Engineering Research Center of Groundwater Pollution Control and Remediation, Ministry of Education, College of Water Sciences, Beijing Normal University, Beijing 100875, China.
South China Institute of Environmental Science, Ministry of Environmental Protection, No.7 West Street, Yuancun, Guangzhou 510655, China.
Chemosphere. 2018 Dec;212:1058-1066. doi: 10.1016/j.chemosphere.2018.09.007. Epub 2018 Sep 3.
Di-n-butylphthalate (DnBP) exhibits alarming thyroid disrupting activities. However, the toxic mechanism of DnBP is not completely understood. In this study, we investigated the mechanism of DnBP in thyroid disruption. Rat pituitary tumor cell lines (GH3) were treated with DnBP in different scenarios, and cell viabilities, target gene transcriptions and protein levels were measured accordingly. The results showed that after treatment with DnBP (20 μmol/L), cell proliferation increased to 114.69% (p < 0.01) and c-fos gene was up-regulated by 1.57-fold (p < 0.01). Both nuclear thyroid hormone receptor β (TRβ) and membrane TR (integrin α and integrin β) genes were up-regulated by 1.31-, 1.08- and 2.39-fold (p < 0.01), respectively, the latter was inhibited by Arg-Gly-Asp (RGD) peptides; the macromolecular DnBP-BSA was unable to bind nuclear TRs, but still promoted cell proliferation to 104.18% and up-regulated c-fos by 2.99-fold (p < 0.01); after silencing TRβ gene, cell proliferation (106.64%, p < 0.05) and up-regulation of c-fos (1.23-fold, p < 0.01) were also observed. All of these findings indicated the existence of non-genomic pathway for DnBP-induced thyroid disruption. Finally, DnBP activated the downstream extracellular regulated protein kinases (ERK1/2) pathway, up-regulating Mapk1 (1.15-, p < 0.05), Mapk3 (1.26-fold, p < 0.01) and increasing protein levels of p-ERK (p < 0.01); notably, DnBP-induced ERK1/2 activation along with c-fos up-regulation were attenuated by PD98059 (ERK1/2 inhibitor). Taken together, it could be suggested that integrin αβ and ERK1/2 pathway play significant roles in DnBP-induced thyroid disruption, and this novel mechanism warrants further investigation in living organisms.
邻苯二甲酸二正丁酯（DnBP）表现出令人震惊的甲状腺破坏活性。然而，DnBP 的毒性机制尚不完全清楚。在这项研究中，我们研究了 DnBP 破坏甲状腺的机制。用不同浓度的 DnBP 处理大鼠垂体瘤细胞系（GH3），并相应地测量细胞活力、靶基因转录和蛋白质水平。结果表明，用 DnBP（20μmol/L）处理后，细胞增殖增加到 114.69%（p<0.01），c-fos 基因上调 1.57 倍（p<0.01）。核甲状腺激素受体 β（TRβ）和膜 TR（整合素 α 和整合素 β）基因分别上调 1.31、1.08 和 2.39 倍（p<0.01），后者被 Arg-Gly-Asp（RGD）肽抑制；大分子 DnBP-BSA 不能与核 TR 结合，但仍能促进细胞增殖至 104.18%，并将 c-fos 上调 2.99 倍（p<0.01）；沉默 TRβ 基因后，细胞增殖（106.64%，p<0.05）和 c-fos 上调（1.23 倍，p<0.01）也观察到。所有这些发现表明 DnBP 诱导的甲状腺破坏存在非基因组途径。最后，DnBP 激活下游细胞外调节蛋白激酶（ERK1/2）通路，上调 Mapk1（1.15，p<0.05）、Mapk3（1.26 倍，p<0.01），并增加 p-ERK 的蛋白水平（p<0.01）；值得注意的是，DnBP 诱导的 ERK1/2 激活以及 c-fos 上调被 PD98059（ERK1/2 抑制剂）减弱。总之，整合素 αβ 和 ERK1/2 通路在 DnBP 诱导的甲状腺破坏中起重要作用，这一新机制值得在生物体中进一步研究。
