Reactive oxygen species (ROS) are involved in several pathological processes, including brain tissue damage. Their possible involvement, whether acute as in stroke, or chronically as in neurodegenerative disease, is of great interest due to the large number of people suffering from these conditions. There is therefore a need to develop in vitro model systems to study the mechanisms involved in ROS damage to neuronal cells. In this investigation MR32 neuroblastoma cells were irradiated with ultra-violet (W) light, a known generator of ROS. W A and UVB light exert their effects in different ways; the former acts via damage secondary to ROS generation, whilst UVB damages DNA directly. In this study the effects of both forms of irradiation on cell cultures were investigated for the purpose of comparison. A novel antibody raised against DNA damaged by ROS was used. This has already been found to detect Winduced DNA damage in immunochemical studies [I]. To establish whether DNA damage was produced by secondary ROS or a direct result of W irradiation, studies were carried out on cells preincubated in the presence and absence of atocopherol or desfemoxamine. a-tocopherol, an isomer of Vitamin E, is a lipid-soluble molecule able to terminate free radical chain reactions at cell membranes and so suppress lipid peroxidation. Desferrioxamine is an iron chelator, and would theoretically decrease damage caused by free radicals produced via the Haber-Weiss reaction, catalysed by reduced iron. Therefore preincubation with these compounds may restrict UV irradiation damage to IMR32 cells mediated by free radicals. either a-tocopherol or desfemoxamine for 24 hours at 37"C, washed with HBSS and irradiated from above with W A (13.6-109.1 d/cm2) or UVB (0.5-2.0 d/cm*) light. Cells were either fixed immediately or 24 hours later. For the latter time point fresh media was added after irradiation and cells kept at 37°C until needed. Cells were fixed and permeabilised by addition of 2% (v/v) paraformaldehyde (pH 7.4) for 1 hour, followed by ice-cold methanol overnight at -2OOC. They were rehydrated with 0.01M PBS, and non-specific binding sites blocked with 1 O O h (vh) normal goat serum (NGS)/PBS for 1 hour before incubation with primary antibody (1 :SO00 dilution (v/v) in 0.2% NGSPBS) overnight at 4 O C . Cells were washed and the secondary antibody (goat anti-rabbit IgG biotin conjugate; 1 :200 dilution (v/v) in 0.2% NGSPBS) added for 1 hour, then avidin-fluorescein isothiocyanate (FITC) conjugate (1 : 100 dilution (v/v) in PBS) for 1 hour in the dark, to detect binding. Cells were washed before fluorescence levels were determined using a Denley Wellfluor Microplate fluorescence reader (measuring and reference filters 485nm and 5 3 0~1 respectively).A dose-related increase in antibody binding was observed in cells fixed immediately after irradiation with both W A and UVB light. Preincubation with a-tocopherol reduced the level of this binding after W A irradiation only, at the highest 2 doses. 24 hours after W A irradiation the increa...
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