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Di(2-ethylhexyl) phthalate (DEHP), an environmental endocrine disruptor, is one of the most common plasticizers and is widely used in various plastic products. DEHP induces apoptosis and oxidative stress and has been shown to have androgenic toxicity. However, the methods to combat DEHP-induced testicular damage and the mechanisms involved remain to be elucidated. In the present study, we used melatonin, which has strong antioxidant properties, to intervene in prepubertal mice and mouse Leydig cells (TM3) treated with DEHP or its metabolite mono(2-ethylhexyl) phthalate (MEHP). The results showed that melatonin protected against DEHP-induced testicular damage in prepubertal mice, mainly by protecting against DEHP-induced structural destruction of the germinal tubules and by attenuating the DEHP-induced decrease in testicular organ coefficients and testosterone levels. Transcriptomic analysis found that melatonin may attenuate DEHP-induced oxidative stress and apoptosis in prepubertal testes. In vitro studies further revealed that MEHP induces oxidative stress injury and increases apoptosis in TM3 cells, while melatonin reversed this damage. In vitro studies also found that MEHP exposure inhibited the expression levels of molecules related to the PI3K/AKT signaling pathway, and melatonin reversed this change. In conclusion, these findings suggest that melatonin protects against DEHP-induced prepubertal testicular injury via the PI3K/AKT signaling pathway, and provide a theoretical basis and experimental rationale for combating male reproductive dysfunction., (© 2023 Wiley Periodicals LLC.)

Di(2-ethylhexyl) phthalate (DEHP) early exposure leads to immature testicular injury, and we aimed to utilize single-cell RNA (scRNA) sequencing to comprehensively assess the toxic effect of DEHP on testicular development. Therefore, we gavaged pregnant C57BL/6 mice with 750 mg/kg body weight DEHP from gestational day 13.5 to delivery and performed scRNA sequencing of neonatal testes at postnatal day 5.5. The results revealed the gene expression dynamics in testicular cells. DEHP disrupted the developmental trajectory of germ cells and the balance between the self-renewal and differentiation of spermatogonial stem cells. Additionally, DEHP caused an abnormal developmental trajectory, cytoskeletal damage and cell cycle arrest in Sertoli cells; disrupted the metabolism of testosterone in Leydig cells; and disturbed the developmental trajectory in peritubular myoid cells. Elevated oxidative stress and excessive apoptosis mediated by p53 were observed in almost all testicular cells. The intercellular interactions among four cell types were altered, and biological processes related to glial cell line-derived neurotrophic factor (GDNF), transforming growth factor-β (TGF-β), NOTCH, platelet-derived growth factor (PDGF) and WNT signaling pathways were enriched after DEHP treatment. These findings systematically describe the damaging effects of DEHP on the immature testes and provide substantial novel insights into the reproductive toxicity of DEHP., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The toxic effect of di(2-ethylhexyl) phthalate (DEHP) on prepubertal testes was examined in this study. We treated 3-week-old male mice with 4.8 mg/kg/day (milligram/kilogram/day) (no observed adverse effect level), 30 mg/kg/day (high exposure dose relative to humans), 100 mg/kg/day (level causing a reproductive system disorder), and 500 mg/kg/day (dose causing a multigenerational reproductive system disorder) of DEHP via gavage. Obvious abnormalities in the testicular organ coefficient, spermatogenic epithelium, and testosterone levels occurred in the 500 mg/kg DEHP group. Ribonucleic acid sequencing (RNA-seq) showed that differentially expressed genes (DEGs) in each group could enrich reproduction and reproductive process terms according to the gene ontology (GO) results, and coenrichment of metabolism pathway was observed by the Reactome pathway analysis. Through the analysis of common genes in the metabolism pathway, we discovered that DEHP exposure at 4.8 to 500 mg/kg or 100 mg/kg caused the same damages to the prepubertal testis. In general, we identified two key transcriptional biomarkers (fatty acid binding protein 3 (Fabp3) and carboxylesterase (Ces) 1d), which provided new insight into the gene regulatory mechanism associated with DEHP exposure and will contribute to the prediction and diagnosis of prepuberty testis injury caused by DEHP., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Di-(2-ethylhexyl) phthalate (DEHP) is an environmental endocrine disruptor that accumulates in organisms in various ways and induces male reproductive system disorders. In this study, we established a testicular injury model by gavage with different concentrations of DEHP. The testes were then collected for RNA sequencing (RNA-seq), and the results were analyzed by bioinformatics and verified by experiments. Our research results show that different concentrations of DEHP interfere with testicular development differently. Weighted gene coexpression network analysis (WGCNA) generated sixteen modules and identified the turquoise module as key. Then, estrogen receptor 1 (ESR1), filamin A (Flna) and Furin were identified as hub genes. qPCR and immunohistochemistry results revealed that all three hub genes were upregulated. We detected the locations of these genes by immunohistochemistry. ESR1 was mainly located in Leydig cells; Flna immunostaining is observed in the Leydig and some germ cells and Furin staining was seen in almost all types of testicular cells. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed enrichment mainly in MAPK signaling pathways, p53 signaling pathways, HIF-1 signaling pathways, protein processing in the endoplasmic reticulum, apoptosis, the cell cycle, RNA degradation, etc. This is the first study using WGCNA to investigate the mechanism of DEHP-induced injury in the prepubertal testis, providing new research angles to further understand the mechanism of DEHP-induced injury in the prepubertal testis., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Di-(2-ethylhexyl) phthalate (DEHP) is the most common phthalate that can affect the male reproductive system. DEHP exposure at the prepubertal stage could lead to the injury of immature testes, but the mechanism has not been fully clarified. In the present study, we elucidated the possible underlying mechanism of DEHP-induced prepubertal testicular injury through stereological analysis and transcriptome profiling. Compared with the control group, the DEHP-treated rats had lower body weight gain and decreased testicular weight and organ coefficient. Moreover, lower serum levels of testosterone and LH were observed in the DEHP group, in contrast to the increased FSH level. Additionally, the serum level of estradiol had no significant difference after DEHP exposure. Stereological analysis showed significant reduction in volumes of most testicular structures, especially in the seminiferous tubule and seminiferous epithelium, along with a vast decrease of spermatogenic cells and obvious structural damages with substantial pathological signs (germ cracks, cytoplasmic vacuolization, sloughing, multinucleated giant cell formation, chromatolysis desquamation and dissolution, pyknosis of nuclei) in the seminiferous tubule upon DEHP exposure at the prepubertal stage. Furthermore, transcriptome profiling identified 5548 differentially expressed genes (DEGs) upon DEHP exposure. Pathway enrichment analysis revealed several crucial signaling pathways related to retinol metabolism, oxidative phosphorylation, steroid hormone biosynthesis, and cell adhesion molecules (CAMs). In addition, seven DEGs selected from RNA-seq data were validated by quantitative real-time polymerase chain reaction (qRT-PCR), and the results showed the same trends as the RNA-seq results. In conclusion, the above findings provide basic morphological data and lay a foundation for systematic research on transcriptome profiling in prepubertal testicular injury induced by DEHP., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Di(2-ethylhexyl) phthalate (DEHP) is a widely-used plasticizer and has long been recognized as an endocrine-disrupting chemical with male reproductive toxicities. DEHP exposure at the prepubertal stage may lead to extensive testicular injury. However, the underlying mechanisms remain to be elucidated. In the present study, we gavaged male C57BL/6 mice with different concentrations of DEHP (0, 250, and 500 mg/kg-bw·d) from postnatal day 22-35, and exposed TM3 Leydig cells with 0, 100, 200, 300, and 400 μM of MEHP (bioactive metabolite of DEHP) for 12-48 h. RNA sequencing was performed both in testicular tissue and TM3 cells. The results showed that DEHP disrupts testicular development and reduces serum testosterone levels in male prepubertal mice. Bioinformatic analysis and experimental verification have revealed that DEHP/MEHP induces cell cycle arrest in TM3 cells and increases apoptosis both in vivo and in vitro. Furthermore, the p53 signaling pathway was found to be activated upon DEHP/MEHP treatment. The inhibition of p53 by pifithrin-α significantly reduced MEHP-induced injuries in TM3 cells. Cumulatively, these findings revealed the involvement of the p53 signaling pathway in DEHP-induced prepubertal testicular injury by promoting cell apoptosis and inhibiting cell proliferation of Leydig cells., (Copyright © 2020 Elsevier B.V. All rights reserved.)

N 6 -methyladenosine (m6A) modification, the most prevalent form of RNA methylation, modulates gene expression post-transcriptionally. Di-(2-ethylhexyl) phthalate (DEHP) is a common environmental endocrine disrupting chemical that induces testicular injury due to the inhibition of the demethylase fat mass and obesity-associated protein (FTO) and increases the m6A modification. How FTO-mediated m6A modification in testicular Leydig cell injury induced by DEHP remains unclear. Here, the TM3 Leydig cell line was treated with mono-(2-ethylhexyl) phthalate (MEHP), the main metabolite of DEHP in the body, as well as FB23-2, an inhibitor of FTO. Decreased levels of testosterone in the culture supernatant, significantly increased apoptosis, and a remarkable upregulation of global m6A modification were found in both TM3 cells treated with MEHP and FB23-2. Transcriptome sequencing showed that both treatments significantly induced apoptosis-associated gene expression. Methylated RNA immunoprecipitation sequencing showed that the Leydig cell injury induced by upregulated m6A modification could be associated with multiple physiological disorders, including histone acetylation, reactive oxygen species biosynthesis, MAPK signaling pathway, hormone secretion regulation, autophagy regulation, and male gonadal development. Overall, the inhibition of FTO-mediated up-regulation of m6A could be involved in MEHP-induced Leydig cell apoptosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)