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. 2013 Apr 5;134(1):111–124. doi: 10.1093/toxsci/kft083

A Hydroxylated Metabolite of Flame-Retardant PBDE-47 Decreases the Survival, Proliferation, and Neuronal Differentiation of Primary Cultured Adult Neural Stem Cells and Interferes with Signaling of ERK5 MAP Kinase and Neurotrophin 3

Tan Li *,†, Wenbin Wang *, Yung-Wei Pan ‡, Lihong Xu †, Zhengui Xia *,‡,1
PMCID: PMC3693129  PMID: 23564643

Abstract

Polybrominated diphenyl ethers (PBDEs) are a group of organobromine compounds widely used as flame retardants. PBDE-47 is one of the most prominent PBDE congeners found in human tissues, and it can be transformed into several metabolites, including 6-OH-PBDE-47. Recent studies have shown that PBDE-47 is neurotoxic to animals and possibly humans. However, the basis for the neurotoxicity of PBDEs and their metabolites is unclear. For example, it is not known whether PBDEs affect adult neurogenesis, a process implicated in learning and memory and in olfactory behavior. In this study, we examined the toxicity of PBDEs for primary adult neural stem/progenitor cells (aNSCs) isolated from the subventricular zone (SVZ) of adult mice. We discovered that 6-OH-PBDE-47, but not its parent compound PBDE-47, is cytotoxic for aNCSs using MTS metabolism and cell number as a measure of cytotoxicity. Interestingly, 6-OH-PBDE-47 induced apoptosis at concentrations above 7.5μM inhibited proliferation at 2.5–5μM while suppressing neuronal and oligodendrocyte differentiation at submicromolar concentrations (≤ 1μM). The effect on proliferation was reversed upon removal of 6-OH-PBDE-47 and correlated with selective but reversible inhibition of ERK5 activation by mitogenic growth factors EGF and bFGF. 6-OH-PBDE-47 also inhibited the proneuronal differentiation effect of neurotrophin 3 (NT3) and NT3 activation of ERK5. Together, these data show that 6-OH-PBDE-47 is more toxic than its parent compound for SVZ-derived aNSCs and that it inhibits multiple aspects of adult neurogenesis. Furthermore, inhibition of ERK5 signaling may underlie the adverse effect of 6-OH-PBDE-47 on proliferation and neuronal differentiation. Our data suggest that exposure to PBDE-based flame retardants could cause neurotoxicity in the adult brain by interfering with adult neurogenesis.

Key Words: PBDE, neurotoxicity, adult neurogenesis, apoptosis, proliferation, neuronal differentiation

Polybrominated diphenyl ethers (PBDEs) are flame retardants widely used by industry in textiles, building materials, electronics, plastics, and furnishings (de Wit, 2002). An estimated 2 million tons of PBDEs have been produced worldwide in the last 3 decades (Shaw and Kannan, 2009). PBDEs are blended with but not chemically bonded to polymers in industrial materials and are therefore likely to leach out of the products and be released into air as dust (Wiseman et al., 2011). PBDEs can also be released into the environment during their production in the factory, from manufacturing facilities that use them, and from the landfill or recycling sites where PBDE-containing commercial products are disposed. The large-scale production and their release into the air, soil, and eventually their accumulation in water have led to the ubiquitous contamination of PBDEs in the natural environment. Although the production of penta- and octa-BDEs has been banned, humans will continue to be exposed to these PBDEs from existing products containing them. In addition, the deca-BDEs are still widely produced and used.

PBDEs are lipophilic and therefore tend to bioaccumulate and persist in living organisms through the food chain (Kuo et al., 2010; Li et al., 2008). Humans are exposed to PBDEs through inhalation and ingestion of house dust and consumption of PBDE-contaminated vegetables, fish, and animal products (Frederiksen et al., 2009; Johnson-Restrepo and Kannan, 2009). Because they are ubiquitous and persistent and they accumulate in the human body, PBDE levels in human bodies have been rising rapidly during the past few decades (Costa et al., 2008). In particular, there are approximately one order of magnitude higher levels of PBDEs in humans and in the environment in North America than in Europe and Asia (Frederiksen et al., 2009). The rising levels of PBDE in human tissue together with their potential toxicity represent a risk to the general public health (Costa et al., 2008).

The tetrabrominated congener PBDE-47 and the pentabrominated congener PBDE-99 are two of the most prominent congeners found in human tissues (Costa et al., 2008). PBDE-47 can be metabolized to 6-OH-PBDE-47 in the body (Huang et al., 2010; Qiu et al., 2009). PBDEs, including PBDE-47, exert a number of toxicities including neurotoxicity in animals (Costa et al., 2008; Dingemans et al., 2008, 2011; Eriksson et al., 2002; Ernest et al., 2012; Fan et al., 2010; Gee et al., 2011; Giordano et al., 2008; Hendriks et al., 2010; Schreiber et al., 2010; Verner et al., 2011; Viberg and Eriksson, 2011). They may also be neurotoxic to humans; pre- and/or postnatal exposure correlates with changes in neural behavior in children, suggesting developmental neurotoxicity (Costa and Giordano, 2007; Dingemans et al., 2011; Eriksson et al., 2002; Herbstman et al., 2010; Verner et al., 2011). Interestingly, PBDE exposure has also been linked to behavior changes in adult humans and rats as well (Fitzgerald et al., 2012; Yan et al., 2012).

Recent studies have led to the exciting idea that functional neurons are continuously generated throughout adult life, by adult neurogenesis in two principal regions of the adult brain: the subgranular zone of the dentate gyrus and the subventricular zone (SVZ) of the lateral ventricle (Hsieh and Eisch, 2010; Ming and Song, 2011; Zhao et al., 2008). These adult born neurons have been implicated in hippocampus-dependent memory formation and olfactory behavior (Li et al., 2013; Pan et al., 2012a, b, c, d; Wang et al., 2013). Many external influences have been shown to modulate adult neurogenesis. For example, stress, drug abuse, and irradiation reduce adult neurogenesis, whereas exercise and environmental enrichment enhance this process (Kempermann et al., 1997; Mak et al., 2007; Mirescu et al., 2006; van Praag et al., 1999). However, little is known about the effect of environmental neurotoxicants on adult neurogenesis. In this study, we examined whether PBDE-47 or its metabolite 6-OH-PBDE-47 is neurotoxic to primary cultured adult neural stem/progenitor cells (aNSCs) and began to elucidate underlying signaling mechanisms.

MATERIALS AND METHODS

Materials.

PBDE-47 was purchased from ChemService (West Chester, PA). 6-OH-PBDE-47 was a gift from Dr Michael H. W. Lam and was synthesized in the Department of Biology and Chemistry of City University of Hong Kong and of > 98% purity as described in the study by He et al. (2009). Both PBDE-47 and 6-OH-PBDE-47 were carefully weighed in a fume hood and dissolved with dimethyl sulfoxide (DMSO) to yield a 20mM stock. Z-VAD-FMK was from R&D Systems and used per instructions of the manufacturer. BrdU was from Sigma. Primary antibodies and dilutions used in immunocytochemistry were mouse anti-SOX2 (1:500, R&D Systems), mouse anti-β-III Tubulin (1:1000, Promega), mouse anti-O4 (1:100, Sigma), rat anti-BrdU (1:500, AbD Serotec), rabbit anti-active caspase-3 (1:200, Cell Signal Technology), rabbit anti-GFAP (1:500, Dako), and rabbit anti-Ki67 (1:200 Novocastra). Hoechst 33342 and Alexa Fluor–conjugated secondary antibodies used in immunocytochemistry were from Invitrogen. Primary antibodies used in Western blot analysis were rabbit antibodies against phospho-ERK5 (Cell Signaling Technology), ERK5 (Cundiff et al., 2009), phospho-Akt Ser473 (Cell Signaling Technology), Akt (Cell Signaling Technology), and mouse anti-β-Actin (Sigma).

Preparation of primary aNSC cultures.

Primary aNSC cultures were prepared from the SVZ of 8-week-old mice in the C57/BL6/SV129 background as previously described (Pan et al., 2012d; Wang et al., 2013). All experimental procedures were approved by the University of Washington Institutional Animal Care and Use Committee. Briefly, tissue samples from the SVZ were microdissected and enzymatically digested with 0.125% trypsin-EDTA for 7min at 37°C followed by incubation with equal volume of 0.014% trypsin inhibitor (Invitrogen). Tissue samples were then centrifuged and resuspended in serum-free DMEM/F12 (Invitrogen) culture media containing 1× N2 supplement (Invitrogen), 1× B27 supplement without retinoic acid (Invitrogen), 2mM L-glutamine (Invitrogen), 100U/ml penicillin/streptomycin (Invitrogen), 2 μg/ml heparin (Sigma), 20ng/ml epidermal growth factor (EGF, EMD Chemicals), and 10ng/ml basic fibroblast growth factor (bFGF, Millipore). Tissue was mechanically triturated and filtered through a 40-μm cell sieve and plated in petri dishes for 7–14 days until primary neurospheres were formed. Primary neurospheres were then dissociated by trituration and continually maintained in petri dishes as neurospheres. All aNSCs used in this study were from neurospheres with no more than 10 passages. Unless specifically indicated, EGF and bFGF were replenished every 3 days.

PBDE-47 and 6-OH-PBDE-47 treatment.

The neurospheres were dissociated and plated as a monolayer aNSC culture on fibronectin- and poly-L-orthinine (BD Biosciences)–coated culture plates or Aclar coverslips (Electron Microscopy Sciences) in the same culture medium as above for neurospheres. To measure MTS metabolism, cell number, apoptosis, and proliferation, cells were treated with PBDE-47 or 6-OH-PBDE-47 in normal culture medium containing EGF and bFGF. Equal final concentrations of DMSO were used as vehicle control for PBDE-47 and 6-OH-PBDE-47 treatments. To measure cell proliferation, BrdU was added to medium (10μM final concentration) for 2h. To induce spontaneous neuronal differentiation, cells were incubated for 5 days in culture medium without EGF/bFGF but supplemented with 1mg/ml of bovine serum albumin (BSA) (Equitech Bio). PBDE-47, 6-OH-PBDE-47, and/or NT3 were also added to the media when their effects on neuronal differentiation were examined.

MTS assay.

The CellTiter 96 aqueous one solution cell proliferation assay (Promega) was used to measure the metabolism of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) as an indicator of cell viability per manufacturer’s instruction. Briefly, aNSCs were plated in fibronectin- and poly-L-orthinine–coated 96-well plates for 48h. Cells were then placed in fresh culture medium containing DMSO, PBDE-47, or 6-OH-PBDE-47 for indicated times, followed by incubation with MTS solution for 2h. Absorption at 490nm was quantified using a plate reader (Molecular Devices).

Immunocytochemistry.

Cells were fixed in PBS containing 4% PFA/4% sucrose at room temperature for 30min. Fixed cells were washed 3×5min in PBS, 5min in 1% SDS for permeabilization, and 3×5min in PBS. Cells were then blocked in 5% BSA/PBST (PBS with 0.1% Triton X-100) for 2h, followed by incubation with primary antibodies overnight at 4°C. For BrdU staining, cells were first subjected to HCl treatment before blocking: cells were sequentially incubated in H2O for 5min, ice-cold 1N HCl for 10min, and 2N HCl for 20min at 37°C, followed by neutralization in 0.5M borate buffer for 2×15min. Cells were then washed 3×10min in PBST, followed by incubation with secondary antibodies for 2h in blocking buffer. Cells were then washed 3×10min in PBST followed by a 10-min incubation in Hoechst 33342 for nuclei visualization and a final wash of 10min in PBST prior to mounting onto slides using antifade Aqua Poly/Mount solution (Polysciences).

Imaging and quantification of immunostained cells.

All images were captured using a Zeiss fluorescence microscope and processed using ImageJ software (NIH). For quantification analysis, at least 500 randomly chosen cells were counted from each sample.

Western blot analysis.

Cells were plated at a density of 106 cells per well in fibronectin- and poly-L-orthinine–coated 12-well plates for 24h. Cells were then treated as described in the figure legends, washed with cold PBS, and lysed with Triton-X lysis buffer. Cell lysates were clarified by centrifugation, and protein concentration was determined using BCA protein assay (Pierce). Samples containing 10 μg protein were separated on 10% SDS-PAGE gel and transferred to PVDF membrane (Millipore), followed by antibody incubation and ECL Plus (GE Healthcare) detection. Image J software (NIH) was used to quantify the intensity of bands.

Statistical analysis.

For all experiments, data were results from at least three independent experiments, each with duplicates (n ≥ 3). All data were expressed as mean ± SD. Comparison of the means was analyzed by Student’s t-test, two-tailed analysis.

RESULTS

Isolation of aNSCs and Confirmation of Stem Cell Properties

We dissected tissue from the SVZ of adult mouse brains (Fig. 1A) and prepared aNSCs as neurospheres. Cells were maintained and passed as neurospheres and used for experimentation within 10 passages. Neurospheres were dissociated and cells plated as monolayer cultures for experimentation. Even at passage 10, more than 98% of the cells expressed SOX2 (Fig. 1B), a stem cell marker, confirming that the cells used in this study were adult neural stem cells.

FIG. 1.

FIG. 1.

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Isolation of aNSCs from the SVZ of adult mouse brain. (A) Schematic diagram of the isolation of aNSCs derived from the SVZ, regions depicted in red rectangles. (B) The SVZ-derived aNSCs, at passage 10, were fixed and immunostained for SOX2 (red), a stem cell marker. Hoechst-stained nuclei (blue) were used to identify all cells. Scale bars represent 100 μm. This figure can be viewed in color online.

6-OH-PBDE-47, but Not Its Parent Form, Is Cytotoxic to aNSCs

To determine whether PBDE-47 or 6-OH-PBDE-47 is cytotoxic to aNSCs, cells were treated with varying concentrations of PBDE-47 or 6-OH-PBDE-47, and cell viability was measured by MTS metabolism. PBDE-47 treatment for 48h, at concentrations ranging from 5 to 40μM, did not decrease MTS metabolism (Fig. 2A). In contrast, treatment with 6-OH-PBDE-47 for 48h decreased MTS metabolism in a dose-dependent manner, starting from 2.5 to 10μM (Fig. 2B), with effective median concentration (EC50) value of approximately 5μM. The decreased MTS metabolism was detectable and statistically significant as early as 3h after treatment with 6-OH-PBDE-47 (Fig. 2C). By 48h, there was very little MTS metabolism left in cells treated with 10μM 6-OH-PBDE-47. Because a decrease in MTS metabolism could result from a loss of cell number and/or a decrease in mitochondrial metabolic activity, we quantified the number of Hoechst-stained nuclei as a measure of total cell number. Treatment of 6-OH-PBDE-47 for 48h decreased the number of cells in a dose-dependent manner (Fig. 2D), in a degree similar to MTS metabolism. There were very few cells left when treated with 7.5 or 10μM 6-OH-PBDE-47 for 48h. The EC50 on cell number reduction was also about 5μM. These data suggest that the metabolite 6-OH-PBDE-47, but not its parent compound PBDE-47, is cytotoxic to primary cultured aNSCs.

FIG. 2.

FIG. 2.

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6-OH-PBDE-47 decreases the overall viability and cell number of aNSCs. (A) MTS metabolism after treatment with PBDE-47. The aNSCs were treated with varying concentrations of PBDE-47 or vehicle control DMSO for 48h. The optical density value for cells with no treatment was set as 1. The media for cells treated with 5 or 10μM PBDE-47 contained 0.05% DMSO, whereas that for cells treated with 20 or 40μM PBDE-47 contained 0.2% DMSO. (B) Relative MTS metabolism after treatment with 6-OH-PBDE-47 for 48h. (C) Kinetics of MTS metabolism after treatment with 5 or 10μM 6-OH-PBDE-47. (D) Relative cell number after treatment with 6-OH-PBDE-47 for 48h. All treatment groups in panels B–D contain 0.05% DMSO. Results from four independent experiments were analyzed. *p < 0.05; **p < 0.01; ***p < 0.001, compared with DMSO control.

6-OH-PBDE-47 Induces Apoptosis in aNSCs

Treatment with 7.5 or 10μM 6-OH-PBDE-47 for 48h, but not with 2.5 or 5μM of 6-OH-PBDE-47, caused nuclear fragmentation and condensation, as well as expression of active caspase-3 (Figs. 3A and B), suggesting apoptosis. A 2-h pretreatment with 20μM of Z-VAD-FMK, a pan-caspase inhibitor, significantly inhibited 6-OH-PBDE-47–induced nuclear fragmentation and condensation (Fig. 3C), as well as the number of active caspase-3+ cells (Fig. 3D). These data suggest that the reduced cell number upon treatment with 7.5 or 10μM 6-OH-PBDE-47 is due to apoptosis.

FIG. 3.

FIG. 3.

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6-OH-PBDE-47 induces apoptosis in aNSCs. (A) Representative fluorescence images of cells stained with Hoechst 33342 (blue) and active caspase-3 (green) after treatment with 7.5μM 6-OH-PBDE-47 or vehicle control for 48h. Arrowheads point to fragmented or condensed apoptotic nuclei, which are also active caspase-3+. Scale bar: 25 μm. (B) Quantification of the percentage of apoptotic nuclei or active caspase-3+ cells after 48-h treatment with 6-OH-PBDE-47. (C and D) Quantification of the percentage of the fragmented or condensed apoptotic nuclei (C), or the percentage of active caspase-3+ cells (D), in the presence of a pan-caspase inhibitor Z-VAD-FMK. The aNSCs were pretreated with 20μM Z-VAD-FMK or vehicle control for 2h, followed by 48-h treatment of 7.5μM 6-OH-PBDE-47. Results from three independent experiments were analyzed. n.s., not significant; *p < 0.05; **p < 0.01; ***p < 0.001, compared with DMSO control or as specifically indicated. This figure can be viewed in color online.

6-OH-PBDE-47 Inhibits Proliferation of aNSCs

Because treatment with 2.5 and 5μM 6-OH-PBDE-47 did not induce apoptosis, we investigated whether the reduced cell number under these treatment conditions is due to inhibition of proliferation. Cell proliferation was measured by BrdU incorporation, which labels actively proliferating, S-phase cells, and by immunostaining for Ki67, a marker for proliferative cells in all phases of the cell cycle (Fig. 4A). Treatment with 2.5 or 5μM 6-OH-PBDE-47 for 48h decreased the percentage of Ki67+ and BrdU+ cells in a dose-dependent manner (Figs. 4B and C). EC50 for inhibition of proliferation is between 2.5 and 5 μM. Greater than 90% of the cells treated with 5μM 6-OH-PBDE-47 were Ki67−, indicating that these cells are in G0 phase and have exited the cell cycle.

FIG. 4.

FIG. 4.

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6-OH-PBDE-47 decreases the proliferation of aNSCs. (A) Representative phase contrast or fluorescence images of cells stained for Ki67 (green) and BrdU (red). Cells were treated with 5μM 6-OH-PBDE-47 or 0.025% DMSO as vehicle control for 48h in regular culture medium containing EGF and bFGF. BrdU was added to media during the last 2h of treatment. Scale bar: 50 μm. (B and C) Quantification of data from panel A as the percentage of Ki67+ cells (B) or BrdU+ cells (C). (D and E) The inhibitory effect of 6-OH-PBDE-47 was reversible. Cells were treated with 0.025% DMSO or 5μM 6-OH-PBDE-47 as in panels A–C for 48h. The culture media were then removed, cells washed with prewarmed culture media, and placed in fresh media containing DMSO or 5μM 6-OH-PBDE-47 as indicated for another 24h. BrdU was added to media during the last 2h of treatment before fixation. The percentages of Ki67+ cells (D) and of BrdU+ cells (E) were quantified. Results from three independent experiments were analyzed. *p < 0.05; **p < 0.01; ***p < 0.001, compared with DMSO control or as specifically indicated. This figure can be viewed in color online.

To determine whether the inhibitory effect on cell proliferation is reversible, cells were treated with 5μM 6-OH-PBDE-47 for 48h as before. The medium was then removed; cells were washed and subsequently placed in fresh culture medium containing either 5μM 6-OH-PBDE-47 or DMSO for an additional 24h. The removal of 6-OH-PBDE-47 significantly increased the percentage of Ki67+ and BrdU+ cells compared with cells continuously treated with 6-OH-PBDE-47 (Figs. 4D and E), suggesting that the effect on cell proliferation is reversible and that the cells have re-entered cell cycle upon removal of 6-OH-PBDE-47.

In contrast, the parent compound of PBDE-47 did not affect BrdU incorporation, thus cell proliferation of aNSCs, from concentrations ranging from 1 to 40μM when treated for 48h (Fig. 5). These data suggest that 6-OH-PBDE-47, but not its parent compound, inhibits cell proliferation of aNSCs.

FIG. 5.

FIG. 5.

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PBDE-47 does not affect the proliferation of aNSCs. (A) Representative immunofluorescence images of cells stained for BrdU (red). Hoechst staining was used to visualize all nuclei (blue). aNSCs were treated with 0.2% DMSO as vehicle control, or with 40μM PBDE-47 for 48h. BrdU was added to the media during the last 2h of the 48-h treatment. Scale bars: 30 μm. (B) Quantification of the percentage of cells that were labeled with BrdU. PBDE-47 treatment groups from 1 to 10μM contain 0.05% DMSO, whereas 20 and 40μM PBDE-47 groups contain 0.2% DMSO. This figure can be viewed in color online.

6-OH-PBDE-47 Selectively Inhibits EGF and bFGF Activation of ERK5

To elucidate signaling mechanisms by which 6-OH-PBDE-47 inhibits cellular proliferation, we examined the effect of 6-OH-PBDE-47 on the activation of ERK5, ERK1/2, and Akt by EGF and bFGF using Western blot analysis. EGF and bFGF are mitogenic growth factors present in the culture medium; their proliferative effect in other cell types is often mediated through the ERK5 and ERK1/2 MAP kinases and/or PI-3 kinase-Akt kinase signaling pathways (Kato et al., 1997; Suhardja and Hoffman, 2003). The aNSCs were incubated in EGF- and bFGF-free medium overnight to reduce background signals. 6-OH-PBDE-47 (5μM) or DMSO vehicle control was also included in the culture medium during the overnight treatment. Cells were then washed and placed in fresh culture medium containing either 5μM 6-OH-PBDE-47 or DMSO vehicle control in the presence or absence of EGF/bFGF cotreatment for 30min as indicated in Figs. 6A–D. Addition of EGF/bFGF to the culture medium induced phosphorylation of ERK5, ERK1/2, and Akt, indicative of activation of these kinase signaling pathways. Pretreatment with 6-OH-PBDE-47 overnight did not reduce the levels of total ERK5, ERK1/2, or Akt. However, it specifically attenuated EGF/bFGF stimulation of ERK5 phosphorylation without affecting the phosphorylation of ERK1/2 or Akt. The inhibitory effect of 6-OH-PBDE-47 on ERK5 phosphorylation was attenuated when 6-OH-PBDE-47 was removed from the medium during the period of EGF/bFGF treatment. Interestingly, when cells were not pretreated with 6-OH-PBDE or only pretreated for 0.5, 1, or 2h instead of overnight, 6-OH-PBDE-47 did not inhibit EGF/bFGF activation of ERK5 (Fig. 6E). These data suggest that overnight pretreatment of 6-OH-PBDE-47 selectively and reversibly inhibits EGF and bFGF activation of ERK5 MAP kinase, a protein kinase required for proliferation, survival, and neuronal differentiation of adult neurogenesis (Li et al., 2013; Pan et al., 2012a, b, c, d; Wang et al., 2013).

FIG. 6.

FIG. 6.

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Pretreatment with 6-OH-PBDE-47 selectively and reversibly attenuates ERK5 activation stimulated by EGF and bFGF. (A–D) aNSCs were incubated in EGF-/bFGF-free medium in the presence or absence of 5μM 6-OH-PBDE-47 overnight. Cells were then washed once with media and incubated for 30min in media with or without EGF/bFGF and 5μM 6-OH-PBDE-47 as indicated. Cell lysates were then subjected to Western blot analysis for p-ERK5, ERK5, p-Akt (Ser473), Akt, and p-ERK1/2. β-Actin was used as a loading control (A) and quantified for p-ERK5 relative to total ERK5 (B), p-Akt relative to total Akt (C), and p-ERK1/2 relative to β-Actin (D). Results from three independent experiments were analyzed. n.s., not significant; *p < 0.05; **p < 0.01, compared with DMSO control or as specifically indicated. (E) aNSCs were incubated in EGF-/bFGF-free medium in the absence or presence of 5μM 6-OH-PBDE-47 for 0.5h, 1h, 2h, or overnight. Cells were then washed incubated for 30min in media with or without EGF/bFGF and 5μM 6-OH-PBDE-47 as indicated. Cell lysates were collected for Western analysis as in panel A.

6-OH-PBDE-47 Inhibits Spontaneous and Neurotrophin-Promoted Neuronal Differentiation

Although treatment with relatively lower concentrations of 6-OH-PBDE-47 (0.5μM) did not cause overt cytotoxicity manifested as a decrease in cell numbers (Fig. 2), it is possible that it could exert other more subtle adverse effects. For example, it could inhibit neuronal differentiation, another important aspect of adult neurogenesis. To address this issue, aNSCs were cultured in EGF- and bFGF-free medium for 5 days to allow spontaneous differentiation in the absence of mitogenic growth factors. Cells were treated with 0.5 or 1μM of 6-OH-PBDE-47 or vehicle control. Neuronal differentiation was assessed by immunostaining of β-III tubulin, a marker for neurons (Fig. 7A). Treatment with 6-OH-PBDE-47 did not affect the total number of cells but decreased the percentage of β-III tubulin+ cells in a dose-dependent manner (Figs. 7B and C). These data suggest that 6-OH-PBDE-47 inhibits spontaneous neuronal differentiation, a phenomenon that is not due to overall reduction of cell numbers.

FIG. 7.

FIG. 7.

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Effect of 6-OH-PBDE-47 and its parent compound on neuronal differentiation of aNSCs. (A) Representative immunofluorescence images of cells stained for β-III tubulin (red). Hoechst staining was used to visualize all nuclei (blue). aNSCs were treated with 0.005% DMSO as vehicle control, or with 1μM 6-OH-PBDE-47, in EGF-/bFGF-free medium for 5 days. Scale bar: 100 μm. (B) The percentage of β-III tubulin+ cells after 6-OH-PBDE-47 treatment was quantified. (C) The relative total cell number after 6-OH-PBDE-47 treatment. (D) aNSCs were treated with 0–10μM of PBDE-47 in EGF-/bFGF-free medium for 5 days. The percentage of β-III tubulin+ cells was quantified. (E) The relative total cell number after PBDE-47 treatment. (F) aNSCs were treated with 100ng/ml NT3, 1μM 6-OH-PBDE-47 as indicated, in EGF-/bFGF-free medium for 5 days. The percentage of β-III tubulin+ cells were quantified. (G) aNSCs were incubated in EGF-/bFGF-free medium in the presence or absence of 5μM 6-OH-PBDE-47 overnight. Cells were then washed once with media and incubated for 1 or 2h in media ± 100ng/ml NT3 and 5μM 6-OH-PBDE-47 as indicated. Cell lysates were then subjected to Western blot analysis for p-ERK5, ERK5, p-Akt (Ser473), and Akt. β-Actin was used as a loading control. Results from three independent experiments were analyzed. n.s., not significant; *p < 0.05; **p < 0.01, compared with DMSO control or as specifically indicated. This figure can be viewed in color online.

To determine whether low concentrations of the parent compound PBDE-47 also interferes with neuronal differentiation, aNSCs were treated with 0.5 to 10μM of PBDE-47 under the otherwise same experimental conditions as those for 6-OH-PBDE-47 treatment. Although no effect on total cell number was observed, 10μM PBDE-47, but not at lower concentrations, inhibited neuronal differentiation by 8% (Figs. 7D and E). This inhibition was statistically significant and to a degree similar to that caused by 0.5μM 6-OH-PBDE-47. Thus, the metabolite 6-OH-PBDE-47 is about 20-fold more potent than its parent compound in inhibiting neuronal differentiation.

Neurotrophic factors such as neurotrophin 3 (NT3) promote neuronal differentiation during brain development. They may also regulate adult neurogenesis in the SVZ (Bath and Lee, 2010). Indeed, treatment with NT3 increased the number of β-III tubulin+ cells generated from aNSCs in culture (Fig. 7F). Interestingly, this increase was attenuated by cotreatment with 1μM of 6-OH-PBDE-47. These data suggest that NT3 promotes neuronal differentiation and that 6-OH-PBDE-47 interferes with this function. We recently reported that ERK5 signaling contributes to NT3-induced neuronal differentiation of adult SGZ-derived aNSCs (Pan et al., 2012d). In addition, endogenous ERK5 activity is required for spontaneous neuronal differentiation of cultured SVZ aNSCs and for prolactin stimulation of neuronal differentiation of SVZ aNSCs both in vitro and in vivo (Li et al., 2013; Wang et al., 2013). To investigate a potential role for ERK5 signaling in 6-OH-PBDE-47 inhibition of neuronal differentiation, we examined whether 6-OH-PBDE-47 interferes with NT3 activation of ERK5 signaling. NT3 treatment for 2h activated ERK5 but not Akt (Fig. 7G). Pretreatment of 6-OH-PBDE-47 attenuated NT3 activation of ERK5. These data suggest that 6-OH-PBDE-47 interferes with NT3-promoted neuronal differentiation and ERK5 activation.

Effect of 6-OH-PBDE-47 on Glial Differentiation

To determine whether 6-OH-PBDE-47 or its parent compound also affects glial differentiation under the same experimental conditions for spontaneous neuronal differentiation, GFAP was used as a marker for astrocytes, and O4 as a marker for oligodendrocytes (Fig. 8). Treatment with 6-OH-PBDE-47 (0.5 and 1μM) or PBDE-47 (0.5–10μM) had no effect on the number of GFAP+ cells (Figs. 8A and B). However, similar to its effect on β-III tubulin+ cells, 6-OH-PBDE-47 caused a dose-dependent reduction on the number of O4+ cells (Fig. 8C). Likewise, 10μM PBDE-47 reduced the number of O4+ cells to a degree similar to that caused by 0.5μM 6-OH-PBDE-47 (Fig. 8D). These data suggest that 6-OH-PBDE-47 and its parent compound inhibit differentiation of oligodendrocytes but not astrocytes. Furthermore, 6-OH-PBDE-47 is about 20-fold more potent than its parent compound in inhibiting oligodendrocyte differentiation.

FIG. 8.

FIG. 8.

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Effect of 6-OH-PBDE-47 and its parent compound on glial differentiation of aNSCs. aNSCs were treated with DMSO as vehicle control, or with different concentrations of 6-OH-PBDE-47 or PBDE-47, in EGF-/bFGF-free medium for 5 days. (A) The percentage of GFAP+ cells after 6-OH-PBDE-47 treatment was quantified. (B) The percentage of GFAP+ cells after PBDE-47 treatment. (C) The percentage of O4+ cells after 6-OH-PBDE-47 treatment. (D) The percentage of O4+ cells after PBDE-47 treatment. *p < 0.05; **p < 0.01, compared with DMSO control.

DISCUSSION

Environmental exposure to PBDEs has raised considerable public health concern in recent years (Costa et al., 2008; Dingemans et al., 2008, 2011; Eriksson et al., 2002; Ernest et al., 2012; Fan et al., 2010; Gee et al., 2011; He et al., 2008b; Hendriks et al., 2010; Schreiber et al., 2010; Verner et al., 2011; Viberg and Eriksson, 2011). High levels of PBDEs are found in human tissues including breast milk and blood including fetal blood (Kalantzi et al., 2004; Mazdai et al., 2003; Morland et al., 2005; Schecter et al., 2003, 2005; Sjödin et al., 2004), posing a threat to the fetus and infants during critical periods of development. Although human evidence is limited, in vitro and animal studies suggest the possibility that PBDEs may be a potential risk factor for developmental neurotoxicity (Costa and Giordano, 2007, 2011; Dingemans et al., 2008; Herbstman et al., 2010; Schreiber et al., 2010; Verner et al., 2011). For example, PBDE exposure may be a risk factor for autism (Hertz-Picciotto et al., 2011; Messer, 2010; Mitchell et al., 2012; Napoli et al., 2013; Woods et al., 2012). Despite a number of studies reporting the effect of PBDEs on the developing brain, there have been few reports concerning the toxic effects of PBDEs for the adult nervous system. Two recent reports suggested that PBDE exposure may cause behavioral changes in adult humans and rats (Fitzgerald et al., 2012; Yan et al., 2012). For example, PBDE-47 causes deficits in spatial learning and memory in adult rats (Yan et al., 2012). Although the sample size was small, a recent study showed that higher serum concentrations of PBDE are associated with lower verbal learning and memory among adult humans who also have relatively high levels of total serum polychlorinated biphenyls (Fitzgerald et al., 2012). Thus, the goal of our study was to examine the potential toxicity of PBDEs to cells in the adult brain, using adult neural stem cells prepared from mouse SVZ as a model system.

Our data showed that treatment with up to 40μM PBDE-47 did not cause cytotoxicity measured by MTS metabolism or loss of cell numbers in primary cultured aNSCs although treatment with PBDE-47 at these concentrations decreases the viability of neuronal cell lines, including human neuroblastoma SH-SY5Y and SK-N-MC cells, and of primary rat hippocampal neurons (He et al., 2008a, 2009; Tagliaferri et al., 2010). However, treatment with 6-OH-PBDE-47 at concentrations as low as 2.5µM decreased MTS metabolism under the same conditions. This suggests that aNSCs are more resistant to PBDE-47 cytotoxicity than SH-SY5Y cells, SK-N-MC cells, and primary rat hippocampal neurons. Furthermore, the metabolite 6-OH-PBDE-47 is more cytotoxic to aNSCs than its parent compound. We also present data that although PBDE-47 inhibits spontaneous neuronal and oligodendrocyte differentiation, it is about 20 times less potent than 6-OH-PBDE-47, further supporting the notion that the metabolite 6-OH-PBDE-47 is more toxic than its parent compound to aNSCs. Interestingly, 6-OH-PBDE-47 is also more potent than PBDE-47 in disturbing Ca2+ homeostasis and neurotransmitter release in PC12 cells (Dingemans et al., 2008). 6-OH-PBDE-47, but not PBDE-47, acts as a partial agonist for GABA(A) receptors and inhibits nicotinic acetylcholine receptors in a Xenopus oocyte model system (Hendriks et al., 2010). Thus, it is important to be mindful when assessing PBDE toxicity that the metabolized forms, such as 6-OH-PBDE-47, may be more toxic than their parent compounds in some assays for toxicology.

Adult neural stem cells can self renew, proliferate, and differentiate into neurons (Hsieh and Eisch, 2010; Ming and Song, 2011; Zhao et al., 2008). Adult neurogenesis can be regulated at multiple levels, including proliferation, neuronal differentiation, and survival. Our data demonstrate that 6-OH-PBDE-47 interferes with several aspects of adult neurogenesis. At relatively low concentrations, it inhibits the differentiation of neurons and oligodendrocytes without any observable effect on astrocytes. As the concentration increases, the toxicity becomes more overt, including inhibition of cell proliferation, which can be reversed upon removal of 6-OH-PBDE-47, to caspase 3–dependent apoptosis. The parent compound PBDE-47 has no adverse effect on proliferation and survival at concentrations as high as 40µM. However, it caused a statistically significant inhibition on the differentiation of neurons and oligodendrocytes at 10µM. Adult neurogenesis is a physiological process in the adult mammalian brain that plays an important role in learning and memory and in olfactory behavior. To our knowledge, our data provide the first evidence suggesting that exposure to PBDE-related environmental toxins may negatively affect adult neurogenesis at multiple steps and thereby impair the normal function of the adult brain. Furthermore, differentiation may be a more sensitive biomarker than proliferation or cell survival.

The BDE-47 concentration in adult human serum ranges from 8 to 29ng/g lipids although concentrations as high as 511 or 540ng/g lipids have been detected in serum obtained from general adult human or foam workers in the United States, respectively (reviewed in Dingemans et al. [2011]). The concentration of 6-OH-PBDE-47 in U.S. adult human serum ranges from 0.1 to 0.5ng/g lipids although concentrations as high as 177 or 62ng/g lipids are found in the adult Korean serum or in the cord blood of U.S. population, respectively. Because they are lipophilic, PBDEs can bioaccumulate up to 140 times in the brain (Viberg et al., 2003). Although it is difficult to know whether the concentrations we used in this study are environmentally relevant to human exposures, the doses we used are comparable to those used by other researchers in the field (Dingemans et al., 2011; Schreiber et al., 2010). It has been estimated that infant exposure could result in a brain concentration of 0.1–1.1µM of PBDEs (Schreiber et al., 2010), a concentration within one order of magnitude of the lowest observable dose, 10µM, of the parent compound PBDE-47 on inhibition of differentiation. Because PBDEs can potentially bioaccumulate in the brain and have long half-life, data presented in this study and in those already published in the literature warrant further investigation on PBDE neurotoxicity.

At the molecular level, PBDEs induce oxidative stress, cause DNA damage, and perturb calcium homeostasis in a number of different cells (Dingemans et al., 2008; He et al., 2008a, 2009; Shao et al., 2008; Tagliaferri et al., 2010). They also interfere with protein kinase signaling including PKC translocation (Kodavanti and Ward, 2005). Interestingly, a recent report showed that DE-71, a commercial penta-PBDE mixture, activates the ERK1/2 MAP kinase in cultured cerebellar granule neurons (Fan et al., 2010). However, 6-OH-PBDE-47 did not activate or inhibit ERK1/2 in aNSCs under our experimental conditions. It is possible that the mode of action of the penta-PBDE mixture DE-71 is different from that of 6-OH-PBDE-47. Alternatively, different signaling mechanisms may underlie the toxicity of PBDEs in postmitotic cerebellar granule neurons compared with aNSCs.

ERK5 is a member of the MAP kinase family including ERK1/2 (Lee et al., 1995). Our recent studies demonstrated that ERK5 plays an important role in neuronal differentiation of embryonic cortical neural stem/progenitor cells in culture (Cundiff et al., 2009; Liu et al., 2006) and in the survival of immature neurons (Liu et al., 2003). ERK5 also regulates the proliferation, differentiation, and survival of olfactory bulb neurons generated from the SVZ in vivo during embryonic development (Zou et al., 2012). Interestingly, ERK5 is specifically expressed in the neurogenic regions in the adult mouse brain where it regulates proliferation and neuronal differentiation during adult neurogenesis and plays an important role in learning and memory, as well as in olfactory behavior (Li et al., 2013; Pan et al., 2012a, b, c, d; Wang et al., 2013). Here, we showed that 6-OH-PBDE-47, at concentrations (5μM) that inhibit cell proliferation, selectively inhibited activation of ERK5 by EGF and bFGF, mitogenic growth factors present in culture medium for aNSCs. In contrast, 6-OH-PBDE-47 did not have any effect on EGF/bFGF activation of ERK1/2 or Akt, kinase signaling pathways commonly implicated in growth factor stimulation of cell proliferation in a variety of cell types (Liang and Slingerland, 2003; Raman et al., 2007; Suhardja and Hoffman, 2003). Furthermore, ERK5 inhibition was reversed upon removal of 6-OH-PBDE-47, consistent with the inhibitory effect of 6-OH-PBDE-47 on proliferation. These data imply that 6-OH-PBDE-47 may inhibit aNSC proliferation through inhibition of ERK5 MAP kinase signaling.

NT3 is one of the neurotrophic factors that promote neurogenesis both during early brain development and later in the adult brain (Ahmed et al., 1995; Bath and Lee, 2010). When SVZ-derived aNSCs were allowed to spontaneously differentiate by removing the mitogenic EGF/bFGF from the medium, addition of NT3 increased the number of β-III tubulin-positive cells. We recently published evidence that endogenous ERK5 activity is required for spontaneous and prolactin-stimulated neuronal differentiation of SVZ aNSCs both in vitro and in vivo (Li et al., 2013; Wang et al., 2013). In addition, we have recently shown that ERK5 is required for spontaneous and NT-stimulated neuronal differentiation of SGZ-derived aNSCs (Pan et al., 2012a, d). In the present study, we demonstrate that 6-OH-PBDE-47 attenuates spontaneous and NT3-stimulated neuronal differentiation. Furthermore, NT3 activates ERK5 but not Akt in these cells. Moreover, OH-PBDE-47 suppresses NT3 activation of ERK5. Although these results are only correlative in the present form, they suggest the possibility that inhibition of ERK5 may underlie 6-OH-PBDE-47 inhibition of neuronal differentiation.

The mechanisms by which 6-OH-PBDE-47 inhibits ERK5 activation are unclear. However, overnight treatment of 6-OH-PBDE-47 does not change the total protein expression level of ERK5. Thus, it seems unlikely due to perturbation of ERK5 transcription, translation, or protein degradation. Furthermore, the inhibitory effect of 6-OH-PBDE-47 on EGF/bFGF activation of ERK5 requires overnight pretreatment and is reversible within 30min upon removal of 6-OH-PBDE-47. These data indicate reversible and indirect mechanisms of inhibition, rather than direct interference with EGF/bFGF receptor signaling to ERK5. Some potential possibilities include internalization of EGF/bFGF receptors away from the cell surface, changes of subcellular localization of specific components of the ERK5 signaling pathway that are not common to Akt or ERK1/2 signaling, leading to temporary uncoupling of receptor signaling to ERK5. The fact that OH-PBDE-47 inhibits only EGF/bFGF activation of ERK5 but not of ERK1/2 or Akt argues against receptor internalization per se.

In summary, we provide evidence that 6-OH-PBDE-47, a metabolite of one of the most prominent PBDE congeners found in human tissues, is more toxic than its parent compound. It inhibits neuronal and oligodendrocyte differentiation, proliferation, and survival of primary cultured aNSCs in a dose-sensitive manner. It also interferes with ERK5 MAP kinase signaling and the function of NT3. These results provide evidence that 6-OH-PBDE-47 disrupts multiple aspects of adult neurogenesis. It is possible that exposure to PBDE-based flame retardants could adversely affect adult neurogenesis and perturb the normal function of adult brain.

FUNDING

This work was supported by the National Institutes of Health (MH95840 to Z.X., T32HD007183 and F31DC011216 to Y.W.P.), the China Scholarship Council (2011632117 to T.L.), and facilitated by grant P30 HD02274 from the National Institute of Child Health and Human Development (P30 HD02274).

ACKNOWLEDGMENTS

We thank Dr Michael H. W. Lam from the State Key Laboratory for Marine Pollution, Department of Biology & Chemistry, City University of Hong Kong, Hong Kong SAR, China for providing us with 6-OH-PBDE 47. We also thank Dr D. R. Storm and members of the Xia laboratory for critical reading of the article. The authors claim no conflict of interest.

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