development before the onset of fetal thyroid function and that even mild deficits in free-T4 in pregnant women produce irreversible neurological effects in their offspring.5,6 Although iodine deficiency is, without doubt, the major cause of severe thyroid gland dysfunction throughout the world, a number of natural and synthetic chemicals also have the ability to inhibit TH synthesis even in iodine-sufficient areas.2,7,8 Some of these thyroid gland function disruptors (TGFD) have a direct effect on thyroid follicles: impairing iodide uptake by the sodiumiodide symporter (NIS), inhibiting iodide organification by thyroid peroxidase (TPO), or inducing selective cytotoxic effects on thyroid follicular cells.9 Other compounds, such as dioxin-like compounds or polybrominated diphenyl ethers, impair thyroid function through indirect mechanisms, mainly by increasing the T4 metabolism and activating the HPT axis in response to the induced hypothyroidal state.10 Women of childbearing age Received: April 13, 2011 Accepted: July 29, 2011 Revised: July 28, 2011 Published: July 29, 2011 7525
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Environmental Science & Technology are exposed daily to chemicals with the potential to impair thyroid function. For instance, many fresh vegetables potentially contain goitrogenic compounds, such as thiocyanate and flavonoids. Thiocyanate and goitrin are also present in milk from dairy cattle grazing on cruciferous plants. Perchlorate and humic substances, for example, resorcinol and phloroglucinol, are potent goitrogens potentially present in drinking water. Cigarette smoking is the most ubiquitous source of exposure to thiocyanate.11 Some drugs widely used as bacteriostatics, diuretics, cutaneous antiseptics, etc., have undesirable antithyroid side-effects. Finally, residues of some fungicides and herbicides, such as mancozeb or amitrole, also reduce TH synthesis by inhibiting TPO. It has been widely reported that, even in situations of high-iodine nutritional status, exposure of women of childbearing age to some individual environmental TGFD leads to hypothyroxinemia.12,13 However, the highest concern has been raised by this broad range of common chemicals and drugs with the potential to impair TH, especially the additive or synergic effects of simultaneous exposure to multiple goitrogenic compounds on the development of mild hypothyroxinemia during early pregnancy.1,2,14 Research identifying compounds capable of impairing thyroid gland function is ongoing, but literally thousands of synthetic and naturally occurring drugs and chemicals require screening. This highlights the need to develop simple methodologies, suitable for screening the potential thyroid-disrupting effect of chemicals. Existing whole-animal testing protocols for identifying goitrogenic compounds are time-consuming, expensive, and wasteful of animals. This has led to increasing activity focused on new, nonanimal-based, short-term tests. Recently, we developed a simple, rapid zebrafish eleutheroembryo bioassay for assessing the potential of chemical pollutants and drugs to disrupt thyroid gland function.15 This bioassay used a T4 immunofluorescence quantitative disruption test (TIQDT) to measure impairment of the thyroid function as a decrease in the intrafollicular T4content (IT4C). Under the EU directive on the protection of animals used for scientific purposes, animal welfare rules only apply to independently feeding fish larvae, but not to eleutheroembryos.16,17 Consequently, the TIQDT assay is an alternative method compliant with the 3R principles (relative replacement of animal tests), a target for many international regulatory bodies.18,19 The purpose of this study was to assess (1) the suitability of TIQDT on zebrafish eleutheroembryos as a predictive vertebrate model of thyroid gland function in Tier 1 screening batteries for detecting molecules that impair TH synthesis; and (2) the use of TIQDT for quantitative estimation of the thyroid disrupting potency and hazard of chemicals, which would provide a useful source of information for risk assessment. Thyroid gland functionality was evaluated with TIQDT after exposure to 25 selected molecules by measuring IT4C in over 4700 individual zebrafish eleutheroembryos.
’ EXPERIMENTAL SECTION Chemicals. Potassium perchlorate (KClO4), potassium thiocyanate (KSCN), sodium nitrate (NaNO3), sodium bromide (NaBr), methimazole (MMI), 6-propyl-2-thiouracil (PTU), genistein, resorcinol, phloroglucinol, sulfamethoxazole (SMX), bisphenol A (BPA), benzophenone-2 (BP2), benzophenone-3 (BP3), linuron, pyrazole, amitrole, 1,10 -bis(4-chlorophenyl)-2,2,2-trichloroethane (4,40 -DDT), 2-imidazolidinethion (ethylenethiourea, ETU), mancozeb, 3,30 ,5,50 -tetrabromobisphenol A (TBBPA),
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pentabromobiphenyl ether (BDE-209), 1,2,3,6,9,10-hexabromocyclododecane (HBCD), heptadecafluorooctanesulfonic acid (PFOS), and pentadecafluorooctanoic acid (PFOA) were purchased from Sigma-Aldrich (St. Louis, MO). 2,20 ,4,40 -tetrabromodiphenyl ether (BDE-47) was a gift from G€oran Marsh. Additional information about the selected chemicals is found in Supporting Information (SI), Table S1, and Figure S1. Zebrafish Embryo Maintenance. Zebrafish (Danio rerio) embryos were obtained by natural mating and raised at 28 °C with a 12 L:12D photoperiod in embryo water [90 μg/mL of Instant Ocean (Aquarium Systems, Sarrebourg, France) and 0.58 mM CaSO4, dissolved in reverse osmosis purified water]. The iodine concentration in Instant Ocean is 7.5 ng/g, according to the manufacturer’s data, so the nominal iodine content in the embryo water was 0.67 μg/L. Animal stages were recorded as hours postfertilization (hpf). Exposure Design. The experimental design used to analyze concordance with mammalian data was described in a recent publication.15 Briefly, 48 hpf eleutheroembryos were exposed for 3 days to freshly prepared test solutions under semistatic conditions. A minimum of eighteen eleutheroembryos were exposed per compound and concentration in at least two independent experiments. For a more detailed description of the experimental design used for the concordance analysis, see SI “Methods”. A second set of experiments analyzed the suitability of TIQDT for assessing the thyroid disrupting potency and hazard of selected chemicals known to disrupt the thyroid function via a direct mechanism (direct-TGFD). While thyroid disrupting potency describes the concentration range over which thyroid gland function is impaired, thyroid disrupting “hazard” is our term for describing the relationship between effective concentrations and systemic toxicity. To obtain accurate concentrationresponse curves, 58 different concentrations were used for each test compound. EC10 and EC50 were the parameters selected to describe thyroid disrupting potency and the thyroid disrupting index (TDI: LC50/ EC50) was used as a descriptor of thyroid disrupting hazard. A third set of experiments analyzed the effects of iodide on thyroid function and its potential role in modulating the impact of the TGFD. The effect of 0.4, 4, 40, 400, and 4000 μM potassium iodide on thyroid follicle function was analyzed using TIQDT. Moreover, the effect of coexposure of KClO4 and MMI was evaluated at two different concentrations (EC50 and maximal effect), combined with three concentrations of iodide (0.4, 4, and 40 μM and 40, 400, and 4000 μM, respectively). All dilutions are reported as nominal concentrations. Stock solutions (1000) of the various compounds were prepared in DMSO on the day of the experiment. Embryos exposed to 0.1% DMSO were used as a vehicle control. All concentrations of direct-TGFD tested were below the water solubility values indicated in the SRC Physprop Database (www.syres.com/esc/ physprop.htm). However, certain highly hydrophobic chemicals that disrupt thyroid gland function indirectly (logKow > 6) were tested above their theoretical water solubility threshold (SI Table S1), although no precipitation was observed at the concentrations used. Thyroxine Immunofluorescence Quantitative Disruption Test (TIQDT). Thyroid gland functionality was evaluated by measuring IT4C in 4768 120 hpf zebrafish eleutheroembryos, following the TIQDT protocol described by Raldua and Babin (2009) with slight modifications (see SI “Methods” for details). Briefly, the protocol is based on whole-mount T4 immunofluorescence detection of 5 dpf control and treated eleutheroembryos 7526
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Figure 1. Screening for the thyroid gland function disrupting activity of drugs, environmental pollutants, and naturally occurring substances in zebrafish eleutheroembryos using intrafollicular T4-content (IT4C) as the end point. Zebrafish eleutheroembryos were exposed to selected compounds at the maximum tolerated concentrations from 48 to 120 hpf and IT4C was measured using TIQDT. Red bars indicate “thyroid gland function disruptors”, with IT4C values significantly lower than control (p < 0.05); Green bars indicate compounds that were not considered “TGFD”, as their IT4C values were not significantly different from control (p > 0.05); Yellow bars indicate “false-positives”, that is, compounds with an IT4C value significantly lower than control (p < 0.05) only in the systemic toxicity range. Data represent mean ( SE from at least two independent experiments (n = 1824).
and quantitative analysis of the T4 immunofluorescence signal assayed on thyroid follicles acts as a measurement of thyroid gland function. Whole-Mount In Situ Hybridization (WISH). Antisense and sense digoxigenin-labeled zebrafish slc5a5 and tg RNA probes were synthesized and WISH was performed on control eleutheroembryos and animals exposed to 500 μM MMI, 200 μM KClO4, and 0.78 μM HBCD, as previously described20 with modifications. See SI “Methods” for a more detailed description of WISH and the semiquantitative analysis. Data Analysis. Estimated effects of the selected chemicals at different concentrations were assessed using one-way ANOVA and regression, as described in detail in SI “Methods”.
’ RESULTS Concordance Analysis of Thyroid Gland Function Disruption in Zebrafish and Mammals. The first criterion for zebra-
fish eleutheroembryos to be considered as a generalized vertebrate model suitable for inclusion in Tier 1 screening batteries for TGFD was to satisfy the concordance criteria, by providing reasonably accurate evaluations of the thyroid gland function disruption caused by chemicals well characterized in mammalian, including human, data. Figure 1 summarizes the results obtained with TIQDT on zebrafish eleutheroembryos for the maximum tolerated concentrations (MTC) of the 25 compounds selected. IT4C in eleutheroembryos exposed to amitrole, KClO4, KSCN, MMI, phloroglucinol, PTU, ETU, BP2, resorcinol, pyrazole, SMX, NaBr, mancozeb, and genistein was significantly lower than in controls (p < 0.05), therefore
those compounds were classified as “TGFD” on zebrafish eleutheroembryos. Although exposure to DDT and TBBPA at MTC also induced a slight, but significant, decrease in IT4C, both chemicals were classified as “false-positives” due to the presence of some systemic toxicity at concentrations where the effect on thyroid follicles had completely disappeared (SI Figure S2). IT4C levels in eleutheroembryos exposed to nitrate, PFOA, BDE-209, PFOS, BPA, HBCD, BDE-47, linuron, and BP3 were similar to controls (p > 0.05), so these chemicals were not classified as “TGFD ”. Figure 2 shows a “heat-map” summarizing reported responses to a variety of known or suspected TGFD in both mammalian in vivo models and various in vitro assays (SI, Table S1 and References), as well as the results obtained with TIQDT on zebrafish eleutheroembryos. No information was found about the effect of BPA or BP3 on thyroid gland function in mammalian models, or that of SMX, mancozeb, and pyrazole on in vitro systems. Primary end points evaluated in mammalian models to determine disruption of thyroid gland function were in vivo iodide uptake, TPO inhibition, histopathology, and enlargement of the thyroid gland. In vitro end points included TPO activity and iodine uptake in various cell models. For those chemicals with a reported direct effect on thyroid gland function, zebrafish eleutheroembryos showed a high degree of concordance with mammalian data (15/16, 93.75%). However, the rate of concordance for chemicals with an indirect effect on the thyroid gland was very low (2/5, 40%). TIQDT also showed a high degree of concordance with in vitro data for direct-TGFD (13/15, 87%), in both correct positives and negatives. Intrafollicular T4-Content Is a Sensitive, Physiologically Relevant End Point for Detecting Thyroid Gland Function 7527
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Figure 2. Heat-map summarizing mammalian versus zebrafish eleutheroembryo responses and in vitro assays using 25 known or suspected TGFDs with different modes of action. Red indicates thyroid gland function disrupting activity; Green indicates that the chemical is not a TGFD; White indicates that no data or contradictory data (BDE-47) on thyroid gland function (mammals), or no data on in vitro systems (in vitro) had been reported for the chemical tested. Bibliographic references concerning the activity of the selected compounds in mammalian and in vitro models are listed in SI, Table S1.
Disruptors in Zebrafish Eleutheroembryos. Although the
thyroid follicles of eleutheroembryos exhibited a dramatic decrease in IT4C after a 3-day exposure to direct-TGFD, this end point was, apparently, not sensitive to chemicals that disrupt thyroid gland function via indirect or secondary mechanisms (indirect-TGFD) (Figures 13, SI Table S2, and Figure S3). An alternative end point commonly analyzed in zebrafish and Xenopus models to identify TGFD is the transcript level of TSH-regulated genes, such as slc5a5 or tg, in thyroid follicular cells. For a direct comparison of the sensitivity of both end points in our short-term assay, we tested the ability of MMI and KClO4, two direct-TGFD with different modes of action (MoA), and HBCD, an indirect-TGFD, to regulate slc5a5 and tg transcript levels. WISH results revealed a restricted expression pattern of both genes in thyroid follicles (Figure 3). Zebrafish eleutheroembryos treated with concentrations of both MMI and KClO4 that induced a strong reduction in IT4C exhibited a significant up-regulation of slc5a5 and tg hybridization signals in thyroid follicles (p < 0.001; see results from WISH in Figure 3). HBCD
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treatment had no effect on IT4C (Figure 1), or the slc5a5 or tg transcript hybridization signals (data not shown). Thyroid Disrupting Potency and Hazard of Chemicals That Have a Direct Effect on Thyroid Gland Function in the Zebrafish Model. Quantitative estimates of thyroid disrupting potency and hazard, based on concentrationresponse analyses, were performed using ten and seven selected direct goitrogenic compounds, respectively (Figure 4). The concentrationresponse relationships of TIQDT (SI Table S2) and lethality (SI Table S3) for the selected compounds exhibited a good fit with an allosteric decay model (SI Figure S3), indicating little or no effect at low concentrations, followed by an accelerating negative response as concentrations increased. All regression equations were statistically significant (p < 0.01), with residuals evenly distributed and r2 ranging from 0.88 for resorcinol to 1.00 for MMI (SI Table S2). Regression-based EC50 and EC10 of individual direct-TGFD differed by more than three and 4 orders of magnitude, respectively. The two NIS inhibitors evaluated, KClO4 and KSCN, exhibited the highest thyroid disrupting potency, whereas BP2 was the most potent TPO inhibitor (Figure 4A). Phloroglucinol and amitrole, with potencies about 100-times lower than BP2, had the least effect on IT4C. In addition, the slope for the various compounds ranged from 0.61 for KClO4 to 6.74 for BP2. TPO inhibitors, which share a similar MoA, also exhibited a wide range of slopes, from 0.66 for resorcinol to 6.74 for BP2. For those with the steepest slopes, doubling the EC50 concentration resulted in an almost 100% decrease in IT4C, whereas for those with the shallowest slope, the T4-content was still about 45% of the control. Thyroid disrupting hazard, a measurement of the specificity of the thyroid disrupting effect, was evaluated for five TPO inhibitors and two NIS inhibitors (Figure 4A). This revealed that, although BP2 was the most potent TPO inhibitor evaluated, it represented the lowest thyroid disrupting hazard. However, the NIS inhibitors perchlorate and thiocyanate not only exhibited the highest potency among the direct-TGFD, but also the highest thyroid disrupting hazard. In addition, for all the compounds tested, except BP2, the slope of the concentrationresponse curve was shallower for the thyroid disrupting effect than for lethality (Figure 4B and SI Tables S2 and S3), illustrating a wider distribution of tolerances to thyroid gland impairment compared to lethality in exposed zebrafish populations. Role of Iodide in Zebrafish Gland Function. The effect of increasing iodide concentrations on the thyroid disrupting effect was evaluated for two prototypic direct TGFD: the NIS inhibitor KClO4 and the TPO inhibitor MMI. Exposing zebrafish eleutheroembryos to water containing 3 and 50 μM KClO4 induced 50% and 100% reductions in IT4C, respectively. However, the effect of KClO4 was completely prevented by adding 0.4 and 40 μM iodide, respectively (Figure 5). Thus, the iodide: KClO4 molar ratio able to recover the IT4C in zebrafish was 0.13:1 at EC50 and 0.8:1 at EC100. On the contrary, iodide concentrations as high as 4000 μM were unable to rescue thyroid gland function in zebrafish eleutheroembryos treated with concentrations of MMI inducing 50% (300 μM) or 100% (500 μM) reduction in IT4C (Figure 5).
’ DISCUSSION This study evaluated the suitability of zebrafish eleutheroembryos as a potential generalized vertebrate model in Tier 1 screening batteries for detecting molecules that impair thyroid 7528
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Figure 3. Short-term exposure to direct TGFD induced a strong decrease in IT4C and up-regulation of selected target gene transcripts in the thyroid follicles of zebrafish eleutheroembryos. (A) Ventral view of heads of representative 120 hpf zebrafish eleutheroembryos at the branchial arch level (ventral view, rostral to the top). The same thyroid follicles were labeled green for T4 and red for TG following double-IHC T4/TG (left and right panels, respectively). WISH was performed on parallel eleutheroembryos using slc5a5 and tg antisense (middle panels) riboprobes. When 48 hpf zebrafish eleutheroembryos were exposed to 200 μM KClO4 or 500 μM MMI for 3 days, IT4C fell to undetectable levels (left panels). Double-IHC T4/TG showed that TG levels decreased compared to control eleutheroembryos (right panels). A faint slc5a5 and tg hybridization signal was detected in the thyroid follicles of control eleutheroembryos, while a strong up-regulation of the signal was detected following TGFD exposure (middle panels). The hybridization signal is colored dark-blue to purple. No staining signal was observed using the sense probes (data not shown). (B) Quantitative analysis of whole-mount immunofluorescence signal. (C) Quantitative analysis of WISH signal. The decrease in integrated density (ID) of the immunofluorescence signal was about 50% of control eleutheroembryos for TG and IT4C disappeared almost completely following exposure to the direct-TGFD used in the experiment. The decrease in T4 and TG content was associated with a significant up-regulation of the slc5a5 and tg hybridization signals in enlarged thyroid follicles, compared to controls. Values shown are mean + SE (n = 10 eleutheroembryos/group for WISH and 18 eleutheroembryos/group for IHC). **p < 0.0001.
Figure 4. TIQDT on zebrafish eleutheroembryos to determine the thyroid disrupting potency and hazard of chemicals. (A) Thyroid disrupting potency (EC50, μM) and hazard (Thyroid Disrupting Index, TDI: LC50/EC50) of selected direct-TGFD compounds. Scale for EC50 has been inverted in order to show chemicals sorted by increasing potency, thus facilitating a direct comparison of potency and hazard for each compound. (B) Concentrationeffect relationship of thyroid gland function disruption (solid lines) and lethality (dotted lines), fitted to an allosteric decay function, for two thyroid peroxidase inhibitors, benzophenone-2 (BP2, open triangles) and methimazole (MMI, filled triangles). Although BP2 exhibited a higher potency than MMI, its thyroid disrupting activity was present only at concentrations very close to lethality (low TDI). Although MMI, an antithyroid drug, exhibited lower potency, the TDI for this compound was much higher. 7529
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Figure 5. The effect of potassium perchlorate (KClO4), but not methimazole (MMI), on thyroid function was prevented by low-micromolar iodide concentrations. A TIQDT assay was performed for both prototypic direct -TGFD at concentrations that induced about 50% (A) or 100% (B) of the effect. Increasing concentrations of iodide in water were added (0.44000 μM) in each condition. While total recovery of IT4C to control levels in eleutheroembryos treated with maximum effective concentration of KClO4 required 40 μM iodide, eleutheroembryos treated with the median effective concentration required 100-times less. None of the iodide concentrations tested (0.44,000 μM) had any protective effect against the thyroid disrupting effect of MMI (A and B). Values shown are mean + SE (n = 3436 eleutheroembryos/group). * p < 0.05; ** p < 0.001.
hormone synthesis. The taxonomic similarity of the screening model organism to humans is not a primary concern for a Tier 1 screening assay,21 so zebrafish eleutheroembryos, commonly used in biomedical research, represent a logical candidate.22 Our teams recently developed TIQDT, a simple, rapid zebrafish eleutheroembryo bioassay for assessing the potential of chemical pollutants and drugs to disrupt thyroid gland function.15 This research consisted of an initial screening of molecules to check whether TIQDT was suited for this purpose and could be usefully integrated into a testing framework. As the purpose of the screening is hazard identification, chemicals may be screened at concentrations higher than those likely to be encountered by wildlife or humans in the environment.21 MTC was selected as the initial screening concentration, in order to minimize the risk of reporting false-negatives. Finally, we minimized the risk of reporting false-positives by testing additional lower concentrations for compounds that produced positive results. The concordance of zebrafish versus mammalian assays was tested to assess the potential utility of optimized TIQDT as a
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screening assay for thyroid disruptors in an endocrine disruptor screening and testing program. End points evaluated in the thyroid gland of mammalian models to detect disruption of thyroid gland function are in vivo iodide uptake, TPO activity, thyroid gland weight, and histology. In vivo and in vitro data support the direct thyroid disrupting effect of NIS inhibitors KClO4, KSCN, bromine, and nitrate, as well as TPO inhibitors MMI, PTU, BP2, resorcinol, phloroglucinol, amitrole and ETU, and the thyroid cytotoxic pyrazole (for references, see SI Table S1). The only compound with a direct thyroid disrupting effect in mammalian models23 that gave negative results in the TIQDT assay was nitrate. Nitrate is a mild human NIS inhibitor, about 240 times less potent than perchlorate.4 Differences in anion selectivity of zebrafish versus rat NIS may justify the negative response in the TIQDT assay. In fact, the relative potency of perchlorate to inhibit human NIS is 15-times that of thiocyanate,24 whereas our TIQDT data indicated that KClO4 and KSCN exhibited similar potencies in zebrafish. Dietary genistein, a classic in vitro TPO inhibitor, is accumulated in the rat thyroid gland, where it induces TPO inhibition, although, paradoxically, no thyroid enlargement or histological changes.25 Zebrafish eleutheroembryos exhibited a slight (35%), but significant, decrease in IT4C, consistent with the observed decrease in TPO activity in rats. Although BPA is also a human TPO inhibitor in vitro,26 no data are available about its potential effects on thyroid gland function in vivo. In the zebrafish eleutheroembryo model, exposure to BPA at MTC did not modify the IT4C, so it was not classified as a TGFD. Pyrazole has been reported to induce necrosis in rat thyroid follicular cells27 and a significant decrease of about 80% in IT4C was observed in zebrafish eleutheroembryos using TIQDT. Finally, BP3 and linuron had no effect in thyroid-relevant in vitro assays28 and no thyroid disrupting effects have been reported in mammalian models. These compounds, selected as negative controls in this concordance analysis, did not disrupt thyroid gland function in our TIQDT assay. Our WISH results on zebrafish eleutheroembryos revealed an up-regulation of tg and slc5a5 mRNA hybridization signals in thyroid follicles after treatment with concentrations of MMI and KClO4 known to impair T4 synthesis. This higher hybridization signal was consistent with published results in perchlorateexposed Xenopus tadpoles.29 Goiter formation was associated with follicular cell hypertrophy and a concurrent reduction in size of the follicular lumina. Perchlorate was very effective in inducing histopathological changes in zebrafish thyroid follicles, including epithelial cell hypertrophy, as an early response to thyroid hormone disruption.30 Our results indicated that both end points, IT4C and thyroid follicle marker gene transcript levels, exhibited similar sensitivity to the goitrogenic compounds. However, a decrease in IT4C is clearly more physiologically relevant than tg or slc5a5 regulation at the transcriptional level. For instance, MMI and KClO4 concentrations that induced upregulation of the tg hybridization signal were correlated to a decrease in TG thyroid follicle content, clearly illustrating the difficulty of predicting the physiological relevance of results at the transcript level in the complex environment of thyroid follicles. Our data show that TIQDT on zebrafish eleutheroembryos is not a suitable assay for detecting indirect-TGFD, as indicated by its very low concordance with mammalian models. On the one hand, both brominated flame retardants and DDT are quite hydrophobic (logKow >6) and aqueous exposure is probably not appropriate for this group of chemicals. Most of the studies investigating the thyroid disrupting potential of highly 7530
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Environmental Science & Technology hydrophobic compounds in mammalian models, or even in the Xenopus model, are based on oral exposure. As TIQDT is an alternative method using only eleutheroembryos, it is not possible to spike their diet with high concentrations of these hydrophobic compounds. These findings on thyroid disrupting potency and hazard highlight the importance of whole-animal assays in thyroid gland function disruption testing. In vitro assays have found BP2, for instance, to be a more potent TPO inhibitor than MMI or PTU. Its thyroid disrupting potency in zebrafish eleutheroembryos was also higher than that of MMI or PTU. Nevertheless, TIQDT, unlike in vitro systems, is capable of determining thyroid disrupting hazard by analyzing the relationship between specific antithyroid effects and systemic toxicity. TIQDT results showed that, although BP2 was the most potent of the TPO inhibitors assayed, its thyroid disrupting hazard was very low, as this compound only impaired thyroid gland function in a range of concentrations very close to lethality. The lowest observed effect concentration (LOEC) of directTGFD provides a measurement of the sensitivity of the TIQDT assay. For example, to compare the sensitivity of rats and zebrafish eleutheroembryos to perchlorate, significant effects on thyroid gland histology were reported at a dose level of 62.5 mg/kg/day in a rat pubertal assay31 and 125 μg/L in our TIQDT assay. However, it is difficult to compare dietary dose levels and aquatic test concentrations in the absence of robust data on uptake, distribution, and clearance of the test chemicals in the respective model organisms.21 Interestingly, the sensitivity of TIQDT to perchlorate was in the range described recently in a 21-day exposure amphibian metamorphosis assay (AMA), that is, 62.5 μg/L.32 However, from a regulatory standpoint, AMA is considered an animal-based assay while TIQDT using eleutheroembryos is a nonanimal based assay, as indicated above. Moreover, one of the requirements of any assay suitable for endocrine screening and testing programs is its rapidity. While AMA required a 21-day exposure to detect the effects of perchlorate on the thyroid gland,32 TIQDT only required 3 days’ exposure. For BP-2, the sensitivity of TIQDT with zebrafish eleutheroembryos (5 mg/L) was also similar to the reported AMA value after 21 days’ exposure (6 mg/L).33 Moreover, AMA indicated that BP2 represented a low goitrogenic hazard using, as the Xenopus tadpoles also exhibited some signs of systemic toxicity at LOEC for thyroid effects.33 MMI, PTU, and ETU also exhibited a similar sensitivity in TIQDT and AMA. MMI and PTU induced histological effects in Xenopus thyroid glands after 8 days’ exposure at 12.5 and 10 mg/L, respectively, while the LOEC for those compounds in our TIQDT were 45.7 and 17 mg/L, respectively.34 Finally, a series of histopathological alterations were observed in the thyroid gland of Xenopus tadpoles exposed to 1 mg/L ETU for 20 days,35 while the LOEC for this chemical using TIQDT was 10.2 mg/L. Although increased duration of exposure to TGFD is likely to result in moderate increases in TIQDT sensitivity, our data suggest that 3 days’ exposure is sufficient to generate a significant decrease in the intrafollicular thyroid hormone content for most of the chemicals, with the probable exception of the highly hydrophobic persistent organic pollutants. It has been reported that increasing iodide concentrations may prevent the inhibitory effect of some direct-TGFD.26,36 These data are important for future validation of the TIQDT assay, suggesting that its sensitivity and, thus, the measured potency of TGFD may be strongly dependent on the iodide content in the
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water. The iodide content of the embryo water used in this study (0.005 μM) was in the low range of the levels commonly found in freshwater systems (0.0040.158 μM).37 Our results demonstrate a high sensitivity of the NIS inhibitor KClO4 to the presence of increasing iodide concentrations. In fact, KClO4 concentrations that had induced a 50% reduction in IT4C (EC50) in our TIQDT assay had no effect on thyroid function when the iodide concentrations in the embryo water reached levels commonly found in seawater (0.4 μM). Although it has been demonstrated in vitro that inactivation of TPO with BP2 recovered when iodide concentrations increased,26 our data revealed that the zebrafish eleutheroembryos did not recover from the effect of MMI on IT4C, even at mM concentrations of iodide. The high iodide:TGFD molar ratio of 3333:1 used to recover TPO activity inhibited with BP226 and the absence of recovery at a 17:1 iodide: MMI molar ratio suggested that TPO inhibitors had a low sensitivity to iodide. The results presented here provide evidence for the suitability of zebrafish eleutheroembryos as a predictive vertebrate model for evaluating thyroid gland function. IT4C is a relevant target organ end point for identifying chemicals and mixtures that have a direct effect on the thyroid gland. Thus, TIQDT on zebrafish eleutheroembryos is ready for validation and represents a promising candidate for inclusion as an intermediate step between cell-based evaluation and conventional animal testing in Tier 1 screening batteries for chemicals that impair TH synthesis. Finally, our data support the use of TIQDT as a source of information for risk assessment, as it provides a quantitative estimation of the thyroid disrupting potency and hazard of chemicals.
’ ASSOCIATED CONTENT
bS
Supporting Information. Details of experimental procedures and further explanation of results: This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Phone: +34-93-4006100; fax: +34-93-2045904; e-mail: drpqam@ cid.csic.es.
’ ACKNOWLEDGMENT This work was partly supported by the Spanish MICINN (CGL2008-01898/BOS and CTM2011-30471-C02-01) and the Generalitat de Catalunya (2010 BE1 00623) to D.R. B.T was supported by a fellowship from MICINN (AP2006-01324). A. T.-S. was supported by a postdoctoral fellowship from the Conseil Regional d0 Aquitaine (CRA). This work was partly supported by the CRA (200881301031/TOD project) to P.J.B. ’ REFERENCES (1) Howdeshell, K. A model of the development of the brain as a construct of the thyroid system. Environ. Health. Perspect. 2002, 110, 337–348. (2) Brown, V. Disrupting a delicate balance: environmental effects on the thyroid. Environ. Health. Perspect. 2003, 111, A642–649. (3) de Escobar, G.; Obregon, M.; del Rey, F. Is neuropsychological development related to maternal hypothyroidism or to maternal hypothyroxinemia? J. Clin. Endocrinol. Metab. 2000, 85, 3975–3987. 7531
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’ NOTE ADDED AFTER ASAP PUBLICATION Corrections were made to the author affiliations and typographical errors in the version published August 9, 2011. The corrected version was reposted on August 12, 2011.
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