Shah Ume-Kulsoom, Mallia Jefferson de Oliveira, Singh Neenu, Chapman Katherine E, Doak Shareen H, Jenkins Gareth J S
In vitro Toxicology Group, Institute of Life Science, College of Medicine, Swansea University,Singleton Park, Swansea, Wales, SA2 8PP, UK.
Faculty of Health Sciences and Life Sciences, School of Allied Health Sciences, De Montfort University, The Gateway, Leicester LE1 9BH, UK.
Mutat Res Genet Toxicol Environ Mutagen. 2018 Jan;825:51-58. doi: 10.1016/j.mrgentox.2017.12.005. Epub 2017 Dec 15.
The liver's role in metabolism of chemicals makes it an appropriate tissue for toxicity testing. Current testing protocols, such as animal testing and two-dimensional liver cell systems, offer limited resemblance to in vivo liver cell behaviour, in terms of gene expression profiles and metabolic competence; thus, they do not always accurately predict human toxicology. In vitro three-dimensional liver cell models offer an attractive alternative. This study reports on the development of a 3D liver model, using HepG2 cells, by a hanging-drop technique, with a focus on evaluating spheroid growth characteristics and suitability for genotoxicity testing. The cytokinesis-blocked micronucleus assay protocol was adapted to enable micronucleus (MN) detection in the 3D spheroid models. This involved evaluating the difference between hanging vs non-hanging drop positions for dosing of the test agents and comparison of automated Metafer scoring with manual scoring for MN detection in HepG2 spheroids. The initial seeding density, used for all experiments, was 5000 cells/20 μl drop hanging spheroids, harvested on day 4, with >75% cell viability. Albumin secretion (7.8 g/l) and both CYP1A1 and CYP1A2 gene expression were highest in the 3D environment at day 4. Exposure to metabolically activated genotoxicants for 24 h resulted in a 6-fold increase in CYP1A1 enzyme activity (3 μM B[a]P) and a 30-fold increase in CYP1A2 enzyme activity (5 μM PhIP) in 3D hanging spheroids. MN inductions in response to B[a]P or PhIP were 2-fold and 3-fold, respectively, and were greater in 3D hanging spheroids than in 2D format, showing that hanging spheroids are more sensitive to genotoxic agents. HepG2 hanging-drop spheroids are an exciting new alternative system for genotoxicity studies, due to their improved structural and physiological properties, relative to 2D cultures.
肝脏在化学物质代谢中的作用使其成为毒性测试的合适组织。当前的测试方案，如动物测试和二维肝细胞系统，在基因表达谱和代谢能力方面与体内肝细胞行为的相似性有限；因此，它们并不总是能准确预测人类毒理学。体外三维肝细胞模型提供了一个有吸引力的替代方案。本研究报告了一种使用HepG2细胞通过悬滴技术构建的三维肝脏模型的开发，重点是评估球体生长特性和对遗传毒性测试的适用性。对胞质分裂阻断微核试验方案进行了调整，以便在三维球体模型中检测微核（MN）。这包括评估测试剂给药时悬滴与非悬滴位置的差异，以及比较在HepG2球体中自动Metafer评分与手动评分用于MN检测的情况。所有实验使用的初始接种密度为5000个细胞/20μl悬滴球体，在第4天收获，细胞活力>75%。在第4天，白蛋白分泌（7.8g/l）以及CYP1A1和CYP1A2基因表达在三维环境中最高。在三维悬滴球体中，暴露于代谢活化的遗传毒性剂24小时导致CYP1A1酶活性增加6倍（3μM苯并[a]芘），CYP1A2酶活性增加30倍（5μM PhIP）。对苯并[a]芘或PhIP的MN诱导分别为2倍和3倍，并且在三维悬滴球体中比二维形式更大，表明悬滴球体对遗传毒性剂更敏感。相对于二维培养，HepG2悬滴球体由于其改善的结构和生理特性，是遗传毒性研究中一个令人兴奋的新替代系统。
