Development of reconstructed intestinal micronucleus cytome (RICyt) assay in 3D human gut model for genotoxicity assessment of orally ingested substances

The micronucleus (MN) assay is widely used as part of a battery of tests applied to evaluate the genotoxic potential of chemicals, including new food additives and novel food ingredients. Micronucleus assays typically utilise homogenous in vitro cell lines which poorly recapitulate the physiology, b...

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Main Authors: Lim, Hui Kheng, Hughes, Christopher Owen, Lim, Michelle Jing Sin, Li, Jia'En Jasmine, Rakshit, Moumita, Yeo, Calvin, Chng, Kern Rei, Li, Angela, Chan, Joanne Sheot Harn, Ng, Kee Woei, Leavesley, David Ian, Smith, Benjamin Paul Chapman
Other Authors: School of Materials Science and Engineering
Format: Article
Language:English
Published: 2022
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Online Access:https://hdl.handle.net/10356/161945
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Institution: Nanyang Technological University
Language: English
id sg-ntu-dr.10356-161945
record_format dspace
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic Engineering::Materials
Micronuclei
DNA Damage
spellingShingle Engineering::Materials
Micronuclei
DNA Damage
Lim, Hui Kheng
Hughes, Christopher Owen
Lim, Michelle Jing Sin
Li, Jia'En Jasmine
Rakshit, Moumita
Yeo, Calvin
Chng, Kern Rei
Li, Angela
Chan, Joanne Sheot Harn
Ng, Kee Woei
Leavesley, David Ian
Smith, Benjamin Paul Chapman
Development of reconstructed intestinal micronucleus cytome (RICyt) assay in 3D human gut model for genotoxicity assessment of orally ingested substances
description The micronucleus (MN) assay is widely used as part of a battery of tests applied to evaluate the genotoxic potential of chemicals, including new food additives and novel food ingredients. Micronucleus assays typically utilise homogenous in vitro cell lines which poorly recapitulate the physiology, biochemistry and genomic events in the gut, the site of first contact for ingested materials. Here we have adapted and validated the MN endpoint assay protocol for use with complex 3D reconstructed intestinal microtissues; we have named this new protocol the reconstructed intestine micronucleus cytome (RICyt) assay. Our data suggest the commercial 3D microtissues replicate the physiological, biochemical and genomic responses of native human small intestine to exogenous compounds. Tissues were shown to maintain log-phase proliferation throughout the period of exposure and expressed low background MN. Analysis using the RICyt assay protocol revealed the presence of diverse cell types and nuclear anomalies (cytome) in addition to MN, indicating evidence for comprehensive DNA damage and mode(s) of cell death reported by the assay. The assay correctly identified and discriminated direct-acting clastogen, aneugen and clastogen requiring exogenous metabolic activation, and a non-genotoxic chemical. We are confident that the genotoxic response in the 3D microtissues more closely resembles the native tissues due to the inherent tissue architecture, surface area, barrier effects and tissue matrix interactions. This proof-of-concept study highlights the RICyt MN cytome assay in 3D reconstructed intestinal microtissues is a promising tool for applications in predictive toxicology.
author2 School of Materials Science and Engineering
author_facet School of Materials Science and Engineering
Lim, Hui Kheng
Hughes, Christopher Owen
Lim, Michelle Jing Sin
Li, Jia'En Jasmine
Rakshit, Moumita
Yeo, Calvin
Chng, Kern Rei
Li, Angela
Chan, Joanne Sheot Harn
Ng, Kee Woei
Leavesley, David Ian
Smith, Benjamin Paul Chapman
format Article
author Lim, Hui Kheng
Hughes, Christopher Owen
Lim, Michelle Jing Sin
Li, Jia'En Jasmine
Rakshit, Moumita
Yeo, Calvin
Chng, Kern Rei
Li, Angela
Chan, Joanne Sheot Harn
Ng, Kee Woei
Leavesley, David Ian
Smith, Benjamin Paul Chapman
author_sort Lim, Hui Kheng
title Development of reconstructed intestinal micronucleus cytome (RICyt) assay in 3D human gut model for genotoxicity assessment of orally ingested substances
title_short Development of reconstructed intestinal micronucleus cytome (RICyt) assay in 3D human gut model for genotoxicity assessment of orally ingested substances
title_full Development of reconstructed intestinal micronucleus cytome (RICyt) assay in 3D human gut model for genotoxicity assessment of orally ingested substances
title_fullStr Development of reconstructed intestinal micronucleus cytome (RICyt) assay in 3D human gut model for genotoxicity assessment of orally ingested substances
title_full_unstemmed Development of reconstructed intestinal micronucleus cytome (RICyt) assay in 3D human gut model for genotoxicity assessment of orally ingested substances
title_sort development of reconstructed intestinal micronucleus cytome (ricyt) assay in 3d human gut model for genotoxicity assessment of orally ingested substances
publishDate 2022
url https://hdl.handle.net/10356/161945
_version_ 1746219674029260800
spelling sg-ntu-dr.10356-1619452022-10-01T23:31:28Z Development of reconstructed intestinal micronucleus cytome (RICyt) assay in 3D human gut model for genotoxicity assessment of orally ingested substances Lim, Hui Kheng Hughes, Christopher Owen Lim, Michelle Jing Sin Li, Jia'En Jasmine Rakshit, Moumita Yeo, Calvin Chng, Kern Rei Li, Angela Chan, Joanne Sheot Harn Ng, Kee Woei Leavesley, David Ian Smith, Benjamin Paul Chapman School of Materials Science and Engineering Innovations in Food and Chemical Safety (IFCS) Programme, A*STAR Skin Research Institute of Singapore (SRIS), A*STAR Singapore Institute of Food and Biotechnology Innovation (SIFBI), A*STAR Nanyang Environment and Water Research Institute Engineering::Materials Micronuclei DNA Damage The micronucleus (MN) assay is widely used as part of a battery of tests applied to evaluate the genotoxic potential of chemicals, including new food additives and novel food ingredients. Micronucleus assays typically utilise homogenous in vitro cell lines which poorly recapitulate the physiology, biochemistry and genomic events in the gut, the site of first contact for ingested materials. Here we have adapted and validated the MN endpoint assay protocol for use with complex 3D reconstructed intestinal microtissues; we have named this new protocol the reconstructed intestine micronucleus cytome (RICyt) assay. Our data suggest the commercial 3D microtissues replicate the physiological, biochemical and genomic responses of native human small intestine to exogenous compounds. Tissues were shown to maintain log-phase proliferation throughout the period of exposure and expressed low background MN. Analysis using the RICyt assay protocol revealed the presence of diverse cell types and nuclear anomalies (cytome) in addition to MN, indicating evidence for comprehensive DNA damage and mode(s) of cell death reported by the assay. The assay correctly identified and discriminated direct-acting clastogen, aneugen and clastogen requiring exogenous metabolic activation, and a non-genotoxic chemical. We are confident that the genotoxic response in the 3D microtissues more closely resembles the native tissues due to the inherent tissue architecture, surface area, barrier effects and tissue matrix interactions. This proof-of-concept study highlights the RICyt MN cytome assay in 3D reconstructed intestinal microtissues is a promising tool for applications in predictive toxicology. Agency for Science, Technology and Research (A*STAR) National Research Foundation (NRF) Published version This study is supported by Industry Alignment Fund (Pre-Positioning) (IAF-PP-H18/01/a0/G14), Biomedical Research Council (BMRC, A*STAR). BPC Smith was also supported by a National Research Foundation Singapore Whitespace Grant (W20W3D0002) administered by A*STAR. 2022-09-27T03:24:43Z 2022-09-27T03:24:43Z 2022 Journal Article Lim, H. K., Hughes, C. O., Lim, M. J. S., Li, J. J., Rakshit, M., Yeo, C., Chng, K. R., Li, A., Chan, J. S. H., Ng, K. W., Leavesley, D. I. & Smith, B. P. C. (2022). Development of reconstructed intestinal micronucleus cytome (RICyt) assay in 3D human gut model for genotoxicity assessment of orally ingested substances. Archives of Toxicology, 96(5), 1455-1471. https://dx.doi.org/10.1007/s00204-022-03228-y 0340-5761 https://hdl.handle.net/10356/161945 10.1007/s00204-022-03228-y 35226136 2-s2.0-85125400212 5 96 1455 1471 en IAF-PP-H18/01/a0/G14 W20W3D0002 Archives of Toxicology © 2022 The Author(s). Open Access. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. application/pdf