目的 初步建立Ames波動試驗酶標儀結(jié)果判定標準，并對染料蘇丹紅I~IV的DNA堿基突變風險進行評價。方法 不同濃度的陽性對照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）分別作用于鼠傷寒沙門氏菌TA98和TA100后，分別經(jīng)人工識別或用酶標儀在492和623 nm波長下進行讀數(shù)，確立基于吸光度的陽性孔判定標準。無或有代謝活化（-S₉）條件下使用鼠傷寒沙門氏菌TA98和TA100開展基于96孔液態(tài)培養(yǎng)法的細菌回復性突變試驗，蘇丹紅I~IV終濃度分別為0.625、1.250、2.500、5.000和10.000 μg/mL。結(jié)果 非S₉代謝條件下酶標儀讀板結(jié)果與人工計數(shù)結(jié)果一致性達98.1%，S₉代謝條件下酶標儀讀數(shù)與菌落生長情況無法建立良好關(guān)聯(lián)。在非代謝活化條件下，蘇丹紅I與IV對TA98的致突變性作用相對較弱；蘇丹紅I~IV對TA100存在明顯的致突變性作用，并呈一定的濃度相關(guān)性。在S₉代謝活化條件下，蘇丹紅III對TA98和TA100均未見致突變作用；蘇丹紅I在最高濃度10 μg/mL時對2者均有明顯的致突變作用，蘇丹紅IV濃度高于2.5 μg/mL、蘇丹紅II濃度高于5 μg/mL時，對2者致突變作用均顯著。結(jié)論 首次使用Ames波動試驗檢出蘇丹紅I~IV的基因突變風險，吸光度讀數(shù)可在非S₉代謝條件下準確判斷Ames波動試驗結(jié)果，為染料的高通量遺傳毒性篩選奠定基礎(chǔ)。

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.

R965.2

國家自然科學基金（81503347）；國家“重大新藥創(chuàng)制”科技重大專項（2018ZX09201017）；