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Neurotoxicity of the pentabrominated diphenyl ether mixture, DE-71, and hexabromocyclododecane (HBCD) in rat cerebellar granule cells in vitro

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Abstract

Polybrominated diphenyl ethers (PBDE) and hexabromocyclododecane (HBCD) are compounds used as additive flame retardants in plastics, electronic equipment, and textiles. The aim of the present study was to investigate the in vitro effects of the pentabrominated diphenyl ether mixture, DE-71, and HBCD on cerebellar granule cells (CGC). Both DE-71 and HBCD induced death of CGC in low micromolar concentrations. The NMDA receptor antagonist MK801 (3 μM), and the antioxidant α-tocopherol (50 μM) significantly reduced the cell death. Incubation of the compounds together with the rat liver post-mitochondrial (S9) fraction reduced cell death by 58 and 64% for DE-71 and HBCD, respectively. No ROS formation and no elevation in intracellular calcium were observed. We further demonstrated apoptotic morphology (Hoechst straining) after exposure to low levels of the two brominated flame retardants and signs of DNA laddering were found after DE-71 exposure. However, other hallmarks of apoptosis, like caspase activity, were absent indicating an atypical form of apoptosis induced by DE-71. After intraperitoneal injection of the two compounds both DE-71 and HBCD were found in significant amounts in brain (559 ± 194 and 49 ± 13 μg/kg, respectively) and liver (4,010 ± 2,437 and 1,248 ± 505 μg/kg, respectively) 72 h after injection. Our results indicate that the lower brominated PBDEs have a higher potency of bioaccumulation than HBCD, and that both compounds have a neurotoxic potential in vitro.

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References

  • Ankarcrona M, Dypbukt JM, Bonfoco E, Zhivotovsky B, Orrenius S, Lipton SA, Nicotera P (1995) Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function. Neuron 15(4):961–973

    Article  PubMed  CAS  Google Scholar 

  • Bezvenyuk Z, Salminen A, Solovyan V (2000) Excision of DNA loop domains as a common step in caspase-dependent and -independent types of neuronal cell death. Brain Res Mol Brain Res 81(1–2):191–196

    Article  PubMed  CAS  Google Scholar 

  • Birnbaum LS, Staskal DF (2004) Brominated flame retardants: cause for concern? Environ Health Perspect 112(1):9–17

    Article  PubMed  CAS  Google Scholar 

  • Branchi I, Alleva E, Costa LG (2002) Effects of perinatal exposure to a polybrominated diphenyl ether (PBDE 99) on mouse neurobehavioural development. Neurotoxicology 23(3):375–384

    Article  PubMed  CAS  Google Scholar 

  • Branchi I, Capone F, Vitalone A, Madia F, Santucci D, Alleva E, Costa LG (2005) Early developmental exposure to BDE 99 or Aroclor 1254 affects neurobehavioural profile: interference from the administration route. Neurotoxicology 26(2):183–192

    Article  PubMed  CAS  Google Scholar 

  • Chaudhuri J (2000) Blood brain barrier and infection. Med Sci Monit 6(6):1213–1222

    PubMed  CAS  Google Scholar 

  • Darnerud PO, Risberg S (2005) Tissue localisation of tetra- and pentabromodiphenyl ether congeners (BDE-47, −85 and −99) in perinatal and adult C57BL mice. Chemosphere 11:11

    Google Scholar 

  • Dreiem A, Fonnum F (2004) Thiophene is toxic to cerebellar granule cells in culture after bioactivation by rat liver enzymes. Neurotoxicology 25(6):959–966

    Article  PubMed  CAS  Google Scholar 

  • Dreiem A, Myhre O, Fonnum F (2003) Involvement of the extracellular signal regulated kinase pathway in hydrocarbon-induced reactive oxygen species formation in human neutrophil granulocytes. Toxicol Appl Pharmacol 190(2):102–110

    Article  PubMed  CAS  Google Scholar 

  • Drejer J, Larsson OM, Schousboe A (1983) Characterization of uptake and release processes for d- and l-aspartate in primary cultures of astrocytes and cerebellar granule cells. Neurochem Res 8(2):231–243

    Article  PubMed  CAS  Google Scholar 

  • Eljarrat E, de la Cal A, Raldua D, Duran C, Barcelo D (2004) Occurrence and bioavailability of polybrominated diphenyl ethers and hexabromocyclododecane in sediment and fish from the Cinca River, a tributary of the Ebro River (Spain). Environ Sci Technol 38(9):2603–2608

    Article  PubMed  CAS  Google Scholar 

  • Eriksson P, Talts U (2000) Neonatal exposure to neurotoxic pesticides increases adult susceptibility. Neurotoxicology 21(1–2):37–47

    PubMed  CAS  Google Scholar 

  • Eriksson P, Viberg H, Jakobsson E, Orn U, 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(1):98–103

    Article  PubMed  CAS  Google Scholar 

  • Gallo V, Ciotti M, Coletti A, Aloisi F, Levi G (1982) Selective release of glutamate from cerebellar granule cells differentiating in culture. Proc Natl Acad Sci USA 79:7919–7923

    Article  PubMed  CAS  Google Scholar 

  • Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260(6):3440–3450

    PubMed  CAS  Google Scholar 

  • Hakk H, Larsen G, Klasson-Wehler E (2002) Tissue disposition, excretion and metabolism of 2,2′,4,4′,5-pentabromodiphenyl ether (BDE-99) in the male Sprague–Dawley rat. Xenobiotica 32(5):369–382

    Article  PubMed  CAS  Google Scholar 

  • Hale RC, La Guardia MJ, Harvey EP, Mainor TM, Duff WH, Gaylor MO (2001) Polybrominated diphenyl ether flame retardants in Virginia freshwater fishes (USA). Environ Sci Technol 35(23):4585–4591

    Article  PubMed  CAS  Google Scholar 

  • Halliwell B, Gutteridge JMC (1999) Free radicals in biology and medicine, 3rd edn. Oxford University Press, New York

    Google Scholar 

  • Kingsbury AE, Gallo V, Woodhams PL, Balazs R (1985) Survival, morphology and adhesion properties of cerebellar interneurones cultured in chemically defined and serum-supplemented medium. Brain Res 349(1–2):17–25

    PubMed  CAS  Google Scholar 

  • Klaassen CD (1995) Casarett & Doull’s toxicology: the basic science of poisons. McGraw-Hill companies, Inc, USA

    Google Scholar 

  • Kodavanti PR, Derr-Yellin EC (2002) Differential effects of polybrominated diphenyl ethers and polychlorinated biphenyls on [3H]arachidonic acid release in rat cerebellar granule neurons. Toxicol Sci 68(2):451–457

    Article  PubMed  CAS  Google Scholar 

  • Kodavanti PR, Ward TR (2005) Differential effects of commercial polybrominated diphenyl ether and polychlorinated biphenyl mixtures on intracellular signaling in rat brain in vitro. Toxicol Sci 85(2):952–962

    Article  PubMed  CAS  Google Scholar 

  • Kuriyama SN, Talsness CE, Grote K, Chahoud I (2005) Developmental exposure to low dose PBDE 99: effects on male fertility and neurobehavior in rat offspring. Environ Health Perspect 113(2):149–154

    Article  PubMed  CAS  Google Scholar 

  • Lau C, Thibodeaux J, Ehresman D, Tanaka S, Froehlich J, Butenhoff J (2005) Evaluation of perfluorooctane (PFOS) in the rat brain. Symposium abstract TOX020LAU, Fluoros 2005, an international symposium an fluorinated alkyl organics in the environment. Toronto

  • Law RJ, Kohler M, Heeb NV, Gerecke AC, Schmid P, Voorspoels S, Covaci A, Becher G, Janak K, Thomsen C (2005) Hexabromocyclododecane challenges scientists and regulators. Environ Sci Technol 39(13):281A–287A

    Article  PubMed  CAS  Google Scholar 

  • Law RJ, Allchin CR, de Boer J, Covaci A, Herzke D, Lepom P, Morris S, Tronczynski J, de Wit CA (2006) Levels and trends of brominated flame retardants in the European environment. Chemosphere (in press)

  • Lindberg P, Sellstrom U, Haggberg L, de Wit CA (2004) Higher brominated diphenyl ethers and hexabromocyclododecane found in eggs of peregrine falcons (Falco peregrinus) breeding in Sweden. Environ Sci Technol 38(1):93–96

    Article  PubMed  CAS  Google Scholar 

  • Lipton SA, Nicotera P (1998) Calcium, free radicals and excitotoxins in neuronal apoptosis. Cell Calcium 23(2–3):165–171

    Article  PubMed  CAS  Google Scholar 

  • Lipton SA, Rosenberg PA (1994) Excitatory amino acids as a final common pathway for neurologic disorders. N Engl J Med 330(9):613–622

    Article  PubMed  CAS  Google Scholar 

  • Madia F, Giordano G, Fattori V, Vitalone A, Branchi I, Capone F, Costa LG (2004) Differential in vitro neurotoxicity of the flame retardant PBDE-99 and of the PCB Aroclor 1254 in human astrocytoma cells. Toxicol Lett 154(1–2):11–21

    Article  PubMed  CAS  Google Scholar 

  • Manchester-Neesvig JB, Valters K, Sonzogni WC (2001) Comparison of polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) in Lake Michigan salmonids. Environ Sci Technol 35(6):1072–1077

    Article  PubMed  CAS  Google Scholar 

  • Mariussen E, Fonnum F (2001) The effect of polychlorinated biphenyls on the high affinity uptake of the neurotransmitters, dopamine, serotonin, glutamate and GABA, into rat brain synaptosomes. Toxicology 159(1–2):11–21

    Article  PubMed  CAS  Google Scholar 

  • Mariussen E, Fonnum F (2003) The effect of brominated flame retardants on neurotransmitter uptake into rat brain synaptosomes and vesicles. Neurochem Int 43(4–5):533–542

    Article  PubMed  CAS  Google Scholar 

  • Mariussen E, Fonnum F (2006) Neurochemical targets and behavioral effects of organohalogen compounds: an update. Crit Rev Toxicol (in press)

  • Mariussen E, Myhre O, Reistad T, Fonnum F (2002) The polychlorinated biphenyl mixture aroclor 1254 induces death of rat cerebellar granule cells: the involvement of the N-methyl-d-aspartate receptor and reactive oxygen species. Toxicol Appl Pharmacol 179(3):137–144

    Article  PubMed  CAS  Google Scholar 

  • Maron DM, Ames BN (1983) Revised methods for the Salmonella mutagenicity test. Mutat Res 113(3–4):173–215

    PubMed  CAS  Google Scholar 

  • Morris S, Allchin CR, Zegers BN, Haftka JJ, Boon JP, Belpaire C, Leonards PE, Van Leeuwen SP, De Boer J (2004) Distribution and fate of HBCD and TBBPA brominated flame retardants in North Sea estuaries and aquatic food webs. Environ Sci Technol 38(21):5497–5504

    Article  PubMed  CAS  Google Scholar 

  • Myhre O, Vestad TA, Sagstuen E, Aarnes H, Fonnum F (2000) The effects of aliphatic (n-nonane), naphtenic (1,2, 4-trimethylcyclohexane), and aromatic (1,2,4-trimethylbenzene) hydrocarbons on respiratory burst in human neutrophil granulocytes. Toxicol Appl Pharmacol 167(3):222–230

    Article  PubMed  CAS  Google Scholar 

  • Myhre O, Andersen JM, Aarnes H, Fonnum F (2003) Evaluation of the probes 2′,7′-dichlorofluorescin diacetate, luminol, and lucigenin as indicators of reactive species formation. Biochem Pharmacol 65(10):1575–1582

    Article  PubMed  CAS  Google Scholar 

  • Orn U, Klasson-Wehler E (1998) Metabolism of 2,2′,4,4′-tetrabromodiphenyl ether in rat and mouse. Xenobiotica 28(2):199–211

    Article  PubMed  CAS  Google Scholar 

  • Reistad T, Mariussen E (2005) A commercial mixture of the brominated flame retardant pentabrominated diphenyl ether (DE-71) induces respiratory burst in human neutrophil granulocytes in vitro. Toxicol Sci 87(1):57–65

    Article  PubMed  CAS  Google Scholar 

  • Reistad T, Mariussen E, 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(1):89–100

    Article  PubMed  CAS  Google Scholar 

  • Remberger M, Sternbeck J, Palm A, Kaj L, Stromberg K, Brorstrom-Lunden E (2004) The environmental occurrence of hexabromocyclododecane in Sweden. Chemosphere 54(1):9–21

    Article  PubMed  CAS  Google Scholar 

  • Reynolds IJ, Hastings TG (1995) Glutamate induces the production of reactive oxygen species in cultured forebrain neurons following NMDA receptor activation. J Neurosci 15(5 Pt 1):3318–3327

    PubMed  CAS  Google Scholar 

  • Schousboe A, Meier E, Drejer J, Hertz L (1989) Preparation of cultures of mouse (rat) cerebellar granule cells. In: Shahar A, de Vellis J, Vernadakis A, Haber B (eds) A dissection and tissue culture manual for the nervous system, Alan R. Liss, New York, pp 203–206

    Google Scholar 

  • Strandberg B, Dodder NG, Basu I, Hites RA (2001) Concentrations and spatial variations of polybrominated diphenyl ethers and other organohalogen compounds in Great Lakes air. Environ Sci Technol 35(6):1078–1083

    Article  PubMed  CAS  Google Scholar 

  • Tan Y, Chen CH, Lawrence D, Carpenter DO (2004) Ortho-substituted PCBs kill cells by altering membrane structure. Toxicol Sci 80(1):54–59

    Article  PubMed  CAS  Google Scholar 

  • Thomsen C, Frøshaug M, Broadwell SL, Eggesbø M (2005) Levels of brominated flame retardants in milk from the Norwegian human milk study: HUMIS. Organohal Comp 67:509–512

    Google Scholar 

  • Urano S, Inomori Y, Sugawara T, Kato Y, Kitahara M, Hasegawa Y, Matsuo M, Mukai K (1992) Vitamin E: inhibition of retinol-induced hemolysis and membrane-stabilizing behavior. J Biol Chem 267(26):18365–18370

    PubMed  CAS  Google Scholar 

  • Viberg H, Fredriksson A, 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(1):104–107

    Article  PubMed  CAS  Google Scholar 

  • Viberg H, Fredriksson A, Jakobsson E, Orn U, Eriksson P (2003) Neurobehavioral derangements in adult mice receiving decabrominated diphenyl ether (PBDE 209) during a defined period of neonatal brain development. Toxicol Sci 76(1):112–120

    Article  PubMed  CAS  Google Scholar 

  • de Wit CA (2002) An overview of brominated flame retardants in the environment. Chemosphere 46(5):583–624

    Article  PubMed  Google Scholar 

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Acknowledgement

The authors are indebted to Mr. Hans Gundersen at NILU for the chemical analysis of the BFRs in the liver and brain extracts, Dr. Avi Ring for help with the calcium measurements and Dr. Yngvar Gundersen for proofreading the manuscript. The authors also acknowledge The Norwegian Defence Research Establishment and Norwegian Research Council, under the PROFO program, for financial support. The experiments were performed within the current national and institutional guidelines for animal research.

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  1. Division for Protection, Norwegian Defence Research Establishment, P. O. Box 25, 2027, Kjeller, Norway

    Trine Reistad, Frode Fonnum & Espen Mariussen

  2. Norwegian Institute for Air Research, P. O. Box 100, 2027, Kjeller, Norway

    Espen Mariussen

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  1. Trine Reistad
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  2. Frode Fonnum
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  3. Espen Mariussen
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Correspondence to Trine Reistad.

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Reistad, T., Fonnum, F. & Mariussen, E. Neurotoxicity of the pentabrominated diphenyl ether mixture, DE-71, and hexabromocyclododecane (HBCD) in rat cerebellar granule cells in vitro. Arch Toxicol 80, 785–796 (2006). https://doi.org/10.1007/s00204-006-0099-8

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