DNA 6 mA修饰在低剂量镉暴露SD孕鼠脐血中的表达研究

Expression of DNA N6-methyladenine modification in umbilical cord blood of SD pregnant rats by low dose cadmium exposure

目的通过研究DNA N6-甲基腺嘌呤(N6-methyladenine,6 mA)修饰及其甲基化修饰酶基因在低剂量镉暴露Sprague Dawley(SD)孕鼠脐血中的表达,探讨6 mA表观遗传修饰对镉发育毒性的影响。方法模拟一代繁殖毒性试验构建低剂量镉暴露的SD孕鼠动物模型,以0、1.0、3.0和9.0 mg/kg·bw剂量对SD雌鼠连续灌胃氯化镉溶液90 d后进行交配,查到阴栓者定为孕第0天,持续染毒至孕第20天进行剖杀,收集脐血,提取DNA和RNA,斑点印迹(Dot blot)检测6 mA修饰水平,RT-qPCR检测6 mA修饰酶相关基因的相对表达水平。结果与对照组相比,各染毒组孕鼠之间的各项繁殖指数未见明显的差异,各染毒组胎鼠体重均明显降低(H=47.82,P<0.01),胎鼠身长、尾长等其他发育指标在个别染毒组出现异常。与对照组相比,各染毒组脐血6 mA修饰水平呈下降趋势,但差异无统计学意义(P>0.05);染毒组6 mA甲基化转移酶N6AMT1表达水平较对照组有不同程度下降,其中9.0 mg/kg剂量组N6AMT1表达与对照组相比差异具有统计学意义(P<0.01);去甲基化酶ALKBH1和ALKBH4的表达水平较对照组有不同程度上升,其中3.0和9.0 mg/kg剂量组ALKBH1表达与对照组相比,差异有统计学意义(P<0.05,P<0.01),9.0 mg/kg剂量组ALKBH4表达与对照组相比差异具有统计学意义(P<0.01)。结论SD孕鼠脐血中6 mA修饰酶的基因表达改变与镉暴露相关,DNA 6 mA表观遗传修饰可能对镉的发育毒性有影响。

Objective To investigate the effect of DNA N6-methyladenine(6 mA)modification on developmental toxicity,we explored the modification level of 6 mA and the expression of its methylating enzyme related genes in umbilical cord blood of Sprague Dawley(SD)pregnant rats exposed to low dose cadmium.Methods The SD pregnant rat model exposed to low-dose cadmium was constructed by simulating the first generation reproductive toxicity test.After 90 days of continuous gavage of cadmium chloride solution at the doses of O,1.0,3.0 and 9.0 mg/kg.bw,mating was carried out.Those who found vaginal suppository were determined as the Oth day of pregnancy.After continuous exposure to the poison until the 20th day of pregnancy,they were dissected and killed.Umbilical cord blood was collected.Then,DNA and RNA were extracted.The modification level of 6 mA was detected by dot blot,and the expression levels of 6 mA modifying enzyme related genes were detected by RT-qPCR.Results Compared with the reproductive and developmental toxicity of the control group,there was no significant difference in various reproductive indexes between the pregnant rats in each exposure group.The body weight of the fetal rats in each exposure group decreased significantly(H=47.82,P<0.01).Other developmental indicators such as the body length and tail length of the fetal rats were abnormal in individual exposure groups.Compared with the control group,the 6 mA modification level of umbilical cord blood in each exposed group showed a downward trend,but there was no statistical significance(P>0.05).The expression level of 6 mA methyltransferase N6AMT1 in the exposed group decreased to different degrees compared with the control group,and the expression of N6AMT1 in the 9.0 mg/kg dose group was significantly different from the control group(P<0.01);The expression levels of demethylase ALKBHI and ALKBH4 increased in different degrees compared with the control group,among which the expression of ALKBH1 in the 3.0 and 9.0 mg/kg dose groups was significantly different from the control group(P<0.05,P<0.01),and the expression of ALKBH4 in the 9.0 mg/kg dose group was statistically significant compared with the control group(P<0.01).Conclusion Thegene expression changes of 6 mA modifying enzyme in umbilical cord blood of SD pregnant rats are related to cadmium exposure.DNA 6 mA epigenetic modification may affect the developmental toxicity of cadmium.