Further evaluation of a flow cytometric in vitro micronucleus assay in CHO-K1 cells: a reliable platform that detects micronuclei and discriminates apoptotic bodies
Further evaluation of a flow cytometric in vitro micronucleus assay in CHO-K1 cells: a reliable platform that detects micronuclei and discriminates apoptotic bodies
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DOI：
10.1093/mutage/gep040
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发表时间：
2010-01-01
期刊：
影响因子：
2.7
通讯作者：
Szkudlinska, Anna
中科院分区：
文献类型：
--
作者：
Shi, Jing;Bezabhie, Rahel;Szkudlinska, Anna
体外微核(MN)试验被广泛用于评估受试物质的遗传毒性潜力，方法是测量培养的哺乳动物细胞中MN的频率。传统上，MN频率是通过显微镜来确定的。近年来，发展了一种用于MN计数的流式细胞术方法，大大缩短了分析时间，提高了检测吞吐量。然而，一个主要的问题是，从流式细胞术获得的MN结果可能会受到细胞凋亡或坏死过程中产生的染色质小体的影响。在这项工作中，我们进一步评估了基于24孔平台的CHO-K-1细胞的基于流式细胞术的体外MN检测。结果表明，用流式细胞术测定的微核率与镜检结果高度相关。重要的是，该系统评估了几种非遗传毒性的细胞凋亡诱导剂或细胞毒素，这些诱导因子或细胞毒素在各种体外遗传毒性测试中都会产生“人工阳性”。因此，当细胞毒性为50+/-10%时，这些非遗传毒性细胞毒素在CHO-K-1细胞的流式细胞仪系统中不会产生假阳性的MN反应。此外，在这项工作中共评价了21种化合物，包括直接或间接致裂物、无性物和非遗传毒性化学物质。从我们测试的化合物中获得了83.3%的敏感性和100%的特异性。最后，在我们的评估中证实了亚二倍体区域事件的显著增加，这是一个非优生特征。结论：流式细胞术体外MN测定是检测受试物在CHO-K-1细胞中的分裂潜能或非分化潜能的可靠方法。
The in vitro micronucleus (MN) assay is widely used to assess genotoxic potential of the test substances by measuring frequency of MN in cultured mammalian cells. Traditionally, MN frequency has been determined by microscopy. In recent years, a flow cytometric method for enumeration of MN has been developed, which significantly shortens analysis time and enhances assay throughput. However, a major concern has been raised that the MN results obtained from flow cytometry can be impacted by chromatin bodies produced during apoptosis or necrosis. In this work, we further evaluated this flow cytometry-based in vitro MN assay with CHO-K-1 cells in a 24-well platform. Our results showed that the MN frequency determined using the flow cytometric method was highly correlated with the microscopy results. Importantly, several non-genotoxic apoptosis inducers or cytotoxins that have been previously reported to produce 'artificial positives' in various in vitro genotoxicity tests were evaluated in this system. As a result, these non-genotoxic cytotoxins did not produce false-positive MN response in the flow cytometric system in CHO-K-1 cells when cytotoxicity was < 50 +/- 10%. Moreover, a total of 21 compounds were evaluated in this work, including direct or indirect clastogens, aneugens and non-genotoxic chemicals. A sensitivity of 83.3% and a specificity of 100% were obtained from the compounds we tested. Finally, significant increase of incidents in the hypodiploid region, an aneugenic signature, was confirmed in our evaluation. In conclusion, the flow cytometric in vitro MN assay is a reliable method that can be used to detect clastogenic or aneugenic potential of the test substances in CHO-K-1 cells.