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Published in final edited form as:
Toxicol Sci. 2006 July ; 92(1): 133–142. doi:10.1093/toxsci/kfj186.

Interactions of Polybrominated Diphenyl Ethers with the Aryl
Hydrocarbon Receptor Pathway
A. K. Peters*,1, S. Nijmeijer*, K. Gradin†, M. Backlund‡, Å. Bergman§, L. Poellinger†, M. S.
Denison¶, and M. Van den Berg** Institute for Risk Assessment Sciences, Utrecht University, PO Box 80176, 3508 TD Utrecht,

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The Netherlands † Department of Cell and Molecular Biology, Institute of Environmental Medicine,
Karolinska Institute, SE-17177, Stockholm, Sweden ‡ Division of Molecular Toxicology, Instituteof Environmental Medicine, Karolinska Institute, SE-17177, Stockholm, Sweden § Department ofEnvironmental Chemistry, Wallenberg Laboratory, Stockholm University, SE-10691 Stockholm,
Sweden ¶ Department of Environmental Toxicology, Meyer Hall, University of California—Davis,
One Shields Avenue, Davis, California 95616

Abstract

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Polybrominated diphenyl ethers (PBDEs) are brominated flame retardants that have been in use asadditives in various consumer products. Structural similarities of PBDEs with otherpolyhalogenated aromatic hydrocarbons that show affinity for the aryl hydrocarbon receptor(AhR), such as some polychlorinated biphenyls, raised concerns about their possible dioxin-like
properties. We studied the ability of environmentally relevant PBDEs (BDE-47, -99, -100, -153,-154, and -183) and the “planar” congener BDE-77 to bind and/or activate the AhR in stablytransfected rodent hepatoma cell lines with an AhR-responsive enhanced green fluorescent protein(AhR-EGFP) reporter gene (H1G1.1c3 mouse and H4G1.1c2 rat hepatoma). 7-Ethoxyresorufin-Odee-thylation (EROD) was used as a marker for CYP1A1 activity. Dose- and bromination-specificinhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced responses was measured bytheir ability to inhibit the induction of AhR-EGFP expression and EROD activity. Individual
exposure to these PBDEs did not result in any increase in induction of AhR-EGFP or CYP1A1
activity. The lower brominated PBDEs showed the strongest inhibitory effect on TCDD-induced
activities in both cell lines. While the highest brominated PBDE tested, BDE-183, inhibited
EROD activity, it did not affect the induction of AhR-EGFP expression. Similar findings wereobserved after exposing stably transfected human hepatoma (xenobiotic response element [XRE]–
HepG2) cells to these PBDEs, resulting in a small but statically significant agonistic effect on
XRE-driven luciferase activity. Co-exposure with TCDD resulted again in antagonistic effects,
confirming that the inhibitory effect of these PBDEs on TCDD-induced responses was not only
due to direct interaction at receptor level but also at DNA-binding level. This antagonism wasconfirmed for BDE-99 in HepG2 cells transiently transfected with a Gal4-AhR construct and thecorresponding Gal4-Luc reporter gene. In addition, a chromatin immunoprecipitation assay further
confirmed that BDE-99 could bind to the AhR and activate the AhR nuclear translocation anddioxin responsive element (DRE) binding in the context of the CYP1A1 promoter. However, thetransactivation function of the BDE-99-activated AhR seems to be very weak. These combined
results suggest that PBDEs do bind but not activate the AhR–AhR nuclear translocator protein–
XRE complex.

1

To whom correspondence should be addressed at Institute for Risk Assessment Sciences, Utrecht University, Yalelaan 2, PO Box
80176, 3508 TD Utrecht, the Netherlands. Fax: +31-30-2535077. l.peters@iras.uu.nl.

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Keywords

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aryl hydrocarbon receptor; PBDE; EROD; AhR-EGFP; XREINTRODUCTION
Polybrominated diphenyl ethers (PBDEs) are a group of brominated flame retardants andhave been in use as additives in various consumer products since the 1970’s. Although
PBDEs are beneficial in that they reduce the chances of ignition and burning of materials,
they have the ability to leach out of the polymer in which they are present. There are threemajor commercial PBDE mixtures that are or have been produced, differing in their degreeof bromination; the penta-, octa-, and deca-mix. Some PBDE congeners have been found tobe lipophilic with log Kow values varying from 6 for penta- to 10 for deca-BDE (Watanabe
and Tatsukawa, 1989). Accordingly, levels of PBDEs have been detected world wide inmany matrices, for example, in fish, wildlife, bird’s eggs (Darnerud et al., 2001; De Wit,
2002; Law et al., 2003; Sjodin et al., 1999), as well as in human blood, adipose tissue(Covaci et al., 2002; Meironyte et al., 1999), and breast milk samples (Noren and
Meironyte, 2000).

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The European Union Community banned the use of penta-BDE and octa-BDE since August
2004 (European Union, Restriction of Hazardous Substances Directive; Directive 2002/95/
EC of the European Parliament and of the Council of 27 January 2003 on the restriction of
the use of certain hazardous substances in electrical and electronic equipment, OJ L37, page19, 13 February 2003), voluntarily followed by one of the major U.S. producers (Great
Lakes Chemical Corporation, IN…) that ceased the production of these two PBDE mixturesby the end of 2004. The continued use of deca-BDE is still under discussion within the
United States, but its use is not restricted in the European Union (Commission Decision of
13 October 2005 amending for the purposes of adapting to the technical progress the Annex
to Directive 2002/95/EC of the European Parliament and of the Council on the restriction of
the use of certain hazardous substances in electrical and electronic equipment, OJ L271, of
15 October 2005, page 48). The current ban on PBDEs will eventually cause thesechemicals to reach a steady state concentration followed by a decline in biota and abiota, as
has already been reported in human breast milk samples from Sweden (Lind et al., 2003).
However, due to the persistent nature of PBDEs, it could take decades before this decline
can be observed in all matrices world wide with continuing exposure through older end
product use and waste incineration.

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Structural similarities of PBDEs with other polyhalogenated aromatic hydrocarbons thatbind to the aryl hydrocarbon receptor (AhR), such as some polychlorinated biphenyls(PCBs; Fig. 1), raised concern about the possible binding of PBDEs to the AhR, leading to
AhR-mediated toxic and biological effects. The AhR can be found in the cytoplasm ofalmost all vertebrate cells and a structurally diverse range of chemicals can bind to and/or
activate AhR-dependent gene expressions, which leads to a variety of biological and toxic
effects (Denison and Heath-Pagliuso, 1998;Denison et al., 2002). This ligand diversity
suggests that the AhR has a rather promiscuous binding pocket and raises questions as to thespectrum of chemicals which can bind to the AhR. Upon binding of a ligand to this receptor,
the ligand-AhR complex migrates into the nucleus of the cell where it binds the AhR nuclear
translocator protein (ARNT) (Whitlock et al., 1996). The AhR and ARNT function together
as a heterodimer and can bind to the xenobiotic response elements (XREs) on the DNA(Reyes et al., 1992;Whitelaw et al., 1993). These XREs have been identified on the 5-prime
promotor regions of several AhR-inducible genes (Hankinson, 2005;Kuramoto et al.,
2002;Lees and Whitelaw, 1999). Binding of the ligand-AhR-ARNT complex to the XRE

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stimulates transcription of adjacent genes; the molecular mechanism of induction of
expression of the phase I biotransformation enzyme cytochrome P4501A1 (CYP1A1) genehas been the most studied response (Nebert and Gonzalez, 1987).
CYP1A1 enhances the detoxification of lipophilic substrates (many of which are AhR
ligands) that might otherwise accumulate to toxic concentrations in a cell. Induction of
CYP1A1 only occurs when necessary; upon removal of the stimulus (ligand) by metabolism,
CYP1A1 gene expression is no longer upregulated. 2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD) is the highest affinity ligand for the AhR, and it is also extremely slowly
metabolized by CYP1A1 and other enzymes. Consequently, the long residence time of
TCDD in the cell results in a sustained induction of gene expression, and it is commonly
thought that most of the potent toxic and biological effects of TCDD and related chemicals
are attributed to the prolonged activation of the AhR-dependent gene expression (Whitlock
et al., 1996). CYP1A1 is inducibly expressed in all vertebrates, although there are large
differences in expression levels in different tissues and among species (Denison and HeathPagliuso, 1998; Denison et al., 2002; Guengerich, 1993). Use of induction of CYP1A1 andCYP1A1-dependent ethoxyresorufin-O-deethylation (EROD) as a biomarker for AhRmediated effects in the risk assessment process developed for dioxin-like compounds has
been proposed and applied (Van den Berg et al., 1998).

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Previously, we exposed rodent hepatoma (H4IIE), human breast carcinoma (MCF-7), andhuman hepatoma (HepG2) cell lines to highly purified PBDEs (BDE-47, -99, -100, -153,-154, -183) (the numbering of the PBDE congeners is adapted from the International Union
of Pure and Applied Chemistry system for numbering PCB congeners [Ballschmiter and
Zell, 1980]) in order to assess their ability to activate the AhR-mediated CYP1A1 induction
using EROD activity as a marker. While these individual PBDEs were not able to induce
CYP1A1, coexposure resulted in a decrease in TCDD-induced CYP1A1 activity (Peters etal., 2004). Similar results were observed for these PBDE congeners using primary
hepatocytes of cynomolgus monkeys (Macaca fascicularis) (Peters et al., 2005). In contrast
to our experiments, a weak but statistically significant CYP1A1 induction has beenpreviously reported for PBDE-77, -100, -153, and -183 in various cell lines (Behnisch et al.,
2003; Chen et al., 2001) and primary rat hepatocytes (Chen and Bunce, 2003; Chen et al.,2001).

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The objectives of our present study were to assess if the observed inhibitory effects of these
PBDEs were caused by direct antagonism of the AhR and/or inhibition of the CYP1A1dependent EROD activity. In addition to measuring CYP1A1 activity, PBDE interactions
with the AhR were also assessed using two stably transfected rodent hepatoma cell lines
containing an AhR-responsive enhanced green fluorescent protein (AhR-EGFP) reporter
gene containing approximately 500 bp of the CYP1A1 promoter including four XRE
sequences (H1G1.1c3 mouse and H4G1.1c2 rat hepatoma cell lines). A human hepatoma(HepG2) cell line containing either a stably transfected construct of two isolated XRE
sequences in front of a heterologous promoter (XRE-HepG2) or transiently transfected witha Gal4-AhR construct were used to investigate the AhR and XRE binding further.

MATERIALS AND METHODS
Chemicals
The chemicals used were obtained from the following companies. TCDD (>99% pure) was
purchased from Cambridge Isotope Laboratories (Woburn, MA); environmentally relevant
PBDE congeners (> 98% pure; Åke Bergman, Sweden) were synthesized and each congener
was subjected to a specific purification on activated charcoal and Celite to remove possible
contamination with dioxin-like compounds such as polybrominated dibenzofurans (Marsh et
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NIH-PA Author Manuscriptal., 1999). Besides using the earlier mentioned environmentally relevant PBDEs, BDE-77
was also included in our experiments. BDE-77 is not environmentally relevant but resembles
PCB-77 due to its lack of ortho-bromine and two adjacent bromine atoms on both aromaticrings and was therefore included. H1G1.1c3 and H4G1.1c2 cell lines were provided by M.
S. Denison (University of California—Davis, CA), and XRE-HepG2 cells were provided by
K. Gradin (University of Stockholm, Stockholm, Sweden); the HepG2 cell line was obtained
from American Type Culture Collection (Manassa, VA). Cell culture media Dulbecco’s
Modified Eagle Medium (DMEM), RPMI1640 with glutamine and phenol red, phosphatebuffered saline (PBS), and fetal calf serum (FCS) were obtained from Gibco BRL (Breda,
The Netherlands). Fugene 6 was purchased from Roche (Mannheim, Germany), luciferase
cell culture lysis buffer (5×) reagent was purchased from Promega (Madison, WI), andluciferase buffer and adenosine triphosphate (ATP) substrate were purchased from
BioThema (Handen, Sweden). Polymerase chain reaction (PCR) reagents as well as
OptiMEM were from Invitrogen (Carlsbad, CA), and the protease inhibitor mix was from
Roche. All other chemicals were obtained from Sigma Chemical Company (St Louis, MO).
Cell culture

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The mouse hepatoma cell line H1G1.1c3 and rat hepatoma cell line H4G1.1c2 were createdby stable transfection of mouse hepatoma (Hepa1c1c7) and rat hepatoma (H4IIE) cells with
the AhR-EGFP reporter plasmid pGreen1 as reported earlier (Nagy et al., 2002). Both celllines were cultured in DMEM, supplemented with 10% heat-inactivated FCS, 100 U/ml
penicillin, and 100 μg/ml streptomycin in an incubator (5% CO2, 37°C).
The HepG2-derived cell line XRE-HepG2 was created by stable transfection with aconstruct containing XRE sequences (Gradin et al., 1993). The human hepatoma cell lines
HepG2 and XRE-HepG2 were cultured in RPMI medium with phenol red and glutamine,
supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, and 100 μg/ml
streptomycin. The XRE-HepG2 medium was additionally supplemented with 800 μg/ml
geneticin.
Enhanced green fluorescent protein assay

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The H1G1.1c3 and H4G1.1c2 cell lines have been genetically modified to produce a
fluorescent protein upon activation of the AhR by ligands. Both cell lines were seeded innormal culture medium in 96-well plates (1 × 104 cells per well) and exposed after 24 h tothe solvent control dimethyl sulfoxide (DMSO) (0.1%), positive control TCDD (0.001–1nM), and the chosen PBDEs (0.01–10μM) or coexposed to both TCDD and PBDEs. After
72 h, the cells were washed twice with PBS (37°C), PBS added to each well, and thefluorescence of intact cells measured using an excitation wavelength of 485 nm andemission wavelength of 510 nm in a Fluostar (BMG Labtechnologies GmbH, Offenburg,
Germany). Dose response relationship analyses for the TCDD, PBDEs, and TCDD/PBDEs
coexposures were carried out, and induced AhR-EGFP activity was determined bysubtracting the background fluorescence in the DMSO sample from the TCDD-, PBDE-, or
TCDD/PBDE-treated samples (Galietta et al., 2001).
7-Ethoxyresorufin-O-deethylase assay
EROD activity was used as a marker for CYP1A1-mediated catalytic activity using amodification of the method described by Burke and Mayer (1974) as reported recently(Peters et al., 2004). Directly following the AhR-EGFP assay, the PBS in the microplate
wells was replaced with serum-free medium supplemented with 5mM MgCl2, 5μM 7ethoxyresorufin (ER), and 10mM dicumarol. The conversion of 7-ER into the fluorescentresorufin was followed over a 10-min period at 37°C using an excitation wavelength of 530

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Page 5nm and emission wavelength of 590 nm. The EROD activity was normalized to the amountof protein per well as measured by the method of Denizot and Lang (1986).

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Transient transfection and luciferase assays

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The Gal4-AhR construct and the corresponding Gal4-Luc reporter gene were created bycloning of the rat AhR into the mammalian expression vector pFA-CMV containing thecDNA for the yeast Gal4 DNA-binding domain as described by Backlund and IngelmanSundberg (2004). Transient transfections into HepG2 cells were carried out in 24-wellplates, using 150 ng of pFA-AhR expression plasmid and 200 ng of pFR-Luc per well in
OptiMEM, as previously reported (Backlund and Ingelman-Sundberg, 2004). Fugene 6transfection reagent was used according to the manufacturer’s instructions (1.5 μl per well).
Approximately 24 h (5% CO2, 37°C) after transfection, the culture medium was exchanged
with fresh media containing solvent control DMSO (0.1%), TCDD (0.001–1nM), BDE-99(0.01–10μM), or a combination of TCDD and BDE-99. After 24 h (5% CO2, 37°C), thetransiently transfected cells (24-well plates) or stable XRE-HepG2 cells (12-well plates)
were rinsed with PBS, and 100–60 μl lysis buffer was added (12–24 well, respectively). The
cell lysates were collected by scraping and transferred to a microcentrifuge tube. After brief
vortexing, cell lysates were spun (2–5 min, 4°C, maximum rpm), and 30 μl of thesupernatant was mixed with 100 μl Luciferin substrate and 100 μl ATP substrate andluciferase activity determined in a luminometer. The values were normalized to the protein
concentrations measured according to the method of Bradford (1976).
Chromatin immunoprecipitation assay

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The procedure for the chromatin immunoprecipitation (ChIP) assay was adopted from that
described by DiRenzo et al. (DiRenzo et al., 2000). HepG2 cells were seeded in cell culture
dishes (15 cm diameter) and grown until 80% confluency. The cells were then treated withDMSO (0.1%), TCDD (1nM), BDE-99 (10 μM), or TCDD and BDE-99 (1nM and 10μM,
respectively). After a 3-h incubation period, cells were washed with PBS. Protein crosslinking was achieved by adding formaldehyde (1%) to the cells and allowing them toincubate at room temperature for 10 min. Cells were washed twice with ice-cold PBS,
collected into a 100mM Tris-HCL (pH 8.7) and 10mM dithiothreitol solution (cold) into acentrifuge tube, incubated for 15 min at 30°C, and subsequently pelleted (5 min, 2000 g).
The pellets were washed sequentially in the following buffers: PBS (ice cold), buffer I(0.25% Triton X-100, 10mM ethylenediaminetetraacetic acid [EDTA], 0.5mM
ethyleneglycol-bis(2-aminoethyl ether)-N,N,N′, N-tetraacetic acid (EGTA), 10mM 4-(2hydroxvethyl)-1-piperazineethanesulfonic acid (HEPES) [pH 6.5]), and buffer II (200mM
NaCl, 1mM EDTA, 0.5mM ethyleneglycol-bis(2-aminoethyl ether)-N,N,N′, N′-tetraacetic
acid (EGTA), 10mM 4-(2-hydroxvethyl)-1-piperazineethanesulfonic acid (HEPES) [pH6.5]). The pellets were resuspended in 0.5 ml lysis buffer (1% SDS, 10mM EDTA, 50mM
Tris-HCl [pH 8.0], protease inhibitor cocktail) and then sonicated on ice (three times for 10s, Branson Sonifier) to yield DNA fragments in the 200- to 900-bp range. Samples were
centrifuged for 10 min at 4°C, and the supernatants were mixed (1:10) with dilution buffer(1% Triton X-100, 2mM EDTA, 150mM NaCl, 20mM Tris-HCl [pH 8.0], protease inhibitor
cocktail). IgG immunoclearing was performed by incubating 1 ml soluble chromatin with 2
μg sheared herring sperm DNA, 6 μg IgG, and 45 μl protein G-sepharose (50% slurry) for 2
h at 4°C with rotation. After centrifugation (15 s, 3000 rpm), the supernatant was incubatedovernight with 5 μg of AhR-specific antibodies or IgG (overnight at 4°C with rotation).
After this, 45 μl of protein G-sepharose slurry containing 2 μg sheared herring sperm DNA
was added to the samples (2 h at 4°C rotation). The sepharose beads were collected bycentrifugation (15 s, 3000 rpm) and washed sequentially in the following buffers (15-minrotation): TSE I (20mM Tris-HCL [pH 8.1], 150mM NaCl, 2mM EDTA, 0.1% SDS, 1%
Triton X-100), TSE II (20mM Tris-HCL [pH 8.1], 500mM NaCl, 2mM EDTA, 0.1% SDS,
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1% Triton X-100), buffer III (10mM Tris-HCL [pH 8.1], 0.5M LiCl, 1% nonidet P-40, 1%
deoxycholate, 1mM EDTA), and twice with TE-buffer (pH 8.0). Immunocomplexes wereextracted three times from the beads with 100 μl 1% SDS-0.1M NaHCO3 and centrifuged at
3000 rpm for 15 s.
Cross-linking was reversed by heating the eluates at 65°C overnight. The eluates were thenpurified with QIAquick Spin Kit, and 1 μl of the purified DNA (0.1 μl for the input sample)
was used for PCR (Platinum Pfx DNA Polymerase; performed according to the
manufacturer’s recommendations) with the primers that would amplify regions containing
the XRE elements of the human CYP1A1 gene (hCYP1A1-XRE 5′-CAC CCT TCG ACAGTT CCT CTC CCT and hCYP1A1-XRE 3′-CTC CCG GGG TGG CTA GTG CTT TGA).
The PCR products were separated and visualized in a 2% agarose gel.
Cell viability

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After incubation with the designated compounds at concentrations used in the experiments
for 72 h (5% CO2, 37C°), H1G1.1c3, H4G1.1c2, HepG2, or XRE-HepG2 cells were
washed, and medium was replaced with a 1 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT, Sigma Chemical Company) solution. The conversion of
MTT to formazan in the presence of the mitochondrial enzymes succinate de-hydrogenase
was allowed to proceed for 30 min at 37°C (Denizot and Lang, 1986). After the incubation
period, cells were washed and the formazan was extracted with 0.1 ml of isopropanol andincubated for 10 min, and the formazan concentration was determined
spectrophotometrically using an absorbance wavelength of 595 nm.
Statistical analysis
In each experiment, exposure to the test compound was carried out in triplicate. EC50 values
for EROD activity and AhR-EGFP response (50% of the maximum activity, calculatedusing the fitted concentration-response curve) were obtained using sigmoidal dose-response
nonlinear regression curve fit (GraphPad Prism 3.0, GraphPad Software Inc., San Diego,
CA). Statistical differences among treatments were determined by a two-tailed Student’s ttest, with a significance level at >95% (p < 0.05).

RESULTS

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Exposure of mouse H1G1.1c3 and rat H4G1.1c2 cells to the positive control agonist TCDD
resulted in a concentration- and time-dependent increase in TCDD-induced CYP1A1(EROD) activity and AhR-EGFP reporter gene expression. No difference in sensitivity ininduction between the H1G1.1c3 and H4G1.1c2 cell lines was observed with the EROD
assay (Fig. 2A), with EC50 values for TCDD of 8.18 × 10− 12M and 4.67 × 10− 12M,
respectively. However, with respect to AhR-EGFP induction (Fig. 2B), the mouse hepatoma
cell line was approximately twofold more sensitive, with EC50 values of 4.03 × 10− 12M for
H1G1.1c3 cells and 9.16 × 10− 12M for H4G1.1c2 cells. These almost similar EC50 values
were caused by the fact that the mouse H1G1.1c3 cell line reached a higher maximum
induction (Fig. 2B). Although these cell lines are exquisitely sensitive bioassays to detect
inducers of AhR-dependent gene expression, no induction of EGFP or CYP1A1/EROD was
observed for any individual PBDE (data not shown).
Antagonistic Effects of PBDEs on AhR-EFGP Expression
Co-incubation of TCDD (0.001–1nM) with 0.1–10μM PBDEs (BDE-47, -99, -100, -153,-154, -183 and -77) resulted in a concentration-dependent decrease in AhR-EGFP
expression (Fig. 3A–3C). This antagonistic effect was observed for almost all PBDEs tested,although the planar BDE-77 exhibited the greatest antagonistic effect in both cell lines (Fig.
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3B). A quantitative difference in antagonistic effects was observed between the PBDEs
tested, which appeared related to their degree of bromination. Lower brominated PBDEs
such as the tetra-brominated BDE-47 and -77 showed stronger antagonism of TCDDinduced AhR-EGFP activity compared to higher brominated PBDEs, while the highest
brominated congener tested (hepta-brominated BDE-183) failed to antagonize AhR-EGFP
reporter gene induction (Fig. 3C).
No striking differences were observed between the two cell lines, though the AhR-EGFP
expression resulted in more significant effects in the rat hepatoma cell line (Tables 1 and 2).
Antagonistic Effects of PBDEs on EROD Activity
Consistent with the above results, a concentration-dependent decrease in EROD activity was
also observed (Fig. 4). These effects occurred at higher concentrations than measured in the
AhR-EGFP assay, resulting in lower EC50 values (Tables 1 and 2). As with the AhR-EGFP
assay, this antagonistic/inhibitory effect was more pronounced with the lower brominated
PBDEs, with the planar BDE-77 being the most potent antagonist in both cell lines (Fig.
4B). No obvious differences were observed between the two cell lines, though the mouse
hepatoma cell line showed a more pronounced effect in the EROD assay as can be seen in
Tables 1 and 2.

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Transfections in HepG2 Cells
The human hepatoma cell line (HepG2) containing a stably transfected XRE-driven
luciferase reporter gene was also used to study the possible AhR-mediated dose- and timedependent activation of transcription by different PBDEs. Exposure of the cells to thepositive control TCDD resulted in a dose-dependent significant induction (EC50 6.7 ×
10− 10M). The PBDEs resulted in a small though not always significant induction of XREdriven luciferase activity (at most 2% of that of TCDD 1nM for BDE-99 10μM) comparedto the solvent control. The planar BDE-77 that proved the most potent antagonist in the
EROD and AhR-EGFP assays did not result in a remarkably higher induction of XREdriven luciferase activity compared to other lower brominated congeners as BDE-47 andBDE-99 (data not shown). BDE-99 resulted in the most consistent significant luciferase
activity.

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After co-incubation of the XRE-HepG2 cells with TCDD and selected PBDEs, the humancell line responded similarly to the rodent cell lines. All PBDEs caused an antagonistic
effect toward the TCDD-induced XRE induction. The AhR-EGFP construct contains 500 bp
of the CYP1A1 promotor and the HepG2-XRE construct contains isolated XRE sequences;
the fact that the PBDEs are able to inhibit TCDD-induced responses in both systems is anindication that the inhibition is mediated via the XRE sequences.
Because the most consistent results in the XRE-HepG2 luciferase assay were obtained with
BDE-99, HepG2 cells were transiently transfected with a Gal4-AhR construct and thecorresponding Gal4-Luc reporter gene and exposed to BDE-99, with and without coexposure to the TCDD. Exposure to TCDD resulted in a dose-dependent increase inluciferase activity in this cell line (EC50 3.7 × 10− 10M). However, exposure to BDE-99(0.01–10μM) alone did not result in a significant induction of AhR expression (Fig. 5). The
Gal4-AhR expression is driven by the CMV-promotor and the activity of the co-transfected
Gal4-dependent luciferase reporter gene is only detected after stimulation of the cells with
AhR activators. Dimerization of the AhR-ARNT complex with the DNA is not needed toobtain a response in cells transiently transfected with this construct. Thus, BDE-99 by itself
did not activate the AhR-XRE complex. Co-exposure of the cells with TCDD and BDE-99

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Page 8resulted again in a dose-dependent decrease in reporter expression, suggesting a similar typeof interaction as observed in the AhR-EGFP cell lines.

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Chromatin Immunoprecipitation Assay
To further assess at what point BDE-99 was able to interfere with the signal transduction
pathway, a ChIP assay was performed with cells treated with DMSO (0.1%), TCDD (1nM),
BDE-99 (10 μM), or TCDD and BDE-99 (1nM and 10μM, respectively). The AhR antibody
resulted in a positive band for both BDE-99 and the co-exposed cells, but the brightest band
appeared with TCDD-exposed cells (Fig. 6). This supports the data obtained in the AhREGFP and transfection assays, further suggesting that some PBDEs can bind to andstimulate AhR DNA binding but that the interaction with DNA and chromatin leads to an
unproductive complex incapable of activating transcription of the CYP1A1 gene. Whilesimilar types of antagonism have been observed for some steroid hormone nuclear receptors,
this would be the first AhR antagonist that exerts its inhibitory effects in this manner. The
control antibody IgG did not result in any unexpected bands on the gel (Fig. 6).

DISCUSSION

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PBDEs have been suggested to act as AhR antagonists based on their ability to inhibit
TCDD-induced CYP1A1 activity in human and rodent cell lines (Peters et al., 2004, 2005).
In the present study, we determined whether these PBDEs could antagonize TCDD-induced
AhR-mediated effects by direct effect on binding and/or activation of the AhR.

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In both AhR-EGFP rodent cell lines, exposure to the PBDEs alone did not result in any
AhR-mediated signals. As could be expected from a mechanistic point of view, this also
resulted in a lack of CYP1A1 induction. However, co-exposure with TCDD resulted in asignificant decrease in TCDD-induced CYP1A1 (EROD) activity in these AhR-EGFP celllines (Fig. 3). These results are in agreement as those observed earlier with the rodent H4IIE
rat hepatoma cell line, human hepatoma HepG2, and breast carcinoma MCF-7 cell lines(Peters et al., 2004). For the lower brominated PBDEs, like BDE-47, this decrease in EROD
activity concurred with a decrease in AhR-EGFP reporter gene expression (Figs 3A and
4A). In these instances, the antagonism in AhR-EGFP expression indicates that theantagonistic effect on TCDD-induced CYP1A1 by PBDEs is related to AhR binding andactivation rather than simple competitive inhibition of EROD activity. This would alsosuggest that PBDEs could bind to the AhR as antagonists and compete with more potent
agonists for binding the AhR in analogy with what was found for PCBs (Bandiera et al.,
1982;Gasiewicz et al., 1996). However, some of the higher brominated PBDEs like
BDE-183 also reduced TCDD-induced CYP1A1 (EROD) activity (Fig. 4C) while notinfluencing the AhR-EGFP gene expression (Fig. 3C). This might suggest that some of these
PBDEs could reduce EROD activity by virtue of their ability to act as competitive CYP1A1
substrates as has been observed for other chemicals such as the PCBs (Bandiera et al.,
1982;Gasiewicz et al., 1996).
In our experiments, the planar BDE-77 showed the strongest inhibitory effect on TCDDinduced CYP1A1 activity and AhR-EGFP expression. This clearly suggests that thesubstitution pattern of PBDE congeners that presumably bind the AhR is of importance forthe observed antagonistic effects (the planarity of the congeners could facilitate binding to
the AhR ligand–binding domain). In analogy with the observed effects of the bromine
substitution pattern, previously Suh et al. (2003) reported that the chlorination substitution
pattern of certain di-ortho–substituted PCB congeners determine their ability to elicit
antagonistic effects on TCDD-induced AhR-mediated effects. Antagonistic effects by thehigher chlorinated PBDEs were also observed at the XRE level. Binding to the XREs on the
DNA was indirectly assessed by the use of synthetic reporter genes, which contain XREToxicol Sci. Author manuscript; available in PMC 2011 February 2.

Peters et al.

Page 9binding sites in a heterologous promotor context, that reports the increased basal activity(Weiss et al., 1996).

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Binding to the XRE on DNA as indirectly measured in our experiments suggests that someof these PBDEs are capable of initiating the AhR transformation as well as the nuclear
localization. All PBDEs tested in our study elicited an inhibitory effect on TCDD-induced
XRE-driven luciferase activity. However, based on our data, it seems that the affinity of thehigher brominated congeners is lower than that of the lower brominated congeners withregard to AhR-mediated effects.
A further analysis of the effects of BDE-99, the congener giving the most consistent andpronounced inhibitory effects in our present study, was performed with the Gal4-AhR
construct. Singular exposure to BDE-99 did not lead to significant effects on the AhR, but
the ChIP assay confirmed the ability of BDE-99 (10μM) to allow the AhR to bind to thepromotor region on the DNA (Fig. 6).

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It has been suggested that certain di-ortho–substituted PCBs bind the AhR but show no
CYP1A1 induction, depending on the substitution pattern (Chen and Bunce, 2004; Merchant
et al., 1992; Suh et al., 2003). However, this ligand binding to the AhR is probably
unproductive as suggested previously for PCB-153 (Chen and Bunce, 2004; Merchant et al.,1992). We suggest that, concerning the antagonistic effects of PBDEs with TCDD, there is asimilar mechanism of action because ligand binding to the AhR alone is not sufficient forachieving later steps in the signaling pathway such as CYP1A1 induction, as was suggested
earlier by Chen and Bunce (2004).
In summary, our results confirm the previous findings that suggest that PBDEs can bind but
not activate the AhR-XRE complex and subsequent transcription processes like that of
CYP1A1 (Peters et al., 2004, 2005). This effect appears to be most pronounced for the lower
brominated congeners, which antagonize TCDD-dependent transcriptional activation by the
AhR. The mechanism for this is unknown, but as BDE-99 can cause nuclear translocation of
the AhR with transcriptional activation not being initiated, this compound could cause an
unfavorable conformational change of the receptor that does not allow binding to cofactors
and/or components of the initiation complex. Observed interactions for some of the higher
brominated PBDEs on CYP1A1 activity may be AhR independent and likely occur bycompetitive inhibition of CYP1A1-dependent EROD activity, but this remains to be
confirmed.

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Because the concentrations of both TCDD and individual PBDEs in our in vitro experiments
far exceed the current levels in human blood or plasma, it is impossible to draw conclusions
from these antagonistic interactions between both groups of compounds for human health.
However, the observed effects might still have implications for the risk assessment as
humans are exposed to a complex mixture with a large number of dioxin-like compounds,
PBDEs and PCBs. As many quantitatively important PBDEs and PCBs can apparently act as
AhR antagonists and most likely act in concert when present in mixtures, this could actually
influence the overall effect of dioxin-like compounds in a downregulating way. Based on
our in vitro results, the question remains to which extent these PBDEs could actually inhibit
or antagonize AhR-mediated toxicological and biological effects in vivo, which should
clearly be assessed further.

Acknowledgments
This study was financially supported by the Bromine Science and Environmental Forum. The rodent AhR-EGFP
cell lines were developed with support by grants from the U.S. National Institutes of Environmental Health
Sciences (ES07685 and ES04699).

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FIG. 1.

Structure of (A) PCBs and (B) PBDEs, with (x + y ≤ 10).

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FIG. 2.

Induction of (A) EROD-activity and (B) AhR-EGFP expression in rat H4G1.1c2 and mouse
H1G1.1c3 cells, after exposure to the positive control TCDD. The data are expressed asmean of three separate experiments ± SEM.

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FIG. 3.

Induction of AhR-EGFP expression in mouse H1G1.1c3 (left) and rat H4G1.1c2 (right) cells
after coexposure to TCDD (0.5pM–1nM) and (A) BDE-47 (0–10μM), (B) BDE-77 (0–10μM), and (C) BDE-183 (0–10μM). The data are expressed as mean of three separate
experiments ± SEM.

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FIG. 4.

Induction of EROD activity in mouse H1G1.1c3 (left) and rat H4G1.1c2 (right) cells after
coexposure to TCDD (0.5pM–1nM) and (A) BDE-47 (0–10μM), (B) BDE-77 (0–10μM),
and (C) BDE-183 (0–10μM). The data are expressed as mean of three separate experiments± SEM.

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FIG. 5.

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Luciferase activity in HepG2 cells transiently transfected with Gal4-AhR expression vector
and the corresponding Gal4-Luc reporter gene. Cells were exposed to solvent control DMSO
(0.1%), TCDD (1nM), BDE-99 (0.01–10μM), and a combination of TCDD (1nM) andBDE-99 (0.01–10μM). Data are expressed as mean of one representative assay ± SEM (n =3, *p < 0.05 compared to TCDD 0.1nM): **p < 0.01 compared to TCDD (1nM); + p < 0.05
compared to DMSO.

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FIG. 6.

End products of the ChIP assay on a 2% agarose gel.

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NIH-PA Author Manuscript

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Toxicol Sci. Author manuscript; available in PMC 2011 February 2.

BDE-154

BDE-153

BDE-100

BDE-99BDE-7763.3 ± 9148.4 ± 9125.0 ± 20

7.81 × 10− 12
6.23 × 10− 12

0.1
1

10

8.72 × 10− 125

1097.4 ± 156.99 × 10− 12

197.7 ± 7

100.1 ± 10

7.91 × 10− 12

0.1

66.5 ± 10130.7 ± 57.13 × 10− 12

107.67 × 10− 12

47.1 ± 52.97 × 10− 115556.0 ± 11

1.49 × 10− 11

196.7 ± 59.30 × 10− 12

1116.7 ± 13

107.8 ± 31

8.89 × 10− 12

0.1

7.16 × 10− 12

14.1 ± 52.02 × 10− 11

10

0.1

24.1 ± 8

2.62 × 10− 115

63.6 ± 1040.8 ± 131.75 × 10− 11

17.37 × 10− 1288.2 ± 28

0.18.92 × 10− 12

31.1 ± 1101.18 × 10− 11

60.8 ± 1254.65 × 10− 11

100.9 ± 22105.8 ± 39

1.86 × 10− 11

1.45 × 10− 111

0.1

BDE-47

Y-max

1.10 × 10− 11

Concentration (μM)

1008.18 × 10− 12

0.1

TCDD

EROD

EC50

Concentration (nM)

0.28

0.03*

0.01*

0.03*

0.74

0.28

0.02*

0.87

0.99

0.00*

0.00*

0.02*

0.55

0.81

0.00*

0.00*

0.01*

0.70

0.00*

0.03*

0.97

0.89

t-Test

p

1

0.1

10

5

1

0.1

10

5

1

0.1

10

5

1

0.1

10

5

1

0.1

10

5

1

0.1

Concentration (μM)

1Concentration (nM)

3.43 × 10− 12

3.95 × 10− 12

4.90 × 10− 12

5.04 × 10− 12

3.80 × 10− 12

3.57 × 10− 12

6.69 × 10− 12

5.39 × 10− 12

3.83 × 10− 12

3.39 × 10− 12

1.08 × 10− 11

7.54 × 10− 12

4.68 × 10− 12

4.33 × 10− 12

2.14 × 10− 11

1.01 × 10− 11

5.33 × 10− 12

3.49 × 10− 12

1.57 × 10− 11

1.02 × 10− 11

4.07 × 10− 12

3.62 × 10− 12

4.03 × 10− 12EC5080.4 ± 3

93.5 ± 1893.4 ± 996.2 ± 1596.0 ± 7

90.6 ± 887.2 ± 1874.7 ± 1594.7 ± 1373.6 ± 4

83.8 ± 1688.9 ± 18102.2 ± 795.2 ± 25

45.5 ± 27

56.4 ± 27

81.3 ± 1771.0 ± 569.7 ± 580.3 ± 5

97.5 ± 1598.2 ± 9

Y-max

100

AhR-EGFP

0.00*

0.81

0.52

0.82

0.61

0.29

0.52

0.17

0.70

0.00*

0.36

0.56

0.77

0.85

0.11

0.18

0.34

0.00*

0.00*

0.02*

0.88

0.85

t-Testp

Effect of PBDEs on TCDD-Induced AhR-EGFP Expression and EROD Activity in Mouse H1G1.c1 Cells

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TABLE 1
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101.9 ± 20
50.1 ± 4
91.4 ± 33
132.2 ± 2398.8 ± 1064.8 ± 35

5.21 × 10− 12
1.16 × 10− 11
7.77 × 10− 128.31 × 10− 129.97 × 10− 12

5
10
0.1
1
5
10

Y-max
7.82 × 10− 12

Concentration (μM)

0.37

0.91

0.23

0.81

0.00*

0.93

t-Test

p

10

5

1

0.1

10

5

Concentration (μM)

1Concentration (nM)

4.40 × 10− 12

5.00 × 10− 12

4.73 × 10− 12

3.91 × 10− 12

7.38 × 10− 12

5.00 × 10− 12

4.03 × 10− 12EC5074.9 ± 7

93.7 ± 1497.2 ± 14102.0 ± 488.1 ± 6

76.8 ± 11

Y-max

100

AhR-EGFP

0.03*

0.29

0.85

0.62

0.11

0.22

t-Testp

Statistically significant compared to the maximum induction by TCDD (p < 0.05).

*

Note. Effects of PBDEs on TCDD-induced AhR-EGFP expression and EROD activity in a stably transfected mouse hepatoma cell line (H1G1.1c3). The data are presented as the mean of three experiments(± SEM), all conducted in triplicate. Y-max: maximum achieved response of the compound compared to the maximal response of TCDD.

BDE-183

TCDD

100

8.18 × 10− 12

0.1

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EROD

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EC50

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Concentration (nM)Peters et al.
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BDE-154

BDE-153

BDE-100

BDE-99BDE-7779.7 ± 2
108.3 ± 9
94.9 ± 590.0 ± 13129.8 ± 15115.1 ± 124.60 × 10− 12
3.83 × 10− 12
3.63 × 10− 12
4.53 × 10− 12
3.96 × 10− 12

1
5
10
0.1
1

1

5.56 × 10− 12

173.8 ± 64

5.09 × 10− 12

0.1

0.1

115.7 ± 36

1.49 × 10− 11

1088.2 ± 1892.1 ± 16

1.03 × 10− 115127.9 ± 37

101.7 ± 156.66 × 10− 12

11.00 × 10− 11

72.7 ± 1

5.60 × 10− 12

0.1

532.6 ± 94.84 × 10− 11101052.3 ± 12

3.00 × 10− 115

140.5 ± 4472.7 ± 13

1.23 × 10− 11

16.85 × 10− 12

105.5 ± 7

0.1

5.25 × 10− 1282.2 ± 185.43 × 10− 12

102.96 × 10− 11

88.7 ± 92.51 × 10− 11

574.1 ± 8
101.7 ± 10

5.45 × 10− 127.95 × 10− 12

0.1

BDE-47

Y-max

1

Concentration (μM)

1004.67 × 10− 12

0.1

TCDD

EROD

EC50

Concentration (nM)

0.29

0.11

0.48

0.39

0.41

0.00*

0.56

0.49

0.41

0.32

0.68

0.65

0.91

0.00*

0.00*

0.02*

0.11

0.45

0.38

0.28

0.87

0.03*

t-Test

p

1

0.1

10

5

1

0.1

10

5

1

0.1

10

5

1

0.1

10

5

1

0.1

10

5

1

0.1

Concentration (μM)

1Concentration (nM)

7.58 × 10− 12

7.46 × 10− 12

7.89 × 10− 12

7.01 × 10− 12

7.19 × 10− 12

7.57 × 10− 12

1.21 × 10− 119.95 × 10− 12

7.55 × 10− 12

7.58 × 10− 12

2.41 × 10− 11

1.71 × 10− 119.97 × 10− 128.96 × 10− 126.75 × 10− 11

4.38 × 10− 11

1.76 × 10− 11

1.05 × 10− 11

4.69 × 10− 11

3.41 × 10− 11

1.38 × 10− 11

1.01 × 10− 11

9.16 × 10− 12EC5083.1 ± 682.0 ± 4

77.3 ± 971.5 ± 5

78.4 ± 1579.8 ± 1160.2 ± 481.0 ± 982.7 ± 7

89.8 ± 1077.8 ± 1380.0 ± 3

89.0 ± 1187.5 ± 1465.0 ± 1765.6 ± 1572.5 ± 480.8 ± 565.2 ± 1466.5 ± 973.2 ± 1270.0 ± 11

Y-max

100

AhR-EGFP

0.06

0.01*

0.07

0.01*

0.22

0.14

0.00*

0.11

0.08

0.37

0.18

0.00*

0.39

0.41

0.12

0.09

0.00*

0.02*

0.06

0.02*

0.09

0.06

t-Testp

Effects of PBDEs on TCDD-Induced AhR-EGFP Expression and EROD Activity in Rat H4G1.c1 Cells

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TABLE 2
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86.6 ± 768.2 ± 26

5.99 × 10− 12

5
10

1

108.8 ± 1277.2 ± 124.87 × 10− 12

0.1

5.15 × 10− 1264.1 ± 13104.71 × 10− 12

89.9 ± 12

4.08 × 10− 12

5

Y-max
4.11 × 10− 12

Concentration (μM)

0.28

0.15

0.50

0.14

0.05*

0.45

t-Test

p

10

5

1

0.1

10

5

Concentration (μM)

1Concentration (nM)

1.26 × 10− 11

1.03 × 10− 11

8.46 × 10− 12

7.86 × 10− 12

1.02 × 10− 11

7.37 × 10− 12

9.16 × 10− 12EC5083.6 ± 5

84.6 ± 8

95.9 ± 892.1 ± 2

67.8 ± 1375.5 ± 9

Y-max

100

AhR-EGFP

0.03*

0.13

0.62

0.02*

0.07

0.05*

t-Testp

Statistically significant compared to the maximum induction by TCDD (p < 0.05).

*

Note. Effects of PBDEs on TCDD-induced AhR-EGFP expression and EROD activity in a stably transfected rat hepatoma cell line (H4G1.1c2). The data are presented as the mean of three experiments (±SEM), all conducted in triplicate. Y-max: maximum achieved response of the compound compared to the maximal response of TCDD.

BDE-183

TCDD

100

4.67 × 10− 12

0.1

NIH-PA Author Manuscript
EROD

NIH-PA Author Manuscript

EC50

NIH-PA Author Manuscript

Concentration (nM)Peters et al.
Page 22

Toxicol Sci. Author manuscript; available in PMC 2011 February 2.