中国药物警戒 ›› 2020, Vol. 17 ›› Issue (8): 455-460.
DOI: 10.19803/j.1672-8629.2020.08.02

• 基础与临床研究 • 上一篇    下一篇

大黄素型单蒽酮基因突变风险评价

文海若1, 王亚楠1, 杨莹1, 赵婷婷1, 马双成2, 汪祺2,*   

  1. 1中国食品药品检定研究院国家药物安全评价监测中心,药物非临床安全评价研究北京市重点实验室,北京 100176;
    2中国食品药品检定研究院中药民族药检定所,北京 100050
  • 收稿日期:2020-07-31 修回日期:2020-07-31 出版日期:2020-08-15 发布日期:2020-07-31
  • 通讯作者: *汪祺,副研究员,中药毒理。E-mail：sansan8251@sina.com
  • 作者简介:文海若,女,博士,副研究员,药理毒理。

Mutagenic Risk Evaluation of Monanthone with Emodin Type

WEN Hairuo1, WANG Yanan1, YANG Ying1, ZHAO Tingting1, MA Shuangcheng2, WANG Qi2,*   

  1. 1National Center for Safety Evaluation of Drugs, National Institutes for Food and Drug Control, Key Laboratory of Beijing for Nonclinical Safety Evaluation Research of Drugs, Beijing 100176, China;
    2Institute for Control of Chinese Traditional Medicine and Ethnic Medicine, National Institutes for Food and Drug Control, Beijing 100050, China
  • Received:2020-07-31 Revised:2020-07-31 Online:2020-08-15 Published:2020-07-31
  • Supported by:
    国家自然科学基金资助项目（81503347, 81503068）; 国家十三五“重大新药创制”专项（2018ZX09201017）

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摘要： 目的 为评价大黄素型单蒽酮潜在遗传毒性致基因突变,分别开展miniAmes试验和体外Pig-a基因突变试验进行研究。方法 miniAmes试验：分别设置对照组（DMSO）,单蒽酮（0.6、1.1、2.3、4.5、9 μg/皿）,阳性剂组,使用鼠伤寒沙门氏菌TA 97、TA 98、TA 100、TA 102、TA 1535、TA 1537和大肠杆菌 WP2 uvrA分别在-S9和S9两种代谢状态下开展基于6孔板培养的细菌回复突变试验,48 h后计数突变菌落数。体外Pig-a基因突变试验：以L5178Y细胞为体系,非S9代谢条件：分别设对照组（1%DMSO）,阳性剂（EMS 500 μg/mL）,单蒽酮（0.2 μg/mL,0.39 μg/mL,0.78 μg/mL,1.56 μg/mL）,受试物处理4 h后计数,去除受试物更换培养,24 h后计数,表达8 d,表达期维持细胞密度在1×106~2×106个/mL,表达结束后进行抗体孵育流式检测。S9代谢条件：分别设对照组（1%DMSO）,阳性剂（B（a）P 5 μg/mL）,单蒽酮（0.2 μg/mL,0.39 μg/mL,0.78 μg/mL,1.56 μg/mL）,受试物处理4 h后,去除受试物更换培养,24 h后计数,表达8 d,表达期维持细胞密度在1×106~2×106个/mL,表达结束后进行抗体孵育流式检测。结果 Ames试验结果：非S9代谢活化条件下,单蒽酮可诱导TA97、TA1537回复突变菌落数与阴性对照组相比有所增加;S9代谢活化条件下,可诱导TA1537回复突变菌落数与阴性对照组相比有所增加。增加倍数超过阴性对照组的2~3倍,且上述结果均存在一定剂量相关变化趋势。体外Pig-a基因突变结果：非S9代谢活化条件下,单蒽酮浓度大于0.78 μg/mL,Pig-a基因突变频率与溶媒对照组相比存在显著性差异（P <0.01）,且存在剂量相关性。结论 本研究条件下,大黄素型单蒽酮存在基因突变风险。

关键词: 何首乌, 单蒽酮, 基因突变, 遗传毒性, Ames试验, 体外Pig-a基因突变

Abstract: Objective To evaluate the gene mutations caused by the potential genotoxicity of monanthone with emodin type by means of the miniAmes test and in vitro Pig-a gene mutation test. Methods The miniAmes test involved the control group (DMSO), monanthone with emodin type (0.6, 1.1, 2.3, 4.5, 9 μg/well), and positive group. Salmonella typhimurium TA97, TA98, TA100, TA102, TA1535, TA1537 bacterial reverse mutation assay based on 6-well plate culture was carried out in E. coli WP2 uvrA under the conditions of -S9 and S9, respectively. The number of mutant colonies was counted after 48 h. The In vitro Pig-a gene mutation test involved L5178Y cells that were used as the system, and metabolic conditions without S9: control group (1% DMSO), positive agent (EMS 500 μg/mL), and monanthone with emodin type (0.2 μg/mL,0.39 μg/mL,0.78 μg/mL,1.56 μg/mL).After the test object was treated for 4 h, the test substance and culture were removed. After 24 h, the cells were counted and expressed for 8 days. The cell density during the expression period was maintained at 1×106~2×106 cells/mL. After the expression was terminated, antibody incubation flow detection was performed. Metabolic conditions with S9: control group (1% DMSO), positive agent (B(a)P 5 μg/mL), and monoterpene ketone (0.2 μg/mL,0.39 μg/mL,0.78 μg/mL,1.56 μg/mL). After the test object was treated for 4 h, the test material was removed and cultured, and counted 24 hours later for 8 days. The cell density during the expression period was 1×106~2×106 cells/mL. After the expression was over, the antibody incubation flow assay was performed. Results In the Ames test, the number of colonies of TA97 and TA1537 reverting mutants induced by monanthone with emodin type increased compared with the negative control group under the metabolic activation conditions without S9. Upon metabolic activation with S9, the number of colonies of TA1537 reverting mutants induced by monanthone with emodin type increased compared with the negative control group. The rate of increase was more than two or three times that of the negative control group, and there was dose-related change in the above results. In vitro Pig-a gene mutation test showed that under metabolic activation conditions without S9, the concentration of monanthone with emodin type exceeded 0.78 μg/mL, and the mutation frequency of Pig-a gene was significantly different from that of the vehicle control group (P <0.01) with a dose correlation. Conclusion Under the conditions described in this study, monanthone with emodin type has the risk of gene mutation.

Key words: Polygonummultiflorum Thunb, monanthone with emodin type, gene mutation, genotoxicity, Ames test, in vitro Pig-a gene mutation

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