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

D. Roy, W. R. Snodgrass

Research output: Contribution to journalArticle

33 Citations (Scopus)

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 - 1990

Fingerprint

Phenytoin
Liver Microsomes
Sulfhydryl Compounds
Proteins
Cysteine
Glutathione
Diamide
Cysteamine
Mercuric Chloride
Ethylmaleimide
Methylcholanthrene
Phenobarbital
Microsomes
Free Radicals
Molecular Weight
Amino Acids
Membranes
Liver
Enzymes

ASJC Scopus subject areas

  • Pharmacology

Cite this

Covalent binding of phenytoin to protein and modulation of phenytoin metabolism by thiols in A/J mouse liver microsomes. / Roy, D.; Snodgrass, W. R.

In: Journal of Pharmacology and Experimental Therapeutics, Vol. 252, No. 3, 1990, p. 895-900.

Research output: Contribution to journalArticle

@article{95727bdcca51487393f9d34f85d5f5d3,
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 > 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.",
author = "D. Roy and Snodgrass, {W. R.}",
year = "1990",
language = "English (US)",
volume = "252",
pages = "895--900",
journal = "Journal of Pharmacology and Experimental Therapeutics",
issn = "0022-3565",
publisher = "American Society for Pharmacology and Experimental Therapeutics",
number = "3",

}

TY - JOUR

T1 - Covalent binding of phenytoin to protein and modulation of phenytoin metabolism by thiols in A/J mouse liver microsomes

AU - Roy, D.

AU - Snodgrass, W. R.

PY - 1990

Y1 - 1990

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0025262246&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025262246&partnerID=8YFLogxK

M3 - Article

C2 - 2319474

AN - SCOPUS:0025262246

VL - 252

SP - 895

EP - 900

JO - Journal of Pharmacology and Experimental Therapeutics

JF - Journal of Pharmacology and Experimental Therapeutics

SN - 0022-3565

IS - 3

ER -