Study On The Effect And Mechanism Of N-Butylparaben On Reproductive And Endocrin System By Utero And Lactational Exposure

Parabens (PB) are a group of alkyl ester compounds of p-hydroxybenzoic acid, widely used as antibacterial agents/preservatives in cosmetic, personal care, pharmaceutical and food products. Formerly PB are thought to be relatively safe compounds with their low toxicity and bioaccumulation. However, recent literatures shown that PB possess weak estrogenic in vitro and in uterotrophic assays in vivo. Epidemiological studies suggest that PB and their metabolites can be measured in human blood and urine, as well as human breast tumor tissue. In view of the possible adverse effect of PB on human, it attached importance to make risk assessment and determine margin of safety of PB. However, it is hard to determine an adequate no observed adverse effect level (NOAEL) value for the observed reproductive effects of n-butylparaben (n-BP) in rodents based on previous studies, and then making it difficult to select and implement appropriate regulatory response to the hazard.The present study was undertaken to investigate the effects and potential mechanism of n-BP on reproductive and endocrine system in male rat offspring following in utero and lacation exposures, which in turn provided toxicity data available for supervision and prevention. It is well known that inappropriately producing or exposureing to exogenous hormone during fetal or neonatal life, such as testosterone (T) and17β-estradiol (E2) could lead to adverse outcomes, affecting reproductive system development and adult reproductive functions. In addittion, exogenous hormones should interact with corresponding receptor to plays its biologic role. Therefore, the effect of n-BP exposure on synthesis and metabolism of hormones and its receptor was studied. Epigenetic modification plays an important part in fetal origins of adult disease. So the changes of DNA methylation was preliminary investigated.1. The effect of n-BP on reproductive and endocrine system by utero and lactational exposureWistar dams were dosed via oral gavage from gestation day (GD)7through postnatal day (PND)21with corn oil (control group) and n-BP at64,160,400,1000, mg/kg/day. Reduced anogenital distance (AGD) and delayed preputial separation (PPS) were observed in male offspring. The weights of testes, epididymis and seminal vesicle were reduced in different degrees. Serum T was significantly decreased, especially on PND49. Serum E2showed increase at each tested point except on PND180. n-BP reduced epididymal cauda sperm counts and daily sperm production in a dose-related manner, statistically significant at400and1000mg/kg/day. The present study strongly suggests that exposure to n-BP in utero and lactational has adverse effects on the reproductive system in male offspring.2. Study on the mechanism of n-BP on synthesis and metabolism of hormones and its receptorSteroid synthesis and metabolism enzymes in testis were detected by RT-PCR and Western blot. The results showed that the expression of steroidogenic acute regulatory protein (StAR) mRNA and protein were significantly decreased. The expression of cytochrome cholesterol side-chain cleavage enzyme (P450scc) mRNA was decreased. The results also demonstrated that Aromatase (Arom) was increased at transcript and protein level, while estrogen sulfotransferase (SultlEl) decreased. And interestingly, n-BP increased estrogen receptor alpha (ERa) level and decreased androgen receptor (AR) in male testis.3. The effect of n-BP on DNA methylation in male ratsThe alterations of DNA methltransferases (Dnmts) mRNA and DNA methylation modification of ERa promoter region in male testis were respectively analyzed by RT-PCR and Bisulfite sequencing PCR (BSP). The results showed that the expression of Dnmt3b mRNA was significantly increased, and Dnmtl was decreased with no statistical significant differences. BSP revealed significant hypomethylation of ERa promoter.Conclusions (1) We found that n-BP could affect the reproduction and endocrine system by utero and lactational exposure, with a NOAEL of160mg/kg/day.(2) Serum E2was evaluated by n-BP exposure, which may be due to downregulation of Sult1E1and upregulation of Arom.(3) n-BP could influence the expression of ERa and AR, indicating both estrogenicity and antiandrogenic effects in vivo.(4) Increasing Dnmt3b expression and hypomethylation of ERa promoter was observed, indicating aberrant DNA methylation in testis as one of the possible mechanisms of n-BP induced adverse effects on reproduction and endocrine system.