Phenytoin-initiated DNA oxidation in murine embryo culture, and embryo protection by the antioxidative enzymes superoxide dismutase and catalase: evidence for reactive oxygen species-mediated DNA oxidation in the molecular mechanism of phenytoin teratogenicity.

A murine embryo culture model was used to investigate phenytoin-initiated embryonic DNA oxidation and dysmorphogenesis and to determine the embryoprotective potential of superoxide dismutase and catalase, which detoxify reactive oxygen species. Gestational day 9.5 CD-1 embryos were cultured for up to 24 hr at 37 degrees in medium containing phenytoin (20 micrograms/ml, 80 microM) or its vehicle (0.002 N NaOH). Embryos cultured for 24 hr were examined for embryotoxicity. After varying durations of incubation, embryonic DNA was isolated and purified, and DNA oxidation was determined from the formation of 8-hydroxy-2'-deoxyguanosine (8-OH-2'-dG). Control embryos showed an early increase in 8-OH-2'-dG levels that was maximal between 2 and 4 hr, followed by a small but significant decrease over 24 hr, with no evidence of embryopathy. Phenytoin-treated embryos within 4 hr also demonstrated maximal 8-OH-2'-dG formation, which was substantially greater than that of controls, with a maximal 3-fold increase over controls at 24 hr (p < 0.05). In wash-out studies, embryos removed from the phenytoin-containing medium after 4 hr and then cultured in phenytoin-free medium for an additional 20-hr period showed no decrease in either 8-OH-2'-dG levels or embryotoxicity, compared with embryos incubated in the presence of phenytoin for 24 hr. Embryos exposed to phenytoin demonstrated substantial dysmorphogenesis, as evidenced by decreases in anterior neuropore closure, turning, yolk sac diameter, crown-rump length, and somite development (p < 0.05). Superoxide dismutase and catalase virtually eliminated phenytoin-initiated 8-OH-2'-dG formation and reduced or completely eliminated all phenytoin-initiated dysmorphological anomalies (p < 0.05). These results suggest that embryonic DNA oxidation constitutes teratologically important molecular target damage, and they provide the first direct evidence that free radical-mediated oxidative stress plays a critical role in phenytoin teratogenesis.