Affordable Access

Publisher Website

Kinetic Analysis of the Activation of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone by Heterologously Expressed Human P450 Enzymes and the Effect of P450- Specific Chemical Inhibitors on This Activation in Human Liver Microsomes

Authors
Journal
Archives of Biochemistry and Biophysics
0003-9861
Publisher
Elsevier
Publication Date
Volume
333
Issue
1
Identifiers
DOI: 10.1006/abbi.1996.0373
Keywords
  • Human Cytochrome P450
  • Cytochrome B5
  • 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone
  • Keto Aldehyde
  • Keto Alcohol

Abstract

Abstract The tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is enzymatically activated by the hydroxylation of the α-methyl and α-methylene groups, leading to the formation of reactive species which can pyridyloxobutylate and methylate DNA, respectively. The present study examined the kinetic parameters of NNK-derived keto alcohol (α-methyl hydroxylation), and keto aldehyde (α-methylene hydroxylation) formation catalyzed by human P450s heterologously expressed by either the baculovirus–insect cell expression system (P450s 2A6, 2D6, 2E1, and 3A4) or by stable expression in CHO cells (P450s 3A4 and 2D6) and human B-lymphoblastoid cells (P450 2D6). Membrane preparations of the expressed P450s catalyzed the α-hydroxylation of NNK, leading to the formation of keto aldehyde and keto alcohol. Human P450 2A6 showed the lowest K M (118 μ m) for the formation of keto aldehyde. A similar K M was observed for keto alcohol formation by expressed P450 2A6, but the k catwas lower than the value obtained for keto aldehyde formation. The addition of exogenous b 5increased the expressed 2A6-dependent NNK hydroxylation activity 2.5-fold for both α-hydroxylation products. Human P450s 2E1 and 2D6 exhibited a high capacity for keto alcohol formation; however, their K M values for this reaction were in the millimolar range. Expressed human P450 3A4 oxidized NNK to keto aldehyde also with a high K M . Ten human liver microsomal samples were each shown to activate NNK to keto aldehyde and keto alcohol. A positive correlation coefficient of 0.74 was found between keto aldehyde formation and both coumarin 7-hydroxylation (P450 2A6) and 6β-testosterone hydroxylation (3A4) activity in characterized human liver microsomes. Keto alcohol formation showed a significant correlation with ethoxyresorufin O-dealkylation (P450 1A2) in human liver microsomes. Both coumarin and troleandomycin, specific inhibitors of P450 2A6 and 3A4, respectively, inhibited the formation of keto aldehyde, but inhibited the formation of keto alcohol only slightly in human liver microsomes. Both furafylline, a P450 1A2 inhibitor, and N-nitrosodimethylamine, a P450 2E1 substrate, inhibited the formation of keto alcohol but not keto aldehyde in human liver microsomes. Quinidine, a specific inhibitor of P450 2D6, was not an effective inhibitor of NNK metabolism. These results demonstrate that P450s 2A6 and 3A4 may be important P450s for the activation of NNK to a DNA-methylating agent and keto aldehyde via the α-methylene hydroxylation pathway. P450s 1A2, 2E1, and 2D6 are shown to be selective for α-methyl hydroxylation of NNK leading to keto alcohol and a DNA-pyridyloxobutylating agent.

There are no comments yet on this publication. Be the first to share your thoughts.