Evaluation of an in vitro three-dimensional HepaRG spheroid model for genotoxicity testing using the high-throughput CometChip platform

Seo, Jeong-Woo; He, Xia; Muskhelishvili, Levan; Malhi, P.K.; Mei, Nan; Manjanatha, Mugimane G.; Bryant, Matthew; Zhou, Tong; Robison, Timothy W.; Guo, Xueyi

doi:10.14573/altex.2201121

Cited by 10 publications

(9 citation statements)

References 67 publications

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“…In addition, there currently is renewed interest in the genotoxicity of NDMA due to its recent identification as an impurity in several popular human drugs used for the treatment of common diseases such as hypertension, heartburn, and diabetes (FDA 2021 ; Seo et al 2023b ). Our previous studies confirmed that NDMA induced significant DNA strand breakage and MN formation following a 24-h treatment of both 2D and 3D HepaRG cell models without the addition of liver S9 fractions (Seo et al 2022 , 2023a , 2023b ). The present study assessed NDMA-induced mutations following a 72-h treatment in both 2D and 3D HepaRG models with further optimized DS and HiFi Sequencing techniques.…”

Section: Introductionsupporting

confidence: 83%

“…Both 2D and 3D HepaRG models were generated as previously described (Seo et al 2022 ). Briefly, human hepatoma HepaRG cells (Cat# HPR101, Biopredic International, Saint Grégoire, France) were cultured for a total of 28 days according to the supplier’s protocol, i.e., first in growth medium for 14 days followed by 14 days of differentiation in differentiation medium, both incubations conducted at 37 °C in a humidified atmosphere of 5% CO 2 in air.…”

Section: Methodsmentioning

confidence: 99%

“…The final concentrations of NDMA were 1, 5, and 10 mM for 2D cultures and 0.1, 0.5, 1, and 2 mM for 3D spheroids. The concentrations were informed by previous studies (Seo et al 2022 , 2023a ) and were chosen to allow observation of a wide range of biological responses.…”

Section: Methodsmentioning

confidence: 99%

“…Currently, the HepaRG 2D/3D systems have been used with the comet and MN assays to assess the genetic toxicity of a variety of compounds, including carcinogens, pyrrolizidine alkaloids, nanomaterials, and food contact recycled paperboard extracts (Allemang et al 2018 ; Barranger and Le Hegarat 2022 ; Buick et al 2020 ; Jalili et al 2022 ; Mandon et al 2019 ; Souton et al 2018 ). HepaRG cells are superior to HepG2 cells for detecting genotoxicants or carcinogens in the comet and MN assays, with three-dimensional (3D) HepaRG spheroids being more sensitive in detecting genotoxicants that require metabolic activation than cells cultured in the 2D format, likely due to the higher levels of cytochrome P450 (CYP) gene expression and enzyme activities in the spheroids compared to its 2D counterpart (Guo et al 2020b ; Seo et al 2022 , 2023a ). Building upon our success performing the MN assay in both 2D and 3D HepaRG cultures (Seo et al 2023a ), we extended our work to measure mutation induction in 2D and 3D HepaRG cell models after chemical exposure.…”

Section: Introductionmentioning

confidence: 99%

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Evaluating the mutagenicity of N-nitrosodimethylamine in 2D and 3D HepaRG cell cultures using error-corrected next generation sequencing

Seo,

Le,

Revollo

et al. 2024

Arch Toxicol

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Human liver-derived metabolically competent HepaRG cells have been successfully employed in both two-dimensional (2D) and 3D spheroid formats for performing the comet assay and micronucleus (MN) assay. In the present study, we have investigated expanding the genotoxicity endpoints evaluated in HepaRG cells by detecting mutagenesis using two error-corrected next generation sequencing (ecNGS) technologies, Duplex Sequencing (DS) and High-Fidelity (HiFi) Sequencing. Both HepaRG 2D cells and 3D spheroids were exposed for 72 h to N-nitrosodimethylamine (NDMA), followed by an additional incubation for the fixation of induced mutations. NDMA-induced DNA damage, chromosomal damage, and mutagenesis were determined using the comet assay, MN assay, and ecNGS, respectively. The 72-h treatment with NDMA resulted in concentration-dependent increases in cytotoxicity, DNA damage, MN formation, and mutation frequency in both 2D and 3D cultures, with greater responses observed in the 3D spheroids compared to 2D cells. The mutational spectrum analysis showed that NDMA induced predominantly A:T → G:C transitions, along with a lower frequency of G:C → A:T transitions, and exhibited a different trinucleotide signature relative to the negative control. These results demonstrate that the HepaRG 2D cells and 3D spheroid models can be used for mutagenesis assessment using both DS and HiFi Sequencing, with the caveat that severe cytotoxic concentrations should be avoided when conducting DS. With further validation, the HepaRG 2D/3D system may become a powerful human-based metabolically competent platform for genotoxicity testing.

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Section: Introductionsupporting

confidence: 83%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Evaluating the mutagenicity of N-nitrosodimethylamine in 2D and 3D HepaRG cell cultures using error-corrected next generation sequencing

Seo,

Le,

Revollo

et al. 2024

Arch Toxicol

Self Cite

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“…It is possible that enzyme expression was not sufficiently present in the cells that yielded a weak response. In a recent study by Seo et al (2022), it was revealed that three‐dimensional spheroids from HepaRG cells had CYP2B6 activity that was 6.7‐ to 25.0‐fold higher than two‐dimensional cell cultures, indicating that three‐dimensional models might be the preferable to studying the genotoxicity of genotoxic chemicals requiring metabolic activation. Furthermore, the cyclophosphamide results highlight the challenge associated with using a generic model in IVIVE application.…”

Section: Discussionmentioning

confidence: 99%

Quantitative in vitro to in vivo extrapolation of genotoxicity data provides protective estimates of in vivo dose

Beal

Audebert

Barton-Maclaren

et al. 2022

Environ and Mol Mutagen

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Genotoxicity assessment is a critical component in the development and evaluation of chemicals. Traditional genotoxicity assays (i.e., mutagenicity, clastogenicity, and aneugenicity) have been limited to dichotomous hazard classification, while other toxicity endpoints are assessed through quantitative determination of points-of-departures (PODs) for setting exposure limits. The more recent higher-throughput in vitro genotoxicity assays, many of which also provide mechanistic information, offer a powerful approach for determining defined PODs for potency ranking and risk assessment.

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Using the HepaCometChip Assay for Broad‐Spectrum DNA Damage Analysis

et al. 2022

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Exposure to DNA damaging agents can lead to mutations that cause cancer. The liver is particularly vulnerable because it contains high levels of Cytochrome P450 enzymes that can convert xenobiotics into DNA reactive metabolites that form potentially carcinogenic bulky DNA adducts. As such, current requirements for preclinical testing include in vivo testing for DNA damage in the liver, which often requires many animals. Given that efforts are underway in many countries to reduce or eliminate the use of animals in research, there is a critical need for fast and robust in vitro tests to discern whether xenobiotics or potential pharmaceutical agents can damage the hepatocyte genome. One possible approach is to leverage the alkaline comet assay, which is used to assess genotoxicity based on the ability of damaged DNA to become free to migrate toward the anode during electrophoresis. The comet assay, however, has several limitations. The assay is (i) slow and (ii) vulnerable to experimental noise, (iii) it is difficult to detect bulky DNA adducts since they do not directly affect DNA migration, and (iv) cell types typically used do not have robust metabolic capacity. To address some of these concerns, we have developed the “HepaCometChip” (a.k.a. the HepaRG CometChip), wherein metabolically competent cells are incorporated into a higher throughput CometChip platform. Repair trapping is used to increase sensitivity for bulky lesions: undetectable bulky lesions are converted into repair intermediates (specifically, single‐strand breaks) that can be detected with the assay. Here, we describe a protocol for performing the HepaCometChip assay that includes handling and dosing of HepaRG cells and performing the CometChip assay. With its higher throughput, ability to capture metabolic activation, and sensitivity to bulky lesions, the HepaCometChip offers a potential alternative to the use of animals for genotoxicity testing. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: HepaRG cell culturing and dosing Basic Protocol 2: CometChip assay

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Evaluation of an in vitro three-dimensional HepaRG spheroid model for genotoxicity testing using the high-throughput CometChip platform

Cited by 10 publications

References 67 publications

Evaluating the mutagenicity of N-nitrosodimethylamine in 2D and 3D HepaRG cell cultures using error-corrected next generation sequencing

Evaluating the mutagenicity of N-nitrosodimethylamine in 2D and 3D HepaRG cell cultures using error-corrected next generation sequencing

Quantitative in vitro to in vivo extrapolation of genotoxicity data provides protective estimates of in vivo dose

Using the HepaCometChip Assay for Broad‐Spectrum DNA Damage Analysis

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