| Microcystins (MCs) is produced by a class of algae in water.MCs is a biological activity monocyclic heptapeptide. Scientists revealed for the first time that microcystins could potently inhibit protein phosphatase 1 and 2A in 1990, which was the most important molecular basis for the toxicity of microcystins. In 1996,Pour reported that at Caruaru dialysis centers in Brazil, human intoxications by MCs (-LR,-YR and-AR) caused deaths of 60 patients. So, after with those reports.,MCs's toxicity and its human health effects has become a hot topic in the world.After MC has been found that the liver was their first target organ. Recently,in fish organs, the gonads has been found the second target organs. So, the MC could had reproductive toxicity. However, at present,there are few reports of the MC's reproductive toxicity studies at home and abroad, The mechanisms are unclear.Objective:In Dong's research,it has found that Kunming male mice were treated with different concentrations of MC-LR by eritoneal injection.The results showed that DNA-protein crosslinks was induced by MC-LR in testicular cells and indicat-ed reproductive damaged. In this study, the primary cultured sertoli cells of rat was exposed to different concentrations of MC-LR,in order to determine MC-LR's role in reproductive system of oxidative stress and it's the toxic mechanisms.Methods:1. A method was set up for obtaining a large number of viable Sertoli cells from Sprague-Dawley 18～20 days's rats. Minced whole testes from SD rats were sequentially treated with 0.25% pancreatin for 15～20 min and 0.1% collagenase for 20～25 min, then washed Sertoli cells sequently, were cultured in DMEM/F-12 media with 10% fetal bovine serum incubated at 37℃in a humidified incubator in an atmosphere of 5% CO2.On the 4th day, cells contaminating spermatogenic were lysed with a hypotonic solution of 20mM Tris-HCL for 3 min. Feulgen staining was used to identify the purification and viability of Sertoli cells. Subsequently the growth curve of Sertoli cells was established in vitro. the maximum concentration of non-cytotoxicity of MC-LR on Sertoli cells in vitro was ascertained via MTT method. Sertoli cells were treated with different concentrations of MC-LR for 24th. The concentration of MC-LR in DMEM/F-12 media were 0,0.15μg/L,1.5μg/L, 15μg/L。2. Sertoli cells was treated with different concentrations (0,0.15μg/L,1.5μg/L, 15μg/L) of MC-LR, after 6,12,24 h, hydroxylamine Determination of SOD, colorimetric determination of LDH.The damage to enzyme system of Sertoli cell line was analyzed.3.Statistical analysis:the use of SPSS 12.0 statistical software for one-way ANOVA (analysis of variance, ANOVA), variance of data for statistical analysis (significance level a=0.05)Results:1 This study supported by isolated and cultured cells grew well, And a relatively high purity of Sertoli cells can be used for in vitro experiments. Different concentrations of MC-LR exposure in vitro the first 4d of the supporting cells, after 24h and found that MC-LR concentrations of up to 15μg/L when the non-toxicity.2 ROS content in each concentration groups the amount of ROS in cells as compared with the control group were increased, and at different exposure time groups, with the increased, dose of cells increased the amount of ROS. the same exposure dose, exposure time, with the extension of cells, the amount of ROS is increased. in 24h after exposure, compared with control cells in the treated group had increased ROS, However, only(1.5,15μg/L) concentration groups compared with the control group statistically significant (P<0.05). There is no change in trend, in the different exposure time in each concentration group exposed cells LDH leakage rate were not statistically significant (P> 0.05).SOD content change, with MC-LR (0.15,1.5,15μg/L) treated cells after 24h, SOD activity in treated cells (1.5,15μg/ L) are higher compared with the control group were statistically significant (P<0.05). The different concentration groups ((0.15,1.5,15μg/L) MC-LR act on after 6,12h, the difference compared with the control group,There was no significant (P>0.05).MDA content in the different exposure time for each group of cells exposed to concentration levels of MDA did not change significantly, there is no change in trend. in the different exposure time in each concentration group exposed cells MDA levels were not statistically significant (P> 0.05).Conclusion:1 After treated with different concentrations (0～15μg/L),it was no significant effects of the survival rate of a rat Sertoli cells in primary culture.2 MC-LR at 0.15μg/L～15μg/L dose range, can make ROS increased, and with the increased role of the extension of time. SOD in the 15μg/L dose was increased, LDH, and MDA is not elevated trends. Note MC-LR can lead to oxidative stress in testicular Sertoli cells, but not observed in lipid peroxidation. |
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