The Study Of Melatonin Intervening The Toxicity Of Atrazine-induced Quail Ovarian Granulosa Cells

Atrazine（ATR）is widely used worldwide as a broad-spectrum environmental herbicide,which has estrogen-like effects and could interfere with reproductive hormone secretion.Diaminochlorotriazine（DACT）is the main metabolite of ATR,which can induce oxida tive stress and disrupt endocrine function.Our previous in vitro study showed that high concentration of ATR and DACT exposure to quail ovarian cells can cause oxidative stress,apoptosis and endocrine disorder.The mechanism of quail ovarian granulosa cell toxicity has been initially understood.Melatonin（MT）,commonly known as"brain nutrients",has been shown to be a potent antioxidant and free radical scavenger secreted by pineal gland.In order to explore the mechanism of MT antagonizing toxicity induced by ATR and DACT in quail ovarian granulosa cell,the primary ovarian granulosa cells of quail at the peak of egg production were selected as the in vitro exposure model.Based on the original experiment,the dosage and action time of ATR,DACT and MT were determined,and then cells were divided into six groups:Con group（control group）,MT group（10μM MT）,A250 group（250μM ATR）,MT+A250 group（10μM MT plus 250μM ATR）,D200 group（200μM DACT）,MT+D200 group（10μM MT plus 200μM DACT）.After 24 hours of treatment,cell morphology,ultrastructure,cell proliferation,Gsk-3βcell cycle signaling pathway,mitochondrial function-related regulatory factors,sex hormone secretion and synthesis regulatory genes and protein expression,and cell c AMP content were observed.The results of this study were as follows:（1）Both ATR and DACT exposure significantly improved the levels of reactive oxygen species（ROS）,hydrogen peroxide（H₂O₂）and lipid peroxide（MDA）,and inhibited antioxidant enzymes activity（GSH-PX and CAT,T-SOD）,decreased total antioxidant capacity（T-AOC）,consequently,induced cell oxidative stress as well as ultrastructural changes（mitochondrial cristae decrease,vacuolization,and mitochondrial damage）.However,MT restored the impaired antioxidant capacity and mitochondril quality,which suggested that MT alleviated ATR and DCT-induced toxicity in quail granulosa cells by reducing oxidative stress.（2）MT could inhibit the changes of immunofluorescence intensity of Gsk-3βinduced by the action of ATR and DACT on ovarian granulocytes,and the expression disturbance of cell cycle-related factors（Cyclin A1,Cyclin B2,Cyclin B3,Cyclin D3）and the change s of protein levels.These results indicated that MT activated Gsk-3βsignaling pathway to alleviate the inhibition of cell cycle.（3）MT could inhibit the expression disorder of mitochondrial function-related genes（Nrf1,CYTC,VDAC1,SIRT1,SIRT3,TFAM）and mitochondrial dysfunction caused by the action of ATR and DACT on ovarian granulosa cells.These results indicated that MT alleviated mitochondrial function induced by exposure to ATR and DACT.（4）MT inhibited the expression of sex hormone synthetase and secretory regulatory factors（St AR,3β-HSD,17β-HSD,P450arom,P450scc,FSHR,LHR,E₂,ERα,ERβ）in ovarian granulosa cells,which indicated that MT alleviated the exposure of ATR and DACT-induced the disorder of sex hormone expression and synthesis.In summary,ATR and DACT caused oxidative stress and mitochondrial damage in ovarian granulocyte cells,thereby inhibiting the occurrence of cell cycle,inducing mitochondrial dysfunction,disorder of expression of regulatory factors-related to sex hormone secretion and disorder of sex hormone synthesis.MT affects cell growth,proliferation and oxidation state,can adversely affect the ATR and DACT-induced granular cell oxidative stress and mitochondrial damage,mediated by activation of Gsk-3 beta cell cycle regulation function and sex hormone synthesis and secretion,in turn,play female function cell protection function,this study as MT antagonism ATR and the molecular mechanism of ovarian granular cell toxicity induced by DACT provides new evidence.This study provides new evidence on the underlying mechanism of MT antagonizing ATR and DACT-induced ovarian granular cell toxicity.