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Harnessing the power of an advanced <i>in vitro</i> 3D liver model and error-corrected duplex sequencing for the detection of mutational signatures
,
Bérénice Chavanel,
François Virard,
Ume-kulsoom Shah ,
Michael Burgum,
Stephen Evans ,
Michael Korenjak,
Laura Thomas ,
Gareth Jenkins ,
Jiri Zavadil,
shareen Doak
Mutagenesis, Start page: geaf015
Swansea University Authors:
Gill Conway , Ume-kulsoom Shah , Michael Burgum, Stephen Evans , Laura Thomas , Gareth Jenkins , shareen Doak
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© The Author(s) 2025. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (CC BY).
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DOI (Published version): 10.1093/mutage/geaf015
Abstract
Genotoxicity testing plays a crucial role in evaluating the hazards posed by various chemicals. Traditional methods, such as the Ames test, mammalian cell mutation assays and the transgenic rodent assay have certain limitations including laborious procedures and/or reliance on animal models. The aim...
| Published in: | Mutagenesis |
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| ISSN: | 0267-8357 1464-3804 |
| Published: |
Oxford University Press (OUP)
2025
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| Online Access: |
Check full text
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| URI: | https://cronfa.swan.ac.uk/Record/cronfa70048 |
| Abstract: |
Genotoxicity testing plays a crucial role in evaluating the hazards posed by various chemicals. Traditional methods, such as the Ames test, mammalian cell mutation assays and the transgenic rodent assay have certain limitations including laborious procedures and/or reliance on animal models. The aim of this study was to determine the potential of using error-corrected next-generation sequencing (ecNGS), specifically duplex sequencing (DS), as an alternative method for the detection of point mutations in conjunction with advanced in vitro models. This study establishes an easy to use, adaptable in vitro 3D HepG2 model, that shows good viability, and liver functionality over 14 days. 3D HepG2 spheroids were exposed to aristolochic acid in a repeated dose regime over 4 days. This was shown to significantly induce micronucleus formation, indicative of fixed DNA damage, in a dose dependent fashion. DS coupled with mutational signature analyses revealed a predominant treatment-specific T:A > A:T-enriched mutational signature explained by COSMIC signature SBS22 derived from human cancers associated with aristolochic acid exposure. De novo extraction provided a stable signature, of which more than 40% were unambiguously explained by SBS22 These results demonstrate that the presented 3D HepG2 spheroid model is appropriate for assessing chemically induced fixed DNA damage. Additionally, we provide evidence that DS applied to the studied in vitro 3D model has the capacity to reveal specific mutational signatures of mutagenic exposures. The modern integrative approach will improve the understanding of mechanisms of carcinogenesis related to chemical exposures by providing a cost-effective and efficient means to assess genotoxicity and mutagenicity. With the inclusion of mutational signature analyses, this approach would see a reduction in reliance on animal models and enhancement of hazard assessment accuracy. |
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| Keywords: |
Next-generation sequencing, duplex sequencing, in vitro, liver, 3D models, genotoxicity |
| College: |
Faculty of Medicine, Health and Life Sciences |
| Funders: |
This work was supported in part from the following sources: Grant(s) n° 47834XM and (815429795 code of UK part), Programme Hubert Curien Alliance, from British Council, UK, and the Ministry for Europe and Foreign Affairs and Ministry of Higher Education Research and Innovation, France. European Union HE project: Twinning for excellence to strategically advance research in carcinogenesis and cancer (CutCancer; 101079113). United Kingdom Environmental Mutagen Society (UKEMS) Small Grants Scheme for Feasibility or Pilot Studies (awarded 2022). |
| Start Page: |
geaf015 |