Acute Arsenic Exposure During Pregnancy Reduces Fetal Brain DNA Hydroxymethylation And Induces Neural Tube Defects

Objective:This study focused on the teratogenic effects of arsenic exposure during pregnancy on fetal rats,and explored the role of changes in fetal brain DNA hydroxymethylation and cell cycle arrest in fetal rat neural tube defects induced by arsenic exposure during pregnancy.Methods:The research is divided into two aspects:animal experiment and in vitro experiment.Animal experiment consists of two parts.Experiment 1:to explore the effects of different doses of arsenic exposure during pregnancy on the pregnancy outcome of fetal rats.GD8 pregnant rats were divided into three groups:the control group was injected with normal saline intraperitoneally,the low-dose group was injected with 10 mg/kg Na As O₂,and the high-dose group was injected with 12 mg/kg Na As O₂.The pregnant rats were killed in the morning of GD18,and the number of live fetuses,dead fetuses,absorbed fetuses and neural tube defects were recorded.Experiment 2:to explore the effects of arsenic exposure during pregnancy on neural tube defects in fetal rats.GD8 pregnant rats were divided into two groups.The control group was injected with normal saline intraperitoneally,and the treatment group was injected with 12 mg/kg Na As O₂.Some pregnant rats were killed in the morning of GD12.Fetal brain tissue were collected and pathological sections were prepared.The levels of 5hm C andα-KG in fetal brain were detected by LC-MS/MS,and the effects of arsenic exposure on the regulation of DNA hydroxymethylation,cell proliferation and cell cycle were detected by RT-PCR.In the morning of GD18,the remaining pregnant rats were killed and the number of live fetuses,dead fetuses,absorbed fetuses and neural tube defects were recorded.C17.2 neural stem cells were used in vitro experiment.The cell viability and proliferation index of C17.2 cells exposed to different doses and different times of arsenic were detected by CCK-8 and RTCA experiments.RT-PCR was used to detect the effects of arsenic exposure at different times on C17.2cell proliferation,cell cycle,DNA hydroxymethylation regulation and reactive oxygen species production.Flow cytometry was used to detect the level of reactive oxygen species after arsenic exposure at different times.Results:The results of animal experiments showed that arsenic exposure during pregnancy could induced neural tube defects in fetal rats.GD12 fetal brain H＆E staining showed that the cells of fetal brain tissue in arsenic exposed group were disorderly and loosely distributed,and the boundary membrane was disordered and irregular.The results of RT-PCR showed that the expression levels of PCNA and ki67m RNA in fetal brain of arsenic exposed group decreased significantly.At the same time,the m RNA levels of G0/G1 cycle regulatory proteins CDK6,Cyclin D,CDK2,and Cyclin E1 were decreased,and the m RNA levels of G2/M cycle regulatory proteins CDK1 and Cyclin B1 also decreased.Further study found that the level of 5hmc in fetal brain decreased significantly and the level ofα-KG in fetal brain decreased in arsenic exposure group during pregnancy.In addition,arsenic also inhibited the expression level of hydroxymethylase TETS series genes.In vitro experiments,the results of CCK-8 and RTCA showed that arsenic exposure at different times or doses could inhibit the proliferation of C17.2 cells.RT-PCR results showed that arsenic inhibited the levels of Cyclin B1 and p16 m RNA related to cell cycle regulation in C17.2 cells.Similarly,arsenic exposure decreased the m RNA levels of cell hydroxymethylase TET1 and TET2in C17.2 cells.The results of flow cytometry showed that arsenic exposure significantly increased the level of intracellular reactive oxygen species.In addition,arsenic exposure also inhibited the m RNA levels of HO-1 and SOD1 in C17.2 cells.Conclusion:Intraperitoneal exposure of pregnant rats to arsenic causes abnormal fetal brain development and induces neural tube defects in fetal rats.The impaired fetal brain development may be caused by arsenic reducing the level of DNA hydroxymethylation in the fetal brain,disturbing the cell cycle and inhibiting the proliferation of the fetal brain.