Mp21-12 Maternal Exposure to Dinbutyl Phthalate Promotes Ripk1regulated Necroptosis of Uroepithelial Cells Through Ampkmtor Signaling Pathway in Newborn Male Hypospadias

You have accessJournal of UrologyPediatrics II (MP21)1 May 2024MP21-12 MATERNAL EXPOSURE TO DINBUTYL PHTHALATE PROMOTES RIPK1REGULATED NECROPTOSIS OF UROEPITHELIAL CELLS THROUGH AMPKMTOR SIGNALING PATHWAY IN NEWBORN MALE HYPOSPADIAS Lei Wu Lei WuLei Wu View All Author Informationhttps://doi.org/10.1097/01.JU.0001008844.84871.17.12AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: We previously demonstrated that maternal exposure to di-n-butyl phthalate (DBP) resulted in hypospadias in male offspring, but the underlying mechanisms remain elusive. Necroptosis is a type of programmed cell death with necrotic morphology, occurring in a variety of biological processes, including inflammation, immune response, embryonic development and metabolic abnormalities. However, the underlying mechanism governing this effect has not been thoroughly elucidated to date. METHODS: DBP (99.5% pure, Sigma Chemical Co., St. Louis, MO, USA) was dissolved in corn oil. Pregnant rats were given a precisely weighed dose of DBP (750 mg/kg/day) by gastric intubation during gestation day 1418. Control rats received only corn oil. The dose of DBP was based on previous publication to effectively induce hypospadias in offspring which resembles the malformation seen in humans. All solutions were freshly prepared before administration. The day of birth was considered postnatal day (PND) 1. On PND 7, male rats and hypospadiac rats were determined as we previously reported. There were 52 male rats from 10 litters in DBP-treated group. In these male rats, there were 22 hypospadiac rats. These rats were identified with numbers ranging from 1 to 22. Eight rats from the pool of 22 hypospadias male rats were randomly assigned into experimental groups using random number table. Similarly, eight rats from the pool of oil-exposed normal rats came from a total of 10 litters were randomly assigned into control group. All animals were anesthetized with pentobarbital sodium (50 mg/kg, i.p.) to collect blood samples before sacrifice. At the end of experiments, animals were euthanized with carbon dioxide (70%/30%, CO2/O2). Genital tubercle tissues were harvested and stored in at −80 °C in RNAlater for subsequent PCR analysis, cryopreserved in liquid nitrogen for Western blotting, or stored in formalin for IHC staining. RESULTS: We hypothesized that DBP exposure induces RIPK1 expression and abnormal activation of necroptosis in uroepithelial cells. Pregnant rats received DBP orally at a dose of 850 mg/kg BW/day during gestational days 14–18. In the DBP-exposed group, immunohistochemistry (IHC) staining and western blot showed increased expression of RIPK1 and necroptosis markers genital tubercle (GT) tissue compared to the control. In rat uroepithelial cells (svHUC), ROS production increased expression levels of RIPK1 and subsequently contributed to the induction of necroptosis. Notably, DBP exposure also promoted AMPK/mTor signaling pathway. In addition, lower testosterone levels and androgen receptor (AR) expression in GT tissue were detected. In vitro studies revealed that impaired AR signaling was involved in DBP-induced necroptosis and autophagy activation furthermore promoted EMT in urethral epithelial cells. DBP combined with necrostatin-1, an necroptosis inhibitor, reduced the expression of necroptosis markers compared with DBP treatment alone. CONCLUSIONS: Taken together, our findings suggest that maternal exposure to DBP promotes necroptosis in uroepithelial cells via upregulation of AMPK/mTor signaling pathway and necroptosis may play important roles in hypospadias development. Source of Funding: National Science Foundation of China © 2024 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 211Issue 5SMay 2024Page: e334 Advertisement Copyright & Permissions© 2024 by American Urological Association Education and Research, Inc.Metrics Author Information Lei Wu More articles by this author Expand All Advertisement PDF downloadLoading ...