基于体外Pig-a基因突变试验的大黄素型蒽醌致突变风险评价  

Mutagenic risk evaluation of emodin-type anthraquinone using in vitro Pig-a mutation assay

作　　者：王亚楠 叶倩 王雪[1] 汪祺[1] 文海若 WANG Yanan;YE Qian;WANG Xue;WANG Qi;WEN Hairuo(National Institutes for Food and Drug Control,Beijing 100050,China)

机构地区：[1]中国食品药品检定研究院,北京100050

出　　处：《药物评价研究》2022年第7期1233-1239,共7页Drug Evaluation Research

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

摘　　要：目的对相同母核结构的8种大黄素型蒽醌类化合物开展体外Pig-a基因突变试验,分析不同大黄素型蒽醌结构与致突变性的关联。方法L1578Y细胞分别与系列浓度的大黄素、芦荟大黄素、大黄素甲醚、大黄酚、大黄酸、羟基大黄素、大黄素-8-O-β-D-葡萄糖苷和芦荟大黄素-8-O-β-D-葡萄糖苷作用4 h(有S9)或24 h(无S9),给药24 h后应用细胞计数板进行计数,计算细胞相对倍增速率(RPD)评价受试物细胞毒性;细胞表达8 d后经APC-anti-CD45和PE-anti-CD90.2标定后,使用流式细胞仪检测突变细胞(CD45+CD90-)发生率。结果所有受试物在有或无S9代谢活化条件下所设浓度组RPD均大于50%,未见明显细胞毒性作用,可排除试验中假阳性结果。在非S9代谢活化条件下芦荟大黄素25μg·mL^(−1)组Pig-a基因突变率与溶媒对照组比较显著升高(P<0.001);S9代谢活化条件下,与溶剂对照组比较,大黄素50μg·mL^(−1)组,羟基大黄素6.25、12.5、25μg·mL^(−1)组,大黄酚25、50、100μg·mL^(−1)组和大黄酸12.5、25、50μg·mL^(−1)组Pig-a基因突变率显著升高(P<0.05、0.01、0.001)。结论羟基取代基的多寡及所在位点是蒽醌类化合物致突变性强弱的决定性因素,其体内致突变性及致癌性作用仍需进行大量体内研究证实。Objective To test eight emodin-type anthraquinone compounds with the same core structure using in vitro Pig-a gene mutation assay,and to analyze the relationship between different emodin-type anthraquinone structures and mutagenicity.Methods L1578Y cells were treated with different concentrations of emodin,aloe-emodin,emodin methyl ether,chrysophanol,rhein,hydroxyemodin,emodin-8-O-β-D-glucoside and aloe-emodin-8-O-β-D-glucoside treatment for 4 h(with S9)or 24 h(without S9).Cell counting plate was used to count 24 h after administration,and the relative cell multiplication rate(RPD)was calculated to evaluate the cytotoxicity of the tested samples.After eight days of expression period,the cells were labelled with APC-anti-CD45 and PE-anti-CD90.2,and the incidence of mutant cells(CD45+CD90−)were detected using flow cytometry.Results The RPD of all tested substances in the concentration group with or without metabolic activation was greater than 50%,and no obvious cytotoxicity was observed,so false positive results in the test could be excluded.The mutation rate of Pig-a gene in aloe emodin 25μg·mL^(−1)group was significantly higher than that in the solvent control group under non-S9 metabolic activation condition(P<0.001).Under S9 metabolic activation condition,compared with solvent control group,the mutation rate of Pig-a gene in emodin 50μg·mL^(−1)group,hydroxyl emodin 6.25,12.5,25μg·mL^(−1)groups,chrysophanol 25,50,100μg·mL^(−1)groups and rhein 12.5,25,50μg·mL^(−1)groups was significantly increased(P<0.05,0.01,0.001).Conclusion The number and position of hydroxyl substituents are the decisive factors for the mutagenicity of anthraquinone compounds.However,in vivo mutagenic and carcinogenic effects of emodintype anthraquinones still need to be confirmed by a large number of in vivo studies.

关 键 词：大黄素型蒽醌 致突变性 L5178Y细胞 体外Pig-a基因突变试验 羟基取代基 黄素 芦荟大黄素 大黄素甲醚 大黄酚 大黄酸 羟基大黄素 大黄素-8-O-β-D-葡萄糖苷 芦荟大黄素-8-O-β-D-葡萄糖苷 

分 类 号：R994[医药卫生—毒理学]