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（0μM,5μM,10μM,20μM,40μM）for different times（4h,8h,12 h,16h,24h）.The cell viability was detected by CCK-8 assay.（2）TM3 cells were incubated with 20μM 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 20μM 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 20μM 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（0μM,5μM,10μM,20μM,40μM）for 4h,the cell viability of 5μM Cd-treated group,10μM Cd-treated group and 20μM Cd-treated group have no difference compared with control group at the same time;the cell viability of 40μM 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（0μM,5μM,10μM,20μM,40μM）for 8h,the cell viability of 5μM Cd-treated group has no difference compared with control group at the same time;the cell viability of 10μM Cd-treated group,20μM Cd-treated group and 40μM 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（0μM,5μM,10μM,20μM,40μM）for 12 h,16h and 24 h,the cell viability of 5μM Cd-treated group,10μM Cd-treated group,20μM Cd-treated group and 40μM Cd-treated group have statistical difference compared with control group at the same time（p < 0.01）.（2）TM3 cells were incubated with 20μM 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 17β-HSD（p < 0.05,p< 0.01）.Moreover,the testosterone biosynthetic enzymes protein expression levels of St AR,P450 SCC,3β-HSD,P45017α,and 17β-HSD were also down-regulated in Cd-treated groups（p < 0.05,p < 0.01）.（3）TM3 cells were incubated with 20μM 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 20μM 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 IF2α 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 IRE1α 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.