* 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
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
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ABSTRACT |
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Key Words: brominated flame retardants (BFR); pentabrominated diphenyl ethers (PeBDE); reactive oxygen species (ROS); lucigenin; calcium; intracellular signaling pathways.
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INTRODUCTION |
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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
) 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 () 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.
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MATERIALS AND METHODS |
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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 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, 1987). The measurements were performed as described elsewhere (Dreiem et al., 2005
). 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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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 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., 1985). 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 59 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.
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RESULTS |
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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., 1993), reduced the DE-71 response by 67%. The selective chelator of intracellular calcium, BAPTA-AM (5 µM) (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-18-CHO3 (5 µM), 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 µM), known as an inhibitor of protein tyrosine kinases (Gazit et al., 1989
). Addition of the isoform nonspecific PI3-kinase inhibitor, LY294002 (25µM) (Vlahos et al., 1995
), 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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DISCUSSION |
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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 (
) 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, 1999
). 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., 1993
), 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, 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 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
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 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 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., 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 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., 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 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)
.
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 (µM 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).
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NOTES |
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ACKNOWLEDGMENTS |
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