大鼠肝彗星试验与骨髓微核试验评价结果比较研究

Comparative study on evaluation results of liver comet assay and bone marrow micronucleus test in rats

目的梳理国家药物安全评价监测中心联合开展的大鼠多终点体内遗传毒性试验数据,比较大鼠肝彗星试验与骨髓微核试验结果的一致性和灵敏性。方法试验分设阴性物质组、作用机制明确的遗传毒性阳性物质组、受试物组,阴性物质包括超纯水、0.9%氯化钠注射液、玉米油、0.5%羧甲基纤维素钠(CMC-Na)、5%蔗糖和聚山梨酯80,给药体积为10 mL·kg^(-1);遗传毒性阳性物质包括200 mg·kg^(-1)甲磺酸乙酯(EMS)、40 mg·kg^(-1)N-乙基-N-亚硝基脲(ENU)、40 mg·kg^(-1)环磷酰胺、75 mg·kg^(-1)甲基苄肼、800 mg·kg^(-1)尿烷、75 mg·kg^(-1)对氯苯胺、40 mg·kg^(-1)1,2-二溴-3-氯丙烷和10 mg·kg^(-1)秋水仙素;受试物包括100、300、1000 mg·kg^(-1)大黄素-8-O-β-D-葡萄糖苷,6.5、65.0、650.0 mg·kg^(-1)单蒽酮和6.5、65.0、650.0 mg·kg^(-1)大黄素甲醚。分别在实验0、24、45 hig给药1次,给药体积为10 mL·kg^(-1)。开展大鼠肝彗星试验和骨髓微核试验,计算每只动物的肝细胞刺猬细胞率和尾DNA百分含量(Tail%DNA),以及每只动物的嗜多染红细胞(PCE)/总红细胞(ERY)比例和嗜多染红细胞微核(MNPCE)率。结果大鼠肝彗星试验可有效检出DNA断裂剂,对多种烷化剂(甲磺酸乙酯、甲基苄肼和尿烷等)有较好的预测性,但对环磷酰胺和多倍体诱导剂不灵敏。大黄素-8-O-β-D-葡萄糖苷、单蒽酮和大黄素甲醚骨髓微核试验结果均为阴性。大黄素-8-O-β-D-葡萄糖苷在1000 mg·kg^(-1)剂量下导致的肝Tail%DNA与0.5%CMC-Na组比较显著升高(P<0.05);单蒽酮的肝彗星试验结果为明确阳性,剂量为650 mg·kg^(-1)时,单蒽酮可导致大鼠肝Tail%DNA显著升高(P<0.05),且作用存在剂量相关性;大黄素甲醚的肝彗星试验结果为阴性。结论大鼠肝彗星试验可与骨髓微核试验互补,有效检出主要作用于肝脏且亲电子性较强的遗传毒性化合物。

Objective The data of multi-endpoint in vivo genotoxicity tests in rats jointly carried out by the National Center for Safety Evaluation of Drugs was analyzed,and the consistency and sensitivity of rat liver comet assay and bone marrow micronucleus test results were compared.Methods The test was divided into negative substance,genotoxic positive substance with clear mechanism of action,and subject group.Negative substances included ultra-pure water,0.9%sodium chloride injection,corn oil,0.5%sodium carboxymethyl cellulose(CMC-Na),5%sucrose and polyssorbide 80,with an administration volume of 10 mL·kg^(−1).Genotoxic substances included 200 mg·kg^(−1) ethyl mesylate(EMS),40 mg·kg^(−1) N-ethyl-N-nitrourea(ENU),40 mg·kg^(−1) cyclophosphamide,75 mg·kg^(−1) methyl benzyl hydrazine,800 mg·kg^(−1) urane,75 mg·kg^(−1) p-chloroaniline,40 mg·kg^(−1)1,2-dibromo-3-chloropropane and 10 mg·kg^(−1) colchicine.The subjects included 100,300 and 1000 mg·kg^(−1) emodin-8-O-β-D-glucoside,6.5,65.0 and 650.0 mg·kg^(−1) monanthrone and 6.5,65.0 and 650.0 mg·kg^(−1) emodin methyl ether.At 0,24 and 45 h of the experiment,the drug was given once ig in a volume of 10 mL·kg^(−1).The rat liver comet test and bone marrow micronucleus test were performed.The hepatocyte hedgehog cell rate and Tail%DNA content(Tail%DNA),polychromatic red blood cell(PCE)/total red blood cell(ERY)ratio and polychromatic red blood cell micronucleus(MNPCE)rate of each animal were calculated.Results Rat liver comet assay could effectively detect chemicals inducing DNA breakage and various alkylating agents(ethyl methanesulfonate,methyl benzylhydrazine and urane,etc.),while it was not insensitive to cyclophosphamide and polyploid inducer.The results of bone marrow micronucleus test were negative for emodin-8-O-β-D-glucoside,single anthrone and emodin-methyl ether.The liver%Tail DNA induced by emodin 8-O-β-D-glucoside at 1000 mg·kg^(−1) was significantly increased compared with 0.5%CMC-Na group(P<0.05).The results of liver comet test of single ananthone were clearly positive.At a dose of 650 mg·kg^(−1),single ananthone could significantly increase liver%Tail DNA(P<0.05),and there was a dose-dependent effect.The liver comet test for emodin was negative.Conclusion As the second in vivo genotoxicity test recommended by the testing guideline,the rat liver comet assay could complement with the bone marrow micronucleus test and effectively detect genotoxic compounds that mainly act on the liver and are highly electrophilic.