title: Covalent binding of phenytoin to protein and modulation of phenytoin metabolism by thiols in A/J mouse liver microsomes.
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 greater than 3-methylcholanthrene greater than saline-treated controls. Low molecular weight thiols (GSH, cysteine and cysteamine), a thiol generator (methylthiazolidine 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.
authors.affiliation: Department of Pharmacology-Toxicology, University of Texas Medical Branch, Galveston.
authors.forename: D
authors.initials: D
authors.lastname: Roy
authors.forename: W R
authors.initials: WR
authors.lastname: Snodgrass
mesh_terms: D000596:Amino Acids / Q000494:pharmacology; D000818:Animals; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D005260:Female; D005978:Glutathione / Q000494:pharmacology; D051379:Mice; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D010672:Phenytoin / Q000378:metabolism* / Q000493:pharmacokinetics; D013438:Sulfhydryl Compounds / Q000494:pharmacology*
chemical_list: D000596:Amino Acids; D013438:Sulfhydryl Compounds; D010672:Phenytoin; D005978:Glutathione