Study On The Combined Toxic Effects Induced By Dibutyl Phthalate And Benzo(a)Pyrene On Primary Cultured Rat Sertoli Cells

Along with the development of human society and expanding process scale of industry and agriculture, more and more pollutants were discharged into surface water. Most of them are organic pollutants, and persistant organic pollutants (POPs) are chemical substances that persist in the environment, bioaccumulate through the food web, and pose a risk of causing adverse effects to human health. The adverse effects include carcinogenesis, mutagenicity, and teratogenic toxicity. It is difficult to evaluate the toxicity of organic pollutants mixture. Many previous studies showed that phthalates and polycyclic aromatic hydrocarbon (PAHs) are the most popular pollutants in surface water. In the present study we chose dibutyl phthalate (DBP) and benzo(a)pyrene (BaP) as the model chemicals of organic pollutants in surface water, and tested the single or combined effect of them on cultured rat Sertoli cells. The purpose of this study is to provide experiment data, and explore a method to evaluate the adverse effect of organic pollutants mixture in water on human health.Our study was divided three parts. The results were as follows:1. Isolation, purification, identification of rat testicular Sertoli cellsTestes of 18-21-day-old SD rats were aseptically removed and directed two steps enzyme digestion, brachytely centrifuge, hypotonic treatment, and twice adherence methods were used for the isolation and purification of Sertoli cells. After 48 h of the Sertoli cells isolation, removal spermatogenic cell by 20 mmol/L Tris-HCl. Sertoli cells were digested and passage after 80% cells conjugated. To check for possible spermatogenic cell, peritubular cell and Leydig cell contamination in Sertoli cell preparations, Sertoli cell cultures were stained histochemically for alkaline phosphatase and oil red O, and HE stain, transmission electron microscope and immunohistochemical study of Fas ligand have been done as well. Sertoli cell cultures were contained < 1% peritubular myoid cells and spermatogenic cell and > 95% staining for the positive Sertoli cell marker oil red O. Borderline of cells were not clear and cell body shape was cylinder or irregularity, and nucleolus are clear, grains and vacuoles can be observed in the cytoplasm with HE staining . The specific structure of Sertoli cells-satellite body were detected by TEM and most of cells were expressed FasL. After tested the growth curve of Sertoli cells, 5-9 days after isolation were the exponential phase of growth and cells in this period were chosen to carry on next experiment.2. The toxic effects induced by DBP, BaP and DBP+BaP on cultured Sertoli cellsThere are 17 groups in MTT assay and cells number count by CASY Cell Counter. They were 0.1,1,10,100,500μg/ml DBP group, 0.01,0.1,1,10,50μg/ml BaP group and DBP+BaP combined treatment groups (combined respectively from low dose to high dose ), 1% DMSO group and control group. The result showed that Sertoli cells proliferation were stimulated by DBP with dose-response relationship tendency although only 500μg/ml DBP group was increased significantly (P<0.01) at 12h.This tendency was continued to 24h, but only 100μg/ml DBP group was increased significantly (P<0.01) and the stimulated effect in 500μg/ml DBP group were disappeared. There were no significant difference between BaP groups and DMSO group at 12h and 24h. DBP+BaP combined treatment groups showed the same tendency as DBP groups, and 500μg/ml DBP +50μg/ml BaP stimulated cell proliferation at 12h and inhibited it at 24h. 100μg/ml DBP stimulated cell proliferation at 24h. The results of cell number count were consistent with that of MTT assay. According to these results, we chose 1,10,100μg/ml DBP, 0.1,1,10μg/ml BaP and DBP+BaP combined treatment groups (combined respectively from low dose to high dose ) to carry on follow experiments.The concentration of lactate in culture medium was tested at 12h, 24h and 48h. The concentration of lactate was increased in 100μg/ml DBP group at 12h , 24h and 48h, and these effect might caused by the increase of cell numbers. Lactate were decreased in BaP groups at 12h and increased at 48 h. In combined treatment groups, lactate were increased in high dose group (100μg/ml DBP+10μg/ml BaP) at 12h and 24h and increased in all three groups at 48h. The combined effect was antagonism at short time (24h) but along with the treatment time extended, it showed an additive action.Vimentin is one kind of interzonal fiber, and it plays an important role in keep cell shape, cell movement and signal transduction in cell. Vimentin in all groups were not changed at 12h. At 24h and 48h, the expression of vimentin decreased in DBP and BaP middle and high dose groups with obvious dose-response relationship. The expression of vimentin was decreased only in high combined treatment group in 24h and in all three groups in 48h. The combined effect was antagonism in 24h and changed to additive action after 48h. DBP and BaP also changed the shape of vimentin, after DBP or / and BaP treatment Sertoli cells shrinked to strand-like or round and the vacant space between cells were enlarged.Injured tubulin will influence the structure of seminiferous epithelium and the support of Sertoli cells to spermatogenic cell.α-tubulin in all groups were not changed in 12h. BaP can stimulated the expression ofα-tubulin in Sertoli cells.10μg/ml BaP stimulated the expression ofα-tubulin in 24h(P<0.05) and 48h(P<0.01),and 0.1μg/ml BaP and 1μg/ml BaP showed the same effect in 48h. In DBP groups, the expression ofα-tubulin only increased in 100μg/ml DBP group in 48h(P<0.05). It was interesting that the expression ofα-tubulin in all combined treatment groups were not significant difference with DMSO group. It clearly showed that DBP and BaP have the antagonism onα-tubulin in Sertoli cells.We also detected the active proteins inhibin B (INH B) and transferrin (Tf) which Sertoli cells secrete because they were very important in adjustment of spermatogenesis。There was no significant difference in the expression of INH B mRNA between DBP groups and DMSO group in 12h. All three doses DBP stimulated the expression of INH B mRNA in Sertoli cells in 24h but 10 and 100μg/ml DBP inhibited it in 48h. BaP inhibited the expression of INH B mRNA in Sertoli cells from 12h to 48h, and there was a dose-response relationship. There was the same tendency as BaP groups in combined treatment groups in 12h, the INH B mRNA were down-regulated in middle and high dose group with a dose-response relationship, but this effect was antagonized by DBP in 24h.However INH B mRNA in all combined treatment groups were down-regulated again In 48h. As to Tf, DBP, BaP and DBP+BaP all showed a tendency which from stimulation to inhibition begin with 24h to 48h, although BaP showed a stronger effect than DBP.3. The changes of MAPK pathway related gene induced by DBP, BaP and DBP+BaP on cultured rat Sertoli cellsWe detected the mRNA variation of MAPK pathway by Rat MAP Kinase Signaling Pathway RT2 Profiler? PCR Array after 10μg/ml DBP,1μg/ml BaP and 10μg/ml DBP+ 1μg/ml BaP treatment, and tested the protein and phosphorylated protein of ERK, JNK and p38 with western blot in all groups.There were 8 genes down-regulated in 10μg/ml DBP group. They were Egr1,c-fos,Ksr1,HPK1,Jnk3,ERK1,NFAT3 and RPLP1. Meanwhile p38δwas up-regulated obviously. This Result showed that 10μg/ml DBP not only influenced three main pathways of MAPK pathway : ERK1/2,JNK/SAPK and p38MAPK, but also changed the upstream molecular of MAPK pathway such as early response genes Egr1,HPK1 and Ksr1.Total ERK2/1 protein and p-p38 were no significant difference between DBP groups and DMSO group respectively. p-ERK1/2 and total p38 were increased and total JNK/SAPK and p-JNK1/2 were decreased in DBP groups with a dose-response relationship.There was only Jnk3 mRNA down-regulated in 1μg/ml BaP group. Total ERK1/2 decreased slightly but p-ERK increased in BaP groups compared with DMSO group respectively. Total JNK/SAPK also was down-regulated slightly and p-JNK decreased with a dose-response relationship. All three doses BaP did not influence the P38 or p-p38.There were 4 genes which expression changed in 10μg/ml DBP+ 1μg/ml BaP group. Cyclin D2 and p38δup-regulated obviusly and Egr1 and c-fos down-regulated significantly. This result demonstrated that the effect of DBP on MAPK pathway in Sertoli cells was alleviated by BaP, because down-regulated 8 genes in 10μg/ml DBP group were reduced to 2 genes. Nevertheless Cyclin D2 was up-regulated not in 10μg/ml DBP group but combined treatment group. This antagonism is consistent with that in IHN B andα-tubulin. p-ERK1/2 were obviously increased in three combined treatment groups but the expression of total ERK1/2 were not changed compare with DMSO group. Total JNK/SAPK in combined treatment groups was lower slightly than DMSO group but p-JNK 1/2 was obviously higher than DBP groups or BaP groups. The tendency of total p38 and p-p38 expression in combined groups were similar to that in DBP groups, and the expression increased along with the dose elevated.In conclusion, DBP stimulated the proliferation of cultured rat Sertoli cells in some dosage, and DBP and BaP both can irritate the lactate secreted andα-tubulin expression, inhibited vimentin expression of Sertoli cells. The mRNA of INH B and Tf were disturbed by DBP and BaP, and the tendency was from stimulation to inhibition with the treatment time extend. DBP can influence the main three pathways in MAPK pathway but BaP only can influence ERK and JNK/SAPK pathway, not the P38 pathway. In this study, most of effects of DBP and BaP combined treatment were antagonistic effects, and others are similar to that of DBP. Considering that the combined effect is complicated, the mechanism of this effect should be carry on in further research.