Avlasevich Svetlana L, Khanal Sumee, Singh Priyanka, Torous Dorothea K, Bemis Jeffrey C, Dertinger Stephen D
Litron Laboratories, Rochester, New York.
Environ Mol Mutagen. 2018 Apr;59(3):176-187. doi: 10.1002/em.22168. Epub 2018 Jan 22.
The current report describes a newly devised method for automatically scoring the incidence of rat hepatocyte micronuclei (MNHEP) via flow cytometry, with concurrent assessments of hepatocyte proliferation-frequency of Ki-67-positive nuclei, and the proportion of polyploid nuclei. Proof-of-concept data are provided from experiments performed with 6-week old male Crl:CD(SD) rats exposed to diethylnitrosamine (DEN) or quinoline (QUIN) for 3 or 14 consecutive days. Non-perfused liver tissue was collected 4 days after cessation of treatment in the case of 3-day studies, or 1 day after last administration in the case of 14-day studies for processing and flow cytometric analysis. In addition to livers, blood samples were collected one day after final treatment for micronucleated reticulocyte (MN-RET) measurements. Dose-dependent increases in MNHEP, Ki-67-positive nuclei, and polyploidy were observed in 3- and 14-day DEN studies. Both treatment schedules resulted in elevated %MNHEP for QUIN-exposed rats, and while cell proliferation effects were subtle, appreciable increases to normalized liver weights were observed. Whereas DEN caused markedly higher %MNHEP when exposure was extended to two weeks, QUIN-induced MNHEP were slightly increased with protracted dosing. Parallel microscopy-based MNHEP frequencies were highly correlated with flow cytometry-based measurements (four study/aggregate R = 0.80). No increases in MN-RET were seen in any of the four studies. Collectively, these results suggest liver micronuclei are amenable to an automated scoring technique that provides objective analyses and higher information content relative to conventional microscopy. Additional work is needed to expand the number and types of chemicals tested, identify the most advantageous treatment schedules, and test the transferability of the method. Environ. Mol. Mutagen. 59:176-187, 2018. © 2018 Wiley Periodicals, Inc.
本报告描述了一种新设计的方法，可通过流式细胞术自动对大鼠肝细胞微核（MNHEP）的发生率进行评分，同时评估肝细胞增殖情况（Ki-67阳性细胞核的频率）和多倍体细胞核的比例。通过对6周龄雄性Crl:CD(SD)大鼠连续3天或14天暴露于二乙基亚硝胺（DEN）或喹啉（QUIN）所进行的实验，提供了概念验证数据。在为期3天的研究中，于停止治疗后4天收集未灌注的肝组织；在为期14天的研究中，于末次给药后1天收集肝组织用于处理和流式细胞术分析。除肝脏外，在末次治疗后1天采集血样用于微核网织红细胞（MN-RET）测量。在为期3天和14天的DEN研究中，观察到MNHEP、Ki-67阳性细胞核和多倍体呈剂量依赖性增加。两种治疗方案均导致喹啉暴露大鼠的MNHEP百分比升高，虽然细胞增殖效应不明显，但观察到正常肝脏重量有明显增加。当暴露延长至两周时，DEN导致的MNHEP百分比明显更高，而喹啉诱导的MNHEP随给药时间延长略有增加。基于显微镜的MNHEP频率与基于流式细胞术的测量高度相关（四项研究/汇总R = 0.80）。在四项研究中的任何一项中均未观察到MN-RET增加。总体而言，这些结果表明肝脏微核适用于一种自动评分技术，该技术相对于传统显微镜检查可提供客观分析和更多信息。需要开展更多工作来扩大测试化学品的数量和类型，确定最有利的治疗方案，并测试该方法的可转移性。《环境与分子突变》，2018年，第59卷，第176 - 187页。© 2018威利期刊公司
