Di-(2-ethylhexyl) Phthalate Disturbs Thyroid Hormones Transport In Human Placental Trophoblastic Cells In Vitro

Background:During pregnancy,thyroid hormones（THs）synthesized by the fetus cannot meet the fetus body’s developmental needs.Before birth,the fetal THs are dependent on maternal placental transport and their physiological level is crucial for normal fetal neurodevelopment.Previous studies have shown that di-（2-ethylhexyl）phthalate（DEHP）has endocrine interference as the most common phthalic acid esters（PAEs）.It can disrupt thyroid function and THs homeostasis in pregnant women and fetuses,and affect placental THs transport.However,the specific effects of DEHP exposure on placental THs transport and the underlying mechanism are poorly understood.Objective:To investigate the specific effects of DEHP exposure on placental THs transport and the underlying mechanism.Methods:Human placental trophoblast cells（JEG-3 cells and HTR-8/SVneocells）were cultured in vitro and treated with 0μM,4μM,40μM,100μM,and 400μM DEHP for24 h.The cell seed plate density and appropriate administration time and the cell proliferation curve after DEHP treatment were measured by RTCA,the cell viability was detected by MTT,and the T₃ and T₄ contents in cell culture fluid and cell cytoplasm were detected by electrochemiluminescence.The effects of DEHP treatment on T₃ uptake function and transthyretin（TTR）internalization function of JEG-3 cells and HTR-8/SVneo cells were measured using isotopic labeling and fluorescent labeling,respectively.RT-PCR and Western-blotting were used to detect related mRNA expression and protein levels,and ELISA was used to detect TTR protein levels in culture medium.Results:（1）The most suitable seed plate density for JEG-3 cells and HTR-8/SVneo cells is 1×10⁵cells/ml cells,while the exposure of DEHP at the doses of 0-400μM for 24 h did not affect cell proliferated and cell viability（P>0.05）.（2）The contents of T₃ and T₄ in the medium and cytoplasmic protein of JEG-3 cells and HTR-8/SVneo cells in the 400μM dose group were higher than that of the control group,and the consumption of T₃ and T₄ in the culture medium of JEG-3 cells and HTR-8/SVneo cell was significantly reduced after DEHP treatment（P<0.05）.（3）DEHP treatment did not affect T₃ uptake,MCT8 and OATP1C1 mRNA expression and protein levels in JEG-3 cells and HTR-8/SVneo cells（P>0.05）.（4）DEHP treatment significantly inhibited TTR internalization,and down-regulated TTR mRNA expression and protein levels of JEG-3 cells and HTR-8/SVneo cells in the 100 and 400μM dose group（P<0.05）.（5）DEHP treatment disrupted deiodinases mRNA expression and protein levels of JEG-3 cells and HTR-8/SVneo cells in the 40,100 and 400μM dose group（P<0.05）.（6）DEHP treatment down-regulated thyroid hormone receptors mRNA expression and protein levels of JEG-3 cells and HTR-8/SVneo cells in the 100 and 400μM dose group（P<0.05）.Conclusion:DEHP can inhibit TTR internalization of placental trophoblast cells,down-regulate TTR expression and affect the expression of deiodinases and thyroid hormone receptor in vitro.These may be the mechanisms by which PAEs affect THs transport through placental.,and provide a scientific basis for further study of fetal thyroid hormone changes mediated by the placental thyroid hormone transport system.