Synergistic metabolic toxicity screening using microsome/DNA electrochemiluminescent arrays and nanoreactors
Synergistic metabolic toxicity screening using microsome/DNA electrochemiluminescent arrays and nanoreactors
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DOI：
10.1021/ac800763r
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发表时间：
2008-07-15
影响因子：
7.4
通讯作者：
Rusling, James F.
中科院分区：
文献类型：
--
作者：
Krishnan, Sadagopan;Hvastkovs, Eli G.;Rusling, James F.
基于酶/DNA薄膜的平台用于两层筛选能够产生毒性的反应性代谢物的化学物质。这些装置首次使用微粒体作为细胞色素（cyt） P450酶的来源。最初的快速筛选涉及电化学发光（ECL）阵列，其特征点含有钌聚（乙烯吡啶），DNA和大鼠肝微粒体或双电性表达的人细胞P450 2E1 （h2E1）。通过NADPH/还原酶循环激活Cyt P450酶。当膜中底物的生物活化产生活性代谢物时，它们通过与DNA碱基的共价附着而被捕获。ECL随酶反应时间的增加反映了相对DNA损伤率。通过在二氧化硅纳米球上使用酶/ DNA膜作为“纳米反应器”，从反应性代谢物中提供核碱基加合物，研究了阵列发现的“毒性撞击”的结构细节。通过估计三种致突变性n -亚硝基化合物和苯乙烯的相对DNA损伤率，证明了这种协同方法的实用性。ECL阵列和LC-UV-MS对这些化合物的相对酶周转率与大鼠肝脏肿瘤形成的TD50值有良好的相关性。结合ECL阵列和纳米反应器/LC-MS技术具有快速、高通量、高成本效益的筛选反应性代谢物的潜力，并提供化学结构信息，是传统毒性生物测定的补充。
Platforms based on thin enzyme/DNA films were used in two-tier screening of chemicals for reactive metabolites capable of producing toxicity. Microsomes were used for the first time as sources of cytochrome (cyt) P450 enzymes in these devices. Initial rapid screening involved electrochemiluminescent (ECL) arrays featuring spots containing ruthenium poly(vinylpyridine), DNA, and rat liver microsomes or bicistronically expressed human cyt P450 2E1 (h2E1). Cyt P450 enzymes were activated via the NADPH/reductase cycle. When bioactivation of substrates in the films gives reactive metabolites, they are trapped by covalent attachment to DNA bases. The rate of increase in ECL with, enzyme reaction time reflects relative DNA damage rates. 'Toxic hits" uncovered by the array were studied in structural detail by using enzyme/ DNA films on silica nanospheres as "nanoreactors" to provide nucleobase adducts from reactive metabolites. The utility of this synergistic approach was demonstrated by estimating relative DNA damage rates of three mutagenic N-nitroso compounds and styrene. Relative enzyme turnover rates for these compounds using ECL arrays and LC-UV-MS correlated well with TD50 values for liver tumor formation in rats. Combining ECL array and nanoreactor/LC-MS technologies has the potential for rapid, high-throughput, cost-effective screening for reactive metabolites and provides chemical structure information that is complementary to conventional toxicity bioassays.