Endocrinol Metab.  2021 Apr;36(2):447-454. 10.3803/EnM.2020.920.

DEHP Down-Regulates Tshr Gene Expression in Rat Thyroid Tissues and FRTL-5 Rat Thyrocytes: A Potential Mechanism of Thyroid Disruption

• Kim MJ
• Kim HH
• Song YS
• Kim OH
• Choi K
• Kim S
• Oh BC
• Park YJ

• KMID: 2515470
• DOI: http://doi.org/10.3803/EnM.2020.920

Abstract

Background
Di-2-ethylhexyl phthalate (DEHP) is known to disrupt thyroid hormonal status. However, the underlying molecular mechanism of this disruption is unclear. Therefore, we investigated the direct effects of DEHP on the thyroid gland.
Methods
DEHP (vehicle, 50 mg/kg, and 500 mg/kg) was administered to Sprague-Dawley rats for 2 weeks. The expression of the thyroid hormone synthesis pathway in rat thyroid tissues was analyzed through RNA sequencing analysis, quantitative reverse transcription-polymerase chain reaction (RT-PCR), and immunohistochemical (IHC) staining. DEHP was treated to FRTL-5 rat thyroid cells, and an RT-PCR analysis was performed. A reporter gene assay containing the promoter of thyroid stimulating hormone receptor (TSHR) in Nthy-ori 3-1 human thyroid cells was constructed, and luciferase activity was determined.
Results
After DEHP treatment, the free thyroxine (T4) and total T4 levels in rats significantly decreased. RNA sequencing analysis of rat thyroid tissues showed little difference between vehicle and DEHP groups. In the RT-PCR analysis, Tshr expression was significantly lower in both DEHP groups (50 and 500 mg/kg) compared to that in the vehicle group, and IHC staining showed that TSHR expression in the 50 mg/kg DEHP group significantly decreased. DEHP treatment to FRTL-5 cells significantly down-regulated Tshr expression. DEHP treatment also reduced luciferase activity in a reporter gene assay for TSHR.
Conclusion
Although overall genetic changes in the thyroid hormone synthesis pathway are not clear, DEHP exposure could significantly down-regulate Tshr expression in thyroid glands. Down-regulation of Tshr gene appears to be one of potential mechanisms of thyroid disruption by DEHP exposure.

Keyword

Diethylhexyl phthalate; Endocrine disruptors; Thyroid gland; Receptors; thyrotropin

Figure

• 

  Fig. 1 Serum thyroid hormones in di-2-ethylhexyl phthalate (DEHP)-treated rats. After DEHP treatments (vehicle, 50 mg/kg, or 500 mg/kg DEHP) for 2 weeks, thyroid function test was performed with obtained rat blood (n=6 to 12). (A) Free thyroxine (T4), (B) total T4, (C) free triiodothyronine (T3), (D) total T3, (E) thyroid stimulating hormone (TSH). One-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test was performed. conc, concentration. aP<0.05 compared to the vehicle group.

• 

  Fig. 2 mRNA sequencing data of rat thyroid tissues treated with di-2-ethylhexyl phthalate (DEHP) for 2 weeks. (A) Principal component analysis. (B) Heatmap of genes related to thyroid hormone synthesis and thyroid differentiation. (C) Box plots of genes related to thyroid hormone synthesis and thyroid differentiation. PC, principal component.

• 

  Fig. 3 Expression of gene related to thyroid hormone synthesis in rat thyroid tissues (n=6 to 12) treated with di-2-ethylhexyl phthalate (DEHP) for 2 weeks. (A) Reverse transcription-polymerase chain reaction (RT-PCR) analysis. (B) Representative image of thyroid stimulating hormone receptor (TSHR) immunohistochemical (IHC) staining (1:100) and quantification of TSHR positive area per total area (%). (C) Representative image of sodium iodide symporter (NIS) IHC staining (1:100) and quantification of TSHR positive area per total area (%). One-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test was performed. conc, concentration. aP<0.05; bP<0.01.

• 

  Fig. 4 Expression of gene related to thyroid hormone synthesis in thyroid cells treated with di-2-ethylhexyl phthalate (DEHP). (A) mRNA expression level of genes related to thyroid hormone synthesis (Tshr, Slc5a5, Tg, and Tpo) in FRTL-5 cells treated with DEHP for 24 hours. (B) Thyroid stimulating hormone receptor (TSHR) luciferase reporter assay. Luciferase activity in Nthy-ori 3-1 cells treated with DEHP for 24 hours. aP<0.05; bP<0.01.

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