In December 2021, Shengde Wu’s group from the Department of Urology, Children's Hospital of Chongqing Medical University, published in a paper at Journal of Hazardous Materials, entitled Di-(2-ethylhexyl) phthalate exposure leads to ferroptosis via the HIF-1α/HO-1 signaling pathway in mouse testes. The primary author of the paper is Yuhao Wu.

Through analyzing DEGs that were related to the HIF-1 signaling pathway, researchers found that Egln1 and Egln3, which encode PHDs, were downregulated after MEHP exposure. PHD complexes generally degrade HIF-1α, suggesting the possible accumulation and stabilization of HIF-1α. To verify the hypothesis, RT-qPCR and Western blotting were conducted. RT-qPCR revealed that after MEHP exposure, the expression of Hif-1α and Hif-1β was upregulated, and the expression of Egln1, Egln2, and Egln3 was downregulated in Leydig and Sertoli cells. By isolating the cytoplasmic and nuclear proteins of Leydig and Sertoli cells, it is found that the expression of nuclear HIF-1α was upregulated, but cytoplasmic HIF-1α was downregulated after MEHP exposure. They also found that HIF-1α translocated into the nucleus after MEHP exposure. ChIP-qPCR identified that HIF- 1α could bind to the promoter of Hmox1, and this combination was induced by MEHP exposure. Altogether, MEHP exposure caused HIF-1α accumulation and stabilization, resulted in HIF-1α translocation into the nucleus, and induced HIF-1/Hmox1 binding in Leydig and Sertoli cells.

Through the above experiments, researchers found a gene related to testicular injury, HIF-1α, and initially discovered its regulatory mechanism. To further determine whether MEHP-induced ferroptosis in a HIF-1α- dependent manner, they knocked out Hif-1α in Leydig and Sertoli cells for subsequent experiments. Deletion of Hif-1α in TM3 Leydig and TM4 Sertoli cell lines was accomplished by Cyagen. Using Sanger sequencing and PCR, we verified the successful knockout of Hif-1α in Leydig and Sertoli cell lines. Western blotting also verified successful knockout of Hif-1α at the protein level.

Compromised cell viability under MEHP exposure was ameliorated after Hif-1α knockout in Leydig cells and Sertoli cells . Meanwhile, lipid peroxidation (Fig. 1 B, I) and ferrous iron overload (Fig. 1 C, J), as a result of MEHP exposure, were also inhibited after Hif-1α knockout. Since MEHP exposure might induce ferroptosis via HIF-1α/Hmox1 binding, the expression of Hmox1 and HO-1 levels was also assessed using qPCR and Western blotting, respectively. They found that the Hif-1α knockout reversed the upregulation of Hmox1 (Fig. 1 D, K) and HO-1 levels (Fig. 1 G, N) due to MEHP exposure. In addition, ROS burst (Fig. 1 E, L) and cell death (Fig. 1 F, M) after MEHP exposure were also attenuated after Hif-1α knockout. Western blotting showed that the Hif-1α knockout restored the upregulation of ACSL4, FTH1, and SLC7A11, and downregulation of GPX4 (Fig. 7 G, N). Overall, these results showed that MEHP exposure led to ferroptosis in a HIF-1α-dependent manner in Leydig and Sertoli cells.

Figure 1. HIF-1α knockout rescued MEHP-induced ferroptosis.

This study revealed that the HIF-1α regulatory gene is related to testicular cell dysfunction through a combination of MEHP exposure phenotype analysis, RNA-Seq analysis, and ChIP-qPCR analysis, etc. They found its regulatory mechanism, MEHP (a major bioactive metabolite of DEHP in vivo), can inhibit PHDs, affect the accumulation and stabilization of HIF-1α, lead to the translocation of HIF-1α to the nucleus, and induce HIF-1/Hmox1 in Leydig and Sertoli cells. When combined, the expression of HO-1 is upregulated. Overexpression of HO-1 leads to ferrous overload, accumulation of ROS, and ultimately ferroptosis, impairing the activity of Leydig and Sertoli cells. This result was further verified in HIF-1α knockout cell lines.