癌变·畸变·突变 ›› 2016, Vol. 28 ›› Issue (4): 277-280.doi: 10.3969/j.issn.1004-616x.2016.04.006

• 论著 • 上一篇    下一篇

体内碱性彗星试验与微核试验联合测定甲磺酸乙酯遗传毒性方法的建立

韩天娇, 周长慧, 常艳   

  1. 中国医药工业研究总院国家上海新药安全评价研究中心, 上海 201203
  • 收稿日期:2015-12-14 修回日期:2016-04-01 出版日期:2016-07-31 发布日期:2016-07-31
  • 通讯作者: 常艳,E-mail:ychang@ncdser.com E-mail:ychang@ncdser.com
  • 作者简介:韩天娇,Tel:021-50800333-208;E-mail:hantianjiaomm@163.com
  • 基金资助:
    国家“十二五重大新药创制”科技重大专项（2012ZX09302002）

Establishment a method for genotoxicity evaluation of ethyl methanesulfonate in rats using Comet and flow micronucleus assays

HAN Tianjiao, ZHOU Changhui, CHANG Yan   

  1. Chinese State Institute of Pharmaceutical Industry, National Shanghai Center for New Drug Safety Evaluation Research, Shanghai 201203, China
  • Received:2015-12-14 Revised:2016-04-01 Online:2016-07-31 Published:2016-07-31

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摘要： 目的：建立彗星试验与微核试验结合的检测方法，应用其评价甲磺酸乙酯（EMS）对大鼠的遗传毒性。方法：试验设置阴性对照组（0.9%的生理盐水）及EMS低（50 mg/kg）、中（100 mg/kg）、高（200 mg/kg）剂量组，共4组，每组5只雄性SD大鼠，分别在0、24、48和69 h经口灌胃1次性给予受试物，在末次给药后3 h进行外周血采样，外周血经固定、洗脱、荧光染色后进行微核试验采用流式细胞术测定网织红细胞微核率；然后处死动物进行肝脏、肾脏和胃组织的取样，样品经单细胞悬液制备、制片、裂解、解旋电泳及染色后，应用Comet Assay IV软件进行彗星试验数据的收集。结果：彗星试验中，50、100和200 mg/kg EMS染毒组肝脏、肾脏和胃的尾DNA百分含量与阴性对照组相比均显著增加，且呈剂量反应关系（r=0.93，P<0.05）；微核试验中，EMS高剂量组骨髓毒性太大未收集到足够的细胞用于检测，仅EMS中剂量组外周血网织红细胞微核率较对照组显著增加（P<0.01）。结论：初步建立了大鼠多靶器官彗星试验方法；在同一试验中彗星试验和微核试验联合可节省动物数量，提高试验效率。

关键词: 甲磺酸乙酯, 体内彗星试验, 微核试验, 多终点

Abstract: OBJECTIVE: To establish a method for genotoxicity evaluation of ethyl methanesulfonate (EMS) in rats by using a combination of Comet and flow micronucleus assays. METHODS: EMS (50, 100 and 200 mg/kg) was administered to rats once daily at 24 h and 45 h intervals. Negative controls received 0.9% physiological saline. Each dose group contained 5 rats. Blood was collected at 3 h after the final administration. From each sample, 2 000 reticulocytes (RET) were counted for MN frequency by flow cytometry. Then, all rats were humanely sacrificed with pentobarbital sodium. Liver, stomach, and kidney were removed to make single cell suspensions. These cells were used for the Comet assay. The Comet parameters were measured using the Comet Assay IV. RESULTS: Based on the Comet assay, EMS induced a significant dose-related increase of the Comet tail DNA percentage in the liver, stomach, and kidney. In the flow micronucleus assay, statistically significant increases in MN frequency were noted in the 100 mg/kg EMS dose group compared with those in the negative controls. CONCLUSION: EMS can cause DNA strand breaks and micronuclei and the combined Comet and MN assays will improve the accuracy of investigations.

Key words: ethyl methanesulfonate, in vivo Comet assay, micronucleus test, multiple endpoints

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