Multi-omics revealed activation of TNF-α induced apoptosis signaling pathway in testis of DEHP treated prepubertal male rat

Di-(2-ethylhexyl) phthalate (DEHP) exposure has been associated with male reproductive damage, but the mechanisms involved remain incompletely defined. This study aims to investigate the effects of DEHP exposure on the testes of prepubertal rats through an integrative analysis of metabolomics and transcriptomics, combined with molecular experiments. DEHP exposure resulted in decreased testis weight and increased oxidative stress level in the testis tissues of prepubertal male rats. Moreover, our findings showed a disordered testis structure, reduced spermatogenic and Sertoli cells as well as destruction of mitochondria structure in the testis tissues of DEHP-treated prepubertal male rats. Transcriptome function analysis together with metabolome function analysis indicated that spermatogenesis, apoptosis, inflammatory, lipid metabolism as well as DNA repair signaling pathway were enriched in the testis of DEHP-treated prepubertal male rats. The integrative omics analysis further suggested that TNF-α induced apoptosis played a crucial role in mediating the detrimental effects of DEHP exposure on the testis of prepubertal rats, which was validated by ELISA, Western blotting and Tunel assays. Validation experiments conducted in vitro using GC-2 cells corroborated these findings, demonstrating that mono-(2-ethylhexyl) phthalate (MEHP), the main active metabolite of DEHP, significantly inhibits cell proliferation and increases apoptosis via activating the TNF-α apoptosis pathway. Overall, these findings provided a novel mechanism of dysregulated spermatogenesis of DEHP exposure on the testes of prepubertal rats.