Department of Nutrition, Food Safety and Toxicology, West China School of Public Health, Sichuan University, Chengdu, China.
Food Safety Monitoring and Risk Assessment Key Laboratory of Sichuan Province, Sichuan University, Chengdu, China.
J Appl Toxicol. 2021 Apr;41(4):595-606. doi: 10.1002/jat.4068. Epub 2020 Oct 16.
The micronucleus test (MNT) is the most widely applied short-term assay to detect clastogens or spindle disruptors. The use of flow cytometry (FCM) has been reported for micronucleated erythrocytes scoring in peripheral blood. The aim of this study was to develop a novel and practical protocol for MNT in rat peripheral blood by FCM, with the method validation. CD71-fluorescein isothiocyanate and DRAQ5 were adopted for the fluorescent staining of proteins and DNA, respectively, to detect micronuclei. To validate the method, groups of male Sprague-Dawley rats (five per group) received two oral gavage doses at 0 and 24 h of six chemicals (four positive mutagens: ethyl methanesulphonate [EMS], cyclophosphamide [CP], colchicine [COL], and ethyl nitrosourea [ENU]; two nongenotoxic chemicals: sodium saccharin and eugenol). Blood samples were collected from the tail vein before and on the five continuous days after treatments; all of which were analyzed for micronuclei presence by both the manual (Giemsa staining) and FCM methods. The FCM-based method consistently demonstrated highly sensitive responses for micronucleus detection at all concentrations and all time points for EMS, CP, COL, and ENU. Sodium saccharin and eugenol could be identified as negative in this protocol. Results obtained with the FCM-based method correlated well with the micronucleus frequencies (r = 0.659-0.952), and the proportion of immature erythrocytes (r = 0.915-0.981) tested by Giemsa staining. The method reported here, with easy operation, low background, and requirement for a regular FCM, could be an efficient system for micronucleus scoring.
微核试验（MNT）是最广泛应用的短期检测断裂剂或纺锤体破坏剂的方法。已经报道了使用流式细胞术（FCM）对外周血中的有微核的红细胞进行评分。本研究旨在通过 FCM 建立一种新的实用的大鼠外周血 MNT 方法，并对其进行方法验证。采用 CD71-异硫氰酸荧光素和 DRAQ5 分别对蛋白质和 DNA 进行荧光染色，以检测微核。为了验证该方法，将雄性 Sprague-Dawley 大鼠（每组 5 只）分为 6 组，每组分别接受两次口服灌胃，在 0 小时和 24 小时给予 6 种化学物质（4 种阳性致突变剂：乙磺酸[EMS]、环磷酰胺[CP]、秋水仙碱[COL]和乙基亚硝脲[ENU]；2 种非遗传毒性化学物质：糖精钠和丁香油）。在处理前和处理后连续 5 天从尾静脉采集血液样本；所有样本均采用吉姆萨染色法和 FCM 法同时分析微核的存在。FCM 法在所有浓度和所有时间点均能灵敏地检测到 EMS、CP、COL 和 ENU 的微核。在该方案中，糖精钠和丁香油可被鉴定为阴性。FCM 法检测到的微核频率与吉姆萨染色法检测到的微核频率（r = 0.659-0.952）和未成熟红细胞比例（r = 0.915-0.981）具有良好的相关性。本研究报道的方法操作简单、背景低，且只需要常规 FCM，可作为一种有效的微核评分系统。
