Swansea University Medical School, Swansea University, Swansea, UK.
GSK R&D, Stevenage, UK.
Arch Toxicol. 2024 Sep;98(9):3137-3153. doi: 10.1007/s00204-024-03801-7. Epub 2024 Jul 12.
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.
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检测方法旨在评估化学物质在早期药物开发中的 DNA 损伤潜力，有助于鉴定具有低遗传损伤风险、导致人类癌症风险的有前途的药物。尽管如此，体外测试会产生大量误导性的阳性结果，其后果可能导致不必要的动物测试和/或有前途的候选药物被放弃。了解化学物质的作用模式（MoA）对于识别物质的真正遗传毒性潜力以及将风险转化为临床应用至关重要。在这里，我们展示了一种简单、稳健的固定人淋巴母细胞 p53 功能正常的 TK6 细胞染色方案，使用针对 γH2AX、p53 和 pH3S28 的抗体以及 DRAQ5™ DNA 染色，通过显微镜方法（如成像流式细胞术）分析未裂解的细胞。在这里，我们使用 Cytek® Amnis® ImageStream®X Mk II，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。使用图像流式细胞仪制造商的软件（IDEAS® 6.2），开发了一种掩蔽策略来自动检测和量化微核事件（MN）并对生物标志物群体进行特征描述。开发的门控策略使能够生成一个模板，该模板能够自动批量处理数据文件，同时定量细胞周期、MN、γH2AX、p53 和 pH3 群体。通过这种方式，我们展示了如何使用未裂解细胞在成像流式细胞仪平台上同时进行 DNA 损伤评估和 MN 鉴定的多重系统。作为概念验证，我们使用工具化学品多菌灵和甲基甲烷磺酸盐（MMS）来证明该测定法能够使用建立的生物标志物谱正确识别断裂剂或非整倍体 MoA。
