TY - JOUR
T1 - Metabolic activation of the tobacco carcinogen 4-(methylnitrosamino)-(3- pyridyl)-1-butanone by cytochrome P450 2A13 in human fetal nasal microsomes
AU - Wong, Hansen L.
AU - Zhang, Xiuling
AU - Zhang, Qing Yu
AU - Gu, Jun
AU - Ding, Xinxin
AU - Hecht, Stephen S.
AU - Murphy, Sharon E.
PY - 2005/6
Y1 - 2005/6
N2 - Among human P450s studied to date, P450 2A13 is the most efficient catalyst of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) α-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 α-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 α-hydroxylation at the methylene and methyl carbons, as well as carbonyl reduction. Antibodies against mouse P450 2A5 inhibited α-hydroxylation by these microsomes greater than 90%. K m and Vmax values for α-methylene hydroxylation were 6.5 ± 1.1 μM and 3.0 ± 0.1 pmol/min/mg; for α-methyl hydroxylation, they were 6.7 ± 0.8 μM and 0.85 ± 0.03 pmol/min/ mg. The Km 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 α-hydroxylation in human fetal nasal mucosa. The results are also the first to demonstrate high efficiency NNK α-hydroxylation in a human tissue.
AB - Among human P450s studied to date, P450 2A13 is the most efficient catalyst of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) α-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 α-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 α-hydroxylation at the methylene and methyl carbons, as well as carbonyl reduction. Antibodies against mouse P450 2A5 inhibited α-hydroxylation by these microsomes greater than 90%. K m and Vmax values for α-methylene hydroxylation were 6.5 ± 1.1 μM and 3.0 ± 0.1 pmol/min/mg; for α-methyl hydroxylation, they were 6.7 ± 0.8 μM and 0.85 ± 0.03 pmol/min/ mg. The Km 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 α-hydroxylation in human fetal nasal mucosa. The results are also the first to demonstrate high efficiency NNK α-hydroxylation in a human tissue.
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U2 - 10.1021/tx0500777
DO - 10.1021/tx0500777
M3 - Article
C2 - 15962925
AN - SCOPUS:21144437181
SN - 0893-228X
VL - 18
SP - 913
EP - 918
JO - Chemical research in toxicology
JF - Chemical research in toxicology
IS - 6
ER -