The early detection of genotoxicity contributes to cutting‐edge drug discovery and development, requiring effective identification of genotoxic hazards posed by drugs while providing mode of action (MoA) information in a high throughput manner. In other words, there is a need to complement standard genotoxicity testing according to the test battery given in ICH S2(R1) with new in vitro tools, thereby contributing to a more in‐depth analysis of genotoxic effects. Here, we report on a proof‐of‐concept MoA approach based on post‐translational modifications of proteins (PTMs) indicative of clastogenic and aneugenic effects in TK6 cells using imaging technology (with automated analysis). Cells were exposed in a 96‐well plate format with a panel of reference (geno)toxic compounds and subsequently analyzed at 4 and 24 hr to detect dose‐dependent changes in PTMs, relevant for mechanistic analysis. All tested compounds that interfere with the spindle apparatus yielded a BubR1 (S640) (3/3) and phospho‐histone H3 (S28) (7/9) positive dose–response reflecting aneugenicity, whereas compounds inducing DNA double‐strand‐breaks were associated with positive FANCD2 (S1404) and 53BP1 (S1778) responses pointing to clastogenicity (2/3). The biomarker p53 (K373) was able to distinguish genotoxicants from non‐genotoxicants (2/4), while the induction of reactive oxygen species (ROS), potentially causing DNA damage, was associated with a positive Nrf2 (S40) response (2/2). This work demonstrates that genotoxicants and non‐genotoxicants induce different biomarker responses in TK6 cells which can be used for reliable classification into MoA groups (aneugens/clastogens/non‐genotoxicants/ROS inducers), supporting a more in‐depth safety assessment of drug candidates.

中文翻译：

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机械蛋白生物标记物的遗传毒性图像表征的图像分析：中子，clastogens和活性氧物种诱导剂。

遗传毒性的早期检测有助于尖端药物的发现和开发，要求有效识别药物造成的遗传毒性危害，同时以高通量方式提供作用模式（MoA）信息。换句话说，有必要用新的体外工具补充根据ICH S2（R1）中给出的测试电池的标准遗传毒性测试，从而有助于更深入地分析遗传毒性作用。在这里，我们报告了一种基于概念证明的MoA方法，该方法基于蛋白质（PTM）的翻译后修饰，表明使用成像技术（具有自动分析功能）对TK6细胞产生了致死性和致瘤性作用。细胞以96孔板格式与一组参考（基因）有毒化合物接触，随后在4和24小时进行分析以检测PTM中剂量依赖性变化，这与机理分析有关。所有测试过的会干扰纺锤体的化合物均产生了BubR1（S640）（3/3）和磷酸组蛋白H3（S28）（7/9）阳性反应，反映了气孔性，而诱导DNA双链断裂的化合物为与积极的FANCD2（S1404）和53BP1（S1778）响应相关，表明发生了致裂性（2/3）。生物标记物p53（K373）能够区分遗传毒性剂和非遗传毒性剂（2/4），而可能导致DNA损伤的活性氧（ROS）的诱导与Nrf2（S40）阳性反应相关（2 / 2）。