Comparisons of the effects of TCDD and hydrocortisone on growth factor expression provide insight into their interaction in the embryonic mouse palate

Abstract

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.

Developmental expression of two members of a new class of transcription factors: I. Expression of aryl hydrocarbon receptor in the C57BL/6N mouse embryo

“…In our studies of AhR-mediated cleft palate, the expression of several growth factors including EGF, TGFcx, and the TGFPs were altered in TCDD-exposed palatal cells with associated changes in proliferation and differentiation of the epithelia (Abbott and Birnbaum, 1989;Abbott et al, 1992). There is no evidence that expression of these growth factors is directly regulated by XREs upstream of their genes, and the effects of TCDD may be part of a secondary cascade of responses to directly regulated genes.…”

“…Embryos exposed to low levels of TCDD, hydrocortisone, or retinoic acid do not develop cleft palate; however, exposure to either compound and TCDD simultaneously produces an incidence of 100% cleft palate (Birnbaum et al, 1986(Birnbaum et al, , 1989. These agents interact to disrupt cellular proliferation and differentiation and these effects are accompanied by altered expression of TGFa, EGF, the TGFPs, and the EGF receptor Birnbaum, 1989, 1990;Abbott et al, 1992). The interaction of A and 8).…”

The Effects of Dioxin on Reproduction and Development.

“…Both hydronephrosis and thymic atrophy are brought on by altered differentiation and proliferation of epithelial cells in the respective tissues. In the case of cleft palates, dioxin inhibits the proper fusion of the palate by inhibiting the decrease in EGF levels and increase in TGFα levels that occur during normal development 24,25) . Hydronephrosis is a more sensitive defect than cleft palate in mice.…”

Developmental Effects of Dioxins

“…The altered proliferation and differentiation is accompanied by changes in the balance of various growth factors and receptors. For example, in the mouse, TCDD treatment both in vivo (38)(39)(40) and in vitro (39) (42)(43)(44). The Ah receptor has also been detected in the developing palate in both mouse and human tissue (44,45).…”