Effects Of Prenatal Exposure To P，p’-DDE On Methylation Of Pancreatic Islet Cells In Offspring And The Regulative Effect Of Folic Acid

ObjectiveThe purpose of this study was to examine the effects of p,p’-DDE exposure during pregnancy on IGF2/H19 gene imprinting and islet function of offspring SD rats and the regulative effect of a methyldonor--folic acid in this process.To explore the role of imprinted gene methylation in the damage of islet cell function induced by p,p’-DDE exposure from the point of view of epigenetics.Methods8~10 week-old sexually mature SD rats were caged and mated.Pregnant rats were randomly divided into three groups: control group,p,p’-DDE exposed group and folic acid regulative group.On the 8th to 15 th day of gestation,the rats in the p,p’-DDE exposed group and folic acid regulative group were given the p,p’-DDE(20 mg/m L)following the volume of 5m L/kg(body weight)by gavage.And the rats in the control group were given the same volume of corn oil.Throughout pregnancy,the folic acid regulative group received a diet with folic acid(3.5 mg/kg).The pregnant rats gave term birth freely and 4 male and female offspring were kept in each litter.Weights of the pups were weighed and analyzed at first week,third weeks and eighth weeks after birth.Eight weeks later,the primary islet cells were isolated by retrograde perfusion and the purity,survival rate and insulin secretion function were identified and the methylation level of IGF2/H19 imprinting regulated region was detected by sodium bisulfite genomic sequencing and the expression of IGF2 and H19 m RNA was analyzed by real-time quantitative PCR.Eight weeks later,the pups were fasted overnight,and then the glucose tolerance test was performed.The blood glucose levels in the caudal vein were measured before and 15,30,60,120 min after intragastric administration.At the same time,the blood of orbit was collected before and 2 hours after intragastric administration,and the serum insulin level was determined by ELISA.Apoptosis in islet cells was determined by TUNEL assay.ResultsThe purity of the extracted primary islet cells was[(90 ±5)%]and the survival rate was higher than 90%.And the primary islet cells possess insulin secretion function.The methylated levels of IGF2/H19 imprinting regulated region in the exposed group were lower than the control group(P<0.05),but there was no significant difference compared with the regulative group.IGF2 m RNA of exposed groupwas higher compared with the control group(P=0.002)and regulative group(P=0.002)and no significant difference was found in H19 m RNA between three groups.There was no significant difference of body weight in the first,third and eighth weeks between three groups.The blood glucose level at 15 min after intragastric administration in the exposed group was higher than the control group(P<0.05)and regulative group(P<0.05).No significant difference was found in serum insulin levels before and 2 hours after intragastric administration between three groups.The results of semi-quantitative analysis of TUNEL cell apoptosis showed that there was no significant difference among the three groups.It is suggested that the gene imprinting level of islet cells decreased,the relative expression of IGF2 increased and the level of blood glucose increased after exposure to p,p’-DDE.Folic acid can uptune the gene imprinting level of islet cells,increase the relative expression of IGF2 and reduce the level of blood glucose.ConclusionPrenatal exposure to p,p’-DDE can reduce the methylation level of imprinting regulated region of IGF2/H19 gene and up-regulate the transcription of IGF2,then impair islet function in the offspring of SD rats and folic acid plays a regulative role in this process.