Polybrominated Diphenyl Ether (PBDE) Effects in Rat Neuronal Cultures: 14C-PBDE Accumulation, Biological Effects, and Structure-Activity Rel
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《毒物学科学杂志》
Neurotoxicology and Experimental Toxicology Divisions, NHEERL, ORD, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
The University of Iowa College of Public Health, Iowa City, IA 52242
ABSTRACT
Polybrominated diphenyl ethers (PBDEs), widely used as flame-retardants, are now recognized as globally distributed pollutants, and are detected in most environmental and biological samples, including human blood, adipose tissue, and breast milk. Due to their wide use in commercial products and their persistent nature, long-term exposure to PBDEs may pose a human health risk, especially to children. Our previous reports showed that the commercial PBDE mixture, DE-71, affected protein kinase C (PKC) and calcium homeostasis in a similar way to those of a structurally-related polychlorinated biphenyl (PCB) mixture. These intracellular signaling events are associated with neuronal development and learning and memory function. The objectives of the present study were to test whether environmentally relevant PBDE congeners, with different position and number of bromines, affected PKC translocation in cerebellar granule neuronal cultures and compare the potency and efficacy of PBDE congeners with their 14C-accumulation. All the tested PBDE congeners increased 3H-phorbol ester (PDBu) binding, and a significant effect was seen as low as 10 μM. Among the congeners tested, 2,2',4,4'-tetrabromodiphenyl ether (PBDE 47) increased 3H-PDBu binding in a concentration-dependent manner and to a greater extent than other congeners. These effects were seen at concentrations and exposure times where no cytotoxicity was observed. The efficacy of PBDE congeners varied with their structural composition, and the effects seen on 3H-PDBu binding with some PBDE congeners are similar to those of PCB congeners. Cerebellar granule neurons accumulated all three PBDE congeners (PBDEs 47, 99, and 153) following exposure. At the lowest concentration (0.67 μM), about 13–18% of the total dose of 14C-PBDE congeners was accumulated by these neurons. There were distinct differences in the pattern of 14C-PBDE accumulation among the PBDE congeners. The 14C-PBDE accumulation, either represented as percent basis or nanomole basis, was much lower for the 30.69 μM PBDE 99 and 10.69–30.69 μM PBDE 153 than at the lower concentrations, which may be due to low solubility of these congeners. The accumulation pattern with PBDE 47 did not vary with concentration. On a nanomole accumulation basis, PBDEs 47, 99, and 153 accumulation was linear with time. While the nanomole accumulation was linear with concentration for PBDE 47, it is nonlinear for PBDEs 99 and 153. The pattern of PBDE accumulation seems to correlate with the effects on PKC translocation, with regression values of 0.773–0.991. These results indicate that PBDEs affected PKC translocation in neurons in a similar way to those of other organohalogens, some PBDE congeners are equally efficacious as the respective PCB congeners, and PBDE accumulation correlated well with PKC translocation, suggesting a common mode of action for this group of chemicals.
Key Words: polychlorinated biphenyls (PCBs); polybrominated diphenyl ethers (PBDEs); neurotoxicity; intracellular signaling; cytotoxicity; protein kinase C; calcium signaling.
INTRODUCTION
Polybrominated diphenyl ethers (PBDEs) were commercially produced with three levels of bromination, i.e., pentaBDE, octaBDE, and decaBDE, indicating the average bromine content. Since direct bromination of aromatic compounds is nonselective, mixtures of homologues and isomers are formed with 209 possible congeners having low vapor pressure at room temperature and a high lipophilicity; log Kow ranges between 4.28 and 9.9 (WHO, 1994). Commercially produced PBDE mixtures contain a limited number of PBDE congeners and are less complex than the corresponding technical polychlorinated biphenyl (PCB) mixtures. PBDEs are extensively used as flame-retardants in the electronic and computer industry in circuit boards, computer housings, televisions, and capacitors. The textile and paint industries also use large amounts of these flame-retardants in furniture, automobile cushions, building materials, and packaging materials. Since PBDEs are used as additive flame-retardants and do not bind chemically to the polymers, they can leach from the surface of the product and easily reach the environment (Birkett and Lester, 2003; de Wit, 2002; Hutzinger et al., 1976; Hutzinger and Thoma, 1987).
PBDEs are structurally similar to polychlorinated biphenyls (PCBs) and other organohalogens (see Fig. 1). Like PCBs, PBDEs are now ubiquitous; they can be found in air, water, fish, birds, marine mammals, and humans, and in many cases, they are increasing over time (Hites, 2004). In spite of their widespread occurrence in the environment, only limited information is available on the toxicology of individual PBDE congeners (Birnbaum and Staskal, 2004). Recent studies showed that several PBDE congeners, including 2,2',4,4'-tetrabromodiphenyl ether (PBDE 47), 2,2',4,4',5-pentabromodiphenyl ether (PBDE 99), 2,2',4,4',5,5'-hexabromodiphenyl ether (PBDE 153), and 2,2',3,3',4,4',5,5',6,6'-decabromodiphenyl ether (PBDE 209) caused aberrations in spontaneous behavior and reduced learning and memory in mice following exposure on postnatal day 10, a period of rapid brain development called "brain growth spurt" (Eriksson et al., 2001, 2002; Viberg et al., 2002, 2003a,b). The developmental effects of PBDEs appear to be as potent in female mice as in male mice, and as potent in C57/Bl mice as in NMRI mice (Viberg et al., 2004), suggesting that PBDE effects were not gender specific or strain dependent. Developmental neurotoxic effects by PBDE 99 have also been reported in rats (Branchi et al., 2002; Lilienthal et al., 2005; Viberg et al., 2005). The behavioral effects of PBDE congeners in mice seem to be similar to those seen after exposure to 1,1,1-trichloro-2,2-bis[p-chlorophenyl]ethane (DDT) or PCB congeners (Eriksson, 1997) when the effects were compared on a molar basis.
We have previously reported that PCBs, which are known to cause neurotoxic effects, affected intracellular signaling pathways including 3H-arachidonic acid (3H-AA) release, calcium homeostasis, and translocation of protein kinase C (PKC) in vitro (Kodavanti and Tilson, 2000) and in vivo during repeated exposure in adults (Kodavanti et al., 1998) and during development (Kodavanti et al., 2000). Considering the role of signal transduction in neural development and learning and memory (Abeliovich et al., 1993; Kater and Mills, 1991; Kodavanti, 2004; Murphy et al., 1987), perturbed intracellular signaling has been proposed as one of the modes of action for PCB-induced neurodevelopmental effects (Kodavanti, 2004). We have previously reported that PBDEs, like PCBs, altered 3H-AA release in neuronal cultures (Kodavanti and Derr-Yellin, 2002). Our recent findings suggest that PBDE mixtures such as DE-71 altered PKC translocation and calcium buffering in neuronal systems, and some of these effects were comparable to the respective PCB mixture such as Aroclor 1254 when the effects were compared on a molar basis (Kodavanti and Ward, 2005). The objectives of the present study were to test (a) whether environmentally relevant PBDE congeners affect PKC translocation in neuronal cultures in a similar way to those of commercial PBDE mixtures and other organohalogens; (b) understand the structure-activity relationships among PBDE congeners on PKC translocation; (c) compare the potency and efficacy of PBDE congeners on PKC translocation with their 14C-accumulation in cerebellar neurons.
MATERIALS AND METHODS
Chemicals.
Radiolabeled 3H-phorbol 12,13-dibutyrate (20 Ci/mmol) was purchased from Dupont NEN Corporation (Boston, MA). Radiolabeled 14C-PBDE congeners (27.8–36.5 μCi/μmol; 96–99% pure) were custom synthesized by NEN Life Sciences Products, Boston, MA. PBDE congener, 2,2',4,4'-tetrabromodiphenyl ether (PBDE 47) was a gift from Great Lakes Chemical Corporation (West Lafayette, IN). PBDE 77 (3,3',4,4'-tetrabromodiphenyl ether) was commercially available from the Biochemical Institute for Environmental Carcinogens, Lurup 4, D-22927 Grosshansdorf, Germany. PBDEs 99 (2,2',4,4',5-pentabromodiphenyl ether), 100 (2,2',4,4'6- pentabromodiphenyl ether), and 153 (2,2',4,4',5,5'-hexabromodiphenyl ether), and PCBs 47 (2,2',4,4'-tetrachlorobiphenyl) and 99 (2,2',4,4',5-pentachlorobiphenyl) were purchased from AccuStandard, Inc, New Haven, CT. All PBDE and PCB congeners were 98–100% pure and dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO in the assay buffer (0.2% v/v) did not significantly affect 3H-phorbol ester binding.
Animals.
Timed pregnant (16 days gestation) Long-Evans hooded rats were obtained from Charles River Laboratory (Raleigh, NC) and housed individually in AAALAC approved animal facilities. Food and water were provided ad libitum. Temperature was maintained at 21 ± 2°C and relative humidity at 50 ± 10% with a 12-h light/dark cycle (700–1900 h). Beginning on gestational day 22, rats were checked twice daily (A.M. and P.M.) for births, and the date when birth was first discovered was assigned postnatal day 0. The litter size for each dam varied between 8 and 14 pups, and litters were left undisturbed until postnatal day 7. All of the experiments were approved in advance by the institutional animal care and use committee of the National Health and Environmental Effects Research Laboratory at U.S. EPA that requires compliance with NIH guidelines.
Cerebellar granule cell culture.
Primary cultures of rat cerebellar granule neurons (CGCs) were prepared from 7- to 8-day-old Long Evans rat pups as outlined by Gallo et al. (1987) with modifications (Kodavanti et al., 1993a,b). Cultures were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS and 30 mM KCl in 12-well plates (Corning Costar), with a plating density of 1.0 x 106 cells/ml. Cytosine arabinoside was added 48 h after plating to prevent the proliferation of nonneuronal cells. Cultures were assayed at 7 days in vitro when they were fully differentiated and exhibiting fasciculation of fibers that interconnect the cells (Kodavanti et al., 1993a). Cell culture made on each day was considered as a single experiment in statistical analysis.
3H-Phorbol ester binding in cerebellar granule cells.
Cerebellar granule cells grown on 12-well culture plates (Costar) were tested at 7 days in culture for 3H-phorbol ester binding as per the method outlined by Vaccarino et al. (1991). Briefly, the monolayers were washed with Locke's buffer (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3, 2.3 mM CaCl2, 5.6 mM D-glucose, 5 mM HEPES, pH 7.4) containing 0.1% fatty-acid-free bovine serum albumin. Following washing, the cells were incubated in Locke's buffer containing 1 nM 4--3H-phorbol 12,13-dibutyrate (3H-PDBu; 0.1 μCi/ml) for 15 min at room temperature with PBDE or PCB congeners (10–50 μM). An equal amount of DMSO was added to controls. After incubation, the medium was aspirated, cells were washed three times with Locke's buffer and suspended with 1 ml of 0.1 M NaOH. An aliquot of this sample (0.7 ml) was added to 9 ml Ultima GoldTM (Packard, Meriden, CT) and the radioactivity was determined using scintillation spectroscopy (Beckman LS6500, Fullerton, CA). A small aliquot (25 μl) was used for protein determination (Bradford, 1976). Nonspecific binding was determined in the presence of 1.6 μM phorbol myristate acetate, which was always <20% and subtracted from all the values. The unit of 3H-PDBu binding was fmol/mg protein/15 min.
14C-PBDE accumulation in cerebellar granule neurons:
After 7 days in culture, the medium (DMEM) was removed from each well containing cerebellar granule neurons, washed twice with 1 ml aliquots of 37°C Locke's buffer, and allowed to equilibrate for 15 min at room temperature. The Locke's buffer was then replaced with 1 ml of 37°C Locke's buffer containing 0.05 μCi of 14C-PBDE congeners (0.67 μM of PBDEs 47, 99, and 153) along with different concentrations of cold PBDEs (0 to 30 μM) and maintained in the 37°C incubator for 15 min to 1 h. After incubation at the respective time periods, a 0.1-ml aliquot of the media was sampled from each well; then, the cells were washed twice with 1 ml cold Locke's buffer, and cells were dissolved in 1 ml NaOH. The entire sample of 1.0 ml was added to 10 ml Ultima GoldTM (Packard, Meriden, CT). The radioactivity in samples from cells, media in the beginning and end of exposure was determined using scintillation spectroscopy (Beckman LS6500, Fullerton, CA). The recovery of radioactivity was 90–95%. The 14C-accumulation of PBDE congeners by cerebellar granule neurons was represented both as percentage and in nanomoles.
Cytotoxicity of cerebellar granule neurons.
Lactic dehydrogenase (LDH) leakage was used as an indicator of cell death in cerebellar granule cells plated and cultured in the same manner as with 3H-phorbol ester binding. As the cell membrane loses integrity, LDH is released, and this activity is measured by the rate of conversion from pyruvate and NADH to lactate and NAD (Loo and Rillema, 1998). After 7 days in culture, the medium (DMEM) was removed from each well, washed twice with 1-ml aliquots of 37°C Locke's buffer, and allowed to equilibrate for 15 min at room temperature. The Locke's buffer was then replaced with 1 ml of 37°C Locke's buffer containing the various test solutions (0 to 100 μM). The cells were maintained in the 37°C incubator when not taking samples. Fifty-μl aliquots were removed from each well at 30, 60, 120, and 240 min after addition of test solutions for determining LDH activity. After collecting the last sample at 240 min, the cells were lysed by adding 50 μl of 5% TritonX-100TM and agitated at room temperature for 20 min. A 50-μl aliquot of lysed cell fraction was also assayed for LDH activity. LDH activity in the lysed fraction plus released activity (samples from medium collected at different time points) represent total LDH in cell culture. LDH leakage was expressed as a percentage of the total LDH capable of leaking into the medium. LDH activity was measured using the Geno Technology, Inc, St. Louis, MO, "CytoscanTM-LDH Cytotoxicity Assay Kit". The data were expressed as percent LDH leakage by test chemicals with chlorpromazine (100 μM) as a positive control.
Statistics.
The data (n = 5–8 experiments, assayed in triplicates) were analyzed by a two-way analysis of variance (ANOVA) with PBDE/PCB as one factor and concentration as the other using SigmaStat software, version 3.1 (SPSS Inc., Chicago, IL). In the case of significant interaction, step-down ANOVAs were used to test for main effects of PBDEs or PCBs. For LDH leakage data, two-way repeated measures ANOVA was performed taking concentration and exposure time as factors. Pair wise comparisons between groups were made using Fisher's LSD test. The accepted level of significance was p < 0.05.
RESULTS
Congener-Specific Effects of PBDEs on PKC Translocation in Cerebellar Granule Neurons
Translocation of PKC from cytosol to the membrane is a key intracellular signaling event and responds to changes in intracellular free calcium levels (Trilivas and Brown, 1989; Vaccarino et al., 1991). This translocation can be successfully measured by determining 3H-PDBu binding. All the tested PBDE congeners increased 3H-PDBu binding, and a significant effect was seen as low as 10 μM (Fig. 2). ANOVA indicated a significant interaction of binding and the concentration of PBDE congeners (F12,120 = 3.786; p < 0.001). A post-hoc test showed that PBDE 47 increased 3H-PDBu binding in a concentration-dependent manner and to a greater extent when compared to other congeners at 30 and 50 μM. The effect of PBDE congeners on 3H-PDBu binding decreased with increasing number of bromines on the molecule (Fig. 2; Table 1). This could be due to low solubility in the buffer or increasing hydrophobicity with increasing number of bromines. In addition, the effects of tetra congeners on 3H-PDBu binding seems to be linear, while the effects were nonlinear for higher bromine congeners. The calculated E50 value (concentration that increases 50% of control activity) for PBDE 47 was 34 ± 2 μM, while it was >50 μM for all other tested congeners (Table 1). PBDE 77, which is a non-ortho-substituted noncoplanar PBDE congener, also increased 3H-PDBu binding in cerebellar granule neurons (Fig. 2).
Comparative Effects of PBDE and PCB Congeners on PKC Translocation
In order to compare the effects between PBDE and PCB congeners, two pairs of congeners with four and five halogens were selected. PBDE 47 and PBDE 99 are the most prevalent congeners in the commercial mixtures as well as in most biological samples (Birnbaum and Staskal, 2004). The results clearly indicate that PBDE 47 increased PKC translocation in a similar way to PCB 47 on a molar basis (Fig. 3). The ANOVA indicated a significant effect of concentration for PBDE 47/PCB 47 (F3,72 = 73.057; p < 0.001), but no significant effect of treatment either for tetra congener pair (F1,72 = 0.0866; p = 0.769). The potency (10 μM), efficacy (72–79% at 50 μM), and E50 values (33–36 μM) were similar for PBDE 47 and PCB 47 (Table 1). A similar observation was also made with penta-halogenated congeners. PBDE 99 increased PKC translocation in a similar way to PCB 99 on a molar basis (Fig. 3). ANOVA indicated a significant effect of concentration for PBDE 99/PCB 99 (F3,56 = 10.764; p < 0.001), but there was no significant effect of treatment for penta congener pair (F1,56 = 0.675; p = 0.415). In addition, PBDE 99 and PCB 99 also have similar potency (10 μM) and efficacy (23–41% at 50 μM) (Table 1).
14C-PBDE Accumulation by Cerebellar Granule Neurons
Cerebellar granule neurons accumulated all three PBDE congeners tested (PBDEs 47, 99, and 153) (Figs. 4–7). When the data were analyzed on a percentage basis, ANOVAs indicated a significant interaction of PBDE congener and concentration at 15-min (F6,36 = 11.025; p < 0.001), 30-min (F6,36 = 10.996; p < 0.001), and 60-min (F6,36 = 12.581; p < 0.001) exposures. At the lowest concentration of PBDEs, about 13–18% of the total exposed concentration was accumulated by these neurons (Figs. 4 and 5). There were distinct differences in the pattern of accumulation among PBDE congeners. The percent 14C-PBDE 47 accumulation was only slightly increased with concentration while percent 14C-PBDEs 99 and 153 accumulation decreased with concentration (Fig. 4). However, the percent accumulation of 14C-PBDE congeners increased with time of exposure at all concentrations, and the accumulation seems to be linear (Fig. 5). ANOVAs indicated a significant effect of exposure time at 0.67 μM (F2,27 = 132.991; p < 0.001), 3.67 μM (F2,27 = 55.898; p < 0.001), 10.67 μM (F2,27 = 49.18; p < 0.001), and 30.67 μM (F2,27 = 85.761; p < 0.001) concentrations. The percent accumulation was much lower for the 30.67 μM PBDE99 and 10.67–30.67 μM PBDE153 than at the lower concentrations. The percent accumulation pattern with PBDE 47 did not vary with concentration (Fig. 5).
When the data was presented on a nanomole accumulation basis, 14C-PBDE 47, but not 14C-PBDEs 99 and 153 accumulation in neurons was linear with concentration (Fig. 6). ANOVAs indicated a significant interaction of PBDE congener and concentration at 15-min (F6,36 = 41.136; p < 0.001), 30-min (F6,36 = 39.118; p < 0.001), and 60-min (F6,36 = 40.475; p < 0.001) exposures. However, the neuronal accumulation of all three 14C-PBDE congeners was linear with exposure time (Fig. 7). ANOVAs indicated a significant effect of exposure time at 0.67 μM (F2,27 = 100.797; p < 0.001), 3.67 μM (F2,27 = 73.443; p < 0.001), 10.67 μM (F2,27 = 51.537; p < 0.001), and 30.67 μM (F2,27 = 87.026; p < 0.001) concentrations.
Correlations between Accumulation of PBDE Congeners and Their Effects on PKC Translocation
The pattern of 14C-PBDE accumulation correlates well with PKC translocation (Fig. 8). Of the three PBDEs tested, PKC translocation was stimulated to the greatest extent with PBDE 47 (Fig. 2), and this congener was also most readily accumulated (Figs. 6–7) by the cerebellar granule neurons. However, the effects of PBDE 99 and 153 on PKC translocation and percent 14C-PBDE accumulation were different when compared to PBDE 47. The dose-response curves for both the stimulation of PKC translocation and 14C-PBDE accumulation for both PBDEs 99 and 153 reached a plateau at 10 μM. Although stimulation of PKC translocation reached a maximum at 10 μM, the accumulation of PBDEs 99 and 153 continued to rise at a much slower rate between 10.67 and 30.67 μM. Since PKC translocation, as measured by 3H-PDBu binding was conducted at 15 min exposure, PKC translocation data was plotted against nmol 14C-PBDE accumulation at 15 min exposure. The analysis indicated a strong correlation for PBDE 47 (r2 = 0.991) as well as for PBDEs 99 (r2 = 0.863) and 153 (r2 = 0.773) (Fig. 8).
Cytotoxicity of PCB 47 or PBDE 47 in Cerebellar Granule Neurons
Chlorpromazine at 100 μM, which was used as a positive control based on our previous reports (Kodavanti et al., 1993a), significantly caused LDH leakage at 4 h of exposure (Table 2). We selected only tetra congeners (PBDE 47 and PCB 47) for cytotoxicity measurements, since these congeners produced greatest effect on PKC signaling (Fig. 3). The ANOVA indicated a significant interaction of exposure time and the concentration for PBDE 47 (F12,45 = 14.464; p < 0.001) and PCB 47 (F12,45 = 66.702; p < 0.001). PCB 47 did not cause a significant LDH leakage up to 30 μM and 120 min exposure. A concentration of 100 μM increased LDH leakage minimally at 120 min of exposure. At the 240 min time point, an increase in LDH leakage was seen at concentrations of 30 μM. (Table 2). Similar to the effects with PCB 47, PBDE 47 also did not cause any significant LDH leakage 50 μM and 120 min exposure. A concentration of 100 μM PBDE 47 increased LDH leakage minimally at 120 min exposure. At the 240 min exposure, a significant increase in LDH leakage was observed at concentrations of 50 μM. (Table 2).
DISCUSSION
Results from the present study demonstrate for the first time that environmentally relevant PBDE congeners increased PKC translocation, which is a critical event in the development of the nervous system and is associated with learning and memory processes (Abeliovich et al., 1993; Kodavanti, 2004), in rat cerebellar granule neurons. In addition, the accumulation of PBDE congeners correlated well with their effects on PKC translocation suggesting a common mode of action for this group of chemicals. Translocation of PKC is a culminating event that responds to a number of intracellular changes, including changes in calcium homeostasis caused by pharmacological agents or environmental chemicals, and is a link between signaling events that occur on cell surface and in the nucleus (Murphy et al., 1987; Trilivas and Brown, 1989; Girard and Kuo, 1990). The observation made with PBDE congeners regarding PKC translocation is consistent with previous studies on commercial PBDE mixtures (Kodavanti and Ward, 2005) and on structurally related chemicals such as PCBs, DDT, and/or polychlorinated diphenyl ethers in neuronal cell cultures (Kodavanti et al., 1995, 1996), human astrocytoma cells (Madia et al., 2004), and human leukemic HL-60 cells (Shin et al., 2002). Recent studies demonstrated the involvement of PKC and calcium homeostasis in the respiratory burst caused by a commercial penta PBDE mixture (Reistad and Mariussen, 2005). A similar effect on PKC was observed with other known developmental neurotoxicants such as alcohol (Hoek and Rubin, 1990), lead, and methylmercury (Haykal-Coates et al., 1998; Nihei et al., 2001) suggesting a key role for PKC signaling pathways in the neurotoxicity of environmental pollutants. In addition, studies using pharmacological agents by Shin et al. (2002) and Kang et al. (2004) clearly indicated that perturbation in calcium homeostasis and activation of PKC are involved in the necrosis of catecholaminergic neurons and apoptosis in human leukemic HL-60 cells by PCB exposure.
We have demonstrated previously that PCBs, known human developmental neurotoxicants and structurally related to PBDEs, perturbed several intracellular signaling pathways including PKC in vitro and in vivo (Kodavanti and Tilson, 1997, 2000; Kodavanti et al., 2000). At comparable doses, PCBs have been reported to cause behavioral changes and cognitive deficits in monkeys (Schantz et al., 1989), rats (Gilbert et al., 2000), and mice (Eriksson and Fredriksson, 1996). PKC translocation and calcium homeostasis are important events in cellular signal transduction; perturbations in these events have been implicated in a variety of physiological, developmental, and pathological processes (Kater and Mills, 1991; Mattson, 1991; Nicotera et al., 1992). A close observation of the structure-activity relationship (SAR) data with more than 50 PCB congeners and analogs revealed that PCB congeners with fewer meta and para chlorine atoms, especially those without para-substitution, and those with ortho-chlorine substitution are the most active PCBs in neuronal preparations (Kodavanti and Tilson, 1997). Studies using other neurochemical endpoints such as brain/PC12 cell dopamine levels (Shain et al., 1991) and ryanodine receptor binding (Schantz et al., 1997) support the hypothesis that PCB-induced neurotoxicity through perturbations in intracellular signaling events is mediated by the noncoplanarity exhibited by ortho- and ortho-, para-substituted congeners.
PBDEs also exist as 209 possible congeners based on the position and number of bromines, however, all these congeners are more noncoplanar than are PCBs. On the other hand, PCBs also have 209 possible congeners where some congeners are more coplanar (approaching about 30 degrees) and some are less coplanar (approaching 90 degrees). Based on our previous SAR results on PCBs about the importance of noncoplanarity (Kodavanti and Tilson, 1997), PBDE congeners were predicted to be active in neurons and our results showed that all the tested congeners increased PKC translocation in cerebellar granule neurons (Fig. 2). Of the congeners studied, PBDE 47 is the most active one and is also found in major quantities in biological and environmental samples. The effect seen with PBDE 47 (E50 = 34 μM) was much greater than that of DE-71 (E50 = >60 μM), which is a pentabrominated diphenyl ether mixture (Kodavanti and Ward, 2005). It is interesting to note that PBDE 77, which is a non-ortho-substituted noncoplanar PBDE congener is active (Fig. 2) while PCB 77, which is a non-ortho-substituted more coplanar congener, was reported to be inactive in cerebellar granule neurons (Kodavanti and Tilson, 1997) suggesting the importance of noncoplanarity of the molecule in the neurotoxicity of this group of pollutants acting via perturbations in intracellular signaling events.
The selected PBDE congeners did not cause cytotoxicity at concentrations where PKC translocation in cerebellar neurons was affected following exposure. Cytotoxicity studies were performed in cerebellar granule neurons after 7 days in vitro, taking LDH leakage as an index. LDH release into the medium has been used as an indicator of cell toxicity (Verity et al., 1990; Sasaki et al., 1992; Kodavanti et al., 1993a,b). Although PBDE 47 and PCB 47 increased LDH leakage indicating cytotoxicity, this response required longer exposures and higher concentrations (Table 2) than the effects seen on PKC translocation suggesting that the effects on PKC translocation precedes the effects on cytotoxicity.
The potency and efficacy between PCB and PBDE congeners were compared with respect to their effects on PKC translocation. Comparisons among tetra- and penta-halogenated congeners indicated that PBDEs 47 and 99 seem to have similar potency and efficacy on a molar basis as PCBs 47 and 99, respectively. This observation is in agreement with our previous studies on commercial penta-brominated mixtures (e.g., DE-71) regarding other signaling events such as 3H-arachidonic acid release (Kodavanti and Derr-Yellin, 2002) and mitochondrial calcium buffering (Kodavanti and Ward, 2005). It is interesting to note that changes in swim-maze performance, spontaneous behavior, and habituation capability with increasing age seen with PBDE exposure were similar to those seen with PCB exposure on a molar basis (Eriksson and Fredriksson, 1996; Eriksson et al., 2001; Viberg et al., 2003b). Among PBDE congener effects in mice, PBDEs 47 and 99 have similar potency on swim-maze performance while PBDE 99 is more potent than PBDE 47 on spontaneous behavior (Eriksson et al., 2001). In addition, there were few reports demonstrating the accumulation of PBDE congeners in mouse brain (Eriksson et al., 2002; Viberg et al., 2003a; Darnerud and Risberg, 2005). These mechanistic and behavioral studies suggest that these two groups of chemicals may be exerting neurotoxic effects through a common mode of action by altering intracellular signaling.
The pattern of PBDE accumulation correlates well with PKC translocation. The accumulation of PBDE congeners increased with time of exposure and at the lowest concentration, about 13–18% of the total dose was accumulated by the cerebellar granule neurons (Figs. 4 and 5). The percent accumulation was much lower for PBDEs 99 and 153 compared to PBDE 47, which may be due to low solubility or increased lipophilicity with increased number of bromines in these congeners. This observation is consistent with previous reports on PBDE-47 accumulation in rat neocortical cells (Mundy et al., 2004). Of the three PBDEs tested, PKC translocation was stimulated to the greatest extent with PBDE 47 and this congener was also most readily accumulated by the cerebellar granule neurons. 14C-PBDE 47 accumulation was linear with concentration and time, both on a percentage or nmole basis (Figs. 4–7). PBDE 47-induced increases in PKC translocation were also linear with concentration (Fig. 3A), suggesting a good correlation between this biological effect and uptake of this congener. The accumulation pattern of PBDEs observed in cerebellar neurons follows the absorption pattern of PBDEs in fish model, where PBDE 47 showed the highest uptake efficiency, and increased bromination of PBDEs resulted in less absorption of these chemicals (Burreau et al., 1997). When PKC translocation is plotted against nmol accumulation, a strong correlation (r2 = 0.991) was found for PBDE 47 (Fig. 8). The effects of PBDE 99 and 153 were different when compared to PBDE 47. The dose-response curves for both the stimulation of PKC translocation and accumulation for both PBDEs 99 and 153 reached plateaus at 10 μM. Although stimulation of PKC translocation reached a maximum at 10 μM, the accumulation of these congeners continued to rise at a much slower rate between 10.69 and 30.69 μM. Although some minor differences exist among the selected PBDE congeners, current results support the hypothesis that PKC translocation is a critical neuronal effect for PBDE congeners, as observed for PCBs.
In summary, PBDEs increased the PKC translocation as do other organohalogens and known neurotoxicants. Some PBDE congeners have similar potency and efficacy as PCBs. PBDE accumulation correlated well with PKC translocation, suggesting a common mode of action for this group of chemicals. In addition, changes seen in some neurochemical endpoints seem to correlate with neurobehavioral endpoints when compared on a molar basis between PCBs and PBDEs. PBDEs are ubiquitous, persistent, and coexist with PCBs in fish (Manchester-Neesvig et al., 2001), human blood, and breast milk samples (Schecter et al., 2003), and the levels of PBDEs are rapidly rising in North Americans (Betts, 2002). PBDE levels found in several biological and environmental samples from the United States were several folds higher than the levels found in European countries (Hale et al., 2003; Hites, 2004; Petreas et al., 2003). Few reports indicate more than additive interaction between PBDEs and PCBs. As seen individually, combination of PBDE and PCB also resulted in impaired spontaneous motor behavior, which worsened with age. In addition, the combination led to the same effect as a 10x higher dose of either chemical alone (Eriksson et al., 2003). Considering the structural similarity of PBDEs with PCBs, coexistence of these two groups of persistent chemicals in the environment, and the known health effects of PCBs in humans and animals, these two groups of chemicals could conceivably work through the same or similar mechanism(s) to cause developmental neurotoxicity. Since these chemicals exist together in almost all the biological samples, understanding the interactions between these chemicals is very important in terms of their risk assessment.
NOTES
The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
ACKNOWLEDGMENTS
The authors thank Great Lakes Chemical Corporation for providing a sample of PBDE 47 and Dr. Tom Burka of NIEHS for providing us 14C-labeled PBDE congeners. Ms. Beth Padnos and Ms. Theresa Freudenrich are acknowledged for their excellent technical assistance and Drs. Hugh Tilson of U.S. EPA, Tom McDonald of Arvesta Corporation, and Maggie Curras-Collazo of University of California at Riverside for their helpful comments on an earlier version of this manuscript. Preliminary findings were presented at the 24th Symposium on Halogenated Environmental Organic Pollutants and POPs (Dioxin2004) meeting in Berlin, Germany (September 5–10, 2004).
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The University of Iowa College of Public Health, Iowa City, IA 52242
ABSTRACT
Polybrominated diphenyl ethers (PBDEs), widely used as flame-retardants, are now recognized as globally distributed pollutants, and are detected in most environmental and biological samples, including human blood, adipose tissue, and breast milk. Due to their wide use in commercial products and their persistent nature, long-term exposure to PBDEs may pose a human health risk, especially to children. Our previous reports showed that the commercial PBDE mixture, DE-71, affected protein kinase C (PKC) and calcium homeostasis in a similar way to those of a structurally-related polychlorinated biphenyl (PCB) mixture. These intracellular signaling events are associated with neuronal development and learning and memory function. The objectives of the present study were to test whether environmentally relevant PBDE congeners, with different position and number of bromines, affected PKC translocation in cerebellar granule neuronal cultures and compare the potency and efficacy of PBDE congeners with their 14C-accumulation. All the tested PBDE congeners increased 3H-phorbol ester (PDBu) binding, and a significant effect was seen as low as 10 μM. Among the congeners tested, 2,2',4,4'-tetrabromodiphenyl ether (PBDE 47) increased 3H-PDBu binding in a concentration-dependent manner and to a greater extent than other congeners. These effects were seen at concentrations and exposure times where no cytotoxicity was observed. The efficacy of PBDE congeners varied with their structural composition, and the effects seen on 3H-PDBu binding with some PBDE congeners are similar to those of PCB congeners. Cerebellar granule neurons accumulated all three PBDE congeners (PBDEs 47, 99, and 153) following exposure. At the lowest concentration (0.67 μM), about 13–18% of the total dose of 14C-PBDE congeners was accumulated by these neurons. There were distinct differences in the pattern of 14C-PBDE accumulation among the PBDE congeners. The 14C-PBDE accumulation, either represented as percent basis or nanomole basis, was much lower for the 30.69 μM PBDE 99 and 10.69–30.69 μM PBDE 153 than at the lower concentrations, which may be due to low solubility of these congeners. The accumulation pattern with PBDE 47 did not vary with concentration. On a nanomole accumulation basis, PBDEs 47, 99, and 153 accumulation was linear with time. While the nanomole accumulation was linear with concentration for PBDE 47, it is nonlinear for PBDEs 99 and 153. The pattern of PBDE accumulation seems to correlate with the effects on PKC translocation, with regression values of 0.773–0.991. These results indicate that PBDEs affected PKC translocation in neurons in a similar way to those of other organohalogens, some PBDE congeners are equally efficacious as the respective PCB congeners, and PBDE accumulation correlated well with PKC translocation, suggesting a common mode of action for this group of chemicals.
Key Words: polychlorinated biphenyls (PCBs); polybrominated diphenyl ethers (PBDEs); neurotoxicity; intracellular signaling; cytotoxicity; protein kinase C; calcium signaling.
INTRODUCTION
Polybrominated diphenyl ethers (PBDEs) were commercially produced with three levels of bromination, i.e., pentaBDE, octaBDE, and decaBDE, indicating the average bromine content. Since direct bromination of aromatic compounds is nonselective, mixtures of homologues and isomers are formed with 209 possible congeners having low vapor pressure at room temperature and a high lipophilicity; log Kow ranges between 4.28 and 9.9 (WHO, 1994). Commercially produced PBDE mixtures contain a limited number of PBDE congeners and are less complex than the corresponding technical polychlorinated biphenyl (PCB) mixtures. PBDEs are extensively used as flame-retardants in the electronic and computer industry in circuit boards, computer housings, televisions, and capacitors. The textile and paint industries also use large amounts of these flame-retardants in furniture, automobile cushions, building materials, and packaging materials. Since PBDEs are used as additive flame-retardants and do not bind chemically to the polymers, they can leach from the surface of the product and easily reach the environment (Birkett and Lester, 2003; de Wit, 2002; Hutzinger et al., 1976; Hutzinger and Thoma, 1987).
PBDEs are structurally similar to polychlorinated biphenyls (PCBs) and other organohalogens (see Fig. 1). Like PCBs, PBDEs are now ubiquitous; they can be found in air, water, fish, birds, marine mammals, and humans, and in many cases, they are increasing over time (Hites, 2004). In spite of their widespread occurrence in the environment, only limited information is available on the toxicology of individual PBDE congeners (Birnbaum and Staskal, 2004). Recent studies showed that several PBDE congeners, including 2,2',4,4'-tetrabromodiphenyl ether (PBDE 47), 2,2',4,4',5-pentabromodiphenyl ether (PBDE 99), 2,2',4,4',5,5'-hexabromodiphenyl ether (PBDE 153), and 2,2',3,3',4,4',5,5',6,6'-decabromodiphenyl ether (PBDE 209) caused aberrations in spontaneous behavior and reduced learning and memory in mice following exposure on postnatal day 10, a period of rapid brain development called "brain growth spurt" (Eriksson et al., 2001, 2002; Viberg et al., 2002, 2003a,b). The developmental effects of PBDEs appear to be as potent in female mice as in male mice, and as potent in C57/Bl mice as in NMRI mice (Viberg et al., 2004), suggesting that PBDE effects were not gender specific or strain dependent. Developmental neurotoxic effects by PBDE 99 have also been reported in rats (Branchi et al., 2002; Lilienthal et al., 2005; Viberg et al., 2005). The behavioral effects of PBDE congeners in mice seem to be similar to those seen after exposure to 1,1,1-trichloro-2,2-bis[p-chlorophenyl]ethane (DDT) or PCB congeners (Eriksson, 1997) when the effects were compared on a molar basis.
We have previously reported that PCBs, which are known to cause neurotoxic effects, affected intracellular signaling pathways including 3H-arachidonic acid (3H-AA) release, calcium homeostasis, and translocation of protein kinase C (PKC) in vitro (Kodavanti and Tilson, 2000) and in vivo during repeated exposure in adults (Kodavanti et al., 1998) and during development (Kodavanti et al., 2000). Considering the role of signal transduction in neural development and learning and memory (Abeliovich et al., 1993; Kater and Mills, 1991; Kodavanti, 2004; Murphy et al., 1987), perturbed intracellular signaling has been proposed as one of the modes of action for PCB-induced neurodevelopmental effects (Kodavanti, 2004). We have previously reported that PBDEs, like PCBs, altered 3H-AA release in neuronal cultures (Kodavanti and Derr-Yellin, 2002). Our recent findings suggest that PBDE mixtures such as DE-71 altered PKC translocation and calcium buffering in neuronal systems, and some of these effects were comparable to the respective PCB mixture such as Aroclor 1254 when the effects were compared on a molar basis (Kodavanti and Ward, 2005). The objectives of the present study were to test (a) whether environmentally relevant PBDE congeners affect PKC translocation in neuronal cultures in a similar way to those of commercial PBDE mixtures and other organohalogens; (b) understand the structure-activity relationships among PBDE congeners on PKC translocation; (c) compare the potency and efficacy of PBDE congeners on PKC translocation with their 14C-accumulation in cerebellar neurons.
MATERIALS AND METHODS
Chemicals.
Radiolabeled 3H-phorbol 12,13-dibutyrate (20 Ci/mmol) was purchased from Dupont NEN Corporation (Boston, MA). Radiolabeled 14C-PBDE congeners (27.8–36.5 μCi/μmol; 96–99% pure) were custom synthesized by NEN Life Sciences Products, Boston, MA. PBDE congener, 2,2',4,4'-tetrabromodiphenyl ether (PBDE 47) was a gift from Great Lakes Chemical Corporation (West Lafayette, IN). PBDE 77 (3,3',4,4'-tetrabromodiphenyl ether) was commercially available from the Biochemical Institute for Environmental Carcinogens, Lurup 4, D-22927 Grosshansdorf, Germany. PBDEs 99 (2,2',4,4',5-pentabromodiphenyl ether), 100 (2,2',4,4'6- pentabromodiphenyl ether), and 153 (2,2',4,4',5,5'-hexabromodiphenyl ether), and PCBs 47 (2,2',4,4'-tetrachlorobiphenyl) and 99 (2,2',4,4',5-pentachlorobiphenyl) were purchased from AccuStandard, Inc, New Haven, CT. All PBDE and PCB congeners were 98–100% pure and dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO in the assay buffer (0.2% v/v) did not significantly affect 3H-phorbol ester binding.
Animals.
Timed pregnant (16 days gestation) Long-Evans hooded rats were obtained from Charles River Laboratory (Raleigh, NC) and housed individually in AAALAC approved animal facilities. Food and water were provided ad libitum. Temperature was maintained at 21 ± 2°C and relative humidity at 50 ± 10% with a 12-h light/dark cycle (700–1900 h). Beginning on gestational day 22, rats were checked twice daily (A.M. and P.M.) for births, and the date when birth was first discovered was assigned postnatal day 0. The litter size for each dam varied between 8 and 14 pups, and litters were left undisturbed until postnatal day 7. All of the experiments were approved in advance by the institutional animal care and use committee of the National Health and Environmental Effects Research Laboratory at U.S. EPA that requires compliance with NIH guidelines.
Cerebellar granule cell culture.
Primary cultures of rat cerebellar granule neurons (CGCs) were prepared from 7- to 8-day-old Long Evans rat pups as outlined by Gallo et al. (1987) with modifications (Kodavanti et al., 1993a,b). Cultures were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS and 30 mM KCl in 12-well plates (Corning Costar), with a plating density of 1.0 x 106 cells/ml. Cytosine arabinoside was added 48 h after plating to prevent the proliferation of nonneuronal cells. Cultures were assayed at 7 days in vitro when they were fully differentiated and exhibiting fasciculation of fibers that interconnect the cells (Kodavanti et al., 1993a). Cell culture made on each day was considered as a single experiment in statistical analysis.
3H-Phorbol ester binding in cerebellar granule cells.
Cerebellar granule cells grown on 12-well culture plates (Costar) were tested at 7 days in culture for 3H-phorbol ester binding as per the method outlined by Vaccarino et al. (1991). Briefly, the monolayers were washed with Locke's buffer (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3, 2.3 mM CaCl2, 5.6 mM D-glucose, 5 mM HEPES, pH 7.4) containing 0.1% fatty-acid-free bovine serum albumin. Following washing, the cells were incubated in Locke's buffer containing 1 nM 4--3H-phorbol 12,13-dibutyrate (3H-PDBu; 0.1 μCi/ml) for 15 min at room temperature with PBDE or PCB congeners (10–50 μM). An equal amount of DMSO was added to controls. After incubation, the medium was aspirated, cells were washed three times with Locke's buffer and suspended with 1 ml of 0.1 M NaOH. An aliquot of this sample (0.7 ml) was added to 9 ml Ultima GoldTM (Packard, Meriden, CT) and the radioactivity was determined using scintillation spectroscopy (Beckman LS6500, Fullerton, CA). A small aliquot (25 μl) was used for protein determination (Bradford, 1976). Nonspecific binding was determined in the presence of 1.6 μM phorbol myristate acetate, which was always <20% and subtracted from all the values. The unit of 3H-PDBu binding was fmol/mg protein/15 min.
14C-PBDE accumulation in cerebellar granule neurons:
After 7 days in culture, the medium (DMEM) was removed from each well containing cerebellar granule neurons, washed twice with 1 ml aliquots of 37°C Locke's buffer, and allowed to equilibrate for 15 min at room temperature. The Locke's buffer was then replaced with 1 ml of 37°C Locke's buffer containing 0.05 μCi of 14C-PBDE congeners (0.67 μM of PBDEs 47, 99, and 153) along with different concentrations of cold PBDEs (0 to 30 μM) and maintained in the 37°C incubator for 15 min to 1 h. After incubation at the respective time periods, a 0.1-ml aliquot of the media was sampled from each well; then, the cells were washed twice with 1 ml cold Locke's buffer, and cells were dissolved in 1 ml NaOH. The entire sample of 1.0 ml was added to 10 ml Ultima GoldTM (Packard, Meriden, CT). The radioactivity in samples from cells, media in the beginning and end of exposure was determined using scintillation spectroscopy (Beckman LS6500, Fullerton, CA). The recovery of radioactivity was 90–95%. The 14C-accumulation of PBDE congeners by cerebellar granule neurons was represented both as percentage and in nanomoles.
Cytotoxicity of cerebellar granule neurons.
Lactic dehydrogenase (LDH) leakage was used as an indicator of cell death in cerebellar granule cells plated and cultured in the same manner as with 3H-phorbol ester binding. As the cell membrane loses integrity, LDH is released, and this activity is measured by the rate of conversion from pyruvate and NADH to lactate and NAD (Loo and Rillema, 1998). After 7 days in culture, the medium (DMEM) was removed from each well, washed twice with 1-ml aliquots of 37°C Locke's buffer, and allowed to equilibrate for 15 min at room temperature. The Locke's buffer was then replaced with 1 ml of 37°C Locke's buffer containing the various test solutions (0 to 100 μM). The cells were maintained in the 37°C incubator when not taking samples. Fifty-μl aliquots were removed from each well at 30, 60, 120, and 240 min after addition of test solutions for determining LDH activity. After collecting the last sample at 240 min, the cells were lysed by adding 50 μl of 5% TritonX-100TM and agitated at room temperature for 20 min. A 50-μl aliquot of lysed cell fraction was also assayed for LDH activity. LDH activity in the lysed fraction plus released activity (samples from medium collected at different time points) represent total LDH in cell culture. LDH leakage was expressed as a percentage of the total LDH capable of leaking into the medium. LDH activity was measured using the Geno Technology, Inc, St. Louis, MO, "CytoscanTM-LDH Cytotoxicity Assay Kit". The data were expressed as percent LDH leakage by test chemicals with chlorpromazine (100 μM) as a positive control.
Statistics.
The data (n = 5–8 experiments, assayed in triplicates) were analyzed by a two-way analysis of variance (ANOVA) with PBDE/PCB as one factor and concentration as the other using SigmaStat software, version 3.1 (SPSS Inc., Chicago, IL). In the case of significant interaction, step-down ANOVAs were used to test for main effects of PBDEs or PCBs. For LDH leakage data, two-way repeated measures ANOVA was performed taking concentration and exposure time as factors. Pair wise comparisons between groups were made using Fisher's LSD test. The accepted level of significance was p < 0.05.
RESULTS
Congener-Specific Effects of PBDEs on PKC Translocation in Cerebellar Granule Neurons
Translocation of PKC from cytosol to the membrane is a key intracellular signaling event and responds to changes in intracellular free calcium levels (Trilivas and Brown, 1989; Vaccarino et al., 1991). This translocation can be successfully measured by determining 3H-PDBu binding. All the tested PBDE congeners increased 3H-PDBu binding, and a significant effect was seen as low as 10 μM (Fig. 2). ANOVA indicated a significant interaction of binding and the concentration of PBDE congeners (F12,120 = 3.786; p < 0.001). A post-hoc test showed that PBDE 47 increased 3H-PDBu binding in a concentration-dependent manner and to a greater extent when compared to other congeners at 30 and 50 μM. The effect of PBDE congeners on 3H-PDBu binding decreased with increasing number of bromines on the molecule (Fig. 2; Table 1). This could be due to low solubility in the buffer or increasing hydrophobicity with increasing number of bromines. In addition, the effects of tetra congeners on 3H-PDBu binding seems to be linear, while the effects were nonlinear for higher bromine congeners. The calculated E50 value (concentration that increases 50% of control activity) for PBDE 47 was 34 ± 2 μM, while it was >50 μM for all other tested congeners (Table 1). PBDE 77, which is a non-ortho-substituted noncoplanar PBDE congener, also increased 3H-PDBu binding in cerebellar granule neurons (Fig. 2).
Comparative Effects of PBDE and PCB Congeners on PKC Translocation
In order to compare the effects between PBDE and PCB congeners, two pairs of congeners with four and five halogens were selected. PBDE 47 and PBDE 99 are the most prevalent congeners in the commercial mixtures as well as in most biological samples (Birnbaum and Staskal, 2004). The results clearly indicate that PBDE 47 increased PKC translocation in a similar way to PCB 47 on a molar basis (Fig. 3). The ANOVA indicated a significant effect of concentration for PBDE 47/PCB 47 (F3,72 = 73.057; p < 0.001), but no significant effect of treatment either for tetra congener pair (F1,72 = 0.0866; p = 0.769). The potency (10 μM), efficacy (72–79% at 50 μM), and E50 values (33–36 μM) were similar for PBDE 47 and PCB 47 (Table 1). A similar observation was also made with penta-halogenated congeners. PBDE 99 increased PKC translocation in a similar way to PCB 99 on a molar basis (Fig. 3). ANOVA indicated a significant effect of concentration for PBDE 99/PCB 99 (F3,56 = 10.764; p < 0.001), but there was no significant effect of treatment for penta congener pair (F1,56 = 0.675; p = 0.415). In addition, PBDE 99 and PCB 99 also have similar potency (10 μM) and efficacy (23–41% at 50 μM) (Table 1).
14C-PBDE Accumulation by Cerebellar Granule Neurons
Cerebellar granule neurons accumulated all three PBDE congeners tested (PBDEs 47, 99, and 153) (Figs. 4–7). When the data were analyzed on a percentage basis, ANOVAs indicated a significant interaction of PBDE congener and concentration at 15-min (F6,36 = 11.025; p < 0.001), 30-min (F6,36 = 10.996; p < 0.001), and 60-min (F6,36 = 12.581; p < 0.001) exposures. At the lowest concentration of PBDEs, about 13–18% of the total exposed concentration was accumulated by these neurons (Figs. 4 and 5). There were distinct differences in the pattern of accumulation among PBDE congeners. The percent 14C-PBDE 47 accumulation was only slightly increased with concentration while percent 14C-PBDEs 99 and 153 accumulation decreased with concentration (Fig. 4). However, the percent accumulation of 14C-PBDE congeners increased with time of exposure at all concentrations, and the accumulation seems to be linear (Fig. 5). ANOVAs indicated a significant effect of exposure time at 0.67 μM (F2,27 = 132.991; p < 0.001), 3.67 μM (F2,27 = 55.898; p < 0.001), 10.67 μM (F2,27 = 49.18; p < 0.001), and 30.67 μM (F2,27 = 85.761; p < 0.001) concentrations. The percent accumulation was much lower for the 30.67 μM PBDE99 and 10.67–30.67 μM PBDE153 than at the lower concentrations. The percent accumulation pattern with PBDE 47 did not vary with concentration (Fig. 5).
When the data was presented on a nanomole accumulation basis, 14C-PBDE 47, but not 14C-PBDEs 99 and 153 accumulation in neurons was linear with concentration (Fig. 6). ANOVAs indicated a significant interaction of PBDE congener and concentration at 15-min (F6,36 = 41.136; p < 0.001), 30-min (F6,36 = 39.118; p < 0.001), and 60-min (F6,36 = 40.475; p < 0.001) exposures. However, the neuronal accumulation of all three 14C-PBDE congeners was linear with exposure time (Fig. 7). ANOVAs indicated a significant effect of exposure time at 0.67 μM (F2,27 = 100.797; p < 0.001), 3.67 μM (F2,27 = 73.443; p < 0.001), 10.67 μM (F2,27 = 51.537; p < 0.001), and 30.67 μM (F2,27 = 87.026; p < 0.001) concentrations.
Correlations between Accumulation of PBDE Congeners and Their Effects on PKC Translocation
The pattern of 14C-PBDE accumulation correlates well with PKC translocation (Fig. 8). Of the three PBDEs tested, PKC translocation was stimulated to the greatest extent with PBDE 47 (Fig. 2), and this congener was also most readily accumulated (Figs. 6–7) by the cerebellar granule neurons. However, the effects of PBDE 99 and 153 on PKC translocation and percent 14C-PBDE accumulation were different when compared to PBDE 47. The dose-response curves for both the stimulation of PKC translocation and 14C-PBDE accumulation for both PBDEs 99 and 153 reached a plateau at 10 μM. Although stimulation of PKC translocation reached a maximum at 10 μM, the accumulation of PBDEs 99 and 153 continued to rise at a much slower rate between 10.67 and 30.67 μM. Since PKC translocation, as measured by 3H-PDBu binding was conducted at 15 min exposure, PKC translocation data was plotted against nmol 14C-PBDE accumulation at 15 min exposure. The analysis indicated a strong correlation for PBDE 47 (r2 = 0.991) as well as for PBDEs 99 (r2 = 0.863) and 153 (r2 = 0.773) (Fig. 8).
Cytotoxicity of PCB 47 or PBDE 47 in Cerebellar Granule Neurons
Chlorpromazine at 100 μM, which was used as a positive control based on our previous reports (Kodavanti et al., 1993a), significantly caused LDH leakage at 4 h of exposure (Table 2). We selected only tetra congeners (PBDE 47 and PCB 47) for cytotoxicity measurements, since these congeners produced greatest effect on PKC signaling (Fig. 3). The ANOVA indicated a significant interaction of exposure time and the concentration for PBDE 47 (F12,45 = 14.464; p < 0.001) and PCB 47 (F12,45 = 66.702; p < 0.001). PCB 47 did not cause a significant LDH leakage up to 30 μM and 120 min exposure. A concentration of 100 μM increased LDH leakage minimally at 120 min of exposure. At the 240 min time point, an increase in LDH leakage was seen at concentrations of 30 μM. (Table 2). Similar to the effects with PCB 47, PBDE 47 also did not cause any significant LDH leakage 50 μM and 120 min exposure. A concentration of 100 μM PBDE 47 increased LDH leakage minimally at 120 min exposure. At the 240 min exposure, a significant increase in LDH leakage was observed at concentrations of 50 μM. (Table 2).
DISCUSSION
Results from the present study demonstrate for the first time that environmentally relevant PBDE congeners increased PKC translocation, which is a critical event in the development of the nervous system and is associated with learning and memory processes (Abeliovich et al., 1993; Kodavanti, 2004), in rat cerebellar granule neurons. In addition, the accumulation of PBDE congeners correlated well with their effects on PKC translocation suggesting a common mode of action for this group of chemicals. Translocation of PKC is a culminating event that responds to a number of intracellular changes, including changes in calcium homeostasis caused by pharmacological agents or environmental chemicals, and is a link between signaling events that occur on cell surface and in the nucleus (Murphy et al., 1987; Trilivas and Brown, 1989; Girard and Kuo, 1990). The observation made with PBDE congeners regarding PKC translocation is consistent with previous studies on commercial PBDE mixtures (Kodavanti and Ward, 2005) and on structurally related chemicals such as PCBs, DDT, and/or polychlorinated diphenyl ethers in neuronal cell cultures (Kodavanti et al., 1995, 1996), human astrocytoma cells (Madia et al., 2004), and human leukemic HL-60 cells (Shin et al., 2002). Recent studies demonstrated the involvement of PKC and calcium homeostasis in the respiratory burst caused by a commercial penta PBDE mixture (Reistad and Mariussen, 2005). A similar effect on PKC was observed with other known developmental neurotoxicants such as alcohol (Hoek and Rubin, 1990), lead, and methylmercury (Haykal-Coates et al., 1998; Nihei et al., 2001) suggesting a key role for PKC signaling pathways in the neurotoxicity of environmental pollutants. In addition, studies using pharmacological agents by Shin et al. (2002) and Kang et al. (2004) clearly indicated that perturbation in calcium homeostasis and activation of PKC are involved in the necrosis of catecholaminergic neurons and apoptosis in human leukemic HL-60 cells by PCB exposure.
We have demonstrated previously that PCBs, known human developmental neurotoxicants and structurally related to PBDEs, perturbed several intracellular signaling pathways including PKC in vitro and in vivo (Kodavanti and Tilson, 1997, 2000; Kodavanti et al., 2000). At comparable doses, PCBs have been reported to cause behavioral changes and cognitive deficits in monkeys (Schantz et al., 1989), rats (Gilbert et al., 2000), and mice (Eriksson and Fredriksson, 1996). PKC translocation and calcium homeostasis are important events in cellular signal transduction; perturbations in these events have been implicated in a variety of physiological, developmental, and pathological processes (Kater and Mills, 1991; Mattson, 1991; Nicotera et al., 1992). A close observation of the structure-activity relationship (SAR) data with more than 50 PCB congeners and analogs revealed that PCB congeners with fewer meta and para chlorine atoms, especially those without para-substitution, and those with ortho-chlorine substitution are the most active PCBs in neuronal preparations (Kodavanti and Tilson, 1997). Studies using other neurochemical endpoints such as brain/PC12 cell dopamine levels (Shain et al., 1991) and ryanodine receptor binding (Schantz et al., 1997) support the hypothesis that PCB-induced neurotoxicity through perturbations in intracellular signaling events is mediated by the noncoplanarity exhibited by ortho- and ortho-, para-substituted congeners.
PBDEs also exist as 209 possible congeners based on the position and number of bromines, however, all these congeners are more noncoplanar than are PCBs. On the other hand, PCBs also have 209 possible congeners where some congeners are more coplanar (approaching about 30 degrees) and some are less coplanar (approaching 90 degrees). Based on our previous SAR results on PCBs about the importance of noncoplanarity (Kodavanti and Tilson, 1997), PBDE congeners were predicted to be active in neurons and our results showed that all the tested congeners increased PKC translocation in cerebellar granule neurons (Fig. 2). Of the congeners studied, PBDE 47 is the most active one and is also found in major quantities in biological and environmental samples. The effect seen with PBDE 47 (E50 = 34 μM) was much greater than that of DE-71 (E50 = >60 μM), which is a pentabrominated diphenyl ether mixture (Kodavanti and Ward, 2005). It is interesting to note that PBDE 77, which is a non-ortho-substituted noncoplanar PBDE congener is active (Fig. 2) while PCB 77, which is a non-ortho-substituted more coplanar congener, was reported to be inactive in cerebellar granule neurons (Kodavanti and Tilson, 1997) suggesting the importance of noncoplanarity of the molecule in the neurotoxicity of this group of pollutants acting via perturbations in intracellular signaling events.
The selected PBDE congeners did not cause cytotoxicity at concentrations where PKC translocation in cerebellar neurons was affected following exposure. Cytotoxicity studies were performed in cerebellar granule neurons after 7 days in vitro, taking LDH leakage as an index. LDH release into the medium has been used as an indicator of cell toxicity (Verity et al., 1990; Sasaki et al., 1992; Kodavanti et al., 1993a,b). Although PBDE 47 and PCB 47 increased LDH leakage indicating cytotoxicity, this response required longer exposures and higher concentrations (Table 2) than the effects seen on PKC translocation suggesting that the effects on PKC translocation precedes the effects on cytotoxicity.
The potency and efficacy between PCB and PBDE congeners were compared with respect to their effects on PKC translocation. Comparisons among tetra- and penta-halogenated congeners indicated that PBDEs 47 and 99 seem to have similar potency and efficacy on a molar basis as PCBs 47 and 99, respectively. This observation is in agreement with our previous studies on commercial penta-brominated mixtures (e.g., DE-71) regarding other signaling events such as 3H-arachidonic acid release (Kodavanti and Derr-Yellin, 2002) and mitochondrial calcium buffering (Kodavanti and Ward, 2005). It is interesting to note that changes in swim-maze performance, spontaneous behavior, and habituation capability with increasing age seen with PBDE exposure were similar to those seen with PCB exposure on a molar basis (Eriksson and Fredriksson, 1996; Eriksson et al., 2001; Viberg et al., 2003b). Among PBDE congener effects in mice, PBDEs 47 and 99 have similar potency on swim-maze performance while PBDE 99 is more potent than PBDE 47 on spontaneous behavior (Eriksson et al., 2001). In addition, there were few reports demonstrating the accumulation of PBDE congeners in mouse brain (Eriksson et al., 2002; Viberg et al., 2003a; Darnerud and Risberg, 2005). These mechanistic and behavioral studies suggest that these two groups of chemicals may be exerting neurotoxic effects through a common mode of action by altering intracellular signaling.
The pattern of PBDE accumulation correlates well with PKC translocation. The accumulation of PBDE congeners increased with time of exposure and at the lowest concentration, about 13–18% of the total dose was accumulated by the cerebellar granule neurons (Figs. 4 and 5). The percent accumulation was much lower for PBDEs 99 and 153 compared to PBDE 47, which may be due to low solubility or increased lipophilicity with increased number of bromines in these congeners. This observation is consistent with previous reports on PBDE-47 accumulation in rat neocortical cells (Mundy et al., 2004). Of the three PBDEs tested, PKC translocation was stimulated to the greatest extent with PBDE 47 and this congener was also most readily accumulated by the cerebellar granule neurons. 14C-PBDE 47 accumulation was linear with concentration and time, both on a percentage or nmole basis (Figs. 4–7). PBDE 47-induced increases in PKC translocation were also linear with concentration (Fig. 3A), suggesting a good correlation between this biological effect and uptake of this congener. The accumulation pattern of PBDEs observed in cerebellar neurons follows the absorption pattern of PBDEs in fish model, where PBDE 47 showed the highest uptake efficiency, and increased bromination of PBDEs resulted in less absorption of these chemicals (Burreau et al., 1997). When PKC translocation is plotted against nmol accumulation, a strong correlation (r2 = 0.991) was found for PBDE 47 (Fig. 8). The effects of PBDE 99 and 153 were different when compared to PBDE 47. The dose-response curves for both the stimulation of PKC translocation and accumulation for both PBDEs 99 and 153 reached plateaus at 10 μM. Although stimulation of PKC translocation reached a maximum at 10 μM, the accumulation of these congeners continued to rise at a much slower rate between 10.69 and 30.69 μM. Although some minor differences exist among the selected PBDE congeners, current results support the hypothesis that PKC translocation is a critical neuronal effect for PBDE congeners, as observed for PCBs.
In summary, PBDEs increased the PKC translocation as do other organohalogens and known neurotoxicants. Some PBDE congeners have similar potency and efficacy as PCBs. PBDE accumulation correlated well with PKC translocation, suggesting a common mode of action for this group of chemicals. In addition, changes seen in some neurochemical endpoints seem to correlate with neurobehavioral endpoints when compared on a molar basis between PCBs and PBDEs. PBDEs are ubiquitous, persistent, and coexist with PCBs in fish (Manchester-Neesvig et al., 2001), human blood, and breast milk samples (Schecter et al., 2003), and the levels of PBDEs are rapidly rising in North Americans (Betts, 2002). PBDE levels found in several biological and environmental samples from the United States were several folds higher than the levels found in European countries (Hale et al., 2003; Hites, 2004; Petreas et al., 2003). Few reports indicate more than additive interaction between PBDEs and PCBs. As seen individually, combination of PBDE and PCB also resulted in impaired spontaneous motor behavior, which worsened with age. In addition, the combination led to the same effect as a 10x higher dose of either chemical alone (Eriksson et al., 2003). Considering the structural similarity of PBDEs with PCBs, coexistence of these two groups of persistent chemicals in the environment, and the known health effects of PCBs in humans and animals, these two groups of chemicals could conceivably work through the same or similar mechanism(s) to cause developmental neurotoxicity. Since these chemicals exist together in almost all the biological samples, understanding the interactions between these chemicals is very important in terms of their risk assessment.
NOTES
The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
ACKNOWLEDGMENTS
The authors thank Great Lakes Chemical Corporation for providing a sample of PBDE 47 and Dr. Tom Burka of NIEHS for providing us 14C-labeled PBDE congeners. Ms. Beth Padnos and Ms. Theresa Freudenrich are acknowledged for their excellent technical assistance and Drs. Hugh Tilson of U.S. EPA, Tom McDonald of Arvesta Corporation, and Maggie Curras-Collazo of University of California at Riverside for their helpful comments on an earlier version of this manuscript. Preliminary findings were presented at the 24th Symposium on Halogenated Environmental Organic Pollutants and POPs (Dioxin2004) meeting in Berlin, Germany (September 5–10, 2004).
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