Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455, United States.
Chem Res Toxicol. 2021 Apr 19;34(4):1004-1015. doi: 10.1021/acs.chemrestox.1c00012. Epub 2021 Mar 15.
The International Agency for Research on Cancer has classified the tobacco-specific nitrosamines '-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as "carcinogenic to humans" (Group 1). To exert its carcinogenicity, NNN requires metabolic activation to form reactive intermediates which alkylate DNA. Previous studies have identified cytochrome P450-catalyzed 2'-hydroxylation and 5'-hydroxylation of NNN as major metabolic pathways, with preferential activation through the 5'-hydroxylation pathway in some cultured human tissues and patas monkeys. So far, the only DNA adducts identified from NNN 5'-hydroxylation in rat tissues are 2-[2-(3-pyridyl)--pyrrolidinyl]-2'-deoxyinosine (Py-Py-dI), 6-[2-(3-pyridyl)--pyrrolidinyl]-2'-deoxynebularine (Py-Py-dN), and -[4-hydroxy-1-(pyridine-3-yl)butyl]-2'-deoxyadenosine (-HPB-dAdo) after reduction. To expand the DNA adduct panel formed by NNN 5'-hydroxylation and identify possible activation biomarkers of NNN metabolism, we investigated the formation of dAdo-derived adducts using a new highly sensitive and specific liquid chromatography-nanoelectrospray ionization-high-resolution tandem mass spectrometry method. Two types of NNN-specific dAdo-derived adducts, -[5-(3-pyridyl)tetrahydrofuran-2-yl]-2'-deoxyadenosine (-Py-THF-dAdo) and 6-[2-(3-pyridyl)--pyrrolidinyl-5-hydroxy]-2'-deoxynebularine (Py-Py(OH)-dN), were observed for the first time in calf thymus DNA incubated with 5'-acetoxyNNN. More importantly, Py-Py(OH)-dN was also observed in relatively high abundance in the liver and lung DNA of rats treated with racemic NNN in the drinking water for 3 weeks. These new adducts were characterized using authentic synthesized standards. Both NMR and MS data agreed well with the proposed structures of -Py-THF-dAdo and Py-Py(OH)-dN. Reduction of Py-Py(OH)-dN by NaBHCN led to the formation of Py-Py-dN both and , which was confirmed by its isotopically labeled internal standard [pyridine-]Py-Py-dN. The NNN-specific dAdo adducts Py-THF-dAdo and Py-Py(OH)-dN formed by NNN 5'-hydroxylation provide a more comprehensive understanding of the mechanism of DNA adduct formation by NNN.
国际癌症研究机构已将烟草特异性亚硝胺 '-亚硝基降烟碱（NNN）和 4-（甲基亚硝氨基）-1-（3-吡啶基）-1-丁酮（NNK）归类为“对人类致癌”（第 1 组）。为了发挥其致癌性，NNN 需要代谢激活形成反应性中间产物，从而使 DNA 烷基化。先前的研究已经确定细胞色素 P450 催化的 NNN 2'-羟化和 5'-羟化是主要的代谢途径，在一些培养的人类组织和食蟹猴中，优先通过 5'-羟化途径激活。到目前为止，从大鼠组织中 NNN 5'-羟化鉴定出的唯一 DNA 加合物是 2-[2-(3-吡啶基)-吡咯烷基]-2'-脱氧肌苷（Py-Py-dI）、6-[2-(3-吡啶基)-吡咯烷基]-2'-脱氧新烟碱（Py-Py-dN）和-[4-羟基-1-（吡啶-3-基）丁基]-2'-脱氧腺苷（-HPB-dAdo），经还原后形成。为了扩展 NNN 5'-羟化形成的 DNA 加合物谱，并鉴定 NNN 代谢的可能激活生物标志物，我们使用新的高灵敏度和特异性液相色谱-纳喷雾电离-高分辨率串联质谱法研究了 dAdo 衍生加合物的形成。首次在小牛胸腺 DNA 中观察到两种类型的 NNN 特异性 dAdo 衍生加合物，-[5-（3-吡啶基）四氢呋喃-2-基]-2'-脱氧腺苷（-Py-THF-dAdo）和 6-[2-(3-吡啶基）-吡咯烷基-5-羟基]-2'-脱氧新烟碱（Py-Py(OH)-dN）。更重要的是，在饮用水中用 racemic NNN 处理 3 周的大鼠的肝和肺 DNA 中也观察到了相对较高丰度的 Py-Py(OH)-dN。使用合成的标准品对这些新的加合物进行了表征。NMR 和 MS 数据与 -Py-THF-dAdo 和 Py-Py(OH)-dN 的建议结构非常吻合。NaBHCN 还原 Py-Py(OH)-dN 可形成 Py-Py-dN 均为 和 ，这通过其同位素标记的内标 [吡啶-]Py-Py-dN 得到证实。NNN 5'-羟化形成的 NNN 特异性 dAdo 加合物 Py-THF-dAdo 和 Py-Py(OH)-dN 为 NNN 引起的 DNA 加合物形成机制提供了更全面的认识。
