Hydroxylation increases the neurotoxic potential of BDE-47 to affect exocytosis and calcium homeostasis in PC12 cells

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+]) 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 PM) 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 mu M). An additional late increase in [Ca2+](i) was often observed at >= 1 mu M 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 mu M. Using the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenythydrazone (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.