In vitro bioactivation of phenytoin to a reactive free radical intermediate by prostaglandin synthetase, horseradish peroxidase, and thyroid peroxidase

S Kubow and PG Wells

Faculty of Pharmacy, University of Toronto, Canada.

Certain toxic effects of phenytoin are thought to result from its cytochrome P-450-catalyzed bioactivation to a reactive arene oxide intermediate that binds covalently to proteins. Using an in vitro system, we examined an alternative hypothesis based upon the cooxidation of phenytoin to a reactive free radical intermediate by prostaglandin synthetase (PGS), horseradish peroxidase, or thyroid peroxidase. Microsomes from hepatic, thyroid, seminal vesicular, or pulmonary tissues, or PGS or horseradish peroxidase, were incubated with the appropriate enzymatic cofactors to study activities of cytochromes P-450 (NADPH), PGS (arachidonic acid), thyroid peroxidase (guiaicol, H2O2), and horseradish peroxidase (H2O2). The production of potentially teratogenic, reactive phenytoin intermediates during in vitro incubations was estimated by the amount of radiolabeled phenytoin bound covalently to microsomal protein or bovine serum albumin and by the detection of a free radical intermediate using ESR spectrometry. Arachidonic acid-dependent bioactivation of phenytoin was demonstrated for purified PGS and ram seminal vesicles (RSV), as well as for liver, lung, and kidney. Optimal arachidonate concentrations varied substantially for different tissues. Arachidonate-dependent binding of phenytoin with PGS and RSV was reduced to baseline levels by coincubation with the cyclooxygenase inhibitor indomethacin. Hydrogen peroxide-dependent covalent binding of phenytoin was observed with thyroid peroxidase and horseradish peroxidase, and binding was significantly reduced in these systems and in PGS and RSV by coincubation with the peroxidase inhibitor methimazole. Glutathione, the antioxidants caffeic acid and butylated hydroxyanisole, and the free radical trapping agent alpha-phenyl-N-t-butylnitrone (PBN) all significantly reduced arachidonate-dependent phenytoin binding. Oxygen uptake was increased in a dose-dependent manner by the arachidonate- dependent bioactivation of phenytoin by PGS. ESR spin-trapping techniques using PBN indicated the generation of a free radical intermediate during the metabolism of phenytoin by PGS. These results suggest that the hydroperoxidase component of PGS, as well as thyroid peroxidase and other peroxidases, can bioactivate phenytoin to a reactive free radical intermediate, which may be toxicologically relevant.

Volume 35, Issue 4, pp. 504-511, 04/01/1989
Copyright © 1989 by American Society for Pharmacology and Experimental Therapeutics

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