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Abstract
Many efforts have been made in the last 30 years to develop more relevant in vitro models to study genotoxic responses of drugs and environmental contaminants. While 2D HepaRG cells are one of the most promising models for liver toxicology, a switch to 3D cultures that integrate both in vivo architecture and cell-cell interactions has occurred to achieve even more predictive models. Preliminary studies have indicated that 3D HepaRG cells are suitable for liver toxicity screening. Our study aimed to evaluate the response of HepaRG spheroids exposed to various genotoxic compounds using the single cell gel electrophoresis assay. HepaRG spheroids were used at 10 days after seeding and exposed for 24 and 48 hours to certain selected chemical compounds (methylmethansulfonate (MMS), etoposide, benzo[a]pyrene (B[a]P), cyclophosphamide (CPA), 7,12-dimethylbenz[a]anthracene (DMBA), 2-acetylaminofluorene (2-AAF), 4-nitroquinoline (4-NQO), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3-methylimidazo[4,5-f]quinolone (IQ), acrylamide, and 2-4-diaminotoluene (2,4-DAT)). After treatment, the comet assay was performed on single cell suspensions and cytotoxicity was determined by the ATP assay. Comet formation was observed for all compounds except IQ, etoposide and 2,4-DAT. Treatment of spheroids with rifampicin increased CYP3A4 activity, demonstrating the metabolic capacity of HepaRG spheroids. These data on genotoxicity in 3D HepaRG spheroids are promising, but further experiments are required to prove that this model can improve the predictivity of in vitro models to detect human carcinogens.
Highlights
Information on genetic toxicity is an essential part of the safety assessment for any type of substance
Considering that (i) 2D HepaRG cells have failed to detect some progenotoxic compounds, (ii) 3D HepaRG spheroids have shown metabolic activities and functional characteristics that are closer to those of the human liver, and (iii) results have shown that the 3D HepG2 model is more sensitive than the 2D model to detect human mutagens, we proposed to evaluate the response of HepaRG spheroids exposed to various genotoxic compounds using the single cell gel electrophoresis assay
HepaRG cells seeded at 2,000 cells/per well in 96-well ultra-low attachment (ULA) plates aggregated from Day 1 to Day 3 and formed a compact spheroid structure at Day 7 (Fig. 2A)
Summary
Information on genetic toxicity is an essential part of the safety assessment for any type of substance. Spheroids maintained a stable phenotype, exhibiting several hallmarks of polarized hepatocyte architecture and functions, including metabolizing enzyme activities, long-term stable albumin secretion, and transporter localization[20,25].Authors showed that 3D HepaRG cells express the three major P450 enzymes (CYP1A2, CYP2B6 and CYP3A4) and active hepatic nuclear receptors (AhR, CAR and PXR)[24]. As this model probably presents the ability to bioactivate xenobiotics due to high levels of CYP enzyme activities maintained for up to 28 days, genotoxicity assays on HepaRG spheroids are worth developing
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