Determining the action spectrum of UVR-induced mitochondrial DNA damage, and related UVR-induced cellular effects in human skin

Abstract

Biological responses of human skin to ultraviolet radiation (UVR) are largely wavelength-dependent as shown by the action spectrum of UVR-induced erythema and nuclear DNA (nDNA) damage. As mitochondrial DNA (mtDNA) damage has been shown to be a reliable and sensitive biomarker of UVR exposure in human skin, the aim of this study was to determine the action spectrum for UVR-induced mtDNA damage in human skin cells. This involved the irradiation of both HaCaT (immortalised keratinocytes) and HDFn (human fibroblasts) cell lines and primary human keratinocyte cells with increasing doses of UVR. A wide variety of UVR sources were chosen for their unique overlapping UVR profiles, covering the entire UVA and UVB spectrum. Overall UVR induced mtDNA damage was assessed using a quantitative long real time PCR to amplify an 11 kb segment of the mitochondrial genome. Specific UVR-induced mtDNA deletions (3895 bp and 4977 bp) were investigated, as was the effect on cellular respiration using the novel Seahorse extracellular flux analyzer. Furthermore investigations sought to determine UVR-induced nDNA damage: This included the induction of p53 mutations, generation of the oxidative stress marker 8-OHdG, photoproduct production as well as differential gene expression. For investigations of general UVR-induced mtDNA damage, dose curves were produced for each of the UVR sources and cell types. From this an action spectrum was determined by a method known as de-convolution. A higher sensitivity was seen in the fibroblast cell type. The overall trend of the action spectrum of the UVR-induced mtDNA damage followed that of various previously determined action spectra with the most detrimental effects occurring over the shorter wavelengths of UVR. This was also shown for the production of photoproducts and with the differential gene expression analysis. UVA and UVB both also affected cellular respiration and gene expression. The UVR exposures used in the study were not effective in inducing detectable levels of either of the mtDNA specific deletions, mutations within the p53 gene or levels of 8-OHdG and this may have been affected by limitations of the analytical techniques. Further experiments saw that the addition of antioxidant type entities provided protection and the possible reversal of some of the UVR damaging effects.