Di-2-Ethylhexyl Phthalate Induced Oxidative Damage Involving Fas L-Associated Apoptotic Pathway In Mouse Spermatogenic GC-2spd Cells

Objective: To dissect the effect of di-2-ethylhexyl phthalate(DEHP)on the apoptosis of mouse spermatogenesis cells(GC-2spd)and its regulation mechanism.Methods: The GC-2spd cells were cultured and exposed to different concentrations of DEHP(0,50,100,200 ?mol/L)for 24 h.Then MTT assay was used to detect cell survival rates,Annexin V-FITC / PI double staining method and flow cytometry assay were used to detect apotosis rates.Real time PCR was used to detect the m RNA expression of apoptosis related gene such as Fas L,caspase-3,caspase-8.The protein expression of apoptosis-associated genes including Fas L,caspase-3,and caspase-8were detected by western blotting method.Spectrophotometer was used to detect oxidative stress related indexes including MDA,SOD,and GSH-PX.Results: The results showed that,cell apoptosis rates increased in a dose-dependent manner after DEHP exposure,the apoptosis rate of 100,200?mol/L dose group was significantly different from that of the control group(P < 0.05).In addition,the activities of relation index of oxidative stress including malondialdehyde(MDA),superoxide dismutase(SOD),and glutatione peroxidase(GSH-PX)changed significantly.Compared with the control group,the activity of GSH-PX was significantly lower than that of the control group at 50,100 ?mol/L dose group(P <0.05),and the SOD activity was significantly lower(P < 0.05)at 200 ?mol/L.The m RNA and protein expression levels of Fas L,caspase-3,and caspase-8 altered obviously,m RNA and protein expression of Fas L were increased,of which 100,200?mol/L exposure group was significantly different(P < 0.05),compared with the control group.The m RNA expression level of caspase-8 in the 200 ?mol/L exposure group increased significantly(P < 0.05),and expression of caspase-8 proenzyme protein in the 100 and 200 ?mol/L exposure group increased(P < 0.05),caspase-3m RNA in 100 ?mol/L group increased(P < 0.05),the proenzyme protein level in the100 and 200 ?mol/L exposure group increased(P < 0.05).Conclusion: DEHP may induce the apoptosis of GC-2spd through oxidative stress,and Fas L pathway,which may affect the normal sperm production.It may be the internal cause of male reproductive dysfunction induced by DEHP.