Preliminary Study On The Role And Mechanisms Of Ferroptosis In Male Reproductive Toxicity Induced By Arsenite

Objective:To explore the effects of arsenite on male reproductive system using the animal and cell model of arsenite exposure.To know whether ferroptosis is involved in the process of male reproductive system damage induced by arsenite,and provide theory evidences for the further investigation on molecular mechanisms of male reproductive toxicity.Methods:The 64 healthy C57BL/6J male mice,aged 7 weeks old,were randomly divided into four groups:the control group,0.5 mg/L,5.0 mg/L and 50.0 mg/L arsenite group?n=16?.After 6 months arsenite exposure via drinking water,the animals were weighed and sacrificed and the testis and epididymis were immediately separated on ice.The body weight and testicular organ coefficient were recorded.The sperm count and malformation rate were calculated.H&E staining and transmission electron microscopy were used to observe histomorphology and ultrastructure of testis.The kits were used to detect the contents of Fe²⁺,MDA,ROS,ATP,GSH and SOD in testis of mice.The RT-PCR and Western blot were applied to detect the expression levels of GPX4?VDAC3?IREB2?SLC7A11?ERK?JNK?CHOP and GRP78 in testis of mice.GC-2 cells were cultured and administrated with arsenite and CCK-8 kit was used to determine cell viability.The kits were adopted to measure the contents of ferroptosis-related indicators and the RT-PCR and Western blot were applied to detect the expression levels of ferroptosis-related genes in GC-2cells.The Ferrostatin-1 was used to comfirm whether ferroptosis inhibitor could inhibit GC-2 cell death,alterations of related indicators and abnormal expressions of genes induced by arsenite.Results:?1?After 6 months of arsenite exposure,compared with control group:?1?There was no significant difference in body weight of male mice,while the testicular weight decreased in mice treated with 50mg/L arsenite?P<0.05?.Testicular organ coefficient of mice in 5 mg/L and50 mg/L arsenite groups decreased significantly?P<0.05?.?2?There was no significant difference in sperm malformation rate,while the sperm count in mice treated with 50.0 mg/L arsenite decreased significantly?P<0.05?.?3?Histological observation showed that the spermatogenic cells were disordered or even absent in testis of male mice,especially in the 50.0 mg/L arsenite group.Ultrastructural observation showed that mitochondrial crista breakage and mitochondrial membrane rupture were obviously observed in testis of mice exposed to arsenite.?4?In testis tissue and mitochondria the contents of Fe²⁺,MDA increased,and the content of ATP decreased?P<0.05?.The ROS content increased in testis of mice exposed to arsenite,while the GSH content and SOD activity drecreased?P<0.05?.?5?The mRNA and protein expression levels of GPX4,SLC7A11 and IREB2 were downregulated,and VDAC3 and CHOP were upregulated?P<0.05?.?2?GC-2 cells viability were significantly inhibited after treated with arsenite for 24 hours.The contents of Fe²⁺and MDA increased in GC-2 cells treated with arsenite?P<0.05?.The proteins expression level of GPX4 and SLC7A11 were down-regulated?P<0.05?.The mRNA expression levels of FPN1,DMT1,ISCU,FTH1 and TFR1 in iron homeostasis pathway altered?P<0.05?.Ferrostatin-1 as the inhibitor of ferroptosis could inhibit these changes.Conclusion:Arsenite exposure can induce ferroptosis in testicular germ cells,damaging testicular tissue and leading to spermatogenic dysfunction.Oxidative damage caused by the abnormal regulation of cystine transport pathway,mitochondrial metabolic pathway and iron homeostasis regulatory pathway may be important mechanisms of ferroptosis in testicular cells induced by arsenite.