TEMPOL protects against lens DNA strand breaks and cataract in the x-rayed rabbit.

Abstract

PURPOSE: To investigate the ability of the nitroxide free radical and superoxide dismutase mimic 4-hydroxy-2,2,6,6-tetramethylpiperidine-n-oxyl (TEMPOL) to protect against x-ray-induced lens DNA damage and cataract formation in the rabbit. METHODS: Eleven gray (Gy) x-rays was delivered twice, with a 48-hour interval, to the same eye of 5-week-old rabbits. Fifteen minutes before each x-ray, 150 microliters aqueous humor was removed from the anterior chamber and replaced with 150 microliters citrate 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 intracameral 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 TEMPOL 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 strand breaks produced by a single 11-Gy dose of x-ray. In vitro studies showed that TEMPOL was reduced rapidly by ascorbic acid but not by reduced glutathione. Oxidized but not reduced TEMPOL (TEMPOL-H) was an effective radioprotector in cultured rabbit lenses, and TEMPOL was nearly completely bioreduced in the plasma and aqueous humor of animals that were fed the compound in drinking water. CONCLUSIONS: TEMPOL was effective in protecting against lens epithelial DNA damage and cataract formation in x-rayed rabbits. Although a number of mechanisms are possible, the protective effect may be associated with the ability of TEMPOL to protect against radiation-produced peroxides by acting as a superoxide dismutase mimic and to oxidize Fe2+ bound to DNA, thus preventing formation of the hydroxyl radical and subsequent DNA damage through the Haber-Weiss mechanism.