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Milou M.L. Dingemans,¹ Aart de Groot,¹ Regina G.D.M. van Kleef,¹ Åke Bergman,² Martin van den Berg,¹ Henk P.M. Vijverberg,¹ and Remco H.S. Westerink¹

¹Toxicology Division, Institute for Risk Assessment Sciences, Utrecht University, Utrecht, the Netherlands; ²Department of Environmental Chemistry, Wallenberg Laboratory, Stockholm University, Stockholm, Sweden

Abstract
Background: Oxidative metabolism, resulting in the formation of hydroxylated polybrominated diphenyl ether (PBDE) metabolites, may enhance the neurotoxic potential of brominated flame retardants.

Objective: Our objective was to investigate the effects of a hydroxylated metabolite of 2,2´,4,4´-tetrabromodiphenyl ether (BDE-47 ; 6-OH-BDE-47) on changes in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and vesicular catecholamine release in PC12 cells.

Methods: We measured vesicular catecholamine release and [Ca²⁺]_i using amperometry and imaging of the fluorescent Ca²⁺-sensitive dye Fura-2, respectively.

Results: Acute exposure of PC12 cells to 6-OH-BDE-47 (5 µM) induced vesicular catecholamine release. Catecholamine release coincided with a transient increase in [Ca²⁺]_i, which was observed shortly after the onset of exposure to 6-OH-BDE-47 (120 µM) . An additional late increase in [Ca²⁺]_i was often observed at ≥ 1 µM 6-OH-BDE-47. The initial transient increase was absent in cells exposed to the parent compound BDE-47, whereas the late increase was observed only at 20 µM. Using the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) and thapsigargin to empty intracellular Ca²⁺ stores, we found that the initial increase originates from emptying of the endoplasmic reticulum and consequent influx of extracellular Ca²⁺, whereas the late increase originates primarily from mitochondria.

Conclusion: The hydroxylated metabolite 6-OH-BDE-47 is more potent in disturbing Ca²⁺ homeostasis and neurotransmitter release than the parent compound BDE-47. The present findings indicate that bioactivation by oxidative metabolism adds considerably to the neurotoxic potential of PBDEs. Additionally, based on the observed mechanism of action, a cumulative neurotoxic effect of PBDEs and ortho-substituted polychlorinated biphenyls on [Ca²⁺]_i cannot be ruled out.

Key words: bioactivation, brominated flame retardants, calcium, catecholamine, exocytosis, intracellular calcium stores, neurotoxicity, neurotransmitter release, persistent organic pollutants, polybrominated diphenyl ether. Environ Health Perspect 116:637–643 (2008) . doi:10.1289/ehp.11059 available via http://dx.doi.org/ [Online 1 February 2008]

Address correspondence to M.M.L. Dingemans, Toxicology Division, Institute for Risk Assessment Sciences (IRAS) , Utrecht University, PO Box 80.177, NL-3508 TD, Utrecht, the Netherlands. Telephone: 31–30–253 4387. Fax: 31–30–253 5077. E-mail: m.dingemans@uu.nl

This study was supported by the Faculty of Veterinary Medicine, Utrecht University.

The authors declare they have no competing financial interests.

Received 12 November 2007 ; accepted 1 February 2008.