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Development of an In Vitro whole cell Micronucleus Multiplex expansion assay: Using DRAQ5™ and the DNA damage biomarkers ɣH2AX, P53, pH3 along with cell cycle relationships and the MN genotoxic endpoint to assess chemical Mode of... / DANIELLE HARTE

Swansea University Author: DANIELLE HARTE

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DOI (Published version): 10.23889/SUthesis.58916

Abstract

Genetic toxicity testing is the assessment of compounds, and their respective metabolites, potential to cause DNA damage either directly or indirectly. There are many genetic toxicology screening assays designed to assess the DNA damaging potential of chemicals in early drug development to help iden...

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Published: Swansea 2021
Institution: Swansea University
Degree level: Doctoral
Degree name: Ph.D
Supervisor: Johnson, George E. ; Cronin, James G.
URI: https://cronfa.swan.ac.uk/Record/cronfa58916
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first_indexed 2021-12-06T13:04:17Z
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spelling 2021-12-06T13:33:18.1192660 v2 58916 2021-12-06 Development of an In Vitro whole cell Micronucleus Multiplex expansion assay: Using DRAQ5™ and the DNA damage biomarkers ɣH2AX, P53, pH3 along with cell cycle relationships and the MN genotoxic endpoint to assess chemical Mode of Action on an imaging flow cytometry platform with template development 8805e2e824cb14037f0273bb085b8353 DANIELLE HARTE DANIELLE HARTE true false 2021-12-06 Genetic toxicity testing is the assessment of compounds, and their respective metabolites, potential to cause DNA damage either directly or indirectly. There are many genetic toxicology screening assays designed to assess the DNA damaging potential of chemicals in early drug development to help identify promising drugs that have a low risk potential of causing damage leading to cancer in humans. Despite this, in vitro tests generate a high number of miss-leading positives, the consequences of which lead to unnecessary animal testing and abandoning promising drug candidates. Understanding chemical Mode of Action (MoA) is vital in identifying true genotoxic potential of substances. Here I demonstrate a simple robust protocol for optimised staining of fixed human lymphoblast P53 proficient TK6 cells with the antibodies; Anti-ɣH2AX, Anti-P53 and Ant-pH3S28 along with DRAQ5™ DNA staining in a whole cell multiplex system that is suitable for analysis via microscopy or imaging flow cytometry. Use of the Amnis FlowSight® and ImageStream X Mark II® platform provided a high content high throughput acquisition platform with the sensitivity of flow cytometry and accuracy of image analysis. Using both optimal and suboptimal lasers for fluorophore excitation demonstrated that a multiplex system for DNA damage assessment including MN was possible in un-lysed cells. IDEAS 6.2 template generation allowed for batch processing of data samples extracting the following metrics: Cell Cycle, Micronucleus (MN), ɣH2AX, P53, PH3, G1 ɣH2AX, G1 P53, S ɣH2AX, S P53, G2 ɣH2AX, G2/M P53, Prophase, Metaphase, Anaphase and Abnormal mitosis. Furthermore, high content nature of the platform using imagery allows for identification of rare cellular event assessment particularly preliminary data on biomarker signal found within MN. The system found differences in the biomarker metric responses between the chemicals; MMS, Carbendazim, ARA-C, Vinblastine, Etoposide and Crizotinib suggesting potential for chemical MoA elucidation. E-Thesis Swansea In Vitro, TK6 cells, DNA, DNA Damage, Cell Cycle, ɣH2AX, P53, pH3, Micronucleus, Multiplex, Aneugen, Clastogen, Mode of Action, Genetic Toxicology, Flow Cytometry, Gating, Imaging, Microscopy, ImageStream, Compensation 6 12 2021 2021-12-06 10.23889/SUthesis.58916 A selection of third party content is redacted or is partially redacted from this thesis due to copyright restrictions. COLLEGE NANME COLLEGE CODE Swansea University Johnson, George E. ; Cronin, James G. Doctoral Ph.D Life Sciences Research Network Wales 2021-12-06T13:33:18.1192660 2021-12-06T13:01:16.2243522 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Medicine DANIELLE HARTE 1 58916__21807__8cefdf1c213344c3b91417a74424a65e.pdf Harte_Danielle_S_G_PhD_Thesis_Final_Redacted.pdf 2021-12-06T13:27:07.5595136 Output 18736236 application/pdf Redacted version - open access true Copyright: The author, Danielle S. G. Harte, 2021. true eng
title Development of an In Vitro whole cell Micronucleus Multiplex expansion assay: Using DRAQ5™ and the DNA damage biomarkers ɣH2AX, P53, pH3 along with cell cycle relationships and the MN genotoxic endpoint to assess chemical Mode of Action on an imaging flow cytometry platform with template development
spellingShingle Development of an In Vitro whole cell Micronucleus Multiplex expansion assay: Using DRAQ5™ and the DNA damage biomarkers ɣH2AX, P53, pH3 along with cell cycle relationships and the MN genotoxic endpoint to assess chemical Mode of Action on an imaging flow cytometry platform with template development
DANIELLE HARTE
title_short Development of an In Vitro whole cell Micronucleus Multiplex expansion assay: Using DRAQ5™ and the DNA damage biomarkers ɣH2AX, P53, pH3 along with cell cycle relationships and the MN genotoxic endpoint to assess chemical Mode of Action on an imaging flow cytometry platform with template development
title_full Development of an In Vitro whole cell Micronucleus Multiplex expansion assay: Using DRAQ5™ and the DNA damage biomarkers ɣH2AX, P53, pH3 along with cell cycle relationships and the MN genotoxic endpoint to assess chemical Mode of Action on an imaging flow cytometry platform with template development
title_fullStr Development of an In Vitro whole cell Micronucleus Multiplex expansion assay: Using DRAQ5™ and the DNA damage biomarkers ɣH2AX, P53, pH3 along with cell cycle relationships and the MN genotoxic endpoint to assess chemical Mode of Action on an imaging flow cytometry platform with template development
title_full_unstemmed Development of an In Vitro whole cell Micronucleus Multiplex expansion assay: Using DRAQ5™ and the DNA damage biomarkers ɣH2AX, P53, pH3 along with cell cycle relationships and the MN genotoxic endpoint to assess chemical Mode of Action on an imaging flow cytometry platform with template development
title_sort Development of an In Vitro whole cell Micronucleus Multiplex expansion assay: Using DRAQ5™ and the DNA damage biomarkers ɣH2AX, P53, pH3 along with cell cycle relationships and the MN genotoxic endpoint to assess chemical Mode of Action on an imaging flow cytometry platform with template development
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author DANIELLE HARTE
author2 DANIELLE HARTE
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doi_str_mv 10.23889/SUthesis.58916
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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 - Medicine{{{_:::_}}}Faculty of Medicine, Health and Life Sciences{{{_:::_}}}Swansea University Medical School - Medicine
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description Genetic toxicity testing is the assessment of compounds, and their respective metabolites, potential to cause DNA damage either directly or indirectly. There are many genetic toxicology screening assays designed to assess the DNA damaging potential of chemicals in early drug development to help identify promising drugs that have a low risk potential of causing damage leading to cancer in humans. Despite this, in vitro tests generate a high number of miss-leading positives, the consequences of which lead to unnecessary animal testing and abandoning promising drug candidates. Understanding chemical Mode of Action (MoA) is vital in identifying true genotoxic potential of substances. Here I demonstrate a simple robust protocol for optimised staining of fixed human lymphoblast P53 proficient TK6 cells with the antibodies; Anti-ɣH2AX, Anti-P53 and Ant-pH3S28 along with DRAQ5™ DNA staining in a whole cell multiplex system that is suitable for analysis via microscopy or imaging flow cytometry. Use of the Amnis FlowSight® and ImageStream X Mark II® platform provided a high content high throughput acquisition platform with the sensitivity of flow cytometry and accuracy of image analysis. Using both optimal and suboptimal lasers for fluorophore excitation demonstrated that a multiplex system for DNA damage assessment including MN was possible in un-lysed cells. IDEAS 6.2 template generation allowed for batch processing of data samples extracting the following metrics: Cell Cycle, Micronucleus (MN), ɣH2AX, P53, PH3, G1 ɣH2AX, G1 P53, S ɣH2AX, S P53, G2 ɣH2AX, G2/M P53, Prophase, Metaphase, Anaphase and Abnormal mitosis. Furthermore, high content nature of the platform using imagery allows for identification of rare cellular event assessment particularly preliminary data on biomarker signal found within MN. The system found differences in the biomarker metric responses between the chemicals; MMS, Carbendazim, ARA-C, Vinblastine, Etoposide and Crizotinib suggesting potential for chemical MoA elucidation.
published_date 2021-12-06T04:15:49Z
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score 11.016258

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