title: CYP2A6 AND CYP2B6 are involved in nornicotine formation from nicotine in humans: interindividual differences in these contributions.
abstract: Nornicotine is an N-demethylated metabolite of nicotine. In the present study, human cytochrome P450 (P450) isoform(s) involved in nicotine N-demethylation were identified. The Eadie-Hofstee plot of nicotine N-demethylation in human liver microsomes was biphasic with high-affinity (apparent K(m) = 173 +/- 70 microM, V(max) = 57 +/- 17 pmol/min/mg) and low-affinity (apparent K(m) = 619 +/- 68 microM, V(max) = 137 +/- 6 pmol/min/mg) components. Among 13 recombinant human P450s expressed in baculovirus-infected insect cells (Supersomes), CYP2B6 exhibited the highest nicotine N-demethylase activity, followed by CYP2A6. The apparent K(m) values of CYP2A6 (49 +/- 12 microM) and CYP2B6 (550 +/- 46 microM) were close to those of high- and low-affinity components in human liver microsomes, respectively. The intrinsic clearances of CYP2A6 and CYP2B6 Supersomes were 5.1 and 12.5 nl/min/pmol P450, respectively. In addition, the intrinsic clearance of CYP2A13 expressed in Escherichia coli (44.9 nl/min/pmol P450) was higher than that of CYP2A6 expressed in E. coli (2.6 nl/min/pmol P450). Since CYP2A13 is hardly expressed in human livers, the contribution of CYP2A13 to the nicotine N-demethylation in human liver microsomes would be negligible. The nicotine N-demethylase activity in microsomes from 15 human livers at 20 microM nicotine was significantly correlated with the CYP2A6 contents (r = 0.578, p < 0.05), coumarin 7-hydroxylase activity (r = 0.802, p < 0.001), and S-mephenytoin N-demethylase activity (r = 0.694, p < 0.005). The nicotine N-demethylase activity at 100 microM nicotine was significantly correlated with the CYP2B6 contents (r = 0.677, p < 0.05) and S-mephenytoin N-demethylase activities (r = 0.740, p < 0.005). These results as well as the inhibition analyses suggested that CYP2A6 and CYP2B6 would significantly contribute to the nicotine N-demethylation at low and high substrate concentrations, respectively. The contributions of CYP2A6 and CYP2B6 would be dependent on the expression levels of these isoforms in any human liver.
authors.affiliation: Drug Metabolism and Toxicology, Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Yamanaka
authors.forename: Miki
authors.initials: M
authors.lastname: Nakajima
authors.forename: Tatsuki
authors.initials: T
authors.lastname: Fukami
authors.forename: Haruko
authors.initials: H
authors.lastname: Sakai
authors.forename: Akiko
authors.initials: A
authors.lastname: Nakamura
authors.forename: Miki
authors.initials: M
authors.lastname: Katoh
authors.forename: Masataka
authors.initials: M
authors.lastname: Takamiya
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Aoki
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Yokoi
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001189:Aryl Hydrocarbon Hydroxylases / Q000502:physiology*; D003342:Corpus Striatum / Q000201:enzymology; D065637:Cytochrome P-450 CYP2A6; D065702:Cytochrome P-450 CYP2B6; D003640:Dealkylation; D006801:Humans; D008862:Microsomes, Liver / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000502:physiology*; D009538:Nicotine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism*; D010089:Oxidoreductases, N-Demethylating / Q000502:physiology*
chemical_list: D009538:Nicotine; C005311:nornicotine; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C585710:CYP2A6 protein, human; C585599:CYP2B6 protein, human; D065637:Cytochrome P-450 CYP2A6; D065702:Cytochrome P-450 CYP2B6; D010089:Oxidoreductases, N-Demethylating