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 prostaglandin H synthase (PHS)-catalyzed bioactivation and resultant oxidative damage to embryonic protein and DNA may constitute a molecular mechanism mediating phenytoin and benzo[a]pyrene teratogenesis. Embryos 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 (embryotoxicity) at 37 degrees C in medium containing either phenytoin (20 micrograms/ml, 80 microM), benzo[a]pyrene (10 microM), or their respective vehicles. As previously observed with phenytoin (Mol. Pharmacol.48: 112-120, 1995), embryos incubated with benzo[a]pyrene showed decreases in anterior neuropore closure, turning, yolk sac diameter, and somite development (p < .05). Addition of the antioxidative enzyme superoxide dismutase (SOD) substantially enhanced embryonic SOD activity (p < .05) and completely inhibited benzo[a]pyrene embryotoxicity (p < .05). Substantial PHS was detected in day 9.5 embryos using SDS/PAGE, anti-PHS antibody, and alkaline phosphatase-conjugated donkey anti-goat IgG. Embryonic protein oxidation was detected by the reaction of 0.5 mM 2,4-dinitrophenylhydrazine with protein carbonyl groups. This method was first validated by using a known hydroxyl radical-generating system consisting of vanadyl sulfate and H2O2, with bovine serum albumin or embryonic protein as the target. Embryonic proteins were characterized by SDS/PAGE, anti-dinitrophenyl antisera, and peroxidase-labeled goat anti-donkey IgG. Using enhanced chemiluminescence, the number and content of oxidized protein bands detected between 25 and 200 kDa were substantially increased by both phenytoin and benzo[a]pyrene. Addition of the reducing agent dithiothreitol, or SOD or catalase, decreased protein oxidation in phenytoin-exposed embryos. Both phenytoin (Mol. Pharmacol.48: 112-120, 1995) and benzo[a]pyrene enhanced embryonic DNA oxidation, determined 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 GSSG disulfide, as measured by HPLC (p < .05). These results provide direct evidence that, in the absence of maternal or placental processes, embryonic PHS-catalyzed bioactivation and reactive oxygen species-mediated oxidation of embryonic protein, thiols, and DNA may constitute a molecular mechanism mediating phenytoin and benzo[a]pyrene teratogenesis.
Winn
L M
LM
Faculty of Pharmacy, University of Toronto, Ontario, Canada.
Wells
P G
PG
D000818:Animals; D001564:Benzo(a)pyrene / Q000633:toxicity*; D001711:Biotransformation; D002417:Cattle; D046508:Culture Techniques; D004249:DNA Damage; D004622:Embryo, Mammalian / Q000187:drug effects* / Q000378:metabolism*; D005260:Female; D005609:Free Radicals / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D046911:Macromolecular Substances; D051379:Mice; D008954:Models, Biological; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects; D010672:Phenytoin / Q000633:toxicity*; D011247:Pregnancy; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D011506:Proteins / Q000187:drug effects / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D013482:Superoxide Dismutase / Q000378:metabolism; D013723:Teratogens / Q000633:toxicity*
D005609:Free Radicals; D046911:Macromolecular Substances; D011506:Proteins; D017382:Reactive Oxygen Species; D013723:Teratogens; D001564:Benzo(a)pyrene; D010672:Phenytoin; D011451:Prostaglandin-Endoperoxide Synthases; D013482:Superoxide Dismutase; D005978:Glutathione