一种体外多终点遗传毒性检测方法  

An in vitro multiple endpoints assay for genotoxicity detection

作　　者：黄鹏程 王敬亭 赵田田 赵泽浩 周长慧 李若婉 朱春梅 郑阳 常艳 HUANG Peng-cheng;WANG Jing-ting;ZHAO Tian-tian;ZHAO Ze-hao;ZHOU Chang-hui;LI Ruo-wan;ZHU Chun-mei;ZHENG Yang;CHANG Yan(Shanghai Innostar Bio-Tech Co.Ltd.,Shanghai 201203,China;China State Institute of Pharma-ceutical Industry,Shanghai 201201,China;School of Pharmacy,Fudan University,Shanghai 201203,China)

机构地区：[1]上海益诺思生物技术股份有限公司,上海201203 [2]中国医药工业研究总院,上海201201 [3]复旦大学药学院,上海201203

出　　处：《中国药理学与毒理学杂志》2021年第3期201-210,共10页Chinese Journal of Pharmacology and Toxicology

基　　金：国家科技重大专项(2018ZX09201017-008);上海市科委研发公共服务平台专项(18DZ2290100)。

摘　　要：目的建立一种操作简单、快速和准确的体外多终点遗传毒性检测方法。方法将组蛋白γ-H2AX、P53蛋白、磷酸化组蛋白H3(p-H3)以及活化多腺苷二磷酸核糖聚合酶(PARP)4个生物标志物整合到γ-H2AX灶点试验中。DNA断裂剂依托泊苷(未加S9条件下,0.2,0.4,0.8和1.6 mg·L^(-1))、环磷酰胺(S9条件下,3.75,7.5,15和30 mg·L^(-1))和非整倍体诱导剂秋水仙素(未加S9条件下,0.0063,0.0125,0.025和0.05 mg·L^(-1))处理TK6细胞24 h,收获细胞、固定,洗涤后加入抗体标记,流式细胞仪检测γ-H2AX、P53蛋白和p-H3荧光强度。选择4种不同作用机制的DNA断裂剂(有S9条件下,丝裂霉素C 0.05,0.1和0.2 mg·L^(-1),甲磺酸甲酯3,6和12 mg·L^(-1),苯并芘0.5,1和2 mg·L^(-1),顺铂2.5,5和10 mg·L^(-1);无S9条件下,分别加丝裂霉素C 0.025,0.05,0.1 mg·L^(-1),甲磺酸甲酯1.5,3和6 mg·L^(-1),苯并芘25,50和100 mg·L^(-1),顺铂1.25,2.5和5 mg·L^(-1))、2种非整倍体诱导剂(S9条件下,长春新碱0.005,0.01和0.02 mg·L^(-1),紫杉醇0.02,0.04和0.08 mg·L^(-1);未加S9条件下,长春新碱0.01,0.02和0.04 mg·L^(-1),紫杉醇0.005,0.01和0.02 mg·L^(-1))和3种不具有遗传毒性的化合物(有/无S9条件下,氯化钠125,250和500 mg·L^(-1),氨苄青霉素G 125,250和500 mg·L^(-1),盐酸普萘洛尔15,30和60 mg·L^(-1)),对该方法进行验证。结果在方法建立阶段,与溶剂对照组相比,无S9依托泊苷组和有S9的环磷酰胺组γ-H2AX和P53蛋白荧光强度增加,且依托泊苷组1.6 mg·L^(-1)和环磷酰胺30 mg·L^(-1)组组荧光强度为溶剂对照组的2倍以上,p-H3阳性细胞比例无显著升高;秋水仙素组γ-H2AX和P53蛋白荧光强度无明显变化,p-H3细胞比例为溶剂对照组2倍以上。在方法验证阶段使用多种不同作用机制的化合物验证得到的灵敏度(6/6)和特异性(3/3)均较高。结论建立了体外多终点遗传毒性检测方法,该方法具有应用于药物早期遗传毒性筛选和遗传毒性评价的潜力。OBJECTIVE To establish a simple,rapid and accurate multi-endpoint in vitro genotoxicity assay.METHODS In this study,γ-H2AX,P53 protein,phosphorylated histone H3(p-H3)and cleaved-PARP were combined into theγ-H2AX assay.TK6 cells were treated with DNA breaking agent etoposide(without S9,0.2,0.4,0.8 and 1.6 mg·L^(-1)),cyclophosphamide(with S9,3.75,7.5,15 and 30 mg·L^(-1))and aneuploidy inducer colchicine for 24 h(without S9,0.0063,0.0125,0.025 and 0.05 mg·L^(-1)).After the cells were harvested,fixed and washed,antibody labeling was added,and the fluorescence intensity ofγ-H2AX,P53 protein and p-H3 was detected by flow cytometry.The method was verified by using four DNA breaking agents(with S9,mitomycin C 0.05,0.1 and 0.2 mg·L^(-1),methyl mesylate 3,6 and 12 mg·L^(-1),benzopyrene 0.5,1 and 2 mg·L^(-1) and cisplatin 2.5,5 and 10 mg·L^(-1);without S9,mitomycin C 0.0025,0.05 and 0.1 mg·L^(-1),methyl mesylate 1.5,3 and 6 mg·L^(-1),benzopyrene 25,50 and 100 mg·L^(-1) and cisplatin 0.5,1 and 2 mg·L^(-1)),two aneuploidy inducers(with S9,vincristine 0.005,0.01 and 0.02 mg·L^(-1),paclitaxel 0.02,0.04 and 0.08 mg·L^(-1);without S9,vincristine 0.01,0.02 and 0.04 mg·L^(-1),paclitaxel 0.005,0.01 and 0.02 mg·L^(-1)),and three non-genotoxic compounds(NaCl 125,250 and 500 mg·L^(-1),ampicillin G 125,250 and 500 mg·L^(-1),propranolol hydrochloride 15,30 and 60 mg·L^(-1)).RESULTS During the establishment of this model,the fluorescence intensities ofγ-H2AX and P53 proteins in the etoposide group without S9 and the cyclophosphamide group with S9 were increased compared with the solvent control group.The fluorescence intensity of etoposide group(1.6 mg·L^(-1))and cyclophosphamide group(30 mg·L^(-1))was more than twice that of the solvent control group,and the proportion of p-H3.During method validation,the sensitivity and(6/6)specificity(3/3)obtained by using a variety of compounds with different mechanisms of action were both high.CONCLUSION An in vitro multi-endpoint genotoxicity assay is established.This method

关 键 词：Γ-H2AX 磷酸化组蛋白H3 P53蛋白 流式细胞术 DNA双链断裂 遗传毒性 

分 类 号：R99[医药卫生—毒理学]