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.
Department of Physiology, Medical College of Nanchang University, 461 Bayi Road, Donghu District, Nanchang, 330006, People's Republic of China.
Xiaomei
X
Liu
Medical Research Center, The First Affiliated Hospital of Nanchang University, Nanchang, 330006, People's Republic of China.
Linlin
L
Xu
Department of Physiology, Medical College of Nanchang University, 461 Bayi Road, Donghu District, Nanchang, 330006, People's Republic of China.
Jingcao
J
Shen
Department of Physiology, Medical College of Nanchang University, 461 Bayi Road, Donghu District, Nanchang, 330006, People's Republic of China.
Jinglei
J
Wang
Department of Physiology, Medical College of Nanchang University, 461 Bayi Road, Donghu District, Nanchang, 330006, People's Republic of China.
Wenli
W
Ruan
Library, Medical College of Nanchang University, Nanchang, 330006, People's Republic of China.
Mei
M
Yu
Department of Physiology, Medical College of Nanchang University, 461 Bayi Road, Donghu District, Nanchang, 330006, People's Republic of China. chenjiaxiang@ncu.edu.cn.
Jiaxiang
J
Chen
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects*; D002478:Cells, Cultured; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007985:Leydig Cells / Q000187:drug effects* / Q000648:ultrastructure; D008297:Male; D051379:Mice; D018384:Oxidative Stress; D010968:Plasticizers / Q000633:toxicity*; D013091:Spermatogenesis / Q000187:drug effects*; D013739:Testosterone / Q000378:metabolism; D018675:Toxicity Tests; D014317:Tritolyl Phosphates / Q000633:toxicity*; D014617:Vacuoles / Q000187:drug effects / Q000648:ultrastructure
D010968:Plasticizers; D014317:Tritolyl Phosphates; D013739:Testosterone; D006861:Hydrogen Peroxide; C025541:tri-o-cresyl phosphate
Autophagy; Leydig cells; Oxidative stress; Tri-ortho–cresyl phosphate