大鼠和小鼠Pig-a基因突变试验历史背景数据采集  

Historical background data of Pig-a gene mutation assay in rat and mouse

作　　者：韩素芹 姜华[1] 叶倩 黄芝瑛[2] 耿兴超[1] 汪祺[1] 文海若 HAN Suqin;JIANG Hua;YE Qian;HUANG Zhiying;GENG Xingchao;WANG Qi;WEN Hairuo(National Institutes for Food and Drug Control,Beijing 100050,China;School of Pharmaceutical Science,Sun Yat-sen University,Guangzhou 510006,China)

机构地区：[1]中国食品药品检定研究院,北京100050 [2]中山大学药学院,广东广州510006

出　　处：《药物评价研究》2023年第5期944-951,共8页Drug Evaluation Research

基　　金：国家十三五“重大新药创制”专项课题(2018ZX09201017);国家自然科学基金资助项目(81503347)。

摘　　要：目的介绍大鼠及小鼠Pig-a基因突变试验方法,并汇总国家药物安全评价监测中心2015—2022年开展的基于免疫磁珠检测法的大鼠及小鼠Pig-a基因突变试验背景数据。方法阴性物质包括超纯水和0.5%羧甲基纤维素钠(CMCNa),雄性C57BL/6J小鼠间隔24 h ig 0.5%CMC-Na,连续7 d;雄性SD大鼠间隔24 h ig 0.5%CMC-Na,连续14 d;大鼠间隔24 h ig超纯水,连续3 d。阳性对照为已知致细菌突变化合物,包括N-乙基-N-亚硝基脲(ENU,10、40 mg·kg^(−1))、盐酸丙卡巴肼(PCZ,60、150 mg·kg^(−1))、乌拉坦(EC,300、800 mg·kg^(−1))、N-亚硝基二甲胺(NDMA,1.5 mg·kg^(−1))、N-亚硝基二乙胺(NDEA,15 mg·kg^(−1))。小鼠间隔24 h ig ENU 40 mg·kg^(−1),连续3 d;间隔24 h ig NDMA 1.5 mg·kg^(−1)、NDEA 15 mg·kg^(−1),连续7 d。大鼠间隔24 h ig PCZ 150 mg·kg^(−1)、EC 800 mg·kg^(−1)、ENU 40 mg·kg^(−1),连续3 d;间隔24 h ig PCZ 60 mg·kg^(−1)、EC 300 mg·kg^(−1)、ENU 10 mg·kg^(−1),连续28 d。分别于给予受试物前,首次给予后14、28 d采集外周血,用流式细胞术检测大鼠红细胞表面CD59蛋白的结合情况,结合免疫磁性计数微球技术计算网织红细胞(RETs)占总红细胞的百分率(%RET)(作为外周血毒性考察指标)、总红细胞中CD59表达为阴性细胞(RBC^(CD59−),即突变的总红细胞)发生率和RETs中CD59表达为阴性细胞(RET^(CD59-),即突变的RETs)发生率。结果各试验%RET数值均无大幅增加。SD大鼠和C57BL/6J小鼠的阴性对照组RBC^(CD59-)和RET^(CD59-)突变率均低于5×10^(−6),小鼠的背景值相对不稳定。连续3 d ig给予小鼠40 mg·kg^(−1)的ENU,RBC^(CD59−)和RET^(CD59−)发生率自给药后2周开始均大幅增加(P<0.05),给药后4周进一步增加(P<0.01、0.001);给予小鼠NDMA后2、4周,RBC^(CD59−)发生率略有增加,但仍在阴性背景范围内,但RET^(CD59−)发生率在给药后第2周大幅增加(P<0.001),给药后第4周则大幅回落;给予小鼠NDEA后2周,RBC^(CD59�Objective The method of Pig-a gene mutation assay in rats and mice was introduced,and the background data based on immunomagnetic beads carried out by the National Center for Safety Evaluation of Drugs from 2015 to 2022 were summarized.Methods The negative substances included ultrapure water and 0.5%carboxymethyl cellulose sodium(CMC-Na).Male C57BL/6J mice received 0.5%CMC-Na at an interval of 24 h for seven consecutive days.Male SD rats were given 0.5%CMC-Na at an interval of 24 h for 14 consecutive days.Rats were given ultrapure water at an interval of 24 hours for three consecutive days.Positive controls were known bacterial mutagenic compounds,including N-ethyl-N-nitrosourea(ENU,10 and 40 mg·kg^(−1)),procarbazide hydrochloride(PCZ,60 and 150 mg·kg^(−1)),uratan(EC,300 and 800 mg·kg^(−1)),N-nitrosodimethylamine(NDMA,1.5 mg·kg^(−1)),and N-nitrosodiethylamine(NDEA,15 mg·kg^(−1)).Mice were given ENU 40 mg·kg^(−1) at an interval of 24 h for three consecutive days,and given NDMA 1.5 mg·kg^(−1) and NDEA 15 mg·kg^(−1) at an interval of 24 h for seven consecutive days.Rats were given PCZ 150 mg·kg^(−1),EC 800 mg·kg^(−1),and ENU 40 mg·kg^(−1) at 24 hour intervals for three consecutive days,and given PCZ 60 mg·kg^(−1),EC 300 mg·kg^(−1),and ENU 10 mg·kg^(−1) at 24 hour intervals for 28 consecutive days.Peripheral blood was collected before the administration of the test substance,14 and 28 days after the first administration,and the binding of CD59 protein on the surface of rat red blood cells was detected by flow cytometry.The percentage of reticulocyte(RETs)in total red blood cells(%RET,as an indicator for peripheral blood toxicity),the incidence of CD59 expression in total red blood cells(RBC^(CD59−),i.e.mutated total red blood cells)and the incidence of CD59 expression in RETs(RET^(CD59-),i.e.mutated RETs)were calculated by using immunomagnetic counting microsphere technology.Results There was no significant increase in the%RET values for each experiment.The spontaneous RBC^

关 键 词：大鼠 小鼠 Pig-a基因突变试验 致突变性 历史背景值 

分 类 号：R965.3[医药卫生—药理学]