The desire for in vitro genotoxicity assays to provide higher information content, especially regarding chemicals’ predominant genotoxic mode of action, has led to the development of a novel multiplexed assay available under the trade name MultiFlow®. We report here on an experimental design variation that provides further insight into clastogens’ genotoxic activity. First, the standard MultiFlow DNA Damage Assay—p53, γ H2AX, phospho-histone H3 was used with human TK6 lymphoblastoid cells that were exposed for 24 continuous hours to each of 50 reference clastogens. This initial analysis correctly identified 48/50 compounds as clastogenic. These 48 compounds were then evaluated using a short-term, ‘pulse’ treatment protocol whereby cells were exposed to test chemical for 4 h, a centrifugation/washout step was performed, and cells were allowed to recover for 20 h. MultiFlow analyses were accomplished at 4 and 24 h. The γ H2AX and phospho-histone H3 biomarkers were found to exhibit distinct differences in terms of their persistence across chemical classes. Unsupervised hierarchical clustering analysis identified three groups. Examination of the compounds within these groups showed one cluster primarily consisting of alkylators that directly target DNA. The other two groups were dominated by non-DNA alkylators and included anti-metabolites, oxidative stress inducers and chemicals that inhibit DNA-processing enzymes. These results are encouraging, as they suggest that a simple follow-up test for in vitro clastogens provides mechanistic insights into their genotoxic activity. This type of information will contribute to improve decision-making and help guide further testing.

中文翻译：

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脉冲处理 TK6 细胞后 γH2AX 和磷酸组蛋白 H3 的动力学提供了对致裂活性的见解

体外遗传毒性试验希望提供更高的信息内容，特别是关于化学品主要的遗传毒性作用模式，这导致开发了一种以商品名 MultiFlow® 可用的新型多重试验。我们在此报告了一种实验设计变异，它提供了对 clastogens 基因毒性活性的进一步了解。首先，将标准的 MultiFlow DNA 损伤分析 - p53、γ H2AX、磷酸组蛋白 H3 与人类 TK6 类淋巴母细胞一起使用，这些细胞连续 24 小时暴露于 50 种参考断裂素中的每一种。该初步分析正确地将 48/50 化合物鉴定为致断裂。然后使用短期“脉冲”处理方案评估这 48 种化合物，其中细胞暴露于测试化学品 4 小时，进行离心/冲洗步骤，并让细胞恢复20小时。MultiFlow 分析在 4 小时和 24 小时完成。发现 γ H2AX 和磷酸组蛋白 H3 生物标志物在其跨化学类别的持久性方面表现出明显差异。无监督层次聚类分析确定了三组。对这些组中的化合物的检查显示一个簇主要由直接靶向 DNA 的烷基化剂组成。其他两组以非 DNA 烷化剂为主，包括抗代谢物、氧化应激诱导剂和抑制 DNA 加工酶的化学物质。这些结果令人鼓舞，因为它们表明对体外断裂剂的简单后续测试提供了对其遗传毒性活性的机制见解。