Triiodothyronine or Antioxidants Block the Inhibitory Effects of BDE-47 and BDE-49 on Axonal Growth in Rat Hippocampal Neuron-Glia Co-Cultures

Abstract

We previously demonstrated that polybrominated diphenyl ethers (PBDEs) inhibit the growth of axons in primary rat hippocampal neurons. Here, we test the hypothesis that PBDE effects on axonal morphogenesis are mediated by thyroid hormone and/or reactive oxygen species (ROS)-dependent mechanisms. Axonal growth and ROS were quantified in primary neuronal-glial co-cultures dissociated from neonatal rat hippocampi exposed to nM concentrations of BDE-47 or BDE-49 in the absence or presence of triiodothyronine (T3; 3-30 nM), N-acetyl-cysteine (NAC; 100 µM), or α-tocopherol (100 µM). Co-exposure to T3 or either antioxidant prevented inhibition of axonal growth in hippocampal cultures exposed to BDE-47 or BDE-49. T3 supplementation in cultures not exposed to PBDEs did not alter axonal growth. T3 did, however, prevent PBDE-induced ROS generation and alterations in mitochondrial metabolism. Collectively, our data indicate that PBDEs inhibit axonal growth via ROS-dependent mechanisms, and that T3 protects axonal growth by inhibiting PBDE-induced ROS. These observations suggest that co-exposure to endocrine disruptors that decrease TH signaling in the brain may increase vulnerability to the adverse effects of developmental PBDE exposure on axonal morphogenesis.

Keywords: PBDE; axonal growth; developmental neurotoxicity; neuronal morphogenesis; reactive oxygen species; thyroid hormone.

Figure 1

T3 supplementation prevented BDE-47 and BDE-49 inhibition of axonal growth in primary hippocampal neurons. Primary neuron-glia co-cultures dissociated from the hippocampi of P0-1 rats were exposed to vehicle (DMSO diluted 1:1000) or varying concentrations of BDE-47 or BDE-49 in the absence or presence of T3 beginning 3 h after plating. After 48 h exposure, cultures were fixed and immunostained for the axon-selective cytoskeletal protein tau-1. (A) Representative photomicrographs of DIV 2 hippocampal neurons exposed to vehicle, BDE 47 at 2 nM ± exogenous T3 at 3 nM. Scale bar = 25 µm. (B) Quantification of axon length in tau-1 immunopositive neurons. Data presented as the mean ± SE (n = 70–90 neurons from three independent dissections). *** Significantly different from vehicle at p < 0.001; # significantly different from the corresponding BDE treatment in the absence of T3 at p < 0.05 as determined by one-way ANOVA followed by Tukey’s post hoc test. (C) Fold-change in transcript levels of Klf9 (as a % of vehicle control). Data are presented as the mean ± SE of Klf9 expression normalized to the average of the reference genes Ppia and Hprt1. * Significantly different from vehicle at p < 0.05 as determined by REST 2009 pairwise randomization test.

Figure 2

T3 did not influence axonal growth. Primary neuron-glia co-cultures dissociated from the hippocampi of P0-1 rat hippocampi were exposed to vehicle (DMSO diluted 1:1000) or T3 and/or BDE-47 or BDE-49 beginning 3 h after plating. After 48 h exposure, cultures were fixed and immunostained for tau-1. Representative photomicrographs (A) and quantification of axon length (B) in tau-1 immunopositive neurons at DIV 2. Data are presented as the mean ± SE (n = 30–40 neurons per group from one dissection; results repeated in 3 independent dissections). There were no significant differences between neurons exposed to vehicle vs. T3 as determined by one-way ANOVA (p < 0.05). Scale bar = 25 µm.

Figure 3

Antioxidants prevented BDE-47 and BDE-49 inhibition of axonal growth and production of ROS. Primary neuron-glia co-cultures dissociated from the hippocampi of P0-1 rat pups were exposed to vehicle, BDE-47 or BDE-49 in the absence or presence of N-acetyl cysteine (NAC) or α-tocopherol. After 48 h exposure, cultures were fixed and immunostained for tau-1. (A) Representative photomicrographs of DIV 2 hippocampal neurons from different experimental groups. Scale bar = 25 µm. (B) Quantification of axon length in tau-1 immunopositive cells (n = 70–90 neurons from three independent dissections). Quantification of ROS levels following exposure to vehicle, BDE-47 or BDE-49 alone (C) or in the presence of an antioxidant (D) (n = three independent dissections). H₂O₂ was included as a positive technical control for the ROS-Glo assay per the manufacturer’s instructions. Data presented as the mean ± SE. * Significantly different from vehicle at * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; # significantly different from PBDE treatment alone at # p < 0.05, ## p < 0.01, as determined by one-way ANOVA followed by Tukey’s post hoc test; †significantly different from individual PBDE treatment at p < 0.05 as determined by Student’s t-test.

Figure 4

T3 normalized ROS levels and mitochondrial substrate metabolism in cultures exposed to BDE-47 or BDE-49. Hippocampal neuron-glia co-cultures were exposed to vehicle, T3, BDE-47 and/or BDE-49 for 1 h on DIV 2. (A) Quantification of ROS production following co-exposure to T3 and PBDEs. (B) Mitochondrial substrate metabolism kinetics immediately following PBDE exposure alone and in the presence of T3. Data presented as the mean ± SE (n = three independent dissections). * Significantly different from vehicle at * p < 0.05, ** p < 0.01, *** p < 0.001; # significantly different from T3 at # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 as determined by one-way ANOVA followed by Dunnett’s post hoc test; †significantly different from individual PBDE treatment at † p < 0.05, ††† p < 0.001 as determined by Student’s t-test.