Phenytoin and related xenobiotics can be bioactivated by embryonic prostaglandin H synthase (PHS) to a teratogenic free radical intermediate. The mechanism of free radical formation was evaluated using photolytic oxidation with sodium persulfate and by EPR spectrometry. Characterization of the products by mass spectrometry suggested that phenytoin photolyzes to a nitrogen-centered radical that rapidly undergoes ring opening to form a carbon-centered radical. PHS-1 was incubated with teratogen (phenytoin, mephenytoin, trimethadione, phenobarbital, and major metabolites) or its vehicle and the free radical spin trap ␣-phenyl-N-tbutylnitrone, and incubations were analyzed by EPR spectrometry. There was no ␣-phenyl-N-t-butylnitrone radical adduct in control incubations. For phenytoin, a putative unstable nitrogen-centered radical adduct and a stable carbon-centered radical adduct were detected. Free radical spin adducts also were detected for all other teratogens and metabolites except carbamazepine. The PHS inhibitor eicosatetraynoic acid abolished the free radical EPR signal. Incubation of 2-deoxyguanosine with phenytoin and PHS-1 resulted in a 5-fold increase in its oxidation to 8-hydroxy-2-deoxyguanosine. This is the first direct chemical evidence for PHS-catalyzed bioactivation of phenytoin and related teratogens to a free radical intermediate that initiates DNA oxidation, which may constitute a common molecular mechanism of teratologic initiation.Phenytoin (diphenylhydantoin; Dilantin) is a widely used anticonvulsant drug that is teratogenic in animals and humans (1-3). Several teratologic mechanisms have been proposed, including the bioactivation of phenytoin by embryonic cytochrome P450 to an electrophilic arene oxide reactive intermediate that covalently binds to embryonic protein, thereby altering cellular function (1-6). However, these hypotheses are not consistent with a number of published observations, including 1) the association of embryopathic activities of the structurally similar, asymmetric hydantoin anticonvulsants mephenytoin (Mesantoin) and its N-demethylated active metabolite nirvanol with the L-isomers that primarily do not form the arene oxide (Fig. 1) (7); 2) the teratogenicity of structurally similar anticonvulsants, such as trimethadione (Tridione) and its N-demethylated pharmacologically active metabolite, dimethadione, that lack the phenyl substituent necessary for the formation of an arene oxide; and 3) the relatively low embryonic activity of most cytochrome P450s during organogenesis (4, 5, 9), including CYP2C9, which is known to bioactivate phenytoin (10).We have investigated an alternative hypothesis involving the bioactivation of proteratogens by peroxidases such as prostaglandin H synthase (PHS) 1 to teratogenic free radical intermediates ( Fig. 2) (3-6, 9). PHS and related potential bioactivating enzymes such as lipoxygenases are present with high activity in the embryo during organogenesis, the period of major teratologic susceptibility. Xenobiotic free radicals can bind cov...
