Evidence for Embryonic Peroxidase-Catalyzed Bioactivation and Glutathione-Dependent Cytoprotection in Phenytoin Teratogenicity: Modulation by Eicosatetraynoic Acid and Buthionine Sulfoximine in Murine Embryo Culture

Abstract

Phenytoin teratogenicity may result from embryonic, peroxidase-catalyzed bioactivation of phenytoin to a toxic reactive free radical intermediate for which embryonic glutathione (GSH) is cytoprotective. This hypothesis was tested in embryo culture using 5.8,11,14-eicosatetraynoic acid (ETYA), a dual inhibitor of two peroxidase systems. prostaglandin synthetase, and lipoxygenases. Embryos from CD-1 mice were explanted on Gestational Day 9.5 (vaginal plug, Day 1) and incubated for 24 hr at 37°C in culture medium (35% male rat serum, 15% fetal bovine serum, and 50% Waymouth′s medium) saturated with 5% CO 2 in air. Initially, a nonembryotoxic concentration of ETYA (0,40,80, or 100 μM) was established within its peroxidase inhibitory range (K, = 4-8 μM). Subsequently, embryos were incubated with vehicle alone, a therapeutic concentration of phenytoin alone (20 μg/ml or 80 μM), ETYA alone (40 μM), or phenytoin and ETYA combined. ETYA alone below 100 μM had no effect on yolk sac diameter (YSD), crown-rump length (CRL), somite development (SD), anterior neuropore closure (ANPC), or turning, but at 100 μM reduced CRL, YSD. and SD ( p ≤ 0.05). Phenytoin alone was embryotoxic, causing reduced CRL, YSD. and SD ( p ≤ 0.0001). Phenytoin and ETYA (40 μM) together resulted in an increase in YSD, SD, and CRE relative to those with phenytoin alone ( p ≤ 0.01), indicating that inhibition by ETYA of embryonic, peroxidase-catalyzed bioactivation of phenytoin is cytoprotective. GSH may play a critical role in detoxifying a phenytoin free radical or subsequent activated oxygen species, thereby reducing covalent binding, lipid peroxidation, and oxidative stress that may initiate embryotoxicity or death. To test this hypothesis, embryos were cultured in the presence or absence of 1 mM buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, for 3 hr, at which time BSO was washed out and the embryos were incubated for 24 hr in fresh culture medium containing 80 μM phenytoin or its vehicle. Soluble thiols, including GSH, and disulfides, including oxidized GSH (GSSG), were measured using high-performance liquid chromatography. Immediately after BSO treatment, there were no differences in the concentrations of GSH or GSSG between BSO-exposed embryos and controls. However, at 24 hr, GSH concentrations in untreated embryos increased almost 17-fold over those at 3 hr concentrations, while GSH in BSO-exposed embryos were reduced to 15% of control values ( p = 0.0008). Incubation with BSO alone reduced embryonic YSD ( p ≤ 0.0001), CRL ( p = 0.03), and ANPC ( p = 0.02) relative to that in untreated embryos. Preincubation with BSO enhanced phenytoin embryotoxicity compared to that in controls, with further reductions in YSD ( p ≤ 0.0001), SD ( p ≤ 0.05), and CRL ( p ≤ 0.05) and a nonsignificant reduction in turning ( p = 0.06), indicating that depletion by BSO of embryonic GSH enhances susceptibility to phenytoin embryotoxicity. These results suggest that embryonic, peroxidase-catalyzed bioactivation of phenytoin and GSH-dependent detoxifying and cytoprotective pathways are critical determinants of phenytoin teratogenicity.