Environmental Toxicology and Chemistry, Vol. 21, No. 11, pp. 2431–2433, 2002
q 2002 SETAC
Printed in the USA
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Short Communication
IN VITRO ASSESSMENT OF POTENTIAL MECHANISM-SPECIFIC EFFECTS OF
POLYBROMINATED DIPHENYL ETHERS
DANIEL L. VILLENEUVE,*† KURUNTHACHALAM KANNAN,† BENJAMIN T. PRIEST,‡ and JOHN P. GIESY†
†National Food Safety and Toxicology Center, Department of Zoology and Institute for Environmental Toxicology, Michigan State University,
East Lansing, Michigan 48824-1311, USA
‡Cambridge Isotope Laboratories, Andover, Massachusetts 01810, USA
( Received 4 February 2002; Accepted 12 May 2002)
Abstract—This study examined the ability of environmentally relevant concentrations of 10 different polybrominated diphenyl
ethers (PBDEs) to induce aryl hydrocarbon receptor (AhR)– and estrogen receptor (ER)–mediated gene expression in vitro. It also
examined the ability of PBDEs to displace steroid hormones from serum proteins in vitro. At concentrations ranging up to 880 ng/
ml, none of the PBDEs significantly displaced tritiated 17b-estradiol (E2) or testosterone from hormone-stripped carp serum. At
concentrations ranging up to 500 ng/ml, 9 of 10 PBDEs tested failed to induce ER- or AhR-mediated gene expression in MVLN
and H4IIE-luc cells, respectively. One congener, 3,39,4,49,5-pentabromodiphenylether (BDE 126), induced significant AhR-mediated
gene expression at 500 ng/ml, but the magnitude of induction was only 13% of that caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD). Overall, the PBDEs tested were found to be at least 200,000 times less potent than TCDD and 50,000 times less potent
than E2 for inducing AhR- and ER-mediated gene expression, respectively.
Keywords—Flame retardants
Aryl hydrocarbon receptor
Estrogen receptor
INTRODUCTION
Serum proteins
METHODS
Worldwide, approximately 31.5 3 106 kg of polybrominated diphenyl ethers (PBDEs) are produced each year [1].
Polybrominated diphenyl ethers are widely used in thermoplastics used in products including electrical appliances, televisions, computers, and building materials [1]. Polybrominated
diphenyl ethers are also found in foams and upholstery used
in home and business furnishings, automobile interiors, and
rugs and draperies [1]. Polybrominated diphenyl ethers have
been widely detected in fish and wildlife [2–9] and have also
been detected in human blood, adipose tissue, and breast milk
[13–16]. Total PBDEs in Swedish human milk increased more
than 50-fold (from 0.7–4.2 ng/g lipid) over a 25-year period
[15]. Despite the widespread and increasing concentrations of
PBDEs in the environment, little is currently known, relative
to other persistent organic pollutants, about their potential toxicological effects, particularly for individual PBDE congeners.
Because PBDEs are structurally similar to polychlorinated diphenyl ethers (PCDEs), polychlorinated biphenyls (PCBs), and
polychlorinated dibenzodioxins (PCDDs), they may cause similar biological effects.
The goal of this study was to employ several in vitro bioassays to examine the potential mechanism-specific potency
of 10 individual PBDEs. In vitro luciferase assay with H4IIEluc recombinant rat hepatoma cells was used to examine AhRmediated (dioxin-like) potency. In vitro luciferase assay with
MVLN recombinant human breast carcinoma cells was used
to examine ER-mediated (estrogenic) potency. Finally, a competitive displacement assay was used to examine the ability
of PBDEs to displace 17b-estradiol (E2) or testosterone (T)
from serum proteins.
Chemicals
All PBDEs used for this study were purchased from Cambridge Isotope Laboratories (Andover, MA, USA). The 10
PBDE congeners tested in this study were 2,29,4,49-tetraBDE
(BDE 47), 3,39,4,49-tetraBDE (BDE 77), 2,29,4,495-pentaBDE
(BDE 99), 2,29,4,49,6-pentaBDE (BDE 100), 2,3,39,4,49pentaBDE (BDE 105), 3,39 ,4,4 9 ,5-hexaBDE (BDE 126),
2,29,4,49,5,59-hexaBDE (BDE 153), 2,29,3,4,49,59,6-heptaBDE
(BDE 183), 2,3,3 9 ,4,4 9 ,5,6-heptaBDE (BDE 190), and
2,29,3,39,4,49,5,59,6,69-decaBDE (BDE 209). They were selected to represent a range of PBDE structures including those
commonly found in biological samples [5–7]. With the exception of 2,3,39,4,49,5,6-heptaBDE (87% pure), all PBDEs
were 99% pure. All stock solutions were 50 mg/ml in n-nonane.
H4IIE-luc and MVLN bioassays
Cells were seeded into the 60 interior wells of 96-well
culture ViewPlatesy (Packard Instruments, Meriden, CT,
USA) and dosed after overnight incubation to allow for attachment. All exposures were 72 h. Dose responses consisted
of six concentrations prepared by threefold serial dilution in
high-purity isooctane (Burdick and Jackson, Muskegon, MI,
USA). A minimum of three replicates of each sample concentration, control, and blank were tested. Nominal PBDE concentrations in the test wells were 500, 167, 55.5, 18.5, 6.17,
and 2.05 ng/ml. Detailed cell culture and assay methods have
been reported previously [14,15]. Responses were expressed
as a percentage of the mean maximum response observed for
a 1,500-pM TCDD standard (%-TCDD-max.) or 1,000-pM E2
standard (%-E2-max.). A significant response was defined as
any response greater than a response equal to three standard
deviations (expressed in %-TCDD/E2-max.) above the mean
solvent control response (set to 0%-TCDD/E2-max.). Details
* To whom correspondence may be addressed
(villene1@msu.edu).
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Environ. Toxicol. Chem. 21, 2002
D.L. Villeneuve et al.
Fig. 1. Response of H4IIE-luc recombinant rat hepatoma cell bioassay
to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) standard and polybrominated diphenyl ether (PBDE) 126 (3,39,4,49,5-pentaBDE). Doses
were expressed as ng/ml media present in the test well. Responses
expressed as a percentage of the maximum response observed for a
1,500-pM TCDD standard (%-TCDD-max.). Significant 5 3 standard
deviations (expressed in %-TCDD-max.) above the mean solvent control response (set to 0%-TCDD-max.).
regarding the calculation of %-TCDD/E2 maximum and bioassay data analysis have been described elsewhere [15,16].
Hormone displacement assays
For hormone displacement assays, the n-nonane was evaporated from a 75-ml subsample PBDE stock solution, under
N2 gas, and each PBDE was reconstituted in dimethylsulfoxide
and diluted to 2.5 mg/ml in TEMG buffer (10 mM Tris-HCl,
1 mM ethylenediaminetetraacetic acid·2H2O, 1 mM 2-mercaptoethanol, 10% glycerol, 0.02% sodium azide, pH 7.5).
Recovery of the PBDEs after evaporation was assumed to be
100%. Four fivefold serial dilutions were prepared by dilution
in TEMG buffer. Five different concentrations (2.5, 0.5, 0.1,
0.02, and 0.004 mg/ml) of PBDE, four concentrations of E2
(20, 4, 0.8, and 0.16 mM), or five concentrations of testosterone
(20, 2, 0.2, 0.02, and 0.002 mM) (150 ml) were incubated for
15 h at 48C with either 15 nM 3H-estradiol (72 Ci/mmol, NEN
Life Sciences, Boston, MA, USA) or 45 nM 3H-testosterone
(96 Ci/mmol, NEN Life Sciences) (150 ml) and hormonestripped carp serum (150 ml: all concentrations equal final
concentration in the entire 450-ml incubation volume). After
15 h, 500 ml dextran-coated charcoal (0.05% w/v dextran, 0.5%
w/v activated carbon, in TEMG) were added. The tubes were
then vortexed for 1 min and centrifuged for 6 min (1,500 g).
Immediately after centrifugation, the supernatant was poured
directly into a liquid scintillation vial in the same order that
the carbon was added, and scintillation cocktail was added.
All procedures were carried out at 48C. Samples were analyzed
using a Packard (Meriden, CT, USA) Tri-Carb 2100 TR liquid
scintillation analyzer. Three replicate tubes were run for each
sample and standard. The same pool of serum was used for
all replicates of each standard and sample. Blanks and total
count controls were also run in at least triplicate with each set
of tubes analyzed. Percentage 3H-steroid bound was calculated
and plotted as a function of dose for each sample and standard.
RESULTS
H4IIE-luc and MVLN bioassays
Ten PBDE congeners, representing those typically found
in biological samples, were analyzed using the H4IIE-luc and
Fig. 2. Displacement of 3H-testosterone (T) (A) or 3H-estradiol (B)
from carp serum proteins using polybrominated diphenyl ether
(PBDE) 126 as an example of the typical sample response. %-bound
5 dpm sample/dpm control (total counts) 3 100. Error bars 5 1
standard deviation. 17b-estradiol (E2).
MVLN bioassays. These included representatives of tetra-,
penta-, hexa-, hepta-, and deca-brominated diphenyl ethers. At
the concentrations tested, only one of the 10 PBDEs,
3,39,4,49,5-penta BDE (PBDE 126), induced a significant response in the H4IIE-luc bioassay (Fig. 1). Exposure to 500
ng/ml PBDE 126 yielded a response magnitude equal to 13
6 1.4 % of the maximum response observed for a 1,500-pM
TCDD standard (%-TCDD-max.; Fig. 1). Exposure to 167 and
55.5 ng/ml PBDE 126 yielded responses of 4.2 6 1.7%TCDD-maximum and 2.4 6 0.1%-TCDD-maximum, respectively, but these responses were not significantly different from
that elicited by the solvent control (Fig. 1). With the exception
of 500 ng/ml 2,3,39,4,49 penta-BDE, which yielded a response
of 1.7%-TCDD-maximum, no other PBDE yielded a response
greater than 1%-TCDD-max. At the concentrations tested in
this study, none of the PBDEs tested induced a significant
response in the MVLN bioassay. All responses were less than
5%-E2-maximum, and no dose dependence was observed.
Hormone displacement assays
At the concentrations tested, none of the 10 PBDE congeners caused significant displacement of 3H-E2 or 3H-T from
carp serum proteins in vitro. Only the E2 and testosterone
standards caused a significant decrease in the %-3H-E2 or
3H-T, respectively, bound to carp serum proteins (Fig. 2). At
In vitro assessment of polybrominated diphenyl ethers
20 mM, the E2 standard caused only a 28 6 1.8% displacement
of 3H-E2 (Fig. 2). The relatively minor displacement caused
by the E2 standard suggests that the carp serum used for this
study may have had relatively few specific binding proteins
for E2. Thus, assay sensitivity for detecting potential displacement of 3H-E2 by PBDEs may have been limited. Assay sensitivity for detecting potential displacement of 3H-T by PBDEs
was greater. At the greatest concentration tested, 20 mM, testosterone caused 61 6 2.2% displacement of 3H-T (Fig. 2).
DISCUSSION
H4IIE-luc and MVLN bioassays
The results of the H4IIE-luc bioassay indicate that the
PBDEs tested were at least 200,000 times less potent (relative
potency [REP] , 2.0 3 1025) than TCDD for inducing AhRmediated gene expression. For comparison, several polycyclic
aromatic hydrocarbons (PAHs: e.g., benzo[k]fluoranthene, indeno[1,2,3-cd ]pyrene) and polychlorinated naphthalenes
(PCNs: e.g., 1,2,3,4,6,7-hxCN, 1,2,3,6,7,8-hxCN, 1,2,3,4,5,6,7hpCN) have been reported to be more potent inducers of AhRmediated gene expression in H4IIE-luc [15,16]. A recent study
by Chen et al. [17] examined the AhR-mediated potency of
12 PBDEs for inducing ethoxyresorufin-O-deethylase (EROD)
activity in six different cell types. Seven of the congeners
tested in this study were also tested by Chen et al. [17]. Among
those, 3,39,4,495-pentaBDE (PBDE 126), 3,39,4,49-tetraBDE
(PBDE 77), and 2,29,4,49,6-pentaBDE (PBDE 100) were reported to have REPs around 1024 to 1025[17]. Thus, the potency of these congeners in several other in vitro bioassays
was greater than that observed in this study. The difference
in potency observed may be due to the shorter exposure period
used by Chen et al. [17] (48 vs 72 h used in this study).
Alternatively, H4IIE-luc cells may simply be less sensitive to
PBDEs than the other cell lines tested. The fact that PBDE
126, the most potent PBDE in the Chen et al. study [17], was
the only PBDE to induce a significant response in the H4IIEluc bioassay supports the hypothesis that the H4IIE-luc assay
(with a 72-h exposure duration) may simply be less sensitive
to PBDEs. Overall, this study and that of Chen et al. [17]
support the conclusion that most PBDEs are at least 10,000
times less potent than TCDD for inducing AhR-mediated responses in vitro.
The results of the MVLN bioassay indicate that the PBDEs
tested were at least 50,000 times less potent (REP , 5.0 3
1024) than E2 for inducing ER-mediated gene expression. This
agrees with results recently reported by Meerts et al. [18].
Their study suggested that PBDEs were at least 250,000 times
less potent than E2 for inducing ER-mediated reporter gene
expression. Together, the results of the in vitro studies suggest
that PBDEs are unlikely to elicit ER-mediated gene expression
at concentrations currently observed in fish and wildlife.
Hormone displacement assays
Polybrominated diphenyl ethers are lipophilic compounds
that would be expected to partition to fatty tissues in exposed
organisms. Nonetheless, PBDE concentrations as great as 6
ng/ml have been detected in salmon blood [4]. Occupationally
exposed humans may have serum concentrations in excess of
20 ng/ml [13]. At concentrations up to 833 ng/ml, the PBDEs
tested in this study did not show an appreciable capacity for
displacing 3H-steroids from carp serum proteins.
Environ. Toxicol. Chem. 21, 2002
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Acknowledgement—This work was supported by U.S. Environmental
Protection Agency (U.S. EPA) Biology Exploratory Grants Program,
Grant R85371-01-0; cooperative agreement CR 822983-01-0 between
Michigan State University and U.S. EPA; and the National Institute for
Environmental Health Sciences Superfund Basic Research Program
NIH-ES-04911. We thank Emily Nitsch for her technical assistance.
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