Reactive oxygen species (ROS) is thought to play an important role in cell signaling of apoptosis, necrosis, and
proliferation. Light irradiation increases mitochondrial reactive oxygen species (ROS) production and mediates its
intracellular signaling by adjusting the redox potential in tumor cells. Mitochondria are the main source of ROS in the
living cell. Superoxide anions (02- are likely the first ROS generated in the mitochondria following radiation damage,
and then convert to hydrogen peroxide (H202), hydroxyl radical (•OH), and singlet oxygen (102), etc. Conventional
methods for research ROS production in mitochondria mostly use isolated mitochondria rather than mitochondria in
living cells. In this study, a highly selective probe to detect mitochondrial 02- in live cells, MitoSOXTM Red, was applied
to quantify the mitochondrial ROS production in human lung adenocarcinoma cells (ASTC-a-1) with laser scanning
microscope (LSM) after ultraviolet C (UVC) and He-Ne laser irradiation. Dichiorodihydrofluoresein diacetate (DCFHDA),
a common used fluorescent probe for ROS detection without specificity, were used as a comparison to image the
ROS production. The fluorescent image of MItoSOXTM Red counterstained with MitoTracker Deep Red 633, a
mitochondria selective probe, shows that the mitochondrial ROS production increases distinctly after UVC and He-Ne
laser irradiation. DCFH-DA diffuses labeling throughout the cell though its fluorescence increases markedly too. In
conclusion, the fluorescent method with MitoSOXTM Red reagent is proved to be a promising technique to research the
role of ROS in radiation induced apoptosis.
KEYWORDS: Chemiluminescence, Oxygen, Ultraviolet radiation, Blood, Organisms, Radiotherapy, Signal detection, Signal processing, Proteins, Biological research
Oxidative stress is mainly mediated by reactive oxygen species (ROS). Evaluation of oxidative stress is helpful for choosing an appropriate method to protect the organism from the oxidative damage. In this study, a highly sensitive and simple chemiluminescence method is presented for the evaluation of radiation-induced oxidative stress in human peripheral lymphocytes. The lymphocytes were irradiated by ultraviolet radiation (320-400nm, UVA) with different doses. The ROS generated by the lymphocytes was detected by chemiluminescence method, using a highly sensitive chemiluminescence probe 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-α] pyrazin-3-one (MCLA). The cell viability was detected with Cell Counting Kit-8 (CCK-8). The malondialdehyde (MDA), a marker of lipid peroxidation and oxidative stress, and the total antioxidant capacity (TAC), a parameter that is taken as evidence of oxidative stress, were measured too. The results show that both chemiluminescence intensity, cell mortality and MDA concentration of lymphocytes grow with the increase of UVA dose range from 0.5 to 8 J/cm2, while the TAC decreases. There exists a positive relationship between cell oxidative damage degree and the chemiluminescence intensity of lymphocytes. This highly sensitive chemiluminescence method would potentially provide an easy way to evaluate the level of UVA-induced oxidative stress readily, sensitively and rapidly
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