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3Rs-friendly approach to exogenous metabolic activation that supports high-throughput genetic toxicology testing.
Environmental and Molecular Mutagenesis ( IF 2.8 ) Pub Date : 2020-02-27 , DOI: 10.1002/em.22361
Shuchang Tian 1 , Aiyana Cyr 1 , Karen Zeise 1 , Steven M Bryce 1 , Nikki Hall 1 , Jeffrey C Bemis 1 , Stephen D Dertinger 1
Affiliation  

MultiFlow® DNA Damage-p53, γH2AX, Phospho-Histone H3 is a miniaturized, flow cytometry-based assay that provides genotoxic mode of action information by distinguishing clastogens, aneugens, and nongenotoxicants. Work to date has focused on the p53-competent human cell line TK6. While mammalian cell genotoxicity assays typically supply exogenous metabolic activation in the form of concentrated rat liver S9, this is a less-than-ideal approach for several reasons, including 3Rs considerations. Here, we describe our experiences with low concentration S9 and saturating co-factors which were allowed to remain in contact with cells and test chemicals for 24 continuous hours. We exposed TK6 cells in 96-well plates to each of 15 reference chemicals over a range of concentrations, both in the presence and absence of 0.25% v/v phenobarbital/β-naphthoflavone-induced rat liver S9. After 4 and 24 hr of treatment cell aliquots were added to wells of a microtiter plate containing the working detergent/stain/antibody cocktail. After a brief incubation robotic sampling was employed for walk-away flow cytometric data acquisition. PROAST benchmark dose (BMD) modeling was used to characterize the resulting dose-response curves. For each of the 8 reference pro-genotoxicants studied, relative nuclei count, γH2AX, and/or p53 biomarker BMD values were order(s) of magnitude lower for 0.25% S9 conditions compared to 0% S9. Conversely, several of the direct-acting reference chemicals exhibited appreciably lower cytotoxicity and/or genotoxicity BMD values in the presence of S9 (eg, resorcinol). These results prove the efficacy of the low concentration S9 system, and indicate that an efficient and highly scalable multiplexed assay can effectively identify chemicals that require bioactivation to exert their genotoxic effects.

中文翻译:

3Rs友好的外源代谢激活方法,支持高通量遗传毒理学测试。

MultiFlow®DNA Damage-p53,γH2AX和Phospho-Histone H3是一种基于流式细胞仪的小型化测定方法,通过区分克拉斯氏菌,无瘤菌和非遗传毒性物质,提供了遗传毒性作用模式信息。迄今为止的工作集中在具有p53能力的人细胞系TK6上。尽管哺乳动物细胞遗传毒性测定法通常以浓缩大鼠肝脏S9的形式提供外源性代谢激活,但出于多种原因(包括3R考虑因素),这不是理想的方法。在这里,我们描述了我们在低浓度S9和饱和辅因子下的经验,这些辅因子可以连续24小时与细胞和测试化学品保持接触。在存在和不存在0的情况下,我们将96孔板中的TK6细胞暴露于15种参考化学品中的每种浓度范围内的浓度。25%v / v苯巴比妥/β-萘黄酮诱导的大鼠肝脏S9。处理4和24小时后,将细胞等分试样加入到含有工作去污剂/污渍/抗体混合物的微量滴定板的孔中。经过短暂的温育后,采用机器人采样进行无人值守流式细胞仪数据采集。使用PROAST基准剂量(BMD)模型来表征所得的剂量反应曲线。对于所研究的8种参考前基因毒性物质中的每一种,相对于0%S9,在0.25%S9条件下,相对核计数,γH2AX和/或p53生物标记物BMD值都降低了数量级。相反,几种直接作用的参考化学品在存在S9的情况下(例如间苯二酚)显示出较低的细胞毒性和/或遗传毒性BMD值。这些结果证明了低浓度S9系统的功效,
更新日期:2020-02-27
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