The Study On Oxidative Stress Effect Of DBP On Urinary System Development

Objective: The oxidative stress effect of di-n-butyl phthalate(DBP)can influence embryo development as well as it anti-androgen activity.The study focused on the oxidative stress effect of DBP on development of urinary system,which provides a new understanding of DBP’s molecular mechanisms and new treatment strategies of related diseases.Methods: Part I: We examined the m RNA expression profiles of genital tubercle(GT)in control and DBP induced hypospadias group by Affymetrix Rat 230 2.0 Array.The result showed that high expression of IP3 R and the abnormalities of epithelial-mesenchymal transition(EMT)may be related to the incidence of hypospadias.Real-time PCR and Western Blot were used to detect the protein and m RNA expression levels of IP3 R and EMT-related molecular markers,such as E-cadherin,α-Catenin,β-Catenin,vimentin,N-cadherin,in the GT of hypospadiac male rats and controls.The results of array was further confirmed in the control and DBP exposed urethral epithelial cells.The changes of intracellular calcium concentration in urethral epithelial cells were detected by Fluo-3-AM before and after DBP treatment.The levels of reactive oxygen species(ROS)in urethral epithelial cells were measured by DCFH-DA with different concentrations of DBP(0,1,10,100 μmol/L)treatment.We identified the differential gene,Hh IP by Affymetrix Rat 230 2.0 Array in GT of undeformed and DBP induced hypospadias group.Western blot and Real-time PCR were used to detect the protein and m RNA expression levels of Hh IP,Gli1,Snail and EMT-related molecular markers,such as E-cadherin,α-Catenin β-Catenin,vimentin and N-cadherin in the genital tubercle of undeformed and hypospadias group.Part II: Kidney dysplasia and renal fibrosis rat models induced by DBP were established.The body weight(BW)and the organ/BW ratios of kidneys were measured in both newborn and adult offspring.The morphological structure of the kidneys in newborn and adult offspring was observed by HE staining.The m RNA expression of renal development related genes Foxd1,Gdnf,Pax2,Wnt11,Cdh11,Calm1 and Ywhab in newborn offspring was detected by Real-time PCR.In DBP exposed adult offspring,histopathological examination and Masson’s trichrome staining revealed the pathological changes of renal fibrosis.Furthermore,expression of transforming growth factor-β(TGF-β),fibronectin and alpha-smooth muscle actin(α-SMA)were also detected by Real-time PCR and Western Blot.Real-time PCR and Western Blot were used to detect the protein and m RNA expression levels of TGF-β,fibronectin and α-SMA in the control and DBP exposed NRK49 F.We analyzed NRK52 E cell arrest at the G2/M phase of the cell cycle by costaining Ki67 with the phosphorylation of histone H3 at Ser10(p-H3)to evaluate cell cycle distribution.The expression of p21 was investigated in NRK52 E cells by Western blot and Real-time PCR.The levels of ROS in NRK49 F and NRK52 E cells were measured by DCFH-DA with different concentrations of DBP(0,1,10,100 μmol / L)treatment.The m RNA expression of the gene responsible for the prevention of oxidative activity Gpx1,Ca,GR,Gst in these cells was detected by Real-time PCR.NRK49 F and NRK52 E cells were pretreated with vitamin C to see if the effect of DBP on NRK49 F and NRK52 E cells can be reversed.Results: Part I: The m RNA expression profiles of GT in control and DBP induced hypospadias group by Affymetrix Rat 230 2.0 Array showed high expression of IP3 R and the abnormalities of EMT.Compared to the control group,in the urethral epithelial cells exposed to DBP,the expression levels of IP3 R,E-cadherin,α-Catenin and β-Catenin increased at both the protein and m RNA levels.However the expression levels of vimentin and N-cadherin decreased.The intracellular calcium concentration increased significantly after DBP treatment.The effect of DBP on urethral epithelial cells was linked to the generation of oxidative stress.We identified the differential gene,Hh IP by Affymetrix Rat 230 2.0 Array in GT of undeformed and DBP induced hypospadias group.Compared to undeformed group,the expression level of Hh IP,E-cadherin,α-Catenin and β-Catenin increased at both the protein and m RNA levels,while the expression level of Gli1,Snail,vimentin and N-cadherin decreased in hypospadias group.Part II: Our studies use an optimized treatment regimen of DBP and are the first to show that maternal exposure to DBP can induce kidney dysplasia in offspring on PND1 and renal fibrosis in adult offspring.Pregnant rats received DBP via oral gavage at a dose of 850 mg/kg BW/day during gestational days 14–18.In vitro,DBP could promote the activation of NRK49 F cells and G2/M arrest in NRK52 E cells at a sublethal dose.The effect of DBP on these cell lines was linked to the generation of oxidative stress.In addition,we determined experimentally that DBP induced oxidative stress in both renal fibroblasts and tubular epithelial cells,whereas vitamin C ameliorated the changes caused by DBP.Conclusion: Part I: DBP can influence the development of GT through its oxidative stress effect,which significantly increases the concentration of calcium and inhibits EMT in urethral epithelial cells,and block the fusion process of urethral groove,causing the occurrence of hypospadias.Some offspring exposed to DBP has low expression of Hh IP in GT,which resulted in activation of the Hh signaling pathway and up-regulation of its target gene Snail by transcription factor Gli1,thereby promoting EMT and avoiding the incidence of hypospadias.The study provides a new understanding of DBP’s molecular mechanisms on hypospadias and new treatment strategies of the disease.part II: These results provide evidence that prenatal exposure to DBP may generate oxidative stress in both renal fibroblasts and tubular epithelial cells,leading to kidney dysplasia and renal fibrosis.According to the DOHAD hypothesis,maternal exposure to DBP may damage the development of the kidneys in utero and have permanent deleterious effects that increase the risk of CKD(renal fibrosis)during adulthood.Thus,it is important to avoid exposure to DBP and other EEDs during pregnancy to decrease the risk of renal fibrosis.In addition,treatment with vitamin C during pregnancy may reduce DBP-induced damage to the developing kidneys.