Drugs To Prevent Fetal Cleft Palate Induced By2,3,7,8-tetrachlorodibenzo-p-dioxin

PART I Be based on the morphological and histological changes to studyoptimal dose of TCDD induced cleft palate in mice embryoObjective: To define the optimal2,3,7,8-tetrachlorodibenzo-p-dioxindose based on the morphological and histological changes of fetal micecleft palate induced by different TCDD doses.Methods: The pregnant mice were randomly divided into five groupsand6in each grouop,and were gavaged on gestation day10(GD10).Thecontrol group were given0.1ml corn oil,and the experimentalgroups Ⅰ, Ⅱ, Ⅲ, Ⅳ were given32,28,24,20μg/kg TCDD respectively.Toweight pregnant mice and embryos,record the number of live, cleft palate,dead and resorption fetal mice on GD17.5. Another15pregnant mice wererandomly divided into five groups(same as above) and3in each group. Thecoronal sections of the fetal mice heads were prepared at GD13.5,14.5and15.5respectively,stained with haematoxylin-eosin staining (HE) and observed by microscopy.Results: No significant differences in embryonic weight and livefetuses weight in each group(P>0.05).Compared with the controlgroup,experimental groupsⅠ~Ⅲ had small palate shelves(PS) and delayedpalae shelves lift;the palate development and elevation in experimentalgroup Ⅳ was similar to the control group. The incidence of cleft palate inthe experimental groups Ⅰ~Ⅳ were97.37%,93.02%,65.12%,56.82%, andno cleft palate in the control group.Conclusion： The optimal dose of TCDD to induce cleft palate inC57BL/6J mice is28μg/kg． PartⅡThe antagonistic effect of folic acid, resveratrol and α-naphthoflavoneon cleft palate in mice induced by TCDDObjective： To evaluate whether or not administration of folicacid,resveratrol and α-naphthoflavone had preventive effects on cleft palateformation.Methods：Pregnant mice were randomly divided into9groups, eachgroup had8mice. The TCDD group mice were dosed with TCDD28μg/kg body weight on GD10; folic acid treated mice weredivided into3groups, respectively dosed with folic acid15,10,5mg/kg andTCDD28μg/kg; α-naphthoflavone treated mice were divided into2groups,respectively dosed with α-naphthoflavone5mg/kg,50μg/kg+TCDD28μg/kg; control group mice were given0.1ml corn oil; resveratroltreated mice were divided into3groups: resveratrol50mg/kg were orallyadministered for6consecutive days, from gestational day GD8to GD13inresveratrol GD8~13group; resveratrol50mg/kg were orally administeredfor6consecutive days, from gestational day GD8to GD13, followed by anoral challenge with TCDD on GD10in resveratrol GD8~13+TCDD group;resveratrol50mg/kg and TCDD28μg/kg were used by gavageadministration at GD10in resveratrol GD10+TCDD group; control micewere treated with the same volume of normal saline for6consecutive daysfrom GD8to GD13and were given a single dose of corn oil on GD10. Torecord pregnant mice and embryos weight, the number of live,cleftpalate,dead and resorption fetal mice on GD17.5. The coronal sections ofthe fetal mice heads were prepared at GD17.5and observed bymicroscopy.Results： The incidence of clefts was92.86%in TCDDgroup,84.00%(15mg),73.08%(10mg),84.00%(5mg) in folic acid+TCDD groups,65.52%(5mg)、74.00%(50μg) in α-naphthoflavone+TCDDgroups,0.00%in resveratrol (GD10) group,74.51%(GD10),57.78% (GD8~13) in resveratrol+TCDD groups. The incidence of clefts was0%inthe control group.Compared with the control and TCDD groups,there weresignificant differences in the number of live,dead and resorption fetal micein TCCD+resveratrol (GD8~13)group(P<0.05).No significant differencesin embryonic weight, live fetuses weight,the number of live,dead andresorption fetal mice in the other groups (P>0.05).Conclusion： All test dose of folic acid,resveratrol andα-naphthoflavone had certain antagonistic effect on cleft palate in miceinduced by TCDD, and folic acid10mg/kg, resveratrol50mg/kg GD10,α-naphthoflavone5mg/kg doses had more strong antagonistic action.Effects of three drugs had no significant difference. Part ⅢDetection of fetal palatal scanning electron microscopy and Rho GDIproteinObjective：To observe the ultrastructure and the expression of RhoGDI protein in test pregnant mice gavaged by TCDD, folic acid, resveratroland α-naphthoflavone. Methods：On gestation day10,15pregnant mice were randomlydivided into5groups. TCDD group(TCDD28μg/kg), control group(cornoil0.1ml/kg), folic acid group (folic acid10mg/kg+TCDD28μg/kg),resveratrol group (resveratrol50mg/kg+TCDD28μg/kg),α-naphthoflavone group (α-naphthoflavone5mg/kg+TCDD28μg/kg)were gavaged. The head coronal sections of the fetal mice were prepared atGD13.5,14.5and15.5,observed by Scanning ElectronMicroscopy(SEM).Expression of Rho GDI protein was detected onGD17.5in fetal mice palatal tissues by immunohistochemistry.Results：The embryonic palatal cells in TCDD group graduallyruptured and shrinkaged, filopodia disappered; the filopodia in the surfaceof the smooth cells gradually increased, extended, and continued until theend stage of the palatal fusion in contral,folic acid, resveratrol,α-naphthoflavone groups.Immunohistochemistry showed that GDI proteinwere negative expression in control group,and in the TCDD group, folicacid group, resveratrol group and alpha-naphthoflavone group werenegative expression.Conclusion：Folic acid, resveratrol and a-naphthoflavone can restorethe ultrastructure of mice embryonic palatal cells and antagonizeTCDD-induced cleft palate.Three drugs can antagonize the cleft palateinduced by TCDD, but can not reverse TCDD inhibition of Rho GDI. Rho GDI protein does not play a significant role in the reversal of palatal fusionprocess.