Metabolism of phenytoin and covalent binding of reactive intermediates in activated human neutrophils

Dennis C. Mays, Lew J. Pawluk, Glen Apseloff, W. Bruce Davis, Zhi Wu She, Arthur L. Sagone, Nicholas Gerber

Research output: Contribution to journalArticle

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Abstract

Activation of neutrophils by phorbol-12-myristate-13-acetate (PMA) causes rapid production of superoxide radical (O2 -), leading to the formation of additional reactive oxygen species, including hydrogen peroxide (H2O2), hypochlorous acid (HOCl), and possibly hydroxyl radical (·OH). These reactive oxygen species have been associated with the oxidation of some drugs. We investigated the metabolism of phenytoin (5,5-diphenylhydantoin) and the covalent binding of reactive intermediates to cellular macromolecules in activated neutrophils. In incubations with 100 μM phenytoin, PMA-stimulated neutrophils from six human subjects produced p-, m-, and o-isomers of 5-(hydroxyphenyl)-5-phenylhydantoin (HPPH) in a ratio of 1.0:2.1:2.8, respectively, as well as unidentified polar products. Analysis of cell pellets demonstrated that phenytoin was bioactivated to reactive intermediates that bound irreversibly to macromolecules in neutrophils. Glutathione, catalase, Superoxide dismutase, azide, and indomethacin all diminished the metabolism of phenytoin and the covalent binding of its reactive intermediates. The iron-inactivating chelators desferrioxamine and diethylenetriaminepentaacetic acid had little or no effect on the metabolism of phenytoin by neutrophils, demonstrating that adventitious iron was not contributing via Fenton chemistry. In an ·OH-generating system containing H2O2 and Fe2+ chelated with ADP, phenytoin was oxidized rapidly to unidentified polar products and to p-, m-, and o-HPPH (ratio 1.0:1.7:1.5, respectively). Reagent HOCl and human myeloperoxidase (MPO), in the presence of Cl- and H2O2, both formed the reactive dichlorophenytoin but no HPPH. However, no chlorinated phenytoin was detected in activated neutrophils, possibly because of its high reactivity. These findings, which demonstrated that activated neutrophils biotransform phenytoin in vitro to hydroxylated products and reactive intermediates that bind irreversibly to tissue macromolecules, are consistent with phenytoin hydroxylation by ·OH generated by a transition metal-independent process, chlorination by HOCl generated by MPO, and possibly cooxidation by neutrophil hydroperoxidases. Neutrophils activated in vivo may similarly convert phenytoin to reactive intermediates, which could contribute to some of the previously unexplained adverse effects of the drug.

Original languageEnglish (US)
Pages (from-to)367-380
Number of pages14
JournalBiochemical Pharmacology
Volume50
Issue number3
DOIs
StatePublished - Jul 31 1995

Fingerprint

Phenytoin
Metabolism
Neutrophils
Hypochlorous Acid
Macromolecules
Peroxidase
Reactive Oxygen Species
Acetates
Iron
Neutrophil Activation
Hydroxylation
Deferoxamine
Azides
Chlorination
Halogenation
Chelating Agents
Superoxides
Indomethacin
Isomers
Pharmaceutical Preparations

Keywords

  • activated neutrophils
  • covalent binding
  • dichlorophenytoin
  • drug oxidation
  • hydroxyl radical
  • hypochlorous acid
  • myeloperoxidase
  • phenytoin

ASJC Scopus subject areas

  • Biochemistry
  • Pharmacology

Cite this

Metabolism of phenytoin and covalent binding of reactive intermediates in activated human neutrophils. / Mays, Dennis C.; Pawluk, Lew J.; Apseloff, Glen; Davis, W. Bruce; She, Zhi Wu; Sagone, Arthur L.; Gerber, Nicholas.

In: Biochemical Pharmacology, Vol. 50, No. 3, 31.07.1995, p. 367-380.

Research output: Contribution to journalArticle

Mays, Dennis C. ; Pawluk, Lew J. ; Apseloff, Glen ; Davis, W. Bruce ; She, Zhi Wu ; Sagone, Arthur L. ; Gerber, Nicholas. / Metabolism of phenytoin and covalent binding of reactive intermediates in activated human neutrophils. In: Biochemical Pharmacology. 1995 ; Vol. 50, No. 3. pp. 367-380.
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