Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269-3060, United States.
Anal Chem. 2010 Dec 15;82(24):10172-8. doi: 10.1021/ac102317a. Epub 2010 Nov 19.
An inexpensive, high-throughput genotoxicity screening method was developed by using magnetic particles coated with cytosol/microsome/DNA films as biocolloid reactors in a 96-well plate format coupled with liquid chromatography-mass spectrometry. Incorporation of both microsomal and cytosolic enzymes in the films provides a broad spectrum of metabolic enzymes representing a range of metabolic pathways for bioactivation of chemicals. Reactive metabolites generated via this process are trapped by covalently binding to DNA in the film. The DNA is then hydrolyzed and nucleobase adducts are collected using filters in the bottom for the 96-well plate of analysis by capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS). The magnetic particles facilitate simple and rapid sample preparation and workup. Major DNA adducts from ethylene dibromide, N-acetyl-2-aminofluorene and styrene were identified in proof-of-concept studies. Relative formation rates of DNA adducts correlated well with rodent genotoxicity metric TD(50) for the three compounds. This method has the potential for high-throughput genotoxicity screening, providing chemical structure information that is complementary to toxicity bioassays.
建立了一种成本低廉、高通量的遗传毒性筛选方法，该方法使用涂有胞质溶胶/微粒体/DNA 膜的磁性颗粒作为生物胶体反应器，在 96 孔板格式中与液相色谱-质谱联用。在薄膜中同时包含微粒体和胞质溶胶酶，提供了一系列代谢酶，代表了化学物质生物活化的一系列代谢途径。通过该过程产生的反应性代谢物通过与薄膜中 DNA 共价结合而被捕获。然后将 DNA 水解，并使用 96 孔板底部的过滤器收集核碱基加合物，然后通过毛细管液相色谱-串联质谱 (LC-MS/MS) 进行分析。磁性颗粒便于简单快速的样品制备和处理。在概念验证研究中鉴定了乙二溴、N-乙酰-2-氨基芴和苯乙烯的主要 DNA 加合物。三种化合物的 DNA 加合物的相对形成率与啮齿动物遗传毒性指标 TD(50)很好地相关。该方法具有高通量遗传毒性筛选的潜力，提供了与毒性生物测定互补的化学结构信息。
