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In vitro profiling of endocrine disrupting potency of 2,20,4,40-tetrabromodiphenylether (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, Chinab Key Laboratory of Reproductive Medicine, Institute of Toxicology, Nanjing Medical University, Nanjing 210029, Chinac Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong, SAR, Chinad Department of Biomedical and Veterinary Biosciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canadae Zoology Department, National Food Safety and Toxicology Center and Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824, USA

a r t i c l e i n f o

Keywords:Receptor reporter gene assayBDE47MetaboliteEndocrine 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-HO-BDE28, 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 or10 lM, 40-HO-BDE17 significantly up-regulated expression of Luc, whereas other four chemicals didnot induce Luc expression under control of the ER. Anti-estrogenic potency was observed for 40-HO-BDE17 (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-HO-BDE49 resulted in greater responses of Luc expression induced by T3. BDE47, 20-HO-BDE28, 6-HO-BDE47 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 transcrip-tional activity.

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1. Introduction

Polybrominated diphenyl ethers (PBDEs) have been widely usedas flame retardant additives for plastics and textiles, electronicequipment, 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 (Biet al., 2006; Chen et al., 2006, 2007; Wurl et al., 2006; Guo et al.,2008; Jin et al., 2008) and these compounds are now ubiquitouscontaminants in aquatic organisms (fish, shrimp, and crabs) fromthe lower Yangtze River, which might pose significant risk tohuman and wildlife (Gao et al., 2009).

The 2,20,4,40-tetrabromodiphenylether congener (BDE47) is thepredominant PBDE found in the environment (Hites, 2004; Valterset al., 2005; Marsh et al., 2006; Streets et al., 2006; Xiang et al.,2007) and humans (Örn and Klasson-Wehler, 1998). BDE47 wasthe most abundant congener, contributing more than 40% of the

PPBDE in all species except for the brown bullhead, for which

2,20,4,40,5-pentabromodiphenyl ether (BDE99) contributed thegreatest proportion (Valters et al., 2005). BDE47 has been reportedto 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 ofPBDE by animals (Stapleton et al., 2004; Marsh et al., 2006; Qiuet al., 2007), recent careful investigations of the biotransformationof PBDE have been suggested that the more likely source ofHO-BDE is biotransformation of methoxylated BDE (MeO-BDE)(Wan et al., 2009). Regardless of their source, whether from naturalproducts or from biotransformation of synthetic BDE flame retar-dants the potential effects of HO-BDE need to be assessed andconsidered.

Both PBDEs and their HO-analogs are toxic to animals (Darne-rud et al., 2001; McDonald, 2002) and concentrations of theHO-PBDE can exceed those of PBDE (Pettersson et al., 2004). Thepharmacokinetic behavior of BDE47 is dose dependent, with rela-tive elimination decreasing as the dose is raised (Staskal et al.,2005). BDE47 is well absorbed after oral, tracheal, and peritonealexposure (>80%) and that dermal absorption is >60%. Relatively

0025-326X/$ - see front matter Crown Copyright � 2011 Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.marpolbul.2011.04.019

⇑ Corresponding author. Tel./fax: +86 25 89680556.E-mail address: [email protected] (H. Yu).

Marine Pollution Bulletin 63 (2011) 287–296

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great bioaccumulation factors for BDE47 have been reported(Gustafsson et al., 1999). BDE47 is known to cause various adverseeffects, including being an ER-agonist (Sanderson et al., 2004).BDE47 inhibited binding of the synthetic androgen R1881 to cyto-solic androgen receptor (AR) from the ventral prostate (Stokeret al., 2005). BDE47 also inhibited dihydrotestosterone induced,human AR-regulated transcriptional activation in a luciferase re-porter gene assay (Stoker et al., 2005). Several studies have re-ported that PBDEs bind competitively with human transthyretin(TTR), a transport protein for the thyroid hormones T3 and thyrox-ine (T4), thereby hampering the transportation of thyroid hormone(Meerts et al., 2000; Zhou et al., 2001). A few studies have alsoaddressed the binding ability of PBDEs with TR (Kitamura et al.,2008), but no PBDEs congeners showed affinity for TR. Somehydroxylated PBDEs (HO-BDEs) metabolites analogs which in-duced or inhibited the activity of aromatase (CYP19) are knownto have endocrine disrupting effects (Cantón et al., 2005, 2006),but information about the potential endocrine activity ofOH-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 inhibi-tors of E2 sulfation by SULT1E1, with IC50 values greater than200 nm (Kester et al., 2002). Also, 2-OH-4,20,40-TrCDE inhibitedSULT1E1 activity at micromolar concentrations (Kester et al.,2002). More PBDEs congeners possessed AR antagonistic potencythan 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 potentialendocrine disrupting effects of BDE47 and four analogousHO-BDEs, 20-HO-BDE28, 6-HO-BDE47, 40-HO-BDE17, and 40-HO-BDE49 on ER-, TR- and AR-mediated anti/estrogenic, anti/thyroidand anti/androgenic effects in the African monkey kidney CV-1 cellline 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-BDE49were synthesized in the Department of Biology and Chemistry ofCity 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 ChemicalCo. (St. Louis, MO, USA). All compounds were dissolved in dimethylsulfoxide (DMSO) and the final DMSO concentration in theexposure medium was 0.1% (v/v) to avoid potential effects on cellyields.

2.2. Plasmids

Recombinant plasmids pERE-TATA-Luc, and pUAS-tk-Luc, withluciferase under control of the estrogen response (ERE), andthyroid response (TRE) elements, respectively, were developed asdescribed (Takeyoshi et al., 2002; Sun et al., 2008).

2.3. Reporter gene assays

2.3.1. ER reporter gene assayGreen monkey kidney fibroblast (CV-1) cells that do not contain

the endogenous receptors (ER and TR) were obtained from theInstitute of Biochemistry and Cell Biology at the Shanghai, ChineseAcademy of Science, and maintained in Dulbecco’s modified Eagle’smedium (DEME) supplemented with 10% fetal bovine serum (FBS),

100 U/ml penicillin and 100 lg/ml streptomycin at 37 �C in anatmosphere containing 5% CO2. Cell culture and exposures wereconducted according to previously described methods (Sun et al.,2008). The host cells were plated into 48-well microplates at a den-sity of 0.5 � 105 cells per well in the phenol red free DMEM med-ium containing 5% charcoal–dextran-stripped FBS (CDS-FBS).Twelve hours after seeding, CV-1 cells were transfected with0.5 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 wasremoved and selected concentrations of chemicals dissolved inmedium. Cells were exposed to E2 (1.0 � 10�10–1.0 � 10�7 M in10-fold dilution steps), solvent-controls or test chemicals for24 h. Dosing solutions of the chemicals were diluted with DMSOto a maximum DMSO concentration of 0.1%. After rinsing threetimes with phosphate-buffered saline (PBS, pH 7.4), cells werelysed with 1� lysis buffer (Promega, Madison, WI, USA, 50 ll/well).Light emission from expression of luciferase was determinedimmediately using the luciferase reporter assay system kit (Prome-ga, Madison, WI, USA) and a 48-well plate luminometer (BertholdDetection System, Pforzheim, Germany). The relative transcrip-tional activity was converted to fold induction relative to thesolvent control (n-fold).

2.3.2. TR reporter gene assayCV-1 cells were transfected with appropriate TR transactivation

vectors (Table 1). CV-1 cells were cultured and plated as describedabove and were transfected after 12 h. After a 12 h incubation, theCV-1 cells were exposed for 24 h to T3 (1.0 � 10�12–1.0 � 10�6 Min 10-fold dilution steps), solvent controls or compounds. Follow-ing cytolysis, cell lysates were analyzed immediately using a 48-well plate luminometer (Berthold Detection System, Pforzheim,Germany).

2.3.3. AR reporter gene assayThe MDA-kb2 cell line (ATCC, USA), stably transformed with

murine mammalian tumor virus (MMTV)-luciferase was culturedin Leibovitz’s L-15 medium with 10% FBS, 100 U/ml penicillin(Sigma), 100 lg/ml streptomycin (Sigma), and 0.25 g/ml ampho-tericin B (Sigma) at 37 �C without CO2. For experiments, cells wereplated at 1 � 104 cells per well in 100 ll of medium in 96-wellluminometer plates. When cells were attached (4–6 h), mediumwas removed and replaced with dosing medium. The MDA-kb2cells were exposed to DHT (Sigma, 1.0 � 10�12–1.0 � 10�7 M in10-fold dilution steps), solvent-control and compounds for 24 h.DHT was tested in a concentration range that had been previouslyshown to not be cytotoxic (Shen et al., 2009). After three rinseswith phosphate-buffered saline (PBS, pH 7.4), the cells were lysedwith 1� passive lysis buffer (Promega). After centrifuged at12,000g for 10 min to remove debris, the cell lysates were analyzedimmediately using a 96-well plate luminometer (BertholdDetection System, Pforzheim, Germany). The amount of luciferasewas measured with the luciferase reporter assay system kit(Promega, Madison, WI, USA) following the manufacturer’s

Table 1Concentrations of plasmids and transfection reagent for estrogenic and thyroidreporter gene assay systems.

Estrogenic activities Thyroid activities

Plasmid pERE-TATA-Luc 0.25 lg/well pUAS-tk-Luc 0.25 lg/wellrERa/pCI 0.10 lg/well pGal4-L-TRb 0.25 lg/well

Transfectionreagent

Sofast™ 0.5 lg/well Sofast™ 0.5 lg/well

288 H. Liu et al. / Marine Pollution Bulletin 63 (2011) 287–296


instructions. The relative luminescence units, which is a surrogatefor transcriptional activity was converted to fold induction abovethe corresponding vehicle control value (n-fold).

2.4. Statistical analysis

All experiments were conducted in duplicate, and within anexperiment, each concentration was tested in triplicate. Valuesare reported as the mean ± standard deviations (SD) (n = 3). Tripli-cate wells were dosed for each bioassay. Calculations were madewith SPSS 12.0. Before parametric analysis, the normality of eachsample set was assessed with the Kolomogrov–Smirnov one-sam-ple test, followed by Duncan’s multiple comparisons, when appro-priate. The level of significance was set at ⁄P < 0.05 and ⁄⁄P < 0.01.For agonists, treatments were compared to the solvent controlgroup; while estrogen antagonists, androgen antagonists, hypothy-roid treatments were compared to the E2, T3, DHT positive controlgroups.

3. Results

3.1. Cell viability and system creditability

Cytotoxicity of chemicals, as assessed by MTT assay andmicroscopic examination, was assessed in cells transfected withthe plasmid. To avoid cytotoxicity caused by the chemicals, allconcentrations were less than 10�5 M. No cytotoxicity wasobserved for BDE47, 20-HO-BDE28, 40-HO-BDE17, 6-HO-BDE47, or40-HO-BDE49 in CV-1 or MDA-kb2 cells, when tested alone or with1 nM E2, 5 nM T3, and 1 nM DHT (P > 0.05 for difference betweencontrol groups and treated groups).

The CV-1 cell and MDA-kb2 cell reporter assay systems exhib-ited appropriate responses to the natural estrogen E2, TR ligandT3 and AR agonist DHT. All of the model test chemicals causedtranscription and expression of luciferase in a concentration-dependent manner (Fig. 1). From the dose–response relationship,E2 induced luciferase activity in the range of 10�9 M–10�7 M(Fig. 1), with 4-fold and maximal induction of 17-fold of controlachieved at a concentration of 10�9, 10�7 M E2, respectively. Themedian effective concentration (EC50) was 2.4 nM E2. For T3, themaximal induction of 11-fold relative to that of solvent controlwas achieved at a concentration of 10�6 M T3, and the EC50 was4.60 nM T3 (Fig. 1). DHT induced luciferase activity in the range

of 10�11 M–10�6 M DHT (Fig. 1) with maximal induction of 16-foldrelative to that of the solvent control achieved at a concentration of10�6 M DHT, and the EC50 was 0.286 nM.

3.2. Estrogenic disrupting potency

3.2.1. Estrogenic effectsWhen BDE47, 20-HO-BDE28, 40-HO-BDE17, 6-HO-BDE47, and

40-HO-BDE49 were administered to the transfected CV-1 cells only40-HO-BDE17 induced luciferase activity (Fig. 2). Induction of lucif-erase activity occurred in a dose-dependent manner. Concentra-tions of 1 and 10 lM 40-HO-BDE17 resulted in 2.3- and 3-foldgreater luciferase relative to control, respectively. This result isconsistent with 40-HO-BDE17 being a relatively weak ER agonistcompared to the E2 standard.

3.2.2. Anti-estrogenic effectsThe anti-estrogenic potential of the test chemicals was deter-

mined by treating CV-1 cells with 0.1–10 lM concentrations ofPBDEs in the presence of 1 nM of E2. In the absence of test chem-icals, this E2 concentration induced luciferase activity that was4.45 ± 0.19-fold greater than the control value (Fig. 1). BDE47, 20-HO-BDE28, 6-HO-BDE47, and 40-HO-BDE17 inhibited the expres-sion of Luc activity induced by E2 (1 nM). IC50 values were21.11, 9.49, 2.65, and 1.14 lM for BDE47, 20-HO-BDE28, 6-HO-BDE47, and 40-HO-BDE17, respectively (Table 2). At 1 lM, 6-HO-BDE47, and 40-HO-BDE17 were both significant ER antagonistsand strongly inhibited the luciferase activity induced by E2 in adose-dependent manner. At the greater concentration of 10 lM,BDE47, and 20-HO-BDE28 also inhibited luciferase activity inducedby E2. Only 40-HO-BDE49 showed no ER interactions at the concen-trations tested (Fig. 3).

The LOEC, EC50 and maximum induction of each chemicalare presented (Table 2). The order of anti-estrogenicity potencywas BDE47 < 20-HO-BDE28 < 6-HO-BDE47 < 40-HO-BDE17. Noanti-estrogenic effect of 40-HO-BDE49 was observed at the testeddosages.

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 greaterthan vehicle control, which suggested that none of the compoundsexhibited any TR potency at concentrations ranging from 10�7 to10�5 M or 5 � 10�5 M.

Effects of BDE47 and OH-analogs on TR-mediated responses inthe presence of 5 nM T3 were determined (Fig. 4). Co-exposureto 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 with5 nM T3 resulted in significantly greater TR-mediated expressionof Luc of 137% and 138% of control, respectively. However, 10 lM40-HO-BDE49 with 5 nM T3 resulted in no change on TR-mediatedexpression of Luc. No effect was observed with the increasing ofexposure concentration to. BDE47, 20-HO-BDE28, 6-HO-BDE47did not affect expression of Luc induced by 5 nM T3.

3.4. Androgenic disrupting potency

BDE47 and all of the OH-analogs, except 40-HO-BDE49 wereantiandrogenic. Exposure to 0.1, 1 and 10 lM BDE47 in the pres-ence of 1 nM DHT resulted in significant (P < 0.05) inhibition ofAR-mediated Luc expression by 33%, 23% and 66% relative to thatof the control, respectively (Fig. 5). The IC50 for AR antagonist po-tency of BDE47 was 3.83 lM (Table 3). There was no inhibition ofAR-mediated Luc expression by 0.1 or 1 lM, 20-HO-BDE28, but

0

5

10

15

20

25

Ctrol -10 -9 -8 -7 -6 -5

Concentration (LogM)

Rel

ativ

e lu

cife

rase

act

ivity

a(n

-fol

d of

con

trol

)

E2

T3

DHT

Fig. 1. Effects of E2, T3 and DHT in reporter gene assays. Estrogenic potency of E2 inER-mediated reporter gene assay with CV-1 cells transfected with pERE-TATA-Lucand rERa/pCI. Thyroid hormone potency of T3 in TR-mediated reporter gene assaywith CV-1 cells transfected with pUAS-tk-luc and pGal4-L-TR. Androgenic potencyof DHT in MDA-kb2 cells stably transfected with MMTV-luciferase.

H. Liu et al. / Marine Pollution Bulletin 63 (2011) 287–296 289


10 lM, 20-HO-BDE28 inhibited Luc expression induced by 1 nMDHT by 25%. The IC50 for 20-HO-BDE28 was 29.22 lM (Fig. 5). 6-HO-BDE47 was an AR antagonist and inhibited the luciferase activ-ity induced by DHT with an IC50 value of 0.34 lM. At 0.1, 1, and10 lM, 40-HO-BDE17 was a significant AR antagonist and inhibitedluciferase expression induced by DHT in a dose-dependent man-ner. Inhibition rates were 23%, 47% and 65%, respectively, withan IC50 of 1.41 lM. 40-HO-BDE49 did not show androgenic tran-

scriptional activity at the tested concentrations. Lowest observedinhibition concentrations for 20-HO-BDE28, 6-HO-BDE47, and 40-HO-BDE17 were 0.10, 10.00, 0.50 and 0.10 lM, respectively (Table3), with IC50 values of 3.83, 29.22, 0.34 and 1.41 lM, respectively.

Endocrine disrupting potency of BDE47, 20-HO-BDE28, 40-HO-BDE17, 6-HO-BDE47, and 40-HO-BDE49 in the reporter gene assayusing the different cells with the deficient in metabolic activity aresummarized in Table 4.

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 standardcurve.



4. Discussion

PBDEs, as well as some other BFRs and their analogs, are consid-ered to be potential endocrine disruptors (Darnerud et al., 2001).Since PBDEs, as well as their OH-analogs, have been reported in hu-mans and wildlife, and their concentrations are increasing, it wasdeemed appropriate to assess their possible effects on endocrineresponses. There is growing concern regarding the potential devel-opmental and endocrine effects of PDBEs, but limited informationis available regarding congener- or analogs-specific effects onmaternal endocrine responses.

The transient ER reporter gene assay in the CV-1 cell line was anexcellent tool for the ER reporter gene assays, as this cell line doesnot express endogenous steroid receptors, and thus littlebackground was observed in these assays. Previous studies hadreported that BDE47 showed estrogenic activity in human T47Dbreast cancer cells (Hamers et al., 2006), while other researchersusing the same methods reported that BDE47 was weakly estro-genic. In the present study, BDE47 did not induce reporter geneexpression but the corresponding analog 40-HO-BDE17 was asignificant ER agonist. BDE47 can inhibit estradiol sulfotransferase(E2SULT) activity, which is the main sulfate metabolism enzyme inthe estrogen pathway (and reduces estrogenic activity) (Hamerset al., 2006). Some BDE can cause up-regulation of phase I meta-bolic enzymes such as CYP17 and CYP19 which are involvedproduction of E2. However, BDE47 has been reported to have noeffect on CYP17 and CYP19 activity (Cantón et al., 2005, 2006). Thisindicates that BDE47 interferes with estrogen through a variety ofways, but the actual mechanism by which it interferes with estro-gen responses is still not clear.

The finding that 40-HO-BDE17 inhibited ER-mediated reportergene expression suggested that this BDE analog was both an ago-nist and an antagonist of estrogen. In contrast to this study where40-HO-BDE17 could induce the expression of Luc under the controlof the estrogen receptor, 40-HO-BDE49 did not, although Mercado-Feliciano and Bigsby (2008) reported that both 40-HO-BDE17 and40-HO-BDE49 show estrogenic activity. This difference may arisefrom differences in concentrations tested, since the concentrationsof 40-HO-BDE49 shown to have estrogenic effects exceeded theconcentration range of the present experiments. However, thestudy by Mercado-Feliciano and Bigsby (2008) focused on whetherHO-PBDEs would bind to the ER. All of the HO-PBDEs were able tobind to ERa with fairly strong affinity.

The fact that 6-HO-BDE47 did not cause up-regulation of theER-mediated response, but antagonized E2-induced Luc expressionis consistent with previous reports (Hamers et al., 2006; Mercado-Feliciano and Bigsby, 2008). The 20-HO-BDE28 analog examined inthis study also had no estrogen activity, and had anti-estrogeneffects only at very great concentrations. The research by Merca-do-Feliciano also showed similar results. Since 20-HO-BDE28 and

6-HO-BDE47 can bind to ERa (Mercado-Feliciano and Bigsby,2008), it can be inferred that 20-HO-BDE28 and 6-HO-BDE47 wouldcompete 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, wefound that the estrogenic activity of an ER agonist and antagonistis stronger in OH-BDE that contain both an ortho-bromide and apara-hydroxyl (40-HO-BDE17). As the numbers of bromide atomsare increased, especially in the meta-position, the estrogenic andanti-estrogenic activity decreases, ultimately to undetectablelevels as with 40-HO-BDE49. For estrogenic PBDEs, Meerts et al.(2001) described the common structural features as being ‘‘twoortho (2,6)-bromine atoms on one phenyl ring, at least onepara-bromine (preferably on the same phenyl ring as the orthobromines), and nonbrominated ortho or meta carbons on the otherphenyl ring.’’ This kind of change in structure makes the chemical abetter fit for the ER and thyroid hormone receptors (TR).

This structure–activity relationship resembles the one sug-gested by Korach et al. (1988) for hydroxylated PCBs, shown in acompetitive binding assay, where congeners with the greatestbinding affinity for the estrogen receptor contained a substitutedphenol ring with a para-hydroxy group (e.g., 4-hydroxy-2́,4́,6́-triCB). However, in the case of the brominated diphenyl ethers,the ortho-hydroxylation of bromine has been found to enhanceestrogen antagonism (e.g., BDE47 < 20-HO-BDE28). Addition of anortho bromine increases estrogen antagonism (e.g., 20-HO-BDE28 < 6-HO-BDE47), which confirmed the general observationby Meerts et al. (2001) that estrogenic activity is associated withlower-brominated PBDEs, while anti-estrogenic activity is associ-ated with greater-brominated PBDEs.

Effects on the function of TH are of concern because they are in-volved in development of the mammalian brain (Kitagawa et al.,2003). Compared to research on estrogens in the environment, lit-tle had been done to evaluate BDE47’s thyroid disrupting effects.Some studies have confirmed that PBDEs can affect the thyroid sys-tem (Hallgren et al., 2001; Zhou et al., 2002; Darnerud et al., 2007),but the mechanism by which this occurs is unclear. Some studieshave shown that BDE47 cannot affect the thyroid system by theway of receptor binding interference. However, several PBDEsinterfere with thyroid function as determined by the T-Screenmethod (Hamers et al., 2006). Together with the T3, BDE47 didnot show TR agonistic effects.

In vivo BDE47 can be metabolized by various metabolicenzymes through hydroxylation, methylation, sulfonation, andaromatization. Because the cells (CV-1) used in this experimentlack of metabolic activation (Sun et al., 2006), tested compoundsdoes not produce metabolites so the effects observed could berestricted to the parent compound to which they were exposed.

Using a recombinant transactivation expression assay with thereporter under the control of the TR a PBDEs mixture and BDE209were found to not have (anti)-thyroid hormone effects (Liu et al.,2008). However, ‘‘metabolites’’ transformed by S9 microsomalfraction or H4IIE cell showed significant (anti-)thyroid hormoneactivities. Seventeen PBDE congeners were shown to not bind tothe 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 whichcan bind to TTR. The structure of 6-OH-BDE47 does fully complywith this optimum prerequisite, which can bind TTR (Hamerset al., 2006). As in the present study, BDE47 has no interfere withthyroid hormone. This may explain why the TTR-binding potencyof this PBDE analog is greater than for non-hydroxylated PBDEs.These results are consistent with proposed prerequisite for TTR-binding PCBs, which is hydroxylation at the para position withone, but preferably two, adjacent halogen substituents. In thisstudy BDE47 was neither an agonist nor an antagonist of the TR.

Table 2Anti-estrogenic potencies of BDE47, 20-HO-BDE28, 40-HO-BDE17, 6-HO-BDE47, and40-HO-BDE49 in the reporter gene assay with CV-1 cells.

Chemical Brominesubstitution

LOEC(lM)

IC50(lM)

Maximum luciferaseinduction (%)

BDE47 2,20 ,4,40 10.00 21.11 35.1320-HO-

BDE282,4,40 10.00 9.49 50.39

6-HO-BDE47

2,20 ,4,40 1.00 2.65 39.79

40-HO-BDE17

2,20 ,4 1.00 1.14 82.44

40-HO-BDE49

2,20 ,4,50 — — —

— Not achieved.



Neither of the two ortho-substituted HO-PBDEs interacted with TH,but two para-substituted HO-PBDEs interacted with T3, leading tothe enhancement of 5 nM T3 induced Luc expression. This resultdemonstrates that HO-PBDEs, especially when para-substitutedwith a hydroxyl group, have stronger thyroid activity than doesBDE47. Para-substituted HO-PBDEs could enhance Luc expressioninduced by T3, which is consistent with HO-PBDEs binding withthe TR.

Some studies have shown that several chemicals may exertantiandrogenic effect by interfering with androgen receptor

(AR) (Sohoni and Sumpter, 1998; Vinggaard et al., 1999). Com-pared to research on environmental estrogens, little had beendone to evaluate androgen effect of BDE47 and its analogs.When MDA-kb2 cells were used to detect compounds anti-androgenic activity, it was found that BDE47 could inhibit Lucexpression induced by DHT (Stoker et al., 2005). BDE47 wasobserved to have anti-androgen potency (Hamers et al., 2006),which was consistent with our results. BDE47 can inhibit bind-ing between artificial androgen R1881 and abdominal prostatecytosol androgen receptor (AR), while inhibition by BDE100

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 tothat of untreated cells (control). Data are (mean ± SD) of three independent experiments. ⁄P < 0.05 vs.Control, ⁄⁄P < 0.01 vs.Control.



was purely competitive (Stoker et al., 2005). This result indicatedthat BDE47 might bind with AR through competition; the spe-cific mechanisms of the anti-androgen effect needed furtherstudy. 20-HO-BDE28 and 6-HO-BDE47 both have an ortho-hydro-xy substituted, with similar structures (Marsh et al., 2006). Thestudy also showed that these two both could inhibit DHTinduced Luc expression. It was confirmed that 6-HO-BDE47

could antagonize the effects of DHT (Hamers et al., 2006). Inaddition PBDEs and HO-BDEs have anti-androgen effects. Thestrength of anti-androgenicity of the tested chemicals was 6-HO-BDE47 > 40-HO-BDE17 > BDE47 > 20-HO-BDE28 > 40-HO-BDE49.More work needs to been done to uncover the structure–activityrelationships of EDCs in terms of their estrogenic/androgenic andanti-estrogenic/anti-androgenic activities.

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 isreported relative to that of untreated cells (control) (mean ± SD) of three independent experiments. Up-regulation of luciferase expression (%) is reported relative to themaximum induction by T3 (5 nM) (100%). Significant differences are indicated by asterisks ⁄P < 0.05 vs. Control, ⁄⁄P < 0.01 vs. Control.



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%). Significantdifferences 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 3Anti-androgenic potency of BDE47, 20-HO-BDE28, 40-HO-BDE17, 6-HO-BDE47, and40-HO-BDE49 in the reporter gene assay with MDA-kb2 cells.


LOEC(lM)

IC50(lM)

Maximumluciferaseinduction(%)

BDE47 2,20 ,4,40 0.10 3.83 66.2420-HO-BDE28 2,4,40 10.00 29.22 25.516-HO-BDE47 2,20 ,4,40 0.50 0.34 65.7040-HO-BDE17 2,20 ,4 0.10 1.41 65.3140-HO-BDE49 2,20 ,4,50 — — —

— Not achieved.

Table 4Summary of effects of Br- and OH-substitution on the endocrine disrupting potency inthe reporter gene assays �, no response;

p, have response.


Estrogenic Anti-estrogenic

Anti-androgenic

Thyroidhormone

BDE47 2,20 ,4,40 � p p �20-HO-BDE28 2,4,40 � p p �6-HO-BDE47 2,20 ,4,40 � p p �40-HO-BDE17 2,20 ,4

p p p p

40-HO-BDE49 2,20 ,4,50 � � � p



5. Concludes

In wildlife and humans, the most frequently detected andpredominant polybrominated diphenyl ether (PBDE) congener is2,20,4,40-tetrabromodiphenyl ether (BDE47). The potential ofBDE47 and its related hydroxylated analogs (20-HO-BDE28, 6-HO-BDE47, 40-HO-BDE17, and 40-HO-BDE49) to modulate estrogenreceptor-(ER), thyroid receptor-(TR) and androgen receptor-(AR)mediated responses were investigated by use of transactivation re-porter gene assays. At concentrations of 1 lM and 10 lM, 40-HO-BDE17 significantly induced the expression of Luc; whereasBDE47, 20-HO-BDE28, 6-HO-BDE47 and 40-HO-BDE49 did not in-duce Luc expression. In contrast, BDE47, 20-HO-BDE28, 6-HO-BDE47, and 40-HO-BDE17 inhibited the expression of Luc activityinduced 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 anti-estrogenic effect of 40-HO-BDE49 was observed at the testeddosages. Although the parent BDE47 and its hydroxylated metabo-lites all can disturb estrogen balance, a number of the HO-PBDEsmay have stronger potency than the parent BDE47 on endocrine-disrupting effects.

Acknowledgements

This work was supported by the fund of National Natural Sci-ence (No. 20737001, 20977047), the National Major Project of Sci-ence & Technology Ministry of China (No. 2008ZX08526-003),Specialized Research Fund for the Doctoral Program of Higher Edu-cation (No. 200802841030) and the fund of Talent Introductionand Cultivation Foundation of Nanjing University. The researchwas supported by a Discovery Grant from the National Scienceand Engineering Research Council of Canada (Project # 326415-07) and a Grant from the Western Economic Diversification Canada(Project # 6578 and 6807). The authors wish to acknowledge thesupport of an instrumentation grant from the Canada Foundationfor Infrastructure. Prof. Giesy was supported by the Canada Re-search Chair program and an at large Chair Professorship at theDepartment of Biology and Chemistry and State Key Laboratoryin Marine Pollution, City University of Hong Kong.

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