Wong Hansen L, Murphy Sharon E, Hecht Stephen S
University of Minnesota Cancer Center, Minneapolis, Minnesota 55455, USA.
Chem Res Toxicol. 2003 Oct;16(10):1298-305. doi: 10.1021/tx0340495.
N-Nitrosopiperidine (NPIP) is a potent rat nasal carcinogen whereas N-nitrosopyrrolidine (NPYR), a hepatic carcinogen, is weakly carcinogenic in the nose. NPIP and NPYR may be causative agents in human cancer. P450-catalyzed alpha-hydroxylation is the key activation pathway by which these nitrosamines elicit their carcinogenic effects. We hypothesize that the differences in NPIP and NPYR metabolic activation in the nasal cavity contribute to their differing carcinogenic activities. In this study, the kinetics of tritium-labeled NPIP or NPYR alpha-hydroxylation mediated by Sprague-Dawley rat nasal olfactory or respiratory microsomes were investigated. To compare alpha-hydroxylation rates of the two nitrosamines, tritiated 2-hydroxytetrahydro-2H-pyran and 2-hydroxy-5-methyltetrahydrofuran, the major NPIP alpha-hydroxylation products, and tritiated 2-hydroxytetrahydrofuran, the major NPYR alpha-hydroxylation product, were quantitated by HPLC with UV absorbance and radioflow detection. These microsomes catalyzed the alpha-hydroxylation of NPIP more efficiently than that of NPYR. K(M) values for NPIP were lower as compared to those for NPYR (13.9-34.7 vs 484-7660 muM). Furthermore, catalytic efficiencies (V(max)/K(M)) of NPIP were 20-37-fold higher than those of NPYR. Previous studies showed that P450 2A3, present in the rat nose, also exhibited this difference in catalytic efficiency. For both types of nasal microsomes, coumarin (100 muM), a P450 2A inhibitor, inhibited NPIP and NPYR alpha-hydroxylation from 63.8 to 98.5%. Furthermore, antibodies toward P450 2A6 inhibited nitrosamine alpha-hydroxylation in these microsomes from 68.8 to 78.4% whereas antibodies toward P450 2E1 did not inhibit these reactions. Further immunoinhibition studies suggest some role for P450 2G1 in NPIP metabolism by olfactory microsomes. In conclusion, olfactory and respiratory microsomes from rat nasal mucosa preferentially activate NPIP over NPYR with P450 2A3 likely playing a key role. These results are consistent with local metabolic activation of nitrosamines as a contributing factor in their tissue-specific carcinogenicity.
N-亚硝基哌啶（NPIP）是一种强效的大鼠鼻腔致癌物，而N-亚硝基吡咯烷（NPYR）作为一种肝脏致癌物，在鼻腔中致癌性较弱。NPIP和NPYR可能是人类癌症的致病因素。细胞色素P450催化的α-羟基化是这些亚硝胺发挥致癌作用的关键激活途径。我们推测，NPIP和NPYR在鼻腔中的代谢激活差异导致了它们不同的致癌活性。在本研究中，我们研究了Sprague-Dawley大鼠鼻腔嗅觉或呼吸微粒体介导的氚标记NPIP或NPYRα-羟基化的动力学。为了比较这两种亚硝胺的α-羟基化速率，通过高效液相色谱结合紫外吸收和放射性流动检测对主要的NPIPα-羟基化产物氚化2-羟基四氢-2H-吡喃和2-羟基-5-甲基四氢呋喃，以及主要的NPYRα-羟基化产物氚化2-羟基四氢呋喃进行了定量。这些微粒体催化NPIP的α-羟基化比催化NPYR的效率更高。与NPYR相比，NPIP的米氏常数（K(M)）值更低（13.9 - 34.7对484 - 7660 μM）。此外，NPIP的催化效率（V(max)/K(M)）比NPYR高20 - 37倍。先前的研究表明，大鼠鼻腔中存在的细胞色素P450 2A3也表现出这种催化效率的差异。对于两种类型的鼻腔微粒体，细胞色素P450 2A抑制剂香豆素（100 μM）可将NPIP和NPYR的α-羟基化抑制63.8%至98.5%。此外，针对细胞色素P450 2A6的抗体可将这些微粒体中的亚硝胺α-羟基化抑制68.8%至78.4%，而针对细胞色素P450 2E1的抗体则不抑制这些反应。进一步免疫抑制研究表明，细胞色素P450 2G1在嗅觉微粒体对NPIP的代谢中发挥了一定作用。总之，大鼠鼻粘膜的嗅觉和呼吸微粒体优先激活NPIP而非NPYR，细胞色素P450 2A3可能起关键作用。这些结果与亚硝胺的局部代谢激活作为其组织特异性致癌性的一个促成因素是一致的。
