Stress responses and the physiological cell fate of human ocular cells

Ball, Simon (2021) Stress responses and the physiological cell fate of human ocular cells. Doctoral thesis, University of East Anglia.

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The eye is under constant attack from external sources, such as UV light and internal sources, such as reactive oxygen species which can give rise to oxidative stress. This insult to the ocular cells can lead to multiple forms of damage that can ultimately result in cell death. In the present study the primary area of interest was DNA damage and the DNA repair systems that are in place to minimise the impact of this trauma, control cell fate and how they can potential be involved in normal physiological processes.

With this central theme in mind, the first area investigated was to establish a distribution profile of DNA repair related proteins within the lens. Non-cultured whole human donor lenses obtained from the East Anglian Eye Bank were fixed in 4% v/v formaldehyde, paraffin embedded and sectioned at 6 µm. Samples were subjected to antigen retrieval before fluorescent immunohistochemistry using Alexa-488 secondary antibody and nuclear counterstaining with DAPI. Sections were visualised using a Zeiss Axioplan fluorescence microscope and digital camera. DNA repair proteins, PARP-1, DNA-PK and Ku80 were present in the cell nucleus of the lens epithelial layer. Within fibre cells, newly laid cells also presented a predominantly nuclear expression before levels rapidly declined. This reduced expression appeared to precede changes in chromatin appearance that could be attributed to lens fibre cell de-nucleation.

Developing tools to assess DNA damage is important. To respond to this need, a second area of focus was to adapt a qPCR method to detect DNA damage to produce a Real Time long amplicon PCR (LA QPCR) assay version that can be used to detect nuclear and mitochondrial DNA lesions The assay was evaluated using DNA extracted from the lens cell line FHL124 treated with the DNA strand break inducing agents Neocarzinostatin and Bleomycin. Following optimisation and validation, the LA QPCR method successfully detected DNA lesions in nuclear and mitochondrial DNA against a range of DNA damaging agents.

The next phase of the experimental study was to assess the putative applications of sulforaphane (SFN), an isothiocyanate abundant in brassicas, to provide benefit for ocular applications. It is important to note that SFN has concentration dependent actions. At low concentrations it is reported to be cytoprotective and at high concentrations cytotoxic. In the present study the aim was to exploit these traits for different needs.

Preservation of the corneal endothelium is vital for successful corneal grafts, therefore the ability of SFN to counter antioxidants was tested. Application of 100uM hydrogen peroxide (to give an oxidative stress) to the corneal endothelial cell line HCEC12 resulted in enhanced cell death. 2µM SFN pre-treatment significantly protected cells. DNA damage was also observed with hydrogen peroxide addition. While the degree of damage observed using the comet assay was not significantly lower with sulforaphane treatment, a significant reduction in nuclear DNA and mitochondrial lesions was detected with LA QPCR.

Higher concentrations of SFN were applied to lens cells, which could provide benefit for the treatment of PCO, that results from a wound healing response induced by cataract surgery. Exposure to 100µM SFN resulted in cell death, increased ROS levels and greater DNA damage. To determine if ROS were responsible for DNA damage and cell death, the ROS scavenger N-acetyl cysteine (NAC) was used. Pre-treatment with 1mM NAC prevented SFN induced DNA damage and cell death suggesting ROS play a key role in mediating SFN responses.

Overall the work presented has advanced our knowledge of DNA damage and repair. In the lens DNA repair proteins are expressed in the nuclei of cells within the epithelium and cortical fibre cells. A loss of these proteins precedes nuclear condensation and fragmentation in fibre cells, suggesting a potential role in this process. In developing the LA QPCR, this provides an additional method to assess DNA damage in nuclear and mitochondrial DNA and appears more sensitive the commonly used comet assay. SFN could serve as an additive to corneal storage medium to increase endothelial cell viability and probability of graft success. SFN when applied at higher concentrations could also provide better PCO management through induction of DNA damage and cell death with ROS playing a critical role in mediating these actions.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Chris White
Date Deposited: 28 Jun 2022 14:03
Last Modified: 28 Jun 2022 14:03

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