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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

Gill Conway Orcid Logo, Bérénice Chavanel, François Virard, Ume-kulsoom Shah Orcid Logo, Michael Burgum, Stephen Evans Orcid Logo, Michael Korenjak, Laura Thomas Orcid Logo, Gareth Jenkins Orcid Logo, Jiri Zavadil, shareen Doak

Mutagenesis, Start page: geaf015

Swansea University Authors: Gill Conway Orcid Logo, Ume-kulsoom Shah Orcid Logo, Michael Burgum, Stephen Evans Orcid Logo, Laura Thomas Orcid Logo, Gareth Jenkins Orcid Logo, shareen Doak

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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...

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Published in: Mutagenesis
ISSN: 0267-8357 1464-3804
Published: Oxford University Press (OUP) 2025
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URI: https://cronfa.swan.ac.uk/Record/cronfa70048
first_indexed 2025-07-29T08:50:20Z
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spelling 2026-02-02T14:02:15.0937177 v2 70048 2025-07-29 Harnessing the power of an advanced <i>in vitro</i> 3D liver model and error-corrected duplex sequencing for the detection of mutational signatures e33e0ee5a076ad91fe6615117caa1800 0000-0002-5991-0960 Gill Conway Gill Conway true false bcef4e069220a4b85f8a2c0cc3059487 0000-0002-0594-580X Ume-kulsoom Shah Ume-kulsoom Shah true false d3fe156a5ee169e586b8bad6ae4cb1d8 Michael Burgum Michael Burgum true false cfca981bdfb8492873a48cc1629def9a 0000-0002-5352-9800 Stephen Evans Stephen Evans true false 6f80a1638d852bd88d37afe3aeb2fb62 0000-0002-8621-5285 Laura Thomas Laura Thomas true false a44095d26187304e903da7ca778697b6 0000-0002-5437-8389 Gareth Jenkins Gareth Jenkins true false 8f70286908f67238a527a98cbf66d387 shareen Doak shareen Doak true false 2025-07-29 MEDS 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. Journal Article Mutagenesis 0 geaf015 Oxford University Press (OUP) 0267-8357 1464-3804 Next-generation sequencing, duplex sequencing, in vitro, liver, 3D models, genotoxicity 6 8 2025 2025-08-06 10.1093/mutage/geaf015 COLLEGE NANME Medical School COLLEGE CODE MEDS Swansea University SU Library paid the OA fee (TA Institutional Deal) 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). 2026-02-02T14:02:15.0937177 2025-07-29T09:44:26.4850091 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Biomedical Science Gill Conway 0000-0002-5991-0960 1 Bérénice Chavanel 2 François Virard 3 Ume-kulsoom Shah 0000-0002-0594-580X 4 Michael Burgum 5 Stephen Evans 0000-0002-5352-9800 6 Michael Korenjak 7 Laura Thomas 0000-0002-8621-5285 8 Gareth Jenkins 0000-0002-5437-8389 9 Jiri Zavadil 10 shareen Doak 11 70048__35275__77fa9797425948c993e25c47ab726f5f.pdf 70048.VOR.pdf 2025-10-07T15:10:20.4978392 Output 1203231 application/pdf Version of Record true © The Author(s) 2025. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (CC BY). true eng https://creativecommons.org/licenses/
title Harnessing the power of an advanced <i>in vitro</i> 3D liver model and error-corrected duplex sequencing for the detection of mutational signatures
spellingShingle Harnessing the power of an advanced <i>in vitro</i> 3D liver model and error-corrected duplex sequencing for the detection of mutational signatures
Gill Conway
Ume-kulsoom Shah
Michael Burgum
Stephen Evans
Laura Thomas
Gareth Jenkins
shareen Doak
title_short Harnessing the power of an advanced <i>in vitro</i> 3D liver model and error-corrected duplex sequencing for the detection of mutational signatures
title_full Harnessing the power of an advanced <i>in vitro</i> 3D liver model and error-corrected duplex sequencing for the detection of mutational signatures
title_fullStr Harnessing the power of an advanced <i>in vitro</i> 3D liver model and error-corrected duplex sequencing for the detection of mutational signatures
title_full_unstemmed Harnessing the power of an advanced <i>in vitro</i> 3D liver model and error-corrected duplex sequencing for the detection of mutational signatures
title_sort Harnessing the power of an advanced <i>in vitro</i> 3D liver model and error-corrected duplex sequencing for the detection of mutational signatures
author_id_str_mv e33e0ee5a076ad91fe6615117caa1800
bcef4e069220a4b85f8a2c0cc3059487
d3fe156a5ee169e586b8bad6ae4cb1d8
cfca981bdfb8492873a48cc1629def9a
6f80a1638d852bd88d37afe3aeb2fb62
a44095d26187304e903da7ca778697b6
8f70286908f67238a527a98cbf66d387
author_id_fullname_str_mv e33e0ee5a076ad91fe6615117caa1800_***_Gill Conway
bcef4e069220a4b85f8a2c0cc3059487_***_Ume-kulsoom Shah
d3fe156a5ee169e586b8bad6ae4cb1d8_***_Michael Burgum
cfca981bdfb8492873a48cc1629def9a_***_Stephen Evans
6f80a1638d852bd88d37afe3aeb2fb62_***_Laura Thomas
a44095d26187304e903da7ca778697b6_***_Gareth Jenkins
8f70286908f67238a527a98cbf66d387_***_shareen Doak
author Gill Conway
Ume-kulsoom Shah
Michael Burgum
Stephen Evans
Laura Thomas
Gareth Jenkins
shareen Doak
author2 Gill Conway
Bérénice Chavanel
François Virard
Ume-kulsoom Shah
Michael Burgum
Stephen Evans
Michael Korenjak
Laura Thomas
Gareth Jenkins
Jiri Zavadil
shareen Doak
format Journal article
container_title Mutagenesis
container_volume 0
container_start_page geaf015
publishDate 2025
institution Swansea University
issn 0267-8357
1464-3804
doi_str_mv 10.1093/mutage/geaf015
publisher Oxford University Press (OUP)
college_str Faculty of Medicine, Health and Life Sciences
hierarchytype
hierarchy_top_id facultyofmedicinehealthandlifesciences
hierarchy_top_title Faculty of Medicine, Health and Life Sciences
hierarchy_parent_id facultyofmedicinehealthandlifesciences
hierarchy_parent_title Faculty of Medicine, Health and Life Sciences
department_str Swansea University Medical School - Biomedical Science{{{_:::_}}}Faculty of Medicine, Health and Life Sciences{{{_:::_}}}Swansea University Medical School - Biomedical Science
document_store_str 1
active_str 0
description 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.
published_date 2025-08-06T05:31:49Z
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