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Jun 25, 2014 - Tetrabromobisphenol A Disrupts Vertebrate Development via. Thyroid Hormone Signaling Pathway in a Developmental Stage-. Dependent ...
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Tetrabromobisphenol A Disrupts Vertebrate Development via Thyroid Hormone Signaling Pathway in a Developmental StageDependent Manner Yin-Feng Zhang,†,‡ Wei Xu,†,§ Qin-Qin Lou,† Yuan-Yuan Li,† Ya-Xian Zhao,† Wu-Ji Wei,§ Zhan-Fen Qin,*,† Hui-Li Wang,*,‡ and Jian-Zhong Li‡ †

State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-environmental Sciences and Department of Environmental Bio-Technology, Research Center for Eco-environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, Beijing 100085, China § College of Environment, Nanjing University of Technology, Nanjing, 211816 Jiangsu, China ‡

S Supporting Information *

ABSTRACT: Data concerning effects of tetrabromobisphenol A (TBBPA) on thyroid hormone (TH)-dependent vertebrate development have been limited, although TBBPA has been demonstrated in vitro to disrupt the TH signaling pathway at the transcriptional level. In this study, we investigated the effects of TBBPA on T3-induced and spontaneous Xenopus laevis metamorphosis, which share many similarities with THdependent development in higher vertebrates. In a 6-day T3induced metamorphosis assay using premetamorphic tadpoles, 10−1000 nM TBBPA exhibited inhibitory effects on T3induced expression of TH-response genes and morphological changes in a concentration-dependent manner, with a weak stimulatory action on tadpole development and TH-response gene expression in the absence of T3 induction. In a spontaneous metamorphosis assay, we further found that TBBPA promoted tadpole development from stage 51 to 56 (preand prometamorphic stages) but inhibited metamorphic development from stage 57 to 66 (metamorphic climax). These results strongly show that TBBPA, even at low concentrations, disrupts TH-dependent development in a developmental stagedependent manner, i.e., TBBPA exhibits an antagonistic activity at the developmental stages when animals have high endogenous TH levels, whereas it acts as an agonist at the developmental stages when animals have low endogenous TH levels. Our study highlights the adverse influences of TBBPA on TH-dependent development in vertebrates.

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INTRODUCTION Thyroid hormone (TH) is essential for normal development, growth, neural differentiation, and metabolic regulation in vertebrates.1,2 It is well-known that TH acts by binding to TH receptors (TRs, TRα, and TRβ), ligand-activated transcription factors, to regulate gene transcription.3,4 In recent years, there is increasing evidence that some chemicals, such as bisphenol A (BPA), polychlorinated biphenyls, and polybrominated diphenyl ethers, can bind to TRs and disrupt the TH signaling pathway because of their similarity in chemical structures to TH.5−7 Tetrabromobisphenol A (TBBPA) is a commonly used brominated flame retardant in many items, including building materials, synthetic textiles, and plastic products.8,9 Because of its widespread use, TBBPA has been found in various environmental samples and biotic samples, including human plasma and breast milk.10−12 The TBBPA levels in the environment present an increasing trend in recent years.8 Thus, TBBPA has aroused concern from scientists and the general public. With a similar chemical structure to TH, TBBPA has been shown to bind to TR © 2014 American Chemical Society

and affect TR-mediated gene expression in vitro, exhibiting antagonistic or agonistic effects on TH actions.8,13−18 However, the effects of TBBPA on TH-dependent development at the morphological and functional levels in vertebrates have yet been unclear because of the lack of in vivo data. Most amphibian species undergo TH-dependent metamorphosis with dramatic changes at the molecular, morphological, and functional levels. Amphibian metamorphosis shares many similarities with vertebrate development, such as intestinal remodeling, bone differentiation, and brain development.19,20 Additionally, free-living tadpoles are favorable over mammalian models because the interferences from maternal TH and the difficulty in manipulating uterus-enclosed embryo can be eliminated. In particular, premetamorphic tadpoles can be Received: Revised: Accepted: Published: 8227

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we conducted a 6-day T3-induced metamorphosis assay using premetamorphic X. laevis. Given that TH-regulated gene transcription through TR is not only necessary but also sufficient for metamorphic development, we chose morphological features and TH-response gene expression as endpoints for assaying TH signaling disrupting activity. Five TH-response genes including TRβ, basic transcription element binding protein (BTEB), stromelysin-3 (ST3), matrix metalloproteinase 2 (MMP2), and type 2 deiodinase (DIO2) were chosen to assay TH signaling disruption in this study. The five genes were used to study TH signaling pathway in previous reports,22,28 and they play important roles in metamorphic development. In detail, BTEB binds the GC-rich basic transcription element and functions as a transcriptional activator on TH-response genes promoters;29 ST3 plays a role in the extracellular matrix remodeling that facilitate apoptotic tissue remodeling or resorption;30 MMP2 affects cell fate and tissue remodeling by participating in postapoptosis extracellular matrix remodeling;31 and DIO2 activates T4 to T3.32 The tadpoles at stage 52 from a clutch were exposed to a series of concentrations of TBBPA (10, 100, 500, 1000 nM) in the presence or absence of 1 nM T3 for 6 days. Three replicate test tanks (10 tadpoles per tank) were employed for each treatment group. Followed by water changes, chemical replacements were performed every 24 h. At the end of exposure, the tadpoles were anesthetized in 100 mg/L MS-222 buffered with 200 mg/L of sodium bicarbonate. The development stage and hind limb length were examined for each tadpole. Among 10 tadpoles per tank, three tadpoles representing the typical stage were fixed in Bouin’s solution for further observation and histological examination (nine tadpoles each treatment), another three tadpoles (small intestine) were fixed in 4% paraformaldehyde (PFA) for apoptosis analysis by TUNEL stain (nine tadpoles each treatment); with the remaining four tadpoles for gene expression analysis (12 tadpoles each treatment). Bouin’s solution-fixed tadpoles were observed under a stereoscopic microscope, and the intestines were sampled for histological examination. With respect to the four tadpoles for gene expression analysis in each tank, their intestines and hindlimbs were pooled separately into two samples and immersed in TRIzol reagent for RNA extraction, with six intestine or hindlimb samples for each treatment. The 6-day assay was repeated three times using tadpoles from different sets of adults, with two replicate tanks in the second and third experiments. Spontaneous Metamorphosis Assay. Based on some observations, we inferred that TBBPA could exhibit an inhibitory action on metamorphic development in the presence of high levels of TH, whereas it possibly has a promoting action in the presence of low levels of TH. To prove this hypothesis, we conducted a spontaneous metamorphosis assay using pre- and prometamorphic tadpoles and the tadpoles at metamorphic climax, respectively. The former have low levels of endogenous TH, whereas the latter have relatively high levels of endogenous TH.33,34 The tadpoles at stage 51 or stage 57 from a clutch were exposed to a series of concentrations of TBBPA (10, 100, 500, 1000 nM), as described above. Three replicate test tanks (10 tadpoles per tank) were employed for each treatment group. Followed by water changes, chemical replacements were performed every 24 h. In the assay for stage 51 tadpoles, the developmental stage, snout−vent length (SVL), and hindlimb length (HLL) of each tadpole were examined on days 7, 14, and 21 after the beginning of exposure. Hindlimb length was normalized by snout−vent length. In the assay for stage 57

induced by TH to metamorphose precociously. Therefore, amphibian metamorphosis serves as an ideal model to study THdependent development and the mechanisms for TH actions in vertebrates.19,21 With increasing concern about thyroid disruptors, amphibian metamorphosis has been used to reveal TH signaling disrupting activity of chemicals in recent years.22,23 Heimeier et al.22 used TH-induced intestinal remodeling in Xenopus laevis, an amphibian model species, to study TH signaling disruption and found that BPA inhibited vertebrate development by disrupting TH signaling. Two research groups reported independently that 1 μM TBBPA suppressed THinduced tail shortening and a decrease in the head area of tadpoles, suggesting that high concentrations of TBBPA inhibited metamorphic development via the TH signaling pathway.13,24 However, another study reported that TBBPA inhibited TH-induced expression of TH-response genes in X. laevis but promoted TH-response gene expression in the absence of TH induction, suggesting that TBBPA could act on the TH signaling pathway as either an antagonist or agonist at different conditions.25 The limited studies showed that effects of TBBPA on TH-dependent development might be complicated. Thus, it is necessary to further clarify this issue to improve the understanding of the effects of TBBPA on vertebrate development via the TH signaling pathway. In this study, we investigated the effects of 10−1000 nM TBBPA on T3-induced and spontaneous metamorphosis in X. laevis. Our results conclusively demonstrate that TBBPA, even at low concentrations, disrupts TH-dependent development in a developmental stage-dependent manner, i.e., TBBPA exhibits TH antagonistic actions on metamorphic development when animals have high endogenous TH levels, whereas it acts as a TH agonist when animals have low endogenous TH levels. Given the similarities between amphibian metamorphosis and higher vertebrates development, our findings from X. laevis highlight the potential adverse influences of TBBPA on the development via the TH signaling pathway in higher vertebrates, including humans.

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MATERIALS AND METHODS Chemicals. T3 and TBBPA were purchased from Geel Belgium (New Jersey, USA). Dimethyl sulfoxide (DMSO) and 3-aminobenzoic acid ethyl ester (MS-222) were from SigmaAldrich (St. Louis, MO, USA). Stock solutions of TBBPA (54.4 mg/mL) and T3 (6.60 mg/mL) were prepared by dissolving in DMSO and distilled water, respectively, and then were stored at −20 °C. TRIzol, RNase-free water, Quantscript RT Kit, RealMasterMix (SYBR Green) Kit, and ethidium bromide (EB) were obtained from Tiangen (Beijing, China). PCR primers were synthesized by Sangon Biotech (Beijing, China). Tissue-Tek OCT compounds were purchased from Sakura Finetek Inc. (Torrance, USA). Poly-L-lysine solution was obtained from ZSGB-BIO (Beijing, China). DeadEnd Fluorometric TUNEL System was obtained from Promega (Madison, USA). 4′,6-Diamidino-2-phenylindole (DAPI) was from Roche (Basel, Switzerland). Animals. X. laevis frogs (Nasco, Fort Atkins, WI, USA) and tadpoles were maintained in glass tanks containing charcoalfiltered tap water. Housing and breeding conditions were reported in our previous study.26 The tadpoles were fed with live Artemia three times every day. The tadpoles were staged according to the Nieuwkoop and Faber system.27 Short-Term T3-Induced Metamorphosis Assay. To determine the effects of TBBPA on TH-dependent development, 8228

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tadpoles, the developmental stage of each tadpole was determined on day 14 after the beginning of exposure, and the time to complete metamorphosis was recorded for each tadpole. The spontaneous metamorphosis assay was repeated twice using the tadpoles from different sets of adults. All animal procedures were conducted according to Regulations for the Administration of Affairs Concerning Experimental Animals.35 RNA Extraction and Quantitative RT-PCR. RNA extraction and gene expression analysis were conducted as described in the Supporting Information (SI “Methods” and Table S1). Histological Examination. Histological examination was conducted as described in the Supporting Information (see “Methods” for details). TUNEL Assay. Apoptotic activity was analyzed by TUNEL assay as described in the Supporting Information (see “Methods” for details). Data Analysis. Data were analyzed by two-way or one-way analysis of variance (ANOVA) or Chi-square test, as described in detail in the Supporting Information “Methods”.

plus TBBPA treatment groups, T3-induced metamorphic features were less pronounced with increasing concentrations of TBBPA compared with T3 treatment alone, showing that TBBPA significantly inhibited T3-induced metamorphosis in a concentration-dependent manner (Figure 1). In the 1000 nM TBBPA plus T3 treatment group, the morphological features of the tadpoles were comparable to the control, except for more developed hindlimbs relative to the control (Figure 1 and SI Table S2). Furthermore, 10 nM TBBPA significantly inhibited T3-induced gill resorption and forelimb emergence. Further, we examined intestinal remodeling at the morphological and histological levels in X. laevis. The intestine of the control tadpole was thin and long, with a deep typhlosole. Histological structure of the intestine was characterized by a thin muscle layer around the exterior, a thin layer of the connective tissue, and a simple inner epithelium (Figure 2A). The typhlosole contained a majority of connective tissue. No morphological and histological changes were observed in the TBBPA-treated tadpoles compared with the controls. Expectedly, T3 resulted in intestinal shortening and thinning, with the disappearance of the typhlosole. Corresponding to morphological changes, T3-

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RESULTS Effects of TBBPA in the Short Term T3-Induced Metamorphosis Assay. TBBPA Inhibits T3-Induced Metamorphosis and TH-Response Gene Expression. To investigate the effects of TBBPA on vertebrate postembryonic development via TH signaling disruption, we conducted a T3-induced metamorphosis assay using X. laevis tadpoles at stage 52. Following 6-day exposure to 1 nM T3, X. laevis tadpoles exhibited remarkable morphological changes, including forelimb protrusion and growth, hindlimb growth, gill resorption, lower jaw protrusion, and abdomen shrinkage (Figure 1). In the T3

Figure 2. Intestine remodeling of stage 52 Xenopus laevis tadpoles following 6-day exposure to tetrabromobisphenol A (TBBPA) in the absence or presence of 1 nM T3. A. Intestinal morphology. Groups of 30 tadpoles were used for each treatment: nine tadpoles representing typical stages in each treatment group were immersed in Bouin’s solution; intestines were removed, photographed, and sampled for histological examination. Ty, typhlosole; CT, connective tissue; M, muscles; Ep, epithelium; Lu, lumen. B. Apoptotic activity assayed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. Intestines from another nine tadpoles were fixed and sectioned at 6 μm thickness. 4′,6-Diamidino-2phenylindole (DAPI) was used for nuclear staining. Red arrow shows positive TUNEL straining. TBBPA alone had little effect on intestine remodeling. In the presence of T3, TBBPA significantly inhibited intestine remodeling compared with T3 treatment in a concentrationdependent manner. The experiment was repeated three times using tadpoles from different sets of adults with similar results, and we show the results from one independent experiment.

Figure 1. Morphological changes of stage 52 Xenopus laevis tadpoles following 6-day exposure to tetrabromobisphenol A (TBBPA) in the absence or presence of 1 nM T3. Groups of 30 tadpoles were used for each treatment: nine tadpoles representing typical stages in each treatment group were immersed in Bouin’s solution and then photographed. Treatment with 500 and 1000 nM TBBPA alone promoted stage development compared with the controls. In the presence of T3, TBBPA significantly inhibited metamorphosis compared with T3 treatment in a concentration-dependent manner. The experiment was repeated three times using tadpoles from different sets of adults with similar results, and we show the result from one independent experiment. LJ, lower jaw; G, gill; FL, forelimb; HL, hindlimb; A, abdomen. 8229

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Figure 3. Relative expression of five thyroid hormone-response genes in the intestines (A) and hindlimbs (B) of stage 52 Xenopus laevis tadpoles following 6-day exposure to tetrabromobisphenol A (TBBPA) in the absence or presence of 1 nM T3. Ribosomal protein L8 (rpl8) is used as a reference gene. Data are shown as mean ± SEM. # and * indicate significant differences between TBBPA treatment and controls and between TBBPA+T3 treatment and T3 treatment, respectively (p < 0.05).The experiment was repeated three times using tadpoles from different sets of adults. The results were consistent among the three independent experiments. Due to different responses of different clutches of tadpoles to T3, we show the data from a clutch rather than combining the three clutches, intestines from 12 tadpoles in each treatment were sampled to six samples for gene expression analysis. TRβ, thyroid hormone receptor beta; BTEB, basic transcription element binding protein; ST3, stromelysin-3; MMP2, matrix metalloproteinase 2; DIO2, type 2 deiodinase. A, Intestine; B, Hindlimb.

induced histological changes of the intestine were observed, including increased thickness of muscle and connective tissue layers, and the appearance and increase of epithelial folds. Similar morphological changes in the intestine were observed in the two lower TBBPA concentrations plus T3 treatment. However, in the 500 nM TBBPA plus T3 treatment groups, the typhlosole just became shallow but did not disappear, and the diameter of the intestine was comparable to that of the control. Furthermore, the intestines following 1000 nM TBBPA plus T3 treatment even exhibited similar morphology with the control intestines, with obvious typhlosole. Histological examination revealed that the intestines in the 10−100 nM TBBPA plus T3 treatment groups had less epithelial folds than T3 treatment, whereas no epithelial folds occurred in the intestines of the 500−1000 nM TBBPA plus T3 treatment groups. Moreover, muscle and connective tissue in the intestines hardly increased in the tadpoles following 500− 1000 nM TBBPA plus T3 treatment compared with the controls. In the 1000 nM TBBPA plus T3 treatment group, the intestines had similar histological features with the controls. TUNEL staining further validated the effects of TBBPA on intestinal remodeling in X. laevis. Apoptosis was hardly observed in the intestines of the TBBPA alone-treated tadpoles and control tadpoles (Figure 2B), as described for in stage 54 X. tropicalis in a previous study.36 However, T3 induced strong apoptosis in the intestine, with significantly higher values for apoptotic nucleus number, TUNEL/DAPI, and IOD compared with the controls (SI Table S3). T3-induced apoptosis in the intestine was in accord with apoptosis described at metamorphic climax by Heimeier.37 In the presence of T3, TBBPA resulted in decreases in the values for apoptotic nucleus number, TUNEL/ DAPI, and IOD in a concentration-dependent manner. Additionally, apoptosis signal was hardly observed in the 500−1000 nM TBBPA plus T3 treatment groups, similar to the control intestines. The above results concerning intestinal remodeling show that TBBPA in the range of 10−1000 nM inhibits TH-induced metamorphosis in a concentration-dependent manner. To

further determine whether the inhibitory effects of TBBPA on TH-induced metamorphosis were mediated by the TH signaling pathway, we examined mRNA expression of five TH-response genes (TRβ, BTEB, ST3, MMP2, and DIO2) in intestines and hindlimbs. TBBPA had no effect on rpl8 expression in the intestines and hindlimbs of the tadpoles following 6-day treatment (data not shown). Therefore, rpl8 was used as a reference gene to normalize mRNA expression of TH-response genes. In general, a chemical will be believed to have TH agonistic activity if it promotes TH-response gene expression. On the contrary, it will be suggested to have TH antagonistic activity if it inhibits spontaneous or TH-induced expression of TH-response genes. As expected, T3 significantly up-regulated expression levels of all the tested genes (Figure 3A, B). In the range of 100−1000 nM, TBBPA inhibited T3-induced upregulation of TRβ, BTEB, DIO2, and MMP2 expression in intestines compared with T3 treatment in a concentrationdependent manner. The observations at the transcriptional level confirmed the results that TBBPA inhibited T3-induced metamorphosis at the morphological, histological, and apoptotic levels (Figure 3A, B). Interestingly, lower concentrations of TBBPA promoted T3-induced ST3 expression, but two higher concentrations had inhibitory effects on ST3 as well as other genes. In the hindlimbs, 10−1000 nM TBBPA also significantly inhibited T3-induced expression of TRβ, ST3, and DIO2 but did not affect MMP2 expression. Similar to the effect in the intestines, the lowest concentration of TBBPA had no effect on T3-induced BTEB expression in the hindlimbs. The results strongly show that the inhibitory effects of TBBPA on THinduced metamorphosis result from TH signaling disruption. High Concentrations of TBBPA Accelerate Tadpole Development in the Absence of T3 Induction. In the absence of T3 induction, high concentrations of TBBPA treatment appeared to accelerate tadpole development compared with the control in terms of the hindlimb length and morphology. With increasing TBBPA concentrations, the hindlimb length of the tadpoles with TBBPA treatment exhibited an increasing trend (p 8230

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= 0.079 for the 500 nM TBBPA group), although only the highest concentrations caused a significant difference from the controls (Figure 1 and SI Table S2). According to hindlimb morphology, the TBBPA-treated tadpoles reached later developmental stages compared with the control tadpoles, with significant differences between higher concentration groups and the control group (SI Figure S1 A, B, C). In accordance with the morphological changes, higher concentrations of TBBPA also promoted expression of some TH-response genes, such as ST3, BTEB, and DIO2, in intestines and hindlimbs (Figure 3). In particular, even 10 nM TBBPA exhibited a promoting action on ST3 expression in the hindlimbs. Overall, these results show that high concentrations of TBBPA promote premetamorphic development of the tadpoles via an agonistic effect on TH action, even during short-term treatment. Given the inhibitory effects of TBBPA on T3-induced metamorphosis and the promoting effects on premetamorphic development in the absence of T3 induction, we inferred that TBBPA could exhibit an inhibitory action on metamorphic development in the presence of high TH levels, whereas it might have a promoting action in the presence of low TH levels. Effects of TBBPA on Spontaneous Metamorphic Development. TBBPA Promotes Pre- and Prometamorphic Development. To prove our hypothesis that effects of TBBPA on TH-dependent metamorphosis could be associated with endogenous TH levels and to further reveal the effects of longterm exposure to TBBPA on metamorphic development, we conducted a spontaneous metamorphosis assay for pre- and prometamorphic tadpoles and the tadpoles at metamorphic climax, respectively. In the spontaneous metamorphosis assay for pre- and prometamorphic tadpoles, all tadpoles developed to stages 52−54 from stage 51 on day 7, but increased numbers of tadpoles at later stages were observed in the treatment groups compared with the controls, with significant differences observed in the 500−1000 nM TBBPA treatment groups (Figure 4). HLL and HLL/SVL also exhibited an increasing association with the TBBPA concentration compared with the control tadpoles, despite only significant difference between the highest concentration group and the controls. The data seem to show a promoting effect of TBBPA on pre- and prometamorphic development of the tadpoles. Following longer exposure, the promoting effect of TBBPA on pre- and prometamorphic development became more pronounced (Figure 4). On day 21, most of the tadpoles in the control group reached stages 54−55 with a few reaching stage 56, whereas in the TBBPA-treated group, stage 56 tadpoles became gradually dominant with the increase in TBBPA concentration. Significant differences in the developmental stage were found between the 100−1000 nM TBBPA treatment groups and the control group. Additionally, the values for HLL and HLL/SVL in the 500−1000 nM TBBPA groups were significantly larger than those of the control group. Taken together, the data from the spontaneous metamorphosis assay for stages 51−56 tadpoles demonstrate that TBBPA promotes the development of tadpoles when exposure starts at the premetamorphic stages. TBBPA Inhibits Development during Metamorphic Climax. We then conducted the spontaneous metamorphosis assay using X. laevis tadpoles at stage 57 to investigate the effects of TBBPA on development during metamorphic climax. The tadpoles at stage 57 were exposed to 10−1000 nM TBBPA. On day 14, the developmental stages of all tadpoles were examined. In the control group, stage 63 was the dominant stage, with several tadpoles at stages 62, 64, and 65. However, some tadpoles before

Figure 4. Percentage of tadpoles at different stages and growth of hind limb length of stage 51 Xenopus laevis tadpoles following 21-day exposure to 10−1000 nM tetrabromobisphenol A (TBBPA). Hind-limb length (HLL) is normalized by snout−vent length (SVL). Data are shown as mean ± SEM. * indicates significant differences between TBBPA treatment and controls (p < 0.05). The experiment was repeated three times using tadpoles from different sets of adults with similar results, and we show the data of 30 tadpoles in each treatment group from one independent experiment.

stage 62 were observed in the TBBPA-treatment groups, and their percentage in each group increased significantly with increased TBBPA concentrations (Figure 5A). The time from stage 57 to stage 66 also increased in a concentration-dependent manner, with mean values of 18.6, 19.8, 19.6, and 22.0 days for the 10, 100, 500, and 1000 nM TBBPA groups, respectively; the latter three of which were significantly longer than 17.6 days for the control (Figure 5B). These results show an inhibitory effect of TBBPA on development during metamorphic climax in a concentration-dependent manner. Collectively, the above findings show that TBBPA disrupts TH-dependent development in a developmental stage-dependent manner, i.e., TBBPA exhibits antagonistic effects on TH actions when animals have high levels of endogenous TH, whereas it acts as an agonist when animals have low levels of endogenous TH.

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DISCUSSION Previous in vitro and in vivo studies demonstrated that TBBPA disrupted the TH signaling pathway at the transcriptional level.13−18,38,39 However, how TBBPA affects TH-dependent vertebrate development at the morphological and functional level is poorly understood. Only three reports have studied this issue. Kitamura et al. reported that 1 μM TBBPA suppressed THinduced tail shortening of Rana rugosa tadpoles.13 Similarly, Fini et al.24 found that 1 μM TBBPA inhibited a TH-induced decrease in head area due to gill resorption in X. laevis. Jagnytsch et al. 8231

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As expected, we found that 100−1000 nM TBBPA in the spontaneous metamorphosis assay promoted X. laevis development from stage 51 to 56, whereas even 10 nM inhibited development from stage 57 to 66. It is well-known that endogenous T3 levels are low in pre- and prometamorphic tadpoles, whereas T3 levels increase dramatically since stage 58, peak at stages 60−62 (several nM in plasma), and decline at the end of metamorphosis.28,33,34 In the T3-induced metamorphosis assay, the exogenous T3 level (1 nM) we used are comparable to the endogenous levels during metamorphic climax.33 The inhibitory effect of TBBPA on spontaneous metamorphosis during metamorphic climax corresponds to the inhibitory effects on 1 nM T3-induced metamorphosis. Thus, the results from the spontaneous metamorphosis assay provide strong support that TBBPA effects on metamorphic development are dependent opon the endogenous TH levels in tadpoles. With differences from our results, Jagnytsch et al.25 reported that 21-day exposure to 2.5−250 μg/L (4.6−460 nM) TBBPA did not affect X. laevis metamorphosis, and only the highest concentration of 500 μg/L (920 nM) TBBPA resulted in a retardation of metamorphosis. This difference might be explained by differences in the tadpole growth rate. In the study by Jagnytsch et al., the tadpoles in the control group developed to stage 58 (±2) and entered metamorphic climax at the end of the 21-day assay, thus the promoting effect of TBBPA on pre- and prometamorphic development might be concealed by the inhibitory effect on development during metamorphic climax. In our spontaneous metamorphosis assay, about 90% tadpoles in the control group were maintained at stages 55−56 at the end of assay owing to a slower growth rate than that observed in the study by Jagnytsch et al. Therefore, we only observed the promoting effect of TBBPA on the development in pre- and prometamorphic tadpoles. Thus, we conclude whether TBBPA acts antagonistic or agonistic effects on TH-dependent development depends on the developmental stage, which is associated with endogenous TH levels in animals. In addition to TH signaling disruption, the effects of TBBPA on spontaneous metamorphosis might be also derived from changes in TH levels in tadpoles following TBBPA treatment because of the disruption on the hypothalamuspituitary-thyroid (HPT) axis. However, the study by Jagnytsch et al. reported a minimal effect of TBBPA on the HPT axis, thus, the effects of TBBPA on spontaneous metamorphosis are mediated mainly by TH signaling disruption. Additionally, our findings that TBBPA exhibited an antagonistic effect on TH actions in the presence of high TH levels, but an agonistic activity in the presence of low TH levels are supported by previous data at the transcriptional level. Jagnytsch et al. reported that short-term exposure to 100−500 μg/L TBBPA antagonized TH-induced TRβ and TH-responsive basic leucine zipper transcription factor (TH/bZIP) expression in X. laevis head tissues, whereas TBBPA alone induced expression of these TH-responsive genes,25 although another study (Hinther et al., 2010) reported no effect on TH-responsive gene transcription in a cultured tadpole tail fin biopsy owing to low responsivity of tail tissue to TH.40 In several in vitro reporter gene systems, TBBPA acted as an agonist on TH action in the absence of TH but as an antagonist in the presence of TH induction.15,16 The biphasic effects of TBBPA on TH actions remind us of NH3, a well-known TH antagonist, which efficiently inhibits THinduced morphological changes and TH-responsive genes in X. laevis tadpoles as well as having partial agonist activity in tadpoles and other animals or cells.28,41−44 An explanation for the biphasic effects of NH3 is its ability to induce some coactivator binding in

Figure 5. A. Percentage of tadpoles at different stages of stage 57 Xenopus laevis tadpoles following 14-day exposure to 10−1000 nM tetrabromobisphenol A (TBBPA) B. Time of each tadpole from stage NF57 to 66 in different treatment groups of stage 57 X. laevis tadpoles following exposure to 10−1000 nM TBBPA. Median, mean (●) and 10, 25, 75, and 95 percentile values are diagrammed. Data are shown as mean ± SEM. *indicates significant differences between TBBPA treatment and controls (p < 0.05). The experiment was repeated three times using tadpoles from different sets of adults with similar results, and we show the data of 30 tadpoles in each treatment from one independent experiment.

reported that 500 μg/L (920 nM) TBBPA inhibited spontaneous metamorphosis of X. laevis in a 21-day metamorphosis assay.25 These studies indicated that high concentrations of TBBPA inhibited TH-dependent metamorphic development. In the present study, we found that TBBPA in the range of 10−1000 nM exhibited obvious inhibitory effects on T3-induced X. laevis metamorphosis in terms of multiple morphological changes, including forelimb protrusion and growth, hindlimb growth, head decrease due to gill resorption, lower jaw protrusion, and abdomen shrinkage due to intestinal remodeling, and was in concentration-dependent manner (Figure 1, 2). Moreover, antagonistic effects of TBBPA on T3 actions were further supported by expression of TH-response genes, including TRβ, BTEB, ST3, DIO2, and MMP2, in the intestine and hindlimb (Figure 3). Overall, these results demonstrate TBBPA antagonized TH-induced development at the gene transcriptional level but also at the morphological level. Additionally, we observed the promoting effects of TBBPA treatment alone on hindlimb development and TH-response gene expression in the 6-day metamorphosis assay. This phenomenon has not been previously reported. Based on the findings from the 6-day T3induced X. laevis metamorphosis assay, we inferred that TBBPA could agonize TH actions and promote metamorphic development when tadpoles have low levels of endogenous TH, whereas it might antagonize TH actions and inhibit metamorphic development when tadpoles have high levels of endogenous TH. Thus, the effects of TBBPA on metamorphic development might depend on the endogenous TH levels in tadpoles. 8232

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addition to release of the corepressor NCoR for TR, resulting in TH agonism.42 Thus, we speculate that TBBPA alone could behave as a weaker TH agonist than T3. When numerous TBBPA molecules coexist with T3, however, TBBPA competes with T3 for TR and results in an inhibitory effect on TH actions. The biphasic effects of TBBPA on TH-dependent development are very interesting and warrant further study. Heimeier et al. (2009) reported that 100 nM and 10 μM BPA, the precursor for TBBPA, inhibited TH-induced morphological changes (in particular intestinal remodeling) and TH-response gene expression (such as TRβ, ST3, TH/bZIP, MMP2) in X. laevis.22 Their results are consistent with our findings of TBBPA in X. laevis in the present study. Whether BPA at lower concentrations than 100 nM acts as a TH antagonist on metamorphic development was not studied by Heimeier et al.22 In our study, 10 nM TBBPA exhibited a significant inhibitory effect on TH-induced metamorphosis in X. laevis. Several in vitro studies showed that TBBPA had higher TH antagonistic activity and binding capacity to TR than BPA.13,18 Therefore, TBBPA might have comparable or more powerful effects than BPA on TH-dependent vertebrate development. In particular, the lowest effective concentration of TBBPA (10 nM, 5.4 μg/L) in our study is comparable to the higher range of environmental concentrations, which generally range from pg/L to several μg/L levels in surface water.45−47 Therefore, it is necessary to investigate further the effects of TBBPA at lower and more environmentally relevant concentrations. Given that TH signaling is conserved across all vertebrate species, in particular, there are many similarities between amphibian metamorphosis and development in higher vertebrates,19,20 our findings from X. laevis highlight potential adverse influences of TBBPA on THdependent development in higher vertebrates, including humans.48−53

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ASSOCIATED CONTENT

S Supporting Information *

Pages S3−S4: details of these methods: RNA extraction and quantitative RT-PCR, histological examination, TUNEL assay, and data analysis. Page S5: Figure S1. Pages S6−S8: Table S1, Table S2, Table S3. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Phone: 86-10-62919177. Fax: 86-10-62923563. E-mail: [email protected]. *Phone: 86-10-62849790. Fax: 86-10-62849358. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from Hi-Tech Research and Development Program of China (2012AA06A302), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB14040102, YSW2013A01), and National Natural Science Foundation of China (21377153).

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