Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, Slovenia; Jozef Stefan International Postgraduate School, Ljubljana, Slovenia.
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
Sci Total Environ. 2021 Feb 10;755(Pt 2):143255. doi: 10.1016/j.scitotenv.2020.143255. Epub 2020 Nov 3.
The major weakness of the current in vitro genotoxicity test systems is the inability of the indicator cells to express metabolic enzymes needed for the activation and detoxification of genotoxic compounds, which consequently can lead to misleading results. Thus, there is a significant emphasis on developing hepatic cell models, including advanced in vitro three-dimensional (3D) cell-based systems, which better imitate in vivo cell behaviour and offer more accurate and predictive data for human exposures. In this study, we developed an approach for genotoxicity testing with 21-day old spheroids formed from human hepatocellular carcinoma cells (HepG2/C3A) using the dynamic clinostat bioreactor system (CelVivo BAM/bioreactor) under controlled conditions. The spheroids were exposed to indirect-acting genotoxic compounds, polycyclic aromatic hydrocarbon [PAH; benzo(a) pyrene B(a)P], and heterocyclic aromatic amine [PhIP]) at non-cytotoxic concentrations for 24 and 96 h. The results showed that both environmental pollutants B(a)P and PhIP significantly increased the level of DNA strand breaks assessed by the comet assay. Further, the mRNA level of selected genes encoding metabolic enzymes from phase I and II, and DNA damage responsive genes was determined (qPCR). The 21-day old spheroids showed higher basal expression of genes encoding metabolic enzymes compared to monolayer culture. In spheroids, B(a)P or PhIP induced compound-specific up-regulation of genes implicated in their metabolism, and deregulation of genes implicated in DNA damage and immediate-early response. The study demonstrated that this model utilizing HepG2/C3A spheroids grown under dynamic clinostat conditions represents a very sensitive and promising in vitro model for genotoxicity and environmental studies and can thus significantly contribute to a more reliable assessment of genotoxic activities of pure chemicals, and complex environmental samples even at very low for environmental exposure relevant concentrations.
当前体外遗传毒性测试系统的主要弱点是指示细胞无法表达代谢酶，这些酶对于遗传毒性化合物的激活和解毒是必需的，这可能导致结果产生误导。因此，人们非常重视开发肝细胞模型，包括先进的体外三维（3D）基于细胞的系统，这些系统更好地模拟体内细胞行为，并为人类暴露提供更准确和可预测的数据。在这项研究中，我们使用动态旋转生物反应器系统（CelVivo BAM/生物反应器）开发了一种方法，用于在受控条件下对人肝癌细胞（HepG2/C3A）形成的 21 天大的球体进行遗传毒性测试。将球体暴露于间接作用的遗传毒性化合物多环芳烃[PAH；苯并（a）芘 B(a)P]和杂环芳香胺[PhIP]下，在非细胞毒性浓度下暴露 24 和 96 小时。结果表明，两种环境污染物 B(a)P 和 PhIP 均显著增加了彗星试验评估的 DNA 链断裂水平。此外，还测定了编码 I 相和 II 相代谢酶和 DNA 损伤反应基因的选定基因的 mRNA 水平（qPCR）。与单层培养相比，21 天大的球体显示出更高的代谢酶编码基因的基础表达。在球体中，B(a)P 或 PhIP 诱导与它们的代谢有关的基因特异性上调，以及与 DNA 损伤和即刻早期反应有关的基因失调。该研究表明，这种利用在动态旋转条件下生长的 HepG2/C3A 球体的模型代表了一种非常敏感和有前途的体外遗传毒性和环境研究模型，因此可以为更可靠地评估纯化学物质和复杂环境样品的遗传毒性活性做出重大贡献，即使在与环境暴露相关的非常低浓度下也是如此。
