De Boeck Marlies, van der Leede Bas-jan, Van Goethem Freddy, De Smedt Ann, Steemans Margino, Lampo Ann, Vanparys Philippe
Johnson & Johnson Pharmaceutical Research & Development--Division of Janssen Pharmaceutica N.V., Genetic and In Vitro Toxicology, Turnhoutseweg 30, B-2340 Beerse, Belgium.
Environ Mol Mutagen. 2005 Jul;46(1):30-42. doi: 10.1002/em.20127.
According to the current Organization of Economic Cooperation and Development (OECD) and International Committee on Harmonization (ICH) guidelines for the mammalian erythrocyte micronucleus (MN) test, analysis of peripheral blood reticulocytes (RETs) for the presence of micronuclei can be performed using flow cytometry. The MicroFlow PLUS method (Litron Laboratories, Rochester, NY) for MN analysis by flow cytometry is based on the binding of FITC-labeled antibodies to the CD71 transferrin receptor of immature RETs, on parallel RNA degradation, and on propidium iodide staining of DNA present as micronuclei. The objective of this study was to assess the sensitivity of this flow cytometry method to detect time- and dose-dependent induction of micronuclei in mouse peripheral blood RETs after treatment with nine chemical agents. Five known clastogens, two known aneugens, and two compounds previously reported to be inactive in the mouse bone marrow MN test were evaluated at three dose levels. Multiple blood sampling of the same animal before and at two time points after treatment was conducted. All known mutagens produced a dose-dependent increase in micronucleated reticulocytes (MN-RETs); the compounds previously shown to be inactive in the in vivo MN test were also negative using the present methodology. The highest frequency of MN-RETs was observed at 48 hr after treatment, except for 5-fluorouracil, which had its peak response at 72 hr. The results indicate that micronuclei can be measured by multiple blood sampling of the same animal before and after treatment without altering the sensitivity of the assay. The results confirm that the flow cytometric assessment of MN-RETs in mouse peripheral blood using MicroFlow PLUS is a sensitive method with high analysis throughput, and robust quality control.
根据经济合作与发展组织（OECD）和国际协调会议（ICH）目前关于哺乳动物红细胞微核（MN）试验的指南，可使用流式细胞术分析外周血网织红细胞（RET）中是否存在微核。用于通过流式细胞术进行MN分析的MicroFlow PLUS方法（Litron Laboratories，纽约罗切斯特）基于异硫氰酸荧光素（FITC）标记的抗体与未成熟RET的CD71转铁蛋白受体的结合、平行的RNA降解以及作为微核存在的DNA的碘化丙啶染色。本研究的目的是评估这种流式细胞术方法检测九种化学试剂处理后小鼠外周血RET中微核的时间和剂量依赖性诱导的敏感性。在三个剂量水平下评估了五种已知的致断裂剂、两种已知的非整倍体诱导剂以及两种先前报道在小鼠骨髓MN试验中无活性的化合物。在处理前和处理后的两个时间点对同一只动物进行多次采血。所有已知的诱变剂均导致微核网织红细胞（MN-RET）呈剂量依赖性增加；先前在体内MN试验中显示无活性的化合物使用本方法也呈阴性。除5-氟尿嘧啶在72小时达到峰值反应外，MN-RET的最高频率在处理后48小时观察到。结果表明，通过在处理前后对同一只动物进行多次采血来测量微核，不会改变检测的敏感性。结果证实，使用MicroFlow PLUS对小鼠外周血中的MN-RET进行流式细胞术评估是一种敏感的方法，具有高分析通量和稳健的质量控制。
