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Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure
Published on: June 5, 2020
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High-throughput micronucleus assay using three-dimensional HepaRG spheroids for in vitro genotoxicity testing.
Ji-Eun Seo1, Xilin Li2, Yuan Le2
1Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR, 72079, USA. Jieun.Seo@fda.hhs.gov.
Archives of Toxicology
|February 27, 2023
Summary
Three-dimensional (3D) HepaRG spheroids enhance the high-throughput (HT) flow-cytometry micronucleus (MN) assay for genotoxicity testing. This 3D model shows improved sensitivity in detecting genotoxicants requiring metabolic activation compared to 2D cultures.
Area of Science:
- Toxicology and Pharmacology
- Cell Biology and Genetics
- Biotechnology and Bioassays
Background:
- The in vitro micronucleus (MN) assay is crucial for assessing genotoxicity.
- Previous work established a high-throughput (HT) flow-cytometry MN assay using HepaRG cells.
- Three-dimensional (3D) HepaRG spheroids exhibit enhanced metabolic capacity over 2D cultures.
Purpose of the Study:
- To compare the performance of the HT flow-cytometry MN assay in 3D HepaRG spheroids versus 2D HepaRG cells.
- To evaluate the sensitivity of 3D spheroids in detecting genotoxicants, particularly those requiring metabolic activation.
- To assess the potential of 3D HepaRG spheroids as part of New Approach Methodologies (NAMs) for genotoxicity testing.
Main Methods:
- Tested 34 compounds (19 genotoxicants/carcinogens, 15 differential responders) using the HT flow-cytometry MN assay.
- Compared 3D HepaRG spheroids and 2D HepaRG cells after 24-hour compound exposure.
- Utilized a 3- or 6-day incubation with human epidermal growth factor to stimulate cell division post-exposure.
Main Results:
- HepaRG spheroids demonstrated higher sensitivity in detecting indirect-acting genotoxicants compared to 2D cultures.
- Compounds like 7,12-dimethylbenzanthracene and N-nitrosodimethylamine induced higher MN formation in 3D spheroids.
- Benchmark dose values for MN induction were significantly lower in 3D spheroids, indicating increased sensitivity.
Conclusions:
- 3D HepaRG spheroids are adaptable to the HT flow-cytometry MN assay for improved genotoxicity testing.
- Integrating MN and comet assays enhances detection of genotoxicants requiring metabolic activation.
- HepaRG spheroids show promise for advancing NAMs in genotoxicity assessment.
