title: In vivo phenytoin-initiated oxidative damage to proteins and lipids in murine maternal hepatic and embryonic tissue organelles: potential molecular targets of chemical teratogenesis.
abstract: The widely used anticonvulsant drug phenytoin may be bioactivated by peroxidases such as prostaglandin H synthase (PHS) to a reactive free radical intermediate that initiates teratogenesis. This in vivo study evaluated the potential molecular targets mediating phenytoin teratogenicity. In vivo phenytoin-induced oxidative tissue damage following bioactivation was quantified in both maternal hepatic and embryonic tissues from pregnant CD-1 mice using lipid peroxidation and protein oxidation and degradation as indices. Pregnant mice were injected with a teratogenic dose of phenytoin, 65 mg/kg ip, during organogenesis on Gestational Day 12. alpha-Phenyl-N-t-butylnitrone (PBN), a free radical spin trapping agent, 41.5 mg/kg, or acetylsalicylic acid (ASA), an inhibitor of the cyclooxygenase component of PHS, 10 mg/kg, were injected ip 2 hr before phenytoin treatment, and maternal hepatic and embryonic tissues were obtained at 0, 3, 6, 8, and 24 hr. Phenytoin enhanced lipid peroxidation in maternal plasma, hepatic microsomes, cytosol, mitochondria, and nuclei and in embryonic microsomes, cytosol, and mitochondria (p < 0.05). Protein oxidation was significantly increased at 3 hr after phenytoin treatment in maternal hepatic and embryonic microsomes. Phenytoin increased protein degradation in maternal plasma, RBCs, hepatic microsomes, cytosol, mitochondria, and nuclei and in embryonic microsomes, cytosol, and nuclei (p < 0.05). In embryonic microsomes, PBN maximally inhibited phenytoin-initiated lipid peroxidation at 6 hr and lipid peroxidation and protein degradation respectively at 24 hr (p < 0.05). PBN also inhibited phenytoin-initiated protein oxidation in maternal hepatic and embryonic microsomes respectively at 24 hr (p < 0.05). These data imply that a free radical generated during phenytoin bioactivation was involved in tissue damage. ASA inhibited phenytoin-induced lipid peroxidation, protein oxidation, and degradation in embryonic microsomes at 24 hr (p < 0.05), indicating that embryonic PHS may be involved in the bioactivation of phenytoin. In maternal hepatic microsomes, however, PBN and ASA did not inhibit lipid peroxidation and protein degradation, suggesting that the lipid and protein damage in maternal liver may be regulated by cytochromes P450 and/or detoxifying pathways that are lacking in embryonic tissues. Such in vivo studies cannot exclude the possibility that protein and lipid oxidation occur as the result rather than the cause of phenytoin cellular damage. However, these results, combined with previous observations, including identical results in vitro and an in vivo reduction in phenytoin teratogenicity by PBN and ASA, suggest that peroxidase-catalyzed bioactivation of phenytoin may initiate oxidative damage to lipids and proteins in embryonic tissues, with teratological consequences.
authors.affiliation: Faculty of Pharmacy, University of Toronto, Ontario, Canada.
authors.forename: L
authors.initials: L
authors.lastname: Liu
authors.forename: P G
authors.initials: PG
authors.lastname: Wells
mesh_terms: D000818:Animals; D004622:Embryo, Mammalian / Q000166:cytology / Q000187:drug effects*; D005260:Female; D050356:Lipid Metabolism; D015227:Lipid Peroxidation / Q000187:drug effects; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D008815:Mice, Inbred Strains; D015388:Organelles / Q000187:drug effects* / Q000378:metabolism; D010084:Oxidation-Reduction; D010672:Phenytoin / Q000633:toxicity*; D011247:Pregnancy; D011506:Proteins / Q000187:drug effects / Q000378:metabolism*; D013723:Teratogens / Q000633:toxicity*
chemical_list: D011506:Proteins; D013723:Teratogens; D010672:Phenytoin