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Updated: Nov 30, 2025
Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure
Published on: June 5, 2020
Hepatocellular carcinoma (HepG2/C3A) cell-based 3D model for genotoxicity testing of chemicals
Martina Štampar1, Helle Sedighi Frandsen2, Adelina Rogowska-Wrzesinska2
1Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, Slovenia; Jozef Stefan International Postgraduate School, Ljubljana, Slovenia.
Abstract:
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.
Insights
This study developed a 3D liver cell model using HepG2/C3A spheroids for genotoxicity testing. The model accurately detects DNA damage from environmental pollutants like benzo(a)pyrene and PhIP.
Area of Science:
- Toxicology
- Cell Biology
- Biotechnology
Background:
- Current in vitro genotoxicity tests lack metabolic enzyme expression, leading to inaccurate results.
- Advanced in vitro hepatic cell models, particularly 3D systems, are crucial for mimicking in vivo behavior and improving human exposure predictions.
- Developing more predictive genotoxicity testing models is essential for assessing environmental chemical safety.
Purpose of the Study:
- To develop and validate a novel in vitro genotoxicity testing approach using human hepatocellular carcinoma (HepG2/C3A) spheroids.
- To evaluate the sensitivity of this 3D cell model in detecting DNA damage induced by indirect-acting genotoxic compounds.
- To assess the expression of metabolic and DNA damage response genes within the spheroid model.
Main Methods:
- Formation of 21-day old HepG2/C3A spheroids using a dynamic clinostat bioreactor system (CelVivo BAM/bioreactor).
- Exposure of spheroids to benzo(a)pyrene [B(a)P] and heterocyclic aromatic amine [PhIP] at non-cytotoxic concentrations for 24 and 96 hours.
- Assessment of DNA strand breaks using the comet assay and gene expression analysis via quantitative polymerase chain reaction (qPCR).
Main Results:
- Exposure to B(a)P and PhIP significantly increased DNA strand breaks in HepG2/C3A spheroids.
- Spheroids exhibited higher basal expression of metabolic enzyme genes compared to monolayer cultures.
- Genotoxic compounds induced compound-specific gene expression related to metabolism, DNA damage, and cellular response in the spheroid model.
Conclusions:
- The HepG2/C3A spheroid model grown under dynamic clinostat conditions is a sensitive and promising in vitro system for genotoxicity and environmental studies.
- This 3D model provides more accurate and predictive data for assessing the genotoxic potential of chemicals and environmental samples.
- The model's ability to detect effects at environmentally relevant low concentrations enhances its utility for risk assessment.
