Abstract

We evaluated the role of microtubule assembly in phenytoin (5-5-diphenylhydantoin) teratogenic activity in the sea urchin embryo. Zygotes were exposed to phenytoin or one of several phenytoin analogs within 15 min of fertilization and the frequency of the resultant malformations was assessed at the cleavage and late gastrula (prism) stages. Concomitant studies of drug uptake into zygotes and drug effects on both microtubule assembly in vitro and spindle morphology in situ were also performed. Phenytoin, 5-p-methylphenyl-5-phenylhydantoin, and 5-p-methoxyphenyl-5-phenylhydantoin were teratogenic (approaching 100% affected embryos) at both developmental stages were concentrated rapidly by the zygotes, and induced a shortened mitotic spindle in situ. In a separate in vitro system using porcine brain microtubular protein, these analogs were shown to inhibit microtubule assembly directly. The major human metabolite of phenytoin, 5-p-hydroxyphenyl-5-phenylhydantoin was teratogenic at the prism stage but induced only a 20% incidence of abnormal embryos at the first cleavage. This was attributed to the slow rate of uptake of this analog. This compound inhibited microtubule assembly in the in vitro assay and also shortened the mitotic spindle to an extent proportional to its observed weak effect on the first cleavage. Another analog, 5-p-hydroxyphenyl-5-p'-methylphenylhydantoin was not teratogenic at concentrations up to the limit of its solubility (285 microM). If this analog were as potent inside the cell as either phenyltoin or 5-p-hydroxyphenyl-5-phenylhydantoin, the intracellular concentrations achieved should have been sufficient to induce abnormal cleavage. Thus, the lack of teratogenic efficacy of this analog was correlated with its observed lack of effects on either microtubule assembly in vitro or spindle formation in situ. The anticonvulsant drug ethotoin was not teratogenic at concentrations up to 2.93 mM, apparently due to either poor uptake or inability to inhibit microtubule assembly or both. Overall, these studies are consistent with a hypothesis that phenytoin may induce abnormal development in this system by a direct inhibition of microtubule assembly.