INVESTIGADORES
GONZALEZ MAGLIO Daniel Horacio
congresos y reuniones científicas
Título:
"Oxidative Damage and Mitochondrial Dysfunction in Ultraviolet B Irradiated Human Keratinocytes"
Autor/es:
PAZ, MARIELA L.; GONZÁLEZ MAGLIO, DANIEL H.; WEILL, FEDERICO S.; BUSTAMANTE, JUANITA; LEONI, JULIANA
Lugar:
Buenos Aires, Argentina.
Reunión:
Congreso; 21st World Congress of Dermatology; 2007
Resumen:
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 (DiOC6) for mitochondrial membrane depolarization,
Hydroethidine for superoxide (O2·-) production; 2´,7´Dichlorodihydrofluorescein
diacetate (DCFH DA) for ROS production; 4,5 Diaminofluorescein diacetate (DAF-2
DA) for nitric oxide (NO) production and Propidium Iodide for nuclear DNA content
to evaluate apoptosis. Apoptotic cell death was also assessed by fluorescence
microscopy with Ethidium Bromide 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 0 hs.) maintaining these high
levels also at 24 hours (70.96 %). O2·- 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 NO production could only be detected 6 hs post-UVB
(38.18 % vs. 26.01 % at 0 hs) and did not increase at the other studied 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.
NO might be involved in these cell death
processes at early times, damaging the electron transport proteins implicated
in the oxidative phosphorylation.