Bioactivation of Phenytoin by Human Cytochrome P450: Characterization of the Mechanism and Targets of Covalent Adduct Formation

Munns, Andrew J., De Voss, James J., Hooper, Wayne D., Dickinson, Ronald G. and Gillam, Elizabeth M. J. (1997) Bioactivation of Phenytoin by Human Cytochrome P450: Characterization of the Mechanism and Targets of Covalent Adduct Formation. Chemical Research In Toxicology, 10 9: 1049-1058. doi:10.1021/tx9700836

Author Munns, Andrew J.
De Voss, James J.
Hooper, Wayne D.
Dickinson, Ronald G.
Gillam, Elizabeth M. J.
Title Bioactivation of Phenytoin by Human Cytochrome P450: Characterization of the Mechanism and Targets of Covalent Adduct Formation
Journal name Chemical Research In Toxicology   Check publisher's open access policy
ISSN 0893-228X
Publication date 1997-09-01
Year available 1997
Sub-type Article (original research)
DOI 10.1021/tx9700836
Open Access Status Not yet assessed
Volume 10
Issue 9
Start page 1049
End page 1058
Total pages 10
Place of publication Washington
Publisher American Chemical Society
Language eng
Subject 03 Chemical Sciences
Abstract The cytochrome P450-dependent covalent binding of radiolabel derived fi om phenytoin (DPH) and its phenol and catechol metabolites, 5-(4'-hydroxyphenyl)-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 incubations and isolated by preparative HPLC. NADPH-dependent covalent binding was demonstrated in incubations of human liver microsomes with HPPH. When CAT was used as substrate, covalent adduct formation was independent of NADPH, was enhanced in the presence of systems generating reactive oxygen species, and was diminished under anaerobic conditions or in the presence of cytoprotective reducing agents. Fluorographic analysis showed that radiolabel derived from DPH and HPPH was selectively associated with proteins migrating with approximate relative molecular weights of 57-59 kDa and at the dye front (molecular weights < 23 kDa) on denaturing gels. Lower levels of radiolabel were distributed throughout the molecular weight range. In contrast, little selectivity was seen in covalent adducts formed from CAT. HPPH was shown to be a mechanism-based inactivator of P450, supporting the contention that a cytochrome P450 is one target of covalent binding. These results suggest that covalent binding of radiolabel derived from DPH in rat and human Liver microsomes occurs via initial P450-dependent catechol formation followed by spontaneous oxidation to quinone and semiquinone derivatives that ultimately react with microsomal protein. Targets for covalent binding may include P450s, though the catechol appears to be sufficiently stable to migrate out of the P450 active site to form adducts with other proteins. In conclusion, we have demonstrated that DPH can be bioactivated in human liver to metabolites capable of covalently binding to proteins. The relationship of adduct formation to DPH-induced hypersensitivity reactions remains to be clarified.
Keyword Chemistry, Medicinal
Chemistry, Multidisciplinary
Endoplasmic-reticulum Autoantibodies
Human Liver-microsomes
Hypersensitivity Reactions
Drug Hypersensitivity
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Unknown

Document type: Journal Article
Sub-type: Article (original research)
Collection: School of Chemistry and Molecular Biosciences
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Citation counts: TR Web of Science Citation Count  Cited 51 times in Thomson Reuters Web of Science Article | Citations
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Created: Tue, 14 Aug 2007, 02:58:22 EST