EVIDENCE FOR EMBRYONIC PROSTAGLANDIN-H SYNTHASE-CATALYZED BIOACTIVATION AND REACTIVE OXYGEN SPECIES-MEDIATED OXIDATION OF CELLULAR MACROMOLECULES IN PHENYTOIN AND BENZO[A]PYRENE TERATOGENESIS

A mouse embryo culture model was used to determine whether embryonic p rostaglandin H synthase (PHS)-catalyzed bioactivation and resultant ox idative damage to embryonic protein and DNA may constitute a molecular mechanism mediating phenytoin and benzo[a]pyrene teratogenesis. Embry os were explanted from CD-1 mouse dams on gestational day 9.5 (vaginal plug = day 1) and incubated for either 4 h (biochemistry) or 24 h (em bryotoxicity) at 37 degrees C in medium containing either phenytoin (2 0 mu g/ml, 80 mu M), benzo[a]pyrene (10 mu M), or their respective veh icles. As previously observed with phenytoin (Mel. Pharmacol.48:112-12 0, 1995), embryos incubated with benzo[a]pyrene showed decreases in an terior neuropore closure, turning, yolk sac diameter, and somite devel opment (p < .05). Addition of the antioxidative enzyme superoxide dism utase (SOD) substantially enhanced embryonic SOD activity (p < .05) an d completely inhibited benzo[a]pyrene embryotoxicity (p < .05). Substa ntial PHS was detected in day 9.5 embryos using SDS/PAGE, anti-PHS ant ibody, and alkaline phosphatase-conjugated donkey anti-goat IgG. Embry onic protein oxidation was detected by the reaction of 0.5 mM 2,4-dini trophenylhydrazine with protein carbonyl groups. This method was first validated by using a known hydroxyl radical-generating system consist ing of vanadyl sulfate and H2O2, with bovine serum albumin or embryoni c protein as the target. Embryonic proteins were characterized by SDS/ PAGE, anti-dinitrophenyl antisera, and peroxidase-labeled goat anti-do nkey IgG. Using enhanced chemiluminescence, the number and content of oxidized protein bands detected between 25 and 200 kDa were substantia lly increased by both phenytoin and benzo[a]pyrene. Addition of the re ducing agent dithiothreitol, or SOD or catalase, decreased protein oxi dation in phenytoin-exposed embryos. Both phenytoin (Mel. Pharmacol.48 :112-120, 1995) and benzo[a]pyrene enhanced embryonic DNA oxidation, d etermined by the formation of 8-hydroxy-2'-deoxyguanosine, as measured by high-performance liquid chromatography (HPLC) (p < .05). Phenytoin also enhanced the oxidation of embryonic glutathione (GSH) to its GSS G disulfide, as measured by HPLC (p < .05). These results provide dire ct evidence that, in the absence of maternal or placental processes, e mbryonic PHS-catalyzed bioactivation and reactive oxygen species-media ted oxidation of embryonic protein, thiols, and DNA may constitute a m olecular mechanism mediating phenytoin and benzo[a]pyrene teratogenesi s. Copyright (C) 1997 Elsevier Science Inc.