Effects Of Reactive Oxygen Species On Genomic DNA Methylation Of Zebrafish ZF4 Cells Under Low Temperature Stress

Cold pressure causes physiological dysfunction,tissue damage,and finally death in fishes,and increasing studies suggest that epigenetic mechanisms play essential roles in cold stress response in fishes.In our previous study,we compared the transcriptome and genome between Antarctic fish and temperate fish and found that gene expression levels in the pathway related to reactive oxygen species(ROS)scavenge are significantly up-regulated in Antarctic fish.Our previous study showed that cold stress induced production of reactive oxygen species(ROS)in zebrafish(Danio rerio)-derived ZF4 cells in a temperature and time-dependent manner,and that genomic DNA methylation level increased under short-term(18°C for 5 days)cold exposure and decreased under long-term cold exposure(18°C for 30 days).However,the relationship between DNA methylation,oxidative damage and cold acclimation remains poorly understood.In this paper,zebrafish ZF4 cell line was used as the research material,and it was exposed to short-term(at 18°C or 10°C for 3 or 5 days)and long-term(18°C for 30 days)cold stresses.At the same time,the growth state of cells was observed under microscope and cell specific growth rate was calculated.Then the cell genome DNA was digested by methylation-sensitive enzyme HpaII and its methylation-insensitive isoenzyme MspI,the ratio of enzyme digestion was calculated to detect the change of DNA methylation level.In addition,we cotreated ZF4 cells with antioxidant NAC(N-acetylcysteine)or ATM(ataxia telangiectasia mutated)inhibitor KU-55933 during and before cold treatment,the expression of γH2A.X was detected by western blot and the methylation level of DNA was detected by enzyme digestion.The results are as follows:1.Cold stress changes cellular morphogenesis: Under normal culture temperature,the cells of zf4 at 28 °C were irregular triangle,the nucleus was oval,the cytoplasm was protruding,and the edge of the cells was clear.After 5 days of low temperature treatment at 18°C,there was no significant change in cell morphology but a small number of cells died.After 30 days of low temperature treatment at 18°C,the edges of the cells were blurred,most of the cells were no longer irregular triangles,and the nuclei were irregular in shape.After treatment of 3d at 10°C,the shape of ZF4 cells contracted from irregular triangle to long strip,and the gap between cells became larger and a part of cells died and floated in the medium.However,after 5 days treatment at 10°C,a large number of cells died and floated in the medium.By measuring the specific growth rate of cells,we found that the cells treated at 28 °C for 30 days and 18°C for 30 days could reach the maximum specific growth rate at 4-5 days after cell passage,which were 0.568 and 0.355,respectively.However,some cells died on the first day after short-term treatment at 18°C,the specific growth rate was negative and the specific growth rate was lower on the day two to day seven after treatment,only about 0.06.2.Compared with the cells cultured at 28°C,we found that global DNA methylation increased remarkably under short-term cold stress(18°C 5 d,10°C 5 d)(P<0.05),but decreased under long-term cold stress(18°C 30 d)(P<0.05).3.We cotreated ZF4 cells with antioxidant NAC(N-acetylcysteine)during and before cold treatment.Results showed that after treated at 18°C or 10°C for 3 days,the expression of γ H2 A.X protein in the cells cotreated with NAC was significantly lower than that in the control group.Besides,we also found that NAC could inhibit global DNA methylation induced by short-term cold stress(18°C 5 d)(P<0.05),4.Furthermore,we cotreated ZF4 cells with ATM(ataxia telangiectasia mutated)inhibitor KU-55933 during and before cold treatment.Results showed that KU-55933 could inhibit global DNA methylation induced by short-term cold stress(18°C 5 d)(P<0.05).Taking together,in this papaer the changes of DNA methylation level of zebrafish cells at different temperatures and time points were studied.It was found that the DNA methylation level of zebrafish cells increased under short-term low cold stress but the DNA methylation level of cells decreased after long-term cold sterss.We presume that short-term cold stress caused ROS production,global DNA damage and activation of ATM,which further induced elevation of global DNA methylation in ZF4 cells,and increased global DNA methylation may protect the genome.The ROS pathway and ATM signal pathway involved in this process need to be further studied.The present study will help understanding of the epigenetic regulation mechanism underlying cold stress in fish.