Solar-induced mitochondrial mutagenesis and dysfunction in human skin

Human skin is exposed to a multiplicity of exogenous damaging agents of which solar radiation is the most important. The ultraviolet (UV) wavelengths of the sun generate accelerated skin ageing (photoageing), and can cause skin cancer. Mitochondria in skin cells are considered to be especially vulnerable to solar radiation and to causatively contribute to skin ageing. The ultraviolet wavelengths reaching earth’s surface are divided into two subgroups. Ultraviolet B (UVB) radiation predominantly causes direct DNA damage such as cyclobutane pyrimidine dimers (CPDs). Ultraviolet A (UVA) is additionally able to generate significant amounts of reactive oxygen species (ROS) which then damage DNA, proteins, and lipids. Mitochondrial research is focused on this oxidative damage in skin. The potential effects of typical UVB-induced damage such as CPDs in mitochondrial DNA (mtDNA) were not researched in detail, although CPDs were shown to persist for several days in mitochondria. Because of its high persistence, this DNA damage type can be expected to be highly mutagenic and to disturb mitochondrial function. In this dissertation, the potential effects of UV-induced persistent damage on mitochondria and their DNA were investigated.We conducted a study including skin samples from exposed and less-exposed body sites to research mitochondrial mutagenesis in vivo. In this study, we measured the levels of the 4,977 base pair mitochondrial common deletion (CD), a CC to TT transition in the chromosomal p53 gene, a CC to TT transition in the mtDNA, and cellular mtDNA content by quantitative real-time PCR. CC to TT transitions are often termed UV fingerprint mutations as they arise from unrepaired CPDs. In nucleus, CPDs are repaired by nucleotide excision repair (NER); nevertheless CC to TT transitions can reach high levels in mutation hotspots such as the p53 gene. In mitochondria, NER is absent so that even higher levels could be expected. Whereas CD levels were significantly higher in sun-exposed skin than in less-exposed skin, no significant increase in the tandem transitions were found. The results for the CD were as anticipated as this deletion is caused by UVA-induced ROS and is known to reach high levels in sun-exposed skin. In contrast, the result for the mitochondrial CC to TT transition is remarkable; even more, as two times more positive samples were found for the p53 transition than for the mtDNA transition. This result is pointing to an unexpected low mutagenicity of mitochondrial CPDs. In addition, we found a significantly higher mtDNA copy number in exposed than in less-exposed skin samples. Elevated mtDNA copy number might be a cellular adaption to solar radiation and makes mtDNA copy number a potential biomarker of sun exposure in photobiological research.To investigate the effects of CPDs on mitochondrial function in detail, we irradiated human skin fibroblasts with increasing doses of ultraviolet C (UVC) or sun simulating light (SSL). UVC was used to efficiently induce CPDs. SSL was applied to simulate the whole solar spectrum with its oxidative stress component. At comparable antiproliferative doses, UVC and SSL induced increased gene expressions of the mitochondrial transcription factor A (TFAM) and the major photoageing-associated collagenase matrix metallopeptidase 1 (MMP1). Only SSL, however, induced a dose-dependent upregulation of mtDNA copy number and a significant dysfunction of mitochondrial respiration. This indicates - again - a low impact of shortwave induced DNA damage as part of the whole solar damage spectrum.Because of the central role of mitochondria in cell physiology, mitochondrial dysfunction potentially interferes with DNA repair. It thereby might causatively contribute to a DNA repair decline and eventually to cancerogenesis. To reveal such potential links, human skin fibroblasts were treated with mitochondrial inhibitors. Then the capacity to functionally restore a UVC-irradiated plasmid via NER was analyzed with a modified host cell reactivation assay (HCRA). All three inhibitors caused an impaired mitochondrial respiration. This resulted for two of the inhibitors in an increased NER capacity suggesting a mitochondrial role in the signaling of this in mitochondria non-existent DNA repair pathway.The results of this thesis indicate only a minor role of shortwave UV-induced DNA damage in mitochondrial mutagenicity and dysfunction. Our data rather point to the important role of long-wave UV-induced ROS which can directly affect mitochondrial respiration complexes. With mtDNA copy number a biomarker of solar exposure was established, which can be measured in smallest skin sample volumes