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 cancer. However, the therapeutic application of retinoids is limited by their teratogenic potency. The limbs are important sites of retinoid-induced malformations in rodents. Therefore, organoid cultures of limb bud mesenchymal cells have been established for screening of the teratogenic potency of retinoids. We have now applied this system to compare the effects of all-trans-retinoyl-beta-D-glucuronide (all-trans-RAG) with those of all-trans-retinoic acid (all-trans-RA) on chondrogenesis, as assessed by the Alcian blue binding assay and by electron microscopic evaluation including quantitative morphometric analysis. First data of retinoid toxicokinetics in the culture media as well as retinoid concentrations in the cultured mesenchymal limb bud cells were established. While all-trans-RA inhibited chondrogenesis at 10(-7) M by ca. 50%, tenfold higher concentrations of all-trans-RAG were necessary to obtain 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 all-trans-RA for the first 2 h of a 6-day incubation period) demonstrated inhibition of chondrogenesis with all-trans-RA, but not with all-trans-RAG. The data indicate that RAG inhibits chondrogenesis upon hydrolysis to RA. Surprisingly, the rather polar RAG isoforms were extensively accumulated in the limb bud mesenchymal cells when compared to the medium. Both all-trans-RAG and all-trans-RA also induced a large increase of retinyl ester concentrations in the chondrocytes compared to vehicle-treated cells. This finding further supports a recent suggestion that RA regulates retinol metabolism via feedback inhibition of retinol oxidation and stimulation of the esterification of retinol.
Sass
J O
JO
Institut für Toxikologie und Embryopharmakologie, Fachbereich Humanmedizin, Freie Universität, Berlin, Germany.
Zimmermann
B
B
Rühl
R
R
Nau
H
H
D000423:Alcian Blue; D000818:Animals; D001846:Bone Development / Q000187:drug effects*; D002356:Cartilage / Q000166:cytology / Q000187:drug effects / Q000648:ultrastructure; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004396:Coloring Agents; D007091:Image Processing, Computer-Assisted; D051379:Mice; D008854:Microscopy, Electron; D012176:Retinoids / Q000378:metabolism*; D014212:Tretinoin / Q000031:analogs & derivatives* / Q000494:pharmacology*
D004396:Coloring Agents; D012176:Retinoids; C026787:retinoyl glucuronide; D014212:Tretinoin; D000423:Alcian Blue