Covalent binding of phenytoin to protein and modulation of phenytoin metabolism by thiols in A/J mouse liver microsomes

Research output: Contribution to journalArticlepeer-review

34 Scopus citations


The role of thiols (nonprotein and protein) in the metabolic activation of phenytoin was examined. In vitro phenytoin covalent binding and metabolite formation were determined in hepatic microsomes from A/J mice. Covalent binding of a phenytoin-reactive intermediate to microsomal protein was linear with respect to time, protein concentration and phenytoin concentration. Covalent binding was inhibited by inhibitors of cytochrome P-450. Inducers of cytochrome P-450 enhanced phenytoin covalent binding as follows: phenobarbital > 3-methylcholanthrene > saline-treated controls. Low molecular weight thiols (GSH, cysteine and cysteamine), a thiol generator (methylthiazoldine carboxylate), and thiol modifying agents (N-ethylmaleimide, mercuric chloride and diamide) significantly inhibited covalent binding. Amino acids other than cysteine did not decrease the covalent binding. Formation of the metabolites, para-hydroxyphenytoin and phenytoin dihydrodiol, was greater following preincubation with GSH or cysteine. In summary, protein thiol groups appear to be important sites for in vitro covalent binding of a reactive intermediate of phenytoin. These data suggest glutathione may protect membrane-bound enzymes responsible for phenytoin metabolism from attack by an electrophilic or free radical reactive intermediate of phenytoin and GSH may inactivate a phenytoin-reactive metabolite by formation of a putative glutathione conjugate.

Original languageEnglish (US)
Pages (from-to)895-900
Number of pages6
JournalJournal of Pharmacology and Experimental Therapeutics
Issue number3
StatePublished - 1990

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology


Dive into the research topics of 'Covalent binding of phenytoin to protein and modulation of phenytoin metabolism by thiols in A/J mouse liver microsomes'. Together they form a unique fingerprint.

Cite this