Arlt Volker M, Schmeiser Heinz H, Osborne Martin R, Kawanishi Masanobu, Kanno Takaharu, Yagi Takashi, Phillips David H, Takamura-Enya Takeji
Institute of Cancer Research, Section of Molecular Carcinogenesis, Sutton, Surrey, United Kingdom.
Int J Cancer. 2006 May 1;118(9):2139-46. doi: 10.1002/ijc.21622.
3-Nitrobenzanthrone (3-NBA) is a potent mutagen and potential human carcinogen identified in diesel exhaust and ambient air particulate matter. Previously, we detected the formation of 3-NBA-derived DNA adducts in rodent tissues by 32P-postlabeling, all of which are derived from reductive metabolites of 3-NBA bound to purine bases, but structural identification of these adducts has not yet been reported. We have now prepared 3-NBA-derived DNA adduct standards for 32P-postlabeling by reacting N-acetoxy-3-aminobenzanthrone (N-Aco-ABA) with purine nucleotides. Three deoxyguanosine (dG) adducts have been characterised as N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone-3'-phosphate (dG3'p-C8-N-ABA), 2-(2'-deoxyguanosin-N2-yl)-3-aminobenzanthrone-3'-phosphate (dG3'p-N2-ABA) and 2-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone-3'-phosphate (dG3'p-C8-C2-ABA), and a deoxyadenosine (dA) adduct was characterised as 2-(2'-deoxyadenosin-N6-yl)-3-aminobenzanthrone-3'-phosphate (dA3'p-N6-ABA). 3-NBA-derived DNA adducts formed experimentally in vivo and in vitro were compared with the chemically synthesised adducts. The major 3-NBA-derived DNA adduct formed in rat lung cochromatographed with dG3'p-N2-ABA in two independent systems (thin layer and high-performance liquid chromatography). This is also the major adduct formed in tissue of rats or mice treated with 3-aminobenzanthrone (3-ABA), the major human metabolite of 3-NBA. Similarly, dG3'p-C8-N-ABA and dA3'p-N6-ABA cochromatographed with two other adducts formed in various organs of rats or mice treated either with 3-NBA or 3-ABA, whereas dG3'p-C8-C2-ABA did not cochromatograph with any of the adducts found in vivo. Utilizing different enzymatic systems in vitro, including human hepatic microsomes and cytosols, and purified and recombinant enzymes, we found that a variety of enzymes [NAD(P)H:quinone oxidoreductase, xanthine oxidase, NADPH:cytochrome P450 oxidoreductase, cytochrome P450s 1A1 and 1A2, N,O-acetyltransferases 1 and 2, sulfotransferases 1A1 and 1A2, and myeloperoxidase] are able to catalyse the formation of 2-(2'-deoxyguanosin-N2-yl)-3-aminobenzanthrone, N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone and 2-(2'-deoxyadenosin-N6-yl)-3-aminobenzanthrone in DNA, after incubation with 3-NBA and/or 3-ABA.
3-硝基苯并蒽酮（3-NBA）是一种在柴油废气和环境空气中的颗粒物中发现的强效诱变剂和潜在的人类致癌物。此前，我们通过32P后标记法在啮齿动物组织中检测到了3-NBA衍生的DNA加合物，这些加合物均来自与嘌呤碱基结合的3-NBA还原代谢产物，但尚未有关于这些加合物结构鉴定的报道。我们现在通过使N-乙酰氧基-3-氨基苯并蒽酮（N-Aco-ABA）与嘌呤核苷酸反应，制备了用于32P后标记法的3-NBA衍生的DNA加合物标准品。已鉴定出三种脱氧鸟苷（dG）加合物，分别为N-（2'-脱氧鸟苷-8-基）-3-氨基苯并蒽酮-3'-磷酸（dG3'p-C8-N-ABA）、2-（2'-脱氧鸟苷-N2-基）-3-氨基苯并蒽酮-3'-磷酸（dG3'p-N2-ABA）和2-（2'-脱氧鸟苷-8-基）-3-氨基苯并蒽酮-3'-磷酸（dG3'p-C8-C2-ABA），一种脱氧腺苷（dA）加合物被鉴定为2-（2'-脱氧腺苷-N6-基）-3-氨基苯并蒽酮-3'-磷酸（dA3'p-N6-ABA）。将体内和体外实验形成的3-NBA衍生的DNA加合物与化学合成的加合物进行了比较。在大鼠肺中形成的主要3-NBA衍生的DNA加合物在两个独立系统（薄层色谱和高效液相色谱）中与dG3'p-N2-ABA共色谱。这也是在用3-NBA的主要人体代谢产物3-氨基苯并蒽酮（3-ABA）处理的大鼠或小鼠组织中形成的主要加合物。同样，dG3'p-C8-N-ABA和dA3'p-N6-ABA与在用3-NBA或3-ABA处理的大鼠或小鼠的各种器官中形成的另外两种加合物共色谱，而dG3'p-C8-C2-ABA与体内发现的任何加合物都不共色谱。利用体外不同的酶系统，包括人肝微粒体和胞质溶胶以及纯化和重组酶，我们发现多种酶[NAD（P）H：醌氧化还原酶、黄嘌呤氧化酶、NADPH：细胞色素P450氧化还原酶、细胞色素P450 1A1和1A2、N，O-乙酰基转移酶1和2、磺基转移酶1A1和1A2以及髓过氧化物酶]在与3-NBA和/或3-ABA孵育后能够催化DNA中2-（2'-脱氧鸟苷-N2-基）-3-氨基苯并蒽酮、N-（2'-脱氧鸟苷-8-基）-3-氨基苯并蒽酮和2-（2'-脱氧腺苷-N6-基）-3-氨基苯并蒽酮加合物的形成。
