Institute for Environmental Research, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany.
Institute for Environmental Research, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany; Department of Evolutionary Ecology and Environmental Toxicology, Goethe University Frankfurt, Max-von-Laue-Str. 13, 60438 Frankfurt am Main, Germany.
Sci Total Environ. 2021 Jan 10;751:142269. doi: 10.1016/j.scitotenv.2020.142269. Epub 2020 Sep 11.
This study presents a high-throughput (HTP) micronucleus assay in multi-well plates with an automated evaluation for risk assessment applications. The evaluation of genotoxicity via the micronucleus assays according to international guidelines ISO 21427-2 with Chinese hamster (Cricetulus griseus) V79 cells was the starting point to develop our methodology. A drawback of this assay is that it is very time consuming and cost intensive. Our HTP micronucleus assay in a 48-well plate format allows for the simultaneous assessment of five different sample-concentrations with additional positive, negative and solvent controls with six technical replicates each within a quarter of the time required for the equivalent evaluation using the traditional slide method. In accordance with the 3R principle, animal compounds should be replaced with animal-free alternatives. However, traditional cell culture-based methods still require animal derived compounds like rat-liver derived S9-fraction, which is used to simulate the mammalian metabolism in in vitro assays that do show intrinsic metabolization capabilities. In the present study, a recently developed animal-free biotechnological alternative (ewoS9R) was investigated in the new high-throughput micronucleus assay. In total, 12 different mutagenic or genotoxic chemicals were investigated to assess the potential use of the animal-free metabolization system (ewoS9R) in comparison to a common rat-derived product. Out of the 12 compounds, one compound did not induce micronuclei in any treatment and 2 substances showed a genotoxic potential without the need for a metabolization system. EwoS9R demonstrated promising potential for future applications as it shows comparable results to the rat-derived S9 for 6 of the 9 pro-genotoxic substances tested. The remaining 3 substances (2-Acetamidofluorene, Benzo[a]pyrene, Cyclophosphamide) were only metabolized by rat-derived S9. A potential explanation is that ewoS9R was investigated with an approx. 10-fold lower enzyme concentration and was only optimized for CYP1A metabolization that may be improved with a modified production procedure. Future applications of ewoS9R go beyond the micronucleus assay, but further research is necessary.
本研究提出了一种高通量（HTP）微核分析方法，该方法采用自动化评估方法进行风险评估应用。根据国际指南 ISO 21427-2 用中国仓鼠（Cricetulus griseus）V79 细胞进行遗传毒性评估是开发我们的方法的起点。该测定法的一个缺点是非常耗时且成本高昂。我们在 48 孔板格式中的 HTP 微核测定法允许同时评估五个不同的样品浓度，另外还有阳性、阴性和溶剂对照，每个对照有六个技术重复，这只需使用传统载玻片方法进行等效评估所需时间的四分之一。根据 3R 原则，动物化合物应被无动物替代物所取代。然而，传统的基于细胞培养的方法仍然需要动物衍生的化合物，如大鼠肝衍生的 S9 级分，该级分用于模拟哺乳动物代谢，因为在体外测定中显示出内在的代谢能力。在本研究中，研究了一种新的高通量微核测定法中最近开发的无动物生物技术替代物（ewoS9R）。总共研究了 12 种不同的诱变或遗传毒性化学物质，以评估无动物代谢系统（ewoS9R）的潜在用途，与常见的大鼠衍生产品相比。在 12 种化合物中，有 1 种化合物在任何处理中均未诱导微核，有 2 种化合物在无需代谢系统的情况下表现出遗传毒性潜力。EwoS9R 显示出有希望的未来应用潜力，因为它对测试的 9 种前遗传毒性物质中的 6 种与大鼠衍生的 S9 具有可比的结果。其余 3 种物质（2-乙酰氨基芴、苯并[a]芘、环磷酰胺）仅由大鼠衍生的 S9 代谢。一种可能的解释是，ewoS9R 的酶浓度约低 10 倍，并且仅针对 CYP1A 代谢进行了优化，这可以通过改进生产工艺来提高。EwoS9R 的未来应用超出了微核测定法，但需要进一步的研究。
