Hydroxylation Increases the Neurotoxic Potential of BDE-47 to Affect Exocytosis and Calcium Homeostasis in PC12 Cells

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′-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.