Sarlauskas J, Dickancaite E, Nemeikaite A, Anusevicius Z, Nivinskas H, Segura-Aguilar J, Cenas N
Institute of Biochemistry, Vilnius, Lithuania.
Arch Biochem Biophys. 1997 Oct 15;346(2):219-29. doi: 10.1006/abbi.1997.0285.
We have synthesized a number of nitrobenzimidazoles containing nitro groups in the benzene ring and found that they acted as relatively efficient substrates for rat liver DT-diaphorase (EC 1.6.99.2), their reactivity exceeding reactivities of nitrofurans and nitrobenzenes. Nitrobenzimidazoles were competitive with NADPH inhibitors of DT-diaphorase in menadione reductase reactions, their inhibition constant being unchanged in the presence of dicumarol and being increased in the presence of 2',5'-ADP. These data indicate that the poor reactivity of nitrobenzimidazoles and other nitroaromatics in comparison to quinones could be determined by their binding in the adenosine-phosphate binding region of the NADPH-binding site, whereas quinones bind at the nicotinamide-binding pocket at the vicinity of FAD of DT-diaphorase. The reduction of 4,5,6-trinitrobenzimidazol-2-one by DT-diaphorase most probably involves reduction of 5-nitro group to 5-nitroso or 5-hydroxylamine derivative at the initial step. A certain parallelism existed between reactivities of nitrobenzimidazoles toward DT-diaphorase and their reactivities in single-electron reduction by Anabaena ferredoxin:NADP+ reductase (EC 1.18.1.2) and Saccharomyces cerevisiae flavocytochrome b2 (EC 1.1.2.3), the latter being determined by electronic factors. However, we suppose that the relatively high reactivity of polinitrobenzimidazoles toward DT-diaphorase was due not only to electronic effects, but also to a sterical crowding of nitrogroups by each other. The toxicity of nitrobenzimidazoles to bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) with a moderate amount of DT-diaphorase (260 U/mg protein) is partly prevented by dicumarol. That points out to partial determination of nitrobenzimidazole cytotoxicity by their reduction by DT-diaphorase. Another important factor of nitrobenzimidazole toxicity to this cell line was oxidative stress, catalyzed by single-electron transferring enzymes.
我们合成了一系列在苯环中含有硝基的硝基苯并咪唑，并发现它们是大鼠肝脏DT-黄递酶（EC 1.6.99.2）相对有效的底物，其反应活性超过硝基呋喃和硝基苯。在甲萘醌还原酶反应中，硝基苯并咪唑与DT-黄递酶的NADPH抑制剂存在竞争性，在双香豆素存在下其抑制常数不变，而在2',5'-ADP存在下其抑制常数增加。这些数据表明，与醌相比，硝基苯并咪唑和其他硝基芳烃反应活性较差可能是由它们在NADPH结合位点的腺苷-磷酸结合区域中的结合所决定的，而醌则结合在DT-黄递酶FAD附近的烟酰胺结合口袋处。DT-黄递酶对4,5,6-三硝基苯并咪唑-2-酮的还原最可能在初始步骤涉及将5-硝基还原为5-亚硝基或5-羟胺衍生物。硝基苯并咪唑对DT-黄递酶的反应活性与其在鱼腥藻铁氧化还原蛋白：NADP +还原酶（EC 1.18.1.2）和酿酒酵母黄素细胞色素b2（EC 1.1.2.3）的单电子还原中的反应活性之间存在一定的平行关系，后者由电子因素决定。然而，我们认为多硝基苯并咪唑对DT-黄递酶相对较高的反应活性不仅归因于电子效应，还归因于硝基之间的空间拥挤。双香豆素可部分防止硝基苯并咪唑对牛白血病病毒转化的羔羊肾成纤维细胞（FLK系）的毒性，该细胞系中DT-黄递酶含量适中（260 U/mg蛋白质）。这表明硝基苯并咪唑的细胞毒性部分由DT-黄递酶对其的还原作用所决定。硝基苯并咪唑对该细胞系毒性的另一个重要因素是由单电子转移酶催化的氧化应激。
