Di-tert-Butylphenol (2,4-DTBP) and its analogs disrupt thyroid hormone signaling via activation of retinoid X receptor α

Di -tert- butylphenol derivatives, including 2,4-di -tert- butylphenol (2,4-DTBP) and its analogs, are commonly used synthetic phenolic antioxidants and have been widely detected in food, environmental matrices, and human samples. In this study, we systematically investigated the disruptive effects of 2,4-DTBP and its analogs on the thyroid hormone (TH) system using molecular, cellular, and in vivo approaches, and elucidated their underlying mechanisms of action. At the molecular level, luciferase reporter assays, coactivator recruitment experiments, and molecular docking analyses demonstrated that 2,4-DTBP, 1,3,5-tri -tert- butylbenzene (1,3,5-TTBB), and 2,4,6-tri -tert- butylbenzene (2,4,6-TTBP) act as agonists of retinoid X receptor α (RXRα). At the cellular level, proliferation assays in rat pituitary GH3 cells showed that these compounds interfere with the TR/RXRα heterodimer signaling pathway by activating RXRα rather than directly targeting the thyroid hormone receptor (TR). At the in vivo level, zebrafish embryo exposure experiments demonstrated that 2,4,6-TTBP disrupts TH levels and the expression of TH-related genes, and transcriptomic analysis further revealed perturbations in multiple key biological processes. Collectively, these findings identify TH-disrupting effects of 2,4-DTBP and its analogs and reveal a novel mechanism mediated through RXRα rather than TR. Furthermore, potential health risks of 2,4-DTBP and 2,4,6-TTBP were evaluated using the hazard quotient approach, indicating that both compounds may pose TH-disrupting risks at current human exposure levels. This study provides mechanistic insights and toxicological evidence supporting the environmental risk assessment of 2,4-DTBP and its analogs.