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Maternal exposure to di-n-butyl phthalate (DBP) has been linked to the induction of hypospadias; however, the underlying mechanism remains unclear. Necroptosis is reported to be implicated in developmental malformations. This study aimed to investigate the underlying mechanism of necroptosis in the development of hypospadias. DBP was dissolved in corn oil, and pregnant rats were administered a precisely measured dose of DBP (750 mg/kg/day) via gastric intubation from gestation day 14-18. Control rats received only corn oil. The day of birth was considered postnatal day (PND) 1. Male hypospadias rats were identified on PND 7. Genital tubercle tissues were collected and stored at -80°C for subsequent PCR analysis, cryopreserved in liquid nitrogen for western blot, or fixed in formalin for immunohistochemistry (IHC) staining. IHC staining and western blot analysis revealed increased expression of RIPK1 and necroptosis markers in genital tubercle (GT) tissue compared to the control group. Additionally, higher levels of EMT and impaired androgen receptor expression were observed in GT tissue. Exposure to increased DBP concentrations in rat primary uroepithelial cells (PUCs) led to elevated ROS production. Necroptosis markers and EMT expression levels were upregulated in PUCs following DBP incubation. Notably, treatment with DBP combined with necrostatin-1, a necroptosis inhibitor, reduced the expression of EMT markers and ROS production compared to DBP treatment alone. In vitro studies further revealed that DBP-induced necroptosis promoted the degradation of E-cadherin through the ubiquitin-proteasome pathway in PUCs. Our findings suggest that maternal exposure to DBP promotes necroptosis in uroepithelial cells by elevating ROS level and EMT status. Thus, necroptosis may play an essential role in the development of hypospadias., 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 © 2024 Elsevier B.V. All rights reserved.)

Prenatal exposure to dibutyl phthalate (DBP) has been reported to cause erectile dysfunction (ED) in adult offspring rats. However, its underlying mechanisms are not fully understood. Previously, we found that DBP activates the RhoA/ROCK pathway in the male reproductive system. This study investigated how prenatal exposure to DBP activates the RhoA/ROCK signalling pathway, leading to ED in male rat offspring. Pregnant rats were stratified into DBP-exposed and NC groups, with the exposed group receiving 750 milligrams per kilogram per day (mg/kg/day) of DBP through gavage from days 14-18 of gestation. DBP exposure activated the RhoA/ROCK pathway in the penile corpus cavernosum (CC) of descendants, causing smooth muscle cell contraction, fibrosis, and apoptosis, all of which contribute to ED. In vitro experiments confirmed that DBP induces apoptosis and RhoA/ROCK pathway activation in CC smooth muscle cells. Treatment of DBP-exposed offspring with the ROCK inhibitor Y-27632 for 8 weeks significantly improved smooth muscle cell condition, erectile function, and reduced fibrosis. Thus, prenatal DBP exposure induces ED in offspring through RhoA/ROCK pathway activation, and the ROCK inhibitor Y-27632 shows potential as an effective treatment for DBP-induced ED., 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 © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Early exposure to dibutyl phthalate (DBP) can cause hypospadias in newborn foetuses. However, the underlying molecular mechanism is not well defined. Aberrant angiogenesis is associated with various dysplasias including urogenital deficits. In vivo and in vitro angiogenesis assays showed reduced angiogenesis in the hypospadias group and DBP exposed group. RNA-sequencing analysis of DBP-treated HUVECs revealed decreased expression of transforming growth factor beta 1-induced transcript 1 (TGFB1I1) and a significantly enriched angiogenesis-associated pathway. Further experiments revealed that decreased TGFB1I1 expression was associated with disrupted tube formation and migration, which resulted in decreased angiogenesis. Functional assays revealed that the overexpression of TGFB1I1 promoted tube formation and migration of HUVECs in the DBP-treated group. Moreover, we showed that the transcription factor AR was regulated by TGFB1I1 through inhibiting its translocation from the cytoplasm to the nucleus. Together, our results identified TGFB1I1 as a component of aberrant angiogenesis in hypospadias rats and its interaction with AR might be a potential target for hypospadias development., 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 © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Di-n-butyl phthalate (DBP) is a plasticizer commonly used in industrial production and is present in our daily life. It has been confirmed that DBP causes genitourinary malformations, especially hypospadias. However, the research of hypospadias mainly focusses on the genital tubercle in previous studies. In this study, we found DBP could affect the exocrine function of the vascular endothelium which disturb the development of genital nodules and induced hypospadias. We used cytokine array to find that vascular endothelium-derived NAP-2 may be a major abnormal secreted cytokine with biological functions. The transcriptomic sequencing analysis showed that abnormal activation of the RhoA/ROCK signaling pathway was the main reason for increased NAP-2 secretion. The expression levels of epithelial-mesenchymal transition (EMT) biomarkers and NAP-2 in hypospadias animal models were detected with Immunohistochemistry, Western blot, Immunofluorescence, and ELISA methods. The expression levels of NAP-2, RhoA/ROCK signaling pathway related proteins, reactive oxygen species (ROS) levels in HUVEC cells, EMT biomarkers and migration capacity of urothelial cells cocultured with HUVEC were measured with ELISA, flow cytometry, Western blot or Transwell assay for further cell experiments. The results showed that DBP leaded to NAP-2 oversecretion from vascular endothelium mainly rely on the activation of RhoA/ROCK signaling pathway and ROS accumulation. The RhoA/ROCK inhibitor fasudil could partially decrease ROS production, and both fasudil and N-acetyl-L-cysteine (NAC) could decrease NAP-2 secretion. Meanwhile, the oversecretion of NAP-2 from HUVEC in coculture system promoted EMT and migration capacity of urothelial cells, and TGF-β inhibitor LY219761 could block the aberrant activation of EMT process. Therefore, it could be concluded that DBP increase NAP-2 secretion from vascular endothelium by RhoA/ROCK/ROS pathway, and further promote EMT in urothelial cells through TGF-β pathway. This study provided a novel direction for studying the occurrence of hypospadias and may provide a hypospadias predictive marker in the future., 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 The Authors. Published by Elsevier Inc. All rights reserved.)

Dibutyl phthalate (DBP) is an endocrine disruptor, which causes male reproductive dysfunction in rodents. Previous researches demonstrated that DBP exposure impaired spermatogenesis, however, the molecular mechanism is largely uncovered. In this study, we demonstrated that prenatal exposure to DBP increased receptor activator of nuclear factor-κB ligand (RANKL) expression in seminiferous tubules, especially in Sertoli cells. Western blot and immunofluorescence assays showed that DBP induced up-regulation of RANKL expression in Sertoli cells. Furthermore, experimental data showed that DBP increased COX-2 and p-p65 expression in Sertoli cells and depleting COX-2 and p-p65 by specific inhibitor NS3-98 and BAY117082 could partially abolish DBP induced up-regulation of RANKL. Moreover, the content of RANKL in Sertoli cells was significantly increased after DBP exposure by conducting enzyme linked immunosorbent assay (ELISA), which promoted spermatogonial stem cells (C18-4 cells) apoptosis in a paracrine manner. Together, our data demonstrated that a novel mechanism for DBP induced impairment of spermatogenesis by activating NF-κB/COX-2/RANKL signaling in Sertoli cells, and provided a diagnostic and therapeutic target., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The objective of this study was to establish a hypospadiac rat model by maternal exposure to di-n-butyl phthalate (DBP) and to evaluate the developmental abnormalities of hypospadiac male rats. Timed-pregnant rats were given DBP by gastric intubation at doses of 0, 250, 500, 750 or 1000 mg/kg body weight (bw)/day from gestation day (GD) 14 to 18 to establish a hypospadiac rat model. The hypospadias was observed in the 500 and 750 mg/kg bw/day groups, the incidence of which was 6.8 and 41.3%, respectively. Transverse serial histological analysis of genitalia of hypospadiac male rats confirmed the malformation. With exposed dose increasing, the serum testosterone (T) levels of male rats inversely decreased, and in the same dosage group the serum T levels of hypospadiac rats were significantly lower than the levels of nonhypospadiac counterparts. The other reproductive lesions such as cryptorchidism and decreased ratio of anogenital distance/body weight (AGD/bw) were also observed. Autopsy analysis revealed the development of reproductive organs (prostate, testes, epididymis, pituitary gland) and nonreproductive organs (adrenal gland, liver, kidney, heart, spleen) of hypospadiac rats and nonhypospadiac counterparts. The results indicated that the reproductive system and developmental condition of hypospadiac male offspring were damaged severely by DBP.

Background: Studies have demonstrated that Sertoli cells are the direct target of Dibutyl phthalate (DBP). However, the role of neurotransmitter receptors is not elucidated. Methods: Based on our previous studies, maternal Sprague–Dawley (SD) rats in Gestation Day (GD) 14–18 and TM4 cells exposure to 750 mg/kg/day and 100 μM DBP were regarded as treated groups. Firstly, qRT-PCR array was used to determine the different expression of neurotransmitter receptors. We examined the OX1R expression on Rats in Control and DBP groups by immunohistochemistry. Real-time PCR and Western Blot were used to detect the protein and mRNA expression levels of OX1R in vivo and in vitro. The potential downstream signaling pathways were explored by analyzing the GSE99690 cohort. In addition, we extracted Primary Sertoli Cells (PSCs) from the testis of control group. The apoptosis-related proteins, AKT signaling pathway-related proteins and mRNA expressions were detected by Western Blot and Real-time PCR in PSCs. The validity of PSCs was measured by CCK-8 assay and flow cytometric analysis was used to demonstrate the apoptotic rates of PSCs after DBP exposure. Results: The Orexin receptor 1 (OX1R) was screened out by qRT-PCR array. Our results showed that DBP could significantly suppress the OX1R expression of Sertoli cells in vivo and in vitro. Functional analysis showed the AKT signaling pathway was mediated by OX1R. The highly expressed apoptosis level and impaired cell activity were observed in PSCs, which can be reversed by Orexin A. Meanwhile, the p-AKT signaling pathway were hindered after DBP exposure while rescued in DBP + Orexin-A group. Conclusions: DBP can induce Sertoli cell apoptosis through its toxicological effect by suppressing OX1R and p-AKT expression, which provide a novel insight on the role of neurotransmitter receptors. [ABSTRACT FROM AUTHOR]