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

Abstract

BackgroundAs 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.MethodsCell 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.ResultsThe 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.ConclusionsThe results show oxidative stress might be involved in TOCP-induced autophagy of mouse Leydig TM3 cells.

Resveratrol protects Leydig cells from nicotine‐induced oxidative damage through enhanced autophagy

“…Some studies have revealed that nicotine can induce oxidative stress, which causes lipid peroxidation accompanied by decreased endogenous antioxidant activity in various organs like testis, central nervous system, kidney, cardiovascular system, liver and lungs . It has been shown that oxidative stress induced cell apoptosis and decreased testosterone production in Leydig cells, which are responsible for producing testicular androgens such as testosterone for the paracrine regulation of spermatogenesis within the testis in adult life . Antioxidant treatment is a widely used therapy for protecting steroidogenesis and/or sperm quality in the testis, avoiding infertility resulting from a low testosterone production due to an imbalance in the production of reactive oxygen species (ROS) and the scavenging ability of cellular antioxidant defense systems.…”

Melatonin appears to protect against steroidogenic collapse in both mice fed with high‐fat diet and H 2 O 2 ‐treated TM3 cells

“…Stress, inflammation and toxic irritation disrupt the mitochondrial balance, resulting in an increase in ROS (Lavranos, Balla, Tzortzopoulou, Syriou, & Angelopoulou, 2012). OS occurs when the internal environment of the testis changes and ROS cannot be scavenged by available antioxidant enzymes, such as superoxide dismutase (SOD2) and glutathione peroxidase (GPx) (Liu et al, 2016). MT has been ER reduction-oxidation (redox) balance is essential for ER function, and increased ROS production and changes in the ROS-scavenging pathways induce ER stress, leading to metabolic dysfunction (Forrester, Kikuchi, Hernandes, Xu, & Griendling, 2018).…”

Tea polyphenols alleviate tri‐ortho‐cresyl phosphate‐induced autophagy of mouse ovarian granulosa cells

“…The homogenized ovarian tissue and mouse primary granulosa cells were harvested in lysis buffer (50 mM Tris pH 7.5, 0.3 M NaCl, 5 mM EGTA, 1 mM EDTA, 0.5% Triton X‐100, 0.5% NP40) containing protease inhibitor cocktail (Huatesheng Biotech, Fushun, Liaoning, China). Equal amounts of cell lysate were utilized for Western blotting analysis as described previously …”

“…Thousands of people have suffered delayed neuropathy caused by TOCP‐contaminated food or beverages in the United States, India, Morocco, and other countries . Apart from the neurotoxicity, immunotoxicity, and liver toxicity, TOCP can also cause severe damage to mouse male reproductive system irreversibly, in which TOCP exposure decreases the sperm density in the epididymis and inhibits viability of spermatogonial stem cells and mouse Leydig TM3 cells . TOCP is also shown to cause ovarian failure .…”

“…Oxidative stress is shown to be induced by many chemicals and plays an important role in reproductive toxicity . It has been reported that oxidative stress is involved in TOCP‐induced liver damage and autophagy of mouse Leydig TM3 cells …”