Department of Chemical and Biomedical Engineering, Cleveland State University, 455 Fenn Hall (FH), 1960 East 24th Street, Cleveland, OH, 44115-2214, USA.
Arch Toxicol. 2018 Aug;92(8):2501-2516. doi: 10.1007/s00204-018-2249-1. Epub 2018 Jul 4.
The US Environmental Protection Agency (EPA) launched the Transform Tox Testing Challenge in 2016 with the goal of developing practical methods that can be integrated into conventional high-throughput screening (HTS) assays to better predict the toxicity of parent compounds and their metabolites in vivo. In response to this need and to retrofit existing HTS assays for assessing metabolism-induced toxicity of compounds, we have developed a 384-pillar plate that is complementary to traditional 384-well plates and ideally suited for culturing human cells in three dimensions at a microscale. Briefly, human embryonic kidney (HEK) 293 cells in a mixture of alginate and Matrigel were printed on the 384-pillar plates using a microarray spotter, which were coupled with 384-well plates containing nine model compounds provided by the EPA, five representative Phase I and II drug metabolizing enzymes (DMEs), and one no enzyme control. Viability and membrane integrity of HEK 293 cells were measured with the calcein AM and CellTiter-Glo kit to determine the IC values of the nine parent compounds and DME-generated metabolites. The Z' factors and the coefficient of variation measured were above 0.6 and below 14%, respectively, indicating that the assays established on the 384-pillar plate are robust and reproducible. Out of nine compounds tested, six compounds showed augmented toxicity with DMEs and one compound showed detoxification with a Phase II DME. This result indicates that the 384-pillar plate platform can be used to measure metabolism-induced toxicity of compounds in high-throughput with individual DMEs. As xenobiotics metabolism is a complex process with a variety of DMEs involved, the predictivity of our approach could be further improved with mixtures of DMEs.
美国环保署（EPA）于 2016 年发起了 Transform Tox Testing 挑战赛，旨在开发实用的方法，将其整合到常规高通量筛选（HTS）测定中，以更好地预测母体化合物及其代谢物在体内的毒性。为了满足这一需求，并对现有的 HTS 测定进行改造以评估化合物代谢诱导的毒性，我们开发了一种 384 柱板，它与传统的 384 孔板互补，非常适合在微尺度上三维培养人类细胞。简要地说，使用微阵列点样器将藻酸盐和 Matrigel 混合物中的人胚肾（HEK）293 细胞打印到 384 柱板上，这些柱板与包含 EPA 提供的 9 种模型化合物、5 种代表性的 I 相和 II 相药物代谢酶（DME）和 1 个无酶对照的 384 孔板相连接。使用 calcein AM 和 CellTiter-Glo 试剂盒测量 HEK 293 细胞的活力和细胞膜完整性，以确定 9 种母体化合物和 DME 生成的代谢物的 IC 值。测定的 Z'因子和变异系数分别高于 0.6 和低于 14%，表明在 384 柱板上建立的测定是稳健且可重复的。在测试的 9 种化合物中，有 6 种化合物与 DME 一起显示出毒性增强，有 1 种化合物与 II 相 DME 一起显示出解毒作用。这一结果表明，384 柱板平台可用于使用单个 DME 以高通量方式测量化合物的代谢诱导毒性。由于外源性化合物代谢是一个涉及多种 DME 的复杂过程，因此我们的方法的预测能力可以通过混合 DME 进一步提高。
