The Effects Of Bisphenol A On Oxidative Stress And DNA Methylation Responsive Mechanism In Grass Carp Ovary Cells

BPA is a common environmental pollutant,which is widely used in the production of industrial and consumer goods.It is ubiquitous in the environment and affects the physiological health of humans and animals.The role of BPA is complex,including endocrine disrupting or toxic effects,as well as oxidative stress and abnormal epigenetic regulation.Our previous research found that BPA exposure affects the overall DNA methylation level of fish ovary,and the response patterns and mechanisms of cells in vitro are unclear.Moreover,under the influence of endocrine disruptors such as BPA,the relationship between oxidative stress and DNA methylation and its molecular mechanism have not yet been clarified and need to be further explored.Therefore,in this study,the GCO cell model was used to perform exposure experiments for 48 h with 0.3,3,and 30μM BPA.To investigate the toxic effects of BPA,the cell proliferation and viability were detected,the cell oxidative responses of intracellular ROS levels examined by flow cytometry and the MDA,H₂O₂,GSH,GSSG,G6PDH levels were investigated.Besides,the 5m C level was detected by dot blot hybridization and immunohistochemistry with the dnmts expression level.The DNMT level,the GSH synthesis and one carbon metabolism-related substrates（MET,SAM,SAH,Hcy,and Cys）contents and enzymes（MAT,GNMT,CBS,andγ-GCS）activity were compared by ELISA to reveal the dose responses of BPA to cellular oxidative stress and genomic DNA methylation.The main results are as follows:1. After 48 h of BPA exposure,the cell proliferation and activity were decreased in a concentration-dependent manner and significantly decreased after 30μM of BPA treatment,indicating that BPA has a toxic effect on the proliferation and growth of GCO cells.Similarly,30μM BPA caused an increase in intracellular ROS,H₂O₂,and MDA content,resulting in increased intracellular oxidative stress,lipid peroxidation,and cell damage.While 0.3μM BPA slightly increased cell viability,decreased intracellular ROS level,increased GSH level and GSH/GSSG ratio,and enhanced G6PDH activity,suggesting that 0.3μM BPA exposure may enhance GSH circulation.2. The results of DNA methylation showed that the 5m C level of GCO cells significantly increased in the 0.3 and 3μM BPA groups,decreased in the 30μM BPA group,showing an inverted U-shaped dose response.The expression levels of dnmt3,dnmt4,dnmt5,dnmt7 and dnmt8 in the cells also showed inverted U-shaped response,and the correlation analysis showed that dnmt5 and dnmt7 were significantly correlated with the change of 5m C.At the same time,the DNMT protein level and the SAM/SAH ratio,which reflects the methylation ability of the cells,also showed inverted U-shaped trends.The difference is that 0.3 and 3μM BPA groups had no effect on the expression levels of dnmt1 and dnmt6,while 30μM BPA could cause the down-regulation of dnmt1 and the up-regulation of dnmt6.3.30μB BPA caused significant increases in MET,SAM,SAH,and Hcy levels,as well as significant increases in the enzyme activity of MAT,GNMT,CBS,andγ-GCS,while 0.3 or3 μM BPA had not affect them.The level of Cys was significantly reduced in the 3μM BPA group,but not significantly changed in the 0.3 and 30μM BPA groups.The results showed that 30 M BPA enhanced de novo synthesis of GSH in GCO cells,which may disrupt the intracellular balance of carbon metabolism and further inhibit DNA methylation activity.In summary,this study found that low-dose BPA treatment could enhance the GSH cycle synthesis in GCO cells and clear ROS in time,thereby creating a relatively reductive intracellular environment and exhibiting hormesis.At the same time,the transcription level of dnmts was up-regulated,resulting in the rise of the overall 5m C level.However,high dose of BPA can significantly induce oxidative stress response in cells,increase intracellular ROS level,and stimulate de novo synthesis of GSH,thereby affecting the intracellular carbon pathway and reducing the level of 5m C.This study facilitates the relationship of oxidative stress and DNA methylation in female fish gonad and promotes further understanding of the molecular mechanisms of BPA exposure,which points out the health risk of BPA to fish and boosts the development of the ecological assessment in aquaculture.