The Mechanism Of Cleft Palate Induced By The 2,3,7,8-tetrachlorodibenzodioxin

BACKGROUND:Cleft palate is a congenital malformation, the incidence of which is increasing. The epidemiologic study in domestic research shows that cleft lip with or without Cleft palate is the first three in congenital defects. The children patients with Cleft palate show dysfunction in eating, respiratory and the language development. Children'facial deformities have influenced their physical and mental health. A lot of research shows that Cleft palate and cleft lip increasing rapidly with environment pollution.2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD),is a toxic environmental pollutant. It is produced during industrial processes such as chemical pulp bleaching and waste incineration. The widespread distribution and stability of TCDD have been resulted in the compound through the food chain. Especially to children, TCDD can cause a wide spectrum of the toxicological responses, including the developmental defects, and significantly related with the occurrence of some congenital diseases, such as cleft palate. However, the precise molecular target for TCDD-affected genitalia toxicity remains unknown. OBJECTIVE: In our study, we established cleft palate mice model by exposing pregnant C57BL/6 mice to TCDD, and investigated the expression of TGF-β3 in fetal palatal shelves and the change of DNA methylamine levels and TGF-β3 methylamine levels. Folic acid, as methylamine donor, is suggested to provide protection from cleft palate through influence TGF-β3 gene expression level. The paper makes a further discussion on the change of DNA methylamine levels and the intervention mechanism and effective dose of folic acid in order to explain the probable role of DNA methylamine and folic acid in the pathogenesis of cleft palate.METHODS:1. We established cleft palate mice model throughexposing pregnant C57BL/6 mice to TCDD. Female mice was mated overnight with a male, and on the GD12(gestational day, GD) mice were randomly divided into the following 2 groups (n=10): Vehicle Control, mice were gavaged with corn oil only; Treated, the mice were respectively gavaged with 64 (TCDD64) ug/Kg/day of TCDD suspended in corn oil. At GD18, pregnant mice were sacrificed, and the fetuses were removed to use. Each fetal mice palatal was dissected under a dissection microscope furnishing with ocular micrometer.2. Obtain cleft palate mice and examine TGF-β3 express level through Immunohistochemical staining, RT-PCR, western blot. 3. We studied the change of DNA methylation levels and the expression of transforming growth factor-β3 (TGF-β3) first exon methylation levels in the TCDD induce the cleft palate mice and the control mice.4. We studied the protection of folic acid. GD12, mice were randomly divided into the following 4 groups (n=10): Vehicle Control, mice were gavaged with 0.1ml corn oil only; Treated 1, the mice were respectively gavaged with 64 (TCDD64) ug/Kg/day of TCDD suspended in corn oil. Treated 2, the mice were gavaged with folic acid (100 mg/kg).Treated 3, the mice were respectively gavaged with 64 (TCDD64) ug/Kg/day and supplemental intake of folic acid (100 mg/kg). At GD18, pregnant mice were sacrificed, and the fetuses were removed to use. We count the number of fetuses and statistics the survival rate. Keep Cleft palate mice for Immunohistochemical staining and histological observations.RESULTS: 1. Cleft palate mice model was established successfullyby exposing pregnant C57BL/6 mice to TCDD. The penetrance of this phenotype increases to 100%.Through the specific expression of cell surface molecules and alterations in the morphology of cells along the medial edge epithelia (MEE), the shelves adhere and form a single structure bisected by a layer of epithelium, known as the medial epithelial seam (MES). The MES subsequently disappears, leading to a continuous palatal shelf consisting of a mesenchymal core bounded by the nasal and oral epithelium. On the contrary, MES don't contact and fusion, leading to cleft plate in our experimental group.The statistical number shows that occurrence rate is higher in experimental group than in control group (P-values<0.01).2. TGF-β3 mRNA and protein expression significantly up-regulated in TCDD treated mice than control, through RT-PCR , western blot and immuno histochemical staining.3. MS-PCR shows that TGF-β3 first exon methylation levels decreased in TCDD treated mice than control. MSAP shows that DNA methylation levels decreased in TCDD treated mice than control.4. As methylation donor, supplemental intake of folic acid is suggested to influence TGF-β3 gene expression level through western blot and immuno histochemical staining. MS-PCR shows that TGF-β3 first exon methylation levels increased in TCDD treated mice than control.CONCLUSION: The DNA methylation levels and TGF-β3 methylation levels were found decreased in TCDD-induced mice than control mice, implying that such differences may be related to the up-regulated express of TGF-β3 express in TCDD induce cleft palate mice. As methylation donor, supplemental intake of folic acid is suggested to provide protection from cleft palate through influence TGF-β3 gene expression level. The results indicated that folic acid play an essential role to reduction in the risk of cleft lip with or without cleft palate (CL/P) through influence TGF-β3 gene expression level.