Role Of Oxidative Stress In Tri-ortho-cresyl Phosphate (TOCP)-Induced Autophagy Of Mice Leydig Cells

Objectives:Tri-ortho–cresyl phosphate(TOCP) is the important one of three isomers(i.e., o-, m-, or p-cresyl) of tricresyl phosphate(TCP), and has been widely used in industry and daily life. TOCP has been shown to induce reproductive toxicology besides neurotoxicity. The aim of this study is to observe the effect of TOCP on the mouse Leydig cells in vitro, and try to investigate the role and mechanism of oxidative stress in TOCP induced toxicity of Leydig cells. Methods:MTT was utilized to observe the effect of TOCP and H2O2 on viability of mouse Leydig cells; ELISA assay was used to test the effect of TOCP on the testosterone content of mouse Leydig cells; Flow cytometry(FCM) and RT-PCR was utilized to investigate the influence of TOCP on cell cycle of mouse Leydig cells; Annexin V-FITC/PI double staining and Hoechst staining were exploited to survey the impact of TOCP on the apoptosis of mouse Leydig cells; Western blot and Transmission electron microscopy(TEM) were used to detect the impact of TOCP and H2O2 on autophagy of mouse Leydig cells; The active oxygen reagents test kits was taken advantage of detecting whether TOCP inducing oxidative stress of mouse Leydig cells. Results:After treatment with different concentration of TOCP(0, 0.125, 0.25, 0.5 m M) for 48 h, the viability and the testosterone levels of mouse Leydig cells was decreased significantly, respectively(P<0.05); but there is no change on the cycle and the apoptosis of mouse Leydig cells between the control and TOCP- treated cells. However, TOCP significantly increased both LC3-II and the ratio of LC3-II/LC3-I, and the contents of autophagy proteins Atg5 and Beclin 1. Transmission electron microscopy(TEM) showed that 0.5 mM TOCP increased autophagic vacuoles of the cytoplasm, indicating that TOCP could induce autophagy of the cells. TOCP significantly increased MDA level in the cells in a dose-dependent manner(P<0.05), whereas the content of GSH, activities of antioxidant enzymes SOD and GSH-PX were dramatically decreased in the TOCP-treated cells, respectively(P<0.05). These results indicated that TOCP could induce 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. Conclusion:TOCP could inhibit viability of mouse Leydig cells and induce autophagy without significant effect on cell cycle and apoptosis of Leydig cells; TOCP could induce oxidative stress of mouse Leydig cells; Involvement of oxidative stress in TOCP induced autophagy of Leydig cells.