Induction of Oxidative Stress by Iron/Ascorbate in Isolated Mitochondria and by UV Irradiation in Human Skin

Two different model systems that have been successfully used in our lab to induce oxidative stress are described. The first is the so-called “iron/ascorbate” system, under appropriate conditions suitable to investigate oxidative stress mediated functional impairment in isolated intact mitochondria in vitro and the second is UV irradiation, which can be used under experimental and clinical conditions in vitro and in vivo to induce oxidative stress and inflammation in human skin. In the presence of iron/ascorbate, the induction of lipid and protein oxidation was investigated in isolated mitochondria. Very early events after treatment of freshly prepared, functionally intact mitochondria consist in the inhibition of active respiration mainly due to an attack of the complex III activity of the respiratory chain. The membrane potential was sustained nearly unchanged for a relatively long time and broke down abruptly at the end of the initiation phase of lipid peroxidation, when sufficient energization and antioxidant protection of mitochondria could not be maintained. UVA irradiation in combination with photosensitizing agents as 8-methoxypsoralen is successfully used as extracorporeal photoimmunotherapy (ECPI) in the treatment of several skin diseases, such as cutaneous T-cell lymphoma and systemic scleroderma. We used this system to determine markers of oxidative stress in plasma and cells from the buffy coat under experimental ECPI-relevant conditions and in patients during ECPI treatment. Only moderate oxidative stress could be demonstrated by a decrease of the antioxidant defense and a dose-dependent augmentation of hydroxyeicosatetraenoic acids and F2-isoprostanes as highly specific and sensitive biomarkers of lipid peroxidation and potential mediators of inflammatory and/or photoimmunomodulatory effects of ECPI. UVB is known as a major skin carcinogen that mainly affects keratinocytes and induces a complex cascade of inflammation, including reactive oxygen species. Effects of moderate UVB doses on the generation of biomarkers of oxidative stress and inflammation in HaCaT keratinocytes in vitro and in microdialysates of human skin in vivo were studied. F2-isoprostanes and prostaglandins were increased UVB dose-dependently, whereas functional integrity of HaCaTs was diminished. As an adaptive response, protein levels of MnSOD were enhanced, but higher UVB doses lead to irreversible cell damage. The microdialysis technique allowed for sensitively quantifying the markers of inflammation and oxidative stress and their kinetic profile in vivo following UVB exposure.