| Nonylphenol (NP) is the biodegradation product of nonylphenol polyethoxylate (NPE), which has been proven to be one of the environmental endocrine disrupting chemicals (EDCs). Sequential studies have reported adverse effects of NP exposed perinatally or directly to adults on the male reproductive system, especially the level of testosterone. Actually, the main source of nonylphenol in the natural environment is technical nonylphenol that contains many kinds of para-substituted isomers. Nowadays, it has been reported that NP isomers varied in their estrogenic potency in vitro. However, there are few studies performed with single NP isomer. The subject measured the distribution, clearance and enrichment of nonylphenol following oral administration in rat, and successfully set up a stable primary culture system of Leydig cells with higher purity, which made it possible to systematically investigate the effect of nonylphenol on testosterone secretion in Vitro and comparatively evaluated the difference between effects of nonylphenol isomers. Part I The distribution and enrichment of Nonylphenol following oral administration in ratObjectiveTo probe the distribution, clearance and enrichment of Nonylphenol following oral administration in rat.Methods1.14C-4-NP111(500mg/kg) was administrated to rats with ad libitum access to feed. The rats were sacrificed at different time point (0,0.5,1,2,4,8,12,24,48,72,96h) and the concentrations of NP in blood, liver, kidney, testis and brain were measured.2.14C-4-NP111(50mg/kg) was administrated to rats with ad libitum access to feed. The rats were sacrificed at different time point (1d,1w,1m) and the concentrations of NP in blood, liver, kidney, testis and brain were measured.Results1. The clearance models of NP in blood, liver, kidney, testis and brain were one-compartment models, and the MRT was42.65,44.32,37.58,49.67and43.51h, respectively.2. After14C-4-NP111(50mg/kg) was administrated for1d,1w or1m, the concentrations of NP in blood, liver, kidney and brain had a mild increase with time, while the concentrations of NP in testis increased largely.ConclutionThere is an apparente distribution of Nonylphenol with a long MRT in testis following oral administration in rat, which suggests an evident enrichment. Part II Primary Culture, Identification and Functional Study of Rat Leydig CellsObjectiveTo set up a stable primary culture system of Leydig cells with higher purity.Methods1. Leydig cells were separated from other testicular cells, such as Sertoli and germ cells, by enzymatic digestion in combination with Percoll density gradient centrifugation.2. The purity of isolated Leydig cells were determined by3β-HSD staining.3. The testosterone-secreting capability of isolated Leydig cells was measured by Testosterone Radioimmunoassay kits.Results1. The purity of Leydig cells isolated by this method could achieved95%or above and the total number of Leydig cells obtained from one testicle was about1×106.2. The cytoplasm of Leydig cells was stained in deep blue by3β-HSD staining. Meanwhile, these cells possessed testosterone-secreting capability.ConclutionLeydig cells can be separated by enzymatic digestion combined with Percoll density gradient centrifugation with high purity. Moreover, the isolated Leydig cells show normal testosterone-secreting capability.Part Ⅲ Effect of Nonylphenol on rat Leydig Cells in VitroObjectiveTo investigate the effect of nonylphenol (NP) on testosterone secretion of Leydig cells in Vitro.Methods1. A primary culture system of Leydig cells was set up as above, followed by identification of Leydig cells with3β-HSD staining.2. Leydig cells were treated with different concentrations (0.005,0.015,0.025,0.05,0.1,0.5,5.0,10.0,15.0,25.0μmol/L) of NP for48h.3. Testosterone secretion was detected and morphological examination was performed.Results1. Following treatment with NP, morphologic changes of Leydig cells were detected, with decreased cell density at high doses of NP.2. Testosterone increased at lower concentrations of NP while decreased at high concentrations of NP.ConclutionLower doses of NP can stimulate the secretion of testosterone, but increased exposure to NP will inhibit testosterone secretion of Leydig cells. Besides, high concentrations of NP can cause death of Leydig cells in vitro.Part IV Comparative evaluation of Nonylphenolisomers on Leydig Cells in VitroObjectiveAs a kind of widespread environmental endocrine disruptors, Nonylphenol has an adverse impact on male fertility in a certain concentration. While, previous studies were usually performed using commercial NP, which is a mixture of many isomers with branched alkylchains. Nowadays, it has been reported that NP isomers varied in their estrogenic potency in vitro. But there are few studies performed with single NP isomer. This study is aimed to evaluate effects of NP isomers on steroidogenesis of rat Leydig Cells in Vitro.Methods1. A primary culture system of Leydig cells was set up as above, followed by identification of Leydig cells with3β-HSD staining.2. To screen for the optimized inhibitory concentration, primary cultured Leydig cells were exposed to E2, t-NP,4n-NP and NP isomers (p33-NP,p262-NP,p353-NP, p363-NP) at concentration-groups(1.0,5.0,10.0and20.0μmol/L). After incubation, testosterone levels in the supernatants and Cell viability were measured.3.Primary cultured Leydig cells were exposed to E2, t-NP,4n-NP,p33-NP,p262-NP, p353-NP and p363-NPat the optimized inhibitory concentration5.0μmol/L for6h. The expression of3β-HSD, Cypl1a1and Star were further measured by RT-PCR and Western blot. Moreover, the apoptosis of Leydig cells were measured by TUNEL assay.Results1. At concentrations higher than5.0μmol/L, there were significant differences of inhibition for all NP isomers. While, there were significant dose-dependent alterations in the viabilities of Leydig cells at concentrations higher than10.0μmol/L. Therefore,5.0μmol/L was chosen as the optimized inhibitory concentration of nonylphenol isomers to investigate effects of NP isomers on steroidogenesis of rat Leydig cells under the condition of no direct cytotoxicity.2. Significant decrease in testosterone concentration was observed in all NP isomers at 5.0μmol/L. But the inhibition varied in their individual degree. The response to p363-NP exposure was the largest.3. Incubation of each NP isomers at5.0μmol/L induced a dramatic inhibition on3β-HSD, Cypl1al and Star gene expression, especially3β-HSD, but varied in their individual degree.4. The effects of NP isomers on Leydig cell apoptosis were examined by TUNEL staining on adherent Leydig cells. Further analysis showed that the response to p353-NP was the weakest, while no dramatic difference was observed between p33-NP, p262-NP and p363-NP.Conclution1. NP isomers at concentrations higher than5.0μmol/L can affect the steroidogenesis of rat Leydig cells, at least in part, through their influence on gene expression and cell apoptosis, but varied in their individual degree.2. The inhibition of NP isomers were not completely coincident with their estrogenic potency tested in vitro, which implies that effects of NP isomers on steroidogenesis appear to be mediated through some other underlying mechanisms besides their various estrogenic potency. Innovative outcome obtained in this study:1. The subject measured the distribution, clearance and enrichment of nonylphenol following oral administration in rat, and successfully set up a stable primary culture system of Leydig cells with higher purity, which made it possible to systematically investigate the effect of nonylphenol on testosterone secretion of Leydig cells in Vitro.2. Since the main source of nonylphenol in the natural environment is technical nonylphenol that contains many kinds of para-substituted isomers, the subject comparatively evaluated the difference between effects of nonylphenol isomers. |
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