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...
| Main Authors: | , , , , , , , , , , , |
|---|---|
| Other Authors: | |
| Format: | Journal Article |
| Language: | English |
| Published: |
2022
|
| Subjects: | |
| Online Access: | https://hdl.handle.net/10356/161945 |
| _version_ | 1824456246892167168 |
|---|---|
| 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 |
| 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 |
| author_sort | Lim, Hui Kheng |
| collection | NTU |
| 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. |
| first_indexed | 2025-02-19T03:51:04Z |
| format | Journal Article |
| id | ntu-10356/161945 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2025-02-19T03:51:04Z |
| publishDate | 2022 |
| record_format | dspace |
| spelling | ntu-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 |
| 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 |
| title | 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_short | 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 |
| topic | Engineering::Materials Micronuclei DNA Damage |
| url | https://hdl.handle.net/10356/161945 |
| work_keys_str_mv | AT limhuikheng developmentofreconstructedintestinalmicronucleuscytomericytassayin3dhumangutmodelforgenotoxicityassessmentoforallyingestedsubstances AT hugheschristopherowen developmentofreconstructedintestinalmicronucleuscytomericytassayin3dhumangutmodelforgenotoxicityassessmentoforallyingestedsubstances AT limmichellejingsin developmentofreconstructedintestinalmicronucleuscytomericytassayin3dhumangutmodelforgenotoxicityassessmentoforallyingestedsubstances AT lijiaenjasmine developmentofreconstructedintestinalmicronucleuscytomericytassayin3dhumangutmodelforgenotoxicityassessmentoforallyingestedsubstances AT rakshitmoumita developmentofreconstructedintestinalmicronucleuscytomericytassayin3dhumangutmodelforgenotoxicityassessmentoforallyingestedsubstances AT yeocalvin developmentofreconstructedintestinalmicronucleuscytomericytassayin3dhumangutmodelforgenotoxicityassessmentoforallyingestedsubstances AT chngkernrei developmentofreconstructedintestinalmicronucleuscytomericytassayin3dhumangutmodelforgenotoxicityassessmentoforallyingestedsubstances AT liangela developmentofreconstructedintestinalmicronucleuscytomericytassayin3dhumangutmodelforgenotoxicityassessmentoforallyingestedsubstances AT chanjoannesheotharn developmentofreconstructedintestinalmicronucleuscytomericytassayin3dhumangutmodelforgenotoxicityassessmentoforallyingestedsubstances AT ngkeewoei developmentofreconstructedintestinalmicronucleuscytomericytassayin3dhumangutmodelforgenotoxicityassessmentoforallyingestedsubstances AT leavesleydavidian developmentofreconstructedintestinalmicronucleuscytomericytassayin3dhumangutmodelforgenotoxicityassessmentoforallyingestedsubstances AT smithbenjaminpaulchapman developmentofreconstructedintestinalmicronucleuscytomericytassayin3dhumangutmodelforgenotoxicityassessmentoforallyingestedsubstances |