Integrative computational toxicology and experimental validation unveils ESR1-mediated steroidogenic disruption in triphenyl phosphate-induced female reproductive toxicity.

Triphenyl phosphate (TPHP), an organophosphorus flame retardant, has emerged as a significant environmental concern due to its persistence and potential health hazards. However, its role in female reproductive toxicity remains poorly understood, necessitating systematic evaluation of its molecular mechanisms. This study integrated network toxicology, transcriptomics, machine learning, adverse outcome pathway (AOP) framework, molecular docking, and in vitro experiments to systematically investigate TPHP-induced reproductive toxicity. Network analysis identified 24 TPHP-infertility shared targets and seven hub genes (AR, CYP19A1, ESR1, ESR2, IGF1, INS, STAR) linked to steroidogenesis and estrogen signalling. Transcriptomic analysis of TPHP-treated KGN cells (GSE241995) revealed downregulated oocyte maturation pathways. Machine learning prioritized ESR1 as the core gene, supported by exploratory external evaluation (AUC = 0.94) in an endometrial dataset (GSE272778). Based on ESR1 prioritization, suppression of estrogen receptor activity was defined as the molecular initiating event (MIE), linking TPHP exposure to downstream STAR suppression, reduced mitochondrial cholesterol transport, impaired progesterone synthesis, and disrupted oocyte maturation. Molecular docking supported the plausibility of TPHP-induced ER activity perturbation. In vitro experiments showed that TPHP significantly suppressed STAR protein expression and progesterone secretion at non-cytotoxic concentrations, whereas total ESR1 protein abundance was not significantly reduced. ESR1 knockdown reduced STAR expression and progesterone production, and TPHP caused no further suppression, supporting an ESR1-related, STAR-dependent mechanism. These findings provide a machine learning-assisted AOP framework and identify ESR1-related steroidogenic disruption as a plausible mechanism of TPHP-induced female reproductive toxicity.