Study On The Mechanism Of Atrazine-induced Cytotoxicity In Quail Ovarian Granulosa Cells

Atrazine(ATR)is a herbicide which is widely used all over the world.It has estrogen-like effects and can interfere with reproductive hormone secretion.The diaminochlorotriazine(DACT)is the main metabolite of ATR in the cells,which causes oxidative stress and destroys endocrine function.A previous in vivo experimental study shown that the different concentrations of ATR can induce developmental disorders of female genital gland and interfere with the hormone regulation of the hypothalamic-pituitary-ovarian axis in female quail after 45 days of exposure.In order to further explore the mechanism of ATR-induced cytotoxicity in quail ovarian granulosa cells,The cell viability,oxidative markers,apoptosis,hormone secretion level and expressions of genes and proteins related to hormone synthesis,and c AMP content were detected following ATR and DACT treatment in the culture system.The results indicated that:(1)Following HE staining of primary quail ovarian granulosa cells isolated from in vitro culture was found complete and uniform in size.The purity of cells was 90% by FSHR-specific fluorescence staining.The secretion of E2 and P were consistent with the cell growth curve,indicating that the primary quail ovarian granulosa cells model was successfully established.(2)ATR and DACT exposure significantly reduced the viability of quail ovarian granulosa cells,increased the levels of ROS and lipid peroxide(MDA)in cells,and inhibited the activities of antioxidant enzymes(GSH-PX,CAT,T-SOD),decreased total antioxidant capacity(T-AOC),and eventually inducing cellular oxidative stress,which indicated that oxidative stress was one of the important mechanisms of ATR-induced cytotoxic damage of quail ovarian granulosa cells.(3)ATR and DACT significantly increased the rate of apoptosis in quail ovarian granulosa cells,and up-regulated the expression of proapoptotic related factors(Fas,Fas L,Bax,Caspase-3 and p53)in a dose-dependent manner,and down-regulated the expression of apoptosis-related factor(Bcl-2),indicated that ATR and DACT-induced apoptosis was one of the important mechanisms of cytotoxicity in quail ovarian granulosa cells.(4)ATR and DACT exposure increased E2 and P levels in culture medium of quail ovarian granulosa cells,and m RNA expression of sex hormone synthesis pathway related factors(St AR,3?-HSD,17?-HSD,P450 scc,P450arom,ER?,FSHR,LHR),and likewise St AR,3?-HSD,P450 scc and P450 arom protein expression levels were also increased.On the other hand,the ER? m RNA expression was decreased,which indicated that ATR can interfere with the secretory function of quail ovarian granulosa cell sex hormones.The results indicated that disorder of the sex hormone secretion was one of the important mechanisms of ATR-induced reproductive toxicity in quail.(5)ATR and DACT can increase the ability of c AMP synthesis induced by FSH and Forskolin in quail ovarian granulosa cells,and ultimately increase intracellular c AMP content and P level in cell culture medium,indicating that ATR can play a toxic role in interfering with the secretion of sex hormones by promoting the synthesis of sex hormones in ovarian granulosa cells.In summary,ATR and its main metabolite DACT can play a role in female reproductive toxicity.They can reduced the activity of ovarian granulosa cells,induce oxidative stress and apoptosis,regulate the synthesis and secretion of cytokines,and interfere with the expression of sex hormone synthesis related factors,and then induced the disorder of sex hormone secretion.