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.
1. Research Highlights
●In vitro and in vivo experiments verified that DEHP exposure can cause testicular damage.
●MEHP exposure accelerates the death of TM3 Leydig and TM4 Sertoli cells
●Exploring the mechanism of HIF-1 α signaling pathway
●HIF-1α knockout in vitro to verify the function of HIF-1α
In vitro and in vivo experiments verified that DEHP exposure can cause testicular injury
To verify if DEHP exposure caused prepubertal testicular injury, male mice were given DEHP by oral gavage for two weeks before puberty. Then researchers noticed exfoliated germ cells in the tubular lumen and fewer layers of seminiferous tubules through HE staining. They performed RNA-seq to screen differentially expressed genes (DEGs) between the control and non DEHP groups. GO analysis revealed that DEGs were enriched in oxidative stress- and iron metabolism-related GO terms. KEGG analysis revealed that DEGs were enriched in the ferroptosis pathway. In addition, the glutathione metabolism pathway, which is closely related to ferroptosis, was also enriched. Next, the researchers carried out a series of experiments, and the results were consistent with the analysis of RNA-sequencing. Collectively, they found that prepubertal DEHP exposure caused testicular injury, which might involve both ferroptosis and HIF-1 signaling pathways.
MEHP exposure accelerates the death of TM3 Leydig and TM4 Sertoli cells
After verifying the correlation between DEHP and testicular injury in vivo, researchers further verified with in vitro experiments and found that the results were consistent with the in vivo. MEHP (MCE, HY-W018392, USA), a major bioactive metabolite of DEHP in vivo, was used for TM3 Leydig and TM4 Sertoli cell treatment in vitro to gain further insights into the molecular mechanisms of DEHP toxicity. It indicated that MEHP exposure accelerated the death of TM3 Leydig and TM4 Sertoli cells. To discover the underlying mechanisms, they performed RNA-seq to screen DEGs between the control and MEHP groups. Gene ontology (GO) analysis revealed that DEGs were enriched in oxidative stress and iron homeostasis-related GO terms. KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis revealed enrichment of DEGs in ferroptosis and HIF-1 signaling pathways and the glutathione metabolism pathway. Therefore, the in vitro experiment showed that ROS in Leydig cells and Sertoli cells were significantly increased after MEHP exposure, and altered expression patterns of genes related to iron metabolism including Hmox1, increased concentrations of ferrous iron, and abnormal mitochondrial morphology.
HIF-1α affects ferrous iron concentration by regulating the expression of HO-1
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.
HIF-1α knockout in vitro to verify the function of HIF-1α
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.
Rigorous research requires a complete experimental process. Generally, omics analysis is used to scientifically screen genes and obtain various experimental results that need to be mutually verified. Knockout is still one of the routine strategies for gene function research, and a successful knockout cell line is crucial to verify the accuracy of the results.
Figure 2. Prepubescent DEHP exposure leads to ferroptosis in mouse testes via the HIF-1α/ Hmox1 signaling pathway.
Wu Y, et al. Di-(2-ethylhexyl) phthalate exposure leads to ferroptosis via the HIF-1α/HO-1 signaling pathway in mouse testes. 2021, 127807. DOI: https://doi.org/10.1016/j.jhazmat.2021.127807