Abstract

N′-nitrosonornicotine (NNN) induces tumors in the rat nasal cavity and esophagus and is believed to be a causative agent for esophageal cancer in tobacco users. To exert its carcinogenic potential, NNN must be metabolically activated by α-hydroxylation at either the 2′- or 5′-carbon. We previously reported that the human cytochrome P450 (P450), 2A6, efficiently and specifically catalyzed NNN 5′-hydroxylation. P450 2A3, which is expressed in the rat nasal cavity and to a small extent in the esophagus, is closely related to P450 2A6. P450 2A3, like 2A6, is a good catalyst of NNN α-hydroxylation (K_m 7 μM; V_max17 nmol/min/nmol). However, in contrast to P450 2A6, 2A3 catalyzed both 5′- and 2′-hydroxylation of NNN. The ratio of 2′- to 5′-hydroxylation was 1:3. These data, both with P450 2A6 and 2A3, were obtained using racemic NNN. P450 2A3 catalyzed metabolism of (S)-NNN occurred exclusively at the 5′-position. The predominant pathway of (R)-NNN metabolism was 2′-hydroxylation, and occurred to a 3-fold greater extent than did 5′-hydroxylation. These data are in contrast to those obtained from a recent study of (R)- and (S)-NNN metabolism by cultured rat esophagus. In that study, (S)-NNN was metabolized predominantly by 2′-hydroxylation and (R)-NNN equally by 2′- and 5′-hydroxylation. Taken together, these data provide strong evidence that P450 2A3 is not the rat esophageal P450 that catalyzes the metabolic activation of NNN. P450 2A3 may be an important catalyst of NNN activation in rat nasal mucosa.