BIOACTIVATION OF PHENYTOIN BY HUMAN CYTOCHROME-P450 - CHARACTERIZATION OF THE MECHANISM AND TARGETS OF COVALENT ADDUCT FORMATION

The cytochrome P450-dependent covalent binding of radiolabel derived f i om phenytoin (DPH) and its phenol and catechol metabolites, 5-(4'-hy droxyphenyl)-5-phenylhydantoin (HPPH) and 5-(3',4'-dihydroxyphenyl)-5- phenylhydantoin (CAT), was examined in liver microsomes. Radiolabeled HPPH and CAT and unlabeled CAT were obtained from microsomal incubatio ns and isolated by preparative HPLC. NADPH-dependent covalent binding was demonstrated in incubations of human liver microsomes with HPPH. W hen CAT was used as substrate, covalent adduct formation was independe nt of NADPH, was enhanced in the presence of systems generating reacti ve oxygen species, and was diminished under anaerobic conditions or in the presence of cytoprotective reducing agents. Fluorographic analysi s showed that radiolabel derived from DPH and HPPH was selectively ass ociated with proteins migrating with approximate relative molecular we ights of 57-59 kDa and at the dye front (molecular weights < 23 kDa) o n denaturing gels. Lower levels of radiolabel were distributed through out the molecular weight range. In contrast, little selectivity was se en in covalent adducts formed from CAT. HPPH was shown to be a mechani sm-based inactivator of P450, supporting the contention that a cytochr ome P450 is one target of covalent binding. These results suggest that covalent binding of radiolabel derived from DPH in rat and human Live r microsomes occurs via initial P450-dependent catechol formation foll owed by spontaneous oxidation to quinone and semiquinone derivatives t hat ultimately react with microsomal protein. Targets for covalent bin ding may include P450s, though the catechol appears to be sufficiently stable to migrate out of the P450 active site to form adducts with ot her proteins. In conclusion, we have demonstrated that DPH can be bioa ctivated in human liver to metabolites capable of covalently binding t o proteins. The relationship of adduct formation to DPH-induced hypers ensitivity reactions remains to be clarified.