ASSESSING THE ROLE OF EPIGENETIC FACTORS IN CHROMOSOME INSTABILITY FOLLOWING COMPOUND EXPOSURE

Abstract

The maintenance of the epigenome is critical for cell survival, with its dysregulation resulting in a multitude of disease states including rheumatoid arthritis, multiple sclerosis and colorectal cancer. There is now substantial evidence that exposure to certain compounds can perturb DNA methylation patterns within the genome. However, there is currently no regulatory requirement to investigate these changes when evaluating the safety of a compound. This thesis presents three complimentary methods, in combination with an assessment of genotoxicity, for the characterisation of changes in DNA methylation following compound exposure. The methods are evaluated on a selection of five compounds (5-azacytidine, colchicine, cadmium chloride, hydroquinone and paclitaxel) in the TK6 and A549 cell lines. The Amplification of Intermethylated Sites (AIMS) 2.0 method was used to highlight changes in genome-wide DNA methylation, as well as at specific fragment sizes. Additionally, genome-wide DNA methylation changes were assessed using Methylation-specific Fluorescence In Situ Hybridisation (meFISH). This method uses changes in the fluorescence intensity of 5-methylcytosine as a proxy for changes in genome-wide DNA methylation. Pyrosequencing was then used to probe the compounds for gene sensitive methylation changes, as a first step towards creating a gene biomarker panel to predict compound epigenotoxicity. The efficacy of the methods was confirmed by a noted reduction in DNA methylation following exposure 5-azacytidine. Conversely, exposure to paclitaxel led to an unexpected increase in DNA methylation, despite having no known direct interaction with DNA. Interestingly, the HRAS gene was more prone to compound-induced demethylation in the TK6 cell line, indicating its potential role as gene biomarker. Relating the changes in DNA methylation across all three assays to other genotoxic endpoints, it was found that DNA methylation changes can occur at low compound doses that do not produce detectable genotoxic end points. This is important because the current genetic toxicology test battery for pharmaceuticals looks for indicators of genotoxic damage only, implicitly assuming that this also functions as a proxy for epigenetic modifications. Understanding that this may not always be the case has key implications for how compound safety is assessed. In summary, these assays could provide a multi-level strategy for evaluating compound epigenotoxicity that could be included in the current regulatory test battery.

Date of Award	1 Aug 2020
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Richard Walmsley (Supervisor) & Gino Poulin (Supervisor)