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A fluorescence-based assay to detect UVA-induced free radical species in human cells.

Research output: Contribution to journalJournal article

Published

Journal publication date06/1999
JournalPhotocchemistry and Photobiology
Journal numberS1
Volume69
PagesS89
Original languageEnglish

Abstract

There is good evidence that the biological effects of UVA are mediated by certain reactive oxygen species (ROS) generated by hitherto unidentified photosensitisers within the cell. With the aim of identifying factors which modify the cellular response to UVA, we have developed an assay to detect such oxidative species 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 2 5nm) dose-dependent increase in fluorescence by flow cytometry following loading of the cells with either dihydrorhodamine (DHR, 10pM) or 2',7'-dichlorofluorescin diacetate (DCFH-DA, 5yM). Spectrofluorimetric measurements demonstrated that the total fluorescence produced in the cells was 7.2 times greater than that produced by irradiating the probe in buffer alone indicating that the majority of probe oxidation is due to ROS generated by UVA. Incubation with buthionine-S,R-sulfoximine (BSO), an inhibitor of A-glutamylcysteine synthetase, reduced cellular glutathione (GSH) levels by upto 74% (mean of 6 measurements) in both cell lines and resulted in an increase in the +UV/-UV ratio (relative to a value of 1.00 for untreated cells) to 1.3320.07(mean) in DCFH-loaded HaCat cells and 1.420.076 in MGH-U1 cells, following treatment with 10kM BSO. Our data therefore point to the involvement of glutathione in modulating the level of UVA-induced free radicals.