Analysis of high dysmorphogenic activity of Ro 13‐6307, a tetramethylated tetralin analog of retinoic acid

Abstract

Certain synthetic retinoids differ widely from retinoic acid (RA) in teratogenic potency, being much more or much less effective than RA. It is assumed that the potency of a retinoid may depend on the nature of its interaction with cellular binding components (nuclear retinoic acid receptors or cytoplasmic binding proteins) and, as in the case of retinoids that are mammalian teratogens, on factors that determine its accessibility to the embryo. To investigate some of the factors that contribute to potency, we used a new synthetic retinoid Ro 13-6307 that differs in structure from RA in having an aromatic ring inserted in its side chain along with gem dimethyl modification of the natural cyclohexenyl ring. Pregnant ICR mice were given a single oral dose (0, 1, or 10 mg/kg) on day 11 of gestation, and the resultant teratogenic outcome was monitored on day 17. Direct effects on cell differentiation were obtained by exposing high density cultures of limb bud mesenchymal cells to a range of concentrations (0.3 ng/ml-3 micrograms/ml) of Ro 13-6307 and scoring for chondrogenic suppression. Concentrations reaching the embryo after maternal administration of Ro 13-6307 were measured by HPLC to quantify the analog for a period of 4 h after administration of the oral dose. We found that this retinoid was 40-fold as active as RA in both inducing teratogenesis and suppressing chondrogenesis, yet its concentration in the affected embryo was only a fraction of that achieved after an equivalent dose of RA was employed in a similar protocol.(ABSTRACT TRUNCATED AT 250 WORDS)

Temporal and spatial expression of alternative splice-forms of the α1(XI) collagen gene in fetal rat cartilage

“…Limb bud mesenchymal cells spontaneously differentiate into chondrocytes when plated at high density as micromass cultures and appear to mimic chondrogenesis in vivo (Caplan, 1970;Umansky, 1966). Like chondrogenesis in vivo, limb bud mesenchymal cells condense into nodules which then differentiate into cartilage-like tissues (Ahrens et al, 1977;Kochhar and Penner, 1992). The appearance of ␣1(XI) splice-forms typical of chondrocytes coincides with strong alcian blue staining of nodules characteristic of cartilage.…”

Cell motility is inhibited by the antiepileptic compound, valproic acid and its teratogenic analogues

“…In order to evaluate teratogenecity of drugs, several in vitro methods are currently employed. Widely used are whole rat or mouse embryo cultures [Andrews et al, 1995;Bojic et al, 1996;Guest et al, 1994;Narotski et al, 1994], high density cultures of rodent limb bud mesenchymal cells, and other so-called micromass teratogen tests [Flint, 1993;Kochhar and Penner, 1992;Regan et al, 1990]. These methods are relatively laborious, and require the sacrifice of laboratory animals.…”

Retinoids and Retinoid Receptors in Teratogenesis*

“…To assess if the differential teratogenicity of retinoids was intrinsic rather than due to maternaYplacenta1 factors, we used an in vitro assay comprising high density "spot" cultures of mouse embryo limb bud rnesenchymal cells (Ahrens et al, 1977;Kistler, 1987;Kochhar and Penner, 1992) (Fig. 4).…”

“…We have undertaken another preliminary study to link receptor function with teratogenesis. Here, we employed RO 13-6307 since its teratogenicity is much higher and its accessibility to the embryo is much lower than RA (Kochhar and Penner, 1992). A fully teratogenic dose of RO 13-6307 (10 mg-kg) given to pregnant mice preferentially elevated the level of RAR-j32 mRNA in susceptible embryonic regions (maximal induction 10-to 12-fold above control limb buds) in a manner comparable to a fully teratogenic dose of all-trans RA (100 mg-kg) ( Fig.…”