Di-(2-ethylhexyl) phthalate induces spermatogenic dysfunction via p38/NCOA4-dependent ferritinophagy and ferroptosis in Sertoli cells

Di-(2-ethylhexyl) phthalate (DEHP) is a ubiquitous environmental endocrine disruptor that leaches from polyvinyl chloride products and metabolizes to mono-(2-ethylhexyl) phthalate (MEHP), causing spermatogenic dysfunction in males.However, the molecular mechanism linking DEHP/MEHP to Sertoli cell damage remains unclear. This study aimed to clarify the role of p38/NCOA4-dependent ferritinophagy in DEHP-induced ferroptosis and validate potential therapeutic targets. Prepubertal male KM mice were exposed to DEHP (250, 500, 1000 mg/kg/day) via oral gavage for 28 days (cyclophosphamide as positive control). Mouse Sertoli TM4 cells were treated with MEHP (200–800 μM) alone or combined with Ferrostatin-1 (Fer-1, ferroptosis inhibitor), dihydrocaffeic acid (DHCA, p38 inhibitor), or NCOA4 siRNA. Testicular histopathology, sperm parameters, serum testosterone, biochemical markers (GSH, Fe²⁺, MDA, ROS), and protein/mRNA expression (NCOA4, FTH1, SLC7A11, GPX4, p-p38) were analyzed. DEHP dose-dependently reduced testicular index, epididymal sperm count, serum testosterone, and Sertoli cell number (all P < 0.05). MEHP induced ferroptosis in TM4 cells, characterized by GSH depletion, Fe²⁺ and MDA accumulation, and ROS overproduction. Concurrently, MEHP upregulated NCOA4 and p38 phosphorylation, while downregulating FTH1, SLC7A11, and GPX4 (all P < 0.05). Fer-1 rescued ferroptosis but not NCOA4/FTH1 expression; NCOA4 knockdown or DHCA inhibited ferritinophagy, restored ferroptosis-related proteins, and attenuated cytotoxicity. These findings demonstrate that DEHP induces Sertoli cell ferroptosis via the p38/NCOA4/ferritinophagy axis. This novel mechanism provides insights into DEHP reproductive toxicity and identifies p38/NCOA4 as potential targets for mitigating pollutant-induced male infertility.