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{dagger}

* Norwegian Defence Research Establishment, Division for Protection, P. O. Box 25, N-2027 Kjeller, Norway, and {dagger} Norwegian Institute for Air Research, P. O. Box 100, N-2027 Kjeller, Norway

1 To whom correspondence should be addressed. Fax: + 47 63807509. E-mail: Trine.Reistad{at}ffi.no.

Received March 15, 2005; accepted June 2, 2005


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 µM), 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 µM) and the selective chelator of intracellular calcium, BAPTA-AM (5 µM), also inhibited NADPH oxidase activation, indicating a calcium-dependent activation of PKC. ROS formation was also inhibited by the tyrosine kinase inhibitor tyrphostin (1 µM), the phospholipase C inhibitor ET-18-OCH3 (5 µM), and the phosphatidylinositol-3 kinase inhibitor LY294002 (25 µM). 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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 penta-brominated diphenyl ethers (PeBDE) accounts for approximately 10% (de Wit, 2002Go). 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, 2002Go).

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., 2003Go). 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, 2004Go; Sjodin et al., 2003Go). 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, 2003Go; Kitamura et al., 2002Go; Meerts et al., 2000Go) and some of the BFR compounds have also a neurotoxic potential comparable to the PCBs (Eriksson et al., 2001Go; Mariussen and Fonnum, 2003Go; Reistad et al., 2002Go). 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., 2002Go; Sweet and Zelikoff, 2001Go). Some of these findings have been attributed to activation of neutrophil granulocytes in vitro (Ganey et al., 1993Go; Reistad et al., 2005Go; Sweet and Zelikoff, 2001Go; Voie et al., 1998Go) and in vivo in fish (Duffy et al., 2002Go, 2003Go).

Neutrophils are major sources of biological oxidants. When stimulated, these cells produce large amounts of superoxide () 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, 2003Go). 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)Go 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (penta-BDE), 3.9% BDE-153 (hexa-BDE), 3.8% BDE-154 (hexa-BDE), 0.2% BDE-183 (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 (99+%), bis-N-methylacridinium-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), ß-nicotineamide adenine dinucleotide phosphate (NADPH), pyruvate, phorbol 12-myristate 13-acetate (PMA), N-formyl-Met-Leu-Phe (fMLP), (±)-{alpha}-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., 1991Go). 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 x 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 x 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 4°C) until use.

Lactate dehydrogenase (LDH) assay.
Leakage of LDH was assessed as an index of cell injury (Koh and Choi, 1987Go). The measurements were performed as described elsewhere (Dreiem et al., 2005Go). In brief, cells (2 x 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 4°C until measured (within 2 h). LDH measurements were performed by transfer of 100 µl aliquots of the supernatant to the wells of a custom made 48 well microplate with glass bottom, and the volume was adjusted to 425 µl with 0.1M KPO4 buffer (pH 7.5). The reactions were started by automated injection of 50 µl of an 846 µM stock solution of NADH (final concentration 84.6 µM) followed by automated injection of 25 µl 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 28°C. 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.


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Table 1 Lactate Dehydrogenase (LDH) Leakage after Exposure to Different Compounds

 
Assay for measuring reactive oxygen species.
Lucigenin and luminol chemiluminescence was used to detect and HOCl in neutrophil granulocytes. The reaction mixture (250 µl) contained 0.1 mM lucigenin/luminol, 2 x 105 cells and different concentrations of the test compounds. Chemiluminescence was measured by a Labsystem Luminoskan luminometer at 37°C for 60 min. PMA (1 x 10–7 M) was included as a positive control in all experiments (n = 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 µl 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 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 . The fluorescence emitted by DCF reflects the general oxidative status of the cell and was determined essentially as described previously (Reistad et al., 2005Go).

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., 2004Go) to ensure that the compounds do not interfere with the chemiluminescence. Wells with 250 µl HBSS containing xanthine (1.1 mM) and lucigenin (0.1 mM) with the different inhibitors were added to 25 µl of a 260 mU/ml xanthine oxidase solution. The rate of superoxide production was monitored for 30 min in a Labsystem Luminoskan luminometer at 37°C.

Measurement of intracellular free calcium in granulocytes.
Intracellular free [Ca2+] was measured by using the fluorescent Ca2+-binding probe fura-2/AM by the method previously described (Grynkiewicz et al., 1985Go). An increase in Ca2+ concentration is indicated by an increase in the fluorescence excitation ratio (I340/I380). Granulocytes (4.5 x 106 cells/ml) in HBSS containing 20 mM HEPES and 5 mM glucose were incubated at 37°C with 5 µM 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 µM 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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, BDE-47, 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 µM 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 µM, data not shown).



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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 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).

 


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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).

 


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FIG. 3. (A) Chemiluminescence used as a measure for formation of ROS in human neutrophil granulocytes after exposure to 12 µM DE-71 in combination with SOD (50 U/ml), the antioxidant vitamin E (20 µM), the NADPH oxidase inhibitor DPI (4 µM), the superoxide dismutase inhibitor DDC (100 µM), and the intracellular calcium binding probe BAPTA-AM (5 µM). 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 µM DE-71 in combination with the PLC-inhibitor ET-18-OCH3 (5 µM), the PKC-inhibitor BIM (0.25 µM), the protein tyrosine kinase inhibitor tyrphostin AG 112 (1 µM), and the PI3-kinase inhibitor LY294002 (25 µM). 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).

 
The Involvement of Different Signaling Pathways in DE-71 Induced ROS Formation
The NADPH oxidase inhibitor DPI (O'Donnell et al., 1993Go) inhibited the ROS formation completely (Fig. 3A). The NADPH oxidase activates formation of . Lucigenin is a sensitive probe for detection of , and 100 µM of the superoxide dismutase inhibitor DDC (Misra, 1979Go) 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 µM BIM, a selective inhibitor of PKC (Bit et al., 1993Go), reduced the DE-71 response by 67%. The selective chelator of intracellular calcium, BAPTA-AM (5 µM) (Strayer et al., 1999Go) reduced the response by 71%, while calcium free buffer, with EGTA (2 mM) had no inhibitory effect on the ROS formation. ET-18-CHO3 (5 µM), which is a PLC-inhibitor (Powis et al., 1992Go), 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 µM), known as an inhibitor of protein tyrosine kinases (Gazit et al., 1989Go). Addition of the isoform nonspecific PI3-kinase inhibitor, LY294002 (25µM) (Vlahos et al., 1995Go), completely abolished the DE-71 induced ROS formation (Fig. 3B).

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 [Ca2+]i were measured using the membrane permeable Ca2+-binding fluorescent probe fura-2/AM. The DE-71 mixture (20 µM) significantly increased the concentration of intracellular free calcium (Fig. 4A). Diphenyl ether (20 µM) had no effect. BDE-47 (20µM) showed a small, but not significant, increase in intracellular calcium. Cells exposed to 20 µM DE-71 in calcium free buffer containing 2 mM EGTA also induced a significant increase in intracellular calcium (Fig. 4B).



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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 [Ca2+] 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.

 
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 peptide fMLP (1 µM) or the PCB congener 153 (5 µM). 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 µM DE-71), whereas higher concentrations on the contrary indicated an antagonistic effect (Table 2).


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Table 2 Lucigenin Amplified Chemiluminescence as a Measure for Formation of ROS

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 environment (Law et al., 2003Go). 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, 1999Go). Phosphorylation of the p47PHOX subunit plays a major role in activation of the NADPH oxidase complex (Nauseef et al., 1991Go), and is responsible for transporting the cytosolic NADPH oxidase complex to the membrane during activation (Babior, 1999Go). Superoxide anion () 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 , and is frequently used to demonstrate activation of respiratory burst in granulocytes (Halliwell and Gutteridge, 1999Go). The SOD inhibitor DDC increased the DE-71 induced chemiluminescence by 240%, indicating production after DE-71 stimulation. DPI, a potent inhibitor of the NADPH oxidase (O'Donnell et al., 1993Go), abolished ROS formation induced by DE-71. Lucigenin must undergo reduction to lucigenin cation to detect . The primary reducing agent in phagocytes is the NADPH oxidase system (Halliwell and Gutteridge, 1999Go). 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 3-kinase (PI3K), protein kinase C (PKC), and mitogen activated protein kinases (MAPK) (Dewas et al., 2000Go; El Benna et al., 1996aGo,bGo; Nauseef et al., 1991Go; Yamamori et al., 2004Go). 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 production by the PCB mixture Aroclor 1242 is dependent on tyrosine kinase activity (Tithof et al., 1997Go). PI3K is involved in activation of PKC, the MAPK pathway and PLC (Bae et al., 1998Go; Bondeva et al., 1998Go; Yamamori et al., 2004Go), 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., 1991Go; Vlahos et al., 1995Go). We found that LY294002 abolished the NADPH oxidase activity induced by DE-71. Yamamori et al. (2004)Go 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., 2005Go), that DE-71 elevates cytosolic free calcium both from the extracellular 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)Go, it has been shown that DE-71 increase translocation of PKC and inhibit 45Ca2+ 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., 2005Go). 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, 1999Go; Eriksson et al., 2003Go). Different combinations of the DE-71 mixture with PCB153 and the bacteria-derived 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., 1998Go). The bacterial chemotactic peptide fMLP is a potent activator of ERK1/2 (Dewas et al., 2000Go), 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., 2004Go). The DE-71 mixture combined with fMLP and PCB153 induced an additive effect (Table 2). In combination with 1µM fMLP, the DE-71 mixture was able to induce respiratory burst already at 1µM, 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)Go.

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, 2004Go), but comparable to what is observed in certain animal tissue (µM concentrations) (Law et al., 2003Go). 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).



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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.

 

    NOTES
 
The authors certify that all research involving human subjects was done under full compliance with all government policies and the Helsinki Declaration.


    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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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