Cellular and Molecular Toxicology Branch, Neurotoxicology Division, NHEERL, ORD, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
1 To whom correspondence should be addressed at Cellular and Molecular Toxicology Branch, Neurotoxicology Division, B 105-06, NHEERL/ORD, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711. Fax: (919) 541-4849. E-mail: kodavanti.prasada{at}epa.gov.
Received February 17, 2005; accepted March 4, 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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Previously, we demonstrated that PCBs, which are known to cause neurotoxic effects in humans and animals (Longnecker et al., 2003; Walkowiak et al., 2001
), 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
). Since these signaling pathways have been associated with learning and memory and neural development (Abeliovich et al., 1993
; Chen et al., 1997
; Girard and Kuo, 1990
; Kater and Mills, 1991
; Murphy et al., 1987
), perturbed signal transduction mechanisms have been proposed as one of the modes of action for PCB neurotoxicity (Kodavanti, 2004
). Our recent findings suggest that PBDE mixtures stimulated 3H-AA release in a concentration-dependent manner in neuronal cultures, and the mechanism by which PBDEs stimulate 3H-AA release seems to be similar to that of PCBs (Kodavanti and Derr-Yellin, 2002
). When PBDE effects were compared with PCB effects, DE-71 (mostly penta-halogenated PBDE mixture) was less efficacious than Aroclor 1254 (penta-halogenated PCB mixture) on a weight basis, but was comparable on a molar basis (Kodavanti and Derr-Yellin, 2002
). Madia et al. (2004)
reported that pentabrominated PBDE congener, PBDE-99, caused translocation of three PKC isoforms (
,
, and
) in human astrocytoma cells. Recent studies by Reistad et al. (2005)
showed the involvement of PKC, calcium, and tyrosine kinase in free radical formation in human neutrophil granulocytes following exposure to a brominated flame retardant, tetrabromobisphenol-A. The objectives of the present study were to extend previous observations and (1) investigate whether commercial PBDE mixtures (DE-71 and DE-79) affected intracellular calcium buffering and PKC translocation in a similar way to that of PCBs and other organohalogens and (2) compare the potency and efficacy of these commercial PBDE and PCB mixtures on these intracellular signaling events.
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MATERIALS AND METHODS |
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Animals.
Timed pregnant (16 days gestation) and adult male (90120 days; 300400 g) Long-Evans hooded rats were obtained from Charles River Laboratory (Raleigh, NC). Pregnant rats were housed individually and adult male rats were housed four per cage in AAALAC-approved animal facilities. Food and water were provided ad libitum. Temperature was maintained at 21 ± 2° C and replative humidity at 50 ± 10% with a 12 hr light/dark cycle (7:0019:00 hrs). 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 a postnatal day 0. The litter size for each dam varied between 8 and 14 pups and was left undisturbed until postnatal day 7. Cerebrellar granule cell cultures were prepared from postnatal day 7 rats. 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 USEPA that requires compliance with NIH guidelines.
Cerebellar granule cell culture.
Primary cultures of rat cerebellar granule neurons (CGCs) were prepared from 6- 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% fetal bovine serum, and 30 mM KCl in 12-well plates (Corning Costar), with a plating density of 1.0 x 10 6 cells/ml. Each well contained 1.5 ml of these cells. 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
). Cultures made on each day were considered as an experiment.
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 BSA. 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 the test chemicals (330 µg/ml). An equal amount of DMSO was added to controls. Glutamate (1060 µM) was used as a positive control. 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, which was always <20%, was determined in the presence of 1.6 µM phorbol myristate acetate and subtracted from all the values. The unit of 3H-PDBu binding was fmol/mg protein/15 min.
Isolation of microsomes and mitochondria.
Different brain regions (frontal cortex, cerebellum, and hippocampus) were excised rapidly from adult (90120 days; 300400 g) male Long Evans rats. The brain regions were homogenized in 20 ml cold homogenizing buffer containing 250 mM sucrose, 5 mM HEPES, 5 mM KCl, 10 mM dithiothreitol, 1 mM MgCl2; pH 7.05. The isolation of microsomes and mitochondria was done by the method described by Gray and Whittaker (1962) and Dodd et al. (1981)
with slight modifications (Kodavanti et al., 1993a
,b
). The homogenate was centrifuged at 1000 x g at 4°C for 10 min to remove cell debris and nuclei, and the resulting supernatant centrifuged at 9000 x g at 4°C for 20 min. The supernatant was further centrifuged at 105,000 g at 4°C for 1 h, to obtain microsomes, and suspended in homogenizing buffer. The pellet obtained after centrifugation at 9000 x g was suspended in 0.32 M sucrose (10 ml) and layered over 1.2 M sucrose (10 ml). After high-speed centrifugation (105,000 x g for 20 min.; Beckman model L8-70, rotor Ti 70 type), the resulting mitochondrial pellet was resuspended in the homogenizing buffer. Protein content in microsomal and mitochondrial fractions was determined by Lowry's method (Lowry et al., 1951
). Freshly isolated microsomes and mitochondria were used for Ca2+ uptake studies. The protein concentration in the fractions was maintained at 2 mg/ml. The fractions isolated from each rat were considered as one experiment.
Measurement of 45Ca2+ uptake.
With slight modifications (Kodavanti et al., 1993a,b
), uptake of 45Ca was measured by the method described earlier by Moore et al. (1975)
. Briefly, the assay mixture (1.5 ml) contained 30 mM histidine-imidazole buffer (pH 6.8), 100 mM KCl, 5 mM MgCl2, with or without 5 mM sodium azide (for microsomes or mitochondria, respectively), 5 mM ammonium oxalate, microsomal/mitochondrial protein (80120 µg), and 0.1 µCi 45CaCl2 containing 5 µM free Ca2+ in calcium-ethylene glycol-bis (ß-aminoethyl ether) N,N,N'N'- tetraacetic acid (Ca2+ -EGTA) buffered medium. Various concentrations of test chemicals (030 µg/ml in DMSO; final concentration) along with protein were preincubated in a water bath maintained at 37°C for 5 min. The reaction was initiated by the addition of ATP (5 mM final concentration) and carried on for 20 min at 37°C in shaking water bath. The reaction was terminated by the addition of 5 ml 10 mM TrisHCl buffer (pH 7.4), and the samples filtered through 0.45 µm Millipore® filters. The filters were then collected in scintillation vials containing 10 ml of Ultima Gold scintillation fluid, and the amount of radioactivity was measured by liquid scintillation spectroscopy. Nonspecific 45Ca2+ uptake in the absence of ATP was subtracted from the total binding, to get the specific 45Ca2+ uptake, and calculated as pmol/mg protein/min.
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 media (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 50 µg/ml). The cells were maintained in the 37°C incubator when not taking samples, and 50-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% Triton X 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 phorbol binding and calcium uptake data were analyzed by a two-way analysis of variance (ANOVA), with chemical as one factor and concentration as the other, using SigmaStat software, version 3.0 (SPSS Inc., Chicago, IL). In the case of significant interaction, step-down ANOVAs were used to test for main effects of PBDEs or PCBs. Pair-wise comparisons between groups were made using Fisher's LSD test. The accepted level of significance was p < 0.05. E50 values were calculated from the regression line fit to the linear portion of the curve and comparisons between groups were made with student's t-test.
The percent LDH leakage data for DE-71 and DE-79 were analyzed by univariate repeated measures analysis of variance (RANOVA) with concentration and time as factors for each chemical. For Aroclor 1254 effects, the 240-min exposure data were separated from the other time points and analyzed independently due to heterogeneity of the variance. The 240-min data were ranked and analyzed by a completely random analysis of variance analysis. The rest of the Aroclor 1254 data were analyzed by RANOVA. If interactions were significant at p 0.05, then further ANOVA were done for each time point and for each concentration. If one time point was selected, a test was done by ANOVA to determine if percent LDH leakage differed with concentration. If these p-values were
0.05, then a least significant differences test was used to determine which concentrations were different in a pair-wise fashion. A similar ANOVA was used at each concentration to test for differences at each time point. This analysis was preformed using SAS.
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RESULTS |
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Based on IC50 values, Aroclor 1254 seems to have a similar effect on microsomal and mitochondrial 45Ca2+ uptake in all three brain regions (Table 2). However, mitochondrial 45Ca2+ uptake seems to be more sensitive to DE-71 than microsomal 45Ca2+ uptake in all the selected brain regions (Table 2). The mostly octa-BDE mixture, DE-79, had no effect on 45Ca2+ uptake by microsomes and mitochondria even at 30 µg/ml in any of the selected three brain regions (Figs. 3 and 4).
Comparative Effects of PCBs and PBDEs on Intracellular Signaling Processes
As observed before on 3H-arachidonic acid release (Kodavanti and Derr-Yellin, 2002), Aroclor 1254 and DE-71 perturbed PKC translocation and calcium buffering processes in a concentration-dependent manner. The potency and efficacy of Aroclor 1254 and DE-71 seem to vary with the endpoint and brain region (Tables 1 and 2; Figs. 3 and 4). For PKC translocation, the E50 value of Aroclor 1254 (5.73 ± 0.24 µg/ml) was significantly lower than that of DE-71 (>30 µg/ml). Although the potency appeared similar among these mixtures, Aroclor 1254 was more efficacious when compared to DE-71 (Table 1). For microsomal 45Ca2+ uptake, the trend was similar to that of PKC translocation. IC50 values were significantly lower for Aroclor 1254 than DE-71, and Aroclor 1254 was more efficacious in all three brain regions than DE-71 (Tables 1 and 2). For mitochondrial 45Ca2+ uptake, the trend appeared different among these mixtures. IC50 values were significantly lower for Aroclor 1254 than DE-71 (Table 2); however, Aroclor 1254 was equally efficacious in all three brain regions as compared to DE-71 (Table 1).
Since we observed differential effects of these mixtures on 3H-arachidonic acid release when compared on weight basis versus molar basis (Kodavanti and Derr-Yellin, 2002), we did similar comparisons for PKC translocation and calcium buffering. Aroclor 1254 was more efficacious than DE-71 on a weight basis (µg/ml) (Figs. 1, 3, and 4; Tables 1 and 2). When the concentrations (µg/ml) were transformed to a molar basis (µM), based on the average/approximate molecular weights of Aroclor 1254 (MW 327, pentachloro mixture) and DE-71 (MW 565, pentabromo mixture), Aroclor 1254 was still more effective in increasing PKC translocation (Fig. 1) and inhibiting microsomal 45Ca2+ uptake (Fig. 3) when compared to DE-71. The E50 values on PKC translocation and IC50 values on microsomal 45Ca2+ uptake for Aroclor 1254 differed significantly from DE-71 when compared on a weight basis or molar basis (Table 2). Aroclor 1254 and DE-71 were equally efficacious on mitochondrial 45Ca2+ uptake when compared on a weight basis or molar basis (Fig. 4). The IC50 values on mitochondrial 45Ca2+ uptake for Aroclor 1254 differed significantly from DE-71 when compared only on a weight basis and not on a molar basis (Table 2).
Cytotoxicity of PCB and PBDE Mixtures in Cerebellar Granule Neurons
Since LDH release into the medium has been used as an indicator of cell toxicity (Kodavanti et al., 1993a,b
; Sasaki et al., 1992
; Verity et al., 1990
), cytotoxicity studies were performed in cerebellar granule neurons after 7 days in vitro, taking LDH leakage as an index. 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 3). Aroclor 1254 did not cause a significant LDH leakage up to 16 µg/ml and 2-h exposure. A concentration of 33 µg/ml increased LDH leakage minimally at 30 min (p = 0.02) and 120 min (p = 0.02) of exposure. While this trend was apparent at 60-min exposure, the corresponding p-value was 0.06; less than the criteria for significance. At the 240-min time point, an increase in LDH leakage was seen at concentrations of 16 and 33 µg/ml. On the other hand, the RANOVAs of both PBDE mixtures (DE-71 and DE-79), showed an increase in LDH leakage at 240 min across doses, but did not show any significant dose effect within that exposure time (Table 3).
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The results from the present study for the first time demonstrate that a penta-brominated PBDE mixture, DE-71, increased PKC translocation in neurons and inhibited 45Ca2+ uptake by both microsomes and mitochondria in selected brain regions at low concentrations (310 µg/ml). These effects were observed at concentrations and exposure times where no significant cytotoxicity was observed. DE-71 was not cytotoxic even at 50 µg/ml up to 4-h exposure. The octa-brominated PBDE mixture, DE-79, did not affect either PKC translocation or 45Ca2+ uptake and did not cause cytotoxicity. While the PCB mixture was not cytotoxic even at 33 µg/ml up to 2-h exposure, cytotoxicity was observed at 16 µg/ml for 4 h of exposure (Table 3). The PBDE mixture, DE-71, consisted of congeners that do not achieve coplanar conformation, and the results are consistent with previous studies on structurally related chemicals such as PCBs in neuronal cells (Kodavanti et al., 1995
), neutrophils (Olivero and Ganey, 2000
), and human leukemic HL-60 cells (Shin et al., 2002
). A number of known neurotoxicants including triorganotins, lead, and methylmercury have been shown to alter these intracellular signaling pathways, which are critical for nervous system development and associated with learning and memory processes (see book chapter, Kodavanti, 2004
).
The potency and efficacy between PCB and PBDE mixtures were compared with respect to their effects on PKC translocation and calcium buffering. On a weight basis (µg/ml), the PCB mixture (Aroclor 1254) has similar or greater potency (based on lowest concentration with a significant effect) over the PBDE mixture (DE-71) on PKC translocation and calcium buffering. On a weight basis, the efficacy of Aroclor 1254 was consistently greater than DE-71 for PKC translocation and microsomal 45Ca2+ uptake, while the efficacy was similar for mitochondrial 45Ca2+ uptake (Table 1). Based on E50 or IC50 values, Aroclor 1254 was 25 times more potent when compared to DE-71 for the selected neurochemical endpoints on a weight basis (Table 2). This trend remained similar even after the data were transformed and represented on a molar basis (µM) (Figs 1, 3, and 4). Previously, we have reported that the effect of Aroclor 1254 on arachidonic acid release was about two times greater than that of DE71 on a weight basis, but was comparable on a molar basis (Kodavanti and Derr-Yellin, 2002). Comparing the effects on a molar basis is more appropriate in terms of understanding the mode of action for the structurally related persistent organic pollutants. These results suggest that PBDEs affected some of the intracellular signaling events with similar potency to that of PCBs and suggest a common mode of action for these structurally related chemicals. It is interesting to note that changes in spontaneous behavior and habituation capability with increasing age seen with PBDE exposure were similar to those seen with PCB exposure on a molar level (Eriksson and Fredriksson, 1996
; Viberg et al., 2003b
).
In summary, results from the present study indicate that PBDEs affected intracellular signaling events, as do other organohalogens and known neurotoxicants, suggesting a common mode of action for neurotoxic effects associated with exposure to these chemicals. PBDEs have similar potency, but the efficacy seems to be different on the neurochemical endpoints when the effects are compared on a weight basis or on a molar basis. Changes seen on some neurochemical endpoints seem to correlate with neurobehavioral endpoints when compared on a molar basis between PCBs and PBDEs. PBDEs are as ubiquitous as PCBs in human blood and breast milk samples (Noren and Meironite, 2000), and the levels of PBDEs are rapidly rising in North Americans (Rayne et al., 2003
; Schecter et al., 2003
). Considering the structural similarity of PBDEs with PCBs and the known health effects of PCBs, these two groups of chemicals could conceivably work through similar mechanism(s), to cause developmental neurotoxicity. Due to the continued use of PBDEs in consumer products and their bioaccumulative nature, attention must be paid to the potential health risks associated with exposure to PBDEs.
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NOTES |
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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.
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ACKNOWLEDGMENTS |
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