Differential Effects of Commercial Polybrominated Diphenyl Ether and Polychlorinated Biphenyl Mixtures on Intracellular Signaling in Rat Bra
http://www.100md.com
《毒物学科学杂志》
Cellular and Molecular Toxicology Branch, Neurotoxicology Division, NHEERL, ORD, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
ABSTRACT
Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants and have been detected in human blood, adipose tissue, and breast milk. Developmental and long-term exposures to these contaminants may pose a human health risk, especially to children. Previously, we demonstrated that polychlorinated biphenyls (PCBs), which are neurotoxic and structurally similar to PBDEs, perturbed intracellular signaling events, including calcium homeostasis and subsequent events such as protein kinase C (PKC), which are critical for the normal function and development of the nervous system. The objective of the present study was to test whether commercial PBDE mixtures (DE-71, a pentabrominated dipheyl ether mixture, and DE-79, a mostly octabromodiphenyl ether mixture) affected intracellular signaling mechanisms in a similar way to that of PCBs and other organohalogens, as an attempt to understand the common mode of action for these persistent chemicals. PKC translocation was studied by determining 3H-phorbol ester (3H-PDBu) binding in rat cerebellar granule cells, and calcium buffering was determined by measuring 45Ca2+ uptake by microsomes and mitochondria isolated from adult male rat brain (frontal cortex, cerebellum, and hippocampus). As seen with PCBs, DE-71 increased PKC translocation and inhibited 45Ca2+ uptake by both microsomes and mitochondria in a concentration-dependent manner. The effect of DE-71 on 45Ca2+ uptake seems to be similar in all three brain regions. Between the two organelles, DE-71 inhibited mitochondrial 45Ca2+ uptake to a greater extent than microsomal 45Ca2+ uptake. DE-79 had no effects on either neurochemical event even at 30 μg/ml. Aroclor 1254 altered both events to a greater extent compared to DE-71 on a weight basis. When the results were compared on a molar basis, Aroclor 1254 altered PKC translocation and microsomal 45CaP2+ uptake to a greater extent than DE-71, however, Aroclor 1254 and DE-71 equally affected mitochondrial 45Ca2+ uptake. These results indicate that PBDEs perturbed intracellular signaling mechanisms in rat brain as do other organohalogen compounds and the efficacy between the commercial PCB and PBDE mixtures seem to vary with different endpoints.
Key Words: polychlorinated biphenyls (PCBs); polybrominated diphenyl ethers (PBDEs); neurotoxicity; intracellular signaling; cytotoxicity; protein kinase C; calcium signaling.
INTRODUCTION
Polybrominated diphenyl ethers (PBDEs) are used as additive flame retardants in electrical equipment, plastics, household textiles, and building materials (Van Esch, 1994). Their global production is in the range of 40 million kilograms annually (Betts, 2002), and they are becoming ubiquitous contaminants because of high production, lipophilic characteristics, and persistence in the environment. PBDEs have similar chemical structure and physicochemical properties to that of other persistent pollutants such as polychlorinated biphenyls (PCBs) and 1,1,1-trichloro,2,2-bis[p-chloropheynyl]-ethane (DDT); rather than containing chlorine, PBDEs contain bromine. PBDEs have been detected in human blood, adipose tissue, and breast milk (Gill et al., 2004; Schecter et al., 2003). Long-term exposure to these contaminants during development may pose a health risk, especially to children (Birnbaum and Staskal, 2004; Hooper and McDonald, 2000). PBDEs have been increasing in the past 20–30 years, while the presence of other persistent organic pollutants, such as PCBs and dioxins, have decreased in environmental and human samples (Noren and Meironite, 2000). In addition, the 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; Mazdai et al., 2003; Petreas et al., 2003; Rayne et al., 2003). In spite of their widespread occurrence in the environment, only limited information is available on the toxicology of PBDEs. Recent studies showed that several PBDE congeners caused aberrations in spontaneous behavior and reduced learning and memory in mice and rats exposed during development (Branchi et al., 2002; 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/B1 mice as in NMRI mice (Viberg et al., 2004), suggesting lack of gender-specific and strain-specific effects of PBDEs. Some of these behavioral effects are reported to be similar to those effects seen after exposure to other structurally related chemicals such as DDT or PCBs (Eriksson, 1997).
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.
MATERIALS AND METHODS
Chemicals.
All radiolabeled chemicals were purchased from Dupont NEN Corporation (Boston, MA). Commercial PBDE mixtures, DE-71 (Lot numbers 7550OK20A and 1550OI18A) and DE-79 (Lot numbers 8525DG01A and 1525DD11A), were a gift from Great Lakes Chemical Corporation (West Lafayette, IN). The compositions of the different lots were reported to contain 58.1% penta-BDE and 24.6% tetra-BDE for DE-71; 30.7% octa-BDE and 45.1% hepta-BDE for DE-79 (Carlson, 1980). PCB mixture (Aroclor 1254; Lot # 124–191) was purchased from AccuStandard (New Haven, CT). The congener composition of Aroclor 1254 has been previously analyzed (Kodavanti et al., 2001). PBDEs and PCBs were dissolved in dimethyl sulfoxide (DMSO), and the final concentration in the assay buffer (0.2 to 0.4% v/v) did not significantly affect any of the assays.
Animals.
Timed pregnant (16 days gestation) and adult male (90–120 days; 300–400 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 (7:00–19:00 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-- 3H-phorbol 12,13-dibutyrate (3H-PDBu; 0.1 μCi/ml) for 15 min at room temperature with the test chemicals (3–30 μg/ml). An equal amount of DMSO was added to controls. Glutamate (10–60 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 (90–120 days; 300–400 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 (80–120 μg), and 0.1 μCi 45CaCl2 containing 5 μM free Ca2+ in calcium-ethylene glycol-bis (-aminoethyl ether) N,N,N'N'- tetraacetic acid (Ca2+ -EGTA) buffered medium. Various concentrations of test chemicals (0–30 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 Tris–HCl 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.
RESULTS
PBDE Effects on PKC Translocation in Cerebellar Granule Cells
PKC translocation is a key intracellular signaling event and responds to perturbations in calcium homeostasis. Translocation of PKC from cytosol to the membrane can be assayed appropriately in cells by measuring 3H-PDBu binding. We selected cerebellar granule neurons to understand this phenomenon, as this cell culture has been well characterized for cell signaling events. Glutamate, which is used as a positive control, significantly increased 3H-PDBu binding (>200% of control) (Fig. 1A insert). ANOVA indicated a significant effect of concentration (F4,14 = 54.355; p < 0.001) for glutamate; a significant effect was observed at 10 μM. Two PBDE mixtures (DE-71 and DE-79) and one PCB mixture (Aroclor 1254), with different congener composition, were tested on 3H-PDBu binding in cerebellar granule neurons. ANOVA indicated a significant interaction of concentration by mixtures (F8,85 = 25.971; p < 0.001). A post hoc test showed that the mostly penta-BDE mixture, DE-71, increased 3H-PDBu binding in a concentration-dependent manner with a significant effect seen at 3–10 μg/ml (Fig. 1A; Table 1). On the other hand, a mostly octa-BDE mixture, DE-79, did not increase 3H-PDBu binding even at 30 μg/ml (Fig. 1A). This lack of effect by DE-79 could be due to its low solubility when compared to DE-71. Aroclor 1254, a penta-PCB mixture, increased 3H-PDBu binding in a concentration-dependent manner and was more efficacious when compared to DE-71 on a weight basis (Table 1). The efficacy of Aroclor 1254 was still higher than DE-71, even when the data were compared on a molar basis (Fig. 1B).
Further experiments were concentrated on comparing the effects of PBDE mixtures with different lot numbers, since our previous work with PCBs clearly showed differential effects with lot numbers on PKC translocation and calcium buffering (Kodavanti et al., 2001). Figure 2 shows the effects of PBDE mixtures with different lot numbers on 3H-PDBu binding. ANOVA indicated a significant interaction of concentration by mixtures (F12,60 = 7.588; p < 0.001). A post hoc test showed that DE-71 significantly increased 3H-PDBu binding, and this increase was not different with lot numbers. DE-79 with both lot numbers had no effect, even at 30 μg/ml (Fig. 2). This observation with PBDE mixtures was different from the one observed with PCB mixtures (Kodavanti et al., 2001). One explanation could be that the number of congeners in PCB mixtures is much greater compared to those of PBDE mixtures, which contain limited numbers of congeners.
PBDE Effects on Intracellular Calcium Buffering in Different Rat Brain Regions
Intracellular Ca2+ buffering by mitochondria and microsomes plays a crucial role in maintaining normal calcium homeostasis in cells. The uptake of 45Ca2+ by microsomes and mitochondria was determined to understand the effects of PBDEs on intracellular Ca2+ buffering mechanisms. Adult brain regions were utilized for obtaining sufficient quantities of mitochondria and microsomes for the assays. As seen previously (Sharma et al., 2000), Aroclor 1254 inhibited both microsomal and mitochondrial 45Ca2+ uptake in a concentration-dependent manner in all three selected brain regions (Figs. 3 and 4). ANOVA indicated a significant interaction of concentration by mixtures for microsomal 45Ca2+ uptake in frontal cortex (F10,48 = 19.366; p < 0.001), cerebellum (F10,48 = 20.186; p < 0.001), and hippocampus (F10,48 = 5.147; p < 0.001). The lowest concentration where a significant effect was observed varied from 3 to 10 μg/ml. Aroclor 1254 inhibited microsomal 45Ca2+ uptake to a similar extent in the selected three brain regions (Table 1), with IC50 values ranging from 2.42 to 3.14 μg/ml (Table 2). Although the IC50 values of DE-71 were similar for the three selected brain regions (Table 2), microsomal 45Ca2+ uptake in frontal cortex seems to be more sensitive than that of cerebellum and hippocampus (Table 1).
ANOVA also indicated a significant interaction of concentration by mixtures for mitochondrial 45Ca2+ uptake in frontal cortex (F10,36 = 5.566; p < 0.001), cerebellum (F10,36 = 3.390; p < 0.003), and hippocampus (F10,36 = 5.973; p < 0.001). The lowest concentration where a significant effect was observed varied from 1 to 3 μg/ml. DE-71 inhibited mitochondrial 45Ca2+ uptake to a similar extent in the selected three brain regions (Table 1), with IC50 values ranging from 4.24 to 6.80 μg/ml (Table 2). Although the IC50 values of Aroclor 1254 were similar (1.29 to 2.59 μg/ml) for the three selected brain regions (Table 2), mitochondrial 45Ca2+ uptake in hippocampus seems to be more sensitive than that of frontal cortex and cerebellum (Table 1).
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).
DISCUSSION
The objective of the present study was to gain further understanding of structural requirements of persistent organic pollutants to cause neurotoxicity and compare the potency and efficacy among structurally related chemicals (e.g., PCBs and PBDEs) on neurochemical end points, such as PKC translocation as determined by 3H-PDBu binding in cerebellar granule neurons and Ca2+ sequestration as determined by 45Ca2+ uptake by microsomes and mitochondria isolated from adult rat brain regions. The underlying molecular mechanisms of the adverse health effects of PCBs have been associated with perturbations in intracellular signaling mechanisms including Ca2+ homeostasis and translocation of PKC (Kodavanti, 2004). Recently, Kang et al. (2004) reported that PCBs perturbed intracellular calcium homeostasis and caused cell death of catecholaminergic cells, while blockers of endoplasmic reticulum calcium release inhibited intracellular calcium elevations and prevented cell death, suggesting the role of calcium signaling in PCB-induced neurotoxicity. PKC translocation and calcium signaling are important events in the cellular signal transduction; perturbations in these events have been implicated in a variety of physiological, developmental, and pathological processes (Felipo et al., 1993; Kater and Mills, 1991; Mattson, 1991; Nicotera et al., 1992). The effects of PCBs on both Ca2+ homeostasis and translocation of PKC have been extensively studied. Structure–activity relationships (SARs) for both measures suggest that PCB congeners with low lateral substitution, especially without para-substitution or lateral content in the presence of ortho-substitution, is the most important structural requirement for the in vitro activity of PCB congeners in neuronal preparations (Kodavanti et al., 1995, 1996). Studies using other endpoints such as brain/PC12 cell dopamine levels (Shain et al., 1991) and ryanodine receptor binding (Schantz et al., 1997) support the observation that the neurotoxicity of PCBs is mediated by noncoplanarity exhibited by ortho- and ortho-, para-substituted congeners.
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 (3–10 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 2–5 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.
NOTES
Preliminary findings were presented at the Third International Workshop on Brominated Flame Retardants in the Environment (BFR2004) meeting in Toronto, Canada (June 6–9, 2004).
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 Lake Chemical Corporation for providing a sample of PBDE mixtures and Mr. Jerry Highfill for statistical analysis of LDH data. Ms. Ping Zhang, Ms. Theresa Freudenrich, and Ms. Beth Padnos are acknowledged for their excellent technical assistance, and Dr. Margarita Curras-Collazo, University of California, Rivierside, CA, Ms. Janet J. Diliberto, and Dr. Karl Jensen of USEPA for their helpful comments on an earlier version of this manuscript.
REFERENCES
Abeliovich, A., Paylor, R., Chen, C., Kim, J. J., Wehner, J. M., and Tonegawa, S. (1993). PKC-gamma mutant mice exhibit mild deficit in spatial and contextual learning. Cell 75, 1263–1271.
Betts, K. S. (2002). Rapidly rising PBDE levels in North America. Environ. Sci. Technol. 36, 50–52A.
Birnbaum, L. S., and Staskal, D. F. (2004). Brominated flame retardants: Cause for concern Environ. Health Perspect. 112, 9–17.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.
Branchi, I., Alleva, E., and Costa, L. G. (2002). Effects of perinatal exposure to a polybrominated diphenyl ether (PBDE 99) on mouse neurobehavioural development. Neurotoxicology 23, 375–384.
Carlson, G. P. (1980). Induction of xenobiotic metabolism in rats by short-term administration of brominated diphenyl ethers. Toxicol. Lett. 5, 19–25.
Chen, S. J., Sweatt, J. D., and Klann, E. (1997). Enhanced phosphorylation of the postsynaptic protein kinase C substrate RC3/neurogranin during long-term potentiation. Brain Res. 749, 181–187.
Dodd, P. R., Hardy, J. A., Oakley, A. E., Edwardson, J. A., Perry, E. K., Delaunoy, J. P. (1981). A rapid method for preparing synaptosomes: Comparison with alternative procedures. Brain Res. 226, 107–118.
Eriksson, P. (1997). Developmental neurotoxicity of environmental agents in the neonate. Neurotoxicology 18, 719–726.
Eriksson, P., and Fredriksson, A. (1996). Developmental neurotoxicity of four ortho-substituted polychlorinated biphenyls in the neonatal mouse. Environ. Toxicol. Pharmacol. 1, 155–165.
Eriksson, P., Jakobsson, E., and Fredriksson, A. (2001). Brominated flame retardants: A novel class of developmental neurotoxicants in our environment Environ. Health Perspect. 109, 903–908.
Eriksson, P., Viberg, H., Jakobsson, E., Orn, U., and Fredriksson A. (2002). A brominated flame retardant, 2,2',4,4',5-pentabromodiphenyl ether: Uptake, retention, and induction of neurobehavioral alterations in mice during a critical phase of neonatal brain development. Toxicol. Sci. 67, 98–103.
Felipo, V., Minana, M.-D., and Grisolia, S. (1993). Inhibitors of protein kinase C prevent the toxicity of glutamate in primary neuronal cultures. Brain Res. 604, 192–196.
Gallo, V., Kingsbury, A., Balazs, R., and Jergensen, O. S. (1987). The role of depolarization in the survival and differentiation of cerebellar granule cells in culture. J. Neurosci. 7, 2203–2213.
Gill, U., Chu I., Ryan, J. J., and Feeley, M. (2004). Polybrominated diphenyl ethers: Human tissue levels and toxicology. Rev. Environ. Contam. Toxicol. 182, 55–96.
Girard, P. R., and Kuo, J. F. (1990). Protein kinase C and its 80-kilodalton substrate protein in neuroblastoma cell neurite outgrowth. J. Neurochem. 54, 300–306.
Gray, E. G., and Whittaker, V. P. (1962). The isolation of nerve endings from brain: An electron microscope study of cell fragments derived by homogenization and centrifugation. J. Anat. (Lond.) 96, 79–88.
Hale, R. C., Alaee, M., Manchester-Neesvig, J. B., Stapleton, H. M., and Ikonomou, M. G. (2003). Polybrominated diphenyl ether flame retardants in the North American environment. Environ. Intl. 29, 771–779.
Hooper, K., and McDonald, T. A. (2000). The PBDEs: An emerging environmental challenge and another reason for breast-milk monitoring programs. Environ. Health Perspect. 108, 387–392.
Kang, J.-H., Park, I.-S., Oh, W.-Y., Lim, H.-K., Wang, S.-Y., Lee S. Y., Choi, K. H., Kim, J.-I., Jung, S.-Y., Suh, C. K., et al. (2004). Inhibition of Aroclor 1254-induced depletion of stored calcium prevents the cell death in catecholaminergic cells. Toxicology 200, 93–101.
Kater, S. B., and Mills, L. R. (1991). Regulation of growth cone behavior by calcium. J. Neurosci. 11, 891–899.
Kodavanti, P. R. S. (2004). Intracellular signaling and developmental neurotoxicity. In Molecular Neurotoxicology: Environmental Agents and Transcription-Transduction Coupling (N. H. Zawia, Ed.), pp. 151–182. CRC Press, Boca Raton, FL.
Kodavanti, P. R. S., and Derr-Yellin, E. C. (2002). Differential effects of polybrominated diphenyl ethers and polychlorinated biphenyls on [3H]arachidonic acid release in rat cerebellar granule neurons. Toxicol. Sci. 68, 451–457.
Kodavanti, P. R. S., Derr-Yellin, E. C., Mundy, W. R., Shafer, T. J., Herr, D. W., Barone, S., Jr., Choksi, N. Y., MacPhail, R. C., and Tilson, H. A. (1998). Repeated exposure of adult rats to Aroclor 1254 causes brain region-specific changes in intracellular Ca2+ buffering and protein kinase C activity in the absence of changes in tyrosine hydroxylase. Toxicol. Appl. Pharmacol. 153, 186–198.
Kodavanti, P. R. S., Kannan, N., Yamashita, N., Derr-Yellin, E. C., Ward, T. R., Burgin, D. E., Tilson, H. A., and Birnbaum, L. S. (2001). Differential effects of two lots of Aroclor 1254: Congener-specific analysis and neurochemical end points. Environ. Health Perspect. 109, 1153–1161.
Kodavanti, P. R. S., Mundy, W. R., Derr-Yellin, E. C., and Tilson, H. A. (2000). Developmental exposure to Aroclor 1254 alters calcium buffering and protein kinase C activity in the brain. Toxicol. Sci. 54 (suppl.), 76–77.
Kodavanti, P. R. S., Mundy, W. R., Tilson, H. A., and Harry, G. J. (1993a). Effects of selected neuroactive chemicals on calcium transporting systems in rat cerebellum and on survival of cerebellar granule cells. Fundam. Appl. Toxicol. 21, 308–316.
Kodavanti, P. R. S., Shin, D., Tilson, H. A., and Harry, G. J. (1993b). Comparative effects of two polychlorinated biphenyl congeners on Ca2+-homeostasis in rat cerebellar granule cells. Toxicol. Appl. Pharmacol. 123, 97–106.
Kodavanti, P. R. S., and Tilson, H. A. (2000). Neurochemical effects of environmental chemicals: In vitro and in vivo correlations on second messenger pathways. Ann. N. Y. Acad. Sci. 919, 97–105.
Kodavanti, P. R. S., Ward, T. R., McKinney, J. D., and Tilson, H. A. (1995). Increased [3H]phorbol ester binding in rat cerebellar granule cells by polychlorinated biphenyl mixtures and congeners: Structure-activity relationships. Toxicol. Appl. Pharmacol. 130, 140–148.
Kodavanti, P. R. S., Ward, T. R., McKinney, J. D., and Tilson, H. A. (1996). Inhibition of microsomal and mitochondrial Ca2+ sequestration in rat cerebellum by polychlorinated biphenyl mixtures and congeners: Structure-activity relationships. Arch. Toxicol. 70, 150–157.
Longnecker, M. P., Wolff, M. S., Gladen, B. C., Brock, J. W., Grandjean, P., Jacobson, J. L., Korrick, S. A., Rogan, W. J., Weisglas-Kuperus, N., Hertz-Picciotto, I., et al. (2003). Comparison of polychlorinated biphenyl levels across studies of human neurodevelopment. Environ. Health Perspect. 111, 65–70.
Loo, D. T., and Rillema, J. R. (1998). Measurement of cell death. Methods Cell Biol. 57, 251–264.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with folin-phenol reagent. J. Biol. Chem. 193, 265–275.
Madia, F., Giordano, G., Fattori, V., Vitalone, A., Branchi, I., Capone, F., and Costa, L. G. (2004). Differential in vitro neurotoxicity of the flame retardant PBDE-99 and of the PCB Aroclor 1254 in human astrocytoma cells. Toxicol. Lett. 154, 11–21.
Mattson, M. P. (1991). Evidence for the involvement of protein kinase C in neurodegenerative changes in cultured human cortical neurons. Exp. Neurol. 112, 95–103.
Mazdai, A., Dodder, N. G., Abernathy, M. P., Hites, R. A., and Bigsby, R. M. (2003). Polybrominated diphenyl ethers in maternal and fetal blood samples. Environ. Health Perspect. 111, 1249–1252.
Moore, L., Chen, T., Knapp, H. R, Jr., and Landon, E. L. (1975). Energy dependent calcium sequestration activity in rat liver microsomes. J. Biol. Chem. 250, 4562–4568.
Murphy, S., McCabe, N., Morrow, C., and Pearce, B. (1987). Phorbol ester stimulates proliferation of astrocytes in primary cultures. Brain Res. 428, 133–135.
Nicotera, P., Bellomo, G., and Orrenius, S. (1992). Calcium-mediated mechanisms in chemically induced cell death. Ann. Rev. Pharmacol. Toxicol. 32, 449–70.
Noren, K., and Meironyte, D., (2000). Certain organochlorine and organobromine contaminants in Swedish human milk in perspective of past 20–30 years. Chemosphere 40, 1111–23.
Olivero, J., and Ganey, P. E. (2000). Role of protein phosphorylation in activation of phospholipase A2 by the polychlorinated biphenyl mixture, Aroclor 1242. Toxicol. Appl. Pharmacol. 163, 9–16.
Petreas, M., She, J., Brown, F. R., Winkler, J., Windham, G., Rogers, E., Zhao, G., Bhatia, R., and Charles, M. J. (2003). High body burdens of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) in California women. Environ. Health Perspect. 111, 1175–1179.
Rayne, S., Ikonomou, M. G., and Antcliffe, B. (2003). Rapidly increasing polybrominated diphenyl ether concentrations in the Columbia river system from 1992 to 2000. Environ. Sci. Technol. 37, 2847–2854.
Reistad, T., Mariussen, E., and Fonnum, F. (2005). The effect of a brominated flame retardant, tetrabromobisphenol-A, on free radical formation in human neutrophil granulocytes: The involvement of the MAP kinase pathway and protein kinase C. Toxicol. Sci. 83, 89–100.
Sasaki, T., Kawai, K., Saijo-Kurita, K., and Ohno, T. (1992). Detergent cytotoxicity: Simplified assay of cytolysis by measuring LDH activity. Toxicol. In Vitro 6, 451–457.
Schantz, S. L., Seo, B. W., Wong, P. W., and Pessah, I. N. (1997). Long-term effects of developmental exposure to 2,2',3,5',6-pentachlorobiphenyl (PCB 95) on locomotor activity, spatial learning and memory and brain ryanodine binding. Neurotoxicology 18, 457–467.
Schecter, A., Pavuk, M., Papke, O., Ryan, J. J., Birnbaum, L., and Rosen, R. (2003). Polybrominated diphenyl ethers in U.S. mothers' milk. Environ. Health Perspect. 111, 1723–1729.
Shain, W., Bush, B., and Seegal, R. (1991). Neurotoxicity of polychlorinated biphenyls: Structure-activity relationships of individual congeners. Toxicol. Appl. Pharmacol. 111, 33–42.
Sharma, R., Derr-Yellin, E. C., House, D. E., and Kodavanti, P. R. S. (2000). Age-dependent effects of Aroclor 1254 on calcium uptake by subcellular organelles in selected brain regions of rat. Toxicology 156, 13–25.
Shin, H.-J., Gye, M.-C., Chung, K.-H., and Yoo, B.-S. (2002). Activity of protein kinase C modulates the apoptosis induced by polychlorinated biphenyls in human leukemic HL-60 cells. Toxicol. Lett. 135, 25–31.
Vaccarino, F. M., Liljequist, S., and Tallman, J. F. (1991). Modulation of protein kinase C translocation by excitatory and inhibitory amino acids in primary cultures of neurons. J. Neurochem. 57, 391–396.
Van Esch, G. J. (1994). Environmental Health Criteria 162: Brominated Diphenyl Ethers. WHO, Geneva.
Verity, M. A., Sarafian, T. S., Guerra, W., Ettinger, A., and Sharp, J. (1990). Ionic modulation of triethyllead neurotoxicity in cerebellar granule cell culture. NeuroToxicology 11, 415–426.
Viberg, H., Fredriksson, A., and Eriksson, P. (2002). Neonatal exposure to the brominated flame Retardant, 2,2',4,4',5-pentabromodiphenyl ether causes altered susceptibility in the cholinergic transmitter system in the adult mouse. Toxicol. Sci. 67, 104–107.
Viberg, H., Fredriksson, A., and Eriksson, P. (2004). Investigations of strain and/or gender differences in developmental neurotoxic effects of polybrominated diphenyl ethers in mice. Toxicol. Sci. 81, 344–353.
Viberg, H., Fredriksson, A., Jakobsson, E., and Eriksson, P. (2003a). Neurobehavioral derangements in adult mice receiving decabrominated diphenyl ether (PBDE 209) during a defined period of neonatal brain development. Toxicol. Sci. 76, 112–120.
Viberg, H., Fredriksson, A., Jakobsson, E., and Eriksson, P. (2003b). Neonatal exposure to polybrominated diphenyl ether (PBDE-153) disrupts spontaneous behaviour, impairs learning and memory, and decreases hippocampal cholinergic receptors in adult mice. Toxicol. Appl. Pharmacol. 192, 95–106.
Walkowiak J., Wiener, J. A., Fastabend, A., Heinzow, B., Kramer U., Schmidt, E., Steingruber, H. J., Wundram S., and Winneke, G. (2001). Environmental exposure to polychlorinated biphenyls and quality of the home environment: Effects on psychodevelopment in early childhood. Lancet 358, 1602–1607.(Prasada Rao S. Kodavanti )
ABSTRACT
Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants and have been detected in human blood, adipose tissue, and breast milk. Developmental and long-term exposures to these contaminants may pose a human health risk, especially to children. Previously, we demonstrated that polychlorinated biphenyls (PCBs), which are neurotoxic and structurally similar to PBDEs, perturbed intracellular signaling events, including calcium homeostasis and subsequent events such as protein kinase C (PKC), which are critical for the normal function and development of the nervous system. The objective of the present study was to test whether commercial PBDE mixtures (DE-71, a pentabrominated dipheyl ether mixture, and DE-79, a mostly octabromodiphenyl ether mixture) affected intracellular signaling mechanisms in a similar way to that of PCBs and other organohalogens, as an attempt to understand the common mode of action for these persistent chemicals. PKC translocation was studied by determining 3H-phorbol ester (3H-PDBu) binding in rat cerebellar granule cells, and calcium buffering was determined by measuring 45Ca2+ uptake by microsomes and mitochondria isolated from adult male rat brain (frontal cortex, cerebellum, and hippocampus). As seen with PCBs, DE-71 increased PKC translocation and inhibited 45Ca2+ uptake by both microsomes and mitochondria in a concentration-dependent manner. The effect of DE-71 on 45Ca2+ uptake seems to be similar in all three brain regions. Between the two organelles, DE-71 inhibited mitochondrial 45Ca2+ uptake to a greater extent than microsomal 45Ca2+ uptake. DE-79 had no effects on either neurochemical event even at 30 μg/ml. Aroclor 1254 altered both events to a greater extent compared to DE-71 on a weight basis. When the results were compared on a molar basis, Aroclor 1254 altered PKC translocation and microsomal 45CaP2+ uptake to a greater extent than DE-71, however, Aroclor 1254 and DE-71 equally affected mitochondrial 45Ca2+ uptake. These results indicate that PBDEs perturbed intracellular signaling mechanisms in rat brain as do other organohalogen compounds and the efficacy between the commercial PCB and PBDE mixtures seem to vary with different endpoints.
Key Words: polychlorinated biphenyls (PCBs); polybrominated diphenyl ethers (PBDEs); neurotoxicity; intracellular signaling; cytotoxicity; protein kinase C; calcium signaling.
INTRODUCTION
Polybrominated diphenyl ethers (PBDEs) are used as additive flame retardants in electrical equipment, plastics, household textiles, and building materials (Van Esch, 1994). Their global production is in the range of 40 million kilograms annually (Betts, 2002), and they are becoming ubiquitous contaminants because of high production, lipophilic characteristics, and persistence in the environment. PBDEs have similar chemical structure and physicochemical properties to that of other persistent pollutants such as polychlorinated biphenyls (PCBs) and 1,1,1-trichloro,2,2-bis[p-chloropheynyl]-ethane (DDT); rather than containing chlorine, PBDEs contain bromine. PBDEs have been detected in human blood, adipose tissue, and breast milk (Gill et al., 2004; Schecter et al., 2003). Long-term exposure to these contaminants during development may pose a health risk, especially to children (Birnbaum and Staskal, 2004; Hooper and McDonald, 2000). PBDEs have been increasing in the past 20–30 years, while the presence of other persistent organic pollutants, such as PCBs and dioxins, have decreased in environmental and human samples (Noren and Meironite, 2000). In addition, the 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; Mazdai et al., 2003; Petreas et al., 2003; Rayne et al., 2003). In spite of their widespread occurrence in the environment, only limited information is available on the toxicology of PBDEs. Recent studies showed that several PBDE congeners caused aberrations in spontaneous behavior and reduced learning and memory in mice and rats exposed during development (Branchi et al., 2002; 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/B1 mice as in NMRI mice (Viberg et al., 2004), suggesting lack of gender-specific and strain-specific effects of PBDEs. Some of these behavioral effects are reported to be similar to those effects seen after exposure to other structurally related chemicals such as DDT or PCBs (Eriksson, 1997).
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.
MATERIALS AND METHODS
Chemicals.
All radiolabeled chemicals were purchased from Dupont NEN Corporation (Boston, MA). Commercial PBDE mixtures, DE-71 (Lot numbers 7550OK20A and 1550OI18A) and DE-79 (Lot numbers 8525DG01A and 1525DD11A), were a gift from Great Lakes Chemical Corporation (West Lafayette, IN). The compositions of the different lots were reported to contain 58.1% penta-BDE and 24.6% tetra-BDE for DE-71; 30.7% octa-BDE and 45.1% hepta-BDE for DE-79 (Carlson, 1980). PCB mixture (Aroclor 1254; Lot # 124–191) was purchased from AccuStandard (New Haven, CT). The congener composition of Aroclor 1254 has been previously analyzed (Kodavanti et al., 2001). PBDEs and PCBs were dissolved in dimethyl sulfoxide (DMSO), and the final concentration in the assay buffer (0.2 to 0.4% v/v) did not significantly affect any of the assays.
Animals.
Timed pregnant (16 days gestation) and adult male (90–120 days; 300–400 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 (7:00–19:00 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-- 3H-phorbol 12,13-dibutyrate (3H-PDBu; 0.1 μCi/ml) for 15 min at room temperature with the test chemicals (3–30 μg/ml). An equal amount of DMSO was added to controls. Glutamate (10–60 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 (90–120 days; 300–400 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 (80–120 μg), and 0.1 μCi 45CaCl2 containing 5 μM free Ca2+ in calcium-ethylene glycol-bis (-aminoethyl ether) N,N,N'N'- tetraacetic acid (Ca2+ -EGTA) buffered medium. Various concentrations of test chemicals (0–30 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 Tris–HCl 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.
RESULTS
PBDE Effects on PKC Translocation in Cerebellar Granule Cells
PKC translocation is a key intracellular signaling event and responds to perturbations in calcium homeostasis. Translocation of PKC from cytosol to the membrane can be assayed appropriately in cells by measuring 3H-PDBu binding. We selected cerebellar granule neurons to understand this phenomenon, as this cell culture has been well characterized for cell signaling events. Glutamate, which is used as a positive control, significantly increased 3H-PDBu binding (>200% of control) (Fig. 1A insert). ANOVA indicated a significant effect of concentration (F4,14 = 54.355; p < 0.001) for glutamate; a significant effect was observed at 10 μM. Two PBDE mixtures (DE-71 and DE-79) and one PCB mixture (Aroclor 1254), with different congener composition, were tested on 3H-PDBu binding in cerebellar granule neurons. ANOVA indicated a significant interaction of concentration by mixtures (F8,85 = 25.971; p < 0.001). A post hoc test showed that the mostly penta-BDE mixture, DE-71, increased 3H-PDBu binding in a concentration-dependent manner with a significant effect seen at 3–10 μg/ml (Fig. 1A; Table 1). On the other hand, a mostly octa-BDE mixture, DE-79, did not increase 3H-PDBu binding even at 30 μg/ml (Fig. 1A). This lack of effect by DE-79 could be due to its low solubility when compared to DE-71. Aroclor 1254, a penta-PCB mixture, increased 3H-PDBu binding in a concentration-dependent manner and was more efficacious when compared to DE-71 on a weight basis (Table 1). The efficacy of Aroclor 1254 was still higher than DE-71, even when the data were compared on a molar basis (Fig. 1B).
Further experiments were concentrated on comparing the effects of PBDE mixtures with different lot numbers, since our previous work with PCBs clearly showed differential effects with lot numbers on PKC translocation and calcium buffering (Kodavanti et al., 2001). Figure 2 shows the effects of PBDE mixtures with different lot numbers on 3H-PDBu binding. ANOVA indicated a significant interaction of concentration by mixtures (F12,60 = 7.588; p < 0.001). A post hoc test showed that DE-71 significantly increased 3H-PDBu binding, and this increase was not different with lot numbers. DE-79 with both lot numbers had no effect, even at 30 μg/ml (Fig. 2). This observation with PBDE mixtures was different from the one observed with PCB mixtures (Kodavanti et al., 2001). One explanation could be that the number of congeners in PCB mixtures is much greater compared to those of PBDE mixtures, which contain limited numbers of congeners.
PBDE Effects on Intracellular Calcium Buffering in Different Rat Brain Regions
Intracellular Ca2+ buffering by mitochondria and microsomes plays a crucial role in maintaining normal calcium homeostasis in cells. The uptake of 45Ca2+ by microsomes and mitochondria was determined to understand the effects of PBDEs on intracellular Ca2+ buffering mechanisms. Adult brain regions were utilized for obtaining sufficient quantities of mitochondria and microsomes for the assays. As seen previously (Sharma et al., 2000), Aroclor 1254 inhibited both microsomal and mitochondrial 45Ca2+ uptake in a concentration-dependent manner in all three selected brain regions (Figs. 3 and 4). ANOVA indicated a significant interaction of concentration by mixtures for microsomal 45Ca2+ uptake in frontal cortex (F10,48 = 19.366; p < 0.001), cerebellum (F10,48 = 20.186; p < 0.001), and hippocampus (F10,48 = 5.147; p < 0.001). The lowest concentration where a significant effect was observed varied from 3 to 10 μg/ml. Aroclor 1254 inhibited microsomal 45Ca2+ uptake to a similar extent in the selected three brain regions (Table 1), with IC50 values ranging from 2.42 to 3.14 μg/ml (Table 2). Although the IC50 values of DE-71 were similar for the three selected brain regions (Table 2), microsomal 45Ca2+ uptake in frontal cortex seems to be more sensitive than that of cerebellum and hippocampus (Table 1).
ANOVA also indicated a significant interaction of concentration by mixtures for mitochondrial 45Ca2+ uptake in frontal cortex (F10,36 = 5.566; p < 0.001), cerebellum (F10,36 = 3.390; p < 0.003), and hippocampus (F10,36 = 5.973; p < 0.001). The lowest concentration where a significant effect was observed varied from 1 to 3 μg/ml. DE-71 inhibited mitochondrial 45Ca2+ uptake to a similar extent in the selected three brain regions (Table 1), with IC50 values ranging from 4.24 to 6.80 μg/ml (Table 2). Although the IC50 values of Aroclor 1254 were similar (1.29 to 2.59 μg/ml) for the three selected brain regions (Table 2), mitochondrial 45Ca2+ uptake in hippocampus seems to be more sensitive than that of frontal cortex and cerebellum (Table 1).
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).
DISCUSSION
The objective of the present study was to gain further understanding of structural requirements of persistent organic pollutants to cause neurotoxicity and compare the potency and efficacy among structurally related chemicals (e.g., PCBs and PBDEs) on neurochemical end points, such as PKC translocation as determined by 3H-PDBu binding in cerebellar granule neurons and Ca2+ sequestration as determined by 45Ca2+ uptake by microsomes and mitochondria isolated from adult rat brain regions. The underlying molecular mechanisms of the adverse health effects of PCBs have been associated with perturbations in intracellular signaling mechanisms including Ca2+ homeostasis and translocation of PKC (Kodavanti, 2004). Recently, Kang et al. (2004) reported that PCBs perturbed intracellular calcium homeostasis and caused cell death of catecholaminergic cells, while blockers of endoplasmic reticulum calcium release inhibited intracellular calcium elevations and prevented cell death, suggesting the role of calcium signaling in PCB-induced neurotoxicity. PKC translocation and calcium signaling are important events in the cellular signal transduction; perturbations in these events have been implicated in a variety of physiological, developmental, and pathological processes (Felipo et al., 1993; Kater and Mills, 1991; Mattson, 1991; Nicotera et al., 1992). The effects of PCBs on both Ca2+ homeostasis and translocation of PKC have been extensively studied. Structure–activity relationships (SARs) for both measures suggest that PCB congeners with low lateral substitution, especially without para-substitution or lateral content in the presence of ortho-substitution, is the most important structural requirement for the in vitro activity of PCB congeners in neuronal preparations (Kodavanti et al., 1995, 1996). Studies using other endpoints such as brain/PC12 cell dopamine levels (Shain et al., 1991) and ryanodine receptor binding (Schantz et al., 1997) support the observation that the neurotoxicity of PCBs is mediated by noncoplanarity exhibited by ortho- and ortho-, para-substituted congeners.
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 (3–10 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 2–5 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.
NOTES
Preliminary findings were presented at the Third International Workshop on Brominated Flame Retardants in the Environment (BFR2004) meeting in Toronto, Canada (June 6–9, 2004).
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 Lake Chemical Corporation for providing a sample of PBDE mixtures and Mr. Jerry Highfill for statistical analysis of LDH data. Ms. Ping Zhang, Ms. Theresa Freudenrich, and Ms. Beth Padnos are acknowledged for their excellent technical assistance, and Dr. Margarita Curras-Collazo, University of California, Rivierside, CA, Ms. Janet J. Diliberto, and Dr. Karl Jensen of USEPA for their helpful comments on an earlier version of this manuscript.
REFERENCES
Abeliovich, A., Paylor, R., Chen, C., Kim, J. J., Wehner, J. M., and Tonegawa, S. (1993). PKC-gamma mutant mice exhibit mild deficit in spatial and contextual learning. Cell 75, 1263–1271.
Betts, K. S. (2002). Rapidly rising PBDE levels in North America. Environ. Sci. Technol. 36, 50–52A.
Birnbaum, L. S., and Staskal, D. F. (2004). Brominated flame retardants: Cause for concern Environ. Health Perspect. 112, 9–17.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.
Branchi, I., Alleva, E., and Costa, L. G. (2002). Effects of perinatal exposure to a polybrominated diphenyl ether (PBDE 99) on mouse neurobehavioural development. Neurotoxicology 23, 375–384.
Carlson, G. P. (1980). Induction of xenobiotic metabolism in rats by short-term administration of brominated diphenyl ethers. Toxicol. Lett. 5, 19–25.
Chen, S. J., Sweatt, J. D., and Klann, E. (1997). Enhanced phosphorylation of the postsynaptic protein kinase C substrate RC3/neurogranin during long-term potentiation. Brain Res. 749, 181–187.
Dodd, P. R., Hardy, J. A., Oakley, A. E., Edwardson, J. A., Perry, E. K., Delaunoy, J. P. (1981). A rapid method for preparing synaptosomes: Comparison with alternative procedures. Brain Res. 226, 107–118.
Eriksson, P. (1997). Developmental neurotoxicity of environmental agents in the neonate. Neurotoxicology 18, 719–726.
Eriksson, P., and Fredriksson, A. (1996). Developmental neurotoxicity of four ortho-substituted polychlorinated biphenyls in the neonatal mouse. Environ. Toxicol. Pharmacol. 1, 155–165.
Eriksson, P., Jakobsson, E., and Fredriksson, A. (2001). Brominated flame retardants: A novel class of developmental neurotoxicants in our environment Environ. Health Perspect. 109, 903–908.
Eriksson, P., Viberg, H., Jakobsson, E., Orn, U., and Fredriksson A. (2002). A brominated flame retardant, 2,2',4,4',5-pentabromodiphenyl ether: Uptake, retention, and induction of neurobehavioral alterations in mice during a critical phase of neonatal brain development. Toxicol. Sci. 67, 98–103.
Felipo, V., Minana, M.-D., and Grisolia, S. (1993). Inhibitors of protein kinase C prevent the toxicity of glutamate in primary neuronal cultures. Brain Res. 604, 192–196.
Gallo, V., Kingsbury, A., Balazs, R., and Jergensen, O. S. (1987). The role of depolarization in the survival and differentiation of cerebellar granule cells in culture. J. Neurosci. 7, 2203–2213.
Gill, U., Chu I., Ryan, J. J., and Feeley, M. (2004). Polybrominated diphenyl ethers: Human tissue levels and toxicology. Rev. Environ. Contam. Toxicol. 182, 55–96.
Girard, P. R., and Kuo, J. F. (1990). Protein kinase C and its 80-kilodalton substrate protein in neuroblastoma cell neurite outgrowth. J. Neurochem. 54, 300–306.
Gray, E. G., and Whittaker, V. P. (1962). The isolation of nerve endings from brain: An electron microscope study of cell fragments derived by homogenization and centrifugation. J. Anat. (Lond.) 96, 79–88.
Hale, R. C., Alaee, M., Manchester-Neesvig, J. B., Stapleton, H. M., and Ikonomou, M. G. (2003). Polybrominated diphenyl ether flame retardants in the North American environment. Environ. Intl. 29, 771–779.
Hooper, K., and McDonald, T. A. (2000). The PBDEs: An emerging environmental challenge and another reason for breast-milk monitoring programs. Environ. Health Perspect. 108, 387–392.
Kang, J.-H., Park, I.-S., Oh, W.-Y., Lim, H.-K., Wang, S.-Y., Lee S. Y., Choi, K. H., Kim, J.-I., Jung, S.-Y., Suh, C. K., et al. (2004). Inhibition of Aroclor 1254-induced depletion of stored calcium prevents the cell death in catecholaminergic cells. Toxicology 200, 93–101.
Kater, S. B., and Mills, L. R. (1991). Regulation of growth cone behavior by calcium. J. Neurosci. 11, 891–899.
Kodavanti, P. R. S. (2004). Intracellular signaling and developmental neurotoxicity. In Molecular Neurotoxicology: Environmental Agents and Transcription-Transduction Coupling (N. H. Zawia, Ed.), pp. 151–182. CRC Press, Boca Raton, FL.
Kodavanti, P. R. S., and Derr-Yellin, E. C. (2002). Differential effects of polybrominated diphenyl ethers and polychlorinated biphenyls on [3H]arachidonic acid release in rat cerebellar granule neurons. Toxicol. Sci. 68, 451–457.
Kodavanti, P. R. S., Derr-Yellin, E. C., Mundy, W. R., Shafer, T. J., Herr, D. W., Barone, S., Jr., Choksi, N. Y., MacPhail, R. C., and Tilson, H. A. (1998). Repeated exposure of adult rats to Aroclor 1254 causes brain region-specific changes in intracellular Ca2+ buffering and protein kinase C activity in the absence of changes in tyrosine hydroxylase. Toxicol. Appl. Pharmacol. 153, 186–198.
Kodavanti, P. R. S., Kannan, N., Yamashita, N., Derr-Yellin, E. C., Ward, T. R., Burgin, D. E., Tilson, H. A., and Birnbaum, L. S. (2001). Differential effects of two lots of Aroclor 1254: Congener-specific analysis and neurochemical end points. Environ. Health Perspect. 109, 1153–1161.
Kodavanti, P. R. S., Mundy, W. R., Derr-Yellin, E. C., and Tilson, H. A. (2000). Developmental exposure to Aroclor 1254 alters calcium buffering and protein kinase C activity in the brain. Toxicol. Sci. 54 (suppl.), 76–77.
Kodavanti, P. R. S., Mundy, W. R., Tilson, H. A., and Harry, G. J. (1993a). Effects of selected neuroactive chemicals on calcium transporting systems in rat cerebellum and on survival of cerebellar granule cells. Fundam. Appl. Toxicol. 21, 308–316.
Kodavanti, P. R. S., Shin, D., Tilson, H. A., and Harry, G. J. (1993b). Comparative effects of two polychlorinated biphenyl congeners on Ca2+-homeostasis in rat cerebellar granule cells. Toxicol. Appl. Pharmacol. 123, 97–106.
Kodavanti, P. R. S., and Tilson, H. A. (2000). Neurochemical effects of environmental chemicals: In vitro and in vivo correlations on second messenger pathways. Ann. N. Y. Acad. Sci. 919, 97–105.
Kodavanti, P. R. S., Ward, T. R., McKinney, J. D., and Tilson, H. A. (1995). Increased [3H]phorbol ester binding in rat cerebellar granule cells by polychlorinated biphenyl mixtures and congeners: Structure-activity relationships. Toxicol. Appl. Pharmacol. 130, 140–148.
Kodavanti, P. R. S., Ward, T. R., McKinney, J. D., and Tilson, H. A. (1996). Inhibition of microsomal and mitochondrial Ca2+ sequestration in rat cerebellum by polychlorinated biphenyl mixtures and congeners: Structure-activity relationships. Arch. Toxicol. 70, 150–157.
Longnecker, M. P., Wolff, M. S., Gladen, B. C., Brock, J. W., Grandjean, P., Jacobson, J. L., Korrick, S. A., Rogan, W. J., Weisglas-Kuperus, N., Hertz-Picciotto, I., et al. (2003). Comparison of polychlorinated biphenyl levels across studies of human neurodevelopment. Environ. Health Perspect. 111, 65–70.
Loo, D. T., and Rillema, J. R. (1998). Measurement of cell death. Methods Cell Biol. 57, 251–264.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with folin-phenol reagent. J. Biol. Chem. 193, 265–275.
Madia, F., Giordano, G., Fattori, V., Vitalone, A., Branchi, I., Capone, F., and Costa, L. G. (2004). Differential in vitro neurotoxicity of the flame retardant PBDE-99 and of the PCB Aroclor 1254 in human astrocytoma cells. Toxicol. Lett. 154, 11–21.
Mattson, M. P. (1991). Evidence for the involvement of protein kinase C in neurodegenerative changes in cultured human cortical neurons. Exp. Neurol. 112, 95–103.
Mazdai, A., Dodder, N. G., Abernathy, M. P., Hites, R. A., and Bigsby, R. M. (2003). Polybrominated diphenyl ethers in maternal and fetal blood samples. Environ. Health Perspect. 111, 1249–1252.
Moore, L., Chen, T., Knapp, H. R, Jr., and Landon, E. L. (1975). Energy dependent calcium sequestration activity in rat liver microsomes. J. Biol. Chem. 250, 4562–4568.
Murphy, S., McCabe, N., Morrow, C., and Pearce, B. (1987). Phorbol ester stimulates proliferation of astrocytes in primary cultures. Brain Res. 428, 133–135.
Nicotera, P., Bellomo, G., and Orrenius, S. (1992). Calcium-mediated mechanisms in chemically induced cell death. Ann. Rev. Pharmacol. Toxicol. 32, 449–70.
Noren, K., and Meironyte, D., (2000). Certain organochlorine and organobromine contaminants in Swedish human milk in perspective of past 20–30 years. Chemosphere 40, 1111–23.
Olivero, J., and Ganey, P. E. (2000). Role of protein phosphorylation in activation of phospholipase A2 by the polychlorinated biphenyl mixture, Aroclor 1242. Toxicol. Appl. Pharmacol. 163, 9–16.
Petreas, M., She, J., Brown, F. R., Winkler, J., Windham, G., Rogers, E., Zhao, G., Bhatia, R., and Charles, M. J. (2003). High body burdens of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) in California women. Environ. Health Perspect. 111, 1175–1179.
Rayne, S., Ikonomou, M. G., and Antcliffe, B. (2003). Rapidly increasing polybrominated diphenyl ether concentrations in the Columbia river system from 1992 to 2000. Environ. Sci. Technol. 37, 2847–2854.
Reistad, T., Mariussen, E., and Fonnum, F. (2005). The effect of a brominated flame retardant, tetrabromobisphenol-A, on free radical formation in human neutrophil granulocytes: The involvement of the MAP kinase pathway and protein kinase C. Toxicol. Sci. 83, 89–100.
Sasaki, T., Kawai, K., Saijo-Kurita, K., and Ohno, T. (1992). Detergent cytotoxicity: Simplified assay of cytolysis by measuring LDH activity. Toxicol. In Vitro 6, 451–457.
Schantz, S. L., Seo, B. W., Wong, P. W., and Pessah, I. N. (1997). Long-term effects of developmental exposure to 2,2',3,5',6-pentachlorobiphenyl (PCB 95) on locomotor activity, spatial learning and memory and brain ryanodine binding. Neurotoxicology 18, 457–467.
Schecter, A., Pavuk, M., Papke, O., Ryan, J. J., Birnbaum, L., and Rosen, R. (2003). Polybrominated diphenyl ethers in U.S. mothers' milk. Environ. Health Perspect. 111, 1723–1729.
Shain, W., Bush, B., and Seegal, R. (1991). Neurotoxicity of polychlorinated biphenyls: Structure-activity relationships of individual congeners. Toxicol. Appl. Pharmacol. 111, 33–42.
Sharma, R., Derr-Yellin, E. C., House, D. E., and Kodavanti, P. R. S. (2000). Age-dependent effects of Aroclor 1254 on calcium uptake by subcellular organelles in selected brain regions of rat. Toxicology 156, 13–25.
Shin, H.-J., Gye, M.-C., Chung, K.-H., and Yoo, B.-S. (2002). Activity of protein kinase C modulates the apoptosis induced by polychlorinated biphenyls in human leukemic HL-60 cells. Toxicol. Lett. 135, 25–31.
Vaccarino, F. M., Liljequist, S., and Tallman, J. F. (1991). Modulation of protein kinase C translocation by excitatory and inhibitory amino acids in primary cultures of neurons. J. Neurochem. 57, 391–396.
Van Esch, G. J. (1994). Environmental Health Criteria 162: Brominated Diphenyl Ethers. WHO, Geneva.
Verity, M. A., Sarafian, T. S., Guerra, W., Ettinger, A., and Sharp, J. (1990). Ionic modulation of triethyllead neurotoxicity in cerebellar granule cell culture. NeuroToxicology 11, 415–426.
Viberg, H., Fredriksson, A., and Eriksson, P. (2002). Neonatal exposure to the brominated flame Retardant, 2,2',4,4',5-pentabromodiphenyl ether causes altered susceptibility in the cholinergic transmitter system in the adult mouse. Toxicol. Sci. 67, 104–107.
Viberg, H., Fredriksson, A., and Eriksson, P. (2004). Investigations of strain and/or gender differences in developmental neurotoxic effects of polybrominated diphenyl ethers in mice. Toxicol. Sci. 81, 344–353.
Viberg, H., Fredriksson, A., Jakobsson, E., and Eriksson, P. (2003a). Neurobehavioral derangements in adult mice receiving decabrominated diphenyl ether (PBDE 209) during a defined period of neonatal brain development. Toxicol. Sci. 76, 112–120.
Viberg, H., Fredriksson, A., Jakobsson, E., and Eriksson, P. (2003b). Neonatal exposure to polybrominated diphenyl ether (PBDE-153) disrupts spontaneous behaviour, impairs learning and memory, and decreases hippocampal cholinergic receptors in adult mice. Toxicol. Appl. Pharmacol. 192, 95–106.
Walkowiak J., Wiener, J. A., Fastabend, A., Heinzow, B., Kramer U., Schmidt, E., Steingruber, H. J., Wundram S., and Winneke, G. (2001). Environmental exposure to polychlorinated biphenyls and quality of the home environment: Effects on psychodevelopment in early childhood. Lancet 358, 1602–1607.(Prasada Rao S. Kodavanti )