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Title: The use of mitochondrial and nuclear DNA to determine the role, mechanism and protective effects of antioxidants in human skin
Author: Oyewole, Anne Oyindamola
Awarding Body: University of Newcastle Upon Tyne
Current Institution: University of Newcastle upon Tyne
Date of Award: 2013
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A key contributor to the development of skin cancer and ageing is damage to the skin caused by an increase in the cellular load of reactive oxygen species (ROS). ROS generation is increased when the skin is exposed to factors, such as ultraviolet radiation (UVR) from the sun. UVR initiates a cascade of biological events including DNA damage, which if allowed to accumulate can accelerate the ageing process. Previously, studies have reported the ROS scavenging capacity of cytosolic antioxidants and demonstrated their ability to abrogate ROS-induced DNA damage. The aim of this study was to further investigate the relative potency of mitochondrial (mt) located antioxidants such as MitoQ (MO) and tiron, because the mitochondria serve as the main cellular location for ROS formation. Using a lesion specific quantitative real-time PCR (OPCR) assay (which follows the principle that DNA lesions block replication of DNA by Taq polymerase) it was found that UVA (9.2 J/cm2", glass filtered TL-09 source) and hydrogen peroxide (H202) (200 IJM) induced widespread mtDNA damage in human dermal fibroblasts (n = 4, P < 0.001). The mt-Iocalised antioxidant tiron was able to offer complete protection (n = 4, P < 0.001) against the deleterious effect of H202. In comparison, treatment with the optimum, sub-lethal dose (as determined by the MTS assay) of 10 cytosolic antioxidants including curcumin and resveratrol were found to provide moderate protection against H20rinduced mtDNA damage. Interestingly, differential responses to UVA were observed, with antioxidants showing a decline in the proportion of protection offered against this potent mutagen, except in the case of tiron; a bi-functional antioxidant with ROS and iron targeting capacity. These findings demonstrate the potential involvement of iron (present in the cytosol and mitochondria) in H202 and UVA-mediated oxidative damage. We sought to further investigate the potency of tiron against ROS-induced nuclear (n) DNA damage using the comet assay. In comparison to a small panel of cytosolic antioxidants which showed a graded response, tiron was able to abrogate ROS-induced nDNA damage in human dermal fibroblasts and primary keratinocytes. It appears that the ability of tiron to detoxify ROS and in particular iron (which in reaction with H202 give rise to the highly reactive hydroxyl radical) enables this antioxidant to inhibit genomic modifications and significantly contribute to cellular protection compared to other antioxidants.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available