大鼠体内肝细胞碱性彗星试验评估3种参考化学物的遗传毒性

Genotoxicity assessment of three reference chemicals in rat hepatocytes by alkaline comet assay

【目的】建立大鼠体内肝细胞碱性彗星试验方法,检测3种参考化学物的遗传毒性。【方法】将6~10周龄的Sprague Dawley大鼠随机分为4组,每组10只,雌雄各半。以生理盐水(0.9%NaCl溶液)为阴性对照组,受试物组分为甲基磺酸乙酯(EMS)200 mg/kg组、N-甲基-N-亚硝基脲(MNU)50 mg/kg组和D-甘露醇2 000 mg/kg组。连续2次(间隔21 h)灌胃染毒,实验期间记录动物临床症状和体质量变化。末次染毒3 h后处死大鼠,称量肝脏重量,取肝脏制备单细胞悬液开展肝细胞碱性彗星试验,测定肝细胞平均尾部DNA含量百分比(DNA%)等指标。【结果】受试物EMS、MNU和D-甘露醇均未对动物整体产生严重的毒性作用。与阴性对照组大鼠肝细胞平均尾部DNA含量百分比[(2.32±1.39)%]相比,EMS和MNU组分别为(60.07±24.69)%和(41.66±22.35)%,差异有统计学意义(P<0.05);D-甘露醇组差异无统计学意义[(3.06±3.30)%,P>0.05]。【结论】本实验室初步建立了大鼠体内肝细胞碱性彗星试验方法,3种参考化学物质中,EMS和MNU有明显遗传毒性,D-甘露醇组未见遗传毒性。

[Objective]To establish a comet test method for detection of genotoxicity of three reference chemicals in rat liver cells.[Methods]6-10 week old Sprague Dawley rats were randomly divided into 4 groups,with normal saline(0.9%NaCl solution)as negative control group.Animals in three test groups were treated,respectively,with ethyl methanesulfonate(EMS)200 mg/kg,N-methyl-Nnitrosourea(MNU)50 mg/kg,and D-mannitol 2000 mg/kg.There were 10 animals in each group,5 males and 5 females.The animals received two times(21 h interval)of test compounds through intragastric administration,and their clinical symptoms and body weight changes were recorded during the experiment.The rats were sacrificed 3 h after the last exposure.The liver was weighed,then used to prepare single-cell suspensions for the alkaline comet test which determines the average tail DNA content percentage(DNA%)of hepatocytes and other comet indicators.[Results](1)D-mannitol,EMS and MNU did not show significant toxicity in the whole animal.(2)The mean values of tail DNA content percentage(DNA%)of rat hepatocytes in EMS[(60.07±24.69)%]and MNU[(41.66±22.35)%]groups were higher than that in the negative control group[(2.32±1.39)%]and the difference was statistically significant(P<0.05).The difference between D-mannitol group[(3.06±3.30)%]and the negative control group was not significant(P>0.05).[Conclusion]This laboratory has established a comet test method using hepatocytes from treated rats.Among three testing chemicals,EMS and MNU have displayed genotoxicity by this assay,but no genotoxicity was observed in D-mannitol treated animals.