Received for publication July 6, 2005.
Revised September 14, 2005.
Accepted for publication September 16, 2005.

Bioactivation of 4-Methylphenol (p-cresol) via CYP-mediated Aromatic Oxidation in Human Liver Microsomes

Abstract

Abstract It has previously been proposed that 4-methylphenol (p-cresol) is metabolically activated by oxidation of the methyl group to form a reactive quinone methide. In present study a new metabolism pathway is elucidated in human liver microsomes. Oxidation of the aromatic ring leads to formation of 4-methyl-ortho-hydroquinone that is further oxidized to a reactive intermediate 4-methyl-ortho-benzoquinone. This bioactivation pathway is fully supported by the following observations: 1), one major and two minor GSH adducts were detected in microsomal incubations of p-cresol in the presence of glutathione; 2), a major metabolite of p-cresol was identified as 4-methyl-ortho-hydroquinone in microsomal incubations; 3), the same GSH adducts were detected in microsomal incubations of 4-methyl-ortho-hydroquinone; 4), the same GSH adducts were chemically synthesized by oxidizing 4-methyl-ortho-hydroquinone followed by the addition of GSH, and the major conjugate was identified by LC/MS/MS and NMR as 3-(glutathione-S-yl)-5-methyl-ortho-hydroquinone. In addition, it was found that 4-hydroxybenzylalcohol, a major metabolite derived from oxidation of the methyl group in liver microsomes, was further converted to 4-hydroxybenzaldehyde. In-vitro studies also revealed that bioactivation of p-cresol was mediated by multiple CYPs, but 2D6, 2E1 and 1A2 are most active enzymes for formation of quinone methide, 4-methyl-ortho-benzoquinone and 4-hydroxybenzaldehyde, respectively. Implications of the newly identified reactive metabolite in p-cresol-induced toxicity remain to be investigated in the future.

Key words: bioactivation, chemical toxicology, cytochrome P450 catalyzed oxidations, glutathione conjugates, liver microsomes, mass spectrometry, metabolite identification, reactive intermediate, reactive metabolites

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