SD大鼠连续灌胃给药对碱性彗星电泳和骨髓微核的影响

发布时间：2018-08-07 16:57

【摘要】：目的:开展SD大鼠3天重复经口灌胃给药毒性实验,联合肝、肾和外周血碱性彗星电泳试验和骨髓微核试验,并对遗传毒性试验常用的溶媒及阳性化合物进行检测和比较。通过对比相同实验条件下开展的以染色体损伤和DNA断裂为检测终点的组合遗传毒性试验结果,为给药和取材时间点以及阳性剂的选择提供参考依据。方法:雄性SD大鼠随机分为去离子水组、0.9%氯化钠注射液组、玉米油组、环磷酰胺(CPA)10 mg·kg~(-1)组、环磷酰胺(CPA)40 mg·kg~(-1)组、N-乙基-N-亚硝基脲(ENU)40 mg·kg~(-1)组和甲磺酸乙酯(EMS)200 mg·kg~(-1)组,每组5只。连续3 d灌胃给药,实验期间记录动物临床症状和体重变化。末次给药后3 h处死大鼠。称量肝、肾脏器重量,并收集肝、肾及外周血制备单细胞悬液开展多脏器碱性彗星试验。样本制片后分别经裂解、解旋、电泳、染色等步骤,使用Komet 6.0软件进行图像分析;取材同时收集骨髓细胞制作涂片,计数2 000个嗜多染红细胞(PCE)的含微核细胞发生率。结果:1)CPA、ENU和EMS均未对动物整体产生严重的毒性作用。2)只有EMS组的肝细胞、肾细胞和淋巴细胞的刺猬细胞百分率明显升高,CPA及ENU对刺猬细胞百分率无明显影响。ENU组在3种组织的彗尾DNA含量百分率及Olive尾矩与溶媒对照组比较为弱致DNA断裂作用;而EMS则在3种组织中均显示了强致DNA断裂作用。3)CPA作为经典的骨髓微核试验阳性剂,其微核结果显示了一定的剂量相关性;ENU的致微核作用相对较弱。结论:将多脏器彗星试验与微核试验与3天重复给药毒性实验整合是可行的;实验流程的标准化、阳性剂的选择及实验设计等需根据具体情况来设计。
[Abstract]:Aim: to carry out a 3-day repeated oral administration of drugs in SD rats, combined with alkaline comet electrophoresis in liver, kidney and peripheral blood, and micronucleus test in bone marrow, and to detect and compare the common solutes and positive compounds in genotoxicity test. By comparing the results of combinatorial genotoxicity test with chromosome damage and DNA breakage as the end point under the same experimental conditions, this paper provides a reference for the selection of drug administration and sampling time points as well as the selection of positive agents. Methods: male Sprague-Dawley rats were randomly divided into three groups: 0.9% sodium chloride injection group, corn oil group, cyclophosphamide (CPA) 10 mg kg ~ (-1) group, cyclophosphamide (CPA) 40 mg kg ~ (-1) group and (ENU) 40 mg kg ~ (-1) group and (EMS) 200 mg kg ~ (-1) group. The clinical symptoms and body weight of the animals were recorded during the experiment. Rats were killed 3 hours after the last administration. To weigh the weight of liver and kidney, and to collect single cell suspensions from liver, kidney and peripheral blood to carry out alkaline comet assay of multiple viscera. The samples were analyzed by image analysis with Komet 6.0 software, and bone marrow cells were collected to make smears, and the incidence of micronucleus cells in (PCE) of 2000 polychromatic erythrocytes was counted. Results: (1) neither CPA nor EMS had serious toxicity on the whole of animals. 2) there were only hepatocytes in EMS group. The percentage of Hedgehog cells in renal cells and lymphocytes was significantly increased. The percentage of DNA in comet tail and Olive tail moment in three tissues were not significantly affected by CPA and ENU. However, EMS showed strong DNA cleavage (3. 3) CPA as a classic micronucleus positive agent in three tissues. The results of micronucleus showed that the micronucleus effect of CPA was relatively weak in a dose-dependent manner. Conclusion: it is feasible to integrate multiple viscera comet assay with micronucleus test with 3-day repeated administration toxicity test, and the standardization of experimental procedure, the selection of positive agents and the design of experiment should be designed according to the specific conditions.
【作者单位】：中国食品药品检定研究院国家药物安全评价监测中心药物非临床安全评价研究北京市重点实验室;中山大学药学院药物安全评价中心;
【基金】：中国食品药品检定研究院中青年发展研究基金课题(2014C1) 国家“重大新药创制”科技重大专项(2015ZX09501004-002)
【分类号】：R965

【参考文献】