Smith T J, Guo Z, Guengerich F P, Yang C S
Laboratory for Cancer Research, College of Pharmacy, Rutgers University, Piscataway, NJ 08855-0789, USA.
Carcinogenesis. 1996 Apr;17(4):809-13. doi: 10.1093/carcin/17.4.809.
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco-specific nitrosamine in animals and has been suggested to play a role in human tobacco-related cancers. Our previous study demonstrated that cytochrome P450 (P450) 1A2 catalyzes the formation of 4-hydroxy-1-(3-pyridyl)-1-butanone (keto alcohol) (an alpha-hydroxylation product) from NNK in human liver microsomes. Phenethyl isothiocyanate (PEITC) inhibits NNK tumorigenesis by blocking the activation of NNK. The purpose of the present study was to elucidate the mechanism of inhibition of P450 1A2-catalyzed NNK activation by PEITC. Human P450 1A2 was expressed in Escherichia coli and purified to homogeneity. In a reconstituted system, P450 1A2 catalyzed the formation of keto alcohol and 4-oxo-1-(3-pyridyl)-1-butanone (keto aldehyde) from NNK, with the keto alcohol being the major metabolite. The apparent Km and Vmax values for keto alcohol formation was 380 microM and 1.7 nmol/min/nmol P450, respectively. For the tobacco-specific nitrosamine N-nitrosonornicotine (NNN), P450 1A2 catalyzed the formation of the derived 4-hydroxy-4-(3-pyridyl)butyric acid (hydroxy acid),4-oxo-4-(3-pyridyl)butyric acid (keto acid) and keto alcohol. In comparison to NNK, NNN had a lower rate of oxidation with P450 1A2. PEITC decreased the formation of the NNK-derived keto alcohol in a concentration-dependent manner, with an IC50 value of 0.14 microM. PEITC was a competitive inhibitor of P450 1A2, exhibiting a Ki value of 0.18 microM. Preincubation of PEITC with NADPH in the reconstituted system resulted in a further decrease (25%) in the catalytic activity of P450 1A2, suggesting that there is a slow metabolism-dependent inhibition of P450 1A2 by PEITC. The formation of keto aldehyde and keto alcohol was inhibited by PEITC in human liver microsomes with IC50 values of 9.5 and 4.6 microM respectively. Methoxyresorufin O-dealkylase activity, a marker for P450 1A2, was decreased by PEITC in a concentration-dependent manner, with an IC50 of 0.34 microM. The results suggest that PEITC itself is a potent inhibitor of P450 1A2 and that a metabolite(s) of PEITC can also inhibit P450 1A2. We conclude that PEITC may be an effective inhibitor of the carcinogenicity or toxicity of chemicals that are activated by P450 1A2.
4-（甲基亚硝基氨基）-1-（3-吡啶基）-1-丁酮（NNK）是一种在动物体内具有强致癌性的烟草特异性亚硝胺，被认为在人类烟草相关癌症中发挥作用。我们之前的研究表明，细胞色素P450（P450）1A2可催化人肝微粒体中NNK生成4-羟基-1-（3-吡啶基）-1-丁酮（酮醇）（一种α-羟基化产物）。苯乙基异硫氰酸酯（PEITC）通过阻断NNK的活化来抑制NNK的肿瘤发生。本研究的目的是阐明PEITC抑制P450 1A2催化的NNK活化的机制。人P450 1A2在大肠杆菌中表达并纯化至均一性。在重组系统中，P450 1A2催化NNK生成酮醇和4-氧代-1-（3-吡啶基）-1-丁酮（酮醛），其中酮醇是主要代谢产物。酮醇生成的表观Km和Vmax值分别为380μM和1.7nmol/（min/nmol P450）。对于烟草特异性亚硝胺N-亚硝基去甲烟碱（NNN），P450 1A2催化生成其衍生物4-羟基-4-（3-吡啶基）丁酸（羟基酸）、4-氧代-4-（3-吡啶基）丁酸（酮酸）和酮醇。与NNK相比，NNN被P450 1A2氧化的速率较低。PEITC以浓度依赖性方式降低NNK衍生的酮醇的生成，IC50值为0.14μM。PEITC是P450 1A2的竞争性抑制剂，Ki值为0.18μM。在重组系统中，PEITC与NADPH预孵育导致P450 1A2的催化活性进一步降低（25%），表明存在PEITC对P450 1A2的慢代谢依赖性抑制。在人肝微粒体中，PEITC抑制酮醛和酮醇的生成，IC50值分别为9.5和4.6μM。甲氧基试卤灵O-脱烷基酶活性是P450 1A2的标志物，PEITC以浓度依赖性方式降低其活性，IC50为0.34μM。结果表明，PEITC本身是P450 1A2的有效抑制剂，且PEITC的一种或多种代谢产物也可抑制P450 1A2。我们得出结论，PEITC可能是P450 1A2活化的化学物质致癌性或毒性的有效抑制剂。
