* Neurotoxicology and Experimental Toxicology Divisions, NHEERL, ORD, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, and
The University of Iowa College of Public Health, Iowa City, IA 52242
1 To whom correspondence should be addressed at Cellular and Molecular Toxicology Branch, Neurotoxicology Division, B 10506, NHEERL / ORD, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711. Fax: (919) 541-4849. E-mail: kodavanti.prasada{at}epa.gov.
Received July 26, 2005; accepted August 12, 2005
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ABSTRACT |
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Key Words: polychlorinated biphenyls (PCBs); polybrominated diphenyl ethers (PBDEs); neurotoxicity; intracellular signaling; cytotoxicity; protein kinase C; calcium signaling.
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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 (7001900 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-ß-3H-phorbol 12,13-dibutyrate (3H-PDBu; 0.1 µCi/ml) for 15 min at room temperature with PBDE or PCB congeners (1050 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 9095%. 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 = 58 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.
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RESULTS |
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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. 67) 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).
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DISCUSSION |
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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 1318% 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. 47
). 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.
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NOTES |
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ACKNOWLEDGMENTS |
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