Changes Of DNA Methylation And Activity Regulated Cytoskeleton Associated Protein In Offspring Hippocampus Exposed To Benzo(α)Pyrene During Pregnancy

Objective: To study the role of DNA methylation in the reduction of learning and memory in offspring rats caused by exposure to benzo(α)pyrene(B[a]P)during pregnancy.Methods: 120 SD(Sprague Dawley)rats were mated with 1:1,and the pregnant females were randomly divided into 5 groups(12 for each group): blank group,olive group,10 mg/kg B[a]P,20 mg/kg B[a]P,and 40 mg/kg B[a]P group.On the 17 th,18th and 19 th days of pregnancy,the female rats were intraperitoneal injection once a day with the B[a]P(0.2m L/kg)according to their body weight.The blank control group was not given any treatment,while the olive oil group was given only olive oil.The immunohistochemistry was used to detect the total level of 5m C in the hippocampus of PND1(Postnatal day 1),PND4,PND7,PND14,PND45 and PND75 and ARC expression levels in PND14,PND45 and PND75.The DNMT3 a and ARC expression were detected by Western blot.RT-PCR was used to detect the m RNA levels of DNMT1,DNMT3 a,DNMT3b and ARC in hippocampus of all time periods.Results: 1.The level of DNMT1 m RNA in the rat hippocampus of PND1-PND75 was measured used RT-PCR.The results showed that the level of DNMT1 m RNA in the exposed groups was increased in PND1,PND4,and PND14 compared with the blank group,and the difference was statistically significant(P<0.05),but no significant difference was observed in other time periods.2.DNMT3 a m RNA level was measured in the hippocampus of PND1-PND75 used RTPCR.The results showed that the expression of DNMT3 a m RNA was significantly increased in all time periods from birth to early adulthood.In PND1 and PND14,the expression of DNMT3 a m RNA in the 20mg/kg and 40mg/kg groups was significantly increased compared with the blank group and the 10mg/kg group,and there was a statistical difference(P<0.05).In the PND4 and PND45 time periods,DNMT3 a m RNA level was significantly increased in the 20mg/kg group compared with the blank group(P<0.05),the 40mg/kg group statistically increased compared with the blank group,10mg/kg and 20mg/kg groups(P<0.05).In PND75,DNMT3 a m RNA levels was increased but no significant difference was observed.3.The level of DNMT3 b m RNA in the PND1-PND75 hippocampus was measured used RT-PCR.We found that the expression of DNMT3 b m RNA was increased in all time periods.Only in PND45,the 40mg/kg group was significant difference compared with blank group,10mg/kg and 20mg/kg(P<0.05),and there was no statistical difference in other time periods.4.We used the Western-blot method to detect the protein level of DNMT3 a.The results showed that exposure to B[a]P during pregnancy caused a decrease in DNMT3 a level in offspring SD rats hippocampus.In all time periods,compared with the blank group,the level of DNMT3 a protein in 20mg/kg and 40mg/kg was significantly decreased(P<0.05).And in PND75,DNMT3 a protein in 40mg/kg group was also significantly reduced compared with the 10mg/kg group(P<0.05).5.The total level of 5m C in the hippocampus was measured by immunohistochemistry.The results showed that the total 5m C level was significantly increased in the 20mg/kg and 40mg/kg groups of PND1-PND75 compared with the blank group(P<0.05).6.The levels of ARC m RNA in different time periods from birth to early adulthood decreased significantly,and there was a dose-dependent manner.Compared with the blank group,ARC m RNA levels in the 40mg/kg group decreased significantly in all time period(P<0.05).In PND1-PND7,the ARC m RNA level was decreased significantly in the 40mg/kg group than that in the 10mg/kg group.In PND45 and PND75,ARC m RNA level was decreased significantly in the 40mg/kg group compared with the blank group,10mg/kg group and 20mg/kg group(P<0.05).7.ARC protein expression in the PND4-PND75 of offspring hippocampu rats was decreased caused by B[a]P exposure during pregnancy.Compared with the blank group,the level of ARC in the 10mg/kg,20mg/kg and 40mg/kg groups was significantly reduced in PND4,PND14 and PND75(P<0.05).In PND7,ARC protein was significantly decreased in the 20mg/kg and 40mg/kg groups compared with the blank group(P<0.05),and the expression of ARC protein was reduced in the 40mg/kg group in PND45(P<0.05).In PND1,no significant difference was observed between the groups.8.We detect the distribution and expression of PND14,PND45 and PND75 ARC in the dentate gyrus(DG).According to the results of immunohistochemistry,ARC is mainly distributed in the cytoplasm,and mainly distributed in the DG and CA1 region of the hippocampus.The level of ARC in PND14,PND45 and PND75 decreased significantly(P<0.05).In PND14 and PND75,ARC significantly decreased in the 40mg/kg group(P<0.05).In PND45,ARC protein was significantly reduced in the 20mg/kg and 40mg/kg groups compared with the blank group(P<0.05).Conclusion: 1.Exposure to B[a]P during pregnancy caused an increase in DNMTs m RNA levels in the hippocampus.2.Exposure to B[a]P during pregnancy caused an decrease in DNMT3 a protein levels.3.Exposure to B[a]P during pregnancy caused an increase in total hippocampal DNA 5m C levels.4.Exposure to B[a]P during pregnancy caused down-regulation of ARC expression in hippocampus.