To explore the mechanism of
cleft palate in mice induced by 2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin (
TCDD).
METHODS: On gestation day 10 (GD 10), 12 pregnant mice were randomly divided into two groups as the treated group and the control group with 6 mice in each group. The mice in the treated group received intragastric administration with 64 microg
TCDD/kg, while the mice in the control group received equivalent
corn oil. The embryos were examined under stereomicroscope to detect the incidence of
cleft palate on GD 18.5. Another 18 pregnant mice were randomly divided into two groups (treated group and control group) on GD 10 with 9 pregnant mice in each group. Then each group was divided into 3 subgroups: GD 13.5, GD 14.5 and GD 15.5, with 3 pregnant mice in each subgroup. The palatal shelves were dissected from the embryos for
RNA and
DNA extraction on GD 13.5, GD 14.5 and GD 15.5. At last the expression of Smad 2-4 and Smad 7
mRNA was investigated by RT-PCR, and the
TGF-beta3 promoter
methylamine levels were investigated by methylation specific PCR (MSP).
RESULTS: The
cleft palate mice model was established successfully by exposing pregnant C57BL/6J mice to
TCDD. Total frequency of clefts was 100% in
TCDD group, and the frequency of clefts was 0 in the control group. The relative expression of Smad 2
mRNA was 0.263 +/- 0.088, 0.296 +/- 0.016 and 0.159 +/- 0.027 in
TCDD group, 0.180 +/- 0.042, 0.282 +/- 0.029 and 0.165 +/- 0.018 in control group. The relative expression of Smad 3
mRNA was 0.453 +/- 0.153, 0.551 +/- 0.160 and 0.328 +/- 0.049 in
TCDD group, 0.375 +/- 0.126, 0.510 +/- 0.145 and 0.259 +/- 0.035 in control group. The relative expression of Smad 4
mRNA was 0.675 +/- 0.174, 0.577 +/- 0.070 and 0.396 +/- 0.066 in
TCDD group, 0.557 +/- 0.138, 0.587 +/- 0.080 and 0.441 +/- 0.054 in control group. The relative expression of Smad 7
mRNA was 0.283 +/- 0.050, 0.320 +/- 0.068 and 0.169 +/- 0.045 in
TCDD group, 0.207 +/- 0.043, 0.288 +/- 0.051 and 0.155 +/- 0.040 in control group. There was no significant difference between the
TCDD treated mice and the control (P > 0.05). The
TGF-beta3 promoters were at the un-methylation state both in the
TCDD treated and control group.
CONCLUSION: It suggests that
TCDD could induce a stable formation of
cleft palate, but it is not through the
TGF-beta/Smad signaling nor through the modification of
TGF-beta3 promoter methylation.