N-亚硝基或尼古丁(NNN)和N-亚硝基anabasine(NAB)都是带有两个杂环氨基的烟草特有的亚硝胺,NAB带有额外的-CH2-基团(赋予六元而不是五元循环),但致癌性显着降低。然而,它们的激活酶和相关的致突变性仍不清楚。在这项研究中,通过分子对接分析了化学-CYP相互作用,因此,人CYP2A6,2A13,2B6,2E1和3A4的NNN的结合能和构象似乎适合作为底物,人CYP1B1、2A6、2A13和2E1的NAB也是如此。每种化合物(62.5~1000μM)暴露48h(双细胞周期)的人肝癌(HepG2)细胞微核试验均为阴性,然而,双酚AF预处理(0.1~100nM,CYPs诱导剂)和乙醇(0.2%v-v,CYP2E1诱导剂)增强了两种化合物的微核形成,whileCITCO(1μM,CYP2B6诱导剂)通过NNN选择性增强。在C3A细胞(内源性CYPs增强超过HepG2)中,两种化合物均诱导微核,被1-氨基苯并三唑(60μM,CYPs抑制剂),而不受8-甲氧基补骨脂素(1μM,CYP2A抑制剂)。始终如一,NNN和NAB分别在V79衍生的表达人CYP2B6/2E1和CYP1B1/2E1的重组细胞系中诱导微核,而在那些表达其他CYPs的阴性。通过免疫荧光测定,两种化合物均选择性诱导C3A细胞中的无着丝粒微核。在HepG2细胞的PIG-A测定中,NNN和NAB呈弱阳性和简单阴性,分别;然而,在C3A细胞中,两种化合物均显着诱导基因突变,NNN稍微更有效。最后,NNN和NAB都具有致突变性和致突变性,取决于部分不同CYP酶的代谢激活。
N-nitrosonornicotine (NNN) and N-nitrosoanabasine (NAB) are both tobacco-specific nitrosamines bearing two heterocyclic amino groups, NAB bearing an extra -CH2- group (conferring a hexa- rather than penta-membered cycle) but with significantly decreased carcinogenicity. However, their activating enzymes and related mutagenicity remain unclear. In this study, the chemical-CYP interaction was analyzed by molecular docking, thus the binding energies and conformations of NNN for human CYP2A6, 2A13, 2B6, 2E1 and 3A4 appeared appropriate as a substrate, so did NAB for human CYP1B1, 2A6, 2A13 and 2E1. The micronucleus test in human hepatoma (HepG2) cells with each compound (62.5-1000 μM) exposing for 48 h (two-cell cycle) was negative, however, pretreatment with bisphenol AF (0.1-100 nM, CYPs inducer) and ethanol (0.2% v:v, CYP2E1 inducer) potentiated micronucleus formation by both compounds, while CITCO (1 μM, CYP2B6 inducer) selectively potentiated that by NNN. In C3A cells (endogenous CYPs enhanced over HepG2) both compounds induced micronucleus, which was abolished by 1-aminobenzotriazole (60 μM, CYPs inhibitor) while unaffected by 8-methoxypsoralen (1 μM, CYP2A inhibitor). Consistently, NNN and NAB induced micronucleus in V79-derived recombinant cell lines expressing human CYP2B6/2E1 and CYP1B1/2E1, respectively, while negative in those expressing other CYPs. By immunofluorescent assay both compounds selectively induced centromere-free micronucleus in C3A cells. In PIG-A assays in HepG2 cells NNN and NAB were weakly positive and simply negative, respectively; however, in C3A cells both compounds significantly induced gene mutations, NNN being slight more potent. Conclusively, both NNN and NAB are mutagenic and clastogenic, depending on metabolic activation by partially different CYP enzymes.