Fluorochloridone Induces Autophagy in TM4 Sertoli Cells:Involvement of ROS-mediated AKT-mTOR Signaling Pathway

Abstract BackgroundFluorochloridone (FLC), a selective pyrrolidone herbicide, had medium persistence in soil and groundwater, indicating that its environmental fate was highly correlated with mammals and human health. FLC has been recognized as a potential endocrine disruptor and reported to induce male reproductive toxicity, but the underlying mechanism is largely unclear. MethodsAdult C57BL/6 mice were raised to divided into one control group (0.5% sodium carboxymethyl cellulose), and four FLC-treated groups (3,15,75,375 mg/kg). The animals (ten mice per group) received gavage for a period of 28 days. After treatment, histological analysis, sperm parameters, the microstructure of autophagy and the expression of autophagy-associated proteins in testis were evaluated. Furthermore, to explore the autophagy mechanism, TM4 Sertoli cells were treated with FLC (0,40,80,160μM) in vitro for 24 h. Cell activity and cytoskeletal changes were measured by MTT assay and F-actin immunofluorescence staining. The formation of autophagosome, accumulation of reactive oxygen species and expression of AKT, mTOR were detected.ResultsIn vivo, it showed that FLC exposure caused testicular injuries, abnormality in epididymal sperm. Moreover, FLC increased the formation of autophagosomes, the accumulation of LC3, Beclin-1 and the expression of P62 protein, which is related to the degradation of autophagy. In vitro, the upregulation of TM4 cells autophagy was confirmed by FLC increased the formation of autophagosomes and upregulation of autophagy marker proteins (LC3, Beclin-1 and P62) levels. In addition, FLC induced ROS production and inhibited the activities of AKT and mTOR kinases. The Inhibition of AKT/mTOR signaling pathways and the activation of autophagy induced by FLC could be efficiently reversed by pretreatment of ROS generation by N-acetylcysteine. SC79, AKT agonist, could restore the autophagy induced by FLC in TM4 cells. Intriguingly, FLC-induced autophagy could be inhibited through AKT agonists, which indicated that FLC-induced autophagy may be pro-death. ConclusionTaken together, our study provided the evidence that FLC promoted autophagy in TM4 Sertoli cells and that this process may involve ROS-mediated AKT/mTOR signaling pathways.