Seo Ji-Eun, Li Xilin, Le Yuan, Mei Nan, Zhou Tong, Guo Xiaoqing
Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR, 72079, USA.
Center for Veterinary Medicine, U.S. Food and Drug Administration, Rockville, MD, 20855, USA.
Arch Toxicol. 2023 Apr;97(4):1163-1175. doi: 10.1007/s00204-023-03461-z. Epub 2023 Feb 27.
The in vitro micronucleus (MN) assay is a component of most test batteries used in assessing potential genotoxicity. Our previous study adapted metabolically competent HepaRG cells to the high-throughput (HT) flow-cytometry-based MN assay for genotoxicity assessment (Guo et al. in J Toxicol Environ Health A 83:702-717, 2020b, https://doi.org/10.1080/15287394.2020.1822972 ). We also demonstrated that, compared to HepaRG cells grown as two-dimensional (2D) cultures, 3D HepaRG spheroids have increased metabolic capacity and improved sensitivity in detecting DNA damage induced by genotoxicants using the comet assay (Seo et al. in ALTEX 39:583-604, 2022, https://doi.org/10.14573/altex.22011212022 ). In the present study, we have compared the performance of the HT flow-cytometry-based MN assay in HepaRG spheroids and 2D HepaRG cells by testing 34 compounds, including 19 genotoxicants or carcinogens and 15 compounds that show different genotoxic responses in vitro and in vivo. 2D HepaRG cells and spheroids were exposed to the test compounds for 24 h, followed by an additional 3- or 6-day incubation with human epidermal growth factor to stimulate cell division. The results demonstrated that HepaRG spheroids showed generally higher sensitivity in detecting several indirect-acting genotoxicants (require metabolic activation) compared to 2D cultures, with 7,12-dimethylbenzanthracene and N-nitrosodimethylamine inducing higher % MN formation along with having significantly lower benchmark dose values for MN induction in 3D spheroids. These data suggest that 3D HepaRG spheroids can be adapted to the HT flow-cytometry-based MN assay for genotoxicity testing. Our findings also indicate that integration of the MN and comet assays improved the sensitivity for detecting genotoxicants that require metabolic activation. These results suggest that HepaRG spheroids may contribute to New Approach Methodologies for genotoxicity assessment.
体外微核（MN）试验是大多数用于评估潜在遗传毒性的试验组合的一个组成部分。我们之前的研究将具有代谢活性的HepaRG细胞应用于基于高通量（HT）流式细胞术的微核试验，以进行遗传毒性评估（Guo等人，《毒理学与环境卫生杂志A》83:702 - 717，2020b，https://doi.org/10.1080/15287394.2020.1822972 ）。我们还证明，与二维（2D）培养的HepaRG细胞相比，3D HepaRG球体具有更高的代谢能力，并且在使用彗星试验检测遗传毒性剂诱导的DNA损伤时具有更高的灵敏度（Seo等人，《ALTEX》39:583 - 604，2022，https://doi.org/10.14573/altex.22011212022 ）。在本研究中，我们通过测试34种化合物，包括19种遗传毒性剂或致癌物以及15种在体外和体内表现出不同遗传毒性反应的化合物，比较了基于HT流式细胞术的微核试验在HepaRG球体和2D HepaRG细胞中的性能。将2D HepaRG细胞和球体暴露于测试化合物24小时，随后再与人表皮生长因子一起孵育3天或6天以刺激细胞分裂。结果表明，与2D培养相比，HepaRG球体在检测几种间接作用的遗传毒性剂（需要代谢活化）时通常具有更高的灵敏度，7,12 - 二甲基苯并蒽和N - 亚硝基二甲胺在3D球体中诱导更高的微核形成百分比，同时微核诱导的基准剂量值显著更低。这些数据表明3D HepaRG球体可应用于基于HT流式细胞术的微核试验进行遗传毒性测试。我们的研究结果还表明，微核试验和彗星试验的结合提高了检测需要代谢活化的遗传毒性剂的灵敏度。这些结果表明HepaRG球体可能有助于遗传毒性评估的新方法学。
