[关键词]
[摘要]
目的 使用Ames波动试验和GADD45a-GFP GreenScreen两种快速筛选方法,对有毒中药芫花和了哥王的主要单体成分大黄素和芫花素的遗传毒性进行评价。方法 (1)Ames波动试验:在非代谢活化(-S9)和代谢活化(+S9)条件下使用鼠伤寒沙门菌TA100开展基于96孔液态培养法的细菌回复性突变试验,大黄素和芫花素(终质量浓度范围均为0.1~10 μg/mL)与菌液充分混合后在37℃下培养72 h。(2)GADD45a GreenScreen高通量筛选试验:在非代谢活化(-S9)和代谢活化条件下(+S9)将表达GADD45a基因的TK细胞(GenM-T01和GenM-C01)与大黄素(0.13~32.0 μg/mL)和芫花素(0.07~16.0 μg/mL)分别作用48 h(-S9)和3 h(+S9),之后通过酶标仪和流式细胞仪检测绿色荧光蛋白(enhanced green fluorescent protein,EGFP)的荧光强度。结果 有无代谢活化条件下,10 μg/mL大黄素均可诱导TA100的回复性突变率显著性升高(P<0.05、P<0.001);代谢活化条件下,10 μg/mL芫花素可诱导TA100的回复性突变率显著性升高(P<0.001),16.0 μg/mL芫花素诱导TK6细胞表达的GADD45a-EGFP荧光强度也超过了遗传毒性阈值(1.3倍)。结论 当前研究提示大黄素和芫花素为可疑遗传毒性,需要开展深入研究明确其遗传毒性及机制。Ames波动试验和GADD45a GreenScreen是良好的高通量筛选备选试验,可极大优化浩繁的药物的毒性筛选工作,尤其适宜诸如中药等成分和配伍复杂的药物。
[Key word]
[Abstract]
Objective To evaluate the genotoxicity of emodin and genkwanin, the mian monomer components of the poisonous traditional Chinese medicines Daphne genkwa and wikstroemia indica, using two rapid screening methods fluctuation Ames test and GADD45a GreenScreen assay. Methods (1) Fluctuation Ames test:A bacterial reversion test based on the 96-well liquid culture method was performed using Salmonella typhimurium TA100 under non-metabolic activation (-S9) and metabolic activation (+S9) conditions. Emodin and genkwanin (final concentrations ranged from 0.1 to 10 μg/mL) were well-mixed with the bacterial broth and incubated at 37℃ for 72 h. (2) GADD45a GreenScreen assay:TK cells expressing GADD45a gene (GenM-T01 and GenM-C01) were treated with emodin (0.13 to 32.0 μg/mL) and genkwanin (0.07 to 16.0 μg/mL) for 48 h (-S9) and 3 h (+S9) respectively. The fluorescence intensity of enhanced green fluorescent protein (EGFP) was detected by microplate reader and flow cytometry. Results In the presence and absence of metabolic activation, the averaged mutated wells induced by 10 μg/mL emodin to TA100 were significantly increase than negative control groups (P<0.05, P<0.001); in the presence of metabolic activation, 10 μg/mL genkwanin also markedly elevated the averaged counts of wells with mutated bacteria (P<0.001), 16.0 μg/mL of genkwanin triggered fluorescence intensity of GADD45a-EGFP expressed by TK6 cells also exceeded the genotoxicity threshold (1.3 folds). Conclusion Both emodin and genkwanin demonstrated as suspicious positive in current genotoxicity study, and investigations on determine their genotoxicity and underlying mechanisms should be followed. The fluctuation Ames test and GADD45a GreenScreen assay are valuable high throughput screening alternatives that could greatly optimize the heavy toxicity screening of drugs, especially for the drugs with complicated compositions and compatibilities, such as the traditional Chinese medicines.
[中图分类号]
[基金项目]
国家“重大新药创制”科技重大专项(2015ZX09501004-002);《中国药典》药品标准提高