Synthesis of polybrominated diphenyl ethers and their capacity to induce CYP1A by the Ah receptor mediated pathway

Article abstract of DOI:10.1021/es0107475

Polybrominated diphenyl ethers (PBDEs) have become widely distributed as environmental contaminants due to their use as flame retardants. Their structural similarity to other halogenated aromatic pollutants has led to speculation that they might share toxicological properties such as hepatic enzyme induction. In this work we synthesized a number of PBDE congeners, studied their affinity for rat hepatic Ah receptor through competitive binding assays, and determined their ability to induce hepatic cytochrome P-450 enzymes by means of EROD (ethoxyre-sorufin-O-deethylase) assays in human, rat, chick, and rainbow trout cells. Both pure PBDE congeners and commercial PBDE mixtures had Ah receptor binding affinities 10^-2-10^-5 times that of 2,3,7,8-tetrachlorodibenzo-p-dioxin. In contrast with polychlorinated biphenyls, Ah receptor binding affinities of PBDEs could not be related to the planarity of the molecule, possibly because the large size of the bromine atoms expands the Ah receptor's binding site. EROD activities of the PBDE congeners followed a similar rank order in all cells. Some congeners, notably PBDE 85, did not follow the usual trend in which strength of Ah receptor binding affinity paralleled P-450 induction potency. Use of the gel retardation assay with a synthetic oligonucleotide indicated that in these cases the liganded Ah receptor failed to bind to the DNA recognition sequence.

Full text of DOI:10.1021/es0107475

Environ. Sci. Technol. 2001, 35, 3749-3756
mercially (PCDDs and PCDFs), tens of thousands of tonnes
Synthesis of Polybrominated
Diphenyl Ethers and Their Capacity
to Induce CYP1A by the Ah
Receptor Mediated Pathway
of PBDEs are presently manufactured annually as flame
retardants for soft furnishings, electronic equipment, and
automobiles (3). The “open” nature of these applications
virtually guarantees that the PBDEs will end up dispersed
into the environment. Levels of PBDEs in biota are currently
rising rapidly and parallel the use pattern of PBDEs since
their introduction in the late 1960s (4-6). Indeed, in one
recent report, PBDE levels in biota surpassed those of PCBs
(2).
The commercial PBDE mixtures presently in use are
nominally deca-, octa-, and penta-brominated. Some 67 000
tonnes were manufactured in 1999 (7), an amount rivalling
PCBs at the height of their production. Congeners 47 and 99
(2,2′,4,4′-tetraBDE and 2,2′,4,4′,5-pentaBDE) are the most
frequently encountered PBDEs in the environment (8); these
are major constituents of the “penta” commercial mixture,
a minor commercial formulation (7). These congeners are
found even in samples taken remotely from known sources
(5), suggesting that, like other HACs having similar vapor
pressures (9), they may undergo atmospheric transport (10,
11).
G U O S H E N G C H E N , †
A L E X A N D R E D . K O N S T A N T I N O V , ‡
B R O C K G . C H I T T I M , ‡
E L I Z A B E T H M . J O YC E , §
N I E L S C . B O L S , § A N D N I G E L J . B U N C E * , †
Department of Chemistry and Biochemistry,
University of Guelph, Guelph, Ontario, Canada N1G 2W1,
Wellington Laboratories, 398 Laird Road,
Guelph, Ontario, Canada N1G 3X7, and
Department of Biology, University of Waterloo,
Waterloo, Ontario, Canada N2L 3G1
Polybrominated diphenyl ethers (PBDEs) have become
widely distributed as environmental contaminants due to
their use as flame retardants. Their structural similarity to
other halogenated aromatic pollutants has led to
Very little is yet known about the toxicology of PBDEs.
The structural similarities between PBDEs and other classes
of HACs, notably the PCBs, suggest that PBDEs might activate
the aryl hydrocarbon receptor (AhR) signal transduction
pathway (12), which is a critical toxicological mechanism for
many HACs. This response leads to the induction of the
cytochrome P-450 isozyme CYP 1A1 (12-14), which can be
assayed as 7-ethoxyresorufin-O-deethylase (EROD) activity.
Induction of CYP 1A1 begins when the HAC binds to the
intracellular AhR, causing the components of the AhR
complex to dissociate and migrate to the nucleus, where the
Ah receptor ligand binding subunit heterodimerizes with the
nuclear protein ARNT. The dimer then associates with
recognition sites on DNA called “dioxin response enhancers”,
triggering the transcription of mRNAs from which CYP 1A1
is translated. This sequence of events is relevant to envi-
ronmental concerns because many HACs exhibit strong rank-
order correlations between strength of Ah receptor binding,
CYP 1A induction, and toxicity (15).
speculation that they might share toxicological properties
such as hepatic enzyme induction. In this work we
synthesized a number of PBDE congeners, studied their
affinity for rat hepatic Ah receptor through competitive binding
assays, and determined their ability to induce hepatic
cytochrome P-450 enzymes by means of EROD (ethoxyre-
sorufin-O-deethylase) assays in human, rat, chick, and
rainbow trout cells. Both pure PBDE congeners and
commercial PBDE mixtures had Ah receptor binding
affinities 10-2-10-5 times that of 2,3,7,8-tetrachlorodibenzo-
p-dioxin. In contrast with polychlorinated biphenyls, Ah
receptor binding affinities of PBDEs could not be related
to the planarity of the molecule, possibly because the large
size of the bromine atoms expands the Ah receptor’s
binding site. EROD activities of the PBDE congeners followed
a similar rank order in all cells. Some congeners, notably
PBDE 85, did not follow the usual trend in which strength
of Ah receptor binding affinity paralleled P-450 induction
potency. Use of the gel retardation assay with a synthetic
oligonucleotide indicated that in these cases the liganded
Ah receptor failed to bind to the DNA recognition sequence.
In the present study, 18 PBDE congeners were synthesized
and evaluated, along with three commercial PBDE mixtures,
for their capacity to bind and activate the AhR in rat
hepatocytes and to induce EROD activity in cells from
rainbow trout, chick, rat, and human.
Synthetic Methodology
Chem icals. 1,2-Dibromobenzene, 1,3-dibromobenzene, 1,4-
dibromobenzene, 1,4-dibromo-2-fluorobenzene, 1,2,4-tri-
bromobenzene, 2-bromophenol, 4-bromophenol, 2,4-di-
bromophenol, 2,4,6-tribromophenol, 2,5-bromoaniline, and
2,6-dibromoaniline were obtained from Aldrich. All solvents
were HPLC Grade (Fisher Scientific, Toronto ON) and were
used as received. Mass spectra were obtained using a
Micromass VG70SE high-resolution mass spectrometer
(HRMS), coupled with Hewlett-Packard 5890 Series II gas
chromatograph, equipped with a DB-5 capillary column
Introduction
Polybrominated diphenyl ethers are ubiquitously present in
the environment, in a manner reminiscent of other highly
halogenated and persistent aromatic compounds (HACs),
such as the polychlorinated and polybrominated biphenyls
(PCBs and PBBs), polychlorinated dibenzo-p-dioxins (PCDDs),
and polychlorinated dibenzofurans (PCDFs) (1, 2). Unlike
these other HACs, which have either been phased out of
production (PCBs and PBBs) or never synthesized com-
1
(J&W, 60 m × 0.25 mm × 0.25 µm). H NMR analyses were
performed using a Bruker AM400. PBDE congeners were
purified by column chromatography on Kieselgel-60 using
hexane as the eluent, followed by a cleanup on a small Celite-
carbon column to remove possible traces of PBDD/ PBDF
and finally crystallized from methanol.
* Corresponding author phone: (519)824-4120 ext. 3962; fax:
(519)766-1499; e-mail: bunce@chembio.uoguelph.ca.
† University of Guelph.
‡ Wellington Laboratories.
Synthesis of Interm ediates. 2-Brom o-4-nitroaniline was
obtained in 89% yield by bromination of 4-nitroaniline with
§
University of Waterloo.
9
10.1021/es0107475 CCC: $20.00
Published on Web 08/14/2001
2001 Am erican Chem ical Society
VOL. 35, NO. 18, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 3 7 4 9

1.01 equivalent of bromine (16), mp 104.5 °C; lit. (17) 104.5
°C; purity 99% by GC-MS. 2,6-Dibrom o-4-nitroaniline was
prepared similarly by bromination of 4-nitroaniline with 2.2
equiv of bromine (89% yield; mp 205 °C; lit. (18) 206 °C; 99%
purity).
anhydrous potassium carbonate. After 4 h at reflux, the
brownish-red mixture was cooled to room temperature, the
acetone was evaporated, and the residue was partitioned
between methylene chloride and water. The organic layer
was washed with 5% NaOH and brine, dried over anhydrous
sodium, the solvent evaporated, and the residue was purified
by flash chromatography over silica gel, eluting with 1:10
hexane:methylene chloride. Other polybromo-4-nitrodiphe-
nyl ethers were prepared similarly except that the product
was eluted with hexane:ethyl acetate (4:1).
3,4,5-Tribrom onitrobenzene was prepared by the Sand-
meyer reaction of diazotized 2,6-dibromo-4-nitroaniline with
CuBr (19) (54% yield; mp 111 °C; lit. (20) 112 °C; purity 99%).
3,4-Dibrom onitrobenzene was prepared similarly from
2-bromo-4-nitroaniline (yield 62%, mp 59 °C; lit. (21) 58-59
°C; purity 98%), 1,2,3-tribrom obenzene from 2,6-dibro-
moaniline (92% yield, mp 88 °C; lit. (22) 87.8 °C; purity 99%),
and 2,4,5-tribrom oiodobenzene by iodination of 2,4,5-
tribromoaniline (72% yield, 99% purity), mp 108-109 °C.
1,2,4-Tribrom o-5-nitrobenzene (1) was prepared by a
modification of the method of Oliver and Ruth (23). 1,2,4-
Tribromobenzene (1.07 g, 3.39 mmol) was dissolved in a
2:2:1 mixture of methylene chloride, trifluoroacetic acid, and
trifluoroacetic anhydride and cooled in an ice/ water bath to
0° C. To the stirred solution was added ammonium nitrate
(0.266 g, 4.15 mmol). The mixture was allowed to warm to
room temperature and stirred overnight, then the solvent
was removed on the rotary evaporator, and the residue,
dissolved in methylene chloride, was eluted on a 40 × 3 cm
silica column (Kieselgel-60) with 1:1 hexane:methylene
chloride. The product crystallized from ethanol to give 1.13
g (92%) of 2,4,5-tribromonitrobenzene (purity >99% by GC-
MS, mp 79-81 °C; lit. (24) 80-81 °C. The analogous method
was used to prepare 2,3,4-tribrom onitrobenzene from 1,2,3-
tribromobenzene (yield 54%, mp 85 °C, lit. (25) 85.4 °C, purity
98.5% by GC-MS) and 2,5-dibrom o-4-fluoro-nitrobenzene
(6) from 2,5-dibromofluorobenzene (89%, brown oil, purity
98.5% by GC-MS).
Preparation of Iodonium Salts from Brom inated Ben-
zenes. The procedure of Jakobsson et al. (34) was followed
at one-fifth scale, giving 2,2′,4,4′-tetrabrom odiphenyliodo-
nium chloride from 1,3-dibromobenzene (5 g), yield 4.59 g,
68%, and 3,3′,4,4′-tetrabrom odiphenyliodonium chloride
from 1,2-dibromobenzene (5 g), yield 1.54 g, 23%.
Synthesis of BPDEs from Iodonium Salts and Bro-
m ophenols. The method of Nilsson et al. (35) was used. The
yields and spectral data of products are provided in Table
1.
Preparation of 4-Methoxyphenyl-2′,4′,5′-tribrom ophen-
yliodonium Brom ide. To hydrogen peroxide (204 µL of 30%,
2 mmol) in a test tube equipped with a magnetic stirrer and
maintained at 0 °C was added 1.56 mL (11.8 mmol) of
trifluoroacetic anhydride during 5 min followed by slow
addition of 2,4,5-tribromoiodobenzene (0.981 g, 2.21 mmol)
in 5 mL of methylene chloride. The mixture was allowed to
warm to room-temperature overnight, then the solvent was
evaporated, and the residue was dissolved in 10 mL of
methylene chloride/ acetic anhydride (1:1 v/ v), containing
170 µL of acetic acid. The solution was cooled to -20 °C and
anisole (482 µL, 4.43 mmol) in 5 mL of acetic anhydride was
slowly added with stirring. The reaction was kept at -20 °C
for 45 min and then at room temperature for 48 h. After
removal of solvents in vacuo below 70 °C, the oily residue
was dissolved in a minimal amount of methanol, and a
saturated solution of sodium bromide was added to pre-
cipitate off-white crystals of the product, 4-methoxyphenyl-
2′,4′,5′-tribromophenyliodonium bromide (yield 0.276 g,
20%).
The method of Krolski et al. (26) was used to reduce the
following nitro compounds to anilines: 3,4-dibrom oaniline
from 3,4-dibromonitrobenzene (yield 62%, mp 81 °C; lit. (27)
81 °C; purity 98% by GC-MS); 2,4,5-tribrom oaniline from
1,2,4-tribromo-5-nitrobenzene (yield 94.5%, mp 79-81 °C;
lit. (28) 80-81 °C; purity >99% by GC-MS); 2,3,4-tribro-
m oaniline from 2,3,4-tribromonitrobenzene (yield 67%, mp
101 °C; lit. (27) 100.6 °C; 99% purity); and 3,4,5-tribrom o-
aniline from 3,4,5-tribromonitrobenzene (yield 88%, mp 123
°C; lit. (29) 123 °C; 99% purity).
2,2′,3,4,4′,5′-Hexabromodiphenyl Ether (BDE-183). 2,3,4-
Tribromophenol (102 mg, 0.31 mmol) was dissolved in a
boiling solution of sodium hydroxide (12.5 mg, 0.31 mmol)
in water (4 mL), and 4-methoxyphenyl-2′,4′,5′-tribromophen-
yliodonium bromide (196 mg, 0.31 mmol) was added in one
portion. The mixture was held at reflux for 2 h, then the pH
was brought to 11, and the water layer was extracted with
3 × 10 mL of methylene chloride. Workup by flash chro-
matography followed by a Celite/ activated carbon column
gave 80 mg (40% yield), purity 98%. Spectral data are provided
in Table 1.
2,4,5-Tribrom ophenol (2). Solid sodium nitrite (77 mg,
1.11 mmol) was slowly added to a stirred solution of 2,4,5-
tribromoaniline (361 mg, 1.09 mmol) in 10 mL of TFA at 0
°C. The resulting light-yellow solution was transferred with
a Pasteur pipet into a boiling solution of sodium sulfate in
50% sulfuric acid, then a little water was added, and the
solution was heated to reflux for 50 min. The product was
steam-distilled, and the distillate was extracted with 3 × 20
mL of methylene chloride. The combined extract was dried
(Na2SO4), the solvent was evaporated, and the product was
purified by flash chromatography (Kieselgel-60), eluting with
1:5 hexane:methylene chloride. Yield 278 mg (78%), mp 86-
87 °C; lit. (30) 87 °C; purity >99% by GC-MS. Analogous
methods were used to prepare 3,4-dibrom ophenol (65%
yield, mp 94 °C; lit. (31) 94 °C; purity 98%), 2,3,4-tribro-
m ophenol from 2,3,4-tribromoaniline (45% yield, mp 95 °C;
lit. (32) 95 °C; purity 98%), 2,5-dibrom ophenol from 2,5-
dibromoaniline (61% yield, mp 73 °C; lit. (30) 73-74 °C, purity
98%), and 3,4,5-tribrom ophenol (68% yield, purity 98%).
2,3,4,6-Tetrabrom ophenol was prepared by reaction of
3-bromophenol with bromine (3.3 equiv) in the presence of
iron filings, as described by Marsh et al. (16) (yield 82%, mp
112 °C; lit. (33) 112-113 °C; purity 98.5% by GC-MS).
Condensation of 2,5-Dibrom o-4-fluoronitrobenzene
with Brom ophenols. In a typical procedure, 1.5 mmol of the
bromophenol was added over 30 min to a boiling mixture
of 6 (1.5 mmol) in 4.00 mL of dry acetone and 600 mg of
Modified Suzuki Coupling to Prepare 3,4,4′,5-Tetra-
brom odiphenyl Ether (BDE-77). The procedure of Chan (36)
was used to react the 4-bromophenyl monoester of boronic
acid (218 mg, 1.09 mmol) and 3,4,5-tribromoiodobenzene
(180 mg, 0.41 mmol) in the presence of copper(II) acetate
(108 mg, 0.59 mmol) and triethylamine (0.245 mL, 1.76 mmol)
in methylene chloride under nitrogen at room-temperature
overnight with the following modifications: the amount of
triethylamine was increased to 4.3 equiv; methylene chloride
and triethylamine were dried over 4 Åmolecular sieves before
the reaction; and 0.5 g of finely ground 4 Å molecular sieves
were present in the reaction slurry. Yield: 40%; purity 98%.
4,4′-Dibromodiphenyl ether (10%) was also formed (sepa-
rated by flash chromatography).
Molecular Modeling. Molecular mechanics calculation
and conformation searches were performed using “Spartan
plus”. The lowest energy and energy of coplanar conformation
for each congener were calculated by the SYBYL method.
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3 7 5 0 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 35, NO. 18, 2001

TABLE 1. Physical Properties of Synthesized PBDE Congeners
yield,
%
M+
congener
Br pattern
2,4,4′-
methoda
(79Br)
spectral details
28
47
I
I
63
45
404
482
7.80, d (J ) 2 Hz); 1H, 7.44, m , 3H, 6.85, dd (J ) 2 Hz, J ) 9 Hz) 3H
7.82, d (J ) 2 Hz) 2H; 7.42, dd (J ) 2 Hz, J ) 9 Hz) 2H; 6.47,
d (J ) 9 Hz) 2H
7.83, d (J ) 2 Hz) 1H; 7.53, d (J ) 8 Hz) 1H; 7.45, dd (J ) 2 Hz,
J ) 8 Hz) 1H; 7.19, dd (J ) 2 Hz, J ) 8 Hz) 1H; 6.93, d
(J ) 2 Hz) 1H; 6.81, d (J ) 8 Hz) 1H
7.81, d (J ) 2 Hz) 1H; 7.58, d (J ) 8 Hz) 1H; 7.46, dd (J ) 2 Hz,
J ) 8 Hz) 1H; 7.19, d (J ) 2 Hz,) 1H; 6.93, d (J ) 9 Hz) 1H;
6.81, dd (J ) 2 Hz, J ) 8 Hz) 1H
2,2′,4,4′-
49
66
2,2′4,5′-
I
I
67
482
482
2,3′,4,4′-
32
71
77
2,3′,4′,6-
3,3′,4,4′-
I
I
41
87
482
482
7.63, d (J ) 8 Hz), 2H; 7.53, d (J ) 8 Hz), 1H; 7.09, d (J ) 2 Hz),
1H; 7.06, t (J ) 8 Hz), 1H; 6.65, dd (J ) 2 Hz, J ) 9 Hz), 1H
7.57, d (J ) 9 Hz), 2H; 7.26, d (J ) 2 Hz), 2H; 6.83,
dd (J ) 2 Hz, J ) 9 Hz), 2H
7.80, s, 1H; 7.42, d (J ) 7 Hz), 1H; 6.71, d (J ) 7 Hz), 1H
8.01, d (J ) 8 Hz), 1H; 7.84, d,(J ) 2 Hz) 1H; 7.39, dd (J ) 2 Hz,
J ) 9 Hz) 1H; 7.28, d(J ) 8 Hz), 1H; 6.51, d (J ) 9 Hz) 1H
7.91, s, 1H; 7.84, d (J ) 2 Hz), 1H; 7.47, dd (J ) 2 Hz,
J ) 9 Hz), 1H; 7.02, s, 1H; 6.84, d (J ) 9 Hz), 1H
7.83, s, 2H; 7.82, d (J ) 2 Hz), 1H; 7.30, dd, (J ) 2 Hz,
J ) 8 Hz), 1H; 6.31, d (J ) 9 Hz), 1H
7.81, s, 2H; 7.55, d (J ) 8 Hz), 1H; 7.09, d (J ) 2 Hz),
1H. 6.69, dd (J ) 2 Hz, J ) 8 Hz), 1H
7.637, d (J ) 9 Hz), 1H; 7.32, d (J ) 2 Hz), 1H; 7.27, s,
2H; 6.90, dd (J ) 2 Hz, J ) 9 Hz) 1H
7.94, s, 2H; 7.13, s, 2H
7.92, s, 1H; 7.83, s, 2H; 6.64, s, 1H
8.02, s, 1H; 7.93, s, 1H; 6.62, s, 1H
81
85
3,4,4′,5-
2,2′,3,4,4′-
III
I
40
74
482
560
99
100
119
126
2,2′,4,4′,5-
2,2′,4,4′,6-
2,3′,4,4′,6-
3,3′,4,4′,5-
I, II
69, 82
51
560
560
560
560
I
I
56
II
84
153
154
183
2,2′,4,4′,5,5′-
2,2′,4,4′,5,6′-
2,2′,3,4,4′,5,5′-
II
II
II, IV
81
48
12, 45
638
638
714
a
I - iodonium salt coupling; II - nucleophilic condensation; III - m odified Suzuki coupling; IV - unsym m etrical iodonium salt coupling.
The “planarization energy” means the energy difference
between the coplanar and minimum energy conformations.
Bioassay Methodology. Supplies. 2,3,7,8-Tetrachloro-
dibenzo-p-dioxin (TCDD) was a generous gift from Well-
ington Laboratories (Guelph, Ontario, Canada). Glycerol,
sodium ethylenediamine tetraacetate (EDTA), dimethyl sulf-
oxide (DMSO), and tris-(hydroxymethyl)-aminomethane
(TRIS) were purchased from Fisher Scientific (Toronto, ON,
Canada); resorufin was purchased from ICN Biomedicals Inc.
(Aurora, OH); sodium pentobarbital (Somnitol) was obtained
from MTC Pharmaceuticals (Cambridge, ON, Canada);
Percoll, Hank’s Balanced Salt Solution, Dulbelcco’s phosphate
buffered saline (PBS), fetal bovine serum, penicillin G,
streptomycin, L-thyroxine (T4), sucrose, EGTA, N-2-hydroxy-
lpiperazine-N-2-ethanesulfonic acid (HEPES), dithioeryth-
ritol (DTE), and 7-ER were purchased from Sigma (St. Louis,
MO); hydroxylapatite was from Bio-Rad Canada (Missisauga,
ON); hepatocyte qualified collagenase, minimal essential
medium, and all components of attachment medium and
serum free medium were purchased from GIBCO BRL
(Burlington, ON, Canada). Immature Sprague-Dawley rats
(100 g) were purchased from Charles River Breeding Labo-
ratories (Canada); 19 day Barred Rock egg embryos were
obtained from the Arkell Research Station, University of
Guelph. Treatment of animals was in accordance with the
University of Guelph Animal Care Policy C5.1.
Rad Protein Assay Dye Reagent with bovine serum albumin
(BSA) as a standard.
Hydroxylapatite (HAP) Ligand Binding Assay (38). One
milliliter aliquots of tissue cytosol were diluted with HEGD
buffer to give a final protein concentration of 2 mg/ mL.
Cytosol was incubated with a 1.0 nM [3H]-TCDD and a range
of concentrations of an unlabeled PBDE. The protein
components of the cytosol were then adsorbed onto hy-
droxylapatite (Ca5(PO4)3OH) and rinsed with a mild detergent
(1.5% v/ v Triton X-100) to remove free and loosely bound
radioligand, and the bound radioactivity was quantitated by
liquid scintillation counting. Nonspecific binding was de-
termined in a parallel incubation of 1.0 nM [3H]-TCDD plus
200 nM unlabeled TCDF and was assumed to be independent
of the concentration of the unlabeled competitor (39). Data
were elaborated by plotting “probit of percent specific
binding” vs log[PBDE]; the EC50 was obtained from the log-
[PBDE] corresponding to probit ) 5.000; 100% specific
binding was the value obtained when no PBDE was present.
Gel Retardation Assay. The following 32-base pair oli-
gonucleotides containing the dioxin response enhancer (DRE)
consensus binding sequence 5′-T-GCGTG-3′ were obtained
from University Core DNA Services, University of Calgary.
Rodent Hepatic Cytosol. Immature male Sprague-
Dawley rats (∼100 g) were euthanised by exposure to carbon
dioxide followed by cervical dislocation. Livers were im-
mediately perfused in situ with ice-cold HEGD buffer via the
hepatic portal vein, then excised. Tissue was homogenized
using three strokes of a Potter Elvhjem Teflon tissue
homogenizer. The homogenate was spun at 9000 × g for 20
min in a Sorvall Superspeed RC2-B centrifuge at 4 °C and
then spun at 100 000 × g for 68 min in a Beckman L7-65
ultracentrifuge at 4 °C. The cytosol was stored in 1 mL aliquots
at -70 °C until required. The protein content of the cytosol
was determined by the method of Bradford (37) using Bio-
The oligonucleotides were [32P]-end labeled by T4 poly-
nucleotide kinase and annealed as follows. A single strand
oligonucleotide was dissolved in TEN buffer (10 mM Tris, 1
mM EDTA, 100 mM NaCl, pH 8.0) to give a concentration
of 150 ng/ µL. A mixture of 3 µL of each oligonucleotide, 3 µL
of 10x T4 polynucleotide kinase reaction buffer (New England
BioLabs), 18 µL of sterile water, 1.5 µL of T4 polynucleotide
kinase (10 000 U/ mL), and 1 µLofγ-[32P]-ATP (7000 Ci/ mmol,
160 mCi/ mL) was incubated at 37 °C for 30 min. An additional
0.5 µL of T4 polynucleotide kinase was added, and the mixture
was incubated for 30 min more. After adding 2.3 µL of 1.4
9
VOL. 35, NO. 18, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 3 7 5 1

M KCl, the microfuge tube was sealed, heated to 85 °C, and
allowed to cool slowly to room temperature to allow the two
complementary oligonucleotides to anneal. The [32P]-labeled
oligonucleotide was then purified on a NICK Sephadex G-50
spin column according to the manufacturer’s instruction and
stored at -20 °C before use. One µL [32P]-labeled oligo-
nucleotide was taken for liquid scintillation counting.
Aliquots of tissue cytosol (16 mg protein / mL) were
incubated with either 1 µL of DMSO (control), 10 nM of TCDD
(reference), or various concentrations of PBDE or mixtures
of TCDD and PBDE at 30 °C for 2 h. Aliquots of liganded
cytosol were incubated at 23 °C with 500 ng of poly (dIdC)
for 15 min; 1 µL of [32P]-oligonucleotide (∼500 000 cpm/ µL)
was then added, and the samples were mixed and incubated
for a further 15 min at 23 °C. After mixing with bromophenol
blue tracking dye, the samples were then electrophoresed in
a 5% polyacrylamide gel in TBE buffer at 11 V/ cm using a
Bio-Rad Protean II x i cell with a Bio-Rad model 200/ 2.0
power supply. Gels were removed and placed on a piece of
Whatman 3MM paper, sealed in Saran Wrap, and loaded
into an autoradiography cassette with an 8” × 10” sheet of
Kodak X-OMAT AR X-ray film. Following overnight exposure
at -20 °C, the film was developed using Kodak Developer/
Replenisher and Kodafix fixing solution.
Preparation of Prim ary Rat Hepatocytes. Cultures of
primary rat hepatocytes were prepared by a modified protocol
of Kreamer (40). An immature male Sprague-Dawley rat
was anaesthetized with sodium pentobarbital, and the liver
was perfused with collagenase, excised, rinsed, and deseg-
regated in a sterile 150 mm Petri dish. The cells were filtered
and resuspended in 25 mL of attachment media (William’s
E medium supplemented with 10 mM HEPES, 2 mM
L-glutamine, and 10% fetal bovine serum) combined with 24
mL of Percoll in Hank’s Balanced Salt Solution. The cells
were then spun at 50 × g for 10 min, the pellet was washed
with 50 mL of attachment media and spun for 3 min at 50
× g, and the pellet was resuspended in 30-40 mL of
attachment media. The cells were then counted using a
hemocytometer. Cells were plated in sterile 48-well collagen-
coated culture plates at a density of 50 000 cells/ well in 0.5
mL of attachment media. After 2 h, the medium was aspirated
away, and 0.5 mL of serum-free media (William’s E medium
supplemented with 10 mM HEPES, 2 mM L-glutamine, 10
mM pyruvate, and 0.35 mM proline) was added to each well.
The cells were then incubated for 24 h at 37 °C (95% air, 5%
CO2).
HepG2 (HB-8065) and intestinal Caco-2 (HTB-37), and of
one rat cell line, hepatoma H4IIE (HB-8065). Caco-2 and
HepG2 were grown at 37 °C in Minimal Essential Medium
(MEM) with 10% fetal bovine serum (FBS). H4IIE and the
rainbow trout liver cell line, RTL-W1, which was developed
in Dr. Bols’ laboratory, were maintained respectively in
Dulbecco’s MEM (DMEM) with 10% FBS at 37 °C and in
Leibovitz’s L-15 with 5% FBS at 22 °C as described previously
(43).
Exposure of Cells to PBDEs. All cells were exposed to
TCDD and PBDEs in microwell cultures. The step-by-step
protocol has been described in detail for TCDD and RTL-W1
(44). The same procedure was followed for the PBDEs in all
cell lines. The mammalian cell lines were exposed at 37 °C;
RTL-W1 was exposed at 22 °C. After 48 h, the medium was
removed, the microwells were rinsed, and the cell monolayers
in the microwells were assayed for EROD activity.
EROD Assays. The measurement of EROD activity on live
cells in the microwell cultures has been described in step-
by-step detail (44). The reaction mixture consisted of DMEM
with 5.2 µM 7-ethoxyresorufin (ER) and 30 µM dicumarol for
the mammalian cell lines and with 0.83 µM ER and no
dicumarol for RTL-W1. Resorufin was measured with fluo-
rometric plate readers, either a CytoFluor 4000 (Framingham,
MA) or a BioRad Fluoromark (Richmond, CA). For each cell
culture type, data are based on the means of at least two
independent experiments, each of which involved 3-5
replicates. After measuring the fluorescence, the cells were
washed and lysed in water at -70 °C overnight, and then the
protein concentration was determined by the fluorescamine
method (45) using bovin serum album (BSA) as standard.
Analysis of EROD Data and Calculation of REPs. PBDE
congeners showed maximum EROD activities that were
different from that of TCDD. The EC50 for each congener was
obtained by assigning its own maximum EROD activity as
100% and plotting the probit of percent maximum activity
against log[PBDE concentration]. Relative potencies (REPs)
of individual PBDE congeners were expressed as EC50 of
TCDD/ EC50 of PBDE congener.
Results and Discussion
Synthesis of PBDE Congeners. Marsh et al. (16) recently
described a general method for synthesizing symmetrical
and unsymmetrical PBDE congeners from substituted bro-
mobenzenes by conversion to an aryliodonium salt, which
is then coupled to a bromophenol.
Preparation of Chicken Em bryo Hepatocytes. Primary
hepatocytes cultures were prepared from 19 day Barred Rock
chicken embryos using the methods described by Kennedy
(41, 42). Thirty eggs were used for one experiment. In brief,
the embryos were decapitated, the livers were removed,
rinsed with Krebs-Ringer buffer, and cut, and the combined
livers were digested with collagenase with shaking for 45
min at 37 °C and then filtered through nylon gauze to remove
undigested tissue. The filtrates were centrifuged at 300 g for
5 min, and the pellets were resuspended. The Percoll/ sucrose
isodensity centrifugation method was used to purify the
hepatocytes. Hepatocytes were cultured in 48-well cell culture
plates in Waymouth’s MD serum free medium (0.5 mL/ well)
that was supplemented with insulin (1 µg/ mL) and thyroxine
(1 µg/ mL). Because the chicken embryo hepatocytes are too
small to be counted under the optical microscope, hepa-
tocytes were plated in sterile 48-well plates at a concentration
that resulted in total protein concentrations of approximately
60 µg/ well. The cells were then incubated for 24 h at 37 °C
(95% air, 5% CO2), and then the medium was aspirated away
and replaced with fresh Waymouth’s MD serum free medium
before the cells were treated with chemicals.
The authors synthesized PBDEs by condensing bromi-
nated phenols with aryliodonium salts having Brn ) unsub-
stituted, 4-bromo-, 2,4-dibromo-, and 3,4-dibromo-. We
followed this method to synthesize PBDE congeners 28, 47,
49, 66, 71, 77, 85, 99, 100, 119, and 126, which were formed
in yields 30-70% overall, and shown in each case to be >98%
1
pure by GC-MS and H NMR at 400 MHz (Table 1). Because
they were to be used, inter alia, for studying Ah receptor
binding affinities, their purification included chromatography
over charcoal, to remove possible impurities of polybromi-
nated dibenzodioxins or dibenzofurans.
We could not synthesize the 2,4,5-substituted iodonium
salt precursor of PBDE congeners 99, 153, 154, and 183, which
are prominent in commercial PBDE mixtures, and so we
devised a new synthetic methodology based on SN2Ar
Cell Lines. The American Type Culture Collection (Ma-
nassas, VA) was the source of two human cell lines, hepatoma
9
3 7 5 2 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 35, NO. 18, 2001

displacement of a halide ion activated by an o- or p-nitro
group. Our initial condensation using 2,4,5-tribromophenol
and 1,2,4-tribromo-5-nitrobenzene was unsatisfactory on
account of excessive reaction times, poor yield (40%), and
the formation of an inseparable mixture of two isomers,
resulting from equal attack of the phenolate o- and p- to the
nitro group.
TABLE 2. Ah Receptor Relative Binding Affinities and
“Planarization Energies” and DRE Binding Induced by PBDE
Congeners
planarity
energy
DRE
binding
AhR EC , µM
50
PBDE no. Br substitution
(RBA)a
kcal/mol activityb
3
15
17
28
47
49
66
71
75
77
85
99
100
119
126
153
154
183
4-
4,4′-
2,2′,4-
2,4,4′-
7.9 (1.3 × 10-4
2.6 (3.8 × 10-4
4.4 (2.3 × 10-4
)
)
)
13.9
14.0
26.9
14.1
26.3
26.5
14.2
26.5
26.8
14.1
26.3
26.3
27.5
26.8
14.0
26.3
27.6
27.8
none
none
none
none
none
none
none
none
0.82 (1.2 × 10-3
)
)
)
2,2′,4,4′-
2,2′,4,5′-
2,3′,4,4′-
2,3′,4′,6-
2,4,4′,6-
3,3′,4,4′-
2,2′,3,4,4′-
2,2′,4,4′,5-
2,2′,4,4′,6-
2,3′,4,4′-
3,3′,4,4′,5-
2,2′,4,4′,5,5′-
2,2′,4,4′,5,6′-
1.8 (5.6 × 10-4
)
15 (6.7 × 10-5
)
0.49 (2.0 × 10-3
28 (13.6 × 10-5
)
)
2.5 (4.0 × 10-4
none
m edium
none
0.46 (2.2 × 10-3
Instead, we synthesized the key precursor 2,5-dibromo-
4-fluoronitrobenzene, by nitrating the commercially available
2,5-dibromofluorobenzene, which under our conditions
occurred in >90% yield and with almost complete re-
giospecificity.
0.053 (1.9 × 10-2
)
7.2 (1.4 × 10-4
)
none
13 (7.7 × 10-5
)
weak
m edium
m edium
weak
none
weak
none
none
0.88 (1.1 × 10-3
0.37 (2.7 × 10-3
)
)
40 (2.5 × 10-5
43 (2.3 × 10-5
)
)
2,2′,3,4,4′,5′,6- 4.0 (2.5 × 10-4
)
)
)
penta m ixture
octa
deca
TCDD
2.4 (4.2 × 10-4
7.9 (1.3 × 10-4
no activity
m ixture
m ixture
none
strong
0.0010 (1.00)
a
2,5-Dibromo-4-fluoronitrobenzene underwent nucleo-
philic displacement of the 4-fluoro substituent by a bro-
mophenolate anion (acetone solvent, K2CO3 as base, 4 h).
Introduction of the final Br substituent involved reduction
of the NO2 group (Sn/ HCl/ HOAc), diazotization, and Sand-
meyer reaction with CuBr.
Values in brackets are binding affinities relative to TCDD; concen-
tration of [3H]-TCDD was 1.0 nM. Qualitative estim ate based on visual
exam ination of autoradiographs.
b
boronic ester to give 4,4′-dibromodiphenyl ether, which must
be separated from the desired product.
Ah Receptor Binding Assays. The EC50 values for Ah
receptor binding of individual PBDE congeners were in the
µM range, indicating weaker affinity than the reference
toxicant TCDD (Table 2). Congener 85, which is not a major
constituent in environmental samples, was the most active,
but its relative binding affinity (RBA) was only 2% that of
TCDD. RBAs of the 18 PBDEs studied spanned about 3 orders
of magnitude, with some tendency for the congeners
analogous to coplanar PCBs (PBDEs 77 and 126) to have
larger RBAs than those with two or more ortho-bromines,
such as 71, 100, 153, and 154.
The most abundant congeners in the commercial mixtures
(PBDEs 47, 99, 153, 154, and 183) all had very small RBAs.
The commercial mixtures also bound weakly to rat hepatic
Ah receptor; their RBAs corresponded closely to those of
their major components (PBDE 47 in the case of the “penta”
mixture, and PBDE 183 in the case of the “octa”). We could
not determine the RBA of the commercial decaBDE because
of its very low solubility. The RBAs of the commercial mixtures
were unaffected by prior passage through a carbon chro-
matography column, a treatment intended to remove possible
traces of highly active minor components such as brominated
dibenzodioxins or dibenzofurans, which would exaggerate
the binding strength of the PBDEs, as is the case for PCBs
(49).
Congener 183 was prepared more successfully by con-
densing the iodosyl triflate with anisole, to give an unsym-
metrical iodonium salt (20% yield) as shown below (cf. refs
46 and 47). Subsequent reaction with 2,3,4,5-tetrabro-
mophenol in alkaline solution led exclusively to the dis-
placement of p-iodoanisole, affording the desired 2,2′,-
3,4,4′,5,5′-heptabromodiphenyl ether in 45% isolated yield.
It is possible that this method may have more general
application.
Congener 81 (3,4,4′,5-tetrabromodiphenyl ether) was
prepared by a modified Suzuki coupling, based on the
chemistry of Evans et al. (48) in 40% yield. This method
requires scrupulous care in drying the reagents, because the
arylboronic ester is highly moisture sensitive; its hydrolysis
gives p-bromophenol, which condenses with additional
For previously examined HAC families, the strength of
binding to the AhR is determined by the planarity and
lipophilicity of the ligand (50), indicating that the AhR has
a nonpolar, planar binding site to which ligands must
accommodate themselves. Recently, Kodavanti et al. (51) used
molecular mechanics calculations to show, for the PCB family,
9
VOL. 35, NO. 18, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 3 7 5 3

a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
FIGURE 1. A typical dose-dependent EROD induction curve for TCDD
and PBDE congeners 77, 119, and 126 in primary rat hepatocytes.
Points represent mean level of triplicate doses and bars represent
standard deviations. Similar dose response curves were obtained
from other cell cultures.
a
a
a
a
a
a
that the “coplanar” congeners, which bind most strongly to
the AhR, exhibited the smallest energy differences between
their equilibrium and coplanar geometries. We used the
SPARTAN software package to carry out similar calculations
for the PBDEs, having first reproduced the “planarization
energies” for PCB congeners reported by Kodavanti et al.
(51). The calculated energies needed to force coplanarity of
the PBDE molecules are larger than for PCBs and appear
unrelated to the RBAs (Table 2): ∆E ∼ 13-14 kcal/ mol for
zero and one ortho Br and ∆E ∼ 26-27 kcal/ mol for two or
three ortho Br. One interpretation is that whereas PCBs (for
example) must accommodate themselves to the geometry of
the AhR binding site, for PBDEs the binding site becomes
distorted by the large atomic volume of bromine and renders
the issue of ligand planarity inconsequential.
EROD Induction. The ability of PBDEs to induce EROD
activity was studied in chick and rat hepatocytes, in liver cell
lines from rainbow trout (RTL-W1), rat (H4IIE) and human
(HepG2), and in a human intestinal cell line (Caco-2). Figure
1 shows a typical set of induction curves, with the data
summarized in Table 3. EROD induction was strongest in all
cell types with PBDEs 77, 100, 119, and 126, although their
maximal EROD activities were less than that of TCDD and
their EC50s were much larger. PBDEs 153 and 183 were weak
inducers in all cells. PBDEs 66 and 85 were very weak inducers
in rat hepatocytes and inactive in the other cells. The
environmentally prominent congeners 47 and 99 were not
inducers in any cell line; neither were PBDEs 28, 47, 99, and
154 and the commercial PBDE mixtures. Declines in EROD
activity at high inducer concentrations have been previously
ascribed to competitive inhibition by the inducer and are
not due to cytotoxicity (52).
b
The strength of EROD induction by HACs frequently
parallels the strength of AhR binding (53), indicating that
induction of EROD activity requires activation of the AhR-
signal transduction pathway. PBDEs congeners were shown
to follow this trend by comparing their EROD activities in
primary cultures of rat hepatocytes with their ability to
activate the AhR to its DNA binding form, as studied by the
gel retardation assay, visualized by autoradiography (Tables
2). The gel retardation assay involves incubating the bound
AhR-ligand complex with a synthetic oligonucleotide that
contains the consensus binding sequence for liganded AhR
and that is [32P] end-labeled using [32P]-ATP. Binding the
massive AhR complex to the oligonucleotide increases its
size and its mass:charge ratio, and it migrates more slowly
in slab gel electrophoresis.
a
a
a
a
a
a
a
a
a
TCDD, the reference ligand, caused induction of a strong
gel-shifted band (Table 2), as repeatedly observed previously
(54, 55). The stronger AhR agonists among the PBDEs, such
as PBDEs 77 and 126, induced moderate intensities of the
a
9
3 7 5 4 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 35, NO. 18, 2001

FIGURE 2. Gel retardation assay for PBDE congener 85 as agonist and antagonist in Ah receptor/Arnt/DRE complex formation. An arrow
indicates the AhR/Arnt/DRE complex. 10-8 M TCDD as positive control. Congeners 100, 66, 183, 153, and 28 were also tested for synthetic
DRE activation.
TABLE 4. Relative Induction Potencies (REPs) for PBDE Congeners in Different Cell Cultures
REP ) EC50 of TCDD/EC50 of PBDE
b
c
d
PBDE no.
RTL-W1a
1.0
CEH
PRH
H4IIE
Caco-2e
1.0
Hep G2f
1.0
TCDD
1.0
1.0
1.0
PBDE 126
PBDE 77
PBDE 119
PBDE 100
PBDE 183
PBDE 153
PBDE 85
PBDE 66
PBDE 154
PBDE 99
PBDE 47
PBDE 28
0.00036
0.00034
0.00019
0.000012
0.0000068
0.0000006
inactive
inactive
inactive
inactive
inactive
inactive
0.0024
0.0032
0.00042
0.00076
0.00014
0.000028
0.0000048
0.0000032
0.00010
0.000036
inactive
inactive
inactive
inactive
0.00041
0.00023
0.00010
0.000013
0.0000039
0.0000034
0.0000099
0.000032
inactive
0.00016
0.00031
0.000068
0.000011
0.0000088
0.0000087
inactive
inactive
inactive
inactive
inactive
inactive
0.00013
0.000086
0.000068
0.000011
0.000010
0.0000093
inactive
inactive
inactive
inactive
inactive
inactive
0.00035
0.00024
inactive
0.000048
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
a
b
c
d
RTL-W1) rainbow trout liver cell line. CEH ) prim ary chick em bryo hepatocytes. PRH) prim ary rat hepatocytes. H4IIE ) rat hepatom a
cell line. Caco-2) hum an intestinal cell line. f Hep G2 ) hum an hepatom a cell line.
e
shifted band, i.e., they activated the AhR to a form that could
bind DNA and also induced EROD greater activity. Con-
versely, weak AhR agonists such as PBDEs 100 and 153 gave
weak activation of the AhR to its DNA binding form and
weak EROD induction. The commercial mixtures and their
major constituents, PBDEs 47 and 99, were almost completely
inactive. PBDE 85 was exceptional: despite its relatively
strong AhR affinity, it showed no evidence of activating the
AhR to its DRE-binding form and was only a very weak EROD
inducer. This behavior is reminiscent of that of PCB 153,
which also binds the AhR but fails to induce EROD activity
due to failure to cause DRE binding (55). To pursue the parallel
with PCB 153 further, the gel shift assay was carried out using
AhR preparations activated with a fixed aliquot of TCDD and
FIGURE 3. Correlation of EC s (M) for PBDE congeners 77, 119, 126,
50
varying concentrations of PBDE 85. Increasing concentrations
of PBDE 85 decreased the intensity of the gel shifted band
and at the highest concentrations completely out-competed
TCDD for AhR binding sites (Figure 2); hence PBDE 85
appeared to act as an AhR antagonist. Similar experiments
carried out with the stronger AhR agonists PBDEs 77 and 126
only partly inhibited DRE activity because these congeners
themselves activate the AhR to its DRE binding form.
100, 153, and 183 from primary chicken embryo hepatocytes (CEH)
and rat hepatoma cell line (H4IIE). Points represent the mean level
of log EC (log M). If TCDD is included in the correlation analysis,
50
the R 2 is 0.9782.
in rat liver cells but not in the other cells, and PBDEs 77 and
126, which were approximately 10-fold more potent in chick
hepatocytes (which have been noted to be particularly
sensitive HACs (59)). The concordance of REP values for a
given congener across species is illustrated in Figure 3, which
shows the correlation between log(EC50) in H4IIE cells and
chick embryo hepatocytes for the six PBDE congeners most
active as EROD inducers. Correlations ranged from a high
of r2 ) 0.96 between H4IIE and Hep G2 cells to a low of 0.71
between primary rat hepatocytes and chick embryo hepa-
tocytes.
The relative potencies (REPs) of individual PBDEs for
EROD induction were expressed as the EC50 for TCDD/ EC50
for PBDE congener, where the EC50 is the concentration
needed to induce 50% of the maximal EROD response (Table
4). The REP values are empirical only because of the variation
of the maximal EROD induction among congeners and cell
lines. Most REPs were similar in cells from different species.
Exceptions were PBDEs 66 and 85, which were weakly active
9
VOL. 35, NO. 18, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 3 7 5 5

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Acknowledgments
Financial support of this work was provided by the Toxic
Substances Research Initiative of Environment Canada and
Health Canada and by the Natural Sciences and Engineering
Research Council of Canada. Commercial penta-, octa-, and
deca-PBDE mixtures were donated by Great Lakes Chemicals.
We thank Dr. Sean Kennedy and his group for advice on
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Received for review March 15, 2001. Revised manuscript
received June 25, 2001. Accepted June 28, 2001.
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