INTERACTIVE DYSMORPHOGENIC EFFECTS OF ALL-TRANS-RETINOL AND ETHANOL ON CULTURED WHOLE RAT EMBRYOS DURING ORGANOGENESIS

Whole rat conceptuses (10.5 gestational days) were explanted into a cu lture medium containing all-trans-retinol (t-retinol, vitamin A(1)), e thanol, or combinations of the two alcohols at various concentrations, and were cultured at 37 degrees C for 24 hr. Parameters emphasized in morphological analyses were branchial arch development, closure of ne ural tube, axial rotation, and development of otic vesicles and of opt ic cup. Additions of t-retinol atone to the culture medium resulted in significant decreases in viability al concentrations of 7.0 mu M and above. A primary target site affected by t-retinol was the second bran chial arch. With initial culture medium concentrations of 3.5 mu M, 28 % of embryos exhibited an underdeveloped second branchial arch, and th e effect was concentration dependent. Incubations with t-retinol alone also caused failure of closure of neural tubes, underdevelopment/abse nce of otic and optic vesicles, and failure of normal axial rotation, but these effects were statistically significant only at the higher co ncentrations (10.5-14.0 mu M). Incubations of conceptuses with ethanol alone resulted in statistically significant decreases in viability an d increases of incidence of embryonic abnormalities at 50 mM but not a t 10- or 20-mM concentrations. The embryotoxicity of ethanol appeared less site-specific than that of t-retinol. However, ethanol-elicited d evelopmental abnormalities included underdevelopment of the first and second branchial arches, abnormally open neural tubes, abnormally smal l or absent otic and optic vesicles, and incomplete axial rotation in common with effects elicited by t-retinol. In general, embryos incubat ed with combinations of t-retinol and ethanol showed lower survival ra tes and higher incidences of developmental abnormalities when compared to the calculated values expected for simple additive effects; i.e., interactive effects were most frequently greater than additive and pro bably synergistic but not antagonistic. To assist in the elucidation o f possible mechanism(s) for the greater than additive/synergistic dysm orphogenic effects observed, concentrations of all-trans-retinoic acid (t-RA) and all-trans-retinal (t-retinal) in cultured conceptal tissue s were determined by high-performance liquid chromatography (HPLC). HP LC analysis showed increases in conceptal tissue levels of both t-RA a nd t-retinal after conceptuses were exposed to t-retinol (10.5 mu M) p lus various quantities of ethanol for 24 hr. These observations, in co mbination with those of previous studies, suggested that the observed greater-than-additive/synergistic dysmorphogenic effects were not due to the inhibition by ethanol of conceptal biosynthesis of t-RA. Whethe r the increased levels of t-RA and t-retinal caused the observed great er than additive/synergistic dysmorphogenic effects remains to be eluc idated. (C) 1996 Wiley-Liss, Inc.