Benzo(a)Pyrene (BaP) is one of the most widespread polycyclic aromatic hydrocarbons (PAHs) with endocrine disrupting properties and carcinogenic effects. In the present study, we tested the effect of BaP on thyroid development and function, using zebrafish as a model system. Zebrafish embryos were treated with 50 nM BaP from 2.5 to 72 h post fertilization (hpf) and compared to 1.2% DMSO controls. The expression profiles of markers of thyroid primordium specification, thyroid hormone (TH) synthesis, hypothalamus-pituitary-thyroid (HPT) axis, TH transport and metabolism, and TH action were analyzed in pools of treated and control embryos at different developmental stages. BaP treatment did not affect early markers of thyroid differentiation but resulted in a significant decrease of markers of TH synthesis (tg and nis) likely secondary to defective expression of the central stimulatory hormones of thyroid axis (trh, tshba) and of TH metabolism (dio2). Consequently, immunofluorescence of BaP treated larvae showed a low number of follicles immunoreactive to T4. In conclusion, our results revealed that the short-term exposure to BaP significantly affects thyroid function in zebrafish, but the primary toxic effects would be exerted at the hypothalamic-pituitary level thus creating a model of central hypothyroidism.
Mutations in thyroid hormone receptor α (TRα), a ligand-inducible transcription factor, cause Resistance to Thyroid Hormone α (RTHα). This disorder is characterised by tissue-specific hormone refractoriness and hypothyroidism, due to inhibition of target gene expression by mutant TRα-corepressor complexes. Using biophysical approaches, we show that RTHα-associated TRα mutants devoid of ligand-dependent transcription activation function, unexpectedly retain the ability to bind thyroid hormone. Visualisation of ligand (T3) within the crystal structure of a prototypic TRα mutant, validates this notion. This finding prompted synthesis of different thyroid hormone analogues, identifying a lead compound (ES08) which dissociates corepressor from mutant human TRα more efficaciously than T3. ES08 rescues developmental anomalies in a zebrafish model of RTHα and induces target gene expression in TRα mutation-containing cells from an RTHα patient, more effectively than T3. Our observations provide proof-of-principle for developing synthetic ligands that can relieve transcriptional repression by the mutant TRα-corepressor complex, for treatment of RTHα.
<b><i>Introduction:</i></b> Resistance to thyroid hormone β (RTHβ) is an inherited syndrome caused by dominant negative variants in the <i>THRB</i> gene (NM_000461.5). The clinical picture of RTHβ is variable, and patients harboring the same variant may display different degrees of disease severity. <b><i>Case Presentation:</i></b> A 30-year-old man presented with thyrotoxicosis and central hyperthyroidism and was found to have a novel variant in the exon 10 of <i>THRB</i> gene (c.C1282G, p.L428V), located within the third hot spot region of the C-terminal of the receptor. Surprisingly, the same variant was found in two other relatives with an apparent normal thyroid function at initial screening. After exclusion of a TSH-secreting adenoma and serum interference in the proband, and the finding that exogenous levothyroxine failed to suppress the TSH in the brother affected by nodular goiter, relatives’ thyroid function tests (TFTs) were reassessed with additional analytical method revealing biochemical features consistent with RTHβ in all carriers of the p.L428V variant. Functional studies showed a slightly impaired in vitro transcriptional activity of p.L428V. Interestingly‚ the expression of the human p.L428V thyroid hormone receptor beta in the zebrafish embryo background generated a phenotype consistent with RTHβ. <b><i>Conclusion:</i></b> Variable results of TFTs on some immunoassays can be a cause of RTHβ diagnostic delay, but the genotype-phenotype correlation in this family and functional studies support p.L428V as a novel <i>THRB</i> variant expanding the spectrum of gene variants causing RTHβ. In vivo, rather than in vitro, functional assays may be required to demonstrate the dominant negative action of <i>THRB</i> variants.
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