title: Nicotine 5'-oxidation and methyl oxidation by P450 2A enzymes.
abstract: In smokers, the primary pathway of nicotine metabolism is P450 2A6-catalyzed 5'-oxidation. The nicotine Delta(5'(1'))-iminium ion product of this reaction is further metabolized to cotinine by aldehyde oxidase. Previous investigators have reported kinetic parameters for cotinine formation using human liver cytosol as a source of aldehyde oxidase. Using [5-(3)H]nicotine and radioflow high-performance liquid chromatography analysis, we determined kinetic parameters for nicotine 5'-oxidation by P450 2A6 and the closely related human extrahepatic P450 2A13 as well as the rodent P450s 2A3, 2A4, and 2A5. The formation of both cotinine and nicotine Delta(5'(1'))-iminium ion was monitored. The K(m) and V(max) values for P450 2A6 were 144 +/- 15 muM and 1.30 +/- 0.05 pmol/min/pmol, respectively. Previously reported K(m) values for cotinine formation by P450 2A6 in the presence of cytosol were much lower, ranging from 11 to 45 muM. P450 2A13 was a somewhat better catalyst of nicotine Delta(5'(1'))-iminium formation, with 2-fold lower K(m) and 2-fold higher V(max) values than P450 2A6. The rat P450 2A3 and the mouse P450 2A5, which are 85 and 84% identical to P450 2A6, were much more efficient catalysts of nicotine 5'-oxidation. P450 2A4 was not an efficient catalyst of nicotine metabolism. Whereas 5'-oxidation was the major pathway of nicotine metabolism for all five P450 2A enzymes, these enzymes also catalyzed methyl oxidation. Nornicotine, the product of this reaction was detected as 5 to 15% of the total nicotine metabolites. Nornicotine is the amine precursor to the esophageal carcinogen N'-nitrosonornicotine. Therefore, methyl oxidation of nicotine by P450 2A6 or P450 2A13 followed by nitrosation of nornicotine are possible endogenous pathways of N'-nitrosonornicotine formation.
authors.affiliation: The Cancer Center, University of Minnesota, Mayo Mail Code 806, 420 Delaware St. SE, Minneapolis, MN 55455, USA. murph062@umn.edu
authors.forename: Sharon E
authors.initials: SE
authors.lastname: Murphy
authors.forename: Vytautas
authors.initials: V
authors.lastname: Raulinaitis
authors.forename: Kathryn M
authors.initials: KM
authors.lastname: Brown
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003367:Cotinine / Q000378:metabolism*; D065637:Cytochrome P-450 CYP2A6; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008862:Microsomes, Liver / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism; D009538:Nicotine / Q000031:analogs & derivatives* / Q000378:metabolism*; D009602:Nitrosamines; D012907:Smoking; D013250:Steroid Hydroxylases / Q000378:metabolism*; D014316:Tritium
chemical_list: D009602:Nitrosamines; D014316:Tritium; D009538:Nicotine; C005311:nornicotine; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C414206:CYP2A13 protein, human; D065637:Cytochrome P-450 CYP2A6; C029009:steroid hormone 7-alpha-hydroxylase; D003367:Cotinine; C008655:N'-nitrosonornicotine