PDF(Pubmed)
Abstract：
Genetic toxicity testing assesses the potential of compounds to cause DNA damage. There are many genetic toxicology screening assays designed to assess the DNA damaging potential of chemicals in early drug development aiding the identification of promising drugs that have low-risk potential for causing genetic damage contributing to cancer risk in humans. Despite this, in vitro tests generate a high number of misleading positives, the consequences of which can lead to unnecessary animal testing and/or the abandonment of promising drug candidates. Understanding chemical Mode of Action (MoA) is vital to identifying the true genotoxic potential of substances and, therefore, the risk translation into the clinic. Here we demonstrate a simple, robust protocol for staining fixed, human-lymphoblast p53 proficient TK6 cells with antibodies against ɣH2AX, p53 and pH3S28 along with DRAQ5™ DNA staining that enables analysis of un-lysed cells via microscopy approaches such as imaging flow cytometry. Here, we used the Cytek® Amnis® ImageStream®X Mk II which provides a high-throughput acquisition platform with the sensitivity of flow cytometry and spatial morphological information associated with microscopy. Using the ImageStream manufacturer\'s software (IDEAS® 6.2), a masking strategy was developed to automatically detect and quantify micronucleus events (MN) and characterise biomarker populations. The gating strategy developed enables the generation of a template capable of automatically batch processing data files quantifying cell-cycle, MN, ɣH2AX, p53 and pH3 populations simultaneously. In this way, we demonstrate how a multiplex system enables DNA damage assessment alongside MN identification using un-lysed cells on the imaging flow cytometry platform. As a proof-of-concept, we use the tool chemicals carbendazim and methyl methanesulphonate (MMS) to demonstrate the assay\'s ability to correctly identify clastogenic or aneugenic MoAs using the biomarker profiles established.
摘要：
遗传毒性测试评估化合物引起DNA损伤的潜力。有许多遗传毒理学筛选试验旨在评估早期药物开发中化学物质的DNA损伤潜力，有助于鉴定具有低风险潜力的有希望的药物，这些药物具有导致人类癌症风险的遗传损伤的风险。尽管如此,体外测试产生大量误导性阳性，其后果可能导致不必要的动物试验和/或放弃有希望的候选药物。了解化学作用模式（MoA）对于识别物质的真正基因毒性潜力至关重要，因此,将风险转化为诊所。这里我们演示一个简单的，染色固定的稳健方案，具有抗H2AX抗体的人淋巴母细胞p53精通TK6细胞，p53和pH3S28以及DRAQ5™DNA染色，可通过显微镜方法（如成像流式细胞术）分析未裂解的细胞。这里,我们使用了Cytek®Amnis®ImageStream®XMkII,它提供了一个高通量采集平台,具有流式细胞术的灵敏度和与显微镜相关的空间形态信息.使用ImageStream制造商的软件(IDEAS®6.2)，我们开发了一种掩蔽策略来自动检测和量化微核事件(MN)并表征生物标志物群体.开发的门控策略可以生成一个模板，该模板能够自动批量处理量化细胞周期的数据文件，MN,H2AX,p53和pH3群体同时存在。这样,我们演示了多重系统如何在成像流式细胞仪平台上使用未裂解的细胞进行DNA损伤评估以及MN鉴定。作为一个概念证明,我们使用工具化学品多菌灵和甲磺酸甲酯（MMS）来证明该测定法能够使用已建立的生物标志物谱正确识别断裂性或不良性MoAs。
