Role of Autophagy in Zinc Oxide Nanoparticles-Induced Apoptosis of Mouse LEYDIG Cells.

Zinc oxide nanoparticles (ZnO NPs) have shown adverse health impact on the human male reproductive system, with evidence of inducing apoptosis. However, whether or not ZnO NPs could promote autophagy, and the possible role of autophagy in the progress of apoptosis, remain unclear. In the current study, in vitro and in vivo toxicological responses of ZnO NPs were explored by using a mouse model and mouse Leydig cell line. It was found that intragastrical exposure of ZnO NPs to mice for 28 days at the concentrations of 100, 200, and 400 mg/kg/day disrupted the seminiferous epithelium of the testis and decreased the sperm density in the epididymis. Furthermore, serum testosterone levels were markedly reduced. The induction of apoptosis and autophagy in the testis tissues was disclosed by up-regulating the protein levels of cleaved Caspase-8, cleaved Caspase-3, Bax, LC3-II, Atg 5, and Beclin 1, accompanied by down-regulation of Bcl 2. In vitro tests showed that ZnO NPs could induce apoptosis and autophagy with the generation of oxidative stress. Specific inhibition of autophagy pathway significantly decreased the cell viability and up-regulated the apoptosis level in mouse Leydig TM3 cells. In summary, ZnO NPs can induce apoptosis and autophagy via oxidative stress, and autophagy might play a protective role in ZnO NPs-induced apoptosis of mouse Leydig cells.

Role of autophagy in di-2-ethylhexyl phthalate (DEHP)-induced apoptosis in mouse Leydig cells.

Di-2-ethylhexyl phthalate (DEHP) has been widely used as a plasticizer in industry. DEHP can cause testicular atrophy, yet the exact mechanism remains unclear. In this study, male mice were intragastrically (i.g.) administered with 0, 100, 200 or 400 mg DEHP/kg/day for 21 days. We found that DEHP caused disintegration of the germinal epithelium and decreased sperm density in the epididymis. Furthermore, there was a significant increase in the levels of cleaved Caspase-8, cleaved Caspase-3 and Bax proteins and a decrease in Bcl2 protein. The results indicated that DEHP could induce apoptosis of the testis tissue. Meanwhile, DEHP significantly induced autophagy in the testis tissues with increases in LC3-II, Atg5 and Beclin-1 proteins. The serum testosterone concentration decreased in the DEHP-treated group, implying that DEHP might lead to Leydig cell damage. Furthermore, oxidative stress was induced by DEHP in the testis. To further investigate the potential mechanism, mouse TM3 Leydig cells were treated with 0-80 µM DEHP for 48 h. DEHP significantly inhibited cell viability and induced cell apoptosis. Oxidative stress was involved in DEHP-induced apoptosis as N-Acetyl-L-cysteine (NAC), an inhibitor of oxidative stress, could rescue the inhibition of cell viability and induction of apoptosis by DEHP. Similar to the in vivo findings, DEHP could also induce cell autophagy. However, inhibition of autophagy by 3-Methyladenine (3-MA) significantly increased cell viability and inhibited apoptosis. Taken together, oxidative stress was involved in DEHP-induced apoptosis and autophagy of mouse TM3 Leydig cells, and autophagy might play a cytotoxic role in DEHP-induced cell apoptosis.

Involvement of oxidative stress in tri-ortho-cresyl phosphate-induced autophagy of mouse Leydig TM3 cells in vitro.

BACKGROUND: As a plasticizer, plastic softener, and flame-retardant, tri-ortho-cresyl phosphate (TOCP) is and has been widely used in industry and reported to have a toxic effect on the male reproductive system in animals besides neurotoxicity and immunotoxicity. We have reported that TOCP inhibits spermatogenesis and induces autophagy of rat spermatogonial stem cells, but it is still unknown whether TOCP induces autophagy of mouse Leydig cells and its potential mechanism. METHODS: Cell viability was observed by MTT assay. Level of testosterone was measured by radioimmunoassay. Apoptosis was observed by AnnexinV-FITC/PI assay. The contents of LC3, Atg5-Atg12, and Beclin 1 were detected by Western blotting analysis. Autophagosomes were investigated by transmission electron microscopy. The contents of MDA and GSH and the activities of SOD, GSH-PX, total antioxidant status (TAS) and total oxidant status (TOS) were measured by oxidative stress kits. RESULTS: The present study shows that TOCP markedly inhibited viability and testosterone output of mouse Leydig TM3 cells but had no effect on apoptosis. However, TOCP significantly increased both LC3-II and the ratio of LC3-II to LC3-I and the contents of autophagy proteins Atg5 and Beclin 1. Transmission electron microscopy (TEM) showed that TOCP increased autophagic vacuoles of the cytoplasm, indicating that TOCP could induce autophagy of the cells. TOCP significantly induced oxidative stress of mouse Leydig TM3 cells. H2O2 also inhibited viability and induced autophagy of the cells; however, inhibition of oxidative stress by N-acetyl-L-cysteine (NAC) could rescue the inhibition of cell viability and induction of autophagy by TOCP. CONCLUSIONS: The results show oxidative stress might be involved in TOCP-induced autophagy of mouse Leydig TM3 cells.