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
| Autor: | Patrick Koch, Theresa J. Smith, Chung S. Yang, Ronald E. Tynes, Christopher J. Patten, Mong Heng Wang, Sharon E. Murphy, Jae Lee |
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| Rok vydání: | 1996 |
| Předmět: |
Insecta
Nitrosamines Furafylline Stereochemistry Biophysics Gene Expression Alcohol CHO Cells In Vitro Techniques Biochemistry Aldehyde Cell Line Hydroxylation chemistry.chemical_compound Cytochrome P-450 Enzyme System Cricetinae Animals Cytochrome P-450 Enzyme Inhibitors Humans Enzyme kinetics Enzyme Inhibitors Molecular Biology Biotransformation chemistry.chemical_classification Metabolism Coumarin Recombinant Proteins Kinetics chemistry Carcinogens Microsomes Liver Microsome Baculoviridae |
| Zdroj: | Archives of Biochemistry and Biophysics. 333:127-138 |
| ISSN: | 0003-9861 |
| Popis: | The tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is enzymatically activated by the hydroxylation of the alpha-methyl and alpha-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 (alpha-methyl hydroxylation), and keto aldehyde (alpha-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 alpha-hydroxylation of NNK, leading to the formation of keto aldehyde and keto alcohol. Human P450 2A6 showed the lowest KM (118 microM) for the formation of keto aldehyde. A similar KM was observed for keto alcohol formation by expressed P450 2A6, but the kcat was lower than the value obtained for keto aldehyde formation. The addition of exogenous b5 increased the expressed 2A6-dependent NNK hydroxylation activity 2.5-fold for both alpha-hydroxylation products. Human P450s 2E1 and 2D6 exhibited a high capacity for keto alcohol formation; however, their KM values for this reaction were in the millimolar range. Expressed human P450 3A4 oxidized NNK to keto aldehyde also with a high KM. 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 6 beta-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 alpha-methylene hydroxylation pathway. P450s 1A2, 2E1, and 2D6 are shown to be selective for alpha-methyl hydroxylation of NNK leading to keto alcohol and a DNA-pyridyloxobutylating agent. |
| Databáze: | OpenAIRE |
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