Di-(2-ethylhexyl) phthalate exposure leads to ferroptosis via the HIF-1α/HO-1 signaling pathway in mouse testes

Di-(2-ethylhexyl) phthalate (DEHP) is an extensively used plasticizer and has been shown to cause reproductive dysfunction in humans and model animals. However, the exact mechanisms of testicular injury induced by DEHP exposure have not been fully clarified. Using gas chromatography-mass spectrometry, we found that mono-2-ethylhexyl ester (MEHP, a major biometabolite of DEHP) and DEHP concentrations were elevated in mouse serum after DEHP exposure. Using RNA-seq, we found that ferroptosis and HIF-1 signaling pathways might be involved in testicular injury due to prepubertal DEHP exposure. Subsequent Western blotting, ferrous iron and MDA measurements, and immunofluorescence of testicular sections verified the RNA-seq findings. Consistently, based on the RNA-seq findings, we found that ferroptosis and HIF-1 signaling pathways might play crucial roles in Leydig and Sertoli cell injury due to MEHP exposure in vitro. Further experiments also confirmed ferroptosis in Leydig and Sertoli cells. Using Western blotting, cellular immunofluorescence and ChIP-qPCR, we found that MEHP exposure caused HIF-1α accumulation and stabilization, resulted in HIF-1α translocation into the nucleus, and induced HIF-1α/Hmox1 binding in Leydig and Sertoli cells. To clarify whether HIF-1α plays a pivotal role in MEHP-induced ferroptosis, we knocked out Hif-1α using the CRISPR/Cas9 technique. We found that Hif-1α knockout rescued MEHP-induced ferroptosis. In summary, our findings certified that prepubertal DEHP exposure led to ferroptosis in mouse testes via the HIF-1α/HO-1 signaling pathway.