Bromination pattern of hydroxylated metabolites of BDE-47 affects their potency to release calcium from intracellular stores in PC12 cells.

Abstract

Brominated flame retardants, including the widely used polybrominated diphenyl ethers (PBDEs), have been detected in humans, raising concern about possible neurotoxicity. Recent research demonstrated that the hydroxylated metabolite 6-OH-BDE-47 increases neurotransmitter release by releasing calcium ions (Ca2+) from intracellular stores at much lower concentrations than its environmentally relevant parent congener BDE-47. Recently, several other hydroxylated BDE-47 metabolites, besides 6-OH-BDE-47, have been detected in human serum and cord blood.
To investigate the neurotoxic potential of other environmentally relevant PBDEs and their metabolites, we examined and compared the acute effects of BDE-47, BDE-49, BDE-99, BDE-100, BDE-153, and several metabolites of BDE-47-6-OH-BDE-47 (and its methoxylated analog 6-MeO-BDE-47), 6 -OH-BDE-49, 5-OH-BDE-47, 3-OH-BDE-47, and 4 -OH-BDE-49--on intracellular Ca2+ concentration ([Ca2+]i), measured using the Ca2+-responsive dye Fura-2 in neuroendocrine pheochromocytoma (PC12) cells.
In contrast to the parent PBDEs and 6-MeO-BDE-47, all hydroxylated metabolites induced Ca2+ release from intracellular stores, although with different lowest observed effect concentrations (LOECs). The major intracellular Ca2+ sources were either endoplasmic reticulum (ER; 5-OH-BDE-47 and 6 -OH-BDE-49) or both ER and mitochondria (6-OH-BDE-47, 3-OH-BDE-47, and 4 -OH-BDE-49). When investigating fluctuations in [Ca2+]i, which is a more subtle end point, we observed lower LOECs for 6-OH-BDE-47 and 4 -OH-BDE-49, as well as for BDE-47.
The present findings demonstrate that hydroxylated metabolites of BDE-47 cause disturbance of the [Ca2+]i. Importantly, shielding of the OH group on both sides with bromine atoms and/or the ether bond to the other phenyl ring lowers the potency of hydroxylated PBDE metabolites.

Full text

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Environmental Health Perspectives • volume 118 | number 4 | April 2010
519
Research
Brominated flame retardants are added to a
wide array of consumer products. Adverse
effects of these compounds on the developing
nervous system give cause for concern (for
review, see Costa and Giordano 2007). Long-
lasting neurobehavioral changes after neo natal
exposure have been detected in rodents after
exposure to poly brominated diphenyl ethers
(PBDEs) (Branchi et al. 2003; Eriksson et al.
2001; Lilienthal et al. 2006; Rice et al. 2007;
Viberg et al. 2003, 2006), as well as various
functional and structural alterations in the
brain (Dingemans et al. 2007; Viberg 2009;
Xing et al. 2009).
During development, acute effects on
neuronal activity may result in long-lasting
changes in proteins, brain function, and behav-
ior. A key regulator for neuronal function is
intracellular concentration of calcium ions,
[Ca2+]i, which regulates many cellular pro-
cesses, including the release of neurotransmit-
ters at the pre synaptic terminal (for reviews, see
Clapham 2007; Laude and Simpson 2009).
PBDEs have been shown to affect Ca2+
homeo stasis in microsomes (Kodavanti and
Ward 2005) and pheochromocytoma (PC12)
cells (Dingemans et al. 2007) and to reduce
the Ca2+ uptake by brain microsomes and
mitochondria (Coburn et al. 2008) at rela-
tively high concentrations. PBDEs have also
been shown to induce oxidative stress in neu-
ronal cells (He et al. 2008; Kodavanti and
Ward 2005). Recently, 6-OH-BDE-47
(6-hydroxy-2,2´,4,4´-tetra bromo diphenyl
ether), a hydroxylated metabolite of BDE-47
(2,2´,4,4´-tetrabromo diphenyl ether), was
demon strated to increase vesicular neurotrans-
mitter release and [Ca2+]i by releasing Ca2+
from intra cellular stores in PC12 cells. The
increase in vesicular neuro transmitter release
was induced by 6-OH-BDE-47 at much
lower concentrations than by the parent
PBDE (Dingemans et al. 2008). Therefore,
hydroxylated PBDEs (OH-PBDEs) may be
more important than the parent compounds
for human risk assessment for neurotoxicity.
Recently, not only PBDEs but also vari-
ous hydroxylated metabolites of PBDEs have
been found to bio accumulate in humans
(Athanasiadou et al. 2008; Qiu et al. 2009).
Therefore, the aim of the present study was
to determine whether mono hydroxylated
metabolites of the abundant BDE-47 affect
[Ca2+]i and to compare this with the effects of
a methoxylated analog and several other envi-
ronmentally relevant PBDE congeners.
Materials and Methods
Chemicals. In the present study, we investigated
the effects of BDE-47, BDE-99 (2,2´,4,4´,5-
penta bromo diphenyl ether), BDE-100
(2,2´,4,4´,6-penta bromo diphenyl ether), and
BDE-153 (2,2´,4,4´,5,5´-hexa bromo diphenyl
ether), the main constituents of the commer-
cial DE-71 pentaBDE mixture (La Guardia
et al. 2006), as well as several metabolites of
BDE-47 [6-OH-BDE-47, 6´-OH-BDE-49
(6´-hydroxy-2,2´,4,5´-tetrabromodiphenyl
ether), 5-OH-BDE-47 (5-hydroxy-2,2´,4,4´-
tetrabromodiphenyl ether), 3-OH-BDE-47
(3-hydroxy-2,2´,4,4´-tetrabromodiphenyl
ether), 4´-OH-BDE-49 (4´-hydroxy-
2,2´,4,5´-tetrabromodiphenyl ether), and
the methoxylated analog 6-MeO-BDE-47
(6-methoxy-2,2´,4,4´-tetrabromodiphenyl
ether)]. During formation of 6´-OH-BDE-49
and 4´-OH-BDE-49, a bromine (Br)-shift takes
place. Consequently, we included BDE-49
(2,2´,4,5´-tetrabromo diphenyl ether) as a con-
trol for possible influences of this change in
bromination pattern.
PBDEs (Figure 1) [see also Supplemental
Material, Table 1 (doi:10.1289/ehp.0901339)]
were synthesized and purified (~ 99%
purity) at the Department of Environmental
Chemistry, Stockholm University (Marsh et al.
1999). Dibenzo-p-dioxins and dibenzofurans
were removed from the PBDEs with a charcoal
column as described by Örn et al. (1996). All
other chemicals, unless otherwise stated, were
obtained from Sigma-Aldrich (Zwijndrecht,
the Netherlands).
Cell culture. Rat pheochromo cytoma
(PC12) cells (Greene and Tischler 1976),
obtained from ATCC (American Type
Culture Collection, Manassas, VA, USA), were
Address correspondence to M.M.L. Dingemans,
Neurotoxicology Research Group, Toxicology
Division, Institute for Risk Assessment Sciences
(IRAS), Utrecht University, P.O. Box 80.178,
NL-3508 TD, Utrecht, the Netherlands. Telephone:
31-30-253-4387. Fax: 31-30-253-5077. E-mail:
m.dingemans@uu.nl
Supplemental Material is available online
(doi:10.1289/ehp.0901339 via http://dx.doi.org/).
We thank A. de Groot for excellent technical
assistance.
This study was supported by the Faculty of
Veterinary Medicine, Utrecht University.
The authors declare they have no competing
financial interests.
Received 17 August 2009; accepted 19 November
2009.
Bromination Pattern of Hydroxylated Metabolites of BDE-47 Affects
Their Potency to Release Calcium from Intracellular Stores in PC12 Cells
Milou M.L. Dingemans,1 Harm J. Heusinkveld,1 Åke Bergman,2 Martin van den Berg,1 and Remco H.S. Westerink1
1Neurotoxicology Research Group, Toxicology Division, Institute for Risk Assessment Sciences, Utrecht University, Utrecht,
the Netherlands; 2Department of Environmental Chemistry, Stockholm University, Stockholm, Sweden
Background: Brominated flame retardants, including the widely used polybrominated diphenyl
ethers (PBDEs), have been detected in humans, raising concern about possible neurotoxicity. Recent
research demonstrated that the hydroxylated metabolite 6-OH-BDE-47 increases neuro transmitter
release by releasing calcium ions (Ca2+) from intra cellular stores at much lower concentrations than
its environmentally relevant parent congener BDE-47. Recently, several other hydroxylated BDE-47
metabolites, besides 6-OH-BDE-47, have been detected in human serum and cord blood.
oBjective and Methods: To investigate the neurotoxic potential of other environmentally
relevant PBDEs and their metabolites, we examined and compared the acute effects of BDE-47,
BDE-49, BDE-99, BDE-100, BDE-153, and several metabolites of BDE-47—6-OH-BDE-47 (and
its methoxylated analog 6-MeO-BDE-47), 6´-OH-BDE-49, 5-OH-BDE-47, 3-OH-BDE-47, and
4´-OH-BDE-49—on intra cellular Ca2+ concentration ([Ca2+]i), measured using the Ca2+-responsive
dye Fura-2 in neuroendocrine pheochromocytoma (PC12) cells.
results: In contrast to the parent PBDEs and 6-MeO-BDE-47, all hydroxylated metabolites induced
Ca2+ release from intracellular stores, although with different lowest observed effect concentrations
(LOECs). The major intracellular Ca2+ sources were either endoplasmic reticulum (ER; 5-OH-
BDE-47 and 6´-OH-BDE-49) or both ER and mitochondria (6-OH-BDE-47, 3-OH-BDE-47, and
4´-OH-BDE-49). When investigating fluctuations in [Ca2+]i, which is a more subtle end point, we
observed lower LOECs for 6-OH-BDE-47 and 4´-OH-BDE-49, as well as for BDE-47.
conclusions: The present findings demonstrate that hydroxylated metabolites of BDE-47 cause
disturbance of the [Ca2+]i. Importantly, shielding of the OH group on both sides with bromine
atoms and/or the ether bond to the other phenyl ring lowers the potency of hydroxylated PBDE
metabolites.
key words: brominated flame retardant, calcium, calcium fluctuations, Fura-2, intracellular calcium
stores, neurotoxicity, PC12, persistent organic pollutant, polybrominated diphenyl ether, structure–
activity relationship. Environ Health Perspect 118:519–525 (2010). doi:10.1289/ehp.0901339 avail-
able via http://dx.doi.org/ [Online 19 November 2009]

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Dingemans et al.
520
volume 118 | number 4 | April 2010 • Environmental Health Perspectives
cultured as described previously [Dingemans
et al. 2008; see also Supplemental Material
(doi:10.1289/ehp.0901339)].
Cell viability assay. To investigate pos-
sible acute effects of the PBDEs on cell viabil-
ity, we used the alamarBlue (AB) and neutral
red (NR) uptake assays with minor modifica-
tions [Magnani and Bettini 2000; Repetto
et al. 2008; see also Supplemental Material
(doi:10.1289/ehp.0901339)].
Intracellular Ca2+ imaging. Changes in
[Ca2+]i were measured using the Ca2+-sensitive
fluorescence ratio dye Fura-2 as described pre-
viously (Dingemans et al. 2007, 2008). For
detailed information on experimental conditions
and calculation of [Ca2+]i, see Supplemental
Material (doi:10.1289/ehp.0901339).
The average and amplitude of [Ca2+]i dur-
ing exposure were determined per cell to inves-
tigate effects of PBDEs on Ca2+ homeo stasis.
The standard deviation during baseline [Ca2+]i
recording ranged from 2% to 74% of average
[Ca2+]i [n = 1,538; see Supplemental Material,
Figure 1 (doi:10.1289/ehp.0901339)]. To
prevent registration of false-positive effects,
an increase in [Ca2+]i to > 175% of baseline
was used to determine no observed effect con-
centrations (NOECs) and lowest observed
effect concentrations (LOECs). A transient
increase within 0–10 min from the start of
exposure is referred to as an “initial increase,”
whereas additional increases are referred to as
“late increases” (Dingemans et al. 2008). At
NOEC levels, based on average and ampli-
tude of [Ca2+]i levels, increases in [Ca2+]i lev-
els to > 175% of baseline during exposure
were scored as fluctuations to investigate
more subtle effects on Ca2+ homeostasis.
We determined the number of cells showing
fluctuations in [Ca2+]i, as well as frequency,
amplitude, and duration of these fluctuations.
Data analysis and statistics. All data are
presented as mean ± SE from the number of
cells or fluctuations in [Ca2+]i. We performed
statistical analyses using SPSS 16 (SPSS,
Chicago, IL, USA). Categorical and con-
tinuous data were compared using Fisher’s
exact test and Student’s t-test, respectively,
paired or unpaired where applicable. Analysis
of variance (ANOVA) and post hoc t-tests
were performed to investigate possible dose–
response relationships. To investigate struc-
ture–activity relationships, we investigated the
possible influence of hydroxylation position
and/or shielding of the OH group by adja-
cent atomic groups on the efficacy to increase
[Ca2+]i. To this aim, we performed a multi-
factorial ANOVA using the mean increase in
basal [Ca2+]i induced by 20 µM of the differ-
ent OH-PBDEs as the dependent variable.
Hydroxylation position (ortho, meta, or para)
and the presence of either one or two shielding
atomic groups (phenyl ring and/or Br atom)
adjacent to the OH group were used as fixed
Figure 1. Molecular structures of (A) the PBDEs (BDE-47, BDE-49, BDE-99, BDE-100, and BDE-153)
and (B) the metabolites of BDE-47 (6-OH-BDE-47, 6´-OH-BDE-49, 5-OH-BDE-47, 3-OH-BDE-47, and
4´-OH-BDE-49) investigated in this study. For the metabolites, the position of the OH group is indicated, as
well as the shielding of this group by occupancy of the adjacent carbon atoms by either phenyl ether and/
or Br atoms.
BrBrBrBr
BrBr
BrBr
Br Br
Br Br
OO
O
O
O
O
BrBrBr
OH
Br
Br
OH
OH
OH
OH
Br
Br
Br
Br
Br
Br
Br
BrBr
BrBr
BrBr
BrBr
O
O
O
O
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
BDE-476-OH-BDE-47
5-OH-BDE-47
3-OH-BDE-47
6’-OH-BDE-49
4’-OH-BDE-49
BDE-49
BDE-99
BDE-100
BDE-153
Molecular structure
Position
OH-groupShieldingMolecular structure
orthoPhenyl ether
Phenyl ether
+
bromine atom
Bromine atom
Bromine atom
Two bromine
atoms
ortho
meta
meta
para
Figure 2. Average and amplitude of [Ca2+]i in PC12 cells exposed to hydroxylated metabolites of BDE-47.
(A) Cells were exposed to 2 or 20 µM 6-MeO-BDE-47, 6-OH-BDE-47, 6´-OH-BDE-49, 5-OH-BDE-47,
3-OH-BDE-47, or 4´-OH-BDE-49; note the change in scale. We tested additional concentrations of 6-OH-
BDE-47 (B) and 5-OH-BDE-47 (C) because the 2-µM dose of these metabolites increased [Ca2+]i to > 175%
of baseline (dashed line). Data are shown from 4–19 experiments per concentration; numbers inside the
bars indicate the number of cells used for data analysis.
10,000
8,000
6,000
4,000
2,000
600
500
400
300
200
100
0
500
400
300
200
100
0
500
400
300
200
100
0
[Ca2+]i (% of baseline)
[Ca2+]i (% of baseline)
[Ca2+]i (% of baseline)
DMSO 20
DMSO0.252 201 DMSO0.252 201
6-MeO-BDE-47
(µM)
6-OH-BDE-47
(µM)
6-OH-BDE-47
(µM)
5-OH-BDE-47
(µM)
5-OH-BDE-47
(µM)
3-OH-BDE-47
(µM)
6’-OH-BDE-49
(µM)
4’-OH-BDE-49
(µM)
20 2020222 20220 2
Average
Amplitude
168
168168
42 53
5850 5286 4853 48 9077 56
50 434048 4964 868790

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Bromination pattern affects potency of OH-PBDEs
Environmental Health Perspectives • volume 118 | number 4 | April 2010
521
variables (Figure 1). A p-value < 0.05 was con-
sidered statistically significant.
Results
Effects of parent PBDEs on [Ca2+]i. Exposure
to the solvent control [dimethyl sulfoxide
(DMSO)] or to 20 µM of BDE-47, BDE-49,
BDE-99, BDE-100, or BDE-153 did not
decrease cell viability (data not shown) or
increase the average or amplitude of [Ca2+]i or
the percentage of cells showing fluctuations in
[Ca2+]i [see Supplemental Material, Figure 2
(doi:10.1289/ehp.0901339)]. However,
the frequency and duration of fluctua tions
in [Ca2+]i increased during exposure to
20 µM BDE-47. At 2 µM BDE-47, simi-
lar effects were observed (NOEC, 1 µM).
No effects could be detected on the fre-
quency, duration, or amplitude of fluctua-
tions during exposure to 20 µM BDE-49,
BDE-99, BDE-100, or BDE-153 [see
Supplemental Material, Table 3 (doi:10.1289/
ehp.0901339)].
Hydroxylated BDE­47 metabolites
increase [Ca2+]i. After 20 min exposure to
6-OH-BDE-47, 5-OH-BDE-47, or 4´-OH-
BDE-49, the NR assay indicated a decrease
in cell viability only at the 20-µM dose,
with means ± SEs of 86 ± 0.3%, 86 ± 1.1%,
and 93 ± 0.8% of control, respectively. We
observed no significant decreases in cell viabil-
ity measured by the NR assay for 6´-OH-
BDE-49 or 3-OH-BDE-47. In the AB assay,
exposure to 6-OH-BDE-47, 6´-OH-BDE-49,
or 3-OH-BDE-47 increased the relative fluo-
rescence intensity dose-dependently above the
control level (data not shown).
Initial increases in [Ca2+]i were observed
during exposure to 6-OH-BDE-47, 6´-OH-
BDE-49, 5-OH-BDE-47, 3-OH-BDE-47,
or 4´-OH-BDE-49 [see Supplemental
Material, Figure 3 and Table 2 (doi:10.1289/
ehp.0901339)]. Exposure to OH-PBDEs
resulted in a dose-dependent increase in
average and amplitude of [Ca2+]i with vary-
ing LOECs. LOECs are determined by the
amplitude of the increase in [Ca2+]i (Figure 2)
and the percentage of cells showing initial and
late increases in [Ca2+]i (Figure 3). No effects
were detected on the average and ampli-
tude of [Ca2+]i during exposure to 20 µM
6-MeO-BDE-47, and we observed no effects
of 6-MeO-BDE-47 on cell viability, the per-
centage of cells with increases in [Ca2+]i to
> 175% of baseline, or the average frequency,
duration, or amplitude of fluctuations.
For 6´-OH-BDE-49, 3-OH-BDE-47,
and 4´-OH-BDE-49, the LOEC for increased
[Ca2+]i is 20 µM. Exposure to 20 µM 6´-OH-
BDE-49 results in an initial increase in 50%
of the cells. We observed late increases less
frequently and with lower amplitude. The
shapes of the increases in [Ca2+]i during expo-
sure to 20 µM 6´-OH-BDE-49 vary widely
(Figure 4B). We observed initial increases
during exposure to 20 µM 3-OH-BDE-47 in
48% of the cells. Exposure to 20 µM 4´-OH-
BDE-49 resulted in a modest initial increase
compared with baseline in 83% of the cells,
after which we observed a larger late increase.
For the OH-PBDEs with an effect at 2 µM
(6-OH-BDE-47 and 5-OH-BDE-47), we also
tested lower concentrations (Figures 2B,C,
and 3B,C), showing that the LOECs for these
OH-PBDEs are 1 µM. We observed late
increases only at concentrations ≥ 2 µM. At
concentrations ≤ 5 µM, this late increase had
an amplitude comparable to that of the initial
increase (~ 200–400% of baseline). At 20 µM,
the late increase induced by 6-OH-BDE-47
or 5-OH-BDE-47 was much larger [see
Supplemental Material, Table 2 (doi:10.1289/
ehp.0901339)], which we also observed for
20 µM 4´-OH-BDE-49. The amplitudes of
the initial and late increases that we observed
during exposure to 6-OH-BDE-47 or 5-OH-
BDE-47 were dose dependent (ANOVAs:
6-OH-BDE-47, initial p < 0.0001, late
p < 0.0001; 5-OH-BDE-47, initial p < 0.01,
late p < 0.0001).
The mean amplitude of [Ca2+]i dur-
ing exposure to 20 µM of the hydroxylated
metabolites was independent of the position
(ortho, meta, or para) of the OH group on the
PBDE molecule (ANOVA, not significant).
However, OH-PBDEs in which the OH
group was shielded on only one side, with
either the other phenyl ring or a Br atom,
induce significantly higher increases in [Ca2+]i
compared with OH-PBDEs in which the OH
group was shielded on both sides (ANOVA,
p < 0.01). However, this influence of the
shielding of the OH group (on one compared
with two sides) was independent of its posi-
tion (ortho, meta, or para) on the PBDE mol-
ecule (ANOVA, not significant).
Hydroxylated BDE­47 metabolites
increase [Ca2+]i by release of Ca2+ from intra­
cellular stores. Although initial increases in
[Ca2+]i induced by 6-OH-BDE-47, 5-OH-
BDE-47, and, to a lesser extent, 4´-OH-
BDE-49 were reduced in Ca2+-free conditions,
increases were still observed for all of the
OH-PBDEs (Figure 4). To identify the respon-
sible Ca2+ stores, endoplasmic reticulum (ER)
or both ER and mitochondrial Ca2+ stores
were depleted by either 1 µM thapsigargin
(TG) or 1 µM TG plus 1 µM carbonyl cyanide
4-(trifluoro methoxy)phenyl hydrazone (FCCP).
TG and TG/FCCP pretreatment, respectively,
Figure 3. Concentration dependence of the occurrence of different types of [Ca2+]i disturbances in PC12
cells during exposure to 2 or 20 µM of hydroxylated metabolites of BDE-47; values shown are the percent-
age of cells showing an initial transient increase or a late increase in [Ca2+]i. (A) Cells were exposed to
6-MeO-BDE-47, 6-OH-BDE-47, 6´-OH-BDE-49, 5-OH-BDE-47, 3-OH-BDE-47, or 4´-OH-BDE-49. We tested
additional concentrations of 6-OH-BDE-47 (B) and 5-OH-BDE-47 (C) because the 2-µM dose of these metabo-
lites showed initial and late increases of [Ca2+]i. Data are from 4–19 experiments per concentration; num-
bers inside the bars indicate the number of cells used for data analysis.
*p < 0.05, **p < 0.01, and #p < 0.001; significant changes in the percentage of cells showing initial or late increases in
[Ca2+]i are indicated at the LOEC.
100
80
60
40
20
0
100
80
60
40
20
0
Percentage of cells
showing increase in [Ca2+]i
Percentage of cells
showing increase in [Ca2+]i
DMSO0.252201DMSO 0.252201
6-OH-BDE-47
(µM)
5-OH-BDE-47
(µM)
Initial increase
Late increase
168
168
168
42
20
#
#
#
#
#
*
**
#
#
#
#
#
#
#
53
20
58
50 52 864853
48 907756
50
2
434048 49
2
6486
2
8790
20
100
80
60
40
20
0
Percentage of cells
showing increase in [Ca2+]i
DMSO
6-MeO-BDE-47
(µM)
6-OH-BDE-47
(µM)
5-OH-BDE-47
(µM)
3-OH-BDE-47
(µM)
6’-OH-BDE-49
(µM)
4’-OH-BDE-49
(µM)
202 20202

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Dingemans et al.
522
volume 118 | number 4 | April 2010 • Environmental Health Perspectives
caused a transient increase in [Ca2+]i of
321 ± 11% (n = 191) and 393 ± 11% of base-
line (n = 195), after which [Ca2+]i stabilized
and the OH-PBDE was applied.
When we depleted ER Ca2+ stores in Ca2+-
free conditions, the initial increase induced by
5 µM 6-OH-BDE-47 was largely diminished
(Figure 4A). Although the amplitude of the late
increase was not different in Ca2+-free condi-
tions, it increased after depletion of the ER Ca2+
stores, but greatly diminished after depletion of
both ER and mitochondrial Ca2+ stores.
We also observed the variation in [Ca2+]i
responses during exposure to 20 µM 6´-OH-
BDE-49 in Ca2+-free conditions (Figure 4B).
After depletion of ER Ca2+ stores, we no lon-
ger observed either initial or late transient
increases in [Ca2+]i.
Figure 4. Increase in [Ca2+]i by OH-PBDEs results from the release of Ca2+ from intracellular stores. Values shown are amplitudes of [Ca2+]i in PC12 cells during
exposure to 5 µM 6-OH-BDE-47 (A), 20 µM 6´-OH-BDE-49 (B), 5 µM 5-OH-BDE-47 (C), 20 µM 3-OH-BDE-47 (D), and 20 µM 4´-OH-BDE-49 (E) measured in external
saline (1.8 mM Ca2+), Ca2+-free saline (0 mM Ca2+), Ca2+-free saline after pretreatment with TG (0 + TG), and Ca2+-free saline after pretreatment with both TG and
FCCP (0 + TG + FCCP). Cells were pretreated with TG and FCCP to deplete Ca2+ stores in ER and mitochondria, respectively. Tops and bottoms of boxes represent
upper and lower quartiles, the line within the box is the median, whiskers represent lowest and highest values, and circles represent outliers (for clarity, outliers
that are more than three interquartile ranges from the boxes are not shown). Data are from three to seven experiments per treatment. Numbers shown in paren-
theses indicate the number of cells used for data analysis; the percentages of responding cells (with increase in [Ca2+]i to > 175% of baseline) are denoted above
each box. Representative traces of [Ca2+]i measurements of individual PC12 cells exposed to OH-PBDE for 10 min (applied as indicated by arrowheads) in external
saline (containing 1.8 mM Ca2+) and in Ca2+-free conditions are shown below.
*p < 0.05, **p < 0.01, and #p < 0.001.
1,000
900
800
700
600
500
400
300
200
100
0
1.8
(61)
0
(42)
0+TG
(34)
0+TG
+FCCP
(42)
1.8
200%
1 min
0
0+TG
0+TG
+FCCP
1.8
200%
1 min
0
0+TG
0+TG
+FCCP
1.8
200%
1 min
0
0+TG
0+TG
+FCCP
1.8
200%
1 min
0
n.a.
0+TG
0+TG
+FCCP
1.8
200%
1 min
0
0+TG
0+TG
+FCCP
1.8
(48)
0
(32)
0+TG
(43)
0+TG
+FCCP
(35)
1.8
(48)
0
(32)
0+TG
(43)
0+TG
+FCCP
(35)
1.8
(61)
0
(42)
0+TG
(34)
0+TG
+FCCP
(42)
1.8
(49)
0
(24)
0+TG
(40)
1.8
(49)
0
(24)
0+TG
(40)
#
##
**
#
Amplitude of [Ca2+]i (% of baseline)
Amplitude of [Ca2+]i (% of baseline)
Amplitude of [Ca2+]i (% of baseline)
Extracellular Ca2+ (mM)Extracellular Ca2+ (mM)Extracellular Ca2+ (mM)
5 µM 6-OH-BDE-47
Initial increase
20 µM 6´-OH-BDE-49
Initial increase
5 µM 5-OH-BDE-47
Initial increase
20 µM 3-OH-BDE-47
Increase (monophasic)
66% 100% 44%
20 µM 4´-OH-BDE-49
Initial increase
83%4%
Late increase
85% 96%
98% 24%65% 40% 41%9%0%
22%
0%
53% 58%0%27% 63% 2.5%
67%34%0%22%9%0%38% 0%
Late increaseLate increase Late increase
1,000
900
800
700
600
500
400
300
200
100
0
1,000
900
800
700
600
500
400
300
200
100
0
**
2,000
1,800
1,600
1,400
1,200
1,000
800
600
400
200
0
1.8
(77)
0
(21)
0+TG
(34)
0+TG
+FCCP
(72)
#
Amplitude of [Ca2+]i (% of baseline)
Extracellular Ca2+ (mM)
78%17%
#
4,000
3,600
3,200
2,800
2,400
2,000
1,600
1,200
800
400
0
1.8
(40)
0
(26)
0+TG
(40)
0+TG
+FCCP
(46)
#
Extracellular Ca2+ (mM)
0%0%
*
1,000
900
800
700
600
500
400
300
200
100
0
1.8
(40)
0
(26)
0+TG
(40)
0+TG
+FCCP
(46)
#
Extracellular Ca2+ (mM)
#
**
#
**
**
#
#
Amplitude of [Ca2+]i (% of baseline)
Amplitude of [Ca2+]i (% of baseline)

Page 5

Bromination pattern affects potency of OH-PBDEs
Environmental Health Perspectives • volume 118 | number 4 | April 2010
523
When the ER Ca2+ stores were depleted,
the amplitudes of both initial and late
increases induced by 5 µM 5-OH-BDE-47
were largely diminished (Figure 4C). After
depletion of both ER and mitochondrial Ca2+
stores, the late increase was further reduced.
We observed mono phasic increases dur-
ing exposure to 20 µM 3-OH-BDE-47 in
Ca2+-free conditions, with similar ampli-
tude (Figure 4D). After depletion of the ER
Ca2+ stores, the amplitude was significantly
decreased, and even further after depletion of
ER and mitochondrial Ca2+ stores.
During exposure to 20 µM 4´-OH-
BDE-49 after depletion of the ER Ca2+ stores,
the amplitude of the initial increase largely
diminished (Figure 4E). The amplitude of the
late increase was not significantly changed in
Ca2+-free conditions. After depletion of the
ER Ca2+ stores, the amplitude significantly
decreased, and even further after depletion of
ER and mitochondrial Ca2+ stores.
Lower LOECs were identified for hydroxy­
lated metabolites when investigating fluctua­
tions. At LOECs and NOECs based on the
amplitude of [Ca2+]i, we investigated fluctua-
tions in [Ca2+]i [Table 1; also see Supplemental
Material, Table 4 (doi:10.1289/ehp.0901339)].
We detected no effects on the percentage of
cells showing fluctuations or the frequency,
duration, and amplitude of these fluctuations
during exposure to 2 µM 6´-OH-BDE-49 or
3-OH-BDE-47. During exposure to 0.2 µM
6-OH-BDE-47, we observed an increase in
the percentage of cells showing fluctuations.
The average duration of fluctuations increased,
without effects on the frequency or ampli-
tude (NOEC, 0.1 µM 6-OH-BDE-47). At
1 µM 5-OH-BDE-47, the number of cells
showing fluctuations increased. Although the
duration increased, the amplitude and fre-
quency were not affected (NOEC, 0.2 µM
5-OH-BDE-47). At 2 µM 4´-OH-BDE-49,
the number of cells showing fluctuations
increased, as well as the average fluctuation
frequency, duration, and amplitude (NOEC,
1 µM 4´-OH-BDE-49).
Discussion
The OH-PBDE metabolite 6-OH-BDE-47
has previously been shown to disrupt [Ca2+]i in
PC12 cells by releasing Ca2+ from intra cellular
stores at lower concentrations than its parent
compound BDE-47 (Dingemans et al. 2008).
The results presented here demon strate that
other hydroxylated metabolites of BDE-47 also
induce Ca2+ release from intra cellular stores,
whereas the methoxylated analog (6-MeO-
BDE-47) and the parent compounds lack this
effect (Figure 2). Experiments in Ca2+-free
conditions (Figure 4) indicate that the initial
increases induced by 6-OH-BDE-47, 5-OH-
BDE-47, and 4´-OH-BDE-49 are partly
caused by influx of extra cellular Ca2+. The
initial and late increases induced by 6-OH-
BDE-47 are caused by release of Ca2+ from
ER and mitochondria, respectively. The
widely varying increase induced by 6´-OH-
BDE-49 was caused primarily by release from
ER Ca2+ stores. Both initial and late increases
induced by 5-OH-BDE-47 and the increase
induced by 3-OH-BDE-47 are caused by
release of Ca2+ mainly from ER, but also from
mitochondria. Both initial and late increases
induced by 4´-OH-BDE-49 are caused by
release of Ca2+ from ER, but the late increases
are also from mitochondria. When investi-
gating fluctuations in [Ca2+]i, we detected
subtle effects of BDE-47 on the Ca2+ homeo-
stasis and observed lower LOECs for several
OH-PBDEs (Table 1).
We detected no or mild effects on cell via-
bility for the investigated PBDEs and MeO/
OH-PBDEs, indicating that the observed
effects on [Ca2+]i were not confounded by
cyto toxicity. The AB assay, which is based on
mitochondrial activity, appeared to be less
useful in determining cell viability because
for 6-OH-BDE-47, 6´-OH-BDE-49, and
3-OH-BDE-47 the relative fluorescence
intensity increased dose dependently above
the control level, suggesting induction of
mitochondrial activity. This may be related to
mitochondrial uncoupling, which was previ-
ously demonstrated for 6-OH-BDE-47 in
isolated zebrafish mitochondria (van Boxtel
et al. 2008).
The low basal [Ca2+]i of PC12 cells is
maintained by the removal of Ca2+ ions by
the plasma membrane Ca2+ ATPases and
Na2+–Ca2+ exchanger (Duman et al. 2008;
for review, see Westerink 2006). Additionally,
Ca2+ can be sequestered into organelles, mainly
ER and mitochondria. Both 3-OH-BDE-47
and 4´-OH-BDE-49 induce increases in
[Ca2+]i even after depletion of both ER and
mitochondria by TG and FCCP. Ca2+ has
also been shown to accumulate in endosomes,
lysosomes, secretory granules, the Golgi appa-
ratus, and nucleus (for review, see Laude and
Simpson 2009). The Golgi apparatus stores
Ca2+ via sarco plasmic/ER Ca2+ ATPase pumps
(Missiaen et al. 2007), which are inhibited by
TG. Therefore, it is unlikely that release of
Ca2+ from the Golgi apparatus caused the addi-
tional increase, whereas this remains unclear for
the other mentioned organelles.
At concentrations not affecting the average
and amplitude of increases in [Ca2+]i, BDE-47,
6-OH-BDE-47, and 4´-OH-BDE-49 caused
an increase in the frequency, amplitude, and/or
duration of fluctuations in [Ca2+]i. These subtle
effects on Ca2+ homeo stasis resulted in lower
NOECs for most of these brominated flame
retardants, particularly BDE-47 (Table 1). Ca2+
signals vary from micro domains to globally
across the cell and from milli seconds to many
hours (Laude and Simpson 2009). Because
the measured [Ca2+]i is an average value for
the entire cytosol, under estimation of mem-
brane- or store-associated high [Ca2+]i or high
[Ca2+]i microdomains (for review, see Cheng
and Lederer 2008) can be expected. The timing
of Ca2+ signals, including frequency and dura-
tion, affects how external stimuli cause (patho)
physiologic results (Boulware and Marchant
2008). Moreover, [Ca2+]i transients trigger
activity-dependent developmental events in
neurons, by activating gene expression, cyto-
skeletal elements, or neuro transmitter release,
whereas the charac teristics of these responses
are determined by amplitude, frequency,
Table 1. LOECs (µM) of BDE-47 and hydroxylated metabolites for cell viability and different parameters of [Ca2+]i in PC12 cells.
Ca2+ homeostasis
Ca2+ release
ERMitochondria
BDE-47———
6-OH-BDE-472011
6´-OH-BDE-49——20
5-OH-BDE-472011
3-OH-BDE-47——20
4´-OH-BDE-49202020
—, not detected. For the mechanisms responsible for effects on Ca2+ homeostasis, under “Ca2+ homeostasis,” values shown are LOEC for effects causing a decrease of > 25% in the
specific Ca2+-free experiments (Figure 4), and resulting NOEC levels. For the investigated parameters of fluctuations in [Ca2+]i, under “fluctuations in [Ca2+]i,” values shown are LOEC
and resulting NOEC levels; NOEC levels for effects on fluctuations in [Ca2+]i that are lower than NOEC values for effects on Ca2+ homeostasis-related processes are indicated by (<).
For values of [Ca2+]i and fluctuation parameters, see Supplemental Material, Tables 2 and 3 (doi:10.1289/ehp.0901339). For all PBDEs and MeO/OH-PBDEs investigated in this study, see
Supplemental Material, Table 4.
aPossible effects on parameters of fluctuations are obscured by release of Ca2+ from intracellular stores. bThe number of data points available to investigate duration and amplitude of
the fluctuations in [Ca2+]i is insufficient to ensure the NOEC.
Fluctuations in [Ca2+]i
Fluctuation
Frequency
2
—
20a
—
20a
2
Decreased
cell viability
Ca2+ influx
(extracellular)
Percent cells
showing fluctuations
—
0.2
20a
1
20a
2
NOEC
1 (<)
0.1 (<)
2b
0.2b
2
1 (<)
NOEC
20
0.2
2
0.2
2
2
Duration
2
0.2
20a
1
20a
2
Amplitude
—
—
20a
—
20a
—
2
—
—
20
202

Page 6

Dingemans et al.
524
volume 118 | number 4 | April 2010 • Environmental Health Perspectives
source, and spatial location of Ca2+ signals
(for review, see Moody and Bosma 2005).
Therefore, the observed effects of OH-PBDEs
at low concentrations can be of relevance for
the development of the nervous system.
None of the parent PBDEs except
BDE-47 showed any effects on Ca2+ homeo-
stasis in PC12 cells. Nonetheless, neuro toxic
effects of BDE-47, BDE-99, BDE-100, and
BDE-153 have been detected at different
biological levels (Costa and Giordano 2007).
Because of the lack of effects by parent PBDEs
in the present study, no effects of bromination
pattern could be investigated. We confirmed
that the activity of the OH-PBDEs depended
on the presence of the OH group, because
no effects were observed during exposure to
the methoxylated analog of 6-OH-BDE-47.
The higher activity of 6-OH-BDE-47 com-
pared with its methoxylated analog is in line
with other studies, mostly on endocrine effects
(Cantón et al. 2008; Kojima et al 2009; van
Boxtel et al. 2008). The mean amplitude of
increases in [Ca2+]i could not be related to
the location of the OH group on the PBDE
mole cule. However, it appeared that when
the OH group was shielded on both sides by
either the other phenyl ring and/or Br atoms
(as in 6´-OH-BDE-49 and 3-OH-BDE-47),
the OH-PBDE increased [Ca2+]i less than
when the OH group was less shielded (as in
6-OH-BDE-47, 5-OH-BDE-47, and 4´-OH-
BDE-49). Also, the OH-PBDEs with only
one shielded side of the OH group induced
release of Ca2+ from ER at the lowest concen-
trations (6-OH-BDE-47 and 5-OH-BDE-47)
or with the highest amplitude in Ca2+-free
conditions (4´-OH-BDE-49). Thus, the tox-
icity of OH-PBDEs appears attenuated by
shielding of the OH group on both sides by
either the other phenyl ring and/or Br atoms.
Several animal studies confirmed the
genera tion of hydroxylated metabolites of
PBDEs in vivo (Hakk et al. 2009; Malmberg
et al. 2005; Marsh et al. 2006), and that
OH-PBDEs were also formed in human liver
cells exposed to BDE-99 (Stapleton et al. 2009).
Interestingly, marine organisms have also been
shown to produce hydroxylated and methoxy-
lated metabolites (Hakk and Letcher 2003).
Only very recently, the occurrence and
accumulation of hydroxylated metabolites
were confirmed in humans (Athanasiadou
et al. 2008), with total OH-PBDE serum con-
centrations up to 120 pmol/g lipids. Another
study also detected OH-PBDEs in U.S. fetal
serum samples and confirmed the bioaccumu-
lation of these metabolites (Qiu et al. 2009).
Moreover, they demon strated that concentra-
tions of OH-PBDEs were similar or some-
times even higher than the concentration of
PBDEs. Fetal total OH-PBDE serum con-
centrations ranged from 2.01 to 899.1 ng/g
lipids (median, 21.96 ng/g lipids). The most
abundant BDE-47 metabolites found in
fetal blood were 5-OH-BDE-47 and 6-OH-
BDE-47 (Qiu et al. 2009). It is concerning
that these metabo lites caused an increase of
[Ca2+]i at much lower concentrations than the
other metabolites of BDE-47 investigated.
All five OH-PBDEs investigated in the
present study caused Ca2+ release from intra-
cellular Ca2+ stores, although with different
LOECs. Likely, depending on the position,
the OH group and adjacent phenyl ether and/
or Br atoms, other hydroxylated metabolites
of tetra and pentaPBDEs have a similar effect
on cellular calcium homeostasis. The median
(21.96 ng/g lipids) and highest (899.1 ng/g
lipids) concentrations of total OH-PBDEs
observed in fetal plasma correspond to
approximately 0.4 and 17.4 nM, respectively,
in blood (calculated with average physiologic
parameters). Thus, the highest concentra-
tion observed in human blood is only two
orders of magnitude lower than the LOEC
for Ca2+ release from intra cellular stores by
OH-PBDEs (1 µM). Moreover, the LOEC
for increased Ca2+ fluctuations is even lower
(0.2 µM), meaning that the margin of expo-
sure is insufficient in some individual expo-
sure situations. Also, because OH-PBDEs are
not associated with lipids—as are the parent
PBDEs—but have a high affinity for plasma
proteins (Verreault et al. 2005), the estimated
blood concentration calculated from expo-
sure values at a lipid-weight–adjusted basis
(nanograms per gram lipids) may be under-
estimated. However, the LOEC (1 µM) used
to calculate the margin of exposure is higher
for other metabolites, and it remains to be
determined whether the observed effects on
fluctuations in [Ca2+]i could result in func-
tional or even adverse effects in vivo.
Because exposure to organo halogen com-
pounds within the time frame of rapid brain
development can result in behavioral defects
in mice (for review, see Costa and Giordano
2007), it is concerning that children are
exposed to these environmental pollutants
prenatally and postnatally. Moreover, several
studies have observed inter actions between
environmental pollutants to enhance (neuro)
toxicity. Additive and synergistic neuro toxic
effects of polychlorinated biphenyls (PCBs)
and PBDEs have been detected in vivo
(Eriksson et al. 2006) and in vitro (Gao et al.
2009). Concern about possible effects on the
developing brain arises from the fact that an
increase in [Ca2+]i by release from intra cellular
stores appears to be a common mechanism for
OH-PBDEs and ortho-PCBs (for review, see
Mariussen and Fonnum 2006). Therefore, a
possible additive effect of these environmental
pollutants with respect to increases in cytosolic
[Ca2+]i, which not only is a trigger for neuro-
transmitter release but affects many cellular
processes (Clapham 2007), is not unlikely.
Because very high concentrations of
PBDEs are occasionally measured in humans,
the voluntary and legislative measures to reduce
the release of PBDEs into the environ ment
appear justified. Also, hydroxylated metabo-
lites of PBDEs, which were recently found to
bioaccumulate in humans (Athanasiadou et al.
2008; Qiu et al. 2009), either from man-made
PBDEs or of natural origin, are currently not
taken into account in regulatory human risk
assessment. The results presented here reveal a
structure–activity relation ship for metabolites
of PBDEs (more shielding of the OH group
reduces the potency of OH-PBDEs) and rein-
force that oxidative metabolism should be
included in human risk assessment of persistent
organic pollutants.
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