The Study On Molecular Mechanisms Of Cadmium-induced Cytotoxicty In Mouse Leydig Cells

Objective To investigate the cytotoxicty of TM3 cells?mouse leydig cell line?induced by cadmium,and explore the molecular mechanisms of Cd-evoked toxic effect of mouse leydig cells in vitro.Methods?1?TM3 cells were incubated with different concentrations of CdCl₂?0?M,5?M,10?M,20?M,40?M?for different times?4h,8h,12 h,16h,24h?.The cell viability was detected by CCK-8 assay.?2?TM3 cells were incubated with 20?M CdCl₂ for different times?4h and 8h?.Testosterone was detected by ELISA assay.Testosterone biosynthetic enzymes m RNA expressions were detected using RT-PCR.Testosterone biosynthetic enzymes protein expressions were detected by western blot.?3?TM3 cells were incubated with 20?M CdCl₂ for different times?4h and 8h?.HO-1and antioxidant enzymes m RNA expressions were detected using RT-PCR.HO-1 and HO-2 protein expressions were detected by western blot.?4?TM3 cells were incubated with 20?M CdCl₂ for different times?4h and 8h?.ER stress-related protein expressions were detected by western blot.ER stress-related m RNA expressions were detected using RT-PCR.Results?1?TM3 cells were incubated with different concentrations of CdCl₂?0?M,5?M,10?M,20?M,40?M?for 4h,the cell viability of 5?M Cd-treated group,10?M Cd-treated group and 20?M Cd-treated group have no difference compared with control group at the same time;the cell viability of 40?M Cd-treated group have statistical difference compared with control group at the same time?p < 0.01?.TM3 cells were incubated with different concentrations of CdCl₂?0?M,5?M,10?M,20?M,40?M?for 8h,the cell viability of 5?M Cd-treated group has no difference compared with control group at the same time;the cell viability of 10?M Cd-treated group,20?M Cd-treated group and 40?M Cd-treated group have statistical difference compared with control group at the same time?p < 0.01?.TM3 cells were incubated with different concentrations of CdCl₂?0?M,5?M,10?M,20?M,40?M?for 12 h,16h and 24 h,the cell viability of 5?M Cd-treated group,10?M Cd-treated group,20?M Cd-treated group and 40?M Cd-treated group have statistical difference compared with control group at the same time?p < 0.01?.?2?TM3 cells were incubated with 20?M CdCl₂ for different times?4h and 8h?,cadmium exposure significantly decreased testosterone level in the culture supernatant fluid?p <0.01?.Compared with the control group,Cd-treated markedly reduced the testosterone biosynthetic enzymes m RNA expression levels of St AR,P450 SCC and 17?-HSD?p < 0.05,p< 0.01?.Moreover,the testosterone biosynthetic enzymes protein expression levels of St AR,P450 SCC,3?-HSD,P45017?,and 17?-HSD were also down-regulated in Cd-treated groups?p < 0.05,p < 0.01?.?3?TM3 cells were incubated with 20?M CdCl₂ for different times?4h and 8h?,Cd-treated significantly induced the m RNA and protein expression levels of HO-1 in a time-dependent manner?p < 0.01?.However,the protein expression level of HO-2 has no difference compared Cd-treated groups with control group.Compared with the control group,Cd-treated declined the antioxidant enzymes m RNA expression levels of SOD1,SOD2,SOD3,GSH-Px and CAT,which have statistical differences compared 8h group with control group?p < 0.05,p < 0.01?.?4?TM3 cells were incubated with 20?M CdCl₂ for different times?4h and 8h?,Cd-treated significantly induced the m RNA and protein expression levels of GRP78?an ER molecular chaperone?in a time-dependent manner?p < 0.01?;Furthermore,Cd-treated also markedly induced the m RNA expression level of GRP94 in a time-dependent manner?p < 0.05,p < 0.01?,showing that the ATF6 signaling pathway was activated by Cadmium.Cd-treated significantly increased TM3 cells PERK phosphorylation?p < 0.01?;And also significantly upregulated the protein expression level of p-e IF2? in a time-dependent manner?p < 0.05,p < 0.01?;In addition,Cd-treated also markedly induced the protein expression level of CHOP in a time-dependent manner?p < 0.05,p < 0.01?,indicating that the PERK signaling pathway was activated by cadmium.Moreover,Cd-treated significantly increased TM3 cells IRE1? and JNK phosphorylation?p < 0.05,p < 0.01?,which indicated that the IRE1 signaling pathway was activated by cadmium.Conclusions?1?Cadmium exposure inhibited the secretion of testosterone in TM3 cells,at least partially,related to downregulating the m RNA and protein levels of testosterone biosynthetic enzymes.?2?Cadmium exposure activated oxidative stress and ER stress in TM3 cells,which maybe play an important role in cellular damage.