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As the most abundantly used phthalate derivative, di-(2-ethylhexyl) phthalate (DEHP) leads to reproductive disorders, especially in males. Testicular injury can be triggered when the testis is exposed to DEHP during the immature stage. However, the potential mechanism is largely unclear. In the present study, Sprague-Dawley rats were exposed to 0, 250 and 500 mg/kg/day DEHP from postnatal day (PND) 20 to PND 30. The spermatogonia cell line GC-1 and spermatocyte cell line GC-2 were exposed to different doses of monoethylhexyl phthalate (MEHP), a metabolite of DEHP. Testicular injury was observed. Oxidative stress was evaluated both in vivo and in vitro. Our results showed that after DEHP exposure, the testicular structure was damaged and spermatogenesis was disturbed. We also found that oxidative stress was increased, as indicated by the upregulation of the important factors in the antioxidant pathway. Furthermore, the expression of autophagy-related proteins was significantly downregulated. Autophagy inhibition led to activation of the pyroptosis pathway. Nucleotide-binding and oligomerisation (NOD) domain-like receptor (NLR) family pyrin domain (PYD)-containing 3 (NLRP3), Caspase-1 and cytokine interleukin-1β (IL-1β) were significantly upregulated. Additionally, an imbalance in self-renewal and differentiation was observed in germ cells after DEHP exposure, causing the cessation of germ cell development. In summary, these data suggest that DEHP exposure enhances oxidative stress, downregulates autophagy, induces NLRP3 inflammasome activation and subsequently triggers pyroptosis in vivo and in vitro, which provides novel insight into DEHP-related injury in immature testes in the context of pyroptosis., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Di‐(2‐ethylhexyl) phthalate (DEHP), a widely used plasticizer, has been shown to cause reproductive toxicity, but the precise mechanism remains unclear. This study aimed to investigate the possible molecular mechanism of DEHP‐induced testicular damage. In vivo study, we administered different doses of DEHP (0, 250, and 500 mg/kg/day) to male C57BL/6 mice from 22 and 35 days after birth. We found that DEHP exposure induced histopathological alterations in prepubertal testes, and testicular lipidomics indicated notable alterations in lipid metabolism and significant enrichment of ferroptosis. Further tests showed that ferrous iron (Fe2+) and malondialdehyde (MDA) levels significantly increased after DEHP exposure. Western blotting revealed that DEHP exposure reduced glutathione peroxidase 4 (GPX4) expression, and elevated acyl coenzyme A synthetase long‐chain member 4 (ACSL4) and lysophosphatidylcholine acyltransferase 3 (LPCAT3) expression. The in vitro results were consistent with the in vivo results. When Leydig cells and Sertoli cells were treated with ferrostatin‐1 and monoethylhexyl phthalate (MEHP), MEHP‐induced increases in Fe2+ and MDA levels, accumulation of lipid reactive oxygen species, downregulation of GPX4, and upregulation of ACSL4 and LPCAT3 were reversed. Collectively, our findings suggested that aberrant lipid metabolism and ferroptosis may be involved in prepubertal DEHP exposure‐induced testicular damage. [ABSTRACT FROM AUTHOR]

Difenoconazole (DFZ) is a broad-spectrum triazole fungicide that is widely utilized in agriculture. Although DFZ has been demonstrated to induce reproductive toxicity in aquatic species, its toxic effects on the mammalian reproductive system have yet to be fully elucidated. In vivo, male mice were administered 0, 20 or 40 mg/kg/d of DFZ via oral gavage for 35 days. Consequently, DFZ significantly decreased testicular organ coefficient, sperm count and testosterone levels, augmented sperm malformation rates, and elicited histopathological alterations in testes. TUNEL assay showed increased apoptosis in testis. Western blotting results suggested abnormally high expression of the sperm meiosis-associated proteins STRA8 and SCP3. The concentrations of retinoic acid (RA), retinaldehyde (RE), and retinol (ROL) were increased in the testicular tissues of DFZ-treated groups. The mRNA expression level of genes implicated in RA synthesis significantly increased while genes involved in RA catabolism significantly decreased. In vitro, DFZ reduced cell viability and increased RA, RE, and ROL levels in GC-2 cells. Transcriptome analysis revealed a significant enrichment of numerous terms associated with the RA pathway and apoptosis. The qPCR experiment verified the transcriptome results. In conclusion, our results indicate that DFZ exposure can disrupt RA signaling pathway homeostasis, and induce testicular injury in mice testes. [ABSTRACT FROM AUTHOR]

Di-(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer that has been shown to impair male reproduction, but the potential mechanism underlying testicular injury caused by DEHP remains unclear. In vivo, rats were gavaged consecutively from postnatal day (PND) 21 to PND 31 with 0, 250, or 500 mg/kg DEHP for 10 days, and impaired mitochondria and increased necroptosis were observed in immature testes. In vitro, the GC-1 and GC-2 cell lines were exposed to monoethylhexyl phthalate (MEHP) at 100, 200 and 400 μM for 24 h, and this exposure induced oxidative stress damage, necroptosis and mitochondrial injury. Necroptosis and mitochondrial fission were inhibited by the reactive oxygen species (ROS) inhibitor acetylcysteine, and the imbalanced mitochondrial dynamics were rescued by the RIPK1 inhibitor necrostatin-1. Colocalization and co-IP analyses confirmed an interaction between dynamin-related protein 1 (DRP1) and phosphoglycerate mutase 5 (PGAM5), indicating that PGAM5 dephosphorylates DRP1 at serine 637 to induce mitochondrial fragmentation and thereby induces germ cell damage. Drug prediction with Connectivity Map (cMap) identified sulforaphane as a therapeutic drug. In summary, our findings indicate that DEHP triggers necroptosis and mitochondrial injury via a ROS storm in immature testes and that the PGAM5–DRP1 interaction is involved in this process. [Display omitted] • Necroptosis-induced mitochondrial damage caused DEHP-induced testicular injury. • ROS may be the driving forces for necroptosis and imbalanced mitochondrial dynamics. • PGAM5 promote mitochondrial fission via dephosphorylation of DRP1 S637. • CMap identified sulforaphane as a potential therapeutic drug. [ABSTRACT FROM AUTHOR]

Copper oxide nanoparticles (CuONPs) are metallic multifunctional nanoparticles with good conductive, catalytic and antibacterial characteristics that have shown to cause reproductive dysfunction. However, the toxic effect and potential mechanisms of prepubertal exposure to CuONPs on male testicular development have not been clarified. In this study, healthy male C57BL/6 mice received 0, 10, and 25 mg/kg/d CuONPs by oral gavage for 2 weeks (postnatal day 22–35). The testicular weight was decreased, testicular histology was disturbed and the number of Leydig cells was reduced in all CuONPs-exposure groups. Transcriptome profiling suggested steroidogenesis was impaired after exposure to CuONPs. The steroidogenesis-related genes mRNA expression level, concentration of serum steroids hormones and the HSD17B3-, STAR- and CYP11A1-positive Leydig cell numbers were dramatically reduced. In vitro, we exposed TM3 Leydig cells to CuONPs. Bioinformatic analysis, flow cytometry analysis and western blotting analysis confirmed that CuONPs can dramatically reduce Leydig cells viability, enhance apoptosis, trigger cell cycle arrest and reduce cell testosterone levels. U0126 (ERK1/2 inhibitor) significantly reversed TM3 Leydig cells injury and testosterone level decrease induced by CuONPs. These outcomes indicate that CuONPs exposure activates the ERK1/2 signaling pathway, which further promotes apoptosis and cell cycle arrest in TM3 Leydig cells, and ultimately leads to Leydig cells injury and steroidogenesis disorders. [Display omitted] • CuONPs decrease the number of Leydig cells in the prepubertal testis. • CuONPs perturb steroidogenesis in vivo and in vitro. • CuONPs induce apoptosis and cell cycle arrest in TM3 Leydig cells. • CuONPs activate the ERK1/2 pathway leading to TM3 Leydig cells injury. • U0126, ERK1/2 inhibitor, can reverse Leydig cell injury and testosterone levels. [ABSTRACT FROM AUTHOR]