Cleft palate (CP) can be induced in embryonic mice by a wide range of compounds, including glucocorticoids and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Hydrocortisone (HC), a glucocorticoid, retards embryonic growth producing small palatal shelves, while TCDD exposure blocks the fusion of normally sized shelves. TCDD induction of CP involves altered differentiation of the medial epithelial cells. Recent studies indicate that growth factors such as EGF, TGF-alpha, TGF-beta 1, and TGF-beta 2 are involved in palatogenesis, regulating proliferation, differentiation, and extracellular matrix production. A synergism has been observed between HC and TCDD in which doses too low to induce CP alone are able to produce greater than 90% incidence when coadministered. In the present study a standard teratology protocol was performed in C57BL/6N mice to examine the synergism at doses lower than those previously published. Data from this study indicate synergistic interactions at doses as low as 3 micrograms TCDD/kg + 1 mg HC/kg. This extreme sensitivity suggests the involvement of a receptor-mediated mechanism possibly resulting in altered regulation of gene expression. Mechanisms of interaction were further studied by comparing growth of the shelves, fate of the medial epithelium, and expression of growth factor mRNAs and peptides. Pregnant mice were dosed on GDs 10-13 with HC (100 mg/kg sc) or with HC (25 mg/kg sc) + TCDD (3 micrograms/kg orally), doses producing 30% and 99% CP, respectively. The interaction between HC and TCDD results in a small HC-like palate, rather than the morphology typical of TCDD-induced clefting. Both compounds inhibited programmed cell death of the medial epithelium, which instead differentiated into an oral-like epithelium. The alterations in growth factor expression after HC or HC + TCDD were similar. Expression of EGF, TGF-beta 1, TGF-beta 2, and EGF receptor increased in specific palatal regions. Increased levels of mRNA were observed only for TGF-beta 1. The effect of TCDD alone on growth factor expression differ from those seen with HC or HC + TCDD. These divergent effects on growth factor expression may contribute to the differences in shelf size and thus to the different mechanisms of HC and TCDD clefting. Thus the synergism between HC and TCDD may involve similar and potentially additive effects on regulators of proliferation and differentiation in the palate, but additional contributing factors cannot be excluded.
