EFFECTS OF ALL-TRANS-RETINOYL-BETA-D-GLUCURONIDE AND ALL-TRANS-RETINOIC ACID ON CHONDROGENESIS AND RETINOID METABOLISM IN MOUSE LIMB BUD MESENCHYMAL CELLS IN-VITRO

Retinoids, derivatives of vitamin A, are essential for many vertebrate functions. Furthermore, several drugs of this class of compounds are valuable in the treatment of certain forms of skin disorders and cance r. However, the therapeutic application of retinoids is limited by the ir teratogenic potency. The limbs are important sites of retinoid-indu ced malformations in rodents. Therefore, organoid cultures of limb bud mesenchymal cells have been established for screening of the teratoge nic potency of retinoids. We have now applied this system to compare t he effects of all-trans-retinoyl-beta-D-glucuronide (all-trans-RAG) wi th those of all-trans-retinoic acid (all-trans-RA) on chondrogenesis, as assessed by the Alcian blue binding assay and by electron microscop ic evaluation including quantitative morphometric analysis. First data of retinoid toxicokinetics in the culture media as well as retinoid c oncentrations in the cultured mesenchymal limb bud cells were establis hed. While all-trans-RA inhibited chondrogenesis at 10(-7) M by ca. 50 %, tenfold higher concentrations of ail-trans-RAG were necessary to ob tain the same effect. This difference reflects the ratio of RA isomers which were found in the medium after incubation with either all-trans -RAG or all-trans-RA. A pulse experiment (10(-5) M all-trans-RAG or al l-trans-RA for the first 2 h of a 6-day incubation period) demonstrate d inhibition of chondrogenesis with all-trans-RA, but not with all-tra ns-RAG The data indicate that RAG inhibits chondrogenesis upon hydroly sis to RA. Surprisingly, the rather polar RAG isoforms were extensivel y accumulated in the limb bud mesenchymal cells when compared to the m edium. Both all-trans-RAG and all-trans-RA also induced a large increa se of retinyl ester concentrations in the chondrocytes compared to veh icle-treated cells. This finding further supports a recent suggestion that RA regulates retinol metabolism via feedback inhibition of retino l oxidation and stimulation of the esterification of retinol.