6PPD and 6PPD Quinone Induce Endometrial Cell Dysfunction via Activating ERα and GPER at Human-Relevant Levels

The widespread environmental prevalence of tire-derived N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) and 6PPD-quinone (6PPD-Q) has provoked public concern about their health risks. This study aimed to investigate the potential of 6PPD and 6PPD-Q to induce endometrial cell dysfunction through nuclear estrogen receptor (ER) and G-protein-coupled estrogen receptor (GPER) signaling pathways. Fluorescence competitive binding and reporter gene assays revealed that 6PPD and 6PPD-Q selectively bound to ERα (not ERβ) and activated ER transcriptional activity, with the lowest observed effective concentrations (LOECs) of 500 and 10 nM, respectively. Calcium mobilization assays further demonstrated that both 6PPD and 6PPD-Q activated the GPER nongenomic pathway in a concentration-dependent manner (LOEC = 1 nM). 6PPD-Q exhibited stronger ERα and GPER activation potency than 6PPD, which was explained well by molecular dynamics simulation. 6PPD and 6PPD-Q stimulated endometrial cell proliferation via ERα/GPER signaling pathways, mechanistically linked to Cyclin D1/Ki67 upregulation. Furthermore, 6PPD/6PPD-Q promoted endometrial cell migration through an ERα/GPER-regulated epithelial-mesenchymal transition and inflammatory responses. Notably, the LOECs for these functional disruptions reached nanomolar levels relevant to human exposure. Collectively, we elucidated the molecular initial events and downstream key events of 6PPD/6PPD-Q-induced endometrial cell dysfunction, which implied their threat to the reproductive system and provided novel perspectives for their health risk evaluation.