Changes of mTOR autophagy pathway and apoptosis in hypospadiac fetal rats induced by di (2-ethylhexyl) phthalate

Abstract

Objective To explore the mechanism of hypospadias induced by di(2-ethylhexyl) phthalate (DEHP) in rats. Methods Twenty adult Sprague-Dawley (SD) rats [aged 10-12 weeks, weight (240±20) g] were randomly divided into 2 groups of control (n=10) and DEHP exposure group (n=10). The animals received oil and 750 mg/kg DEHP by gavage from gestation day (GD) 8.5 to GD 18.5 respectively.Genital tubercles (GTs) were collected from male fetuses on GD 19.5.Anogenital distance (AGD) and anogenital index (AGI) of male fetuses were measured and gross imaging changes of GTs observed by scanning electron microscope (SEM) and hematoxylin eosin staining.The level of autophagy in GTs was examined by transmission electron microscope (TEM) and autophagy-related proteins (LC3, p62, Beclin1, Akt, mTOR, p-Akt, p-mTOR) and apoptotic-related proteins (cleaved-caspase 3, Bax & Bcl2) in GTs were analyzed by Western blot.And apoptosis in GTs was measured by TUNEL. Results Compared with control group, the incidence of DEHP-induced hypospadias was 27.58% in male fetuses while AGD and AGI decreased in hypospadias group (P<0.05). SEM and HE staining revealed abnormal fusion of urethral folds and ectopia of urethral opening.Autophagosome was found in hypospadias group, but not in control group.Compared with non-hypospadias, the protein expression of LC3 and ratio of LC3Ⅱ/LC3Ⅰsignificantly increased in GTs of hypospadiac rats (P<0.05) while p62 expression decreased (P<0.05). The expressions of cleaved-caspase3 and Bax increased while Bcl2 decreased (P<0.05). Apoptotic cells significantly decreased in GTs of hypospadiac rats (P<0.05). Conclusions DEHP is absorbed by body as an exogenous disruptor and Akt/mTOR signaling pathway becomes activated in GTs, Phosphorylations of Akt and mTOR decrease and enhanced autophagy leads to an imbalance of apoptosis and anti-apoptotic system in GTs.With an abnormal increase of apoptosis, it causes the occurrence of hypospadias. Key words: Hypospadias; Apoptosis; Autophagy; Diethylhexyl Phthalate