The Effects Of 2,3,7,8-tetrachlorodibenzo-p-dioxin On Apoptosis Of Rat Leydig Cells

ObjectivesTo explore the effects of TCDD on apoptosis of SD rat leydig cells and the mechanism.Methods8-10 weeks old male SD rats were bred in barrier environment for 2 weeks and then leydig cells were isolated and cultured for the third generation cells.The third generation cells were divided in to five groups and treated with different dose of TCDD(0,1,5,10,50 nM)for 24 hours,respectively.MTT assay was used to determine the effect of TCDD on proliferation of leydig cells at 24 h.The ROS level of leydig cells were examined by DCFH-DA method and the effect of TCDD on apoptosis were detected by confocal microscopy combined Annexin V-FITC/PI.The mRNA and protein expression levels of Bax,Bcl-2,and Caspase-3 were detected by RT-PCR and Western blot,respectively.ResultsThe result of 3?-HSD showed that all cells were dyed dark blue which suggested that leydig cells were in high purity.Compared with the control group,the inhibitory effects,ROS levels and apoptosis positive signal ratio showed a rising trend with increasing of the dose of TCDD(P<0.05).Furthermore,the results from Western blot and RT-PCR showed that TCDD could up-regulate the expression level of Bax and Caspase-3 proteins and mRNA,and down-regulate the expression level of Bcl-2 of mRNA and protein.ConclusionTCDD can significantly inhibit the proliferation of leydig cells,induce apoptosis,and increase the ROS level.The apoptosis related factors of Bax,Caspase-3,and Bcl-2 are participated in apoptosis of Leydig cells induced by TCDD.