"Oxidative Damage and Mitochondrial Dysfunction in Ultraviolet B Irradiated Human Keratinocytes".

PAZ, MARIELA L.; GONZÁLEZ MAGLIO, DANIEL H.; WEILL, FEDERICO S.; BUSTAMANTE, JUANITA; LEONI, JULIANA.

Buenos Aires

Congreso; 21st World Congress of Dermatology,; 2007

Society Investigative of Dermatology

6964 OXIDATIVE DAMAGE AND MITOCHONDRIAL DYSFUNCTION IN ULTRAVIOLET B IRRADIATED HUMAN KERATINOCYTES ML Paz, DH Gonzalez Maglio, FS Weill, Bustamante, J Leoni Facultad de Farmacia y Bioqulrnica, Universidad de Buenos Aires, Buenos Aires. Argentina.   Objectives · To evaluate mitochondrial alterations and subsequent reactive oxygen species (ROS) production after an acute dose of UVB irradiation. • To determine the simultaneous reactive nitrogen species production. • To relate the oxidative and mitochondrial damage with apoptotic cell death.   Materials and Methods Human keratinocytes cell line (HaCaT) were irradiated with 50 mj/cm2 of UVB light and evaluated 6, 17 and 24 hours after the stimulus, non-irradiated cells were used as control {0 hs). Several parameters were analyzed by flow cytometry using different probes: 3,3’ Dihexyloxacarbocyanine iodide (DiOC 6} for mitochondrial membrane depolarization, Hydroethidine for superoxide (02*) production; 2',7’ Dichlorodihydrofluorescein diacetate (DCFH DA) for ROS production; 4,5 Diaminofluorescein diacetate {DAF-2 DA) for nitric oxide (N0) production and Propidium Iodide for nuclear DNA content to evaluate apoptosis. Apoptotic cell death was also assessed by fluorescence . microscopy with Ethidium Bromlde and Acridine Orange staining. For each probe the proper positive control was used.   Results Mitochondrial membrane depolarization began 17 hs post-irradiation (72.44% vs. 22.20% at O hs} maintaining these high levels also at 24 hours (70.96%l. 02** and ROS production showed a similar pattern, increasing towards 17 hs post-UVB (40.32% and 6.10% respectively vs. 10.95% and 2.62% at 0 hs) and reaching the maximum detected level at 24 hs (51.90% and 15.37% respectively). Low intensity N0 production could only be detected 6 hs post-UVB (38.18% vs. 26.01% at 0 hs) and did not increase at the other mudied times. Apoptotic cell death rose in a time dependent manner, finding a high percentage of apoptotic cells 24 hs post-irradiation.   Conclusions After an acute dose of UVB irradiation keratinocytes rapidly undergo apoptosis, apparently due to mitochondrial independent mechanisms, since in the meantime the mitochondria appear not to be altered. However, 17 hs post-irradiation the mitochondria lose its membrane potential (depolarization) with the subsequent oxidative phosphorylation impairment and ROS production. Following these alterations apoptosis levels increase even more, probably as a result of the release of cytochrome c from the damaged mitochondria, making all cells become apoptotic. N0 might be involved in these cell death processes at early times, damaging the electron transport proteins implicated in the oxidative phosphorylation.