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

D. Roy, W. R. Snodgrass

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Abstract

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
Volume252
Issue number3
StatePublished - Jan 1 1990

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ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

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