FREE-RADICAL INTERMEDIATES OF PHENYTOIN AND RELATED TERATOGENS - PROSTAGLANDIN-H-SYNTHASE-CATALYZED BIOACTIVATION ELECTRON-PARAMAGNETIC-RESONANCE SPECTROMETRY, AND PHOTOCHEMICAL PRODUCT ANALYSIS

Phenytoin and related xenobiotics can be bioactivated by embryonic pro staglandin H synthase (PHS) to a teratogenic free radical intermediate . The mechanism of free radical formation was evaluated using photolyt ic oxidation with sodium persulfate and by EPR spectrometry. Character ization 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 te ratogen (phenytoin, mephenytoin, trimethadione, phenobarbital, and maj or metabolites) or its vehicle and the free radical spin trap alpha-ph enyl-N-t-butylnitrone, and incubations were analyzed by EPR spectromet ry. There was no alpha-phenyl-N-t-butylnitrone radical adduct in contr ol incubations. For phenytoin, a putative unstable nitrogen-centered r adical adduct and a stable carbon-centered radical adduct were detecte d. Free radical spin adducts also were detected for all other teratoge ns and metabolites except carbamazepine. The PHS inhibitor eicosatetra ynoic acid abolished the free radical EPR signal. Incubation of 2'-deo xyguanosine 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 re lated teratogens to a free radical intermediate that initiates DNA oxi dation, which may constitute a common molecular mechanism of teratolog ic initiation.