Multiple bioanalytical methods reveal a thyroid-disrupting mechanism related to the membrane receptor integrin αvβ3

Di(2-ethylhexyl) phthalate (DEHP), a manufactured chemical, is suitable for large-scale production and has extensive applications. Although restricted for use, DEHP is still ubiquitous in the environment and shows potential to disrupt the structure or function of the thyroid system. However, its toxic mechanism is complex and not clearly understood. In this study, a battery of methods was employed to investigate DEHP-induced thyroid-disrupting effects and their mechanism of action, focusing on a newly discovered membrane receptor-mediated mechanism. The results showed that DEHP promoted rat pituitary tumor (GH3) cell proliferation and c-fos gene expression at environment level concentrations (2 and 5 μmol/L) in a manner similar to that of the natural thyroid hormone 3,3′,5-triiodo-L-thyronine (T3). The macromolecule DEHP-BSA cannot pass through the cell membrane to interact with nuclear receptors but upregulated the c-fos gene expression when administered at concentrations comparable to DEHP concentrations; molecular docking demonstrated that DEHP has affinity for the membrane receptor integrin αvβ3; DEHP at 2 μmol/L upregulated the β3 gene expression in GH3 cells; after the addition of integrin αvβ3-inhibiting RGD peptide, DEHP-induced c-fos gene upregulation decreased. All of these findings support the supposition that DEHP-induced thyroid-disrupting effects might be mediated by the membrane receptor integrin αvβ3. Moreover, DEHP activated the downstream extracellular regulated protein kinase (ERK1/2) pathway, upregulating the gene expression of raf-1, MEK-1 and MAPK1 and increasing the protein levels of p-ERK; interestingly, ERK1/2 activation and c-fos upregulation induced by DEHP were attenuated by PD98059 (an ERK1/2 inhibitor). Taken together, the data suggest that the membrane receptor integrin αvβ3 and the downstream ERK1/2 pathway might be involved in DEHP-induced thyroid-disrupting effects. This study provides new insight into the thyroid-disrupting effect and the underlying mechanism and will advance the effort to construct adverse outcome pathways of DEHP and other thyroid hormone disrupting chemicals.