Following advancements in the field of genotoxicology, it has become widely accepted that 3D models are not only more physiologically relevant but also have the capacity to elucidate more complex biological processes that standard 2D monocultures are unable to. Whilst 3D liver models have been developed to evaluate the short-term genotoxicity of chemicals, the aim of this study was to develop a 3D model that could be used with the regulatory accepted in vitro micronucleus (MN) following low-dose, longer-term (5 days) exposure to engineered nanomaterials (ENMs). A comparison study was carried out between advanced models generated from two commonly used liver cell lines, namely HepaRG and HepG2, in spheroid format. While both spheroid systems displayed good liver functionality and viability over 14 days, the HepaRG spheroids lacked the capacity to actively proliferate and, therefore, were considered unsuitable for use with the MN assay. This study further demonstrated the efficacy of the in vitro 3D HepG2 model to be used for short-term (24 h) exposures to genotoxic chemicals, aflatoxin B1 (AFB1) and methyl-methanesulfonate (MMS). The 3D HepG2 liver spheroids were shown to be more sensitive to DNA damage induced by AFB1 and MMS when compared to the HepG2 2D monoculture. This 3D model was further developed to allow for longer-term (5 day) ENM exposure. Four days after seeding, HepG2 spheroids were exposed to Zinc Oxide ENM (0–2 µg/ml) for 5 days and assessed using both the cytokinesis-block MN (CBMN) version of the MN assay and the mononuclear MN assay. Following a 5-day exposure, differences in MN frequency were observed between the CBMN and mononuclear MN assay, demonstrating that DNA damage induced within the first few cell cycles is distributed across the mononucleated cell population. Together, this study demonstrates the necessity to adapt the MN assay accordingly, to allow for the accurate assessment of genotoxicity following longer-term, low-dose ENM exposure.

中文翻译：

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使用支持长期暴露持续时间的3D肝模型，对体外微核试验进行遗传毒性测试的调整。

随着基因毒理学领域的发展，人们已广泛接受3D模型不仅在生理上更相关，而且具有阐明标准2D单培养无法实现的更复杂的生物学过程的能力。虽然已经开发了3D肝模型来评估化学品的短期遗传毒性，但本研究的目的是开发可以与体外公认的法规一起使用的3D模型。低剂量，长期（5天）暴露于工程纳米材料（ENM）之后的微核（MN）。在由两种常用的肝细胞系（即HepaRG和HepG2）以椭球体形式生成的高级模型之间进行了比较研究。尽管两种球体系统均在14天内显示出良好的肝功能和生存能力，但HepaRG球体缺乏主动增殖的能力，因此被认为不适合用于MN分析。这项研究进一步证明了体外的功效3D HepG2模型可用于短期（24 h）暴露于遗传毒性化学物质，黄曲霉毒素B1（AFB1）和甲磺酸甲酯（MMS）。与HepG2 2D单培养相比，显示3D HepG2肝球体对AFB1和MMS诱导的DNA损伤更敏感。对该3D模型进行了进一步开发，以允许长期（5天）的ENM暴露。接种后四天，将HepG2球体暴露于氧化锌ENM（0–2 µg / ml）5天，并使用MN分析的胞质分裂阻滞MN（CBMN）版本和单核MN分析进行评估。暴露5天后，在CBMN和单核MN分析之间观察到MN频率的差异，表明在前几个细胞周期内诱导的DNA损伤分布在单核细胞群中。一起，