Three-dimensional HepaRG spheroids as a liver model to study human genotoxicity in vitro with the single cell gel electrophoresis assay

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.

Hepato(Geno)Toxicity Assessment of Nanoparticles in a HepG2 Liver Spheroid Model

“…Several studies have shown differences in sensitivity to induced toxicity in 3D and 2D cultures [25,[27][28][29]73,78]. We have previously found similar cytotoxicity in 2D and 3D HepG2 cultures, but higher sensitivity in the 3D culture for induced DNA damage by MMS [27].…”

“…We have previously found similar cytotoxicity in 2D and 3D HepG2 cultures, but higher sensitivity in the 3D culture for induced DNA damage by MMS [27]. Increased sensitivity in genotoxicity was also seen after exposure to 11 chemicals in a HepaRG spheroid model [29]. In agreement with this, benzo(a)pyrene and 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine, which both require metabolic activation for induction of genotoxicity, induced a higher micronucleus frequency in HepG2 spheroids compared to monolayer cultures [28].…”

“…Interestingly, we found differences in sensitivity between the 2D and 3D models [27]. The comet assay has also been performed with Ag-, ZnO-and TiO 2 -NPs and carbon nanotubes on a commercialized spheroid model with primary liver cells [6], and it has been shown to work well with different 3D models [6,27,29,30]. However, the comet assay has-to our knowledge-not yet been applied in HepG2 spheroids for genotoxicity testing of NPs.…”

Genotoxicity of Nanomaterials: Advanced In Vitro Models and High Throughput Methods for Human Hazard Assessment—A Review

“…For genotoxicity assessment, robust protocols for 3D models have been established for skin, airways and liver tissue equivalents [ 10 , 197 ]. Many of the 3D cell culture systems applied in genotoxicity testing (of NMs) have been spheroids, such as liver spheroids constructed from primary hepatocytes, HepG2 hepatocellular carcinoma cells or the HepaRG cell line applied to the comet assay [ 14 , 31 , 34 , 198 , 199 ] and micronucleus assay [ 200 ]. Human 3D airway models usually consist of a functional and differentiated respiratory epithelium with cilia and mucus and are cultured on the physiological relevant air-liquid-interface (ALI) [ 19 ].…”

Utility of Three-Dimensional Cultures of Primary Human Hepatocytes (Spheroids) as Pharmacokinetic Models

“…Further, cells in spheroid cultures secrete an extracellular matrix in which they reside, and they can interact with cells from their original microenvironment [ 31 ]. Spheroids can also be formed from human cancer-derived hepatocytes, such as HepaRG cells [ 32 , 33 , 34 ], HepG2 cells [ 35 , 36 , 37 ], and Huh-7 cells [ 38 ]. Cultured human cancer-derived hepatocytes have long been used for human-specific toxicity assessments, but they generally have lower expression levels of drug-metabolizing enzymes than PHHs [ 39 , 40 ].…”