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′-tetra-bromodiphenyl ether (BDE-47; 6-OH-BDE-47) on changes in the intracellular Ca(2+) concentration ([Ca(2+)](i)) and vesicular catecholamine release in PC12 cells. METHODS: We measured vesicular catecholamine release and [Ca(2+)](i) using amperometry and imaging of the fluorescent Ca(2+)-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 [Ca(2+)](i), which was observed shortly after the onset of exposure to 6-OH-BDE-47 (120 μM). An additional late increase in [Ca(2+)](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 Ca(2+) stores, we found that the initial increase originates from emptying of the endoplasmic reticulum and consequent influx of extracellular Ca(2+), whereas the late increase originates primarily from mitochondria. CONCLUSION: The hydroxylated metabolite 6-OH-BDE-47 is more potent in disturbing Ca(2+) 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 [Ca(2+)](i) cannot be ruled out.