Krishnan Sadagopan, Hvastkovs Eli G, Bajrami Besnik, Schenkman John B, Rusling James F
Department of Chemistry, University of Connecticut, Storrs, CT 06269-3060, USA.
Mol Biosyst. 2009 Feb;5(2):163-9. doi: 10.1039/b815910f. Epub 2008 Dec 12.
Electrochemiluminescent (ECL) arrays containing polymer (Ru(bpy)(2)(PVP)(10), PVP = polyvinylpyridine), DNA, and selected enzymes were employed to elucidate cytochrome (cyt) P450 dependent metabolism of the tobacco specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Bioactivated NNK metabolites formed upon H(2)O(2)-enzymatic activation were captured as DNA adducts and detected simultaneously from 36 spot arrays by capturing and quantifying emitted ECL with an overhead CCD camera. Increased ECL emission was dependent on NNK exposure time. Of the enzymes tested, the activity toward NNK bioactivation was cyt P450 1A2 > 2E1 > 1B1 approximately chloroperoxidase (CPO) > myoglobin (Mb) in accordance with reported in vivo studies. Cyt P450/polyion films were also immobilized on 500 nm diameter silica nanospheres for product analysis by LC-MS. Analysis of the nanosphere film reaction media provided ECL array validation and quantitation of the bioactivated NNK hydrolysis product 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) confirming production of reactive metabolites in the films. Chemical screening in this fashion allows rapid clarification of enzymes responsible for genotoxic activation as well as offering insight into cyt P450-related toxicity and mechanisms.
含有聚合物（Ru(bpy)(2)(PVP)(10)，PVP = 聚乙烯吡啶）、DNA和特定酶的电化学发光（ECL）阵列被用于阐明细胞色素（cyt）P450对烟草特异性致癌物4-(甲基亚硝胺基)-1-(3-吡啶基)-1-丁酮（NNK）的依赖性代谢。H(2)O(2)酶促活化后形成的生物活化NNK代谢产物被捕获为DNA加合物，并通过用顶置式电荷耦合器件（CCD）相机捕获和定量发射的ECL，从36个斑点阵列中同时进行检测。ECL发射的增加取决于NNK的暴露时间。在所测试的酶中，对NNK生物活化的活性为细胞色素P450 1A2 > 2E1 > 1B1 ≈ 氯过氧化物酶（CPO）> 肌红蛋白（Mb），这与已报道的体内研究一致。细胞色素P450/聚离子膜也被固定在直径500 nm的二氧化硅纳米球上，用于通过液相色谱-质谱联用（LC-MS）进行产物分析。对纳米球膜反应介质的分析为ECL阵列提供了验证，并对生物活化的NNK水解产物4-羟基-1-(3-吡啶基)-1-丁酮（HPB）进行了定量，证实了膜中产生了反应性代谢产物。以这种方式进行化学筛选能够快速明确负责遗传毒性活化的酶，同时深入了解细胞色素P450相关的毒性和机制。
