Role And Mechanism Of Autophagy In Di-(2-ethylhexyl) Phthalate-induced Apoptosis Of Mouse Leydig Cells

Objectives:As one of the phthalate esters(PAEs),di-2-ethylhexyl phthalate(DEHP)has been widely used as a plasticizer in industry.DEHP has been shown to induce malereproductive toxicity in addition to embryonic developmental toxicity,immunot oxicity,liver toxicity and neurotoxicity,yet the exact mechanism remains unclear.The aim of this study is to investigate the role and mechanism of autophagy in DEHP-induced apoptosis of mouse Leydig cells by in vivo and in vitro experiments.Methods:We used hematoxylin and eosin(HE)staining to observe the effect of DEHP on mouse testicular and epididymal damage.ELISA assay was used to test the testosterone content of mouse serum.Western blot was utilized to investigate the effect of DEHP on apoptosis and autophagy in mouse testicular tissue.The reactive oxygen species test kits were used to detect whether DEHP induces oxidative stress of mouse testicular tissue and Leydig cells.MTT assay was utilized to observe the effect of DEHP on viability of mouse Leydig cells.Western blot and Annexin V-FITC/PI double staining were exploited to test the impact of DEHP on the apoptosis of mouse Leydig cells.Western blot and Transmission electron microscopy(TEM)were used to detect the impact of DEHP on autophagy of mouse Leydig cells.Results:Male mice were intragastrically(i.g.)administered with 0,100,200 or 400 mg DEHP/kg/day for 21 days.Compared with the control group,DEHP has no obvious effect on the testicular and epididymal coefficient in mice(P > 0.05).HE staining showed that the testicular spermatogenic cells in the control group were orderly and compact.Compared with the control group,the spermatogenic cells in the 100 mg/kg DEHP-treated group were arranged in a relatively orderly manner,and the spermatozoon cells did not fall off significantly.However,the spermatogenic cellswere significantly disorganized,evacuated and dislodged to a certain degree in the200 and 400 mg/kg DEHP treatment groups.There was a significant decrease in sperm density following exposure to either 100,200 or 400 mg DEHP/kg/day in mice compared with the control group.The contents of apoptosis-related proteins cleaved Caspase-8,cleaved Caspase-3 and Bax and the protein level of Bcl-2 were increased and decreased in the DEHP-treated testis tissue,respectively,which showed that DEHP could induce apoptosis of mice testis tissue.Meanwhile,DEHP obviously increased both the protein level of LC3-II and the ratio of LC3-II/LC3-I,as well as the contents of autophagy proteins Atg5 and Beclin1.These results revealed that DEHP could induce autophagy of the testis tissue.DEHP significantly increased MDA level in the testis tissue,whereas the content of GSH,activities of antioxidant enzymes SOD and GSH-PX were dramatically decreased in a dose-dependent manner.These results indicated that DEHP could induce oxidative stress in the mouse testis tissue.According to the ELISA result,DEHP inhibited testosterone output in a dosedependent manner,implying that DEHP could affect the function of Leydig cells.In vitro studies showed that DEHP inhibited viability of mouse Leydig TM3 cells in a dose-dependent manner(P<0.05)and markedly increased the contents of cleaved Caspase-8,cleaved Caspase-3 and Bax,as well as decreased the protein level of Bcl-2,respectively,which indicated that DEHP could induce apoptosis of TM3 cells.Induction of apoptosis of the TM3 cells by DEHP was further confirmed by AnnexinV-FITC and PI double staining.DEHP can induce oxidative stress in TM3cells(P<0.05),while inhibition of oxidative stress by N-Acetyl-L-cysteine(NAC)could rescue the inhibition of cell viability and induction of apoptosis by DEHP.These results indicated that oxidative stress might be involved in DEHP-induced apoptosis of TM3 cells.Meanwhile,DEHP were shown to induce autophagy of TM3cells;while inhibition of oxidative stress could also restrain autophagy,which indicated that oxidative stress was also involved in DEHP-induced autophagy of TM3 cells.However,inhibition of autophagy by 3-Methyladenine(3-MA)could rescue inhibition of viability of TM3 cells and induction of apoptosis by DEHP.Conclusion:These results showed that DEHP induced apoptosis,autophagy and oxidative stress of mice testis tissue.Oxidative stress was involved in DEHP-induced apoptosis and autophagy of mouse Leydig TM3 cells;inhibition of autophagy could decrease DEHP-induced apoptosis of TM3 cells.