SD大鼠连续灌胃给药对碱性彗星电泳和骨髓微核的影响

Effects of repeated intragastric administration on alkaline comet assay and micronucleus test in SD rats

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 compare the effects of commonly used solvent and positive controls in SD rats by gavage for 3-day repeated dosed toxicity study, combined with alkaline comet assay for liver, kidney and peripheral blood cells and bone marrow micronucleus test. By comparing the results of combined genotoxicity tests for detecting chromosomal damage and DNA breakage under the same experimental conditions, this study aims to provide reference for identifying the dosing and sampling time points and the selection of appropriate positive controls. Methods： Male SD rats were randomly divided into deionized water group, 0.9% NaCl injection group, corn oil group, cyclophosphamide （ CPA ） 10 mg kg-1 group, cyclophosphamide （ CPA ） 40 mg kg-1 group, N-ethyl- N-nitrosourea （ ENU ） 40 mg kg-1 group and ethyl methanesulfonate （ EMS ） 200 mg kg-1 group, 5 rats in each group. The clinical observation and body weight were recorded during the experiment. Animals were sacrificed 3 hours after the last dosing, and the weights of liver and kidney were measured. Single cell suspensions were made from liver, kidney and peripheral lymphocytes for comet assay. The samples were subsequently proceeded to lysis, unwinding, electrophoresis, dyeing, and the results were analyzed using Komet 6.0 software. Bone marrow cells were also prepared smear in parallel for micronucleus test, and the incidence of micronucleated cells were counted in 2 000 PCE. Results ： 1 ） No significant toxic effect was observed in animals treated with CPA, ENU or EMS in this study ; 2 ） The percentage of hedgehog cells in hepatocytes, kidney cells and lymphocytes were significantly increased in only EMS group, and CPA and ENU had no significant effect on the percentage of hedgehog cells. Compared with the solvent control group, the percentage of tail DNA content and Olive tail moment （ OTM ） in all three tissues were the weak DNA fracture by ENU, while EMS in all three tissued showed strong DNA fracture.3 ） As a classic bone marrow micronucleus test positive agent, its micronucleus results of CPA showed a certain dose correlation; ENU induced micronucleus was relatively weak. Conclusion： It is feasible to integrate multiple organ comet assay and micronucleus test with 3-day repeated drug toxicity test. However, the standardization of the experimental procedure, the selection of the positive agent and the design of the experiment should be designed according to the specific situation.