[關(guān)鍵詞]
[摘要]
目的 使用Ames波動(dòng)試驗(yàn)和GADD45a-GFP GreenScreen兩種快速篩選方法,對(duì)有毒中藥芫花和了哥王的主要單體成分大黃素和芫花素的遺傳毒性進(jìn)行評(píng)價(jià)。方法 (1)Ames波動(dòng)試驗(yàn):在非代謝活化(-S9)和代謝活化(+S9)條件下使用鼠傷寒沙門菌TA100開展基于96孔液態(tài)培養(yǎng)法的細(xì)菌回復(fù)性突變?cè)囼?yàn),大黃素和芫花素(終質(zhì)量濃度范圍均為0.1~10 μg/mL)與菌液充分混合后在37℃下培養(yǎng)72 h。(2)GADD45a GreenScreen高通量篩選試驗(yàn):在非代謝活化(-S9)和代謝活化條件下(+S9)將表達(dá)GADD45a基因的TK細(xì)胞(GenM-T01和GenM-C01)與大黃素(0.13~32.0 μg/mL)和芫花素(0.07~16.0 μg/mL)分別作用48 h(-S9)和3 h(+S9),之后通過酶標(biāo)儀和流式細(xì)胞儀檢測(cè)綠色熒光蛋白(enhanced green fluorescent protein,EGFP)的熒光強(qiáng)度。結(jié)果 有無代謝活化條件下,10 μg/mL大黃素均可誘導(dǎo)TA100的回復(fù)性突變率顯著性升高(P<0.05、P<0.001);代謝活化條件下,10 μg/mL芫花素可誘導(dǎo)TA100的回復(fù)性突變率顯著性升高(P<0.001),16.0 μg/mL芫花素誘導(dǎo)TK6細(xì)胞表達(dá)的GADD45a-EGFP熒光強(qiáng)度也超過了遺傳毒性閾值(1.3倍)。結(jié)論 當(dāng)前研究提示大黃素和芫花素為可疑遺傳毒性,需要開展深入研究明確其遺傳毒性及機(jī)制。Ames波動(dòng)試驗(yàn)和GADD45a GreenScreen是良好的高通量篩選備選試驗(yàn),可極大優(yōu)化浩繁的藥物的毒性篩選工作,尤其適宜諸如中藥等成分和配伍復(fù)雜的藥物。
[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.
[中圖分類號(hào)]
[基金項(xiàng)目]
國(guó)家“重大新藥創(chuàng)制”科技重大專項(xiàng)(2015ZX09501004-002);《中國(guó)藥典》藥品標(biāo)準(zhǔn)提高