Potential molecular targets mediating chemical teratogenesis : in vitro peroxidase-catalyzed phenytoin metabolism and oxidative damage to proteins and lipids in murine maternal hepatic microsomes and embryonic 9000g supernatant

Phenytoin and related proteratogens may be bioactivated by peroxidases to a reactive free radical intermediate that initiates teratogenesis. This study evaluated the potential molecular targets mediating phenytoin teratogenicity. In vitro phenytoin-induced oxidative tissue damage following metabolic activation by prostaglandin H synthase (PHS) and lipoxygenases (LPOs) was quantified in both maternal hepatic microsomes and embryonic 9000g supernatant (S-9) from CD-1 mice, using lipid peroxidation and protein oxidation and degradation as indices. Phenytoin (0-0.2 mM) and the PHS/LPO cofactor arachidonic acid (AA, 0-1.0 mM) were incubated with maternal hepatic microsomes or embryonic S-9 (2 mg) for 0-60 min. Lipid peroxidation was measured by the formation of thiobarbituric acid-reactive substance, protein oxidation quantified by the formation of carbonyl groups in proteins, and protein degradation measured by the release of primary amines. Phenytoin was cooxidized by peroxidases in both maternal hepatic microsomes and embryonic S-9 to initiate oxidative damage to tissue lipids and proteins. The lipid peroxidation and protein oxidation and degradation were dependent on incubation time, AA concentration, and phenytoin concentration (p < 0.05), indicating that peroxidases were involved in phenytoin metabolic activation. Phenytoin-initiated oxidative tissue damage in maternal hepatic microsomes was inhibited by the cyclooxygenase inhibitor indomethacin, confirming that PHS was involved in the reactions. Phenytoin-induced oxidative damage in embryonic S-9 was not reduced by indomethacin, but was by nordihydroguaiaretic acid, an antioxidant and LPO inhibitor, indicating that additional enzymes containing peroxidase activity, such as LPO, may play a substantial role in phenytoin metabolic activation in embryonic tissues. Phenytoin-initiated lipid peroxidation and protein oxidation were early events, preceding protein degradation (10 min versus 60 min), and a lower phenytoin concentration was needed for lipid peroxidation (0.04 mM) and protein oxidation (0.01 mM) than for protein degradation (0.08 mM), suggesting that phenytoin-initiated protein and lipid oxidation may be two potential causes for protein cleavage. These results provide an in vitro basis for the in vivo evidence that embryonic lipid and protein may constitute important molecular targets mediating phenytoin teratogenicity, wherein peroxidase-catalyzed metabolic activation initiates early macromolecular oxidation by reactive oxygen species.