Ultrastructural characterization of normal and abnormal chondrogenesis in micromass rat embryo limb bud cell cultures.
Inhibition of chondrogenesis in limb bud cell micromass cultures has been proposed as a short-term teratogen detection test. Validation studies were performed by testing large series of reference compounds and comparing their teratogenic potential with their ability to inhibit chondrogenesis; however, there are few reports describing the histological and ultrastructural changes associated with inhibition of chondrogenesis in vitro. The objective of this study was to provide a qualitative description of the histological and ultrastructural alterations induced by three chondrogenesis inhibitors: retinoic acid (RA) and 6-aminonicotinamide (6AN), two teratogens, and doxylamine succinate (DS), a nonteratogen compound. In addition, in order to have a basis for the interpretation of the morphological alterations induced by the test compounds, the histological and ultrastructural changes which occur during the time course of chondrogenesis in control cultures were described and compared with those in rat embryo limb buds. We found that RA at 0.5 micrograms/ml led to a marked decrease in the number and size of cartilaginous foci; most cells lacked morphological signs of differentiation but their ability to proliferate was unaffected. At concentrations of 2 micrograms/ml and more, 6AN delayed cell proliferation, reduced staining of the extracellular matrix, and induced the formation of endoplasmic cisternae. DS at 50 micrograms/ml affected both differentiation and proliferation; pigment deposits were observed in chondrocytes, suggesting phospholipid metabolism disorders. In conclusion, this study showed that inhibition of chondrogenesis in this simple cell culture system can be associated with different types of histological and ultrastructural alterations. Examination of these alterations can provide useful information about the teratogenic potential of tested compounds and their mechanism of action.
Renault
J Y
JY
Drug Safety Department, Rhône-Poulenc Rorer S.A., Vitry-sur-Seine, France.
Caillaud
J M
JM
Chevalier
J
J
D015120:6-Aminonicotinamide / Q000633:toxicity; D000818:Animals; D002356:Cartilage / Q000002:abnormalities / Q000196:embryology*; D002478:Cells, Cultured; D004319:Doxylamine / Q000031:analogs & derivatives / Q000633:toxicity; D004622:Embryo, Mammalian; D005314:Embryonic and Fetal Development / Q000187:drug effects; D005260:Female; D005552:Forelimb / Q000002:abnormalities / Q000187:drug effects / Q000196:embryology; D006634:Histamine H1 Antagonists / Q000633:toxicity; D051381:Rats; D017207:Rats, Sprague-Dawley; D013723:Teratogens / Q000633:toxicity*; D014212:Tretinoin / Q000633:toxicity
D006634:Histamine H1 Antagonists; D013723:Teratogens; D015120:6-Aminonicotinamide; D014212:Tretinoin; D004319:Doxylamine; C035385:doxylamine succinate