Wen-Lan Sun

Prenatal exposure to di-n-butyl phthalate induces anorectal malformations in male rat offspring

The objectives of this study were to investigate the dysplasia, histological malformations, and genetic abnormalities in male rats induced by maternal exposure to di-n-butyl phthalate (DBP). Here we report novel findings concerning developmental abnormalities resulting from prenatal exposure to DBP, which leads to significant anorectal malformations (ARMs) in male rat offspring. The incidence of ARMs was 39.5% in male offspring and all abnormal pups were complicated with secondary megacolon. General images, histological analysis and anatomy examination confirmed the malformation. The development abnormalities such as decreased bodyweight (BW) and anogenital distance (AGD), shortened body lengths (with tail removed), as well as increased abdominal circumference were observed at different developmental stages of ARMs in male rat. The developmental abnormalities in both solid organs (brain, heart, liver, spleen, lung and kidney) and reproductive organs (testes and epididymis) of abnormal pubs on PND35 were also investigated. In addition, the serum testosterone (T) level of ARMs in male rats on PND1 was significantly lower than that of controls with accompanying reduced expression of androgen receptor (AR), sonic hedgehog (Shh) and bone morphogenetic protein 4 (Bmp4) mRNA from tissues of the terminal rectum. These results conclusively demonstrate for the first time that in utero exposure to DBP leads to an increased likelihood for the development of ARMs and subsequent complicating megacolon in male rat offspring.

The role of androgen-induced growth factor (FGF8) on genital tubercle development in a hypospadiac male rat model of prenatal exposure to di-n-butyl phthalate.

Fibroblast growth factor 8 (FGF8) is an androgen-induced growth factor (AIGF) that is crucial for embryonic development. This study was developed to investigate the role of FGF8 in developmental abnormalities of the genital tubercle (GT) in hypospadiac male rats when prenatally exposed to di-n-butyl phthalate (DBP). DBP was administered to timed-pregnant rats to establish the hypospadiac rat model where the incidence of hypospadias in male offspring was 43.6%. On postnatal day (PND) 7, decreased mRNA and protein expression levels for androgen receptor (AR) and FGF8 were observed in the GT of hypospadiac rats. Decreased serum testosterone (T) levels were observed in groups displaying hypospadias, which was confirmed using histological analysis. Further anatomical examination using digital photography helped to reveal visualized expression of dysplasia in organs strongly associated with hypospadias. In addition, changes in body weight (BW) and anogenital distance (AGD) were recorded, showing definitive decreases. Collectively, these data clearly demonstrate an interaction between androgen and FGF8, which might play an important role in the occurrence of hypospadias and abnormal organ development induced by DBP.

Prenatal exposure to di-n-butyl phthalate (DBP) differentially alters androgen cascade in undeformed versus hypospadiac male rat offspring

This study was to compare the alterations of androgen cascades in di-n-butyl phthalate (DBP)-exposed male offspring without hypospadias (undeformed) versus those with hypospadias. To induce hypospadias in male offspring, pregnant rats received DBP via oral gavage at a dose of 750mg/kg BW/day during gestational days 14-18. The mRNA expression levels of genes downstream of the androgen signaling pathway, such as androgen receptor (AR) and Srd5a2, in testes of undeformed rat pups were similar to those in controls; in hypospadiac rat pups these levels were significantly lower than those of control pups. In contrast, both undeformed and hypospadiac rats had decreased serum testosterone levels, reduced mRNA expression of key enzymes in the androgen synthetic pathway in the testes, and ablated genes of developmental pathways, such as Shh, Bmp4, Fgf8, Fgf10 and Fgfr2, in the genital tubercle (GT) as compared to those in DBP-unexposed controls, albeit hypospadiac rats had a more severe decrement than those of undeformed rats. Although other possibilities cannot be excluded, our findings suggest that the relatively normal levels of testosterone-AR-Srd5a2 may contribute to the resistance to DBP toxicity in undeformed rats. In conclusion, our results showed a potential correlation between decreased testosterone levels, reduced mRNA expression of AR and Srd5a2 and the occurrence of hypospadias in male rat offspring prenatally exposed to DBP.

Maternal exposure to di-n-butyl phthalate (DBP) induces combined anorectal and urogenital malformations in male rat offspring.

Anorectal malformations in combination with hypospadias (ARMs & hypospadias) are a type of complex congenital malformations. The underlying mechanisms of this deformity are largely unknown. In this study, we comprehensively characterized the dysplasia, histological malformations, and genetic changes of ARMs & hypospadias in male rats after maternal exposure to di-n-butyl phthalate (DBP) by gastric intubation at doses of 850mg/kg bw/day during GD11-15. On postnatal day 1, anatomical and histopathological analysis confirmed combined malformations of the genital tubercle (GT), terminal rectum (TR) and testes. DBP-induced dysplasia was also seen in the kidney, lung, spleen, heart and liver of ARMs & hypospadias male rats. Moreover, decreased levels of serum testosterone, as well as reduced expression of genes related to the androgen signaling pathway (Cyp11a1, Hsd3b, Scarb1, Star, AR, Srd5a2) were found in the testes of ARMs & hypospadias male rats after DBP exposure as compared to untreated controls. Further, decreased mRNA levels of Shh, Fgf10, Gli2, Gli3, Bmp4, Wnt5a, Hoxa13, Hoxd13, Fgfr2 and AR were observed in TR and GT in the ARMs & hypospadias group. These results provide evidence that prenatal exposure to DBP can lead to combined anorectal and urogenital malformations as well as dysplasia of the testes.

Reduced Fgf10/Fgfr2 and androgen receptor (AR) in anorectal malformations male rats induced by di-n-butyl phthalate (DBP): A study on the local and systemic toxicology of DBP.

Previous study have demonstrated that not only the anorectal development but also the general conditions of anorectal malformations (ARMs) male rats are severely affected by di-n-butyl phthalate (DBP) maternal exposure. However, the mechanisms underlying DBP-induced congenital defects remain elusive. Reportedly, Fgf10/Fgfr2 and androgen receptor (AR) are pivotal for the development of multiple organs. In this study, we therefore investigated the expression of Fgf10/Fgfr2 together with AR in the terminal rectum and multiple organs of ARM male rats induced by in utero exposure to DBP. DBP was administered to pregnant rats to establish the model and the incidence of ARMs in male offspring was 39.5%. On postnatal day(PND)1, the gross photograph and histopathological staining confirmed the abnormal manifestations in these organs of newborn ARMs. Decreased anogenital distance, body weight and serum testosterone level were observed in ARM male offspring. The reduced expression of Fgf10/Fgfr2 mRNA and protein was seen in terminal rectum and kidney, spleen, liver, heart in ARM male rats, whereas the reduced expression of AR was only observed in the kidney and terminal rectum. Our findings suggest the potential involvement of altered Fgf10/Fgfr2 signaling and AR in pathogenesis of local and systemic development defects in ARMs male rats induce by DBP.

Genetic research and structural dysplasia assessment of anorectal malformations in neonatal male rats induced by di(n-butyl) phthalate.

This study was the first to investigate the genetic abnormalities and structural dysplasia of anorectal malformations (ARMs) in male rats induced by di(n‐butyl) phthalate (DBP). DBP was administered to timed‐pregnant rats to establish the ARM rat model. The incidence of ARMs in male offspring was 39.5%. In neonatal period, decreased body weight and anogenital distance were observed. The general image and histological analysis of male offspring confirmed the presence of ARMs. Anatomical examination of the ARM male rats revealed the dysplasia in solid organs (heart‐lung, liver, spleen, and kidney). The decreases of serum testosterone concentration and androgen receptor expression in terminal rectum were indicative of the antiandrogenic effects of DBP. Moreover, significant decreased mRNA expressions of these androgen‐related genes such as sonic hedgehog, Gli2, Gli3, bone morphogenetic protein 4, Wnt5a, Hoxa13, Hoxd13, fibroblast growth factor 10, and fibroblast growth factor receptor 2 were found in terminal rectum of the ARM male pubs. These results demonstrated that development of ARM rats was impaired by maternal exposure to DBP. The antiandrogenic effects of DBP disturbing the androgen‐related signaling networks might play an important role in the occurrence of ARMs.

Maternal exposure to di-n-butyl phthalate (DBP) induces renal fibrosis in adult rat offspring

This study was to determine the impact of maternal exposure to di-n-butyl phthalate (DBP) on renal development and fibrosis in adult offspring. Pregnant rats received DBP at a dose of 850 mg/kg BW/day by oral perfusion during gestational days 14–18. In DBP exposed newborn offspring, gross observation and histopathological examination revealed the dysplasia of kidney. The expression of genes related to renal development was also changed. In DBP exposed adult offspring, histopathological examination and Massons trichrome staining revealed the pathological changes of renal fibrosis. Furthermore, higher expression levels of transforming growth factor- β (TGF-β) and alpha-smooth muscle actin (α-SMA) were also detected. In vitro studies reveal that DBP promoted the activation of NRK49F cells and G2/M arrest in NRK52E cells at a sublethal dose. The effect of DBP on these cell lines was linked to the generation of oxidative stress. In addition, DBP induced oxidative stress in both renal fibroblasts and tubular epithelial cells, whereas vitamin C ameliorated the changes caused by DBP. In conclusion, our results showed that prenatal exposure to DBP may generate oxidative stress in both renal fibroblasts and tubular epithelial cells, leading to kidney dysplasia and renal fibrosis.

Expression of Wnt5a during development of anorectal malformations in a rat model of prenatal exposure to di(n-butyl) phthalate.

Abstract Mounting evidence has indicated the crucial role of Wnt5a in the embryonic development including guts. However, the Wnt5a involvement in the process of anorectal malformations (ARMs) remains unclear. In this study, we examined the expression of Wnt5a during ARMs development in the offspring of di(n-butyl) phthalate (DBP)-treated pregnant rats. During the neonatal period, Wnt5a expression was evaluated in the terminal rectum of ARM offspring, non-ARM littermates and controls. Using real-time polymerase chain reaction (real-time PCR), western-blot analysis and immunohistochemistry approaches, we found a significant decrease of Wnt5a expression in DBP-induced ARMs rats. Collectively, our results demonstrate the aberrant expression of Wnt5a during anorectal development, which suggests that Wnt5a might be involved in DBP-induced ARMs.

Expression of the Shh/Bmp4 signaling pathway during the development of anorectal malformations in a male rat model of prenatal exposure to di(n-butyl) phthalate

Sonic hedgehog (Shh)/bone morphogenetic protein 4 (Bmp4) is an androgen-regulated signaling pathway that has been shown to be crucial for embryonic development. This study was designed to investigate the expression of the Shh/Bmp4 signaling pathway during the development of anorectal malformations (ARMs) in male rats when prenatally exposed to di(n-butyl) phthalate (DBP). We found decreased serum testosterone concentration and reduced expression of the androgen receptor in the terminal rectum induced by DBP. In addition, significant decreases in mRNA and protein expression levels of Shh, GLI family zinc finger 2 (Gli2), GLI family zinc finger 3 (Gli3) and Bmp4 in the terminal rectum of male offspring induced by DBP were observed. Collectively, this study shows that the antiandrogenic effects of DBP disturb the androgen-related Shh/Bmp4 signaling pathway, which plays an important role in the occurrence of ARMs induced by DBP.

Maternal exposure to di-n-butyl phthalate (DBP) promotes epithelial-mesenchymal transition via regulation of autophagy in uroepithelial cell

Maternal exposure to di-n-butyl phthalate (DBP) induces hypospadias, but the underlying mechanisms remain elusive. Here we hypothesize that aberrant activation of autophagy and epithelial-mesenchymal transition (EMT) are the leading cause of DBP-related hypospadias. Pregnant rats received DBP orally at a dose of 750 mg/kg/day during gestational days 14-18. In DBP-induced hypospadiac male offspring, immunohistochemistry (IHC) staining and Western blot showed increased expression of autophagy and EMT markers in genital tubercle (GT) tissue compared to the control. 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 autophagy and autophagy activation furthermore promoted EMT in urethral epithelial cells. DBP combined with chloroquine, an autophagy inhibitor, reduced the expression of EMT markers compared with DBP treatment alone, while DBP combined with the autophagy inducer rapamycin elevated the expression of EMT markers. The autophagy-lysosomal pathway inhibitor CQ but not proteasome inhibitor MG-132 rescued the decrease of E-cadherin after DBP treatment, which indicated autophagy-induced E-cadherin degradation contributes to DBP-related EMT. Taken together, our findings show that prenatal exposure to DBP induces abnormal autophagy and EMT that may play important roles in hypospadias development.