N-Nitrosobenzylmethylamine Hydroxylation and Coumarin 7-Hydroxylation: Catalysis by Rat Esophageal Microsomes and Cytochrome P450 2A3 and 2A6 Enzymes

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• 作者：Linda B. von Weymarn ; Nadia D. Felicia ; Xinxin Ding ; and Sharon E. Murphy
• 刊名：Chemical Research in Toxicology
• 出版年：1999
• 出版时间：December 1999
• 年：1999
• 卷：12
• 期：12
• 页码：1254 - 1261
• 全文大小：95K
• 年卷期：v.12,no.12(December 1999)
• ISSN：1520-5010

文摘

N-Nitrosobenzylmethylamine (NBzMA) is a potent and selective esophageal carcinogen inthe rat and may be a causative agent for human esophageal cancer. This nitrosamine, likemost, must be metabolically activated to exert its carcinogenic potential. NBzMA may bemetabolized by P450-catalyzed methyl or methylene hydroxylation; the latter is believed to bethe activation pathway. The sensitivity of the esophagus to NBzMA-induced tumorigenesis isbelieved to be due, at least in part, to the presence of efficient P450 catalysts in this tissue.However, while it was reported almost 20 years ago that the rat esophagus catalyzes themethylene hydroxylation of NBzMA, the P450 that catalyzes this reaction has yet to beidentified. We report here that human P450 2A6 and the closely related extrahepatic rat enzymeP450 2A3 both efficiently catalyze NBzMA methylene hydroxylation, characterized asbenzaldehyde formation. The catalytic efficiency of P450 2A3 in this reaction was 3-fold greaterthan that of P450 2A6, 7.6 (K_m = 0.63 ± 0.18 ages/entities/mgr.gif">M and the V_max = 4.8 nmol min^-1 nmol ofP450^-1) versus 2.3 (K_m = 6.7 ± 2.9 ages/entities/mgr.gif">M and the V_max = 15.7 nmol min^-1 nmol of P450^-1),respectively. Both enzymes catalyzed methylene hydroxylation at least 4-fold more efficientlythan methyl hydroxylation. In addition, P450 2A6, but not P450 2A3, catalyzed benzyl ringhydroxylation, generating N-(p-hydroxybenzyl)methylamine. The identity of this metabolitewas confirmed by synthesis of a standard and LC/MS and LC/MS/MS analysis. P450 2A6 isan efficient coumarin 7-hydroxylase, and we report here that P450 2A3 is an equally goodcatalyst of this reaction (K_m = 1.7 ± 0.41 ages/entities/mgr.gif">M and V_max = 1.7 ± 0.08 nmol min^-1 nmol of P450^-1).Rat esophageal microsomes (REM), like P450 2A3, were efficient catalysts of NBzMA methylenehydroxylation. However, in contrast to P450 2A3, the major product of this reaction was theproduct of benzaldehyde oxidation, benzoic acid. Antibody to the closely related mouse P450,2A5, did not inhibit REM-catalyzed NBzMA metabolism, and most importantly, REM did notcatalyze the 7-hydroxylation of coumarin. Therefore, P450 2A3 does not appear to be the P450in the rat esophagus responsible for catalyzing the methylene hydroxylation of NBzMA.