Parallel in vivo and in vitro transcriptomics analysis reveals calcium and zinc signalling in the brain as sensitive targets of HBCD neurotoxicity

V Reffatto,T S Carroll,M Hershfinkel,I Sekler,C Hogstrand,T Ganay,J D Rasinger,A.-K Lundebye

doi:10.1007/s00204-017-2119-2

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https://doi.org/10.1007/s00204-017-2119-2

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Hexabromocyclododecane (HBCD) is a brominated flame retardant (BFR) that accumulates in humans and affects the nervous system. To elucidate the mechanisms of HBCD neurotoxicity, we used transcriptomic profiling in brains of female mice exposed through their diet to HBCD (199 mg/kg body weight per day) for 28 days and compared with those of neuronal N2A and NSC-19 cell lines exposed to 1 or 2 µM HBCD. Similar pathways and functions were affected both in vivo and in vitro, including Ca2+ and Zn2+ signalling, glutamatergic neuron activity, apoptosis, and oxidative stress. Release of cytosolic free Zn2+ by HBCD was confirmed in N2A cells. This Zn2+ release was partially quenched by the antioxidant N-acetyl cysteine indicating that, in accordance with transcriptomic analysis, free radical formation is involved in HBCD toxicity. To investigate the effects of HBCD in excitable cells, we isolated mouse hippocampal neurons and monitored Ca2+ signalling triggered by extracellular glutamate or zinc, which are co-released pre-synaptically to trigger postsynaptic signalling. In control cells application of zinc or glutamate triggered a rapid rise of intracellular [Ca2+]. Treatment of the cultures with 1 µM of HBCD was sufficient to reduce the glutamate-dependent Ca2+ signal by 50%. The effect of HBCD on zinc-dependent Ca2+ signalling was even more pronounced, resulting in the reduction of the Ca2+ signal with 86% inhibition at 1 µM HBCD. Our results show that low concentrations of HBCD affect neural signalling in mouse brain acting through dysregulation of Ca2+ and Zn2+ homeostasis.

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1,2,5,6,9,10-Hexabromocyclododecane (HBCD) is a brominated flame retardant (BFR), mainly used in thermal insulation foams in building and construction, as well as in plastics and textiles
HBCD is a lipophilic compound (American Chemistry Council 2001) that bioaccumulates through food webs, and concentrations are high in oily fish, such as salmon (Chain EPoCitF 2012; Fromme et al 2016)
Caspase-3 activity was measured as a marker of apoptosis in cells exposed for 24 h to HBCD at concentrations ranging from 1 to 8 μM

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1,2,5,6,9,10-Hexabromocyclododecane (HBCD) is a brominated flame retardant (BFR), mainly used in thermal insulation foams in building and construction, as well as in plastics and textiles. The main sources of human exposure to HBCD are contaminated food, breast milk, and inhalation from polluted indoor dust and air (Chain EPoCitF 2012; Fromme et al 2016). HBCD has the potential to cause histological changes in liver, thymus and thyroid (Maranghi et al 2013; van der Ven et al 2006). 28-day oral exposure to HBCD increased thyroid weight and activity of the thyroxine (T4) metabolising enzyme T4-UGT, and decreased total circulating thyroxin in female Wistar rats (van der Ven et al 2006). These effects are accompanied by evidence of suppressed thyroid hormone receptor-mediated transcription (Ibhazehiebo et al 2011). Along with effects on the thyroid hormone system, the most sensitive end-points in animal studies have related to neurodevelopment (Chain EPoCitF 2012)

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