In the present study, we found that neonatal exposure to BDE-47 causes developmental effects consisting of a reduction of PTP and LTP, as well as specific reductions of key post-synaptic proteins involved in glutamate receptor signaling. Presynaptic parameters were not affected ex vivo. In vitro experiments on PC12 cells show an increase in intracellular Ca2+ and spontaneous vesicular release, only at the highest concentration BDE-47 (20 μM). The combined results suggest that presynaptic changes do not directly contribute to the observed defect in synaptic plasticity.
The exposure to BDE-47 took place during a period of rapid brain growth, which in mice takes place during the first 3–4 weeks of life, reaching its peak around PND10 (Davison and Dobbing 1968
). The multitude and complexity of processes during this rapid development makes the developing brain particularly vulnerable to the effects of xenobiotics, like the adverse effect of BDE-47 on spontaneous behavior and habituation (Eriksson et al. 2001
). Interestingly, exposure to BDE-47 does not affect performance in the Morris water maze test (Eriksson et al. 2001
), commonly used as a learning task to detect effects in the hippocampus. This suggests that habituation is a more sensitive parameter for BDE-47 effects in the hippocampus.
We observed a specific reduction of key proteins in the PSD (i.e., GluR1, NR2B, and p286-αCaMKII). Because no changes were observed in total hippocampus homogenate, the specific decrease in the PSD is therefore attributed to changes in glutamate receptor subunit trafficking or clustering in the PSD instead of a reduced protein translation.
A study in GluR1-knockout mice showed that approximately 10% of the normal amount of GluR1 is sufficient for LTP (Mack et al. 2001
). Also, a GluR1-independent form of LTP has been observed in juvenile GluR1-knockout mice (Jensen et al. 2003
). Therefore, major effects on LTP as a consequence of the observed reduction of AMPA subunit GluR1 by approximately 30% are not expected.
The observed reduction of NR2B subunits results in an increased NR2A/NR2B ratio. The majority of NMDA receptors consist of 2 NR1 and 2 NR2A or 2 NR2B subunits. NR2A-NMDA receptors gate smaller Ca2+
currents, have a lower affinity for glutamate, and desensitize faster than NR2B-NMDA receptors (Kutsuwada et al. 1992
). Therefore, an increased NR2A/NR2B ratio is likely to result in a higher threshold for LTP induction, which could explain the reduction of PTP and LTP.
In mice exposed to BDE-47, the auto-phosphorylated-active form of αCaMKII was significantly reduced. Because CamKII autophosphorylation is essential for hippocampal NMDA-dependent LTP (Giese et al. 1998
), this specific effect may lead to reduced synaptic plasticity resulting in behavioral impairments.
To ascertain the absence of presynaptic effects, we investigated neurotransmitter release from chromaffin cells from BDE-47–exposed [68 mg (140 μmol)/kg bw] mice. Because PPR and chromaffin neurotransmitter release remained unchanged after developmental exposure to BDE-47, and because modest acute effects on free intracellular Ca2+
and spontaneous vesicular catecholamine release in PC12 cells were only detected at a concentration of 20 μM BDE-47, we propose that presynaptic changes do not contribute considerably to the observed functional defect in synaptic plasticity. Based on tissue distribution data for 1 mg/kg bw 14
C-BDE-47 orally given to C57Bl/6 mice on PND10 (Staskal et al. 2006
), brain concentration at sacrifice after exposure to 6.8 mg (14 μmol)/kg bw BDE-47 is estimated to be 0.43–0.81 μM and the peak brain concentration, reached 8 hr after exposure, is estimated to be 1.1 μM. These estimated concentrations are at least one order of magnitude lower than the lowest effective concentration in the in vitro
experiments described here.
As with PCBs (for review, see Fonnum et al. 2006
), in vitro
exposure to the commercial penta-BDE mixture DE-71, which contains (on a weight basis) 31.8% BDE-47 (Reistad and Mariussen 2005
), affects several other transmitter systems. Previous studies reported cell death of cerebellar granule cells, alterations of Ca2+
homeostasis in human neutrophils and brain microsomes, and arachidonic acid release and protein kinase C translocation in cerebellar granule cells; inhibition of dopamine reuptake in rat brain synaptic vesicles has been reported after in vitro
exposure to DE-71 in the micromolar range (2–20 μM) (Kodavanti and Ward 2005
; Mariussen and Fonnum 2003
; Reistad et al. 2002
; Reistad and Mariussen 2005
). Interestingly, addition of the NMDA receptor antagonist MK801 protects cerebellar granule cells against DE-71-induced cell death (Reistad et al. 2006
). No other effects of PBDEs on glutamate receptors have yet been published.
Pure (~ 99%) BDE-47, which has been used in only a few experiments, has revealed formation of reactive oxygen species in human neutrophils and increased 3
H-phorbol ester binding in primary rat cerebellar granule neurons, also at micromolar concentrations (Kodavanti et al. 2005
; Reistad and Mariussen 2005
). The effects of BDE-47 in PC12 cells reported here occur at concentrations in the same order of magnitude.
In the 1990s, an association between delayed human neurodevelopment and pre-natal or early exposure to PCBs was reported by cohort studies. These results were corroborated by experiments demonstrating the developmental neurotoxicity of PCBs. The observed interaction with the thyroid hormone system is usually considered part of the underlying mechanism (for review, see Winneke et al. 2002
). For hazard characterization of PCBs and the structurally related PBDEs, it is relevant to investigate whether they induce similar effects through similar mechanisms. This is of particular importance because, in neonatal mice, the effects of a combined dose of PCB-52 and BDE-99 on spontaneous motor behavior and habituation capability appear to be additive or perhaps even synergistic (Eriksson et al. 2006
High human serum concentrations of BDE-47 were measured in female inhabitants of California by Petreas et al. (2003)
; the concentration of BDE-47 in serum ranged from 5 to 510 ng/g lipid weight, with a median of 16.5 ng/g lipid weight. High concentrations (> 100 ng/g lipid weight) have also been reported in Californian children (Fisher et al. 2006). The highest and median values correspond (using average physiologic values) to blood concentrations of approximately 11.5 nM and approximately 0.37 nM. Using the tissue distribution data for 1 mg/kg bw 14
C-BDE-47 (Staskal et al. 2006
), the dose used in the current study corresponds to an estimated blood concentration of approximately 2.6 μM after 3 hr and to approximately 0.6 μM after 10 days (i.e., ~ 50–200 times higher than in the worst, and ~ 1,600–7,000 times higher than in the median human situation described above). For risk assessment, the difference between the animal dose level causing an adverse effect and the highest human dose levels is relatively small, considering safety factors for species extrapolation and intra-species variability. Additional uncertainty comes from the fact that humans are exposed to multiple flame retardants over a lifetime. Accumulation of BDE-47, as demonstrated in primary rat cerebellar granule neurons and primary rat neocortical cells (Kodavanti et al. 2005
; Mundy et al. 2004
), is another reason for concern about the neurotoxic potential of PBDEs.
No tolerable daily intake is assigned to PBDEs because sufficient data are not available. However, the limited toxicity data suggest that adverse effects induced by exposure to the more toxic congeners in rodents occur at doses of at least 100 μg/kg bw per day [Joint FAO/WHO Expert Committee on Food Additives (JECFA) 2005
]. The combination of quantitative molecular data with functional neurophysiologic effects reported here provides strong functional support for the previously reported neurobehavioral effects (Eriksson et al. 2001
) and is essential for characterization of the neurotoxic hazard of brominated flame retardants, particularly for rational risk assessment, which is required in response to the general concern about the vulnerability of the developing brain.