Biomarkers of DNA damage response improve in vitro micronucleus assays by revealing genotoxic mode of action and reducing the occurrence of irrelevant positive results.

IF 2.5 4区 医学 Q3 GENETICS & HEREDITY
Mutagenesis Pub Date : 2021-11-29 DOI:10.1093/mutage/geab039
Svetlana Avlasevich, Tina Pellegrin, Manali Godse, Steven Bryce, Jeffrey Bemis, Peter Bajorski, Stephen Dertinger
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引用次数: 1

Abstract

We have previously described two flow cytometry-based in vitro genotoxicity tests: micronucleus (MN) scoring (MicroFlow®) and a multiplexed DNA damage response biomarker assay (MultiFlow®). Here, we describe a strategy for combining the assays in order to efficiently supplement MN analyses with a panel of biomarkers that comment on cytotoxicity (i.e. relative nuclei count, relative increased nuclei count, cleaved PARP-positive chromatin and ethidium monoazide-positive chromatin) and genotoxic mode of action (MoA; i.e. γH2AX, phospho-histone H3, p53 activation and polyploidy). For these experiments, human TK6 cells were exposed to each of 32 well-studied reference chemicals in 96-well plates for 24 continuous hours. The test chemicals were evaluated over a range of concentrations in the presence and absence of a rat liver S9-based metabolic activation system. MultiFlow assay data were acquired at 4 and 24 h, and micronuclei were scored at 24 h. Testing 32 chemicals in two metabolic activation arms translated into 64 a priori calls: 42 genotoxicants and 22 non-genotoxicants. The MN assay showed high sensitivity and moderate specificity (90% and 68%, respectively). When a genotoxic call required significant MN and MultiFlow responses, specificity increased to 95% without adversely affecting sensitivity. The dose-response data were analysed with PROAST Benchmark Dose (BMD) software in order to calculate potency metrics for each endpoint, and ToxPi software was used to synthesise the resulting lower and upper bound 90% confidence intervals into visual profiles. The BMD/ToxPi combination was found to represent a powerful strategy for synthesising multiple BMD confidence intervals, as the software output provided MoA information as well as insights into genotoxic potency.

DNA损伤反应的生物标志物通过揭示基因毒性作用模式和减少不相关阳性结果的发生来改善体外微核检测。
我们之前描述了两种基于流式细胞术的体外遗传毒性测试:微核(MN)评分(MicroFlow®)和多重DNA损伤反应生物标志物测定(MultiFlow®)。在这里,我们描述了一种结合分析的策略,以便有效地补充MN分析与一组生物标志物,这些生物标志物评论细胞毒性(即相对细胞核计数,相对增加的细胞核计数,裂解parp阳性染色质和单叠氮乙啶阳性染色质)和基因毒性作用模式(MoA;即γ - h2ax,磷酸化组蛋白H3, p53激活和多倍体)。在这些实验中,人类TK6细胞在96孔板中连续24小时暴露于32种充分研究的参比化学物质中。在存在和不存在以大鼠肝脏s9为基础的代谢激活系统的情况下,测试化学品在一定浓度范围内进行了评估。在4和24小时获得MultiFlow分析数据,并在24小时对微核进行评分。在两个代谢激活分支中测试32种化学物质,转化为64种先验调用:42种基因毒物和22种非基因毒物。MN检测显示高灵敏度和中等特异性(分别为90%和68%)。当基因毒性呼叫需要显著的MN和MultiFlow反应时,特异性增加到95%,而不会对敏感性产生不利影响。使用PROAST基准剂量(BMD)软件对剂量-反应数据进行分析,以计算每个终点的效价指标,并使用ToxPi软件将所得的下限和上限90%置信区间合成为可视化曲线。研究发现,BMD/ToxPi组合是合成多个BMD置信区间的有力策略,因为软件输出提供了MoA信息以及对遗传毒性效力的见解。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Mutagenesis
Mutagenesis 生物-毒理学
CiteScore
5.90
自引率
3.70%
发文量
22
审稿时长
6-12 weeks
期刊介绍: Mutagenesis is an international multi-disciplinary journal designed to bring together research aimed at the identification, characterization and elucidation of the mechanisms of action of physical, chemical and biological agents capable of producing genetic change in living organisms and the study of the consequences of such changes.
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