Oxy radicals in the eye tissues of rabbits after diquat in vivo

It is our hypothesis that oxygen free radicals are the triggering agents in cataractogenesis. However, besides H2O2 there is no direct evidence of generation of oxy radicals in the eye tissues. Due to extremely short life of O2-. and OH. it is not possible to measure their cellular steady state levels. We found that indirect spectrophotometric techniques based on superoxide dismutase (SOD)-inhibitable cytochrome c reduction for estimation of O2-., salicylate hydroxylation for OH. and peroxidase catalysed reoxidation of 2,6-dichlorophenolindophenol for H2O2, were suitable, sensitive and reproducible for measurements of the reactive species of O2 produced in the eye tissues by oxy radical enhancer, diquat in the rabbit eye in vivo. After a single intravitreal injection of 60, 120 or 300 nmole diquat in the right eyes, there was a dose-dependent rise in O2. levels, 106-265 fold in the aqueous humor, 34-87 fold in the vitreous humor, 6-19 fold in the lens, and 43-88 fold in the retina as compared to 0.16 microM, 0.21 microM, 2.47 nmole/g and 5.56 nmole/g in tissues of the normal eyes, respectively. There were similar increases of OH. in the eye tissues, and of H2O2 in the aqueous humor and vitreous humor after diquat injection. We propose that endogenous reductants of the eye tissues univalently reduce diquat to its free radical which spontaneously reacts with O2 generating O2. in excessive amounts, further giving rise to H2O2 and OH. triggering cataractogenesis.