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 culture medium containing all-trans-retinol (t-retinol, vitamin A1), ethanol, 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 neural tube, axial rotation, and development of otic vesicles and of optic cup. Additions of t-retinol alone to the culture medium resulted in significant decreases in viability at concentrations of 7.0 microM and above. A primary target site affected by t-retinol was the second branchial arch. With initial culture medium concentrations of 3.5 microM, 28% of embryos exhibited an underdeveloped second branchial arch, and the effect was concentration dependent. Incubations with t-retinol alone also caused failure of closure of neural tubes, underdevelopment/absence of otic and optic vesicles, and failure of normal axial rotation, but these effects were statistically significant only at the higher concentrations (10.5-14.0 microM). Incubations of conceptuses with ethanol alone resulted in statistically significant decreases in viability and increases of incidence of embryonic abnormalities at 50 mM but not at 10- or 20-mM concentrations. The embryotoxicity of ethanol appeared less site-specific than that of t-retinol. However, ethanol-elicited developmental abnormalities included underdevelopment of the first and second branchial arches, abnormally open neural tubes, abnormally small or absent otic and optic vesicles, and incomplete axial rotation in common with effects elicited by t-retinol. In general, embryos incubated with combinations of t-retinol and ethanol showed lower survival rates 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 probably synergistic but not antagonistic. To assist in the elucidation of possible mechanism(s) for the greater than additive/synergistic dysmorphogenic effects observed, concentrations of all-trans-retinoic acid (t-RA) and all-trans-retinal(t-retinal) in cultured conceptal tissues were determined by high-performance liquid chromatography (HPLC). HPLC analysis showed increases in conceptal tissue levels of both t-RA and t-retinal after conceptuses were exposed to t-retinol (10.5 microM) plus various quantities of ethanol for 24 hr. These observations, in combination 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. Whether the increased levels of t-RA and t-retinal caused the observed greater than additive/synergistic dysmorphogenic effects remains to be elucidated.