KINETIC-ANALYSIS OF THE ACTIVATION OF 4-(METHYLNITROSAMINO)-1-(3-PYRIDYL)-1-BUTANONE BY HETEROLOGOUSLY EXPRESSED HUMAN P450 ENZYMES AND THEEFFECT OF P450-SPECIFIC CHEMICAL INHIBITORS ON THIS ACTIVATION IN HUMAN LIVER-MICROSOMES

The tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is enzymatically activated by the hydroxylation of the alpha-met hyl and alpha-methylene groups, leading to the formation of reactive s pecies which can pyridyloxobutylate and methylate DNA, respectively. T he present study examined the kinetic parameters of NNK-derived keto a lcohol (alpha-methyl hydroxylation), and keto aldehyde (alpha-methylen e hydroxylation) formation catalyzed by human P450s heterologously exp ressed by either the baculovirus-insect cell expression system (P450s 2A6, 2D6, 2E1, and 3A4) or by stable expression in CHO cells (P450s 3A 4 and 2D6) and human B-lymphoblastoid cells (P450 2D6). Membrane prepa rations of the expressed P450s catalyzed the alpha-hydroxylation of NN K, leading to the formation of keto aldehyde and keto alcohol. Human P 450 2A6 showed the lowest K-M (118 mu M) for the formation of keto ald ehyde. A similar K-M was observed for keto alcohol formation by expres sed P450 2A6, but the k(cat) was lower than the value obtained for ket o aldehyde formation. The addition of exogenous b(5) increased the exp ressed 2A6-dependent NNK hydroxylation activity 2.5-fold for both Lu-h ydroxylation products. Human P450s 2E1 and 2D6 exhibited a high capaci ty for keto alcohol formation; however, their K-M values for this reac tion were in the millimolar range. Expressed human P450 3A4 oxidized N NK to keto aldehyde also with a high K-M. Ten human liver microsomal s amples were each shown to activate NNK to keto aldehyde and keto alcoh ol. 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 correlat ion with ethoxyresorufin O-dealkylation (P450 1A2) in human liver micr osomes. Both coumarin and troleandomycin, specific inhibitors of P450 2A6 and 3A4, respectively, inhibited the formation of keto aldehyde, b ut inhibited the formation of keto alcohol only slightly in human live r microsomes. Both furafylline, a P450 1A2 inhibitor, and N-nitrosodim ethylamine, a P450 2E1 substrate, inhibited the formation of keto alco hol but not keto aldehyde in human liver microsomes. Quinidine, a spec ific inhibitor of P450 2D6, was not an effective inhibitor of NNK meta bolism. These results demonstrate that P450s 2A6 and 3A4 may be import ant P450s for the activation of NNK to a DNA-methylating agent and ket o aldehyde via the alpha-methylene hydroxylation pathway. P450s 1A2, 2 E1, and 2D6 are shown to be selective for alpha-methyl hydroxylation o f NNK leading to keto alcohol and a DNA-pyridyloxobutylating agent. (C ) 1996 Academic Press, Inc.