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Ultraviolet Radiation (UVR) is a major contributing factor to the worldwide incidence of skin cancers, and causes additional effects in the skin including photo ageing and DNA damage. The damage caused by UVR is frequently linked to the increased production of reactive oxygen species (ROS). The major source of cellular ROS is the mitochondrial electron transport chain (ET C) and its individual complexes. Although these complexes are accepted as a major source of cellular ROS, the relative roles of each complex in cellular ROS production is a matter for considerable scientific debate. This study aimed to quantify ROS production from the mitochondrial electron transport chain complexes, primarily in skin cells. It is important, when assessing cellular ROS production, to ensure that the methods used are appropriate for the applications, as unexpected interactions between two of the most commonly used ROS probes, DHR123 and DCF-DA, were found. The effects of loading protocol and diluents upon probe function and fluorescence were assessed and it was concluded that probes may be loaded into cells before or after UV A as long as appropriate controls are employed. This study used specific electron transport chain complex inhibitors to report a significant role in UVA-induced ROS production for complexes I and Ill, but also a significant increase in ROS production from complex 11. The increase in complex 11 specific ROS production was greater than complex I or complex III ROS production in some circumstances, and was more important than suggested by current literature. This is a novel finding and indicates that complex 11 may be a useful target for future studies of cellular ROS production. Furthermore, complex- specific ROS production profiles were shown to differ depending upon cell type and time of complex inhibition. In order to further investigate complex 11 as a novel source of ROS production, spectrophotometric assays were carried out. These allowed quantification of respiration and inhibition of complex 11 using the specific inhibitors 3- Nitropropionic Acid (3NP) and 2-thenoyltrifluoroacetone (TTF A). Both complex 11 activity and percentage inhibition were found to vary considerably between cell types, and it was also found that ROS production did not necessarily reflect the proportion of inhibition exerted by a particular inhibitor.

NF-KB signalling is known to be involved in skin homeostasis, with aberrant expression within diseased skin. NF-KB has also been shown to be a key regulator of aged gene expression both within the skin and globally, being involved in ROS production, a key mediator of ageing, and interacting with sirtuins, expression of which is linked to longevity. The NF-KB family consists offive subunits, ReIA, ReIB, c-Rel, NF-KB 1 and NF- KB2 which act as both homo and heterodimers. They have distinct biological functions, however, the action of individual subunits remains largely unexplored. Immunohistochemical analysis of p50 (NF-KBI) and c-Rel subunits within sclerotic and psoriatic skin revealed abnormal expression patterns while previously published data associates SNPs in c-Rel with psoriasis and SNPs in NF-KB I with scleroderma.

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