Study On The Mechanism Of Zearalenone-induce Testicular Stromal Tumor In Vitro

Zearalenone (ZEA), an estrogenic mycotoxin detected in grain, beer, milk and other foods, can threaten the health of animals and human. The studies found that ZEA led to not only the reproductive toxicity, liver toxicity, genetic toxicity andimmunotoxicity, but also tumorigenesis including testicular stromal tumor. However, the mechanism of ZEA-induced testicular stromal tumor is still unknown. In order to reveal this mechanism, we observed the influence on cells viability, cell cycle and its related regulatory protein, DNA damage, oncogenes and gap junction intercellular communication (GJIC) of mice Leydig cells (TM3 cells). The main contents are as following.1. Effects of zearalenone on cell proliferation, cell cycle and apoptosis in TM3 cells. At first, different concentrations of ZEA (0,5,10,20μg/L)were treated with TM3 cells for 72h, then the MTT method was used to estimate cells viability. AnnexinV-FITC/PI double fluorescent was applied to dye the TM3 cells which was detected by flow cytometry to access apoptosis. Furthermore, PI fluorescent staining method was used to observe cell cycle changes. Western blot assay was applied to analyse the protein expression level of Cyclin D1 and its kinase Cdk4 and Bcl-2 gene family members. The results showed that low concentration of ZEA could signicantly promote the growth of TM3 cells. A dose-effect relationship could be found that, the relative proliferation activity was dramatically improved after being exposured to 20ug/L ZEA for 72h (P<0.05). Through the experimentation of cell cycle, we discovered that TM3 cells was gradually transferred from G1/G0 phase to S phase after being treated with ZEA. The cell cycle regulatory protein Cyclin D1 and its kinase Cdk4 levels increased obviously with the increasing concentration of the ZEA. The Apoptotic detection result showed that the apoptotic rate decreased significantly with 10、20μg/L of ZEA (P< 0.01). Compared with the control group, the apoptosis related protein Bax/Bcl-2 ratio was decreased evidently (P< 0.05).2. Effects of zearalenone on DNA damage, proto-oncogene and anti-oncogene regulation of TM3 cells. The protein expression of yH2AX was analysed by the western blot method after being treated with ZEA for 72h. Meanwhile, the protein expression levels of proto-oncogene c-Myc, c-Fos, c-Jun and anti-oncogene p53 and PTEN were also detected by using western blot method. The situation of the formation of the fluorescence focus was detected by the immunofluorescence. The results showed that the numbers of the γH2AX fluorescent focus which formed in TM3 cells as well as the cells forming the fluorescent focus increased when the concentration of ZEA increased. In the 20μg/L ZEA group only 26.21 percent of TM3 cells did not form fluorescent focus, while the rest suffered different levels of DNA damage. Compared with the control group, the γH2AX protein expression of all the ZEA groups all raised significantly (P<0.05). With the increasing of the concentration of ZEA, the protein levels of proto-oncogene c-Myc, c-Fos, c-Jun increased (P<0.05), but anti-oncogene p53, PTEN decreased significantly (P<0.01).3. Zearalenone induced the inhibition of gap junction intercellular communication and connexin of TM3 cells. The changes of the function of the gap junction intercellular communication (GJIC) between cells exposed in ZEA was detected by the scrape-loading and dye transfer technique (SLDT) and the distribution of Cx43 was observed by immunofluorescence. The results showed that the protein levels of Cx32 and Cx43 analysed by western blot method were inhibited with the increasing concentration of ZEA (P<0.01). Meanwhile, we found that the normal Cx43 showing dot linear structure distributed mostly around the cytomembrane. On contrast, the structure was destroyed and the fluorescence intensity was weakened after being treated with ZEA, which led the distribution to converge to the cytoplasm and cell nuclear membrane. The results of the SLDT technique showed that the diffusion distance of the small molecule yellow dyes fluorescent (LY) between adjoining cells was shortened when ZEA increased. Compared with the control group, the obvious difference was showed with the concentration of ZEA over than 10μg/L, which indicated that the GJIC function could be restrained by ZEA.All in all, low concentration of ZEA could lead to DNA damage, the up-regulation of proto-oncogene, the down-regulation of anti-oncogene and the dysregulation of Bax/Bcl-2 ratio as well as the inhibiton of GJIC in TM3 cells. These changes are alwalys regarded as the key mechanism of tumor development. ZEA could induce the genomic instability, the cell growth imbalance such as the cell proliferation, differentiation and abnormal changes. The sustained cellular proliferation and the malignant transformation may eventually result in the occurrence of tumor.