Regulation Of Mitochondrial Damage In Lycopene Against Atrazine-Induced Testicular Toxicity In Mice

Atrazine is one of the most widely used agricultural herbicide all over the world that has been classified as a potential environmental endocrine disrupting chemical(EDC)by World Health Organization and cause deleterious effects on male reproductive system.Atrazine can invade testis tissue,cause reproductive dysfunction,result in testicular hypoplasia,interfere with spermatogenesis.However,the exact molecular mechanism of atrazine-induced testicular toxicity remains to be elucidated.Lycopene,known as "plant gold",is a kind of carotenoid with the most potent antioxidant activity,distributing abundantly in testis,which provide some theoretically basis of antagonizing atrazine-induced testis toxicity.In this study,the functionary mechanisms and the effect action target of lycopene against atrazine-induced testicular toxicity were explored in adolescent male mice during reproductive development.350 male SPF kunming mice randomly divided into seven groups(50 mice/group)and treated as follows: Con group(control group),Vcon group(vehicle control group),LYC group(5 mg/kg lycopene group),ATRI group(50 mg/kg atrazine group),ATRII group(200 mg/kg atrazine group),ATRI+LYC group(50 mg/kg atrazine+5 mg/kg lycopene group)and ATRI+LYC group(200 mg/kg atrazine+5 mg/kg lycopene group)by oral gavage administration for 21 days.The alterations of testicular morphology and ultrastructure of germ cells,sertoli cells and leydig cells were observed,the sperm parameters and spermatogenesis associated factors,the changes of testosterone secretion related regulatory factors,the changes of tight junction protein,mitochondrial dynamics and function,antioxidant function,SIRT3 mediated mitochondrial health regulation,mitophagy and apoptosis were detected.Furthermore,to verify the antagonistic mechanism of lycopene against the toxicity of atrazine and its major metabolite DACT(diaminochlorotriazine),TM3 and TM4 cells as models were used in vitro experiment.After screening the concentration and reaction time of atrazine/DACT and lycopene,TM3 and TM4 cells were divided into six groups: Con group(control group),LYC group(1 ?M lycopene),ATR group(100 ?M atrazine),DACT group(100 ?M DACT),ATR+LYC group(100 ?M atrazine+1 ?M lycopene),DACT+LYC group(100 ?M DACT+1 ?M lycopene).After 24 hours of treatment,cell activity,mitochondrial dynamics and function,oxidative stress level,SIRT3 mediated mitochondrial health regulation,mitophagy and apoptosis were examined.The results show that:(1)At high doses of atrazine,the residual amount of DACT in testis is ten times greater than that of atrazine,indicating that DACT is the major metabolite which induced testicular toxicity;lycopene promotes the metabolism of atrazine by activating P450 enzymes(APND,ERND,AH,NCR)and the excretion of atrazine and DACT from testis tissue.(2)Lycopene alleviates atrazine exposure-induced oxidative stress,inhibits the separation of mitochondria from endoplasmic reticulum in germ cells(including spermatocyte and spermatocyte),reduces mitophagy and apoptosis level,and improves the meiosis-related factors(Cyclin A1?Cyclin A2?Cyclin B1?Cyclin B2?Cyclin B3?Cyclin D1?Cyclin D2?Cyclin D3)disorders during spermatogenesis,inhibited the decrease of sperm activity and the damage of sperm morphology,motility and DNA integrity,which indicated that lycopene antagonized atrazine-induced testicular germ cell toxicity and spermatogenesis disturbance.(3)Lycopene inhibits atrazine-induced mitochondrial damage(decreased mitochondrial volume density,decreased mitochondrial cristae,increased mitochondrial vacuolation,increased percentage of damaged mitochondria),decreases the expression of testosterone synthesis related factors(FSH?,ER?,ER?,LHR,StAR,3?-HSD,17?-HSD,CYP11A1),and increases the expression of cholesterol synthesis related factors(HMG-CoA synthase,HMG-CoA reductase,LDL-R,ARB1 and SHBG),inhibited the decrease of serum testosterone content.Lycopene protects the structure and function of mitochondria in testicular leydig cells and activates the cholesterol synthesis pathway to increase cholesterol,which is the raw material of testosterone synthesis process.It's indicates that lycopene can antagonize atrazine exposure-induced testicular leydig cells cytotoxicity and testosterone synthesis disorder.(4)Lycopene inhibits atrazine-induced mitochondrial damage(decreased mitochondrial volume density,decreased mitochondrial cristae,increased percentage of damaged mitochondria)in testicular sertoli cells,inhibits the expression of tight junction protein Occludin,and antagonizes atrazine-induced destructive effect of testicular Sertoli cells tight junction,thus maintaining spermatogenesis process smoothly.Lycopene antagonizes atrazine-induced testicular sertoli cells toxicity and tight junction damage.(5)Lycopene activates SIRT3 signaling pathway,antagonizes atrazine-induced the increase of acetylation level(Ac-Lysine),promotes the deacetylation of SIRT3,thereby reducing the oxidative stress level in testis tissue,promotes mitochondrial biogenesis via activating PGC1?,and antagonizes the increase and expression of mitochondrial fusion proteins(Mfn1,Mfn2,OPA1).The decrease of fission proteins(Drp1 and Fis1);activating Parkin/PINK signaling pathway to antagonized atrazine-induced testicular mitophagy and apoptosis,thereby antagonizing atrazine-induced SIRT3-mediated mitochondrial health regulation damage in order to protect the structure and function of mitochondria in germ cells,sertoli cells and leydig cells,and maintain spermatogenesis.(6)Atrazine and DACT induce significant cytotoxicity of TM3 and TM4,including decreased cell viability,increased reactive oxygen species,decreased ATP,decreased mitochondrial membrane potential,mitochondrial biogenesis disorders,elevated levels of autophagy and apoptosis,and the toxic effects of TM3 and TM4 cells induced by DACT were more significant,indicating that DACT is the most significant metabolite of testicular leydig cells and sertoli cells toxicity.(7)Lycopene antagonizes atrazine and DACT-induced the the proliferation of TM3 and TM4 cells inhibition and the number of apoptotic cells decreases in a dose-dependent manner.Lycopene activates SIRT3 signaling pathway in TM3 and TM4 cells,then activates PGC1? expression,promotes mitochondrial biogenesis,inhibits the decrease of mitochondrial membrane potential,and antagonizes atrazine and DACT-induced mitophagy and apoptosis in TM3 and TM4 cells by activating Parkin/PINK.Lycopene antagonizes atrazine and DACT-induced the dysfunction of TM3 and TM4 cells by activating SIRT3-mediated mitochondrial health regulation mechanism.In summary,these results provide new evidence for the molecular mechanism of lycopene antagonizing atrazine-induced testicular toxicity.Atrazine leads to oxidative stress in testis and causes mitochondrial damage in germ cells,leydig cells and sertoli cells,which induces mitophagy and ultimately result in apoptosis.However,lycopene can promote the excretion of atrazine and DACT from testis tissue and antagonize the oxidative stress and spermatogenesis disorder caused by atrazine,which may play a vital role in male reproductive protection by activating SIRT3-mediated mitochondrial health regulation.