Marine Pollution Bulletin 62 (2011) 2356–2361
Contents lists available at SciVerse ScienceDirect
Marine Pollution Bulletin
journal homepage: www.elsevier.com/locate/marpolbul
Endocrine effects of methoxylated brominated diphenyl ethers in three in vitro
models
Wei Hu a, Hongling Liu a,⇑, Hong Sun b, Ouxi Shen b, Xinru Wang b, Michael H.W. Lam c, John P. Giesy a,c,d,e,f,
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 Special Administrative Region
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
f
Zoology Department, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia
b
c
a r t i c l e
i n f o
Keywords:
20 -MeO-BDE-68
6-MeO-BDE-47
Reporter gene assay
Receptor-mediated pathway
Estrogen receptor
Androgen receptor
Thyroid receptor
a b s t r a c t
Methoxylated brominated diphenyl ethers (MeO-BDEs) in aquatic environments have been found to be
primarily of natural origin in the marine environment and not from biotransformation of synthetic
PBDEs. Two of the eight MeO-PBDEs (20 -MeO-BDE-68 and 6-MeO-BDE-47) that were detected in anchovy
from the Yangtze River Delta, were natural products from marine organisms. So 20 -MeO-BDE-68 and
6-MeO-BDE-47 were chosen to study the potential to modulate androgen, estrogen, or thyroid hormone
receptor- (AR, ER, ThR) mediated responses by use of reporter gene assays. 20 -MeO-BDE-68 was
antiandrogenic at 50 lM, estrogenic at 10 lM and antiestrogenic at 10 and 50 lM (IC50 = 4.88 lM). 20 MeO-BDE-68 enhanced luciferase expression by 5 nM T3 at 50 lM. 6-MeO-BDE-47 exhibited potent
antiandrogenicity at 1 lM and greater (IC50 = 41.8 lM) and possessed estrogenic activity at 10 lM and
antiestrogenic activity at 10 and 50 lM (IC50 = 6.02 lM).
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Methoxylated brominated diphenyl ethers (MeO-BDEs), which
are structurally related to PBDEs, have been known for decades
(Sharma and Vig, 1972; Carté and Faulkner, 1981). Polybrominated
diphenyl ethers (PBDEs) are commonly used as flame retardants in
construction materials, textiles, and polymers in electronic equipment (Alaee et al., 2003; Brown et al., 2004). They have been
widely found in soil, sediment, water, and air (Alaee et al., 2003),
biological samples (fish, seashell, birds, earth mammals and sea
mammals) (Lindström et al., 1999; Johnson and Olson, 2001; Wolkers et al., 2004), as well as in human blood serum, adipose tissue,
breast milk, placental tissue and the brain (Sjödin et al., 2004;
Athanasiadou et al., 2008; Domingo et al., 2008). Concentrations
of PBDEs in the environment are increasing (Sellström et al.,
1993; Meironyté et al., 1999; She et al., 2002; Schecter et al., 2005).
Recently MeO-BDEs have drawn more and more attention because they have been identified to be bioaccumulated by biota
(Haglund et al., 1997) in top predators, such as polar bears (Ursus
maritimus) from Norwegian Arctic (Verreault et al., 2005), whales
from Virginia (Teuten et al., 2006), whales and dolphins from the
⇑ Corresponding authors. Tel.: +86 25 89680356 (H. Liu).
E-mail address: hlliu@nju.edu.cn (H. Liu).
0025-326X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.marpolbul.2011.08.037
Mediterranean Sea (Pettersson et al., 2004; Teuten et al., 2006),
sea lions (Zalophus californianus) from California (Stapleton et al.,
2006), cetaceans from Australian waters (Vetter et al., 2002; Melcher et al., 2005), pike from Swedish waters (Kierkegaard et al.,
2004), fish and guillemot from Baltic, Atlantic and Arctic environments (Sinkkonen et al., 2004), pinnipeds from the Baltic Sea
(Haglund et al., 1997), harbor porpoises and harbor seals from
the North Sea (Weijs et al., 2009a–c), marine mammals from Japan
(Marsh et al., 2005) and beluga whales from the Canadian Arctic
(Kelly et al., 2008). The distribution of MeO-BDEs is ubiquitous at
concentrations sometimes greater than those of PBDE congeners
(Teuten et al., 2005). Since MeO-BDEs are neither a commercial
product nor reported to be byproducts in industrial processes
(Vetter, 2006), it had been postulated that MeO-BDEs could be
formed by direct methoxylation or in a two-step process by a
hydroxylation followed by a methylation, either of which is a viable biochemical transformation (Haglund et al., 1997; Hakk and
Letcher, 2003). It had been suggested that MeO-BDEs might be biotransformation products of PBDEs, possibly through hydroxy-BDEs
(HO-BDEs) intermediates. The pathways of formation have been
elucidated and it is now evident that some MeO-BDEs are not
formed from HO-BDEs and PBDEs but rather that the MeO-BDEs
are natural products, especially in the marine environment (Teuten
et al., 2005; Wan et al., 2009; Su et al., 2010). MeO-BDEs are
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W. Hu et al. / Marine Pollution Bulletin 62 (2011) 2356–2361
biotransformed to HO-BDEs and in fact are the primary source of
HO-BDEs and little of the HO-BDEs formed from synthetic PBDEs
(Wan et al., 2010a,b).
Nuclear hormone receptors are ligand-dependent transcription
factors that regulate a variety of important physiologic processes
(McKenna et al., 1999). Several studies have reported that PBDEs
can disrupt the endocrine system via a variety of nuclear hormone
receptors, such as estrogen receptor (ER) and androgen receptor
(AR) (Meerts et al., 2001; Stoker et al., 2005; Hamers et al., 2006).
In the ER-CALUX assay using a human T47D breast cancer cells,
the ER was activated by less-brominated PBDEs and inactivated by
more-brominated PBDEs (Meerts et al., 2001; Hamers et al., 2006).
Consistent with these findings is the recent observation that BDE28, -47, and -100 were estrogenic while BDE-99 and -100 were
antiestrogenic to Chinese hamster ovary (CHOK1) cells (Kojima
et al., 2009). Although none of the tested PBDE congeners showed
any androgenic activity, BDE-100 has been reported to be an antiandrogen (Stoker et al., 2005). Some PBDEs are AR antagonists (Hamers
et al., 2006; Kojima et al., 2009). PBDEs bind competitively with human transthyretin (TTR), a transport protein for the thyroid hormones triiodothronine (T3) and thyroxine (T4), thereby affecting
transport of thyroid hormone (Meerts et al., 2000; Zhou et al.,
2001; Richardson et al., 2008). No PBDEs have been reported to bind
to the thyroid hormone receptor (ThR) (Kitamura et al., 2008).
There is little information on the toxicology of MeO-BDEs, especially their endocrine disrupting properties. Since MeO-BDEs are
structurally similar to PBDEs which have the potency to disrupt
the balance of endocrine system, and are present in relatively great
concentrations in marine organisms, including food eaten in Asia
(Wan et al., 2010b) it was decided to determine if MeO-BDEs can
affect endocrine homeostasis. In order to test this hypothesis,
endocrine modulating potential of 6-MeO-BDE-47 and 20 -MeOBDE-68, which are most frequently observed MeO-BDEs in animal
tissue (Kierkegaard et al., 2004; Teuten et al., 2006), were assessed.
Concentrations of MeO-PBDE in dolphins of the continental shelf
(CS) of Brazil are among the greatest detected in cetaceans (up to
250 lg/g lw) (Dorneles Paulo et al., 2010). The greatest concentration of 20 -MeO-BDE-68 previously reported for marine mammal
tissues was 3760 ng/g wet weight found in the blubber of a pygmy
sperm whale from Queensland, northeast Australia (Vetter et al.,
2002). The lipid content of blubber in marine mammals ranges
from 30% to 90% (Reijnders and Aguilar, 2002). Considering the lesser of the lipid percentages (30%) a maximum value of 12,500 ng/g
lipids can be calculated for 20 -MeO-BDE-68 in the pygmy sperm
whale. Concentrations of the sum of MeO-triBDEs were less, with
a maximum value (260 ng/g lw) determined in a false killer whale
(Pseudorca crassidens), which contained also the greatest concentrations of 20 -MeO-BDE-68 and 6-MeO-BDE-47 (sum 250 lg/g lw)
(Dorneles Paulo et al., 2010). 6-MeO-BDE-47 and 20 -MeO-BDE-68
were two out of eight MeO-PBDEs, which were detected in Anchovy (Coilia sp.) from the lower Yangtze River in the region around
Jiangsu Province of China (Su et al., 2010).
Promoter-reporter gene assays have been used as in vitro methods for clarifying agonistic and antagonistic potency of various
chemicals against nuclear hormone receptors. AR-, ER- and ThRmediated transactivation reporter gene assays were used to assess
anti/androgenic, anti/estrogenic and anti/thyroid hormone properties of these two MeO-BDEs.
2. Materials and methods
Br
Br
OCH3
O
OCH3
O
Br
Br
Br
Br
Br
Br
2'-MeO-BDE-68
6-MeO-BDE-47
Fig. 1. Structure of 20 -MeO-BDE-68 and 6-MeO-BDE-47.
MO, USA). L-3,5,30 -triiodothyronine (T3) with a purity of over
98% was also purchased from Sigma Chemical Co. (St. Louis, MO,
USA). 20 -MeO-BDE-68 and 6-MeO-BDE-47 (Fig. 1) were synthesized in the Department of Biology and Chemistry of City University of Hong Kong and have been confirmed to have purities of
greater than 98% (Wan et al., 2010a). Stock solutions of the chemicals were prepared in dimethyl sulphoxide (DMSO, Sigma), stored
at 20 °C, and diluted to desired concentrations in phenol red-free
Dulbecco’s modified Eagle medium (DMEM, Sigma) or red-free L15 medium (Sigma) immediately before use.
2.2. Plasmids
The luciferase reporter plasmid pERE-TATA-Luc and rat estrogen receptor a (ERa) expression vector rERa/pCI were provided
by Dr. Takeyoshi (Chemicals Assessment Center, Chemicals Evaluation and Research Institute, Oita, Japan). The plasmids were constructed as previously described (Takeyoshi et al., 2002). The
expression plasmid pGal4-L-TRb and Gal4 responsive luciferase reporter plasmid pUAS-tk-Luc containing four copies of the Gal4
binding site were obtained from Professor Ronald M. Evans (Gene
Expression Laboratory, Howard Hughes Medical Institute, San Diego, CA, USA), and their structures had been described before (Chen
and Evans, 1995).
2.3. Luciferase reporter gene assay
2.3.1. AR reporter gene assay
MDA-kb2 cells (ATCC, USA), stably transformed with murine
mammalian tumor virus (MMTV)-luciferase were cultured in
Leibovitz’s L-15 medium with 10% fetal bovine serum (FBS, Gibco),
100 U/ml penicillin (Sigma), 100 lg/ml streptomycin (Sigma), and
0.25 lg/ml amphotericin B (Sigma) at 37 °C without CO2. The
MMTV is not specific for androgen. However, this stable cell line
has been used for androgen screening (Wilson et al., 2002). Cells
were plated at 1 104 cells per well in 100 ll of medium in 96well luminometer plates. When cells were attached (4–6 h), medium was removed and replaced with dosing medium, which contained the test chemicals, including MeO-BDEs and or model
chemicals of known potency. The MDA-kb2 cells were exposed to
DHT (Sigma, 1.0 1011–1.0 106 M in 10-fold dilution steps),
solvent-controls or test chemicals for 24 h. The model compounds
were tested at a concentration range that had been shown to be
noncytotoxic. After rinsing three times with phosphate-buffered
saline (PBS, pH 7.4), cells were lysed with 1 passive lysis buffer
(Promega). After centrifuging at 12,000g for 10 min to remove debris, luciferase activities in cell lysates were analyzed immediately
using a 96-well plate luminometer (Berthold Detection System,
Pforzheim, Germany). The amount of luciferase was measured by
use of the luciferase reporter assay system kit (Promega) following
the manufacturer’s instructions.
2.1. Chemicals
5a-Dihydrotestosterone (DHT), 17b-estradiol (E2) with purities
of over 99% were purchased from Sigma Chemical Co. (St. Louis,
2.3.2. ER reporter gene assay
When green monkey kidney fibroblast (CV-1) (Chinese Cell Center, Beijing) cells were not transfected with hormone responsive
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W. Hu et al. / Marine Pollution Bulletin 62 (2011) 2356–2361
elements (HRE), E2 and T3 cannot induce Luc activity. CV-1 cells
were from monkey kidney, which do not contain the endogenous
receptors (ER and ThR). CV-1 cells were maintained in Dulbecco’s
modified Eagle’s medium (DMEM, Sigma) supplemented with 5%
FBS, 100 U penicillin/ml and 100 streptomycin lg/ml, at 37 °C in
an atmosphere containing 5% CO2. Cells were cultured and exposed
according to previously published methods (Sun et al., 2008a,b).
Briefly, host cells were plated in 48-well microplate at a density
of 0.5 105 cells per well in DMEM medium containing 5% charcoal-dextran-stripped FBS that was free of phenol red (CDS-FBS).
After 12 h cells were transfected. The concentration of plasmids
and transfection reagent for ER activity and ThR activity was described before (Zhang et al., 2011). After an incubation period of
12 h, cells were treated with test chemicals for 24 h to E2
(1.0 1010–1.0 107 M in 10-fold dilution steps), solvent-controls and compounds. Then, luciferase activity was determined as
described above.
2.3.3. ThR reporter gene assay
CV-1 cells were cultured and plated as described above. After an
initial incubation of 12 h, cells were transfected. After an incubation period of 12 h, cells were exposed to T3 (1.0 1012–
1.0 106 M in 10-fold dilution steps), solvent controls and test
chemicals for 24 h. Cell lysates were analyzed immediately using
a 96-well plate luminometer (Berthold Detection System, Pforzheim, Germany).
2.4. Cell viability assessed by MTT assay
The MTT assay was performed to detect the cytotoxicity of test
chemicals. Both MDA-kb2 and CV-1 cells attached to culture dishes
were collected and plated at a density of 1 104 cells/100 ll in
each well of 96-well plates using DMEM with 5% DCC serum and
L-15 with 10% DCC serum, respectively. After 24 h, cells were treated with vehicle or various concentrations (0.1, 1, 10 and 50 lM) of
test chemicals alone or with 1.0 nM DHT, 1.0 nM E2, or 5.0 nM T3
for 24 h. Then, 25 ll 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg/ml 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 ll DMSO and shaken for 10 min
2'-MeO-BDE-68
6-MeO-BDE-47
Androgenic activity
(% of 1nM DHT androgenicity)
140
120
to solubilize crystals. Absorbance was measured by automatic
microplate reader (EL808, Bio-Tek, Winooski, VT, USA) at 570 nm.
2.5. Statistical analysis
All experiments were conducted in triplicate and within an
experiment; each concentration was tested in triplicate. Values
are reported as the mean ± standard deviations (SD) (n = 3). Results
were analyzed by one-way analysis of variance (ANOVA), followed
by Duncan’s multiple comparisons test (SPSS 11.5; SPSS Inc., Chicago, IL, USA). The level of significance was set at p < 0.05. For agonists, treatments were compared to the vehicle control group, and
the relative transcriptional activity was converted to fold induction
relative to that of the vehicle control. For antagonists, treatments
were compared to the activity caused by 1.0 nM DHT, 1.0 nM E2
or 5.0 nM T3 as positive controls, and percent of the activity caused
by 1.0 nM DHT, 1.0 nM E2 or 5.0 nM T3. The median inhibitory
concentration (IC50) or median effective concentration (EC50) was
calculated with SPSS software (SPSS Inc., Chicago, IL, USA).
3. Results
3.1. Cell viability and responsiveness
There was no statistically significant difference between vehicle-treated groups and those treated alone or with 1.0 nM DHT,
1.0 nM E2, 5.0 nM T3 (data not shown) for neither MDA-kb2 nor
CV-1 cells. No cytotoxicity was observed at the concentrations of
chemicals tested. The assays displayed appropriate responses to
the known androgen receptor (AR) agonist 5a-dihydrotestosterone
(DHT), estrogen receptor (ER) agonist estradiol (E2) and thyroid
hormone receptor (ThR) agonist triiodothronine (T3). The method
responsiveness was described before (Zhang et al., 2011).
3.2. Antiandrogenicity
Chemicals were tested for their inhibitory effects on transcriptional activity induced by co-exposure to DHT. 20 -MeO-BDE-68 did
not reduce luciferase expression at concentrations from 0.1 to
10 lM, but significantly less luciferase activity was observed at
50 lM. The IC50 for 20 -MeO-BDE-68 was 41.8 lM (Fig. 2). 6-MeOBDE-47 was a potent antiandrogen that significantly inhibited the
luciferase activity induced by 1 nM of DHT at concentration of
1 lM and greater (Fig. 2). The IC50 value of 6-MeO-BDE-47 was
4.3 lM. 6-MeO-BDE-47 was a stronger antagonist than 20 -MeOBDE-68.
100
3.3. Estrogenic and antiestrogenicity
*
80
60
*
*
40
*
20
0
Ctrol
-7
-6
-5
Concentration (Log M)
-4.3
Fig. 2. Antiandrogenic potency of 20 -MeO-BDE-68 and 6-MeO-BDE-47 measured by
reporter gene assay with MDA-kb2 cells stably transfected with MMTV-luciferase.
Chemicals were added along with 1 nM 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 DHT. Significant differences
between the chemicals and the DHT treatment were tested for by use of ANOVA,
followed by Dunnett’s test. Significant differences are indicated by asterisks ⁄,
P < 0.05 vs. the value of 1 nM DHT (100%).
Neither 2’-MeO-BDE-68 nor 6-MeO-BDE-47 resulted in induction of luciferase activity that was greater than that of the vehicle
control at 0.1 or 1.0 lM, while both of them displayed weak estrogenic potency with maximal induction of 1.47- and 1.58-fold,
respectively at a concentration of 10 lM (Fig. 3).
Neither 0.1 nor 1.0 lM of either 20 -MeO-BDE-68 or 6-MeO-BDE47 resulted in less expression of luciferase in the presence of E2
(Fig. 4). Concentrations of both 20 -MeO-BDE-68 and 6-MeO-BDE47 greater than 10 lM significantly antagonized the effect of E2
on ER-mediated luciferase activity with IC50 values of 4.88 and
6.02 lM, respectively.
3.4. Thyroid and antithyroid potencies
Effects of the two MeO-BDEs on ThR-mediated luciferase
activity were determined in the presence of 5.0 nM T3. While
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W. Hu et al. / Marine Pollution Bulletin 62 (2011) 2356–2361
E2
18
180
2'-MeO-BDE-68
14
12
10
8
6
*
140
120
100
80
60
40
20
0
4
*
2
6-MeO-BDE-47
160
Thyroid activity
(% of 5 nM T3 activity)
Relative luciferase induction
(n-fold of control)
16
2'-MeO-BDE-68
Ctrol
-7
-6
-5
-4.3
Concentration (Log M)
0
Ctrol -10
-9 -8 -7 -6 -5
Concentration (Log M)
Fig. 5. Thyroid hormone potency of 20 -MeO-BDE-68 and 6-MeO-BDE-47 measured
by reporter gene assay with CV-1 cells. Chemicals were added along with 5 nM T3.
Data were (mean ± SD) of three independent experiments 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 vs. the value of 5 nM T3
(100%).
E2
18
6-MeO-BDE-47
Relative luciferase induction
(n-fold of control)
16
14
4. Discussion
12
10
8
6
4
*
2
0
Ctrol -10
-9 -8 -7 -6
Concentration (Log M)
-5
Fig. 3. Estrogenic potency of E2, 20 -MeO-BDE-68 and 6-MeO-BDE-47 measured by
use of the CV-1 reporter gene assay. 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.
2'-MeO-BDE-68
6-MeO-BDE-47
Estrogenic activity
(% of 1nM E2 estrogenicity)
120
100
80
60
* *
40
*
20
0
*
Ctrol
-7
-6
-5
Concentration (Log M)
-4.3
Fig. 4. Antiestrogenic potency of 20 -MeO-BDE-68 and 6-MeO-BDE-47 measured by
reporter gene assay with CV-1 cells. These chemicals were added along with 1 nM
E2. Data were (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, vs. the value of 1 nM E2
(100%).
50 lM, 20 -MeO-BDE-68 significantly increased luciferase activity
(Fig. 5), no significant antagonistic activity was detectable at the
tested dosages for 6-MeO-BDE-47 (Fig. 5). Neither of the two
MeO-BDEs exhibited ThR agonistic activity at concentrations from
0.1 to 10 lM (Data not shown).
Transactivation, reporter gene assays are useful to characterize
receptor-mediated endocrine activity and elucidate mechanisms of
action (Freyberger and Schmuck, 2005). In the present study, three
reporter gene assays were used to investigate potency of two PBDE
methoxylated derivatives to modulate AR-, ER- and ThR-mediated
responses. Both MeO-BDEs possessed mixed antiandrogenic activity, estrogenic/antiestrogenic activities.
A novel stable cell line, MDA-kb2, was used to determine the
potency of androgen antagonists. The assay was specific and sensitive to AR antagonistic chemicals. Maximal induction of about 9fold relative to that of vehicle control has been reported to occur
at 10 nM DHT (Wilson et al., 2002). The assay displayed appropriate sensitivity to the known AR agonist, DHT. The maximal induction of 11.2-fold relative to vehicle control which was slightly
greater that which had been previously reported (Wilson et al.,
2002), but exhibited a similar dose–response relationship. Detection of androgen antagonist activity of compounds was done by
measuring their ability to decrease DHT-induced luciferase activity. In the studies upon which we report here, concentrations of
DHT within the linear concentration–response range were used.
Antagonist activity can usually be detected in competition against
1.0 nM DHT, which is equivalent to the concentration observed in
men of 20–40 years of age (Lewis et al., 1976), and can induce an
8.08-fold change in luciferase activity. In vitro antiandrogenicities
of MeO-BDEs have been reported previously (Kojima et al., 2009).
The results of that study indicated that, all MeO-BDEs tested were
potent antiandrogens.
Both estrogenic and antiestrogenic potencies of MeO-BDEs were
determined by use of a sensitive reporter gene assay based on CV-1
cells, which had been transiently transfected with expression vectors for ER-a (rERa) along with a plasmid encoding for the reported
gene, luciferase. While the sensitivity of this system has been demonstrated previously (Sun et al., 2008b), in this study a maximum
fold-change of 17.02-fold relative to the vehicle control was observed. In the present study, 20 -MeO-BDE-68 and 6-MeO-BDE-47
were weakly estrogenic with 1.47-, and 1.58-fold greater ER-mediated luciferase activity relative to the vehicle control. Both MeOBDEs antagonized the ERa at greater doses (10 and 50 lM) in the
presence of 1.0 nM E2. Taken together, the results suggested that
MeO-BDEs might act as ERa agonists and/or antagonists, depending on concentration, which is similar to the results observed previously (Kojima et al., 2009). However, all previous studies were
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W. Hu et al. / Marine Pollution Bulletin 62 (2011) 2356–2361
conducted with para-MeO-BDEs (Kojima et al., 2009). Until now
there was no information about ortho-MeO-BDEs. Our results also
indicated that some MeO-PBDEs exhibit agonistic and antagonistic
interactions with the ER.
PBDEs and HO-BDEs can bind competitively to human transthyretin (TTR), a transport protein for the thyroid hormones T3 and
thyroxine (T4), thereby interfering with functioning of thyroid hormone (Meerts et al., 2001; Zhou et al., 2001, 2002; Richardson
et al., 2008). However, few studies have addressed the effects of
MeO-BDEs on thyroid hormone function except for the results for
reporter gene assays using Chinese hamster ovary cells by (Kojima
et al., 2009).
6-MeO-BDE-47 exhibited endocrine disrupting effects similar to
those of BDE-100, which is structurally similar, with the only difference being that the methoxy group on 6-MeO-BDE-47 is replaced by a bromine atom in BDE-100. In vivo and in vitro
antiandrogenicity of BDE-100 have been reported previously (Stoker et al., 2005; Hamers et al., 2006; Kojima et al., 2009). BDE-100
is a weak ER agonist (Meerts et al., 2001; Hamers et al., 2006; Kojima et al., 2009). Alternatively BDE-100 can antagonize the effect
of E2 (Hamers et al., 2006; Kojima et al., 2009), but was not an agonist or antagonist of T3 (Kojima et al., 2009). This result indicates
that MeO-BDEs have endocrine-disrupting potential similar to
those of PBDEs.
MeO-BDEs act as agonist and/or antagonist via ERa and AR in
in vitro reporter gene assays. Based on these results, the predominant MeO-BDEs found in human tissues have multiple endocrinedisrupting effects modulated via nuclear hormone receptors. Ligand-dependent transcription of nuclear hormone receptors requires association of protein cofactors and basal transcription
factors, expression levels of which differ from cell to cell (McKenna
et al., 1999). Furthermore, there is some discrepancy in the cellular
metabolic ability against different cells. Thus, there is a possibility
that different cells could respond differently. The CV-1 and MDAkb2 cells used in our study were deficient in metabolic activity,
especially compared with hepato carcinoma cells which have some
metabolic activity that could affect the test result. Thus, the results
of our study reflected the effects of 20 -MeO-BDE-68 and 6-MeOBDE-47 and not their biotransformation products.
Most of the effects observed in this study occurred at concentrations of 10 or 50 lM, which is greater than relevant concentrations
that have been observed in humans and the environment. In addition, the results of previous studies have suggested that MeO-BDEs
have the potential to interfere with CYP17 and CYP19 activities,
both of which catalyze key steps in the production of sex hormones
in humans (Cantón et al., 2005, 2006; He et al., 2008; Song et al.,
2008). Therefore, MeO-BDEs may also indirectly disrupt the endocrine system by affecting the expression of relevant genes other
than interacting with nuclear hormone receptors. Furthermore,
information about the potential endocrine activity of MeO-PBDEs
is limited to several MeO-BDEs (Song et al., 2008; Kojima et al.,
2009).
5. Conclusions
Few studies have been done to characterize the endocrine-disrupting potential of MeO-BDEs, some of which are natural products
especially in marine organisms that are consumed by humans. In
the present study, the antiandrogenic activity of two frequently
detected MeO-BDEs (20 -MeO-BDE-68 and 6-MeO-BDE-47) was
determined by use of a stably transfected cell line (MDA-kb2).
The (anti)estrogenic and (anti)thyroid activity was determined by
use of the rat estrogen receptor a (rERa) and human thyroid hormone receptor (hThR) mediated luciferase reporter gene assay in
African green monkey kidney cells (CV-1). The assays displayed
appropriate responses to the known androgen receptor (AR) agonist 5a-dihydrotestosterone (DHT), estrogen receptor (ER) agonist
estradiol (E2) and thyroid hormone receptor (ThR) agonist triiodothronine (T3). 20 -MeO-BDE-68 was antiandrogenic only at
50 lM, but was estrogenic at 10 lM and antiestrogenic at 10 and
50 lM. 20 -MeO-BDE-68 was found to enhance luciferase expression by 5 nM T3 at 50 lM. The result also showed that 6-MeOBDE-47 exhibited potent antiandrogenic activity at the concentration of 10 lM and higher. 6-MeO-BDE-47 possessed estrogenic
activity at 10 lM and antiestrogenic activity at 10 and 50 lM.
These results suggest that these two MeO-BDEs can cause multiple
endocrine-disrupting effects through interfering with several hormonal signaling pathways simultaneously.
Acknowledgements
This work was supported by the fund of National Natural Science (Nos. 20737001, 20977047), the National Major Project of Science & Technology Ministry of China (No. 2012ZX07506-001),
Specialized Research Fund for the Doctoral Program of Higher Education (No. 200802841030) and the Fundamental Research Funds
for the Central Universities (No. 1116021104). The research was
supported by a Discovery Grant from the National Science and
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 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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