2. Academic Validation
  3. Di-n-butyl phthalate induces oversecretion of vascular endothelium-derived NAP-2 and promotes epithelial-mesenchymal transition of urothelial cells in newborn hypospadias rats

Di-n-butyl phthalate induces oversecretion of vascular endothelium-derived NAP-2 and promotes epithelial-mesenchymal transition of urothelial cells in newborn hypospadias rats

  • Ecotoxicol Environ Saf. 2023 Apr 12;256:114892. doi: 10.1016/j.ecoenv.2023.114892.
Shan Hua 1,  Fei Shi 1,  Zhiwen Xie 1,  Lei Wu 2,  Mengqiao Dai 3,  Yongqing Zhang 1,  Xinyu Xu 1,  Yiping Zhu 4,  Juntao Jiang 5
Affiliations

Affiliations

  • 1 Department of Urology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, China.
  • 2 Department of Urology, Shanghai General Hospital of Nanjing Medical University, Shanghai 200080, China.
  • 3 Shanghai University of Traditional Chinese Medicine, School of Nursing, Shanghai 201203, China.
  • 4 Department of Urology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, China. Electronic address: zhuypurologist@163.com.
  • 5 Department of Urology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, China. Electronic address: jjturologist@126.com.
PMID: 37059017 DOI: 10.1016/j.ecoenv.2023.114892
Abstract

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.

Keywords

Dibutyl phthalate; Endothelial cells; Epithelial-mesenchymal Transition; Hypospadias; Neutrophil-activating peptide 2.

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