Bryce Steven M, Bemis Jeffrey C, Avlasevich Svetlana L, Dertinger Stephen D
Litron Laboratories, 200 Canal View Blvd., Rochester, NY, United States.
Mutat Res. 2007 Jun 15;630(1-2):78-91. doi: 10.1016/j.mrgentox.2007.03.002. Epub 2007 Mar 19.
This laboratory has previously reported on the development of a flow cytometry-based method for scoring in vitro micronuclei in mouse lymphoma (L5178Y) cells [S.L. Avlasevich, S.M. Bryce, S.E. Cairns, S.D. Dertinger, In vitro micronucleus scoring by flow cytometry: differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability, Environ. Molec. Mutagen. 47 (2006) 56-66]. With this method, necrotic and mid/late stage apoptotic cells are labeled with the fluorescent dye ethidium monoazide. Cells are then washed, stripped of their cytoplasmic membranes, and incubated with RNase plus a pan-nucleic acid dye (SYTOX Green). This process provides a suspension of free nuclei and micronuclei that are differentially stained relative to chromatin associated with dead/dying cells. The current report extends this line of investigation to include the human cell line TK6. Additionally, methods are described that facilitate simultaneous quantitative analysis of cytotoxicity, perturbations to the cell cycle, and what we hypothesize is aneuploidization. This comprehensive cytogenetic damage assay was evaluated with the following diverse agents: etoposide, ionizing radiation, methyl methanesulfonate, vinblastine, ethanol, and staurosporine. Cells were harvested after 30h of continuous treatment (in the case of chemicals), or following graded doses of radiation up to 1Gy. Key findings include the following: (1) Significant discrepancies in top dose selection were found for five of the six agents studied when relative survival measurements were based on Coulter counting versus flow cytometry. (2) Both microscopy- and flow cytometry-based scoring methods detected dose-dependent micronucleus formation for the four genotoxic agents studied, whereas no significant increases were observed for the presumed non-genotoxicants ethanol and staurosporine when top dose selection was based on flow cytometric indices of cytotoxicity. (3) SYTOX and ethidium monoazide fluorescence signals conveyed cell cycle and cell death information, respectively, and appear to represent valuable aids for interpreting micronucleus data. (4) The frequency of hypodiploid nuclei increased in response to each of the genotoxic agents studied, but not following exposure to ethanol or staurosporine. Collectively, these results indicate that a comprehensive assessment of genotoxicity and other test article-induced toxicities can be acquired simultaneously using a simple two-color flow cytometry-based technique.
本实验室之前报道过一种基于流式细胞术的方法，用于对小鼠淋巴瘤（L5178Y）细胞中的体外微核进行评分[S.L. 阿夫拉谢维奇、S.M. 布莱斯、S.E. 凯恩斯、S.D. 德廷格，通过流式细胞术进行体外微核评分：微核与凋亡和坏死染色质的差异染色提高了检测的可靠性，《环境分子突变》47 (2006) 56 - 66]。使用这种方法，坏死细胞和中/晚期凋亡细胞用荧光染料单叠氮乙锭标记。然后洗涤细胞，去除其细胞质膜，并与核糖核酸酶和一种泛核酸染料（SYTOX Green）一起孵育。这个过程提供了游离细胞核和微核的悬浮液，并相对于与死亡/濒死细胞相关的染色质进行差异染色。本报告将这一研究方向扩展到包括人类细胞系TK6。此外，还描述了有助于同时对细胞毒性、细胞周期扰动以及我们假设的非整倍体化进行定量分析的方法。用以下不同试剂评估了这种全面的细胞遗传学损伤检测方法：依托泊苷、电离辐射、甲基磺酸甲酯、长春碱、乙醇和星形孢菌素。在连续处理30小时后（对于化学物质），或在高达最大剂量1Gy的分级辐射剂量后收获细胞。主要发现如下：（1）当基于库尔特计数与流式细胞术进行相对存活测量时，在所研究的六种试剂中的五种中，在最大剂量选择上发现了显著差异。（2）基于显微镜和流式细胞术的评分方法均检测到了所研究的四种遗传毒性试剂的剂量依赖性微核形成，而当基于细胞毒性流式细胞术指标进行最大剂量选择时，对于假定的非遗传毒性剂乙醇和星形孢菌素未观察到显著增加。（3）SYTOX和单叠氮乙锭荧光信号分别传达了细胞周期和细胞死亡信息，并且似乎是解释微核数据的有价值辅助手段。（4）响应所研究的每种遗传毒性试剂，亚二倍体核的频率增加，但在暴露于乙醇或星形孢菌素后未增加。总体而言，这些结果表明，使用一种简单的基于双色流式细胞术的技术可以同时获得对遗传毒性和其他受试物诱导毒性的全面评估。
