Estrogenic and anti-estrogenic assessment of the flame retardant, 2-ethylhexyl diphenyl phosphate (EHDPP), and its metabolites: Evidence from in vitro, in silico, and transcriptome studies.

2-Ethylhexyl diphenyl phosphate (EHDPP) is a replacement flame-retardant commonly found in several environmental matrices and human biospecimens. Although some adverse effects of EHDPP have been identified, the endocrine-disrupting effects of EHDPP and its key metabolites on the human estrogen receptor (ER) are largely unknown. Herein, we report for the first time that EHDPP, at concentrations found in the environment and humans, significantly promoted estrogenic activity and synergized with 17β-estradiol-induced ER transactivation. However, two major EHDPP metabolites 2-ethyl-3-hydroxyhexyl diphenyl phosphate (3-OH-EHDPP) and 2-ethyl-5-hydroxyhexyl diphenyl phosphate (5-OH-EHDPP), inhibited the ER through a non-competitive binding mechanism. Molecular docking showed that Pi-Pi stacking, hydrogen, and hydrophobic bonds primarily stabilize intermolecular interactions between EHDPP and the binding pockets of human ERα and ERβ. Moreover, transcriptome analysis confirmed the estrogenic effects of EHDPP, revealing notable enrichments in ER-mediated signaling and breast cancer pathways, consistent with the validated estrogenic gene expression profile. Intriguingly, EHDPP markedly promoted the clonogenic growth of two ER+ breast cancer cell lines, corroborating the expression levels of ERα protein. Our findings indicate that the common flame-retardant EHDPP activates the ER and downstream signaling, providing far-reaching implications for environmental and health risks associated with estrogen-related adversities such as the development of ER+ breast cancer.