Validation of an in vitro teratology system using chiral substances: stereoselective teratogenicity of 4-yn-valproic acid in cultured mouse embryos.
In vitro systems are important for toxicity testing as well as for investigating the mechanism of action of xenobiotics. The validation of such in vitro systems is often incomplete and extrapolation to the in vivo situation is equivocal. In the present study, we studied the effects of enantiomers of an analogue of the antiepileptic drug valproic acid (VPA): R(+)- and S(-)-4-yn-VPA (R- and S-2-n-propyl-4-pentynoic acid), which have previously been shown to induce selective teratogenicity in mice after in vivo administration, in mouse whole-embryo culture (WEC). Aqueous solutions of the sodium salts of the pure R- and S-enantiomers as well as R,S-4-yn-VPA (racemic mixture) or VPA itself were added to the culture medium at 0, 0.075, 0.15, 0.3, 0.6, or 1.2 mmol/liter and embryos were evaluated 24 hr later. The S-4-yn-VPA enantiomer induced clear concentration-dependent dysmorphogenesis that was evident even at the lowest concentration. The primary anomalies were neural tube defects, erratic neural seams, blisters, and rotational defects. Embryolethality was observed at 1.2 mmol/liter. The R-4-yn-VPA enantiomer was neither embryotoxic nor dysmorphogenic at any tested concentration. The lack of biological activity over 24 hr in WEC with the R-enantiomer suggests also that, as previously shown in vivo, there was no racemization of this isomer to the more active S-enantiomer. The racemic mixture of R and S isomers appeared to be slightly more embryolethal and dysmorphogenic than VPA. Overall, the potency of the S-enantiomer was approximately four times that of VPA. Therefore, the rank order of the four chemicals tested was S(-) >> S(-), R(+) > VPA >>> R(+), which is in agreement with the effects observed in in vivo exposed mice. These data demonstrate a direct stereoselective effect of these compounds on the embryo. This is the first illustration of the stereoselectivity of a xenobiotic in the WEC in vitro test system. Pure and stable enantiomers, which induce stereoselective toxicity in vivo, are demonstrated to be valuable for validation of this in vitro system.
Andrews
J E
JE
Developmental Toxicology Division, United States Environmental Protection Agency, Research Triangle Park, North Carolina 27711, USA.
Ebron-McCoy
M
M
Bojic
U
U
Nau
H
H
Kavlock
R J
RJ
D000818:Animals; D004622:Embryo, Mammalian / Q000187:drug effects*; D005314:Embryonic and Fetal Development / Q000187:drug effects; D005260:Female; D051379:Mice; D008815:Mice, Inbred Strains; D009924:Organ Culture Techniques; D011247:Pregnancy; D013237:Stereoisomerism; D013723:Teratogens / Q000633:toxicity*; D014635:Valproic Acid / Q000633:toxicity*
D013723:Teratogens; D014635:Valproic Acid