Prenatal exposure to di-n-butyl phthalate promotes RIPK1-regulated necroptosis of uroepithelial cells and induces hypospadias through the epithelial-mesenchymal transition process in newborn male rats

IF 4.8 3区医学 Q1 PHARMACOLOGY & PHARMACY Toxicology Pub Date : 2024-10-24 DOI:10.1016/j.tox.2024.153982

Lei Wu , Zhiwen Xie , Tiewen Li , Xincan Chen , Juntao Jiang , Fei Shi , Yongqing Zhang , Xinyu Xu , Shujie Xia , Wenlan Sun

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Abstract

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.

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产前暴露于邻苯二甲酸二正丁酯会促进 RIPK1 调节的尿路上皮细胞坏死，并通过上皮-间质转化过程诱发新生雄性大鼠尿道下裂。

母体接触邻苯二甲酸二正丁酯（DBP）与尿道下裂的诱发有关，但其根本机制仍不清楚。据报道，坏死与发育畸形有关。本研究旨在探究尿道下裂发生的坏死机制。将 DBP 溶解在玉米油中，从妊娠第 14 天到第 18 天，通过插胃给妊娠大鼠注射精确计量的 DBP 剂量（750 毫克/千克/天）。对照组大鼠只摄入玉米油。雄性尿道下裂大鼠在出生后第 7 天进行鉴定。收集生殖器结节组织并保存在 -80°C 温度下，用于随后的 PCR 分析；在液氮中冷冻保存，用于 Western 印迹；或在福尔马林中固定，用于免疫组织化学 (IHC) 染色。IHC染色和Western印迹分析显示，与对照组相比，生殖器结节（GT）组织中RIPK1和坏死标志物的表达增加。此外，在 GT 组织中观察到更高水平的 EMT 和受损的雄激素受体表达。大鼠原发性尿路上皮细胞（PUCs）暴露于浓度增加的 DBP 会导致 ROS 生成增加。在 DBP 培养后，PUC 中坏死标志物和 EMT 表达水平上调。值得注意的是，与单独使用 DBP 处理相比，DBP 与坏死抑制剂 necrostatin-1 结合使用可减少 EMT 标记物的表达和 ROS 的产生。体外研究进一步发现，DBP 诱导的坏死促进了 E-cadherin 通过泛素-蛋白酶体途径在 PUC 中降解。我们的研究结果表明，母体暴露于 DBP 会通过提高 ROS 水平和 EMT 状态来促进尿路上皮细胞的坏死。因此，坏死可能在尿道下裂的发展过程中扮演着重要角色。

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来源期刊

Toxicology 医学-毒理学

CiteScore

7.80

自引率

4.40%

发文量

222

审稿时长

23 days

期刊介绍： Toxicology is an international, peer-reviewed journal that publishes only the highest quality original scientific research and critical reviews describing hypothesis-based investigations into mechanisms of toxicity associated with exposures to xenobiotic chemicals, particularly as it relates to human health. In this respect "mechanisms" is defined on both the macro (e.g. physiological, biological, kinetic, species, sex, etc.) and molecular (genomic, transcriptomic, metabolic, etc.) scale. Emphasis is placed on findings that identify novel hazards and that can be extrapolated to exposures and mechanisms that are relevant to estimating human risk. Toxicology also publishes brief communications, personal commentaries and opinion articles, as well as concise expert reviews on contemporary topics. All research and review articles published in Toxicology are subject to rigorous peer review. Authors are asked to contact the Editor-in-Chief prior to submitting review articles or commentaries for consideration for publication in Toxicology.