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 μM) for the formation of keto aldehyde. A similar K(M) was observed for keto alcohol formation by expressed P450 2A6, but the k(cat) 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 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.
Bibliographical noteFunding Information:
*Department of Drug Metabolism and Pharmacokinetics, Sandoz Pharmaceuticals Corporation, Sandoz Research Institute, East Hanover, New Jersey 07936; ²Rutgers University, Laboratory for Cancer Research, College of Pharmacy, Piscataway, New Jersey 08855; ³American Health Foundation, Dana Naylor Institute for Disease Prevention, Valhalla, New York 10595; and §Drug Safety Department, Sandoz Pharmaceuticals Ltd., Basel, Switzerland
- 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanone
- Human cytochrome P450
- cytochrome b
- keto alcohol
- keto aldehyde