Di(2-ethylhexyl) phthalate induces male reproductive toxicity through mitophagy-dependent ferroptosis of spermatocytes in mice.

Di(2-ethylhexyl) phthalate (DEHP), a widely utilized plasticizer, impairs male reproductive function; however, the precise mechanisms underlying this effect have yet to be fully elucidated. This study investigates DEHP-induced spermatocyte toxicity and identifies therapeutic strategies. Integrated network toxicology and proteomics delineated testicular toxicity mechanisms through multi-dimensional analyses. We demonstrate that DEHP exposure induces spermatocyte ferroptosis via PINK1/Parkin-mediated mitophagy. Mechanistically, the bioactive metabolite MEHP promotes NRF2 degradation through the ubiquitin-proteasome pathway, inducing excessive mitochondrial clearance. This process mediates mitochondrial Fe2+ efflux, causing iron dysregulation and lipid peroxidation. Pharmacological inhibition of mitophagy by CsA attenuated ferroptosis and restored iron homeostasis, confirming ferroptosis dependence on mitophagic activation. Crucially, NRF2 activation concurrently suppresses both mitophagic flux and ferroptotic execution. MEHP-induced NRF2 degradation initiates pathological mitophagy and facilitates mitochondrial iron efflux, resulting in dysregulated iron metabolism within spermatocytes. This cascade culminates in spermatocyte ferroptosis mediated by Fe2+ accumulation and lipid peroxidation. This work provides definitive evidence linking environmental toxicant-induced mitophagy to germ cell ferroptosis, identifies NRF2 as a central regulator of this pathway, and proposes targeted mitophagy inhibition combined with NRF2 stabilization as therapeutic interventions.