TOXICOLOGICAL SCIENCES 87(1), 57–65 (2005)
doi:10.1093/toxsci/kfi222
Advance Access publication June 15, 2005
A Commercial Mixture of the Brominated Flame Retardant
Pentabrominated Diphenyl Ether (DE-71) Induces Respiratory
Burst in Human Neutrophil Granulocytes In Vitro
Trine Reistad*,1 and Espen Mariussen†
*Norwegian Defence Research Establishment, Division for Protection, P. O. Box 25, N-2027 Kjeller, Norway,
and †Norwegian Institute for Air Research, P. O. Box 100, N-2027 Kjeller, Norway
Received March 15, 2005; accepted June 2, 2005
Polybrominated diphenyl ethers (PBDEs) are widely used
brominated flame retardants (BFRs), which have become ubiquitous in the environment. This study investigates the effects of the
pentabrominated diphenyl ether mixture, DE-71, on human
neutrophil granulocytes in vitro. DE-71 enhanced production of
reactive oxygen species (ROS) in a concentration-dependent
manner measured as lucigenin-amplified chemiluminescence.
Octabrominated diphenyl ether (OBDE), decabrominated diphenyl ether (DBDE), and the non-brominated diphenyl ether did not
induce ROS formation at the concentrations tested. DPI (4 mM),
an inhibitor of the NADPH oxidase completely inhibited DE-71
induced ROS formation, highlighting a role for NADPH oxidase
activation. The protein kinase C inhibitor BIM (0.25 mM) and the
selective chelator of intracellular calcium, BAPTA-AM (5 mM),
also inhibited NADPH oxidase activation, indicating a calciumdependent activation of PKC. ROS formation was also inhibited
by the tyrosine kinase inhibitor tyrphostin (1 mM), the phospholipase C inhibitor ET-18-OCH3 (5 mM), and the phosphatidylinositol-3 kinase inhibitor LY294002 (25 mM). Alterations in
intracellular calcium were measured using fura-2/AM, and
a significant increase was measured after exposure to DE-71 both
with and without extracellular calcium. The tetra brominated
compound BDE-47 also enhanced ROS formation in a concentration dependent manner. The combination of DE-71 with the
bacteria-derived N-formyl peptide fMLP and PCB153 induced an
additive effect in the lucigenin assay. We suggest that tyrosine
kinase mediated activation of PI3K could result in enhanced
activation of calcium-dependent PKC by enhanced PLC activity,
followed by intracellular calcium release leading to ROS formation in neutrophil granulocytes.
Key Words: brominated flame retardants (BFR); pentabrominated diphenyl ethers (PeBDE); reactive oxygen species (ROS);
lucigenin; calcium; intracellular signaling pathways.
The authors certify that all research involving human subjects was done
under full compliance with all government policies and the Helsinki
Declaration.
1
To whom correspondence should be addressed. Fax: þ 47 63807509.
E-mail: [email protected].
The brominated flame retardants (BFRs) accounts for
approximately 20–30% of the annual demand of nearly
600,000 metric tons of fire preventives. About 80 different
BFRs are registered for use for fire preventing purposes. Within
this group the polybrominated diphenyl ethers (PBDE),
tetrabromobisphenol-A (TBBPA), and hexabromocyclododecane (HBCD) are the dominating compounds. Some of the
BFRs, especially the PBDEs, have chemical and physical
properties resembling the more established environmental
contaminants, such as the polychlorinated biphenyls (PCBs).
The annual demand of PBDEs is approximately 65,000 metric
tons, of which the commercial decabrominated diphenyl ethers
(DBDE) accounts for more than 85%, the octabrominated
diphenyl ethers (OBDE) accounts for 5%, and the pentabrominated diphenyl ethers (PeBDE) accounts for approximately 10% (de Wit, 2002). The PBDEs have been cause for
considerable concern the last two decades due to their potential
as environmental toxicants. Recent research has revealed that
the PBDEs have become ubiquitous in the environment at
levels comparable to what is found for the PCBs. PBDEs have
been detected in human blood, adipose tissue, breast milk, and
wild life (de Wit, 2002).
Generally it is the lower brominated PBDE congeners that
are found in substantial amounts in the environment and seem
to have the highest potential for bioaccumulation. The
European Union has therefore prohibited further use of this
compound group, and there are some indications that the
environmental levels in Europe have stabilized (Sjodin et al.,
2003). Recent studies from the U.S. have, however, revealed
that the levels of PBDEs are increasing and are at least one
order of magnitude higher in mother’s milk collected from U.S.
women compared to their European counterparts (Hites, 2004;
Sjodin et al., 2003). Due to their persistency in the environment
and their bioaccumulative properties, humans and wildlife will
be subjected to PBDE exposure for a long time in the future.
Several studies have shown that the lower brominated
PBDEs have a toxic potential similar to the PCBs. The PBDEs
can elicit thyroidogen- and estrogen-like activity in vitro
(Darnerud, 2003; Kitamura et al., 2002; Meerts et al., 2000)
The Author 2005. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
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REISTAD AND MARIUSSEN
and some of the BFR compounds have also a neurotoxic
potential comparable to the PCBs (Eriksson et al., 2001;
Mariussen and Fonnum, 2003; Reistad et al., 2002). Previously
a correlation has been found between wildlife animals’
exposure to environmental contaminants, such as PCB and
methyl mercury, and effects on immune-parameters (Duffy
et al., 2002; Sweet and Zelikoff, 2001). Some of these findings
have been attributed to activation of neutrophil granulocytes
in vitro (Ganey et al., 1993; Reistad et al., 2005; Sweet and
Zelikoff, 2001; Voie et al., 1998) and in vivo in fish (Duffy
et al., 2002, 2003).
Neutrophils are major sources of biological oxidants. When
stimulated, these cells produce large amounts of superoxide
(O2 ) that dismutates to hydrogen peroxide (H2O2). The
enzyme myeloperoxidase utilizes H2O2 and chloride to produce hypochlorous acid (HOCl), a potent oxidant that reacts
with a wide range of biological targets and is implicated as
a cause of inflammatory tissue damage. The same arsenal and
processes used by neutrophils for host defense may also be very
deleterious for an organism when deregulation occurs (Girard,
2003). The role of chemicals of environmental concern in the
cellular physiology of human neutrophils is an area of research
that has gained increasing attention during the past few years.
Fowles et al. (1994) investigated the immunotoxic effects of
the commercial PBDE mixture, DE-71, in mice. In their 14-day
subchronic study, the highest dose (72 mg/kg body weight per
day) significantly suppressed the antibody response to sheep
red blood cells, indicating a modest immunotoxic response.
Because of the high production volume of PBDEs, their
presence in biotic samples and the close resemblance to other
environmental contaminants, we have examined the effect of
the three main commercial PBDE mixtures and HBCD on
human neutrophil granulocytes. By knowing the importance of
neutrophils in the inflammatory process, research conducted in
this field will greatly increase our understanding of the
potential role of environmental pollutants in the development
of inflammatory disorders.
d
MATERIALS AND METHODS
Chemicals. Commercial mixtures of pentabrominated diphenyl ether
(PeBDE mixture, DE-71, Great Lakes), octabromo diphenyl ether (OBDE,
Lot GL10 B, Great Lakes), decabromo diphenyl ether (DBDE, Lot D102A 03,
Great Lakes) and hexabromocyclododecane (HBCD, CD-75P, Great Lakes)
were purchased from Promochem, Sweden. The DE-71 mixture was analyzed
on GC/LRMS (gas chromatography/low resolution mass spectrometry) in the
NCI (negative chemical ionization) mode (HP5890 GC coupled to a HP MSD)
monitoring at m/z 79 and 81 with methane as the chemical ionization gas. The
DE-71 mixture contained on a weight basis 0.23% BDE-28 (tri-BDE), 31.8
BDE 47 (tetra-BDE), 50.9% BDE-99 (penta-BDE), 9.1% BDE-100 (pentaBDE), 3.9% BDE-153 (hexa-BDE), 3.8% BDE-154 (hexa-BDE), 0.2% BDE183 (Hepta-BDE). BDE-47 (~99% puriry) was a gift from Gerhardt Rimkus,
Official food and Veterinary Institute (LVUA), Neumunster Germany. PCB153
(~99% purity) was obtained from AccuStandard Inc (New Haven, CT).
Stock solutions were prepared by dissolving the compounds in DMSO. The
final DMSO concentration in the samples was always less than 1%.
Bisindolylmaleimide (BIM), bromphenol blue, diphenyl ether (99þ%), bis-Nmethylacridinium-nitrate (lucigenin), 5-amino-2,3-dihydro-1,4-phthalazinedione
(luminol) 2,7-dichlorofluorescein diacetate (DCFH-DA), tyrphostin AG 112, dimethyl sulfoxide (DMSO), diphenyleneiodonium (DPI), diethyldithio-carbamic acid
(DDC), EGTA, 1-O-octadecyl-2-O-methyl-sn-glycerol-3-phosphorylcholine
(ET-18-OCH3), phosphate-buffered saline (PBS), b-nicotineamide adenine
dinucleotide phosphate (NADPH), pyruvate, phorbol 12-myristate 13-acetate
(PMA), N-formyl-Met-Leu-Phe (fMLP), (±)-a-tocopherol (vitamin E), xanthine, and xanthine oxidase were all from Sigma-Aldrich (St. Louis, MO).
LY294002 were obtained from Promega Corporation (Madison, WI). Hanks
Balanced Salt Solution (HBSS) and HEPES buffer were purchased from
GibcoBRL (U.K.). Lymphoprep was purchased from Nycomed Pharma (Oslo,
Norway). BAPTA/AM, and Fura-2/AM were from Calbiochem Novabiochem
Corp. (San Diego, CA). All other reagents used were analysis grade laboratory
chemicals from standard commercial suppliers.
Isolation of human neutrophil granulocytes. Human venous blood was
obtained from nonsmoking healthy adult male volunteers in the morning.
Neutrophil granulocytes were separated from EDTA blood by dextran
sedimentation followed by a standard density-gradient centrifugation as
previously described (Boyum et al., 1991). In brief, EDTA blood from
individual donors (30 ml) was mixed with 3 ml 6% dextran and left for
sedimentation at room temperature for 30 min. The supernatant, containing the
granulocytes, was subjected to Lymphoprep density gradient centrifugation at
600 3 g for 15 min. The pellet was washed in 0.9% NaCl and then resuspended
in 7 ml 0.83% NH4Cl for 7 min to lyse the erythrocytes, and then centrifuged
for 7 min (600 3 g). This was repeated if proper lysis was not obtained. Cells
were then resuspended in HBSS and the number of granulocytes was
determined in an AVIDA 60 hematology system. The cells were kept on ice
(approximately 4C) until use.
Lactate dehydrogenase (LDH) assay. Leakage of LDH was assessed as an
index of cell injury (Koh and Choi, 1987). The measurements were performed
as described elsewhere (Dreiem et al., 2005). In brief, cells (2 3 106/ml) were
exposed to the test compounds for the indicated times (5 or 30 min). Cells were
then spun down and the supernatant from each sample was transferred to
sample tubes and stored at 4C until measured (within 2 h). LDH measurements
were performed by transfer of 100 ll aliquots of the supernatant to the wells of
a custom made 48 well microplate with glass bottom, and the volume was
adjusted to 425 ll with 0.1M KPO4 buffer (pH 7.5). The reactions were started
by automated injection of 50 ll of an 846 lM stock solution of NADH (final
concentration 84.6 lM) followed by automated injection of 25 ll of a 13.6 mM
stock solution of pyruvate (final concentration 0.68 mM). The LDH activity
was measured, using a BMG FLUOstar Optima fluorimeter, from the decay rate
of NADH fluorescence for 30 min at 28C. The LDH activity was calculated off
line and is given as the rate constant of the decrease in fluorescence emission at
460 nm (excitation wavelength 340 nm). The LDH activity (fluorescence units/s)
is not a direct measure of the number of dead cells but gives a qualitative
measure of the relative amount of cell necrosis. 100% cell death was estimated
by administration of 0.01% triton and corresponded to a NADH fluorescence
decay rate of approximately 95 units/s (control values, 11 units/s). In Table 1
the values are shown as percent of triton ± SEM.
Assay for measuring reactive oxygen species. Lucigenin and luminol
chemiluminescence was used to detect O2 and HOCl in neutrophil granulocytes. The reaction mixture (250 ll) contained 0.1 mM lucigenin/luminol, 2 3
105 cells and different concentrations of the test compounds. Chemiluminescence was measured by a Labsystem Luminoskan luminometer at 37C for
60 min. PMA (1 3 107 M) was included as a positive control in all experiments
(n ¼ 5–7). The cells and reagents were prepared in HEPES-buffered (20 mM)
HBSS with 5 mM glucose. When calcium free buffer was used, 2 mM EGTA
was added. The reaction was started by adding 100 ll of the cell suspension to
each well. Results are presented as area under the curve (AUC). Formation of
ROS was also measured with use of the fluorescent probe DCFH-DA. The
method is based on the incubation of the granulocytes with DCFH-DA, which
d
59
DE-71 AND HUMAN NEUTROPHIL GRANULOCYTES
TABLE 1
Lactate Dehydrogenase (LDH) Leakage after Exposure to
Different Compounds
LDH activity (% of triton)
5-Min exposure
Control
DMSO
PeBDE (20 lM)
30-Min exposure
Control
DMSO
PeBDE (12 lM)
PCB 153 (5 lM)
fMLP (1 lM)
PeBDE (12 lM) þ PCB 153 (5 lM)
PeBDE (12 lM) þ fMLP (1 lM)
13.5 ± 1.8
15.3 ± 1.5
13.4 ± 1.5
13.8
15.3
14.5
16.5
17.1
14.9
15.7
±
±
±
±
±
±
±
1.2
1.3
2.3
1.6
1.4
1.9
1.3
Note. Lactate dehydrogenase (LDH) leakage measured in neutrophil
granulocytes after exposure to different compounds used in this study. The
results are presented as % of maximal LDH activity as given by exposure to
0.01% triton. Values are mean ± SEM, 4–6 experiments in triplicate. Student’s
t-test (paired, two tail distribution) was performed to indicate statistical
significant differences between each exposure group with DMSO as control.
FIG. 1. Chemiluminescence was used as a measure for formation of ROS
in human neutrophil granulocytes after exposure to different concentrations of
the brominated flame retardant DE-71. Chemiluminescence was measured in
a Labsystem Luminoskan luminometer for 60 minutes. All values are presented
as area under the curve (AUC). Control value: 14 ± 3. Values are mean ± SEM,
5–9 experiments in triplicate. One-way ANOVA followed by Dunnett’s twosided post hoc test was performed to indicate statistical significant differences
between exposure groups with DMSO as control (**p 0.01, ***p 0.001).
RESULTS
diffuses passively through the cellular membrane. Intracellular esterase activity
results in the formation of DCFH, which emits fluorescence when oxidized to
2#,7#-dichlorofluorescein (DCF). It is reported to detect several types of
reactive molecules such as H2O2, in presence of cellular peroxidases, OONO
and OH , but have no sensitivity towards O2 . The fluorescence emitted by
DCF reflects the general oxidative status of the cell and was determined
essentially as described previously (Reistad et al., 2005).
Compounds used for blocking intracellular signaling pathways leading
to ROS formation were tested in a cell free xanthine/xanthine oxidase system
with lucigenin (Fernandes et al., 2004) to ensure that the compounds do not
interfere with the chemiluminescence. Wells with 250 ll HBSS containing
xanthine (1.1 mM) and lucigenin (0.1 mM) with the different inhibitors were
added to 25 ll of a 260 mU/ml xanthine oxidase solution. The rate of superoxide
production was monitored for 30 min in a Labsystem Luminoskan luminometer
at 37C.
d
d
Measurement of intracellular free calcium in granulocytes. Intracellular
free [Ca2þ] was measured by using the fluorescent Ca2þ-binding probe
fura-2/AM by the method previously described (Grynkiewicz et al., 1985).
An increase in Ca2þ concentration is indicated by an increase in the
fluorescence excitation ratio (I340/I380). Granulocytes (4.5 3 106 cells/ml)
in HBSS containing 20 mM HEPES and 5 mM glucose were incubated at 37C
with 5 lM Fura-2/AM for 20 min. The cells were washed and resuspended
in HEPES-buffered HBSS with glucose. Measurements of fura-2 mediated
fluorescence were performed on a computerized Shimadzu RF-5301PC
Spectrofluorophotometer, using excitation wavelength ranging between
340 and 380 nm and emission wavelength 510 nm. All data are results of
5–9 separate measurements. Cell free experiments with Fura acid was
done and no interference with fura fluorescence was found after addition of
12 lM DE-71.
Statistical analyses. Differences between controls and treated groups were
evaluated using a two-way Student’s t-test (paired, two tail distribution), or by
one-way ANOVA followed by Dunnett’s two-sided post hoc test. The
calculations were performed using SPSS 11.5.
The Effect of Different BFRs on Human
Neutrophil Granulocytes
The DE-71 mixture induced a concentration dependent
increase in lucigenin-amplified chemiluminescence in human
neutrophil granulocytes (Fig. 1). The compound had no effect
on the DCF fluorescence, or luminol chemiluminescence
indicating primarily extracellular formation of ROS (data not
shown). Addition of superoxide dismutase (50 U/ml) showed
an almost total inhibition of ROS formation, strengthening the
assumption that ROS formation primarily was extracellular.
Similar to DE-71, the tetra brominated diphenyl ether, BDE47, induced ROS in a concentration dependent manner (Fig. 2).
DMSO, which was used for dilution of the test compounds,
showed no significant effect. In the presence of 20 lM of the
anti-oxidant vitamin E the superoxide anion was barely
detectable, strengthening the assumption of ROS formation
induced by DE-71 (Fig. 3A). The neutrophils were also
exposed to the commercial mixtures of OBDE, DBDE, and
HBCD without any effect (data not shown). Table 1 shows that
none of the different compounds used in this study had any
effect on cell viability at the times and concentrations tested.
The non-brominated diphenyl ether did not induce ROS
formation at the concentrations tested (1–12 lM, data
not shown).
The Involvement of Different Signaling Pathways in
DE-71 Induced ROS Formation
The NADPH oxidase inhibitor DPI (O’Donnell et al., 1993)
inhibited the ROS formation completely (Fig. 3A). The
60
REISTAD AND MARIUSSEN
FIG. 2. Chemiluminescence was used as a measure for formation of ROS
in human neutrophil granulocytes after exposure to different concentrations of
the tetra brominated diphenyl ether, BDE-47. Chemiluminescence was
measured in a Labsystem Luminoskan luminometer for 60 minutes. All values
are presented as area under the curve (AUC). Control value: 15 ± 2. Values are
mean ± SEM, 5–9 experiments in triplicate. One-way ANOVA followed by
Dunnett’s two-sided post hoc test was performed to indicate statistical
significant differences between exposure groups with DMSO as control
(**p 0.01, ***p 0.001).
NADPH oxidase activates formation of O2 . Lucigenin is
a sensitive probe for detection of O2 , and 100 lM of the
superoxide dismutase inhibitor DDC (Misra, 1979) increased
the DE-71 induced lucigenin chemiluminescence by 266%,
demonstrating superoxide anion radical formation.
Several signaling pathways may activate the NADPH
oxidase. Incubation of the granulocytes with 0.25 lM BIM,
a selective inhibitor of PKC (Bit et al., 1993), reduced the
DE-71 response by 67%. The selective chelator of intracellular
calcium, BAPTA-AM (5 lM) (Strayer et al., 1999) reduced
the response by 71%, while calcium free buffer, with EGTA
(2 mM) had no inhibitory effect on the ROS formation. ET-18CHO3 (5 lM), which is a PLC-inhibitor (Powis et al., 1992),
inhibited the ROS formation by 91%. Two of the major PLC
isoforms are activated by tyrosine kinases, and the DE-71
induced response was significantly reduced (48%) by tyrphostin AG 112 (1 lM), known as an inhibitor of protein tyrosine
kinases (Gazit et al., 1989). Addition of the isoform nonspecific
PI3-kinase inhibitor, LY294002 (25lM) (Vlahos et al.,
1995), completely abolished the DE-71 induced ROS formation
(Fig. 3B).
d
d
Intracellular Calcium Measurements with Fura-2/AM
Human neutrophil granulocytes were exposed to DE-71, the
non-brominated diphenyl ether and BDE-47. Changes in
[Ca2þ]i were measured using the membrane permeable Ca2þbinding fluorescent probe fura-2/AM. The DE-71 mixture
(20 lM) significantly increased the concentration of intracellular
free calcium (Fig. 4A). Diphenyl ether (20 lM) had no effect.
BDE-47 (20lM) showed a small, but not significant, increase
in intracellular calcium. Cells exposed to 20 lM DE-71 in
FIG. 3. (A) Chemiluminescence used as a measure for formation of ROS in
human neutrophil granulocytes after exposure to 12 lM DE-71 in combination
with SOD (50 U/ml), the antioxidant vitamin E (20 lM), the NADPH oxidase
inhibitor DPI (4 lM), the superoxide dismutase inhibitor DDC (100 lM), and
the intracellular calcium binding probe BAPTA-AM (5 lM). Chemiluminescence was measured in a Labsystem Luminoskan luminometer for 60 min. All
values are presented as area under the curve (AUC). Control value: 14 ± 5.
Values are mean ± SEM, 5–9 experiments in triplicate. Two-way Student’s t-test
(paired, two tail distribution) was performed to indicate statistical significant
differences between each exposure group treated with or without the indicated
inhibitor (*p 0.05, **p 0.01, ***p 0.001). (B) Chemiluminescence used
as a measure for formation of ROS in human neutrophil granulocytes after
exposure to 12 lM DE-71 in combination with the PLC-inhibitor ET-18-OCH3
(5 lM), the PKC-inhibitor BIM (0.25 lM), the protein tyrosine kinase inhibitor
tyrphostin AG 112 (1 lM), and the PI3-kinase inhibitor LY294002 (25 lM).
Chemiluminescence was measured in a Labsystem Luminoskan luminometer
for 60 min. All values are presented as area under the curve (AUC). Control
value: 14 ± 5. Values are mean ± SEM, 5–9 experiments in triplicate. Two-way
Student’s t-test (paired, two tail distribution) was performed to indicate
statistical significant differences between each exposure group treated with or
without the indicated inhibitor (*p 0.05, **p 0.01, ***p 0.001).
calcium free buffer containing 2 mM EGTA also induced
a significant increase in intracellular calcium (Fig. 4B).
Effects of DE-71 in Combination with fMLP or PCB153
The granulocytes were exposed to different concentrations
of DE-71 in combination with the bacterial chemotactic
61
DE-71 AND HUMAN NEUTROPHIL GRANULOCYTES
FIG. 4. Intracellular calcium measured in human neutrophil granulocytes after exposure to (A) DE-71, diphenyl ether and BDE-47 and (B) DE-71 with and
without extracellular calcium. Buffer without calcium contained 2 mM EGTA. Intracellular [Ca2þ] was measured using the calcium binding fluorescent probe
fura-2/AM. Measurements of fura-2 mediated fluorescence were performed on a computerized Perkin-Elmer LS50 luminescence spectrometer, using excitation
wavelength ranging between 340 and 380 nm and emission wavelength 510 nm. Results are presented as mean ratio (340 nm/380 nm) ± SEM, of 5–8 separate
measurements. Student’s t-test (paired, two tail distribution) was performed to indicate statistical significant differences between each exposure group with DMSO
as control (* 0.05, ** 0.01, *** 0.001). There was also a statistical difference between the PeBDE with and without extracellular calcium (**). All statistics
was performed on values after 250 s.
peptide fMLP (1 lM) or the PCB congener 153 (5 lM). fMLP
and PCB153 had both a significant effect on the ROS
formation. fMLP in combination with different DE-71 concentrations showed an additive effect at all concentrations
tested. DE-71 in combinations with PCB153 was additive in
combination with lower concentrations of the DE-71 mixture
(1–4 lM DE-71), whereas higher concentrations on the
contrary indicated an antagonistic effect (Table 2).
DISCUSSION
Our investigation demonstrates that the BFRs DE-71 and
BDE-47 induce ROS formation in human neutrophil granulocytes as shown with lucigenin-amplified chemiluminescence
in vitro. The commercial pentaBDE mixture, DE-71, reflects
the PBDE-congener pattern in environmental samples, whereas
BDE-47 is generally the dominating BDE congener in the
62
REISTAD AND MARIUSSEN
TABLE 2
Lucigenin Amplified Chemiluminescence as a Measure for Formation of ROS
PeBDE
fMLP
1 lM
PCB 153
5 lM
10.2 ± 1.9
12.7 ± 2.7
þfMLP
þPCB 153
1 lM
2 lM
4 lM
6 lM
12 lM
3.9 ± 1.7
15.9 ± 2.1**
16.6 ± 2.9*
9.3 ± 3.5
20.7 ± 2.7*
20.8 ± 4.4*
16.1 ± 2.6
37.1 ± 1.3***
36.1 ± 3.7**
24.7 ± 3.9
43.5 ± 2.5**
30.3 ± 2.8
23.8 ± 3.7
49.9 ± 4.1***
29.9 ± 3.8
Note. Lucigenin amplified chemiluminescence as a measure for formation of ROS in human neutrophil granulocytes after exposure to 1 lM fMLP or 5 lM
PCB153, in combination with different concentrations of the DE-71 mixture. All values are presented as area under the curve (AUC) with the control value
subtracted. Control value: 9.4 ± 0.9. Values are mean ± SEM, 5–8 experiments in triplicate. Student’s t-test (paired, two-tail distribution) was performed to
indicate statistical significant differences between the combination experiment and each DE-71 exposure group (*p 0.05, **p 0.01, ***p 0.001).
environment (Law et al., 2003). The non-brominated diphenyl
ether did not induce ROS formation neither did the commercial
mixtures of the OBDE and DBDE. This indicates that oxidative
stress induced by PBDEs is dependent on bromination pattern.
DE-71 induced chemiluminescence was inhibited by superoxide dismutase (SOD), indicating that the signals measured in
our experiments were mainly due to superoxide production.
This also strengthens the assumption that ROS formation
primarily was extracellular. We propose that DE-71’s effect
is mediated through activation of tyrosine kinases, PI3-kinase,
PKC, and PLC leading to activation of the NADPH oxidase,
and production of ROS in neutrophil granulocytes in vitro.
Formation of ROS in granulocytes may be induced by
different mechanisms, of which activation of the NADPH
oxidase is the most important. In resting neutrophils this
complex consists of unassembled cytosolic and membrane
components. Following activation, the cytosolic components,
p40PHOX, p47PHOX, p67PHOX, and Rac-2, translocate to the
plasma membrane where they associate with flavocytochrome
b558 and Rap1A to form the active oxidase (Babior, 1999).
Phosphorylation of the p47PHOX subunit plays a major role in
activation of the NADPH oxidase complex (Nauseef et al.,
1991), and is responsible for transporting the cytosolic NADPH
oxidase complex to the membrane during activation (Babior,
1999). Superoxide anion (O2 ) formed by NADPH oxidase
activation can be reduced to hydrogen peroxide (H2O2), either
spontaneously or by SOD. Lucigenin is a sensitive probe for the
detection of the O2 , and is frequently used to demonstrate
activation of respiratory burst in granulocytes (Halliwell and
Gutteridge, 1999). The SOD inhibitor DDC increased the DE71 induced chemiluminescence by 240%, indicating O2 production after DE-71 stimulation. DPI, a potent inhibitor of the
NADPH oxidase (O’Donnell et al., 1993), abolished ROS
formation induced by DE-71. Lucigenin must undergo reduction to lucigenin cation to detect O2 . The primary reducing
agent in phagocytes is the NADPH oxidase system (Halliwell
and Gutteridge, 1999). This suggests that the effect of DE-71 is
mediated through activation of the NADPH oxidase complex.
Various protein kinases have been involved in the regulation
of NADPH oxidase activity, including phosphatidylinositol
d
d
d
d
3-kinase (PI3K), protein kinase C (PKC), and mitogen activated
protein kinases (MAPK) (Dewas et al., 2000; El Benna et al.,
1996a,b; Nauseef et al., 1991; Yamamori et al., 2004).
Upstream, some of these kinases may be activated by tyrosine
kinases and the tyrosine kinase inhibitor tyrphostin reduced the
DE-71 induced ROS response by 48%. Previously it has been
demonstrated that activation of neutrophil O2 production by
the PCB mixture Aroclor 1242 is dependent on tyrosine kinase
activity (Tithof et al., 1997). PI3K is involved in activation of
PKC, the MAPK pathway and PLC (Bae et al., 1998; Bondeva
et al., 1998; Yamamori et al., 2004), all of which are major
regulatory pathways in the activation of the NADPH oxidase.
Several studies report that PI3K is involved in phosphorylation
of p47PHOX, but it has not been fully understood how PI3K
regulates it. Pharmacological inhibition of PI3K with wortmannin and LY294002 reduces the NADPH oxidase activity
stimulated by chemoattractants, such as fMLP, in human
neutrophils (Bonser et al., 1991; Vlahos et al., 1995). We
found that LY294002 abolished the NADPH oxidase activity
induced by DE-71. Yamamori et al. (2004) reported that PLC
and DAG-dependent PKC are regulated in a PI3K-dependent
manner in differentiated HL-60 cells (neutrophil-like phenotype). Similar to LY294002, the PLC-inhibitor ET-O18-CH3
almost totally abolished the DE-71 induced ROS formation in
our assay. PLC induces formation of DAG and IP3, which is
followed by calcium release from intracellular stores and
PKC activation.
By chelating intracellular calcium with BAPTA-AM, the
DE-71-induced ROS response was lost (Fig 3A) while removal
of extracellular calcium had no effect. The DE-71 mixture was
further shown to increase the intracellular concentration of
calcium, measured with the fluorescent probe Fura-2/AM. We
also observed an increase in intracellular calcium when
extracellular calcium was removed and EGTA was added.
The single congener, BDE-47, did not induce a significant
increase in intracellular calcium, suggesting that a mixture of
different PBDEs increase its tentative toxicity. Taken together,
this indicates, as shown for the brominated flame retardant
TBBPA in a previous paper (Reistad et al., 2005), that DE-71
elevates cytosolic free calcium both from the extracellular
d
DE-71 AND HUMAN NEUTROPHIL GRANULOCYTES
environment and through release from intracellular compartments. An increase in intracellular calcium can result in an
activation of important isoforms of PKC. BIM, which is
a nonselective inhibitor of the different PKC isoforms, reduced
the DE-71 induced ROS formation by 80%. In a study by
Kodavanti and Ward (2005), it has been shown that DE-71
increase translocation of PKC and inhibit 45Ca2þ uptake by
both microsomes and mitochondria in cerebellar granule cells
in culture. These results support our findings that PKC
activation is involved in the DE-71 induced respiratory burst.
Previously it has been shown that exposure of neutrophils to
the brominated flame retardant TBBPA leads to an activation of
the NADPH oxidase primarily by an ERK1/2 stimulated
pathway, which was followed by a potent intracellular and
extracellular ROS formation (Reistad et al., 2005). There was,
however no clear indications of an involvement of ERK1/2 in
the formation of PeBDE induced ROS, shown by Western blot
by use of a phospho specific ERK antibody. A striking
difference between TBBPA and PeBDEs mechanism of action
is TBBPA’s ability to induce both intracellular and extracellular
ROS, which may explain the different mechanism of action.
As the concentration of one single toxicant often does not
reach a level with an anticipated effect an important issue in
environmental toxicology is the effect of mixtures of contaminants. It has previously been reported that environmental
toxicants even may act synergistically when combined (Bemis
and Seegal, 1999; Eriksson et al., 2003). Different combinations of the DE-71 mixture with PCB153 and the bacteriaderived N-formyl peptide fMLP were therefore investigated.
The PCBs are still one of the most dominating groups of
toxicants in environmental samples, and the ortho chlorinated
PCBs have previously been shown to induce respiratory burst
in granulocytes (Voie et al., 1998). The bacterial chemotactic
peptide fMLP is a potent activator of ERK1/2 (Dewas et al.,
2000), even at very low concentrations, but it induces less
potent respiratory burst on its own, showing the characteristic
of a priming agent (Brown et al., 2004). The DE-71 mixture
combined with fMLP and PCB153 induced an additive effect
(Table 2). In combination with 1lM fMLP, the DE-71 mixture
was able to induce respiratory burst already at 1lM, indicating
that a priming agent, such as fMLP in combination with DE-71
may considerably increase its toxicity. This may indicate
a higher susceptibility of environmental toxicants when
exposed in stress related situations such as an infection.
However, one interesting observation was that beyond a certain
high DE-71 concentration, combination with PCB153 appeared to have antagonistic effect. The LDH assay showed
that this was not due to a cytotoxic effect (Table 1). The reason
for this may be due to a threshold limit for response reaching
maximum response beyond a certain degree of stimulation.
Environmental contaminants, such as PCBs and PBDEs, are
also lipophilic making it probable that high concentrations will
influence the cells membrane properties affecting the cellular
responses as suggested by Tan et al. (2004).
63
FIG. 5. Proposed pathways leading to activation of the NADPH oxidase
after stimulation with the brominated flame retardant DE-71. PMA and fMLP
are included in the figure. We propose that DE-71s effect is mediated through
activation of protein tyrosine kinases, followed by activation of PI3 kinase,
PKC, and PLC, leading to activation of the NADPH oxidase, and production of
ROS in neutrophil granulocytes.
The present article demonstrates that the BFR DE-71
activates the NADPH oxidase followed by respiratory burst
in granulocytes. The DE-71 mixture in combination with the
ortho-PCB153 and fMLP induced an additive effect. This is of
concern since the levels of BFR in certain areas are rapidly
increasing, both in human and environmental samples. The
concentrations used in this study are higher than what is
detected in human samples (nM concentrations) (Hites, 2004),
but comparable to what is observed in certain animal tissue
(lM concentrations) (Law et al., 2003). Our investigation
indicates that the DE-71 induced respiratory burst was
primarily due to activation of tyrosine kinases followed by
PI3-kinase, PKC and PLC activation, and an increase in
intracellular calcium concentration. We suggest that tyrosine
kinase mediated activation of PI3K could result in enhanced
activation of calcium-dependent PKC by enhanced PLC
activity, followed by intracellular calcium release. Finally,
based on our findings we propose a model for signal transduction pathway leading to NADPH activation stimulated by
DE-71. PMA and fMLP are included in the figure (Fig. 5).
ACKNOWLEDGMENTS
The authors wish to thank Frode Fonnum for helpful discussions, Dr. Avi
Ring for assistants in the LDH measurements, Yngvar Gundersen for proof
reading and the blood donors for making this work possible. The authors also
acknowledge The Norwegian Defence Research Establishment and Norwegian
Research Council, under the PROFO program, for financial support. Conflict
of interest: none declared.
64
REISTAD AND MARIUSSEN
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