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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Marine Pollution Bulletin 63 (2011) 287–296 Contents lists available at ScienceDirect Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul In vitro profiling of endocrine disrupting potency of 2,20 ,4,40 -tetrabromodiphenyl ether (BDE47) and related hydroxylated analogs (HO-PBDEs) Hongling Liu a, Wei Hu a, Hong Sun b, Ouxi Shen b, Xinru Wang b, Michael H.W. Lam c, John P. Giesy a,c,d,e, Xiaowei Zhang a, Hongxia Yu a,⇑ a State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210093, China Key Laboratory of Reproductive Medicine, Institute of Toxicology, Nanjing Medical University, Nanjing 210029, China Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong, SAR, China d Department of Biomedical and Veterinary Biosciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada e Zoology Department, National Food Safety and Toxicology Center and Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824, USA b c a r t i c l e i n f o Keywords: Receptor reporter gene assay BDE47 Metabolite Endocrine disruption a b s t r a c t The potential of 2,20 ,4,40 -tetrabromodiphenyl ether (BDE47) and its related hydroxylated analogs (20 -HOBDE28, 6-HO-BDE47, 40 -HO-BDE17, and 40 -HO-BDE49) to modulate estrogen/thyroid/androgen receptor(ER, TR, AR), mediated responses were investigated by use of reporter gene assays. Exposure to 1 or 10 lM, 40 -HO-BDE17 significantly up-regulated expression of Luc, whereas other four chemicals did not induce Luc expression under control of the ER. Anti-estrogenic potency was observed for 40 -HOBDE17 (IC50 = 1.14 lM) > 6-HO-BDE47 (IC50 = 2.65 lM) > 20 -HO-BDE28 (IC50 = 9.49 lM) > BDE47 (IC50 = 21.11 lM). No anti-estrogenic effect of 40 -HO-BDE49 was observed. Both 40 -HO-BDE17, 40 -HOBDE49 resulted in greater responses of Luc expression induced by T3. BDE47, 20 -HO-BDE28, 6-HOBDE47 did not show any effect on the expression of Luc induced by 5 nM T3. 6-HO-BDE47 (IC50 = 0.34 lM) > 40 -HO-BDE17 (IC50 = 1.41 lM) > BDE47 (IC50 = 3.83 lM) > 20 -HO-BDE28 (IC50 = 29.22 lM) exhibited anti-androgenic potency, while 40 -HO-BDE49 did not show androgenic transcriptional activity. Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved. 1. Introduction Polybrominated diphenyl ethers (PBDEs) have been widely used as flame retardant additives for plastics and textiles, electronic equipment, and building materials (de Wit, 2002; Brown et al., 2004). PBDEs can be found in wildlife (Hale et al., 2001; de Wit, 2002; Pettersson et al., 2004) and humans (Noren and Meironyte, 2000; Akutsu et al., 2003; Johnson-Restrepo et al., 2005; Bi et al., 2007). In China, PBDEs have been detected in many media (Bi et al., 2006; Chen et al., 2006, 2007; Wurl et al., 2006; Guo et al., 2008; Jin et al., 2008) and these compounds are now ubiquitous contaminants in aquatic organisms (fish, shrimp, and crabs) from the lower Yangtze River, which might pose significant risk to human and wildlife (Gao et al., 2009). The 2,20 ,4,40 -tetrabromodiphenylether congener (BDE47) is the predominant PBDE found in the environment (Hites, 2004; Valters et al., 2005; Marsh et al., 2006; Streets et al., 2006; Xiang et al., 2007) and humans (Örn and Klasson-Wehler, 1998). BDE47 was the most abundant congener, contributing more than 40% of the ⇑ Corresponding author. Tel./fax: +86 25 89680556. E-mail address: yuhx@nju.edu.cn (H. Yu). P PBDE in all species except for the brown bullhead, for which 2,20 ,4,40 ,5-pentabromodiphenyl ether (BDE99) contributed the greatest proportion (Valters et al., 2005). BDE47 has been reported to be formed by debromination of BDE99 in common carp (Cyprinus carpio) (Stapleton et al., 2004). Hydroxylated analogs of PBDEs are found in the environment. While HO-BDEs have been suggested to be biotransformation of PBDE by animals (Stapleton et al., 2004; Marsh et al., 2006; Qiu et al., 2007), recent careful investigations of the biotransformation of PBDE have been suggested that the more likely source of HO-BDE is biotransformation of methoxylated BDE (MeO-BDE) (Wan et al., 2009). Regardless of their source, whether from natural products or from biotransformation of synthetic BDE flame retardants the potential effects of HO-BDE need to be assessed and considered. Both PBDEs and their HO-analogs are toxic to animals (Darnerud et al., 2001; McDonald, 2002) and concentrations of the HO-PBDE can exceed those of PBDE (Pettersson et al., 2004). The pharmacokinetic behavior of BDE47 is dose dependent, with relative elimination decreasing as the dose is raised (Staskal et al., 2005). BDE47 is well absorbed after oral, tracheal, and peritoneal exposure (>80%) and that dermal absorption is >60%. Relatively 0025-326X/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2011.04.019 Author's personal copy 288 H. Liu et al. / Marine Pollution Bulletin 63 (2011) 287–296 great bioaccumulation factors for BDE47 have been reported (Gustafsson et al., 1999). BDE47 is known to cause various adverse effects, including being an ER-agonist (Sanderson et al., 2004). BDE47 inhibited binding of the synthetic androgen R1881 to cytosolic androgen receptor (AR) from the ventral prostate (Stoker et al., 2005). BDE47 also inhibited dihydrotestosterone induced, human AR-regulated transcriptional activation in a luciferase reporter gene assay (Stoker et al., 2005). Several studies have reported that PBDEs bind competitively with human transthyretin (TTR), a transport protein for the thyroid hormones T3 and thyroxine (T4), thereby hampering the transportation of thyroid hormone (Meerts et al., 2000; Zhou et al., 2001). A few studies have also addressed the binding ability of PBDEs with TR (Kitamura et al., 2008), but no PBDEs congeners showed affinity for TR. Some hydroxylated PBDEs (HO-BDEs) metabolites analogs which induced or inhibited the activity of aromatase (CYP19) are known to have endocrine disrupting effects (Cantón et al., 2005, 2006), but information about the potential endocrine activity of OH-PBDEs is limited to a small number of HO-BDEs (Marsh et al., 1998; Meerts et al., 2000, 2001; Kester et al., 2002; Stoker et al., 2005). 4-HO-20 ,40 ,60 -TrBDE, 4-HO-3,20 ,40 ,60 -TeBDE, 4-HO-3,5,20 ,40 , 60 -PeBDE, 2-HO-4,20 ,40 -TrCDE tested were relatively weak inhibitors of E2 sulfation by SULT1E1, with IC50 values greater than 200 nm (Kester et al., 2002). Also, 2-OH-4,20 ,40 -TrCDE inhibited SULT1E1 activity at micromolar concentrations (Kester et al., 2002). More PBDEs congeners possessed AR antagonistic potency than those that have been shown to cause ER-mediated effects (Hamers et al., 2006; Kojima et al., 2009). The objective of the present study was to assess the potential endocrine disrupting effects of BDE47 and four analogous HO-BDEs, 20 -HO-BDE28, 6-HO-BDE47, 40 -HO-BDE17, and 40 -HOBDE49 on ER-, TR- and AR-mediated anti/estrogenic, anti/thyroid and anti/androgenic effects in the African monkey kidney CV-1 cell line and a stably transfected cell line (MDA-kb2). 2. Materials and methods 2.1. Test chemicals BDE47 was purchased from Sigma Chemical Co. (St. Louis, MO, USA). 20 -HO-BDE28, 40 -HO-BDE17, 6-HO-BDE47, and 40 -HO-BDE49 were synthesized in the Department of Biology and Chemistry of City University of Hong Kong with purities >98%. 17b-Estradiol (E2), triiodothyronine (T3), and 5a-dihydrotestosterone (DHT) with a purity of over 99% were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All compounds were dissolved in dimethyl sulfoxide (DMSO) and the final DMSO concentration in the exposure medium was 0.1% (v/v) to avoid potential effects on cell yields. 2.2. Plasmids Recombinant plasmids pERE-TATA-Luc, and pUAS-tk-Luc, with luciferase under control of the estrogen response (ERE), and thyroid response (TRE) elements, respectively, were developed as described (Takeyoshi et al., 2002; Sun et al., 2008). 2.3. Reporter gene assays 2.3.1. ER reporter gene assay Green monkey kidney fibroblast (CV-1) cells that do not contain the endogenous receptors (ER and TR) were obtained from the Institute of Biochemistry and Cell Biology at the Shanghai, Chinese Academy of Science, and maintained in Dulbecco’s modified Eagle’s medium (DEME) supplemented with 10% fetal bovine serum (FBS), Table 1 Concentrations of plasmids and transfection reagent for estrogenic and thyroid reporter gene assay systems. Estrogenic activities Plasmid Transfection reagent pERE-TATA-Luc rERa/pCI Sofast™ 0.25 lg/well 0.10 lg/well 0.5 lg/well Thyroid activities pUAS-tk-Luc pGal4-L-TRb Sofast™ 0.25 lg/well 0.25 lg/well 0.5 lg/well 100 U/ml penicillin and 100 lg/ml streptomycin at 37 °C in an atmosphere containing 5% CO2. Cell culture and exposures were conducted according to previously described methods (Sun et al., 2008). The host cells were plated into 48-well microplates at a density of 0.5 105 cells per well in the phenol red free DMEM medium containing 5% charcoal–dextran-stripped FBS (CDS-FBS). Twelve hours after seeding, CV-1 cells were transfected with 0.5 lg of pERE-TATA-Luc + 0.2 lg of rERa/pCI using 2.5 lg Sofast™ (Sunma Company, Xiamen, China) transfection reagent per well (Table 1). After 12 h incubation, the transfection medium was removed and selected concentrations of chemicals dissolved in medium. Cells were exposed to E2 (1.0 1010–1.0 107 M in 10-fold dilution steps), solvent-controls or test chemicals for 24 h. Dosing solutions of the chemicals were diluted with DMSO to a maximum DMSO concentration of 0.1%. After rinsing three times with phosphate-buffered saline (PBS, pH 7.4), cells were lysed with 1 lysis buffer (Promega, Madison, WI, USA, 50 ll/well). Light emission from expression of luciferase was determined immediately using the luciferase reporter assay system kit (Promega, Madison, WI, USA) and a 48-well plate luminometer (Berthold Detection System, Pforzheim, Germany). The relative transcriptional activity was converted to fold induction relative to the solvent control (n-fold). 2.3.2. TR reporter gene assay CV-1 cells were transfected with appropriate TR transactivation vectors (Table 1). CV-1 cells were cultured and plated as described above and were transfected after 12 h. After a 12 h incubation, the CV-1 cells were exposed for 24 h to T3 (1.0 1012–1.0 106 M in 10-fold dilution steps), solvent controls or compounds. Following cytolysis, cell lysates were analyzed immediately using a 48well plate luminometer (Berthold Detection System, Pforzheim, Germany). 2.3.3. AR reporter gene assay The MDA-kb2 cell line (ATCC, USA), stably transformed with murine mammalian tumor virus (MMTV)-luciferase was cultured in Leibovitz’s L-15 medium with 10% FBS, 100 U/ml penicillin (Sigma), 100 lg/ml streptomycin (Sigma), and 0.25 g/ml amphotericin B (Sigma) at 37 °C without CO2. For experiments, cells were plated at 1 104 cells per well in 100 ll of medium in 96-well luminometer plates. When cells were attached (4–6 h), medium was removed and replaced with dosing medium. The MDA-kb2 cells were exposed to DHT (Sigma, 1.0 1012–1.0 107 M in 10-fold dilution steps), solvent-control and compounds for 24 h. DHT was tested in a concentration range that had been previously shown to not be cytotoxic (Shen et al., 2009). After three rinses with phosphate-buffered saline (PBS, pH 7.4), the cells were lysed with 1 passive lysis buffer (Promega). After centrifuged at 12,000g for 10 min to remove debris, the cell lysates were analyzed immediately using a 96-well plate luminometer (Berthold Detection System, Pforzheim, Germany). The amount of luciferase was measured with the luciferase reporter assay system kit (Promega, Madison, WI, USA) following the manufacturer’s Author's personal copy H. Liu et al. / Marine Pollution Bulletin 63 (2011) 287–296 289 instructions. The relative luminescence units, which is a surrogate for transcriptional activity was converted to fold induction above the corresponding vehicle control value (n-fold). of 1011 M–106 M DHT (Fig. 1) with maximal induction of 16-fold relative to that of the solvent control achieved at a concentration of 106 M DHT, and the EC50 was 0.286 nM. 2.4. Statistical analysis 3.2. Estrogenic disrupting potency All experiments were conducted in duplicate, and within an experiment, each concentration was tested in triplicate. Values are reported as the mean ± standard deviations (SD) (n = 3). Triplicate wells were dosed for each bioassay. Calculations were made with SPSS 12.0. Before parametric analysis, the normality of each sample set was assessed with the Kolomogrov–Smirnov one-sample test, followed by Duncan’s multiple comparisons, when appropriate. The level of significance was set at ⁄P < 0.05 and ⁄⁄P < 0.01. For agonists, treatments were compared to the solvent control group; while estrogen antagonists, androgen antagonists, hypothyroid treatments were compared to the E2, T3, DHT positive control groups. 3.2.1. Estrogenic effects When BDE47, 20 -HO-BDE28, 40 -HO-BDE17, 6-HO-BDE47, and 0 4 -HO-BDE49 were administered to the transfected CV-1 cells only 40 -HO-BDE17 induced luciferase activity (Fig. 2). Induction of luciferase activity occurred in a dose-dependent manner. Concentrations of 1 and 10 lM 40 -HO-BDE17 resulted in 2.3- and 3-fold greater luciferase relative to control, respectively. This result is consistent with 40 -HO-BDE17 being a relatively weak ER agonist compared to the E2 standard. 3. Results 3.1. Cell viability and system creditability Cytotoxicity of chemicals, as assessed by MTT assay and microscopic examination, was assessed in cells transfected with the plasmid. To avoid cytotoxicity caused by the chemicals, all concentrations were less than 105 M. No cytotoxicity was observed for BDE47, 20 -HO-BDE28, 40 -HO-BDE17, 6-HO-BDE47, or 40 -HO-BDE49 in CV-1 or MDA-kb2 cells, when tested alone or with 1 nM E2, 5 nM T3, and 1 nM DHT (P > 0.05 for difference between control groups and treated groups). The CV-1 cell and MDA-kb2 cell reporter assay systems exhibited appropriate responses to the natural estrogen E2, TR ligand T3 and AR agonist DHT. All of the model test chemicals caused transcription and expression of luciferase in a concentrationdependent manner (Fig. 1). From the dose–response relationship, E2 induced luciferase activity in the range of 109 M–107 M (Fig. 1), with 4-fold and maximal induction of 17-fold of control achieved at a concentration of 109, 107 M E2, respectively. The median effective concentration (EC50) was 2.4 nM E2. For T3, the maximal induction of 11-fold relative to that of solvent control was achieved at a concentration of 106 M T3, and the EC50 was 4.60 nM T3 (Fig. 1). DHT induced luciferase activity in the range Relative luciferase activity a (n-fold of control) 25 E2 T3 DHT 20 15 10 3.2.2. Anti-estrogenic effects The anti-estrogenic potential of the test chemicals was determined by treating CV-1 cells with 0.1–10 lM concentrations of PBDEs in the presence of 1 nM of E2. In the absence of test chemicals, this E2 concentration induced luciferase activity that was 4.45 ± 0.19-fold greater than the control value (Fig. 1). BDE47, 20 HO-BDE28, 6-HO-BDE47, and 40 -HO-BDE17 inhibited the expression of Luc activity induced by E2 (1 nM). IC50 values were 21.11, 9.49, 2.65, and 1.14 lM for BDE47, 20 -HO-BDE28, 6-HOBDE47, and 40 -HO-BDE17, respectively (Table 2). At 1 lM, 6-HOBDE47, and 40 -HO-BDE17 were both significant ER antagonists and strongly inhibited the luciferase activity induced by E2 in a dose-dependent manner. At the greater concentration of 10 lM, BDE47, and 20 -HO-BDE28 also inhibited luciferase activity induced by E2. Only 40 -HO-BDE49 showed no ER interactions at the concentrations tested (Fig. 3). The LOEC, EC50 and maximum induction of each chemical are presented (Table 2). The order of anti-estrogenicity potency was BDE47 < 20 -HO-BDE28 < 6-HO-BDE47 < 40 -HO-BDE17. No anti-estrogenic effect of 40 -HO-BDE49 was observed at the tested dosages. 3.3. Thyroid hormone disrupting potency For BDE47, 40 -HO-BDE17, 20 -HO-BDE28, 6-HO-BDE47 and 40 HO-BDE49, the induction of luciferase was not significantly greater than vehicle control, which suggested that none of the compounds exhibited any TR potency at concentrations ranging from 107 to 105 M or 5 105 M. Effects of BDE47 and OH-analogs on TR-mediated responses in the presence of 5 nM T3 were determined (Fig. 4). Co-exposure to 1 or 10 lM 40 -HO-BDE17 with 5 nM T3 resulted in significantly (P < 0.05) greater Luc expression of 137% and 157% of control, respectively. Co-exposure to 0.1 lM and 1 lM 40 -HO-BDE49 with 5 nM T3 resulted in significantly greater TR-mediated expression of Luc of 137% and 138% of control, respectively. However, 10 lM 40 -HO-BDE49 with 5 nM T3 resulted in no change on TR-mediated expression of Luc. No effect was observed with the increasing of exposure concentration to. BDE47, 20 -HO-BDE28, 6-HO-BDE47 did not affect expression of Luc induced by 5 nM T3. 5 3.4. Androgenic disrupting potency 0 Ctrol -10 -9 -8 -7 -6 -5 Concentration (LogM) Fig. 1. Effects of E2, T3 and DHT in reporter gene assays. Estrogenic potency of E2 in ER-mediated reporter gene assay with CV-1 cells transfected with pERE-TATA-Luc and rERa/pCI. Thyroid hormone potency of T3 in TR-mediated reporter gene assay with CV-1 cells transfected with pUAS-tk-luc and pGal4-L-TR. Androgenic potency of DHT in MDA-kb2 cells stably transfected with MMTV-luciferase. BDE47 and all of the OH-analogs, except 40 -HO-BDE49 were antiandrogenic. Exposure to 0.1, 1 and 10 lM BDE47 in the presence of 1 nM DHT resulted in significant (P < 0.05) inhibition of AR-mediated Luc expression by 33%, 23% and 66% relative to that of the control, respectively (Fig. 5). The IC50 for AR antagonist potency of BDE47 was 3.83 lM (Table 3). There was no inhibition of AR-mediated Luc expression by 0.1 or 1 lM, 20 -HO-BDE28, but Author's personal copy 290 H. Liu et al. / Marine Pollution Bulletin 63 (2011) 287–296 Fig. 2. Estrogenic activities of chemicals measured with reporter gene assay with CV-1 cells. Estrogenic potency is reported as expression relative to that of untreated cells (control) (mean ± SD) of three independent experiments. ⁄P < 0.05 vs. Control, ⁄⁄P < 0.01 vs. Control. Dashed line is meant to show hypothetical parallelism to the E2 standard curve. 10 lM, 20 -HO-BDE28 inhibited Luc expression induced by 1 nM DHT by 25%. The IC50 for 20 -HO-BDE28 was 29.22 lM (Fig. 5). 6HO-BDE47 was an AR antagonist and inhibited the luciferase activity induced by DHT with an IC50 value of 0.34 lM. At 0.1, 1, and 10 lM, 40 -HO-BDE17 was a significant AR antagonist and inhibited luciferase expression induced by DHT in a dose-dependent manner. Inhibition rates were 23%, 47% and 65%, respectively, with an IC50 of 1.41 lM. 40 -HO-BDE49 did not show androgenic tran- scriptional activity at the tested concentrations. Lowest observed inhibition concentrations for 20 -HO-BDE28, 6-HO-BDE47, and 40 HO-BDE17 were 0.10, 10.00, 0.50 and 0.10 lM, respectively (Table 3), with IC50 values of 3.83, 29.22, 0.34 and 1.41 lM, respectively. Endocrine disrupting potency of BDE47, 20 -HO-BDE28, 40 -HOBDE17, 6-HO-BDE47, and 40 -HO-BDE49 in the reporter gene assay using the different cells with the deficient in metabolic activity are summarized in Table 4. Author's personal copy H. Liu et al. / Marine Pollution Bulletin 63 (2011) 287–296 Table 2 Anti-estrogenic potencies of BDE47, 20 -HO-BDE28, 40 -HO-BDE17, 6-HO-BDE47, and 40 -HO-BDE49 in the reporter gene assay with CV-1 cells. Chemical Bromine substitution LOEC (lM) IC50 (lM) Maximum luciferase induction (%) BDE47 20 -HOBDE28 6-HOBDE47 40 -HOBDE17 40 -HOBDE49 2,20 ,4,40 2,4,40 10.00 10.00 21.11 9.49 35.13 50.39 2,20 ,4,40 1.00 2.65 39.79 2,20 ,4 1.00 1.14 82.44 2,20 ,4,50 — — — — Not achieved. 4. Discussion PBDEs, as well as some other BFRs and their analogs, are considered to be potential endocrine disruptors (Darnerud et al., 2001). Since PBDEs, as well as their OH-analogs, have been reported in humans and wildlife, and their concentrations are increasing, it was deemed appropriate to assess their possible effects on endocrine responses. There is growing concern regarding the potential developmental and endocrine effects of PDBEs, but limited information is available regarding congener- or analogs-specific effects on maternal endocrine responses. The transient ER reporter gene assay in the CV-1 cell line was an excellent tool for the ER reporter gene assays, as this cell line does not express endogenous steroid receptors, and thus little background was observed in these assays. Previous studies had reported that BDE47 showed estrogenic activity in human T47D breast cancer cells (Hamers et al., 2006), while other researchers using the same methods reported that BDE47 was weakly estrogenic. In the present study, BDE47 did not induce reporter gene expression but the corresponding analog 40 -HO-BDE17 was a significant ER agonist. BDE47 can inhibit estradiol sulfotransferase (E2SULT) activity, which is the main sulfate metabolism enzyme in the estrogen pathway (and reduces estrogenic activity) (Hamers et al., 2006). Some BDE can cause up-regulation of phase I metabolic enzymes such as CYP17 and CYP19 which are involved production of E2. However, BDE47 has been reported to have no effect on CYP17 and CYP19 activity (Cantón et al., 2005, 2006). This indicates that BDE47 interferes with estrogen through a variety of ways, but the actual mechanism by which it interferes with estrogen responses is still not clear. The finding that 40 -HO-BDE17 inhibited ER-mediated reporter gene expression suggested that this BDE analog was both an agonist and an antagonist of estrogen. In contrast to this study where 40 -HO-BDE17 could induce the expression of Luc under the control of the estrogen receptor, 40 -HO-BDE49 did not, although MercadoFeliciano and Bigsby (2008) reported that both 40 -HO-BDE17 and 40 -HO-BDE49 show estrogenic activity. This difference may arise from differences in concentrations tested, since the concentrations of 40 -HO-BDE49 shown to have estrogenic effects exceeded the concentration range of the present experiments. However, the study by Mercado-Feliciano and Bigsby (2008) focused on whether HO-PBDEs would bind to the ER. All of the HO-PBDEs were able to bind to ERa with fairly strong affinity. The fact that 6-HO-BDE47 did not cause up-regulation of the ER-mediated response, but antagonized E2-induced Luc expression is consistent with previous reports (Hamers et al., 2006; MercadoFeliciano and Bigsby, 2008). The 20 -HO-BDE28 analog examined in this study also had no estrogen activity, and had anti-estrogen effects only at very great concentrations. The research by Mercado-Feliciano also showed similar results. Since 20 -HO-BDE28 and 291 6-HO-BDE47 can bind to ERa (Mercado-Feliciano and Bigsby, 2008), it can be inferred that 20 -HO-BDE28 and 6-HO-BDE47 would compete with E2 and have some type of estrogen interaction. When we compared the differences in structure among BDE47, 20 -HO-BDE28, 40 -HO-BDE17, 6-HO-BDE47, and 40 -HO-BDE49, we found that the estrogenic activity of an ER agonist and antagonist is stronger in OH-BDE that contain both an ortho-bromide and a para-hydroxyl (40 -HO-BDE17). As the numbers of bromide atoms are increased, especially in the meta-position, the estrogenic and anti-estrogenic activity decreases, ultimately to undetectable levels as with 40 -HO-BDE49. For estrogenic PBDEs, Meerts et al. (2001) described the common structural features as being ‘‘two ortho (2,6)-bromine atoms on one phenyl ring, at least one para-bromine (preferably on the same phenyl ring as the ortho bromines), and nonbrominated ortho or meta carbons on the other phenyl ring.’’ This kind of change in structure makes the chemical a better fit for the ER and thyroid hormone receptors (TR). This structure–activity relationship resembles the one suggested by Korach et al. (1988) for hydroxylated PCBs, shown in a competitive binding assay, where congeners with the greatest binding affinity for the estrogen receptor contained a substituted phenol ring with a para-hydroxy group (e.g., 4-hydroxy-2́,4́,6́triCB). However, in the case of the brominated diphenyl ethers, the ortho-hydroxylation of bromine has been found to enhance estrogen antagonism (e.g., BDE47 < 20 -HO-BDE28). Addition of an ortho bromine increases estrogen antagonism (e.g., 20 -HOBDE28 < 6-HO-BDE47), which confirmed the general observation by Meerts et al. (2001) that estrogenic activity is associated with lower-brominated PBDEs, while anti-estrogenic activity is associated with greater-brominated PBDEs. Effects on the function of TH are of concern because they are involved in development of the mammalian brain (Kitagawa et al., 2003). Compared to research on estrogens in the environment, little had been done to evaluate BDE47’s thyroid disrupting effects. Some studies have confirmed that PBDEs can affect the thyroid system (Hallgren et al., 2001; Zhou et al., 2002; Darnerud et al., 2007), but the mechanism by which this occurs is unclear. Some studies have shown that BDE47 cannot affect the thyroid system by the way of receptor binding interference. However, several PBDEs interfere with thyroid function as determined by the T-Screen method (Hamers et al., 2006). Together with the T3, BDE47 did not show TR agonistic effects. In vivo BDE47 can be metabolized by various metabolic enzymes through hydroxylation, methylation, sulfonation, and aromatization. Because the cells (CV-1) used in this experiment lack of metabolic activation (Sun et al., 2006), tested compounds does not produce metabolites so the effects observed could be restricted to the parent compound to which they were exposed. Using a recombinant transactivation expression assay with the reporter under the control of the TR a PBDEs mixture and BDE209 were found to not have (anti)-thyroid hormone effects (Liu et al., 2008). However, ‘‘metabolites’’ transformed by S9 microsomal fraction or H4IIE cell showed significant (anti-)thyroid hormone activities. Seventeen PBDE congeners were shown to not bind to the TR at maximum concentrations of 0.25 lM (Meerts et al., 2001). The structure of HO-PBDEs is similar to T2, T3, T4 all of which can bind to TTR. The structure of 6-OH-BDE47 does fully comply with this optimum prerequisite, which can bind TTR (Hamers et al., 2006). As in the present study, BDE47 has no interfere with thyroid hormone. This may explain why the TTR-binding potency of this PBDE analog is greater than for non-hydroxylated PBDEs. These results are consistent with proposed prerequisite for TTRbinding PCBs, which is hydroxylation at the para position with one, but preferably two, adjacent halogen substituents. In this study BDE47 was neither an agonist nor an antagonist of the TR. Author's personal copy 292 H. Liu et al. / Marine Pollution Bulletin 63 (2011) 287–296 Fig. 3. Anti-estrogenic potency of chemicals measured by pERE-aug-Luc and rER/pCI plasmid by reporter gene assay with CV-1 cells. Estrogenic potency is reported relative to that of untreated cells (control). Data are (mean ± SD) of three independent experiments. ⁄P < 0.05 vs.Control, ⁄⁄P < 0.01 vs.Control. Neither of the two ortho-substituted HO-PBDEs interacted with TH, but two para-substituted HO-PBDEs interacted with T3, leading to the enhancement of 5 nM T3 induced Luc expression. This result demonstrates that HO-PBDEs, especially when para-substituted with a hydroxyl group, have stronger thyroid activity than does BDE47. Para-substituted HO-PBDEs could enhance Luc expression induced by T3, which is consistent with HO-PBDEs binding with the TR. Some studies have shown that several chemicals may exert antiandrogenic effect by interfering with androgen receptor (AR) (Sohoni and Sumpter, 1998; Vinggaard et al., 1999). Compared to research on environmental estrogens, little had been done to evaluate androgen effect of BDE47 and its analogs. When MDA-kb2 cells were used to detect compounds antiandrogenic activity, it was found that BDE47 could inhibit Luc expression induced by DHT (Stoker et al., 2005). BDE47 was observed to have anti-androgen potency (Hamers et al., 2006), which was consistent with our results. BDE47 can inhibit binding between artificial androgen R1881 and abdominal prostate cytosol androgen receptor (AR), while inhibition by BDE100 Author's personal copy H. Liu et al. / Marine Pollution Bulletin 63 (2011) 287–296 293 Fig. 4. Thyroid hormone potency of chemicals measured with pUAS-tk-Luc and pGal4-L-TRb plasmid by reporter gene assay with CV-1 cells. Thyroid hormone potency is reported relative to that of untreated cells (control) (mean ± SD) of three independent experiments. Up-regulation of luciferase expression (%) is reported relative to the maximum induction by T3 (5 nM) (100%). Significant differences are indicated by asterisks ⁄P < 0.05 vs. Control, ⁄⁄P < 0.01 vs. Control. was purely competitive (Stoker et al., 2005). This result indicated that BDE47 might bind with AR through competition; the specific mechanisms of the anti-androgen effect needed further study. 20 -HO-BDE28 and 6-HO-BDE47 both have an ortho-hydroxy substituted, with similar structures (Marsh et al., 2006). The study also showed that these two both could inhibit DHT induced Luc expression. It was confirmed that 6-HO-BDE47 could antagonize the effects of DHT (Hamers et al., 2006). In addition PBDEs and HO-BDEs have anti-androgen effects. The strength of anti-androgenicity of the tested chemicals was 6HO-BDE47 > 40 -HO-BDE17 > BDE47 > 20 -HO-BDE28 > 40 -HO-BDE49. More work needs to been done to uncover the structure–activity relationships of EDCs in terms of their estrogenic/androgenic and anti-estrogenic/anti-androgenic activities. Author's personal copy 294 H. Liu et al. / Marine Pollution Bulletin 63 (2011) 287–296 Fig. 5. Anti-androgenic potency in MDA-kb2 cells stably transformed with MMTV-luciferase. Anti-androgenic potency is reported relative to that of untreated cells (control) (mean ± SD) of three independent experiments. Up-regulation of luciferase expression (%) is reported relative to the maximum induction by DHT (1 nM) (100%). Significant differences between the chemicals and the DHT treatment were tested using ANOVA, Dunnett’s test. Significant differences are indicated by asterisks ⁄P < 0.05 vs. Control, ⁄⁄ P < 0.01 vs. Control. Table 3 Anti-androgenic potency of BDE47, 20 -HO-BDE28, 40 -HO-BDE17, 6-HO-BDE47, and 40 -HO-BDE49 in the reporter gene assay with MDA-kb2 cells. Chemical BDE47 20 -HO-BDE28 6-HO-BDE47 40 -HO-BDE17 40 -HO-BDE49 — Not achieved. Bromine substitution LOEC (lM) IC50 (lM) Maximum luciferase induction (%) 2,20 ,4,40 2,4,40 2,20 ,4,40 2,20 ,4 2,20 ,4,50 0.10 10.00 0.50 0.10 — 3.83 29.22 0.34 1.41 — 66.24 25.51 65.70 65.31 — Table 4 Summary of effects of Br- and OH-substitution on the endocrine disrupting potency in p the reporter gene assays , no response; , have response. Chemical BDE47 20 -HO-BDE28 6-HO-BDE47 40 -HO-BDE17 40 -HO-BDE49 Bromine Estrogenic Antisubstitution estrogenic p 0 0 2,2 ,4,4 p 0 2,4,4 p 2,20 ,4,40 p p 2,20 ,4 2,20 ,4,50 Antiandrogenic p p p p Thyroid hormone p p Author's personal copy H. Liu et al. / Marine Pollution Bulletin 63 (2011) 287–296 5. Concludes In wildlife and humans, the most frequently detected and predominant polybrominated diphenyl ether (PBDE) congener is 2,20 ,4,40 -tetrabromodiphenyl ether (BDE47). The potential of BDE47 and its related hydroxylated analogs (20 -HO-BDE28, 6-HOBDE47, 40 -HO-BDE17, and 40 -HO-BDE49) to modulate estrogen receptor-(ER), thyroid receptor-(TR) and androgen receptor-(AR) mediated responses were investigated by use of transactivation reporter gene assays. At concentrations of 1 lM and 10 lM, 40 -HOBDE17 significantly induced the expression of Luc; whereas BDE47, 20 -HO-BDE28, 6-HO-BDE47 and 40 -HO-BDE49 did not induce Luc expression. In contrast, BDE47, 20 -HO-BDE28, 6-HOBDE47, and 40 -HO-BDE17 inhibited the expression of Luc activity induced by 1 nM E2, showing a significant anti-estrogenic effect, with IC50 of 21.11, 9.49, 2.65, and 1.14 lM, respectively. No antiestrogenic effect of 40 -HO-BDE49 was observed at the tested dosages. Although the parent BDE47 and its hydroxylated metabolites all can disturb estrogen balance, a number of the HO-PBDEs may have stronger potency than the parent BDE47 on endocrinedisrupting effects. Acknowledgements This work was supported by the fund of National Natural Science (No. 20737001, 20977047), the National Major Project of Science & Technology Ministry of China (No. 2008ZX08526-003), Specialized Research Fund for the Doctoral Program of Higher Education (No. 200802841030) and the fund of Talent Introduction and Cultivation Foundation of Nanjing University. The research was supported by a Discovery Grant from the National Science and Engineering Research Council of Canada (Project # 32641507) and a Grant from the Western Economic Diversification Canada (Project # 6578 and 6807). The authors wish to acknowledge the support of an instrumentation grant from the Canada Foundation for Infrastructure. Prof. Giesy was supported by the Canada Research Chair program and an at large Chair Professorship at the Department of Biology and Chemistry and State Key Laboratory in Marine Pollution, City University of Hong Kong. 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