Objective: To investigate the correlation between CpG islands methylation statuses of TGF-β₃, Dnmts and their expression during TCDD-induced mouse embryonic palatal development. Methods: Eithtteen pregnant C57BL/6J mice were randomly divided into 2 groups: the control group(n=9) and TCDD-exposure group(n=9). At gestation day 10.5(GD10.5), the mice in TCDD-group were orally administrated with TCDD 28 μg/kg, while the mice in the control group received equivalent corn oil. The pregnant mice were sacrificed at GD13.5, GD14.5, GD15.5, fetal palates were collected. CpG island methylation statuses were analysed by methylation specific polymerase chain reaction(MSP). IBM SPSS 20.0 software was applied for statistical analysis. Kolmogorov-Smirnov test was used for normal distribution check, and the distributions were normal. Independent t-test was carried out between two groups. P<0.05 was considered statistically significant. Results: CpG island in promoter region of gene TGF-β₃ were all at low methylation level at all GDs of both groups, there were no differences at same GD between two groups [GD13.5: (8.6±0.8)% vs (8.7±0.8)%, P>0.05; GD14.5: (11.5±1.4)%vs (11.7±1.0)%, P>0.05; GD15.5: (12.0±0.7)% vs (12.1±0.5)%, P>0.05]. CpG island in promoter region of gene Dnmt1 were all highly methylated with no differences showed at same GD between two groups [GD13.5: (73.9±1.1)%vs (72.6±0.8)%, P>0.05; GD14.5: (70.8±1.7)% vs (70.7±1.0)%, P>0.05; GD15.5: (69.4±2.2)% vs (69.7±0.5)%, P>0.05]. The methylation level of CpG island 1 in promoter region of gene Dnmt3a in TCDD group was higher than that in control group at GD13.5 and GD15.5 [(21.9±1.1)% vs (8.1±0.6)%, P<0.01, (43.4±0.4)% vs(32.9±0.7)%, P<0.01], while lower at GD14.5[(33.2±0.5)% vs (42.9±0.3)%, P<0.01]. The methylation level of CpG island 2 in promoter region of gene Dnmt3a in control group was higher than that in TCDD group at all GDs [GD13.5: (82.0±0.7)% vs (32.3±0.6)%, P<0.01; GD14.5: (62.7±1.0)%vs (25.5±1.4)%, P<0.01; GD15.5: (47.2±0.4)% vs (30.3±1.4)%, P<0.01]. Conclusions Low methylation level of CpG island 2 which is close to the first exon in promoter region of gene Dnm3a may be the cause of highly expressed Dnmt3a mRNA at GD13.5 during mice palatogenesis induced by TCDD, thus the global DNA methylation is extremely high to induce cleft palate. TCDD-treatment doesn′t influence the CpG methylation statuses in promoter region of TGF-β₃ and Dnmt1.

Objective: To explore the common differentially expressed proteins in 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin(TCDD) and retinoic acid-induced cleft palate of fetal mice by isobaric tags for relative and absolute quantitation(iTRAQ) combined with mass spectrometry. Methods: Thirty-six pregnant C57BL/6J mice were randomly divided into 3 groups, 12 cases in each group. C57BL/6J pregnant mice were given a gavage of TCDD 28 μg/kg or retinoic acid 80 mg/kg on gestational day 10.5(GD10.5) as experimental groups, while the control group received equivalent corn oil. Anatomical and histological changes of palates in fetal mice were observed on GD17.5. Total proteins were extracted from palates of fetal mice in each group on GD17.5. Differentially expressed proteins were identified in experimental groups as well as in control group by iTRAQ combined with two-dimensional liquid chromatography/tandem mass spectrometry. Western Blot was used for validation of the differentially expressed proteins of Annexin A1 and 14-3-3 sigma. All statistical analyses were measured with SPSS software(version 17.0). Chi-square test was used to compare the incidence of cleft palate. One-way ANOVA was carried out for comparison of the relative expression levels of three groups, homogeneity of variance was analyzed by Levene test, and Turkey HSD test was used for comparison between two groups. P values were judged as significant difference if they were less than 0.05. Results: ①Model of cleft palate in fetal mice were successfully established with incidence of cleft palate of 97.1%(68/70)in TCDD group and 98.6%(70/71) in retinoic acid group, respectively(χ²=0.00, P>0.05), without significant difference between two groups. However, they were similar on the phenotype. ② A total of 2 996 proteins were identified. Compared with control group, 75 and 90 differentially expressed proteins were screened out from TCDD group and retinoic acid group respectively. There were 18 differentially expressed proteins in common both in two experimental groups. ③Western Blot assay indicated that the expression of Annexin A1 protein was 0.52±0.11 in control group, while in TCDD group was 0.99±0.34 and in retinoic acid group was 0.98±0.31, with significant difference between any of two experimental groups and control group(P<0.05). The expression of 14-3-3 sigma protein in control group was 0.55±0.15, while in TCDD group was 0.86±0.17 and in retinoic acid group was 0.93±0.13, with significant difference between any of two experimental groups and control group(P<0.05). These results were consistent with the results of iTRAQ experiment. Conclusions Using iTRAQ technology can quickly and effectively filtrate the common differentially expressed proteins in fetal mice with cleft palate induced by TCDD and retinoic acid. These proteins may have closely related relationship with the occurrence of cleft palate induced by TCDD or retinoic acid.

Objective To investigate global DNA methylation and DNA methyhransferases participation in the mechanism of cleft palate induced by maternal exposure to 2,3,7,8-tetrachlrodibenzo-p-dioxin (TCDD)in mice.Methods 40 pregnant C57BL/6J mice were randomly divided into 2 groups:the control group(n =20) and TCDD-exposure group(n =20).On gestation day 10.5 (GD10.5),the mice in TCDD-group were orally administrated with TCDD 28 μg/kg,while the mice in the control group received equivalent corn oil.The pregnant mice were sacrificed on GD13.5,GD14.5,GD15.5,GD16.5,GD17.5,fetal palates were collected for analysis.Global DNA methylation levels were detected by MethylampTM Global DNA Methylation Quantification Ultra Kit through an ELISA-like reaction.The expression levels of DNA methyltransferases were examined by quantitative real-time PC R(q-PCR).IBM SPSS 20.0 software was applied for statistical analysis.Kolmogorov-Smirnov test was used for normal distribution check,and the distribution was normal.Independent t-test was carried out among two groups.P ＜ 0.05 was considered statistically significant.Results The global DNA methylation level in TCDD-exposure group was significantly higher than that in control group on GD13.5 (49.52％ ±4.03％ vs 33.42％ ± 6.78％,P ＜ 0.01),whilelower on GD14.5 (24.10％ ±2.29％ vs 30.12％ ±3.92％,P ＜0.05) and on GD16.5 (32.77％ ±0.98％ vs 36.45％ ± 3.27％,P ＜ 0.05).The expression level of Dnmt1 mRNA in TCDD-exposure group was higher than that in control group on GD13.5(1.28±0.11 vs 1.01 ±0.10,P＜0.05) and on GD16.5(1.04 ±0.05 vs 0.81 ±0.01,P ＜0.01).The expression level of Dnmt3a mRNA in TCDD-exposure group was higher than that in control group on GD13.5 (1.15 ±0.17 vs 0.81 ±0.02,P ＜0.05)and on GD16.5 (1.11 ± 0.06 vs 0.96 ± 0.06,P ＜ 0.05).The expression level of Dnmt3b mRNA in TCDD-exposure group was higher than that in control group on GD14.5(0.97 ±0.06 vs 0.72 ±0.06,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 increased DNA methylation level on GD13.5 resulting from up-expression of Dnmt1 and Dnmt3a may be one of the epigenetic mechanisms which cause palate malformation in fetal mice induced by maternal exposure to TCDD.

Objective To investigate global DNA methylation and DNA methyhransferases participation in the mechanism of cleft palate induced by maternal exposure to 2,3,7,8-tetrachlrodibenzo-p-dioxin (TCDD)in mice.Methods 40 pregnant C57BL/6J mice were randomly divided into 2 groups:the control group(n =20) and TCDD-exposure group(n =20).On gestation day 10.5 (GD10.5),the mice in TCDD-group were orally administrated with TCDD 28 μg/kg,while the mice in the control group received equivalent corn oil.The pregnant mice were sacrificed on GD13.5,GD14.5,GD15.5,GD16.5,GD17.5,fetal palates were collected for analysis.Global DNA methylation levels were detected by MethylampTM Global DNA Methylation Quantification Ultra Kit through an ELISA-like reaction.The expression levels of DNA methyltransferases were examined by quantitative real-time PC R(q-PCR).IBM SPSS 20.0 software was applied for statistical analysis.Kolmogorov-Smirnov test was used for normal distribution check,and the distribution was normal.Independent t-test was carried out among two groups.P ＜ 0.05 was considered statistically significant.Results The global DNA methylation level in TCDD-exposure group was significantly higher than that in control group on GD13.5 (49.52％ ±4.03％ vs 33.42％ ± 6.78％,P ＜ 0.01),whilelower on GD14.5 (24.10％ ±2.29％ vs 30.12％ ±3.92％,P ＜0.05) and on GD16.5 (32.77％ ±0.98％ vs 36.45％ ± 3.27％,P ＜ 0.05).The expression level of Dnmt1 mRNA in TCDD-exposure group was higher than that in control group on GD13.5(1.28±0.11 vs 1.01 ±0.10,P＜0.05) and on GD16.5(1.04 ±0.05 vs 0.81 ±0.01,P ＜0.01).The expression level of Dnmt3a mRNA in TCDD-exposure group was higher than that in control group on GD13.5 (1.15 ±0.17 vs 0.81 ±0.02,P ＜0.05)and on GD16.5 (1.11 ± 0.06 vs 0.96 ± 0.06,P ＜ 0.05).The expression level of Dnmt3b mRNA in TCDD-exposure group was higher than that in control group on GD14.5(0.97 ±0.06 vs 0.72 ±0.06,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 increased DNA methylation level on GD13.5 resulting from up-expression of Dnmt1 and Dnmt3a may be one of the epigenetic mechanisms which cause palate malformation in fetal mice induced by maternal exposure to TCDD.