Effects of all-trans-retinoic acid and all-trans-retinoyl glucuronide in two in vitro systems of distinct biological complexity

In vitro systems are widely used to evaluate the embryotoxic potential of r etinoids. The effective concentrations of these retinoids, however, are not consistent in the various in vitro systems used in evaluating embryotoxici ty. This may be explained by the different level of complexity for each ind ividual system. which may lead to different concentrations of the substance s in the target tissues. To verify this hypothesis we have compared two in vitro systems of distinct biological complexity: the rat whole embryo cultu re system, and the mouse limb bud organ culture system. The lipid soluble, teratogenic retinoid all-trans-retinoic acid (ATRA), and all-trans-retinoyl -beta -D-glucuronide (ATRAG). an endogenous, water-soluble and biologically active retinoid with limited placental transfer, were compared with regard to their embryotoxic potential in vitro. In both in vitro systems, ATRAG s howed a lower degree of embryotoxicity than ATRA. In the limb bud organ cul ture, ATRAG revealed only slightly less toxicity than ATRA, whereas the eff ective concentrations of the two compounds in the whole embryo culture syst em differed by almost two orders of magnitude. During incubation with ATRAG , ATRA is generated by hydrolysis and is found in culture media and exposed tissues. The presence of membrane barriers around the developing embryo in the whole embryo culture system possibly prevents the transfer of ATRAG to the embryo and, therefore, its exposure to the active hydrolysis product A TRA. From these results we conclude that analysis of retinoid concentration s in the culture media and in the exposed tissues is essential for the inte rpretation of results obtained from in vitro toxicity testing.