title: Metabolic activation of the tobacco carcinogen 4-(methylnitrosamino)-(3-pyridyl)-1-butanone by cytochrome P450 2A13 in human fetal nasal microsomes.
abstract: Among human P450s studied to date, P450 2A13 is the most efficient catalyst of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) alpha-hydroxylation. This reaction is a key bioactivation pathway in NNK-induced carcinogenesis. P450 2A13 mRNA has been detected in human tissues, but it is unknown whether the enzyme is functional in vivo. Therefore, we studied NNK alpha-hydroxylation in human fetal nasal mucosal microsomes, which have been shown to contain high levels of P450 2A protein, presumed to be a mixture of P450 2A6 and 2A13. The microsomes efficiently catalyzed NNK alpha-hydroxylation at the methylene and methyl carbons, as well as carbonyl reduction. Antibodies against mouse P450 2A5 inhibited alpha-hydroxylation by these microsomes greater than 90%. K(m) and V(max) values for alpha-methylene hydroxylation were 6.5 +/- 1.1 muM and 3.0 +/- 0.1 pmol/min/mg; for alpha-methyl hydroxylation, they were 6.7 +/- 0.8 microM and 0.85 +/- 0.03 pmol/min/mg. The K(m) values agree closely with those for NNK metabolism by P450 2A13. Using a new technique, we separated P450 2A13 from P450 2A6 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Quantitative immunoblot analysis indicated that the level of P450 2A13 in the pooled fetal nasal microsome sample used for kinetic analysis was approximately 1.6 pmol/mg protein. In the same sample, P450 2A6 was not detected (detection limit, 67 fmol/mg protein). These kinetic, immunoinhibition, and immunoblot data confirm that P450 2A13 is a functional enzyme and the catalyst of NNK alpha-hydroxylation in human fetal nasal mucosa. The results are also the first to demonstrate high efficiency NNK alpha-hydroxylation in a human tissue.
authors.affiliation: The Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Hansen L
authors.initials: HL
authors.lastname: Wong
authors.forename: Xiuling
authors.initials: X
authors.lastname: Zhang
authors.forename: Qing-Yu
authors.initials: QY
authors.lastname: Zhang
authors.forename: Jun
authors.initials: J
authors.lastname: Gu
authors.forename: Xinxin
authors.initials: X
authors.lastname: Ding
authors.forename: Stephen S
authors.initials: SS
authors.lastname: Hecht
authors.forename: Sharon E
authors.initials: SE
authors.lastname: Murphy
mesh_terms: D000328:Adult; D001711:Biotransformation; D002273:Carcinogens / Q000378:metabolism / Q000493:pharmacokinetics*; D005260:Female; D005333:Fetus; D005865:Gestational Age; D006801:Humans; D006900:Hydroxylation; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008875:Middle Aged; D009297:Nasal Mucosa / Q000196:embryology / Q000201:enzymology*; D009602:Nitrosamines / Q000378:metabolism / Q000493:pharmacokinetics*; D014026:Nicotiana / Q000378:metabolism
chemical_list: D002273:Carcinogens; D009602:Nitrosamines; C016583:4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone