Patten C J, Smith T J, Murphy S E, Wang M H, Lee J, Tynes R E, Koch P, Yang C S
Department of Drug Metabolism, Sandoz Pharmaceuticals Corporation, Sandoz Research Institute, East Hanover, New Jersey 07936, USA.
Arch Biochem Biophys. 1996 Sep 1;333(1):127-38. doi: 10.1006/abbi.1996.0373.
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.
烟草致癌物4-（甲基亚硝基氨基）-1-（3-吡啶基）-1-丁酮（NNK）通过α-甲基和α-亚甲基的羟基化进行酶促活化，导致形成可分别使DNA发生吡啶氧基丁酰化和甲基化的反应性物种。本研究检测了由杆状病毒-昆虫细胞表达系统（P450s 2A6、2D6、2E1和3A4）或在CHO细胞（P450s 3A4和2D6）以及人B淋巴细胞样细胞（P450 2D6）中稳定表达的人P450催化形成NNK衍生的酮醇（α-甲基羟基化）和酮醛（α-亚甲基羟基化）的动力学参数。所表达的P450的膜制剂催化NNK的α-羟基化，导致形成酮醛和酮醇。人P450 2A6在形成酮醛时显示出最低的KM（118 microM）。对于所表达的P450 2A6形成酮醇，观察到类似的KM，但kcat低于形成酮醛所获得的值。添加外源性b5使所表达的依赖2A6的NNK羟基化活性对于两种α-羟基化产物均增加2.5倍。人P450s 2E1和2D6表现出形成酮醇的高能力；然而，它们对于该反应的KM值在毫摩尔范围内。所表达的人P450 3A4也将NNK氧化为酮醛，且KM也很高。十个肝微粒体样品均显示可将NNK活化为酮醛和酮醇。在特征明确的人肝微粒体中，酮醛形成与香豆素7-羟基化（P450 2A6）和6β-睾酮羟基化（3A4）活性之间发现正相关系数为0.74。酮醇形成与人肝微粒体中的乙氧异羟肟酸O-脱烷基化（P450 1A2）显示出显著相关性。香豆素和三乙酰夹竹桃霉素分别是P450 2A6和3A4的特异性抑制剂，它们抑制人肝微粒体中酮醛的形成，但仅轻微抑制酮醇的形成。P450 1A2抑制剂呋拉茶碱和P450 2E1底物N-亚硝基二甲胺均抑制人肝微粒体中酮醇的形成，但不抑制酮醛的形成。P450 2D6的特异性抑制剂奎尼丁不是NNK代谢的有效抑制剂。这些结果表明，P450s 2A6和3A4可能是通过α-亚甲基羟基化途径将NNK活化为DNA甲基化剂和酮醛的重要P450。P450s 1A2、2E1和2D6显示对NNK的α-甲基羟基化具有选择性，导致形成酮醇和DNA-吡啶氧基丁酰化剂。
