Glutathione modulates the level of free radicals produced in UVA-irradiated cells.
We have developed an assay to detect reactive oxygen species (ROS) generated by UVA radiation utilising chemical probes which become fluorescent upon oxidation. Using a human bladder carcinoma cell line (MGH-U1) and spontaneously immortalised keratinocytes (HaCaT), we have shown a UVA (narrow band 365+/-5 nm) dose-dependent increase in fluorescence by flow cytometry following loading of the cells with either dihydrorhodamine 123 (DHR) or 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The UVA response of both DHR and DCFH was enhanced by elevation of intracellular levels of the photosensitiser protoporphyrin IX by incubation for 2.5 h with 5-aminolaevulinic acid. Depletion of the antioxidant glutathione (GSH) using the inhibitor D,L-buthionine-sulphoximine (BSO), resulted in an increase in the UVA-induced fluorescence of DCF but not of rhodamine 123. Conversely, raising intracellular GSH levels with N-acetyl cysteine (NAC) had relatively little protective effect in terms of degree of induced fluorescence.
Tobi
S E
SE
Department of Biological Sciences, IENS, Lancaster University, UK.
Paul
N
N
McMillan
T J
TJ
D000111:Acetylcysteine / Q000378:metabolism / Q000494:pharmacology; D000975:Antioxidants / Q000378:metabolism*; D004307:Dose-Response Relationship, Radiation; D005452:Fluoresceins; D005456:Fluorescent Dyes; D016166:Free Radical Scavengers / Q000378:metabolism / Q000494:pharmacology; D005609:Free Radicals / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D015603:Keratinocytes / Q000528:radiation effects; D010084:Oxidation-Reduction; D012235:Rhodamines / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
D000975:Antioxidants; D005452:Fluoresceins; D005456:Fluorescent Dyes; D016166:Free Radical Scavengers; D005609:Free Radicals; D012235:Rhodamines; C058319:dihydrorhodamine 123; C029569:diacetyldichlorofluorescein; D005978:Glutathione; D000111:Acetylcysteine