RIPK1-driven calcium overload and lysosomal-mitochondrial dysfunction induce testicular necroptosis following DEHP exposure

Di-(2-ethylhexyl) phthalate (DEHP) is a plasticizer widely used to enhance the flexibility and durability of plastic products. As an environmental endocrine disruptor, DEHP impairs male reproductive function. Its metabolite, mono-(2-ethylhexyl) phthalate (MEHP), mediates many toxic effects, but the mechanisms remain unclear. We hypothesized that DEHP induces testicular necroptosis through MEHP-mediated calcium overload and the RIPK1-regulated lysosomal-mitochondrial axis. Here, we reveal this novel mechanism. This study investigated DEHP-induced testicular damage, focusing on necroptosis and calcium (Ca²⁺) signaling pathways. Sprague-Dawley rats were exposed to 250 and 750 mg/kg DEHP for 5 weeks. Testicular damage was assessed via histopathology, testosterone measurement, and RNA sequencing (RNA-seq). A common Sertoli cell line was treated with MEHP to study Ca²⁺ overload, lysosomal membrane permeabilization (LMP), mitochondrial dysfunction, and necroptosis. Pharmacological inhibitors were employed to explore pathway involvement, including CA-074 Me (cathepsin B inhibitor), BAPTA-AM (Ca²⁺ chelator), and Nec-1 (RIPK1 inhibitor). DEHP caused testicular damage, including seminiferous tubule disorganization and reduced plasma testosterone. RNA-seq revealed necroptosis pathway enrichment, with upregulated RIPK1, RIPK3, MLKL, and PGAM5. MEHP induced Ca²⁺ overload, LMP, and mitochondrial dysfunction in Sertoli cells. CA-074 Me attenuated mitochondrial damage, while BAPTA-AM mitigated LMP. Nec-1 suppressed necroptosis-related proteins and restored blood-testis barrier integrity by upregulating ZO-1, Cx-43 and Claudin-11. DEHP exposure induced testicular necroptosis via MEHP-mediated Ca²⁺ overload-lysosomal-mitochondrial axis, regulated by RIPK1. These findings provide insights into DEHP reproductive toxicity.