Microcystin-LR Inhibits Testosterone Synthesis Via Reactive Oxygen Species-mediated GCN2/eIF2α Pathway In Mouse Testis

Objective Microcystins are widely distributed in irrigation waters and enter into human body mainly through contaminated food chain and water.Recently,numerous data demonstrate that microcystins cause toxicological effects in liver,kidney,nerve,immunity,and even reproduction systems.Microcystins have a lot of variants and microcystin-leucine-arginine(MC-LR)is the broadest distributed and most toxic.Previous studies demonstrated that MC-LR disrupted testosterone(T)synthesis,but the underlying mechanisms are not entirely elucidated.This study aims to explore the role of reactive oxygen species(ROS)-mediated GCN2/eIF2α activation on MC-LR-induced disruption of testicular T synthesis.Methods Twenty-four male ICR mice were randomly divided into control and MC-LR groups.They were exposed to normal saline and MC-LR(20μg/kg)by intraperitoneal injection for 35 days.Serum and testes were taken to detect testosterone levels,and testosterone synthesas such as St AR,CYP11A1 and CYP17A1 were detected in testicular tissues.TM3 cells were used to explore underlying mechanisms in vitro.Testosterone synthase expression levels,GCN2/eIF2α signaling pathway related protein and ROS levels were detected.In addition,GCN2 inhibitors(GCN2i B)and free radical scavengers(PBN)were used to verify the role of GCN2/eIF2α signaling pathway and ROS in the testicular testosterone synthesis disorder caused by MC-LR in male ICR miceResults Serum and testicular T levels were decreased in MC-LR-exposed mice.Steroidogenic proteins and synthases,such as St AR,CYP11A1 and CYP17A1,were downregulated in MC-LR-exposed mouse testes and TM3 cells.Mechanistically,p-GCN2 and p-eIF2α were elevated in MC-LR-exposed TM3 cells.GCN2 i B,a GCN2 kinase inhibitor,attenuated MC-LR-induced GCN2 and eIF2α phosphorylation in TM3 cells.Moreover,GCN2 i B attenuated MC-LR-induced downregulation of steroidogenic proteins and synthases in TM3 cells.Further analysis found that cellular ROS were elevated and HO-1 was upregulated in MC-LR-exposed TM3 cells.PBN,a radical scavenger,rescued MC-LR-induced activation of GCN2/eIF2α signaling in TM3 cells.In addition,pretreatment with PBN attenuated MC-LR induced downregulation of steroidogenic proteins and synthases in TM3 cells.Conclusion In summary,these results suggest that ROS-mediated GCN2/eIF2αactivation contributes partially to MC-LR-caused downregulation of steroidogenic proteins and synthases.The present study provides a new clue for understanding the mechanism of MC-LR-induced endocrine disruption.