Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Večna pot 111, 1000, Ljubljana, Slovenia.
Jozef Stefan International Postgraduate School, Ljubljana, Slovenia.
Arch Toxicol. 2019 Nov;93(11):3321-3333. doi: 10.1007/s00204-019-02576-6. Epub 2019 Sep 21.
The evaluation of genotoxicity plays an important role within hazard identification and risk assessment of chemicals and consumer products. For genotoxicity assessment, in vitro hepatic cells are often used as they have retained certain level of xenobiotic metabolic activity. However, current protocols are designed for the use on 2D monolayer models that are associated with several limitations due to the lack of numerous biological functions, which results in the loss of many hepatic properties. In this respect, an attractive alternative are three-dimensional (3D) models. The aim of our study was to develop physiologically more relevant 3D cell model (spheroids) from the human hepatocellular carcinoma (HepG2) cell line for genotoxicity testing. The spheroids were prepared by the forced floating method, which had been optimized for the production of a large number of uniform spheroids. The sensitivity of the spheroids to detect genotoxicity was determined by the comet assay after the exposure of spheroids to non-cytotoxic concentrations of model indirect acting genotoxic compounds, namely polycyclic aromatic hydrocarbon (B(a)P), mycotoxin (AFB1), two heterocyclic aromatic amines (PhIP and IQ) and a direct acting etoposide (ET). All five tested compounds concentration dependently induced DNA damage. Higher sensitivity of 3D cell model compared to 2D monolayer culture was noticed particularly for detection of the genotoxicity of the heterocyclic aromatic amines and BaP. Deregulation of mRNA expression (qPCR) by genotoxic compounds revealed that HepG2 cells in 3D express important genes encoding phase I and II metabolic enzymes, as well as DNA damage responsive genes in an inducible form. The newly developed HepG2 3D model shows improved sensitivity for detecting genotoxic compounds compared to 2D cultures and can provide a suitable experimental model for genotoxicity assessment.
遗传毒性评价在化学品和消费品的危害识别和风险评估中起着重要作用。对于遗传毒性评估，通常使用体外肝细胞，因为它们保留了一定水平的外源性代谢活性。然而，目前的方案是为 2D 单层模型设计的，由于缺乏许多生物学功能，这些模型存在一些局限性，导致许多肝脏特性丧失。在这方面，三维（3D）模型是一种很有吸引力的替代方法。我们的研究目的是开发更具生理相关性的三维细胞模型（球体），用于遗传毒性测试。球体是通过强制漂浮法制备的，该方法已经过优化，可用于大量生产均匀的球体。通过彗星试验测定球体对非细胞毒性浓度模型间接作用遗传毒性化合物（多环芳烃（B(a)P）、霉菌毒素（AFB1）、两种杂环芳香胺（PhIP 和 IQ）和直接作用依托泊苷（ET））的遗传毒性敏感性。所有五种测试化合物均表现出浓度依赖性的 DNA 损伤。与 2D 单层培养相比，3D 细胞模型的敏感性更高，特别是对杂环芳香胺和 BaP 的遗传毒性检测。遗传毒性化合物对 mRNA 表达（qPCR）的调控表明，3D 培养中的 HepG2 细胞以诱导形式表达编码 I 相和 II 相代谢酶以及 DNA 损伤反应基因的重要基因。与 2D 培养相比，新开发的 HepG2 3D 模型对遗传毒性化合物的检测敏感性更高，可为遗传毒性评估提供合适的实验模型。
