[关键词]
[摘要]
目的 初步建立Ames波动试验酶标仪结果判定标准,并对染料苏丹红I~IV的DNA碱基突变风险进行评价。方法 不同浓度的阳性对照2-(2-呋喃基)-3-(5-硝基-2-呋喃基)丙烯酰胺(AF-2)、2-氨基蒽(2-AA)分别作用于鼠伤寒沙门氏菌TA98和TA100后,分别经人工识别或用酶标仪在492和623 nm波长下进行读数,确立基于吸光度的阳性孔判定标准。无或有代谢活化(-S9)条件下使用鼠伤寒沙门氏菌TA98和TA100开展基于96孔液态培养法的细菌回复性突变试验,苏丹红I~IV终浓度分别为0.625、1.250、2.500、5.000和10.000 μg/mL。结果 非S9代谢条件下酶标仪读板结果与人工计数结果一致性达98.1%,S9代谢条件下酶标仪读数与菌落生长情况无法建立良好关联。在非代谢活化条件下,苏丹红I与IV对TA98的致突变性作用相对较弱;苏丹红I~IV对TA100存在明显的致突变性作用,并呈一定的浓度相关性。在S9代谢活化条件下,苏丹红III对TA98和TA100均未见致突变作用;苏丹红I在最高浓度10 μg/mL时对2者均有明显的致突变作用,苏丹红IV浓度高于2.5 μg/mL、苏丹红II浓度高于5 μg/mL时,对2者致突变作用均显著。结论 首次使用Ames波动试验检出苏丹红I~IV的基因突变风险,吸光度读数可在非S9代谢条件下准确判断Ames波动试验结果,为染料的高通量遗传毒性筛选奠定基础。
[Key word]
[Abstract]
Objective To preliminary establish a determination criteria for the results of the Ames fluctuation test microplate reader and to evaluate the risk of DNA base mutation of the dye Sudan Red I~IV. Methods After different concentrations of AF-2 or 2-AA were applied to Salmonella typhimurium TA98 and TA100, respectively, they were manually identified or read at 492 nm and 623 nm using a microplate reader to establish a positive well determination based on absorbance. A 96-well liquid culture-based bacterial reversion mutation test was performed using Salmonella typhimurium TA98 and TA100 in the absence or presence of metabolic activation (-S9). The final concentrations of Sudan I-IV were 0.625, 1.250, 2.500, 5.000 and 10.000 μg/mL, respectively. Results The results of the microplate reader readings and manual counting results were 98.1% under non-S9 metabolic conditions. The microplate reader readings and colony growth conditions under S9 metabolic conditions could not establish a good correlation. Under the condition of non metabolic activation, the mutagenic effect of Sudan I and IV on TA98 was relatively weak; Sudan I-IV had obvious mutagenic effect on TA100, and there was a certain concentration correlation. Under the condition of S9 metabolic activation, Sudan III had no mutagenic effect on TA98 and TA100; Sudan I had obvious mutagenic effect on both of them at the highest concentration of 10 μg/mL; when the concentration of Sudan IV was higher than 2.5 μg/mL and the concentration of Sudan II was higher than 5 μg/mL, the mutagenicity of both was significant. Conclusion This study was the first to use the Ames fluctuation test to detect the risk of genetic mutations in Sudan Red I~IV, and for the first time to establish a positive well standard based on absorbance readings. The absorbance reading can accurately judge the results of the Ames fluctuation test under non-S9 metabolic conditions, laying a foundation for future high-throughput genotoxicity screening of dyes.
[中图分类号]
R965.2
[基金项目]
国家自然科学基金(81503347);国家“重大新药创制”科技重大专项(2018ZX09201017);