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Oct 30, 2012 - (18) The hypothalamus–pituitary–thyroid (HPT) axis, also known as thyroid homeostasis or thyrotropic feedback control, is part of the e...
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Accumulation and Biotransformation of BDE-47 by Zebrafish Larvae and Teratogenicity and Expression of Genes along the Hypothalamus−Pituitary−Thyroid Axis Xinmei Zheng,† Yuting Zhu,† Chunsheng Liu,† Hongling Liu,*,† John P. Giesy,†,‡,§ Markus Hecker,⊥ Michael H. W. Lam,§ and Hongxia Yu*,† †

State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210023, China ‡ Department of Veterinary Biomedical Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada § Department of Biology and Chemistry and State Key Laboratory for Marine Pollution, City University of Hong Kong, Kowloon, Hong Kong, SAR, China ⊥ School of Environment and Sustainability and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S Supporting Information *

ABSTRACT: Accumulation and effects of BDE-47 and two analogues, 6-OH-BDE47 and 6-MeO-BDE-47, on ontogeny and profiles of transcription of genes along the hypothalamus−pituitary−thyroid (HPT) axis of zebrafish (Danio rerio) embryos exposed from 4 h post fertilization (hpf) to 120 hpf were investigated. The 96 h-LC50 of the most toxic compound, based on teratogenicity, was 330 μg of 6-OH-BDE-47/ L. 6-OH-BDE-47 significantly down-regulated expression of mRNA of thyroid stimulating hormone receptor (TSHR), thyroid hormone receptors (TRs, including TRα and TRβ), sodium/iodide symporter (NIS), and transthyretin (TTR) while upregulating expression of thyroglobulin (TG) and thyrotropin-releasing hormone (TRH). Spontaneous movement was affected by 1 mg of 6-OH-BDE-47/L or 5 mg of 6-MeO-BDE-47/L. BDE-47 did not alter activity of larvae at any concentration tested. 6-MeO-BDE-47 significantly up-regulated expression of mRNA of TRH, TRα, TRβ and NIS. Both 6-OH-BDE-47 and 6-MeO-BDE-47 affected the thyroid hormone pathway. BDE-47 and 6-MeO-BDE-47 were accumulated more than 6-OH-BDE-47. 6-MeO-BDE-47 was transformed into 6-OH-BDE-47, but BDE-47 was not transformed into it. In summary, the synthetic brominated flame retardant, BDE-47, did not elicit the adverse effects caused by the other two analogues and appeared to have less toxicological relevance than the two natural product analogues 6-OH- and 6-MeO-BDE-47.

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dietary intake of fish was positively correlated with concentrations of PBDEs in human milk. BDE-47 was the predominant congener observed among fishes, with a maximum concentration of 1000 pg/g wet weight (dw).5 Two structural analogues of BDE-47, 6-OH-BDE-47 and 6-MeO-BDE-47, were also detected in aquatic biota such as blood and tissues of fish and marine mammals and algae and have been shown to be accumulated through the food chain into top predators.6−10 6-OH-BDE-47 and 6-MeO-BDE-47 were originally thought to be biotransformation products or byproducts during synthesis of PBDEs.11 However, recent studies have confirmed that 6-OH-BDE-47 and 6-MeO-BDE-47 are likely naturally occurring compounds, with interconversion between 6-MeO-

INTRODUCTION

As one of the predominant polybrominated diphenyl ethers (PBDEs), 2,2′,4,4′-tetrabromodiphenylether (BDE-47) is a contaminant of concern due to its ubiquity and potential to bioaccumulate, especially in aquatic systems.1,2 Concentrations of BDE-47 in water and sediments in south China were 21 pg/ L and greater than 100 ng/g dw, respectively.3 BDE-47 was detected in electrical and electronic equipment waste (e-waste) recycling sites at concentrations of 0.83 ng/L in the dissolved phase and 4.3 ng/L in the particulate phase. 4 Mean concentrations of BDE-47 in the Little River sewage treatment plant (LRSTP) in Windsor, Ontario, Canada were 102 ± 83 ng/L in the influent to the primary sedimentation tanks, 36 ± 29 ng/L in primary sedimentation tank effluent, 14 ± 4 ng/L in final effluent, 586 ± 207 ng/g dry weight (dw) in primary sludge, and 963 ± 415 ng/g dw in waste activated sludge. Concentrations of BDEs in fish, vegetables, meat, and human milk in Japan were determined, and it was concluded that © 2012 American Chemical Society

Received: Revised: Accepted: Published: 12943

August 13, 2012 October 25, 2012 October 30, 2012 October 30, 2012 dx.doi.org/10.1021/es303289n | Environ. Sci. Technol. 2012, 46, 12943−12951

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MA, USA). MeO-PBDEs and OH-PBDEs (12 MeO-PBDEs: 6MeO-BDE-47, 6′-MeO-BDE-17, 2′-MeO-BDE-28, 5-MeOBDE-47, 4′-MeO-BDE-49, 2′-MeO-BDE-68, 6-MeO-BDE-85, 6-MeO-BDE-90, 4-MeO-BDE-90, 3-MeO-BDE-100, 6′-MeOBDE-123, 6-MeO-BDE-137; 12 OH-PBDEs: 6-OH-BDE-47, 2′-OH-BDE-7, 3′-OH-BDE-7, 2′-OH-BDE-17, 2′-OH-BDE-25, 2-OH-BDE-28, 4′-OH-BDE-49, 2′-OH-BDE-66, 2′-OH-BDE68, 6-OH-BDE-85, 6-OH-BDE-90, 6-OH-BDE-137) were synthesized in the Department of Biology and Chemistry at City University of Hong Kong, and purities of greater than 98% have been confrmed.30 Stock solutions of chemicals (6-OHBDE-47, BDE-47, and 6-MeO-BDE-47) were prepared in dimethyl sulfoxide (DMSO, Generay Biotech, Shanghai, China) and diluted with embryonic rearing water (60 mg/L instant ocean salt in aerated distilled water) to the final concentrations immediately before use. The final concentration of solvent (DMSO) in the test solution did not exceed 0.1%. RNAlater RNA Stabilization Reagents, RNeasy Mini Kit, and Omniscript RT Kit were purchased from Qiagen (Hilden, Germany). SYBR Real time PCR Master Mix Plus Kits were obtained from Toyobo (Tokyo, Japan). Maintenance of Zebrafish and Exposure. Adult zebrafish (4-month old) were obtained from the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China), and maintained in a semiautomatic system with treated tap water (no residual ammonia, chlorine, chloramines, and disinfected with UV light) under 14/10 h light/dark photoperiod. Fish were fed frozen blood worms or dry food twice a day. Nylon nets were used at the bottom of each tank to isolate eggs and adult fish. Spawning was induced in the morning when the light was turned on. Collected embryos were rinsed with embryonic rearing water and examined under an inverted stereomicroscope. The majority of embryos developed normally at the early cleavage stage with cytoplasm streams toward animal pole to form the blastodisc as determined by means of a stereomicroscope at magnification of 50×. 6-well cell culture plates (Corning Inc. Steuben, New York, USA) were used in the experiments. Twenty normally shaped fertilized embryos were randomly assigned to each well including 10 mL of exposure or vehicle control (DMSO) solution and a medium control each with three replicates at 4 h post fertilization (hpf). The experiment was terminated at 120 hpf. In order to directly compare potencies of the three test chemicals (6-OH-BDE-47, BDE-47, and 6-MeO-BDE-47), fertilized embryos were exposed to the same nominal concentrations of each compound (0, 8, 40, 200, 1000, or 5000 μg/L). Wells were covered with foil to avoid evaporation of test solutions. Development of the embryos was not affected by the covering of foil on the wells during the exposure since mortality of embryos was less than 20%. Embryos were examined under a multipurpose zoom microscope (Nikon AZ 100) at different developmental stages (4, 8, 12, 24, 48, 72, 96, and 120 hpf). Coagulated embryos before hatching are opaque, milky white, and appear dark under the microscope. Toxicological endpoints included whether embryos were clear or opaque, edema at 48, 72, or 96 hpf, structural malformations at 72 or 96 hpf, and body lengths measured after hatching until 120 hpf. Malformations of the crooked spine were defined as scoliosis and curvature of the tail. Mortalities included coagulated embryos before hatching and dead larvae after hatching until 120 hpf. Each exposure experiment was replicated three times. The mRNA expression studies were conducted under the same conditions described above with

BDE47 and 6-OH-BDE47 observed in Japanese medaka (Oryzias latipes).12,13 Several studies of effects of BDE-47 on aquatic organisms have been conducted. Inflated swim bladder and dorsal curvature of zebrafish (Danio rerio) larva were observed when zebrafish embryos were exposed to 25 mg BDE-47/L.14 25−50 nM (12−24 μg/L) of 6-OH-BDE-47 caused a range of developmental defects such as pericardial edema, yolk sac deformations, lesser pigmentation, lessened heart rate, and delayed development.15 PBDEs, OH-PBDEs, and MeO-PBDEs are structural analogues to chlorinated biphenyls (PCBs), dioxins (PCDD), and furans (PCDF), some of which have structure similarities to thyroid hormones (THs). However, PBDEs are larger and contain a flexible ether linkage such that they are not agonists of the aryl hydrocarbon receptor (AhR).16 Therefore, PBDEs were hypothesized to potentially affect thyroid hormone homeostasis. Concentrations of BDE-47 were negatively correlated with circulating concentrations of free T4 (FT4) in blood plasma of white whales (Delphinapterus leucas) from Svalbard.17 BDE-47 has been reported to alter thyroid status and thyroid hormone-regulated gene transcription in pituitary and brain of adult fathead minnows. 18 The hypothalamus−pituitary−thyroid (HPT) axis, also known as thyroid homeostasis or thyrotropic feedback control, is part of the endocrine system that is responsible for regulation of metabolism and early life-stage development.19,20 In addition to the gonads, the HPT axis is the target of endocrine-disrupting chemicals (EDCs).21 Altered function of the HPT axis is associated with endocrine and developmental effects.22 Regulation of expression of genes was found to be affected in embryos exposed to 0.625 ppm 6-OH-BDE 47 from 24 to 28 h post fertilization (hpf) due to potential disruption of the cholinergic system and thyroid hormone homeostasis.23 Mostly, in vitro studies have shown that PBDEs can have potential endocrine disrupting properties, a number of which can be attributed to the hydroxylated metabolites.24 OHPBDEs can bind competitively to transthyretin (TTR), the thyroid hormone transport protein, and also can cause estrogenic effects through interaction with the estrogen receptor.25,26 However, to our knowledge, there has been no published information on effects of 6-MeO-BDE-47 on thyroid function in fishes. Comparison of the developmental toxicity of these three compounds in fish is also limited. Zebrafish embryos/larvae previously have been shown to be a good model to study effects of chemicals on the HPT axis, and polymerase chain reaction (PCR) methods have been used to measure expression of mRNA of genes in the thyroid system.27−29 In the study upon which we report here, an HPTPCR array for zebrafish larvae was used to investigate effects of 6-OH-BDE-47, BDE-47, and 6-MeO-BDE-47 on the HPT axis and on pathways of thyroid synthesis and to compare these effects with developmental and behavioral effects. Furthermore, accumulation and biotransformation of 6-OH-BDE-47, BDE47, and 6-MeO-BDE-47 by zebrafish larvae were assessed by liquid−liquid extraction coupled with GC/MS.

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MATERIALS AND METHODS Chemicals. BDE-47 (98% purity) was purchased from Chem Service, Inc. (West Chester, PA, USA). All other PBDEs (11 PBDEs: BDE-17, BDE-28, BDE-71, BDE-66, BDE-100, BDE-99, BDE-85, BDE-154, BDE-138, BDE-183, BDE-190), 13 C-PCB-178, 13C-2′-OH-BDE-99, and 13C-BDE-139 were purchased from Cambridge Isotope Laboratories (Andover, 12944

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Figure 1. Photomicrographs demonstrating changes in morphology at several stages of development after zebrafish embryos were exposed to 6-OHBDE-47 and 6-MeO-BDE-47. (A) Normal developed embryo (36 hpf). (B) Delayed development embryo exposed to 1000 μg of 6-OH-BDE-47/L (36 hpf). (C) Normal hatched larva (72 hpf). (D) Abnormal embryo exposed to 1000 μg of 6-OH-BDE-47/L (72 hpf). (E) Normal hatched larva (72 hpf). (F) Abnormal larva with pericardial edema after exposure to 5000 μg of 6-MeO-BDE-47/L (72 hpf). (G) Normal hatched larva (96 hpf). (H) Abnormal larva with edema and malformed spine after exposure to 5000 μg of 6-MeO-BDE-47/L (96 hpf).

exposure concentrations of 0, 8, 40, or 200 μg/L for each of the three chemicals. Duration of exposure was chosen on the basis of the fact that most genes expressed along the HPT axis can be detected before 96 hpf and that thyroid follicles continue to grow until 120 hpf in zebrafish.31,32 At 120 hpf, larvae were randomly sampled and stored in RNAlater solution at −20 °C for subsequent gene assays. A subset of larvae was analyzed for bioaccumulation of BDEs and presence of metabolites. Isolation of RNA, Reverse Transcription, and Quantitative Real-Time Polymerase Chain Reaction (RT-PCR). Effects of 6-OH-BDE-47, BDE-47, and 6-MeO-BDE-47 on relative transcription of RNA of key genes involved in HPT axis were determined by RT-PCR as described previously.33 Whole bodies of larvae were used as the samples because they were only 3−4 mm long. Isolation of total RNA was performed using the RNeasy Mini Kit (Hilden, Germany), and quantification and verification were performed by use of a previously reported protocol.33 The Omniscript RT Kit was used to synthesize cDNA following the manufacturers’ instructions. Quantitative real-time PCR was performed using the SYBR Green PCR kit under Applied Biosystems Stepone Plus Real-time PCR System (Applied biosystems inc. Foster city, CA, USA). The online Primer 3 program (http://frodo.wi.mit.edu) was used to design the primer sequences for the selected genes (Table S1 of Supporting Information). Conditions of the RT-PCR reaction were as follows: initial denaturation step at 95 °C for 2 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Melt curves were derived during RT-PCR to validate that all cDNA samples amplified only a single product. The expression of mRNA for each target gene was normalized to the mRNA content of housekeeping gene (rpl8), and the change in the mRNA expression of the relevant genes was analyzed by the 2−ΔΔCt method.29 Each concentration was measured in triplicate in a composite sample containing 15 larvae. Instrumental Analysis. Zebrafish larvae were collected at 120 hpf, rinsed with Milli-Q water, and gently dried. Larvae from each exposure were composited, weighed, and stored separately at −80 °C until analysis. Detailed protocols for

extraction, clean up, identification, and quantification and quality assurance and quality control (QA/QC) are provided in Wen et al.34 Internal dose and potential products of transformation of the three chemicals were determined as follows: Briefly, concentrations of individual OH-PBDEs, MeOPBDEs, and PBDEs were quantified by liquid−liquid extraction combined with gas chromatography and mass spectrometry (GC/MS). Approximately 0.1 g of larvae sample was homogenized and transferred into amber serum bottles. The sample was then spiked with the surrogate recovery standard, and 2 mL of Milli-Q water, 50 μL of hydrochloric acid (HCl, 2 M), and 2 mL of 2-propanol were added. The thus prepared sample was then extracted three times with 10 mL of n-hexane/ methyl tert-butyl ether (MTBE) (1:1, v/v). Extracts were concentrated by rotary evaporation and dried under nitrogen. The derivation of PBDEs, MeO-PBDEs, and OH-PBDEs and their purification were conducted in accordance with the method of Wen et al.34 Concentrations of 12 PBDEs, 12 OHPBDEs, and 12 MeO-PBDEs were determined by use of a TSQ Quantum GC/MS (Thermo Scientific, USA) coupled with an Agilent DB-XLB column (15 m × 0.25 mm × 0.25 μm, J&W Scientific, USA) in 3 separate runs. Identification of specific PBDEs, OH-PBDEs, and MeO-PBDEs was performed by comparing relative retention times versus internal standard and product ions in SRM mode with the standard chemicals. QA/QC and Statistical Analysis. Quality assurance and quality control were performed by regular analysis of procedural blanks (RSD < 22.6% for 6 replicates). Rates of recovery for standard compounds ranged between 93−146%, 67−113%, and 103−133% for PBDEs, OH-PBDEs, and MeOPBDEs, respectively. The limit of detection (LOD) for each compound was defined as three times the SD of the laboratory blanks. For congeners not detected in the blanks, the LOD was set to the instrumental limit of quantification (LOQ). Method LODs ranged between 9.2 × 10−2 and 2.9 × 101 ng/g wet weight (ww) for individual PBDEs, OH-PBDEs, and MeOPBDEs congeners. Concentrations less than the method LOD were assumed to be not detectable. PBDEs, OH-PBDEs, and 12945

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hormone (TRH) was significantly greater (1.77-fold) in larvae exposed to 40 μg of 6-OH-BDE-47/L and up-regulated by 1.60- and 1.72-fold in larvae exposed to 40 or 200 μg of 6MeO-BDE-47/L, respectively, while no significant alterations were observed in larvae exposed to BDE-47 (Figure 2A). Transcription of thyrotropin-releasing hormone 1 (TRHR1) and thyroid stimulating hormone β (TSHβ) genes was not affected by any of the three chemicals at the selected concentrations (Figure 2B,C). Expression of mRNA of thyroid hormone receptors (TRs, including TRα and TRβ) showed contrary effects in larvae exposed to 6-OH-BDE-47 and 6MeO-BDE-47 (Figure 2D,E). Exposure of 200 μg of 6-OHBDE-47/L larvae resulted in lesser expression of TRα and TRβ mRNA by 2.24- and 3.14-fold, respectively, while 6-MeO-BDE47 resulted in up-regulation of expression of TRα and TRβ mRNA by 1.62- and 2.01-fold, respectively. mRNA expression of TRα and TRβ after exposure to BDE-47 decreased slightly but the difference was not statistically significant. Transcriptional profiles of Na+/I− symporter (NIS) and transthyretin (TTR) genes were both significantly affected by exposure to 6-OH-BDE-47 and 6-MeO-BDE-47 (Figure 3). The effects of 6-OH-BDE-47 and 6-MeO-BDE-47 on the expression of the NIS genes were concentration dependent (Figure 3A). Expression of the NIS gene was down-regulated by 1.43- and 3.33-fold in larvae exposed to 40 or 200 μg of 6OH-BDE-47/L, respectively. However, expression of the NIS gene was 3.05-fold greater in larvae exposed to 200 μg of 6MeO-BDE-47/L. 6-OH-BDE-47 significantly affected expression of the TTR gene (Figure 3B). Exposure to 40 or 200 μg of 6-OH-BDE-47/L resulted in 1.48- and 1.50-fold downregulation of mRNA of TTR, respectively, while 6-MeOBDE-47 did not significantly affect expression of TTR. No significant effect on expression of mRNA of either the NIS or the TTR gene was observed after exposure to BDE-47. Transcription of the thyroid stimulating hormone receptor (TSHR) gene was significantly down-regulated by 7.88-fold in larvae exposed to 200 μg of 6-OH-BDE-47/L. However, there was no significant effect of 6-MeO-BDE-47 or BDE-47 (Figure 4A). Expression of thyroglobulin (TG) mRNA was significantly affected by only 6-OH-BDE-47 (Figure 4B), expression of which was up-regulated by 1.52- and 1.71-fold in larvae exposed to 8 or 40 μg/L, respectively. Transcriptions of Dio1 and Dio2 were not significantly affected by any of the three chemicals (Figure 4C,D). Accumulation and Biotransformation. A concentrationdependent bioconcentration of 6-OH-BDE-47, BDE-47, and 6MeO-BDE-47 was observed in larvae after 5 days of exposure (Figure 5). Larvae exposed to 200 μg of 6-MeO-BDE-47/L, accumulated 117 ng of 6-OH-BDE-47/g ww (Figure 5). There were also small percentages of other isomers detected in zebrafish exposed to each compound. These included 2’-OHBDE-28, BDE-28, BDE-85 and BDE-99 in zebrafish larvae exposed to BDE-47; 2’-OH-BDE-28, 6-OH-BDE-85, 6-MeOBDE-47 and BDE-47 observed in zebrafish larvae exposed to 6OH-BDE-47; and 2’-OH-BDE-28, 2’-MeO-BDE-28 and BDE47 observed in zebrafish larvae exposed to 6-MeO-BDE-47. The percentages were all very small and due exclusively to impurities in test compounds. In larvae exposed to 200 μg of 6MeO-BDE-47/L, 117 ng of 6-OH-BDE-47/g ww were observed (Figure 5). No 6-OH-BDE-47 or 6-MeO-BDE-47 was observed in zebrafish exposed to BDE-47, and no 6-MeOBDE-47 occurred in individuals exposed to 6-OH-BDE-47. In

MeO-PBDEs were all quantified in samples extracts relative to 13 C-PCB-178. Recoveries of surrogate standards 13C-BDE-139 and 13C-2-OH-BDE-99 averaged 67.3−118.5% and 77.7− 121%, respectively. Statistical analyses were performed with the SPSS 12.0 for Windows. The Kolmogorov−Smirnov test was used to verify the normality of the data, and homogeneity of variances was analyzed by the Levene’s test. If the data failed the Kolmogorov−Smirnov test, logarithmic transformation was performed and then checked again for homogeneity of variances. Once the data satisfied the assumptions of homogeneity of variances, one-way analysis of variance (ANOVA) followed by LSD test was used to evaluate the differences between the variables. A value of p < 0.05 was considered as statistically significant. The probit model was used to calculate the LC50 for the endpoints used to characterize teratogenic effects at the different developmental stages of zebrafish embryos. All values were expressed as mean ± standard error (SEM).

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RESULTS Developmental Toxicity. (Raw data of morphological effects are shown in Tables S2−S4, Supporting Information.) Zebrafish embryos grew well in culture medium with or without carrier solvent (0.1% DMSO). On the basis of nominal concentrations, 6-OH-BDE-47 was more toxic to zebrafish embryos than was 6-MeO-BDE-47 or BDE-47. Development of embryos exposed to 1000 μg of 6-OH-BDE-47/L was slower, and embryos had significantly less melanin at 36 hpf compared with controls (Figure 1). Development of these larvae was arrested at 72 hpf. By 96 hpf, all embryos exposed to 1000 or 5000 μg/L had died. LC50 values were 1550 and 330 μg 6-OHBDE-47/L after 48 and 96 h, respectively (Table 1). Table 1. Lethal Concentrations (LC50; μg/L) of 6-OH-BDE47 to Developing Zebrafish Embryos (μg/L) duration of development (hpf)

LC50 (μg/L)

confidence interval (95%)

24 36 48 54 60 72 96

4620 1690 1550 1500 1190 540 330

2810−13760 820−5720 870−3710 880−3270 890−1720 340−940 160−990

Concentrations less than 200 μg of 6-OH-BDE-47/L did not cause significantly mortality compared with the controls after 120 hpf. Pericardial edema was observed after 96 hpf in embryos exposed to 5000 μg of 6-MeO-BDE-47/L. None of the tested concentrations of 6-MeO-BDE-47 or BDE47 affected mortality or body lengths in 96 hpf larvae. However, at 120 hpf, edema and curved spine occurred in embryos exposed to 5000 μg of 6-MeO-BDE-47/L (Figure 1). Lengths of embryos were significantly less when exposed to 5000 μg of 6-MeO-BDE-47/ L (3568 ± 167 μm) compared with controls (4241 ± 76 μm). The three target BDE congeners ranked as follows regarding their toxic potencies (values from greater to lesser potency): 6OH-BDE-47 > 6-MeO-BDE-47 > BDE-47. Transcriptional Responses. Exposure to 6-OH-BDE-47 or 6-MeO-BDE-47 but not BDE-47 resulted in significant changes in gene expression profiles of genes along the HPT axis (Figures 2−4). Expression of mRNA of thyrotropin-releasing 12946

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Figure 2. Expressions of (A) TRH, (B) TRHR1, (C) TSHβ, (D) TRα, and (E) TRβ, determined by real-time PCR in zebrafish larvae after exposure to 8, 40, or 200 μg/L of individual chemicals. Results are expressed as means ± SEM of three replicates. *P < 0.05 and **P < 0.01 indicate significant difference between exposure groups and the control.

Figure 3. Expression of (A) NIS and (B) TTR in zebrafish larvae determined by real-time PCR after exposure to 8, 40, or 200 μg/L individual chemicals. Results are means ± SEM of three replicate samples. *P < 0.05 and **P < 0.01 indicate significant difference between exposure groups and the control.

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Figure 4. Expression of (A) TSHR, (B) TG, (C) Dieo1, and (D) Dieo2 in zebrafish larvae determined by real-time PCR after exposure to 8, 40, or 200 μg/L of individual chemicals. Results expressed as means ± SEM of three replicate samples. *P < 0.05 and **P < 0.01 indicate significant difference between exposure groups and the control.

nominal concentrations of 8, 40, and 200 μg 6-OH-BDE-47/L, respectively. Estimated BCFs were 13, 4, and 22, when larvae were exposed to 8, 40, or 200 μg BDE-47/L, respectively, and 7, 13, and 70 when exposed to 8, 40, or 200 μg 6-MeO-BDE47/L, respectively.

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DISCUSSION On the basis of the small percentage of each compound present as an impurity in BDE-47, the concentrations of 2′-OH-BDE28, BDE-28, BDE-85, and BDE-99 observed in zebrafish larvae exposed to BDE-47 probably resulted from impurities in the original BDE-47, which indicates that zebrafish might not transform BDE-47 into OH-PBDEs or MeO-PBDEs. This result is consistent with that of a previous study in which BDE47 was not converted to 6-OH-BDE-47 or 6-MeO-BDE-47.36 In larvae exposed to 6-OH-BDE-47, 2′-OH-BDE-28, 6-OHBDE-85, 6-MeO-BDE-47, and BDE-47 were observed, which might also come from impurities in the parent compounds. In zebrafish larvae exposed to 6-MeO-BDE-47, in addition to a large amount of the precursor, small amounts of 2′-OH-BDE28, 2′-MeO-BDE-28, and BDE-47 were also observed because of the impurities in the precursor material. In addition, 117 ng of 6-OH-BDE-47/g ww was determined in embryos exposed to 200 μg/L, so it indicated that 6-OH-BDE-47 could be metabolized from 6-MeO-BDE-47. This study, for the first time, demonstrated that both 6-OHBDE-47 and 6-MeO-BDE-47 can affect expression of specific genes along the HPT axis as well as resulted in teratogenic effects in zebrafish embryos, while the man-made BDE-47 did not result in molecular or pathological effects at exposure concentrations up to 200 or 5000 μg of BDE-47/L, respectively. Previous studies demonstrated that changes

Figure 5. Bioconcentration and metabolism of 6-OH-BDE-47, BDE47, and 6-MeO-BDE-47 measured in zebrafish larvae after exposure to nominal concentrations of the three chemicals (0, 8, 40, or 200 μg/L) in water with DMSO at 0.1% (v/v) for 5 days.

the control group, neither BDE-47 nor any of the analogues or products of biotransformation were detected (Figure 5). Concentrations of BDE-47 and 6-MeO-BDE-47 were approximately 10- to 100-fold greater than concentrations of 6-OH-BDE-47 in larvae that were exposed to similar concentrations of the three compounds at a nominal concentration of 200 μg/L. The log Kow for 6-OH-BDE-47 is known to be less than that of the other compounds,35 which might make its excretion more likely to potentially contribute to its lower concentration in zebrafish larvae. The estimated bioconcentration factors (BCF) for 6-OH-BDE-47 based on measured concentrations in larvae divided by the nominal concentration in water were 0, 3, and 3 with treatment of 12948

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of the thyroid gland. Its primary function is to serve as a macromolecular substrate for coupling of iodide to its tyrosine residues during synthesis of TH.46 THs bind to and activate nuclear TRs, via 5′ deiodination of T4 in several organs, including brain, pituitary, brown adipose tissue (BAT), skin, placenta, human heart, muscle, and thyroid.47 Modulation of transcription of TRH and TSH genes is also influenced by concentrations of THs via negative feedback.29 In this study, most genes including TSHR, TRα, TRβ, NIS, and TTR were down-regulated after exposure to 6-OH-BDE-47. This is in accordance with the significant impact of greater concentrations of this congener on larval development and is indicative of the involvement of the disruptions along the thyroid axis in the pathologies observed. Further studies are required, however, to elucidate the specific mechanism by which 6-OH-BDE-47 causes developmental effects through the thyroid axis in fish and to help establish specific adverse outcome pathways for this chemical. As members of the nuclear receptor superfamily, thyroid hormone receptors (TRs) have been shown to be associated with postnatal development of birds, metamorphosis of amphibians, and smoltification of fish.48 TRs including TRα and TRβ can bind to specific DNA sequences (thyroid hormone response elements) on promoters to regulate target genes, which are involved in resistance to thyroid hormone (TRH).49 The TRα isoform is predominantly expressed in adipocytes and mediates actions of thyroid hormone in these cells.50 Thus, up-regulation of TRs could reduce concentrations of THs. TSH enhances the ability of the thyroid gland to trap iodide. So when TSH is decreased, expression of NIS is downregulated. TTR is a carrier protein for TH in blood and regulates the supply of the TH to various target tissues.51 Concentrations of THs in blood were directly proportional to expression of mRNA of TTR, and lesser expression of TTR was accompanied by lesser THs in blood plasma.51 The significant decrease in TTR gene expression observed in this study indicates that concentrations of THs in zebrafish were likely decreased by 6-OH-BDE-47. In contrast, the significant increase in the expression of TRH mRNA could also have resulted in an increase of THs in larvae exposed to 6-OH-BDE47. Considering the negative impacts exposure to 6-OH-BDE47 had on larval development, however, it is hypothesized that the increase in TRH mRNA is more likely a compensatory effect in an attempt to offset the negative impacts along the HPT axis. Accumulation and toxic potencies varied among the three compounds tested. While 6-OH-BDE-47 was the least accumulated into larvae, it exhibited the greatest toxicity. 6MeO-BDE-47 was accumulated more than the other two chemicals, and significant amounts of 6-MeO-BDE-47 were transformed to 6-OH-BDE-47, even though the biotransformation capability of larvae was limited (approximately 0.1% after 120 h). This study revealed significant conversion of 6-MeOBDE-47 but not BDE-47 to 6-OH-BDE-47, which is in accordance with other studies on different fish species that hypothesized that natural MeO-BDEs and not the man-made BDEs are the main source of the more toxic OH-congeners.13 Considering the greater concentration of 6-MeO-BDE-47 in marine environments and its greater potential for bioaccumulation and biotransformation of 6-MeO-BDE-47, it is the likely source of 6-OH-BDE-47 observed in fishes. In conclusion, this study demonstrated that 6-OH-BDE-47, and to a lesser extent 6-MeO-BDE-47, had significant effects at

along the HPT axis were often related to specific pathological phenomena. For instance, when zebrafish embryos were exposed to 500 μg/L microcystin-LR, changes in gene expression along the HPT axis could be correlated with a significant decrease of body length.37 All BDE-47, TBBPA and BPA were shown to induce alteration of genes along the HPT axis of zebrafish larvae as well as cause acute toxicity.38 In this study, 6-OH-BDE-47 was acutely toxic with a 96 h-LC50 of 330 μg/L while reduced body length and teratogenic effects were observed at 5000 μg/L at 120 hpf. These results further suggest a relationship between early molecular-level effects and subsequent morphological changes. The observed impact on development of zebrafish is in accordance with the critical role of the HPT axis in early life-stage development of vertebrates.19,20 In female C57BL/6 mice, BDE-47 had previously been reported to decrease total serum T4 concentrations by 43% at 100 mg/kg/day treatment, activate the nuclear receptor, CAR, and decrease Mdr1a mRNA expression at 3 mg/kg/day.39 An earlier study indicated that 96 h-EC50 values of BDE-47 for zebrafish embryos based on hatching success were 20.30 mg/L between 2.03 and 15.23 mg/L, significantly affecting expression of some genes along the HPT.38 In this study, BDE-47 did not show developmental or molecular toxicity at exposure concentrations up to 5000 or 200 μg/L, respectively. Perhaps, because the concentrations used in those studies were greater than concentrations tested in our experiments. It could also have been due to differences in exposures and different organisms. The environmental relevance of such great exposures, however, would be negligible. In contrast, 6-OHBDE-47, and to a lesser extent 6-MeO-BDE-47, had significant effects at the molecular level on the HPT axis in larval zebrafish that were indicative of developmental effects in this species. The fact that most genes along the HPT axis, such as TSHR, TRα, TRβ, NIS, and TTR, were down-regulated and only TRH and TG were up-regulated by 6-OH-BDE-47, while exposed to 6-MeO-BDE-47, resulted in up-regulation of the expression of TRH, TRα, TRβ, and NIS, indicating that these two chemicals affect the HPT axis of zebrafish through different toxic pathways (Figure S1 of Supporting Information). These results are consistent with the findings of a previous study that reported that cytotoxicity of 6-OH-BDE-47 to E. coli was via a mechanism that was different from that of either BDE-47 or 6MeO-BDE-47.40 In mammals, the hypothalamic tripeptide TRH stimulates thyroid stimulating hormone (TSH) synthesis and release from the anterior pituitary to bind to TSHR in the thyrocytes.41 TRH increases locomotor activity and intake of food, both of which are associated with increases in the hypothalamic expressions of OX and CART in goldfish.42 TSH is a member of a glycoprotein hormone family which is composed of common α- and specific β-subunits. TSHR is a key protein in the control of thyroid function, by stimulating TH synthesis after binding its ligand, the thyrotropin. This pathway is involved with both growth and functional characteristics, by acting via the cAMP pathway.43 TSHR plays a critical role in certain thyroid diseases, including Graves’ disease (GD), multinodular thyroid goiter (MTG), and Hashimoto’s thyroiditis (HT).44 When TSH was transferred to thyroid, T4 was produced with the assistant of NIS and TG. As an integral plasma membrane glycoprotein, NIS is localized in the baso-lateral membrane of thyrocytes. It is necessary for the transport of iodide for the biosynthesis of TH.45 TG is a large glycoprotein produced and stored in the follicular lumen 12949

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pollutants resulted from e-wastes recycling and bioaccumulation of polybrominated diphenyl ethers in Chinese loach (Misgurnus anguillicaudatus). J. Environ. Sci. (China) 2009, 21 (12), 1695−1701. (5) Ohta, S.; Ishizuka, D.; Nishimur, H.; Nakao, T. Comparison of polybrominated diphenyl ethers in fish, vegetables, and meats and levels in human milk of nursing women in Japan. Chemosphere 2002, 46 (5), 689−696. (6) Valters, K.; Li, H.; Alaee, M.; D’Sa, I.; Marsh, G.; Bergman, A.; Letcher, R. J. Polybrominated diphenyl ethers and hydroxylated and methoxylated brominated and chlorinated analogues in the plasma of fish from the Detroit River. Environ. Sci. Technol. 2005, 39 (15), 5612− 5619. (7) Verreault, J.; Gabrielsen, G. W.; Chu, S.; Muir, D. C.; Andersen, M.; Hamaed, A.; Letcher, R. J. Flame retardants and methoxylated and hydroxylated polybrominated diphenyl ethers in two Norwegian Arctic top predators: glaucous gulls and polar bears. Environ. Sci. Technol. 2005, 39 (16), 6021−6028. (8) Pettersson, A.; van Bavel, B.; Engwall, M.; Jimenez, B. Polybrominated diphenylethers and methoxylated tetrabromodiphenylethers in cetaceans from the Mediterranean Sea. Arch. Environ. Contam. Toxicol. 2004, 47 (4), 542−550. (9) Teuten, E. L.; Johnson, C. G.; Mandalakis, M.; Asplund, L.; Gustafsson, O.; Unger, M.; Marsh, G.; Reddy, C. M. Spectral characterization of two bioaccumulated methoxylated polybrominated diphenyl ethers. Chemosphere 2006, 62 (2), 197−203. (10) Zhang, K.; Wan, Y.; Jones, P. D.; Wiseman, S.; Giesy, J. P.; Hu, J. Occurrences and fates of hydroxylated polybrominated diphenyl ethers in marine sediments in relation to trophodynamics. Environ. Sci. Technol. 2012, 46 (4), 2148−2155. (11) Hakk, H.; Huwe, J. K.; Murphy, K.; Rutherford, D. Metabolism of 2,2’,4,4’-tetrabromodiphenyl ether (BDE-47) in chickens. J. Agric. Food Chem. 2010, 58 (15), 8757−8762. (12) Wan, Y.; Wiseman, S.; Chang, H.; Zhang, X.; Jones, P. D.; Hecker, M.; Kannan, K.; Tanabe, S.; Hu, J.; Lam, M. H.; Giesy, J. P. Origin of hydroxylated brominated diphenyl ethers: Natural compounds or man-made flame retardants? Environ. Sci. Technol. 2009, 43 (19), 7536−7542. (13) Wan, Y.; Liu, F.; Wiseman, S.; Zhang, X.; Chang, H.; Hecker, M.; Jones, P. D.; Lam, M. H.; Giesy, J. P. Interconversion of hydroxylated and methoxylated polybrominated diphenyl ethers in Japanese medaka. Environ. Sci. Technol. 2010, 44 (22), 8729−8735. (14) Wahl, M.; Guenther, R.; Yang, L.; Bergman, A.; Straehle, U.; Strack, S.; Weiss, C. Polybrominated diphenyl ethers and arylhydrocarbon receptor agonists: Different toxicity and target gene expression. Toxicol. Lett. 2010, 198 (2), 119−126. (15) van Boxtel, A. L.; Kamstra, J. H.; Cenijn, P. H.; Pieterse, B.; Wagner, J. M.; Antink, M.; Krab, K.; van der Burg, B.; Marsh, G.; Brouwer, A.; Legler, J. Microarray analysis reveals a mechanism of phenolic polybrominated diphenylether toxicity in zebrafish. Environ. Sci. Technol. 2008, 42 (5), 1773−1779. (16) Wahl, M.; Guenther, R.; Yang, L.; Bergman, A.; Straehle, U.; Strack, S.; Weiss, C. Polybrominated diphenyl ethers and arylhydrocarbon receptor agonists: Different toxicity and target gene expression. Toxicol. Lett. 2010, 198 (2), 119−126. (17) Villanger, G. D.; Lydersen, C.; Kovacs, K. M.; Lie, E.; Skaare, J. U.; Jenssen, B. M. Disruptive effects of persistent organohalogen contaminants on thyroid function in white whales (Delphinapterus leucas) from Svalbard. Sci. Total Environ. 2011, 409 (13), 2511−2524. (18) Lema, S. C.; Dickey, J. T.; Schultz, I. R.; Swanson, P. Dietary exposure to 2,2’,4,4’-tetrabromodiphenyl ether (PBDE-47) alters thyroid status and thyroid hormone-regulated gene transcription in the pituitary and brain. Environ. Health Perspect. 2008, 116 (12), 1694−1699. (19) Perello, M.; Cakir, I.; Cyr, N. E.; Romero, A.; Stuart, R. C.; Chiappini, F.; Hollenberg, A. N.; Nillni, E. A. Maintenance of the thyroid axis during diet-induced obesity in rodents is controlled at the central level. Am. J. Physiol. Endocrinol. Metab. 2010, 299 (6), E976− E989.

the molecular level on the HPT axis in larval zebrafish that were indicative of developmental effects in this species. In contrast, exposure to the man-made BDE-47 did not result in any effects either at the molecular or the pathological level. Furthermore, this study revealed significant conversion of 6-MeO-BDE-47 but not BDE-47 to 6-OH-BDE-47, which is in accordance with other studies on different fish species that hypothesized that natural MeO-BDEs and not the man-made BDEs are the main source of the more toxic OH-congeners. Although 6-OH-BDE47 was less bioaccumulative than BDE-47 and 6-MeO-BDE-47, the significant transformation of the highly bioaccumulative MeO-BDE is likely to represent a continuous internal source for the toxic OH-BDE.

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ASSOCIATED CONTENT

S Supporting Information *

Figure S1, pathway of hypothalamic-pituitary-thyroid (HPT) axis in zebrafish; Table S1, primer sequences for the quantitative reverse transcription-polymerase chain reaction (q-PCR); Table S2, raw data of morphological effect of 6-OHBDE-47; Table S3, raw data of morphological effect of 6-MeOBDE-47; Table S4, raw data of morphological effect of BDE-47. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 86-25-89680356. Fax: 86-25-89680356. E-mail: hlliu@ nju.edu.cn (H.L.); [email protected] (H.Y.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was jointly funded by the National Natural Science Foundation of China (Nos. 20977047, 20737001), Major National Science and Technology Projects (Nos. 2012ZX07506-001, 2012ZX07529-003-02), and the Environmental Monitoring Research Foundation of Jiangsu Province (No. 1114). The research was supported by a Discovery Grant from the Natural Science and Engineering Research Council of Canada (Project # 326415-07). J.P.G. and M.H. were supported by the Canada Research Chair program. Furthermore, J.P.G. was supported by an at large Chair Professorship at the Department of Biology and Chemistry and State Key Laboratory in Marine Pollution, City University of Hong Kong, and the Einstein Professor Program of the Chinese Academy.

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REFERENCES

(1) Valters, K.; Li, H. X.; Alaee, M.; D’Sa, I.; Marsh, G.; Bergman, A.; Letcher, R. J. Polybrominated diphenyl ethers and hydroxylated and methoxylated brominated and chlorinated analogues in the plasma of fish from the Detroit River. Environ. Sci. Technol. 2005, 39 (15), 5612− 5619. (2) Haraguchi, K.; Kotaki, Y.; Relox, J. R.; Romero, M. L. J.; Terada, R. Monitoring of naturally produced brominated phenoxyphenols and phenoxyanisoles in aquatic plants from the Philippines. J. Agric. Food Chem. 2010, 58 (23), 12385−12391. (3) He, M. J.; Luo, X. J.; Chen, M. Y.; Sun, Y. X.; Chen, S. J.; Mai, B. X. Bioaccumulation of polybrominated diphenyl ethers and decabromodiphenyl ethane in fish from a river system in a highly industrialized area, South China. Sci. Total Environ. 2012, 419, 109− 115. (4) Qin, X.; Xia, X.; Li, Y.; Zhao, Y.; Yang, Z.; Fu, S.; Tian, M.; Zhao, X.; Qin, Z.; Xu, X.; Yang, Y. Ecotoxicological effects of mixed 12950

dx.doi.org/10.1021/es303289n | Environ. Sci. Technol. 2012, 46, 12943−12951

Environmental Science & Technology

Article

(20) Schaefer, J. S.; Klein, J. R. Immunological regulation of metabolisma novel quintessential role for the immune system in health and disease. FASEB J. 2011, 25 (1), 29−34. (21) Klammer, H.; Schlecht, C.; Wuttke, W.; Schmutzler, C.; Gotthardt, I.; Kohrle, J.; Jarry, H. Effects of a 5-day treatment with the UV-filter octyl-methoxycinnamate (OMC) on the function of the hypothalamo-pituitary-thyroid function in rats. Toxicology 2007, 238 (2−3), 192−199. (22) Spachmo, B.; Arukwe, A. Endocrine and developmental effects in Atlantic salmon (Salmo salar) exposed to perfluorooctane sulfonic or perfluorooctane carboxylic acids. Aquat. Toxicol. 2012, 108, 112− 124. (23) Usenko, C. Y.; Hopkins, D. C.; Trumble, S. J.; Bruce, E. D. Hydroxylated PBDEs induce developmental arrest in zebrafish. Toxicol. Appl. Pharmacol. 2012, 262 (1), 43−51. (24) Canton, R. F.; Scholten, D. E.; Marsh, G.; de Jong, P. C.; van den Berg, M. Inhibition of human placental aromatase activity by hydroxylated polybrominated diphenyl ethers (OH-PBDEs). Toxicol. Appl. Pharmacol. 2008, 227 (1), 68−75. (25) Meerts, I. A. T. M.; Letcher, R. J.; Hoving, S.; Marsh, G.; Bergman, A.; Lemmen, J. G.; van der Burg, B.; Brouwer, A. In vitro estrogenicity of polybrominated diphenyl ethers, hydroxylated PBDEs, and polybrominated bisphenol A compounds. Environ. Health Perspect. 2001, 109 (4), 399−407. (26) Liu, H.; Hu, W.; Sun, H.; Shen, O.; Wang, X. In vitro profiling of endocrine disrupting potency of 2,2’,4,4’-tetrabromodiphenyl ether (BDE47) and related hydroxylated analogues (HO-PBDEs). Mar. Pollut. Bull. 2011, 63, 287−296. (27) Liu, C.; Zhang, X.; Deng, J.; Hecker, M.; Al-Khedhairy, A.; Giesy, J. P.; Zhou, B. Effects of prochloraz or propylthiouracil on the cross-talk between the HPG, HPA, and HPT axes in zebrafish. Environ. Sci. Technol. 2011, 45 (2), 769−775. (28) Shi, X.; Liu, C.; Wu, G.; Zhou, B. Waterborne exposure to PFOS causes disruption of the hypothalamus-pituitary-thyroid axis in zebrafish larvae. Chemosphere 2009, 77 (7), 1010−1018. (29) Yu, L.; Deng, J.; Shi, X.; Liu, C.; Yu, K.; Zhou, B. Exposure to DE-71 alters thyroid hormone levels and gene transcription in the hypothalamic-pituitary-thyroid axis of zebrafish larvae. Aquat. Toxicol. 2010, 97 (3), 226−233. (30) Marsh, G.; Stenutz, R.; Bergman, A. Synthesis of hydroxylated and methoxylated polybrominated diphenyl ethers - Natural products and potential polybrominated diphenyl ether metabolites. Eur. J. Org. Chem. 2003, 14, 2566−2576. (31) Alt, B.; Reibe, S.; Feitosa, N. M.; Elsalini, O. A.; Wendl, T.; Rohr, K. B. Analysis of origin and growth of the thyroid gland in zebrafish. Dev. Dyn. 2006, 235 (7), 1872−1883. (32) Elsalini, O. A.; von Gartzen, J.; Cramer, M.; Rohr, K. B. Zebrafish hhex, nk2.1a, and pax2.1 regulate thyroid growth and differentiation downstream of Nodal-dependent transcription factors. Dev. Biol. 2003, 263 (1), 67−80. (33) Liu, C.; Yan, W.; Zhou, B.; Guo, Y.; Liu, H.; Yu, H.; Giesy, J. P.; Wang, J.; Li, G.; Zhang, X. Characterization of a bystander effect induced by the endocrine-disrupting chemical 6-propyl-2-thiouracil in zebrafish embryos. Aquat. Toxicol. 2012, 118−119, 108−115. (34) Wen, Q.; Liu, H.; Su, G.; Wei, S.; Feng, J.; Yu, H. Method and application of polybrominated diphenyl ethers (PBDEs) and their derivate analysis in zebrafish eggs. J. Anal. Chem. 2012, 40 (11), 1698− 1702. (35) Yu, Y.; Yang, W.; Gao, Z.; Lam, M. H. W.; Liu, X.; Wang, L.; Yu, H. RP-HPLC measurement and quantitative structure-property relationship analysis of the noctanol-water partitioning coefficients of selected metabolites of polybrominated dyphenyl ethers. Environ. Chem. 2008, 5 (5), 332−339. (36) Wan, Y.; Liu, F. Y.; Wiseman, S.; Zhang, X. W.; Chang, H.; Hecker, M.; Jones, P. D.; Lam, M. H. W.; Giesy, J. P. Interconversion of hydroxylated and methoxylated polybrominated diphenyl ethers in Japanese medaka. Environ. Sci. Technol. 2010, 44 (22), 8729−8735. (37) Yan, W.; Zhou, Y.; Yang, J.; Li, S.; Hu, D.; Wang, J.; Chen, J.; Li, G. Waterborne exposure to microcystin-LR alters thyroid hormone

levels and gene transcription in the hypothalamic-pituitary-thyroid axis in zebrafish larvae. Chemosphere 2012, 87 (11), 1301−1307. (38) Chan, W. K.; Chan, K. M. Disruption of the hypothalamicpituitary-thyroid axis in zebrafish embryo-larvae following waterborne exposure to BDE-47, TBBPA and BPA. Aquat. Toxicol. 2012, 108, 106−111. (39) Richardson, V. M.; Staskal, D. F.; Ross, D. G.; Diliberto, J. J.; DeVito, M. J.; Birnbaum, L. S. Possible mechanisms of thyroid hormone disruption in mice by BDE 47, a major polybrominated diphenyl ether congener. Toxicol. Appl. Pharmacol. 2008, 226 (3), 244−250. (40) Su, G.; Zhang, X.; Liu, H.; Giesy, J. P.; Lam, M. H. W.; Lam, P. K. S.; Siddiqui, M. A.; Musarrat, J.; Al-Khedhairy, A.; Yu, H. Toxicogenomic mechanisms of 6-HO-BDE-47, 6-MeO-BDE-47, and BDE-47 in E. coli. Environ. Sci. Technol. 2012, 46, 1185−1191. (41) Davies, T.; Marians, R.; Latif, R. The TSH receptor reveals itself. J. Clin. Invest. 2002, 110 (2), 161−164. (42) Abbott, M.; Volkoff, H. Thyrotropin releasing hormone (TRH) in goldfish (Carassius auratus): Role in the regulation of feeding and locomotor behaviors and interactions with the orexin system and cocaine- and amphetamine regulated transcript (CART). Horm. Behav. 2011, 59 (2), 236−245. (43) Shao, L.; Jiang, H.; Liang, J.; Niu, X.; Teng, L.; Zhang, H. Study on the relationship between TSHR gene and thyroid diseases. Cell Biochem. Biophys. 2011, 61 (2), 377−382. (44) Tomer, Y. Genetic susceptibility to autoimmune thyroid disease: Past, present, and future. Thyroid 2010, 20 (7), 715−725. (45) Carvalho, D. P.; Ferreira, A. C. The importance of sodium/ iodide symporter (NIS) for thyroid cancer management. Arq. Bras. Endocrinol. Metab. 2007, 51 (5), 672−682. (46) Noguchi, Y.; Harii, N.; Giuliani, C.; Tatsuno, I.; Suzuki, K.; Kohn, L. D. Thyroglobulin (Tg) induces thyroid cell growth in a concentration-specific manner by a mechanism other than thyrotropin/cAMP stimulation. Biochem. Biophys. Res. Commun. 2010, 391 (1), 890−894. (47) Martinez de Mena, R.; Scanlan, T. S.; Obregon, M. J. The T3 receptor beta1 isoform regulates UCP1 and D2 deiodinase in rat brown adipocytes. Endocrinology 2010, 151 (10), 5074−5083. (48) An, K. W.; An, M. I.; Nelson, E. R.; Habibi, H. R.; Choi, C. Y. Gender-related expression of TRalpha and TRbeta in the protandrous black porgy, Acanthopagrus schlegeli, during sex change processes. Gen. Comp. Endrocrinol. 2010, 165 (1), 11−18. (49) Bassett, J. H.; Harvey, C. B.; Williams, G. R. Mechanisms of thyroid hormone receptor-specific nuclear and extra nuclear actions. Mol. Cell. Endocrinol. 2003, 213 (1), 1−11. (50) Cai, Z. W.; Sheng, Y. F.; Zhang, L. F.; Wang, Y.; Jiang, X. L.; Lv, Z. Z.; Xu, N. Y. Mapping, expression and regulation of the TRalpha gene in porcine adipose tissue. Genet. Mol. Res. 2011, 10 (3), 1320− 1330. (51) Power, D. M.; Elias, N. P.; Richardson, S. J.; Mendes, J.; Soares, C. M.; Santos, C. R. Evolution of the thyroid hormone-binding protein, transthyretin. Gen. Comp. Endrocrinol. 2000, 119 (3), 241− 255.

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