AVT-Chair for Biochemical Engineering, RWTH Aachen University, Forckenbeckstraße 51, 52074 Aachen, Germany.
Institute for Environmental Research, Department of Ecosystem Analysis, Worringerweg 1, 52074 Aachen, Germany.
Sci Total Environ. 2020 May 15;717:137168. doi: 10.1016/j.scitotenv.2020.137168. Epub 2020 Feb 7.
The Ames test is one of the most widely used mutagenicity tests. It employs histidine auxotrophic bacteria, which can mutate back to histidine prototrophy and, thus, grow on a histidine deficient medium. These mutants develop predominantly after adding a mutagenic compound during an initial growth phase on 1 mg/L histidine. In the established test systems, an endpoint determination is performed to determine the relative number of mutants. An alternative Ames test, the Ames RAMOS test, has been developed, which enables the online detection of mutagenicity by monitoring respiration activity. The reproducibility of the newly developed test system was investigated. A strong dependence of the test results on the inoculum volume transferred from the preculture was found. The more inoculum was needed to reach the required initial OD, the more mutagenic a positive control was evaluated. This effect was attributed to the histidine transfer from the preculture to the original Ames RAMOS test. The same problem is evident in the Ames fluctuation test. High reproducibility of the Ames RAMOS test could be achieved by performing the preculture on minimal medium with a defined histidine concentration and termination after histidine depletion. By using 5 mg/L initial histidine within the minimal medium, a higher separation efficiency between negative control and mutagenic samples could be achieved. This separation efficiency could be further increased by lowering the cultivation temperature from 37 to 30 °C, i.e. lowering the maximum growth rate. The optimized Ames RAMOS test was then transferred into a 48-well microtiter plate format (μRAMOS) for obtaining a high throughput test. The online detection of mutagenicity leads to a reduction of working time in the laboratory. Due to the optimization of reproducibility and the increase in separation efficiency, a sound mutagenicity evaluation, even of weak mutagenic compounds, can be achieved.
Ames 试验是最广泛使用的致突变性试验之一。它采用组氨酸营养缺陷型细菌，这些细菌可以突变回组氨酸原养型，从而在缺乏组氨酸的培养基上生长。这些突变体主要在初始生长阶段添加诱变剂化合物后在 1mg/L 组氨酸上生长时发展。在建立的试验系统中，进行终点测定以确定突变体的相对数量。已经开发了一种替代的 Ames 试验，即 Ames RAMOS 试验，它通过监测呼吸活性来实现致突变性的在线检测。研究了新开发的试验系统的重现性。发现试验结果强烈依赖于从预培养物转移的接种物体积。需要更多的接种物来达到所需的初始 OD，评价阳性对照的致突变性就越高。这种效应归因于从预培养物向原始 Ames RAMOS 试验转移的组氨酸。在 Ames 波动试验中也存在同样的问题。通过在含有定义浓度组氨酸的最小培养基上进行预培养，并在耗尽组氨酸后终止，可以实现 Ames RAMOS 试验的高重现性。在最小培养基中使用 5mg/L 的初始组氨酸，可以在阴性对照和致突变样品之间实现更高的分离效率。通过将培养温度从 37°C 降低至 30°C，即降低最大生长速率，可以进一步提高这种分离效率。然后将优化的 Ames RAMOS 试验转移到 48 孔微量滴定板格式（μRAMOS）中，以获得高通量测试。致突变性的在线检测可减少实验室的工作时间。由于重现性的优化和分离效率的提高，可以实现对弱致突变化合物的可靠致突变性评价。
