Cellular and Molecular Toxicology Branch, Neurotoxicology Division, MD 74B, NHEERL, ORD, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
Received February 13, 2002; accepted April 22, 2002
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ABSTRACT |
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Key Words: polybrominated diphenyl ethers; brominated flame retardants; arachidonic acid release; polychlorinated biphenyls; phospholipases; neurotoxicity.
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INTRODUCTION |
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Previously, we demonstrated that PCBs, which are structurally similar to PCBs and DDT and are known to cause neurotoxic effects, including changes in learning and memory in rats, perturbed intracellular signaling mechanisms (Kodavanti and Tilson, 2000), including stimulation of [3H]arachidonic acid ([3H]AA) release by a cPLA2/iPLA2-dependent mechanism (Kodavanti and Derr-Yellin, 1999
) in an in vitro neuronal culture model. However, it is not known whether these mechanisms are related to the developmental neurotoxic effects observed in humans or nonhuman primates. Membrane phospholipids of the brain contain high concentrations of AA (arachidonic acid), and AA release is involved in synaptic plasticity, such as long-term potentiation and other cell-signaling systems (Farooqui et al., 1997b
; Katsuki and Okuda, 1995
). AA can be released by activation of phospholipases, predominantly by PLA2. AA itself affects the signaling pathway as a retrograde messenger, causing release of calcium from microsomal and mitochondrial stores (Huang and Chueh, 1996
), alterations in neurotransmitter release and uptake (Roseth et al., 1998
, Cunha and Ribeiro, 1999
), and stimulation of protein kinase C (Luo and Vallano, 1995
). PLA2 activity has been associated with learning and memory, and AA has been identified as a second messenger involved in synaptic plasticity (Wolf et al., 1995
). The objectives of the present study are: to(1) test whether the available commercial PBDE mixtures, DE-71 (penta-mixture) and DE-79 (octa-mixture), have an action on [3H]AA release similar to that of PCBs and other organohalogens; (2) delineate the nature of PLA2 responsible for the release of [3H]AA by PBDEs; (3) and compare the effects of commercial PBDE and PCB mixtures on [3H]AA release. The selected PBDE mixtures DE-71 and DE-79 contain mostly penta- and octa-brominated diphenyl ether congeners, respectively. The composition of different commercial mixtures of PBDEs can be found in Sjodin (2000). The prominent congeners found in most human and biotic samples range from tetra to deca (2,2',4,4'-tetrabromodiphenyl ether, 2,2',4,4',5-pentabromodiphenyl ether, 2,2',4,4',5,5'-hexabromodiphenyl ether, 2,2',3,4,4',5',6-heptabromodiphenyl ether, and decabromodiphenyl ether; Noren and Meironyte, 2000
; Ryan and Patry, 2000
; Sjodin et al., 2001
; She et al., 2002
). DE-71 and DE-79 contain the prominent congeners found in biological samples and are representatives of lightly versus heavily brominated congeners. These two mixtures were tested to compare the effects of penta- versus octa-BDE mixtures.
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MATERIALS AND METHODS |
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Animals.
Timed-pregnant female (16 days of gestation) Long-Evans rats were obtained from Charles River Laboratory (Raleigh, NC) and housed individually in animal facilities approved by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Food and water were provided ad libitum. Temperature was maintained at 21 ± 2°C, relative humidity at 50 ± 10% with a 12-h light/dark cycle.
Cerebellar granule cell culture.
Primary cultures of rat cerebellar granule neurons were prepared from 68-day-old Long-Evans rat pups as outlined by Gallo et al. (1987) with modifications, Kodavanti et al.(1993). Cultures were grown in Dulbeccos modified Eagles medium (DMEM) with 10% fetal bovine serum and 30 mM KCl in 12-well plates (Corning Costar), with a plating density of 1.5 x 106 cells/ml. Cytosine arabinoside was added 48 h after plating to prevent the proliferation of nonneuronal cells. Cultures were assayed for [3H]AA release at 7 days in vitro, when they are fully differentiated and exhibiting fasciculation of fibers that interconnect the cells (Fig. 2).
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Statistics.
All the data (mean ± SEM of 36 preparations, assayed in triplicate) were expressed as a percentage of total cellular radioactivity incorporation per well. The data were analyzed by a 2-way analysis of variance (ANOVA) with SigmaStat software, version 2.03 (SPSS Inc., Chicago, IL). In the case of significant interaction, step-down ANOVAs were used to test for main effects of PBDEs or pharmacological agents. Pair-wise comparisons between groups were made using Fishers LSD test. Since the data represented on a molar basis (Fig. 7C) could not be analyzed by ANOVA, we used the analysis of covariance to determine whether the linear regressions for Aroclor 1254 and DE-71 were parallel. If they were parallel, we then tested to see if the means of Aroclor 1254 and DE-71 were different (SAS, 1989
). The accepted level of significance was p < 0.05.
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RESULTS |
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DISCUSSION |
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Further experiments focused on the mechanism by which PBDE mixture caused [3H]AA release. Liberation of AA from membrane phospholipids mainly occurs by two pathways: through the activation of phospholipase A2 (PLA2) or by activation of phospholipase C (PLC). Although phospholipase D (PLD) is considered an alternative pathway, the involvement of PLD in the generation of AA has not been confirmed in the nervous system (Katsuki and Okuda, 1995). Previous studies from our laboratory indicated that ortho-substituted PCBs (noncoplanar PCBs), which are structurally similar to PBDEs, stimulated [3H]AA release in cerebellar neurons by activation of PLA2, which is both calcium-dependent and -independent (Kodavanti and Derr-Yellin, 1999
). These studies also indicated that the major factor of PCB-stimulated [3H]AA release consists of a calcium-independent mechanism. A similar activation of calcium-dependent and -independent PLA2 has also been demonstrated for PCBs (Brown and Ganey, 1995
; Tithof et al., 1996
) as well as organochlorine pesticides in rat neutrophils (Tithof et al., 2000
). Therefore in the present study, we examined the role of calcium and tested the effects of a PLA2 inhibitor on DE-71-stimulated [3H]AA release. Results from the present study indicated that DE-71-induced [3H]AA release also requires the presence of calcium. In addition, MAFP, which inhibits both Ca2+-dependent and -independent cytosolic phospholipase A2 (cPLA2/iPLA2 (Basavarajappa et al., 1998
; Lio et al., 1996
) attenuated DE-71stimulated release of [3H]AA completely, suggesting the involvement of cPLA2/iPLA2. We have demonstrated that structurally related chemicals such as PCBs increased intracellular free calcium (Kodavanti and Tilson, 1997
), which is dependent upon the presence of extracellular calcium (Mundy et al., 1999
) and have demonstrated that removal of extracellular Ca2+ caused a slight, but significant, decrease in PCB-stimulated [3H]AA release (Kodavanti and Derr-Yellin, 1999
). We have also shown that polychlorinated diphenyl ethers (PCDEs) have similar effects on intracellular second messengers as noncoplanar PCBs (Kodavanti et al., 1996
), demonstrating the role of noncoplanarity. PBDEs are also noncoplanar in nature as PCDEs and noncoplanar PCBs. Both calcium-independent and -dependent cPLA2 are present in the rat cerebellum (Molloy et al., 1998
) and one or both are probably involved in the stimulation of [3H]AA release in rat cerebellar granule cells by PCBs, PBDEs, and other organohalogens.
These results from this study indicate that PBDEs stimulated [3H]AA release by activating the PLA2 pathway in neuronal cells, as do other organohalogen mixtures in neurons and neutrophils. Although the efficacy of PBDEs seems to be lower when compared to PCBs on a weight basis, they are almost equally potent on a molar basis. For example, mostly the pentachlorinated mixture of PCBs (Aroclor 1254) at 10 µg/ml stimulated [3H]AA release during a 20-min incubation by 589735% of control, while a similar mixture of PBDEs (DE-71) stimulated only 275336% of control. These results indicate that PCBs are 2.2 times more potent than PBDEs on a weight basis. However, the approximate average molecular weight of Aroclor 1254 is 1.7 times less than DE-71. Hence, the difference in efficacy between PCBs and PBDEs decreased considerably on a molar basis as compared to weight basis. Additional studies with specific congeners are needed to address this differential efficacy/potency between PBDEs and PCBs. Although there are minor quantitative differences in the effects of PBDEs and PCBs, the effects were qualitatively similar. Since PBDEs are as ubiquitous as PCBs in human tissues (Ryan and Patry, 2000) and the levels of PBDEs are rapidly rising in North Americans (Betts, 2002
), PBDEs might pose a greater health risk in the future. Considering the structural similarity of PBDEs with PCBs (Fig. 1
) and the known health effects of PCBs, these two groups of chemicals may be working together, through the same mechanisms, to cause developmental neurotoxicity. Due to continued use of PBDEs in consumer products and the bioaccumulative nature of the PBDE congeners, toxicological consequence of exposure to PBDEs should be evaluated.
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ACKNOWLEDGMENTS |
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NOTES |
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2 Present address: Cancer Research Institute, Wells Research Center, Room 432, 1044 West Walnut Street, Indianapolis, IN 46202-5254.
This manuscript 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. Some of the data included in this report were presented at the Society of Toxicology annual meeting, March 2001, at San Francisco, CA.
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