Identifying targets and mechanisms of endocrine disruptor-induced sex-specific toxicity: Integration of network toxicology and medaka experimental evidence.

Endocrine-disrupting chemicals (EDCs), including di(2-ethylhexyl) phthalate (DEHP) and propylparaben (PrP), are widespread environmental contaminants with adverse effects on reproductive health. However, the mechanisms underlying their sex-specific toxicity, particularly under combined exposure, remain incompletely understood. In this study, experimental toxicology was integrated with network-based analyses to investigate the reproductive toxicity of DEHP and PrP in Japanese medaka (Oryzias latipes), with particular emphasis on the brain-gonad-liver axis. Following 28 days of exposure, DEHP exposure reduced the gonadosomatic index (GSI) by 60.45% and increased gonadal 17β-estradiol (E2) and testosterone (T) levels to 203.57 pg/g and by 2.39-fold for females, respectively (p < 0.05). In males, DEHP exposure increased gonadal E2 and T concentrations to 87.52 and 38.73 pg/g, respectively, whereas combined exposure increased hepatic VTG levels by 1.37-fold. In contrast, hepatic VTG concentrations decreased by 735.45 ng/g in DEHP-exposed females (p < 0.05). Network toxicology analysis identified PTGS2, BCL2, EGFR, ESR1, and IL10 as major DEHP-associated targets, whereas PrP was predicted to affect AKT1 and STAT3 through the EGFR tyrosine kinase inhibitor resistance pathway. Alterations in hormone levels and gene expression across the brain, gonads, and liver indicated systemic dysregulation of the hypothalamic-pituitary-gonadal axis. Despite their distinct molecular targets, DEHP and PrP converged on shared pathways, including chemical carcinogenesis-receptor activation and PI3K/Akt signaling. These findings demonstrate sex-specific disruption of the brain-gonad-liver axis by DEHP and PrP and highlight the importance of considering combined EDC exposure in reproductive hazard assessment.