Disruption of thyroid hormone receptor-mediated transcription and thyroid hormone-induced Purkinje cell dendrite arborization by polybrominated diphenyl ethers

Abstract

Background: Polybrominated diphenyl ethers (PBDEs) have been used as flame retardants and are becoming a ubiquitous environmental contaminant. Adverse effects in the developing brain are of great health concern.

Objective: We investigated the effect of PBDEs/hydroxylated PBDEs (OH-PBDEs) on thyroid hormone (TH) receptor (TR)-mediated transcription and on TH-induced dendrite arborization of cerebellar Purkinje cells.

Methods: We examined the effect of PBDEs/OH-PBDEs on TR action using a transient transfection-based reporter gene assay. TR-cofactor binding was studied by the mammalian two-hybrid assay, and TR-DNA [TH response element (TRE)] binding was examined by the liquid chemiluminescent DNA pull-down assay. Chimeric receptors generated from TR and glucocorticoid receptor (GR) were used to identify the functional domain of TR responsible for PBDE action. The change in dendrite arborization of the Purkinje cell in primary culture of newborn rat cerebellum was also examined.

Results: Several PBDE congeners suppressed TR-mediated transcription. The magnitude of suppression correlated with that of TR-TRE dissociation. PBDEs suppressed transcription of chimeric receptors containing the TR DNA binding domain (TR-DBD). We observed no such suppression with chimeras containing GR-DBD. In the cerebellar culture, PBDE significantly suppressed TH-induced Purkinje cell dendrite arborization.

Conclusions: Several PBDE congeners may disrupt the TH system by partial dissociation of TR from TRE acting through TR-DBD and, consequently, may disrupt normal brain development.

Figure 1

Congener-specific suppression of TR-mediated transcription by PBDEs/OH-PBDEs. Expression plasmids encoding TRβ1 (A–C, E–G) or TRα1 (D; 10 ng) were cotransfected with F2-TRE (A, D–G), DR4-TRE (B), or Pal-TRE (C; 100 ng) into CV-1 cells. Cells were incubated with or without 10⁻⁷ M T₃ and indicated amounts of PBDEs/OH-PBDEs. (H) Expression plasmids encoding GR (10 ng) were cotransfected with MMTV (GRE)-LUC reporter plasmids (100 ng) into CV-1 cells. Cells were cultured in the absence or presence of Dex (10⁻⁷ M) and indicated concentrations of BDE209. Total amounts of DNA for each well were balanced by adding vector pcDNA3. Data are mean ± SE of experiments performed in triplicate. *p < 0.01, and ^#p < 0.05 by ANOVA, compared with TRβ1 (+), T₃ (+), and BDE209 (−) in A, B, and D and DE-71 (−) in G.

Figure 2

Effects of PBDE congeners (A) or OH-PBDEs and a PBDE mixture (B) on TR-mediated transcription and TR–TRE binding. Transient transfection-based reporter gene assays (Figure 1A) and LCDPA (Figure 3E) were carried out using each chemical. Results of minimum effective dose, maximum suppression (%) and dose, and maximum dissociation (%) and dose are listed. —, no effect.

Figure 3

The effect of PBDE on TR cofactor or TR–TRE binding. (A) Schematic diagram of Gal4–SRC-1–NBD-1. Functionally active LXXLL motifs (Leu-Xaa-Xaa-Leu-Leu) are located in central (residues 633–637, 690–694, and 749–753) and C-terminal (residues 1434–1438) regions termed NBD-1 and NBD-2, respectively. Gal4–SRC-1–NBD-1 contains amino acid residues 595–780 of SRC-1. (B) BDE209 did not affect SRC-1 binding to liganded TR in CV-1 cells. Expression plasmids encoding Gal4–DBD–fused SRC-1–NBD-1 (10 ng) were cotransfected with VP16 constructs (50 ng) and 5× UAS-TK-LUC reporter plasmids (170 ng) into CV-1 cells. Cells were incubated with or without T₃ (10⁻⁷ M) and indicated concentrations of BDE209. (C) Schematic diagram of Gal4–N-CoR, which contains amino acid residues 1579–2454 of N-CoR. (D) BDE209 did not recruit N-CoR to TR in CV-1 cells. Expression plasmids harboring Gal4-DBD-fused N-CoR (100 ng) were cotransfected with VP16-TRβ1-LBD (50 ng) and 5× UAS-TK-LUC (100 ng) into CV-1 cells. Cells were incubated with or without T₃ (10⁻⁷ M) and indicated concentrations of BDE209. (E) Schematic diagram showing the LCDPA method. GST-TRβ1 bound to Sepharose beads was incubated with DIG-labeled F2-TRE in protein–DNA binding buffer with or without T₃ (10⁻⁶ M) and indicated concentrations of PBDE. (F) The effect of BDE209 on TRβ1–TRE binding using LCDPA. In B, D, and F, data represent mean ± SE of experiments performed in triplicate. *p < 0.01 by ANOVA, compared with GST-TRβ1 (+), T₃ (+), and PBDE (−).

Figure 4

TR-mediated transcription is altered by PBDE through TR-DBD. (A) Schematic diagram of chimeric protein used in the present study. Abbreviations: G, GR; T, TR; N, N-terminal domain. (B,C) Representative examples of PBDE actions on chimeric receptor-induced transcription (B, GTG; C, TGT). (D,E) Effects of BDE209 on transcription through chimeric receptors containing TR-DBD (D) or GR-DBD (E). For B–E, chimeric receptors (10 ng) were cotransfected with F2-TRE (B,D) or MMTV (GRE)-LUC (C,E) reporter plasmid (100 ng) into CV-1 cells and incubated with or without Dex (10⁻⁷ M) or T₃ (10⁻⁷ M; C), and 10⁻¹⁴ to 10⁻⁹ M BDE209. Data represent mean ± SE of experiments performed in triplicate. *p < 0.01, and ^#p < 0.05 by ANOVA, compared with GTG (+), Dex (+), and BDE209 (−) in B, and compared with chimeric receptors (+), ligand (+), and BDE209 (−) in D.

Figure 5

Effect of PBDEs on dendrite arborization of Purkinje cell (17 days in culture). (A,B) Photomicrographs showing the effect of PBDEs on Purkinje cell morphology. BDE209 (10⁻¹⁰ M; A) or BDE47 (10⁻¹⁰ M; B) was added to the culture in the absence or presence of T₄ (10⁻⁸ M), and immunocytochemistry was performed using anti-calbindin antibody to visualize Purkinje cells. Bars = 50 μm. (C,D) Change in dendritic areas of Purkinje cells by BDE209 (C)or BDE47 (D). In C and D, data are expressed as mean ± SE (n = 10 determinations) from one experiment and represent at least three independent experiments. *p < 0.01 by ANOVA, for T₄ (+)/BDE209 (−) compared with T₄ (−)/BDE209 (−). ^#p < 0.01 by ANOVA, for T₄ (+)/BDE209 (+) compared with T₄ (+)/BDE209 (−).

Figure 6

Increasing the concentration of T₄ partially reverses the suppressive effects of BDE209 on Purkinje cell dendrite arborization (17 days in culture). (A) Photomicrographs showing the effect of BDE209 and T₄ on Purkinje cell morphology. BDE209 (10⁻¹⁰ M) was added to the culture in the absence or presence of T₄ (10⁻⁸ M to 10⁻⁶ M), and immunocytochemistry was performed using anti-calbindin antibody to visualize Purkinje cells. Bars = 50 μm. (B) Effect of BDE209 and T₄ on dendritic area of Purkinje cells. Data are mean ± SE (n = 10 determinations) from one experiment and represent at least three independent experiments. *p < 0.01, and ^#p < 0.05 by ANOVA, compared with T₄ (10⁻⁸ M)/BDE209 (−).