Evidence for embryonic peroxidase-catalyzed bioactivation and glutathione-dependent cytoprotection in phenytoin teratogenicity: modulation by eicosatetraynoic acid and buthionine sulfoximine in murine embryo culture.
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 degrees C in culture medium (35% male rat serum, 15% fetal bovine serum, and 50% Waymouth's medium) saturated with 5% CO2 in air. Initially, a nonembryotoxic concentration of ETYA (0,40,80, or 100 microM) was established within its peroxidase inhibitory range (Ki = 4-8 microM). Subsequently, embryos were incubated with vehicle alone, a therapeutic concentration of phenytoin alone (20 micrograms/ml or 80 microM), ETYA alone (40 microM), or phenytoin and ETYA combined. ETYA alone below 100 microM had no effect on yolk sac diameter (YSD), crown-rump length (CRL), somite development (SD), anterior neuropore closure (ANPC), or turning, but at 100 microM reduced CRL, YSD, and SD (p < or = 0.05). Phenytoin alone was embryotoxic, causing reduced CRL, YSD, and SD (p < or = 0.0001). Phenytoin and ETYA (40 microM) together resulted in an increase in YSD, SD, and CRL relative to those with phenytoin alone (p < or = 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 microM 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).(ABSTRACT TRUNCATED AT 400 WORDS)
Miranda
A F
AF
Department of Pharmacology, University of Toronto, Ontario, Canada.
Wiley
M J
MJ
Wells
P G
PG
D015117:5,8,11,14-Eicosatetraynoic Acid / Q000494:pharmacology*; D000818:Animals; D000963:Antimetabolites / Q000627:therapeutic use*; D019328:Buthionine Sulfoximine; D002478:Cells, Cultured; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D004220:Disulfides / Q000378:metabolism; D004347:Drug Interactions; D004622:Embryo, Mammalian; D005260:Female; D005978:Glutathione / Q000378:metabolism; D008297:Male; D008717:Methionine Sulfoximine / Q000031:analogs & derivatives* / Q000627:therapeutic use; D051379:Mice; D010544:Peroxidases / Q000037:antagonists & inhibitors; D010672:Phenytoin / Q000037:antagonists & inhibitors / Q000633:toxicity*; D051381:Rats; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013723:Teratogens / Q000633:toxicity*
D000963:Antimetabolites; D016861:Cyclooxygenase Inhibitors; D004220:Disulfides; D013438:Sulfhydryl Compounds; D013723:Teratogens; D015117:5,8,11,14-Eicosatetraynoic Acid; D008717:Methionine Sulfoximine; D019328:Buthionine Sulfoximine; D010672:Phenytoin; D010544:Peroxidases; D005978:Glutathione