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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。
OBJECTIVE: To carry out oral administration of oral toxicity test in SD rats for 3 days, combined with alkaline comet assay and bone marrow micronucleus test in liver, kidney and peripheral blood and detection of common solvents and positive compounds in genotoxicity test Compare By comparing the results of genotoxicity test with chromosomal damage and DNA fragmentation under the same experimental conditions, this study provides a reference for the time of administration and the selection of positive agents. Methods: Male SD rats were randomly divided into deionized water group, 0.9% sodium chloride injection group, corn oil group, cyclophosphamide (CPA) 10 mg · kg -1, cyclophosphamide (CPA) 40 mg · kg -1, 40 mg · kg -1 of N-ethyl-N-nitrosourea (ENU) and 200 mg · kg -1 of ethyl methanesulfonate (EMS) -1) group, 5 in each group. The rats were administered intragastrically for 3 days, and the clinical symptoms and body weight were recorded during the experiment. Rats were sacrificed 3 h after the last administration. The weights of liver and kidney were weighed, and single cell suspensions of liver, kidney and peripheral blood were collected to carry out multi-organ alkaline comet assay. After the samples were prepared, the cells were lysed, untwisted, electrophoresed and stained, respectively. The Komet 6.0 software was used for image analysis. Meanwhile, bone marrow cells were collected and smeared, and 2 000 polychromatic erythrocytes (PCE) -containing micronucleated cells Occurrence rate. Results: 1) CPA, ENU and EMS did not cause serious toxic effects on the whole animal. 2) Only the percentage of hedgehog cells of hepatocytes, kidney cells and lymphocytes in EMS group was significantly increased, while the percentage of hedgehog cells was not affected by CPA and ENU. ENU group in the three kinds of tissue DNA percentage and Olive tail tail moment compared with the vehicle control group was weakly caused by DNA cleavage; EMS in all three tissues showed strong DNA fragmentation. 3) CPA as a classic positive bone marrow micronucleus test showed micronucleus results showed a dose-related; ENU micronuclei relatively weak. Conclusion: It is feasible to integrate the multi-organism comet assay and micronucleus test with the three-day repeated dose toxicity experiment. The standardization of experimental procedure, selection of positive agent and experimental design should be designed according to the specific situation.