Di-2-ethylhexylphthalate promotes thyroid cell proliferation and DNA damage through activating thyrotropin-receptor-mediated pathways in vitro and in vivo

Phthalates are being suggested to be associated with altered thyroid function and proliferative changes, but detailed mechanisms remain unclear. Here, we examined the effects of di-(2-ethylhexyl) phthalate (DEHP) on DNA damage and proliferation in thyroid using thyroid carcinoma cell line, 8505C, in vitro and the rats orally treated with DEHP at 0, 0.3, 3, 30 and 150 mg/kg for 90 days from post-natal day 9 in vivo. Exposure to DHEP (1-50 mu M) induced cellular proliferation, as evidenced by increased cell viability and DNA synthesis. Activation of gamma H2AX, a sensitive biomarker for DNA damage was observed following the exposure to DHEP (from 5 to 50 mu M) with increased comet tail moment (5-100 mu M) in comet assay, reflecting that DNA damage also occurred. When upstream signaling was examined, both thyrotropin receptor (TSHR)-ERK1/2 axis and TSHR-AKT axis were activated with upregulation of Pax8, a master transcriptional factor for thyroid differentiation and proliferation. Thyroid tissue from juvenile rats orally exposed to DEHP also confirmed DNA damage responses and the activation of TSHR signaling, which was evident from 0.3 to 3 mg/kg respectively. Notably, deletion of TSHR through siRNA attenuated these DEHP-induced events in vitro. Collectively these results suggest that DEHP induces DNA damage and cellular proliferation in thyroid, which appears to be from TSHR activation, providing an important insight into endocrine disrupting activities of phthalates on thyroid.