Experimental Research On Dna Methylation During Fetal Cleft Palate Induced By 2,3,7,8-tetrachlorodibenzo-p-dioxin

Part ? Further discussion on optimal dose of TCDD which inducescleft palate in C57BL/6J miceObjective: To investigate the correlation between the incidence of cleft palate,stillbirth rate and TCDD dosage with the observation of palatal development and stillbirth of C57BL/6J fetal mice induced by different dose of TCDD.Methods: Pregnant mice were confirmed by weight and divided into different groups based on the TCDD given orally at GD10.5.All dams were sacrificed at GD17.5,the embryos were taken from the uteruses,and stillbirth number was checked by naked eye.Cleft palate numbers were checked through dissecting microscope.Results: No significant changes were observed in dam weight gain and numbers of embryos per litter among all groups(P<0.05),the phenotype of fetal palates in TCDD groups differed little.There were no cleft palates or stillbirth in control group.The incidence of cleft palate in TCDD groups was significantly higher than control group(P<0.01),but there was no statistical difference in the incidence of cleft palate between TCDD18?g/kg group and 28?g/kg group(P>0.05).The stillbirth rate was different only between TCDD 28?g/kg group and control or lower dosage groups(P<0.05).Conclusion: 20?g/kg~30?g/kg of TCDD can cause stable and high incidence of cleft palate,and this dosage range is optimal for C57BL/6J fetal mice to occure obvious cleft palate.Part ? Preliminary research of global DNA methylation changesduring murine palatogenesis induced by TCDDObjective: To investigate global DNA methylation,DNA methyl-transferases and ten-eleven translations participation in the mechanism of cleft palate induced by maternal exposure to 2,3,7,8-tetrachlrodibenzo-pdioxin(TCDD)in mice.Methods: 40 pregnant C57BL/6J mice were randomly divided into two groups: the control group(n=20)and TCDD-exposure group(n=20).At gestation day 10.5(GD10.5),the mice in TCDD-group were orally adminis-trated with TCDD 28?g/kg,while the mice in the control group received equivalent corn oil.The pregnant mice were sacrificed at GD13.5,GD14.5,GD15.5,GD16.5,GD17.5,fetal palates were collected for analysis.Global DNA methylation levels were detected by MethylampT.M.Global DNA 9 Methylation Quantification Ultra Kit through an ELISA-like reaction.The expression levels of Dnmts and TETs were examined by quantitative real-time PCR(q-PCR).Results: The normalized global DNA methylation ratio in TCDD-exposure group was significantly higher than that in control group at GD13.5(49.52�4.03 vs 33.42�6.78,P<0.01),while lower at GD14.5(24.10 �2.29 vs 30.12�3.92,P<0.05)and at GD16.5(32.77�0.98 vs 36.45�3.27,P<0.05).The expression level of Dnmt1 m RNA in TCDD-exposure group was higher than that in control group at GD13.5(1.28�0.11 vs 1.01�0.10,P<0.05)and at GD16.5(1.04�0.05 vs 0.81�0.01,P<0.01).The expression level of Dnmt3 a m RNA in TCDD-exposure group was higher than that in control group at GD13.5(1.15�0.17 vs 0.81�0.02,P<0.05)and at GD16.5(1.11�0.06 vs 0.96�0.06,P<0.05).The expression level of Dnmt3 b m RNA in TCDD-exposure group was higher than that in control group at GD14.5(0.97�0.06 vs 0.72�0.06,P<0.01).The expression level of TET3 m RNA in TCDD group was significantly than that of control group at GD13.5(1.00�0.04 vs 1.29�0.01,P<0.01).Conclusions: It is supposed that complicated mechanisms are exist to regulate global DNA methylation levels in palatal tissue of fetal mice.The significant change of DNA methylation level at GD13.5 resulting from up-expression of Dnmt1 and Dnmt3 a and down-expression of TET3 may be one of the epigenetic mechanisms which cause palate malformation in fetal mice induced by maternal exposure to TCDD.Part ? Experimental research on methylation status of Cp G islandsin promoter region of gene TGF-?3,Dnmt1 and Dnmt3aObjective: To investigate the correlation between Cp G islands methy-lation status of TGF-?3,Dnmts gene promoter regions and TCDD-induced mouse embryonic palatal development disorder.Methods: 18 pregnant C57BL/6J mice were randomly divided into two groups: the control group(n=9)and TCDD-exposure group(n=9).On gestation day 10.5(GD10.5),the mice in TCDD-group were orally administrated with TCDD 28?g/kg,while the mice in the control group received equivalent corn oil.The pregnant mice were sacrificed on GD13.5,GD14.5,GD15.5,fetal palates were collected.Cp G methylation statuses were analysed by methylation specific polymerase chain reaction(MSP).Results: Cp G island in promoter region of gene TGF-?3 were all at low methylation level at all GDs of both groups,there was no difference at same GD between two groups(P>0.05).Cp G island in promoter region of gene Dnmt1 were all highly methylated with no differences showed at same GD between two groups(P>0.05).The methylation level of Cp G island 1 in promoter region of gene Dnmt3 a in TCDD group was higher than that in control group at GD13.5 and GD15.5(P<0.01,P<0.01),while lower at GD14.5(P<0.01).The methylation level of Cp G island 2 in promoter region of gene Dnmt3 a in control group was higher than that in TCDD group at all GDs(P<0.01,P<0.01,P<0.01).Conclusions: Low methylation level of Cp G island which is close to the first exon in promoter region of gene Dnm3 a may be the cause of highly expressed Dnmt3 a m RNA at GD13.5 during mice palatogenesis induced by TCDD,thus the global DNA methylation was extremely high to induce cleft palate.