TEMPOL PROTECTS AGAINST LENS DNA STRAND BREAKS AND CATARACT IN THE X-RAYED RABBIT

PURPOSE. To investigate the ability of the nitroxide free radical and superoxide dismutase mimic 4-hydroxy-2,2,6,6-tetramethylpiperidine-n-o xyl (TEMPOL) to protect against x-ray-induced lens DNA damage and cata ract formation in the rabbit. METHODS. Eleven gray (Gy) x-rays was del ivered twice, with a 48-hour interval, to the same eye of the 5-week-o ld rabbits. Fifteen minutes before each x-ray, 150 mu l aqueous humor was removed fom the anterior chamber and replaced with 150 mu l citrat e buffer containing 0 mM or 100 mM TEMPOL. The development of cataract was classified into seven stages by slit-lamp examination. DNA strand breaks were measured in the lens epithelium of x-rayed rabbits using a single-cell gel electrophoresis method. RESULTS. The level of total TEMPOL in the aqueous humor of rabbits at 15 minutes after intracamera l injection of the compound was 21 mM with 84% present in the oxidized form (determined by electron paramagnetic resonance spectroscopy). At 19 weeks after x-ray, rabbits irradiated without TEMPOL showed either stage 5 (complete posterior subcapsular opacity) or stage 6 (mature) cataracts, whereas the animals that had first been injected with TEMPO L developed only stage 2 to stage 4 cataracts (the difference between the two groups was significant at P < 0.01). Intracameral injection of TEMPOL resulted in a decrease of nearly 70% in the level of DNA stran d breaks produced by a single 11-Gy dose of x-ray. In vitro studies sh owed that TEMPOL was reduced rapidly by ascorbic acid by not by reduce d glutathione. Oxidized but not reduced TEMPOL (TEMPOL-H) was an effec tive radioprotector in cultured rabbit lenses, and TEMPOL was nearly c ompletely bioreduced in the plasma and aqueous humor of animals that w ere fed the compound in drinking water. CONCLUSIONS. TEMPOL was effect ive in protecting against lens epithelial DNA damage and cataract form ation in x-rayed rabbits. Although a number of mechanisms are possible , the protective effect may be associated with the ability of TEMPOL t o protect against radiation-produced peroxides by acting as a superoxi de dismutase mimic and to oxidize Fe2+ bound to DNA, thus preventing f ormation of the hydroxyl radical and subsequent DNA damage through the Haber-Weiss mechanism.