Received for publication August 29, 2005.
Revised November 21, 2005.
Accepted for publication November 22, 2005.

Selective Pathways for the Metabolism of Phencyclidine by Cytochrome P450 2B Enzymes: Identification of Electrophilic Metabolites, Glutathione and N-Acetyl cysteine Adducts

Abstract

The metabolism of phencyclidine (PCP) has been studied previously in P450-containing microsomal systems. However, the reactive intermediate(s) that covalently binds to the P450 and leads to inactivation or leaves the active site to modify other proteins has not been identified. In this study two electrophilic intermediates of PCP were identified by mass spectrometry and by trapping with reduced glutathione (GSH) or N-acetyl cysteine (NAC). The tentative structures of these electrophilic intermediates were determined using mass spectrometry. P450s 2B1 and 2B4 formed a metabolite that exhibited an m/z of 240 corresponding to the mass of the 2,3-dihydropyridinium species of PCP or its conjugate base, the 1,2-dihydropyridine. Chemical reduction of the incubation mixture using NaBH₄ resulted in the disappearance of the signal at m/z of 240, consistent with reduction of the 2,3-dihydropyridinium species. Furthermore, the reactive metabolite trapped by GSH resulted in an adduct exhibiting an m/z of 547 consistent with the mass of the 2,3-dihydropyridinium species of PCP (m/z 240) that has reacted with a molecule of GSH (m/z 308). However, P450 2B6 formed a different reactive intermediate of PCP that was isolated as a GSH adduct exhibiting an m/z of 581 and NAC adduct with an m/z of 437. LC-MS/MS analysis of these adducts suggested that a di-oxygenated iminium metabolite of PCP could be the reactive intermediate formed by P450 2B6 but not by the other 2B isoforms. These data suggest that P450 2B6 favors oxidation pathways for PCP metabolism that are different from those of P450s 2B1 and 2B4.

Key words: CYP2B, cytochrome P450 catalyzed oxidations, glutathione conjugates, human CYP enzymes, metabolite identification, reactive intermediate

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