13
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C
0
0
a
c a
a
acdef
b
b
a
b
* a
Received 3rd November 2010, Accepted 25th February 2011
DOI: 10.1039/c0em00654h
Polybrominated diphenyl ethers (PBDEs) constitute an important group of flame retardants.
2,2 0 ,4,4 0 ,6-Pentabromodiphenylether (BDE100) is a prominent PBDE congener in some human populations. The potential of BDE100 to modulate responses mediated by the estrogen (ER), thyroid hormone (ThR) or androgen receptors (AR) were investigated by use of transactivation reporter gene assays. The African green monkey kidney CV-1 cell transiently transfected with the constructed reporter gene plasmid ERE-TATA-Luc and pUAS-tk-Luc with luciferase (Luc) under control of the estrogen response (ERE), or thyroid hormone response (ThRE) elements were used to evaluate
(anti)estrogen and thyroid effects of BDE100. The (anti)androgenic potency of BDE100 was also evaluated by use of MDA-kb2 cells, which were stably transfected with MMTV-luciferase. The assays displayed appropriate responses to known natural estrogen 17 b -estradiol (E2), ThR ligand triiodothyronine (T3), and the AR agonist 5 a -dihydrotestosterone (DHT). 10 or 50 m M BDE100 significantly up-regulated expression of Luc under control of the ER. Antiestrogenic potency was observed for BDE100 (IC50 ¼ 6.21
m M). Co-exposure to 50 m M BDE100 significantly enhanced expression of Luc caused by 5 nM T3. BDE100 was antiandrogenic at 10 and 50 m M with an IC50 of
28.60
m M BDE100. These results suggest that BDE100 can modulate the endocrine system in multiple ways by interfering with several hormonal signaling pathways simultaneously.
a State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210093, China. E-mail: hlliu@nju.edu.cn; Tel: +86-25-89680356 b Key Laboratory of Reproductive Medicine, Institute of Toxicology,
Nanjing Medical University, Nanjing 210029, China c 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,
Center for Integrative Toxicology, Michigan State University, East
Lansing, 48824, USA f Zoology Department, College of Science, King Saud University, P.O. Box
2455, Riyadh 11451, Saudi Arabia
† Part of a themed issue featuring work presented at the 2010 SETAC
Asia/Pacific Meeting held in Guangzhou, China, 4–7 June, 2010.
Polybrominated diphenyl ethers (PBDEs) are an important series of flame retardants (BFRs) that are persistent and can be transported long-distances in the environment.
1–4 PBDEs are lipophilic and can be bioaccumulated and biomagnified and have the potential to cause adverse effects.
5,6 Penta-BDE products have been reported to account for 12% of total PBDE production.
7 Tetra- to hexabrominated diphenyl ether (BDE) congeners (BDE47, -99, -100, -153 and
-154) are the most frequently reported BDE congeners in wildlife and humans.
8–12 2,2
0
,4,4
0
,6-Pentabromodiphenylether (BDE100) is a prominent congener in some wildlife and human populations.
13
More and more attention has been focused on BDE100. The U.S.
Environmental Protection Agency 1614 draft methodology lists
PBDEs are now ubiquitous contaminants in many media, which might pose significant risk to human and wildlife. BDE100 is a prominent congener in some wildlife and human populations. More and more attention has been focused on BDE100. The degradation rate of BDE100 is far slower than BDE99 or BDE209. Since BDE100 degrades more slowly, it will be in the environment even longer. In this study, we study the comprehensive endocrine disrupting activity of BDE100. These results suggest that
BDE100 can modulate the endocrine system in multiple ways by interfering with several hormonal signaling pathways simultaneously.
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BDE100 as one of the eight PBDEs of primary concern in the environment.
14–16 In human breast milk from 13 different areas in
Japan, BDE100 accounted for 11% of the total concentration of
PBDEs.
17 In lake trout BDE100 accounted for 8% of the total concentration of PBDEs.
16 BDE100 accounted for 7.7% of the total concentration of PBDE in fish and fish-eating mammals.
16
Concentrations of BDE100 in lake trout from Lake Michigan were even greater than those of BDE47, BDE66 and BDE99.
16 PBDEs can be transformed in the environment. Even though they are both penta-BDEs, BDE100 is more slowly transformed than BDE99.
18
Recently evidence of PBDEs affecting the endocrine system has been reported. The DE-71 mixture of PBDE, which includes
BDE100 has been reported to be an endocrine disruptor.
18–20
Although penta-BDE in commercial products was banned from the EU market (2002/95/EU), it is expected to remain in the environment for a long time. Since BDE100 degrades more slowly, it will be in the environment even longer.
Reporter gene assays are useful tools for screening for aggregate effects on various aspects of the endocrine system.
5 The potencies of BDE100 to modulate estrogen receptor- (ER), thyroid hormone receptor- (ThR) and androgen receptor- (AR) mediated responses were investigated by use of transactivation reporter gene assays.
Chemicals
Test chemicals were the highest grade available for sale or specifically prepared for use in analysis of environmental samples.
17 b -Estradiol (E2, purity > 99%), 5 a -dihydrotestosterone (DHT, purity > 99%), and triiodothyronine (T3, purity > 99%) were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
BDE100 was synthesized in the Department of Biology and
Chemistry of City University of Hong Kong and purity of greater than 98% has been demonstrated. Stock solutions of the chemicals were prepared in dimethyl sulfoxide (DMSO, Sigma) at a concentration of 10 2 M, stored at 20 C, and diluted to desired concentrations in Dulbecco’s modified Eagle medium (DMEM,
Sigma) immediately before use. To avoid solvent effects, final concentrations of DMSO in culture media did not exceed 0.1% (v/v).
Plasmids and cells
The luciferase reporter plasmid pERE-aug-Luc and rat estrogen receptor a (ER a ) expression vector rER a /pCI were provided by Dr
M. Takeyoshi (Chemicals Assessment Center, Chemicals Evaluation and Research Institute, Oita, Japan). The expression plasmid pGal4-
L-TR b and Gal4 responsive luciferase reporter plasmid pUAS-tk-
Luc were obtained from Prof. Ronald M. Evans (Gene Expression
Laboratory, Howard Hughes Medical Institute, San Diego, CA,
USA). African green monkey kidney cells (CV-1) were purchased at the Chinese Academy of Medicine. Stable integration pMMTV-Lucneo plasmid MDA-kb2 cells were purchased from United States
American Type Culture Collection (Rockville, Maryland, USA).
Cell culture and transfection
CV-1 cells were maintained in phenol red-free Dulbecco’s modified Eagle medium (DMEM, Sigma) supplemented with
This journal is ª The Royal Society of Chemistry 2011
10% fetal bovine serum (FBS), 100 U ml
100 m g ml 1 streptomycin at 37
1 penicillin and
C in an atmosphere containing
5% CO
2.
Host cells were plated in 48-well microplates (Nunc,
Denmark) at a density of 0.5
10 5 cells per well in DMEM medium containing 10% charcoal-dextran-stripped FBS (CDS-
FBS) by adding a volume of 0.25 ml per well. Following 12 h of incubation, different endocrine-disrupting activity systems were transfected with different plasmids and transfection reagent
(Table 1). After an incubation period of 24 h, the transfection medium was removed before various concentrations of test chemicals dissolved in medium were added for measurement of estrogenic activities and ThR antagonist activities. Medium with
0.1% dimethyl sulfoxide (DMSO, Sigma) was used as a vehicle control.
MDA-kb2 cells were cultured in L15 medium with 10% fetal bovine serum (FBS) at 37 C in a 100% air incubator. Cells were plated at 1 10 4 cells per well in 100 m l of medium in 96-well luminometer plates. Without plasmid transfection, the medium was removed before various concentrations of test chemicals dissolved in medium were added for measurement of antiandrogenic activity after 12 h. Medium with 0.1% DMSO was also used as a vehicle control.
The MTT assay was performed to detect the cytotoxicity of test chemicals. Both CV-1 and MDA-kb2 cells attached to culture dishes were collected and plated at a density of 1 10 4 cells per 100 m l in each well of 96-well plates using DMEM with
5% DCC serum and L15 with 10% DCC serum, respectively.
After 24 h, cells were treated with vehicle or various concentrations (0.1, 1.0, 10, or 50 m M) of test chemicals alone or with 5 nM
T3, 1.0 nM E2, or 1.0 nM DHT for 24 h. Then, 25 m l 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT,
5 mg ml 1 in PBS, Sigma) was added to each well and continuously incubated at 37 C for 4 h, after which MTT solutions were replaced with 150 m l DMSO and shaken for 10 min to solubilize the crystals. Absorbance was measured by automatic microplate reader (EL808, Bio-Tek, Winooski, VT, USA) at 570 nm.
Reporter gene assays
Cells were harvested 24 h after dosing. After rinsing three times with phosphate-buffered saline (PBS, pH 7.4), cells were lysed with 1 lysis buffer (Promega, Madison, WI, USA, 50 m l per well). After centrifuging at 12 000 g for 10 min to remove debris, cell lysates were either analyzed immediately or quick-frozen at
70 C. Light emission of cell lysates was analyzed using a 96-well plate luminometer (Berthold Detection System,
Pforzheim, Germany).
Statistical analysis
All experiments were conducted in triplicate, and within an experiment, each concentration was tested in triplicate. The values shown are mean SD. Data were analyzed by one-way analysis of variance (ANOVA), followed by Duncan’s multiple comparisons test when appropriate. The level of significance was set at * p < 0.05 and ** p < 0.01. For agonists, treatments were compared to the vehicles control group; while estrogen antagonists, androgen antagonists and hypothyroid treatments were
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Table 1 Concentration of plasmids and transfection reagent for ER activity and ThR activity
Plasmid
Transfection regent
ER activity pERE-aug-Luc
250 ng per well rER a /pCI
100 ng per well
15 m l per well
ThR activity pUAS-tk-Luc
200 ng per well pGal4-L-TR b
75 ng per well
15 m l per well compared to the 1 nM E2, 1 nM DHT and 5 nM positive control groups.
21
Cell viability and responsiveness
Cytotoxicity of BDE100, as assessed by the MTT assay and microscopic examination, was reported for cells transfected with plasmid. No significant ( P > 0.05) cytotoxicity of BDE100 to
CV-1 or MDA-kb2 cells was observed when tested alone or with
1.0 nM E2, 5.0 nM T3, or 1.0 nM DHT.
The CV-1 and MDA-kb2 cell reporter assay systems for ER-,
ThR-, and AR-mediated effects, exhibited appropriate responses to E2, T3 and DHT, respectively. All model chemicals caused transcription and expression of Luc in a concentrationdependent manner (Fig. 1). From the dose-response relationship it was determined that E2 induced Luc activity in the range of
10 9 –10 7 M (Fig. 1), with 4-fold and maximal induction of
17-fold of control achieved at a concentrations of 10
10 7
9 M and
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 10 6 M T3, and the EC50 was 4.60 nM T3 (Fig. 1). DHT induced luciferase activity in the range of 10 11 –10 6 M DHT
(Fig. 1) with maximal induction of 16-fold relative to that of the solvent control was achieved at a concentration of 1.0
m M DHT, and the EC50 was 0.286 nM.
Estrogenic effects
When BDE100 was administered to CV-1 cells that had been transfected with the ERE, induction of Luc activity occurred in a dose-dependent manner (Fig. 2). Concentrations of 1.0
m M and
10 m M BDE100 resulted in 1.31- and 2.10-fold greater Luc activity relative to control, respectively.
Antiestrogenic effects
When BDE100 was co-administered with 1.0 nM E2 (Fig. 3), there were no statistically significant antiestrogenicity detectable at 0.1 or 1.0
m M BDE100. Exposure to 10 or 50 m M BDE100 significantly antagonized E2-induced Luc expression with inhibition rates of 68.36% and 87.76%, respectively. The median inhibitory concentration (IC50) value was 6.21
m M BDE100.
Thyroid hormone disrupting effects
Within the concentration range from 10 7 M to 10 5 M, there was no ThR agonist activity (results not shown). Effects of BDE100 on ThR-mediated responses in the presence of 5.0 nM T3 were also determined. Co-exposure to 50 m M BDE100 with 5.0 nM T3 resulted in significantly ( P < 0.05) greater Luc expression of
202.49% (Fig. 4).
Antiandrogenic effects
Co-exposure of BDE100 with DHT did not reduce Luc expression at concentrations of 0.1 or 1.0
m M BDE100, but significantly less Luc activity was observed at 10 and 50 m M BDE100 (Fig. 5).
Inhibition of AR-mediated Luc expression was 66.07% and
66.35%, respectively relative to that of the control. The median inhibitory concentration (IC50) value was 7.18
m M.
Since BDE100 has been reported to occur in tissues of humans and wildlife, and their concentrations are increasing, it was deemed appropriate to assess the possible effects of BDE100 on endocrine responses. There is growing concern regarding the potential developmental and endocrine effects of PBDEs, but limited information is available regarding thyroid hormone effects on maternal endocrine responses. In the present study, three reporter gene assays were used to investigate potency of
BDE100 to modulate ER-, ThR- and AR-mediated responses.
BDE100 possessed mixed estrogenic/anti-estrogenic activities, thyroid effects, and anti-androgenic activity. The transient ER reporter gene assay in CV-1 cells was an excellent system for examining ER-mediated responses, since this cell line does not
Fig. 1 Effects of E2, T3 and DHT in reporter gene expression. Estrogenic potency of E2 in ER-mediated reporter gene assay with CV-1 cells transfected with pERE-TATA-Luc and rER a /pCI. Thyroid hormone potency of T3 in ThR-mediated reporter gene assay with CV-1 cells transfected with pUAS-tk-Luc and pGal4-L-TR b . Androgenic potency of DHT in MDA-kb2 cells stably transfected with MMTV-luciferase.
852 | J. Environ. Monit.
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Fig. 2 Estrogenic activities of BDE100 measured by 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. Significant differences are indicated by asterisks *,
P < 0.05
vs.
control, **, P < 0.01
vs.
control.
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Fig. 3 Antiestrogenic potency of BDE100 measured by reporter gene assay with CV-1 cells. BDE100 was added along with 1 nM E2. Data are
(mean SD) of three independent experiments and are presented as percent induction, with 100% activity defined as the activity achieved with
1 nM E2. Significant differences are indicated by asterisks *, P < 0.05, **,
P < 0.01
vs.
the value of 1 nM E2 (100%).
Fig. 4 Thyroid hormone potency of BDE100 measured by reporter gene assay with CV-1 cells. BDE100 was added along with 5 nM T3. Data are
(mean SD) of three independent experiments and are presented as percent induction, with 100% activity defined as the activity achieved with
T3 (5 nM). Significant differences are indicated by asterisks *, P < 0.05,
**, P < 0.01
vs.
the value of 5 nM T3 (100%).
Fig. 5 Antiandrogenic potency of BDE100 measured by reporter gene assay with MDA-kb2 cells stably transformed with MMTV-luciferase.
BDE100 was added along with 1 nM of DHT. Values are mean SD of three independent experiments and are presented as percent induction, with 100% activity defined as the activity achieved with 1 nM of DHT.
Significant differences are indicated by asterisks *, P < 0.05, **, P < 0.01
vs.
the value of 1 nM DHT (100%).
Effects on the function of TH are of concern because thyroid hormone is involved in development of the mammalian brain.
23
Compared to research on environmental estrogens, little had been done to evaluate the effects of BDE100 on thyroid hormone. In the presence of T3, BDE100 did not cause agonistic effects on the ThR. BDE100 interacted with T3, leading to enhancement in Luc expression caused by 50 nM T3 significantly. Some studies have confirmed that PBDEs can affect the thyroid system.
21,24,25 Almost all PBDEs in commercial products are able to interfere with the balance of thyroid hormones.
24,25
But the mechanism by which this occurs is unclear. BDE100 was shown to interfere with thyroid function in the T-Screen.
22 The structure of BDE100 is similar to that of thyroid hormones T3 and T4. The amount of information on the effects of PBDE on thyroid hormone is relatively less than that for AR and ER.
18,26
Several articles have shown the order of anti-androgenic activity to be BDE100 (2,2,4,4,6) > BDE47 (2,2,4,4) > BDE99
(2,2,4,4,5).
23,26 A study by Hamers confirmed that BDE100 exhibits anti-androgenicity.
22 Stoker et al.
used MDA-kb2 cells to detect compounds having anti-androgen effects and found that BDE100 suppressed the effects of DHT.
18,26 These results are consistent with those observed in the study upon which we report here, where BDE100 was found to be a potent competitive inhibitor of AR agonists, relative to other environmental compounds.
26
BDE100 is a weak ER agonist.
20,22 Alternatively BDE100 can antagonize the effect of E2, 22 but was not an agonist or antagonist of T3.
27 The results of this study demonstrated that
BDE100 was an ER agonist at concentrations of 1 m M or greater and an ER antagonist at 10 m M. It also interacted with T3, leading to enhancement of the effects of 5.0 nM T3 on Luc expression at 50 m M. BDE100 was antiandrogenic at 10 m M or greater. The results from the present study indicate a possible effect of BDE100 on hormone receptors that could have endocrine consequences within the m M concentration range. BDE100 has recently been reported to be present in various animals including freshwater fish, shrimps, crabs, 28 wildlife 29–31 humans.
32–34 and
In all these cases, BDE100 concentrations were in the pM and nM range, which is more than three orders of magnitudes less than the concentrations (1–10 m M range) that caused significant endocrine-disrupting effects of hormone receptors in in vitro experiments, upon which we report here. The physical and chemical properties of BDE100 are such that it can be biomagnified. Assuming that median concentrations in our in vitro experiments can be compared with our maximal estimated blood concentrations in humans, our calculations indicate that in vivo endocrine interaction by BDE100 would not be expected at current concentrations in the environment. These results suggest that BDE100 can cause multiple endocrine-disrupting effects through interfering with several hormonal signaling pathways simultaneously.
express endogenous steroid receptors, and thus little background was observed in these assays. In the present study, BDE100 was a moderate ER agonist and ER antagonist. Previous studies had reported that BDE100 exhibited estrogenic effects on human
T47D breast cancer cells, 19,20,22 while using the same methods no anti-estrogenic effects of BDE100 were observed.
19,20 BDE100 exhibited anti-estrogen activity in CV-1 cells used in this study, which might respond differently from the cells used by
Meerts et al.
19,20
This journal is ª The Royal Society of Chemistry 2011
This work was supported by the fund of National Natural
Science (No.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
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Introduction and Cultivation Foundation of Nanjing University.
Prof. Giesy was supported by the Canada Research Chair program 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 Visiting
Professor Program of King Saud University.
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