The overall objective of this project is to evaluate the role of oxidative stress in the development of human senile cataract. The hypothesis that a breakdown in antioxidant defense systems in the lens can lead to increased levels of prooxidants, irreversible modification of lens proteins and eventual loss of lens transparency will be tested. Long-term goals are to develop an animal model to investigate the formation of nuclear cataract and to contribute to the development of antioxidant therapies to delay the onset of senile cataract. Lenses or lens epithelial cells will be challenged oxidatively with hyperbaric O2 in vitro and in vivo, H2O2 and X-irradiation. Hyperbaric O2 will be used to study the relationship which exists in the lens between the glutathione redox cycle and ascorbic acid (AA) and to determine whether a function of GSH in the lens is to maintain AA in the reduced state. Sensitive methods involving HPLC, electrochemistry and electron spin resonance (ESR) will be employed to measure lens and aqueous humor levels of reduced and oxidized glutathione, AA, dehydroascorbate and ascorbyl free radical (AFR). Changes occurring in the levels and oxidation states of the two antioxidants will bc correlated with possible modifications to lens proteins including alterations in crystallin composition, formation of acidic protein species and increases in the levels of protein carbonyls and mixed disulfides. Methods of protein analysis will include HPLC, SDS-PAGE, isoelectric focussing and difference-absorption spectroscopy. Observed 02-induced protein modifications will bc compared to those which have been reported to occur in the aging human lens, and which are believed to be related to metal-catalyzed oxidative attack. Studies with an experimental diet, deficient in AA, will be employed to investigate the function of this antioxidant in the aqueous humor and lens, as well as to evaluate a possible role for oxidative stress in the formation of sugar cataract. Studies of the mechanism of H202-induced damage in rabbit and human lens epithelial cells will bc performed with the use of the antioxidants butylated hydroxytoluene and TEMPOL. The possible catalytic role of transition metals in lens oxidative processes will also bc investigated. The contribution of iron and copper to the formation of AFR in the aqueous humor will be determined with the use of ESR, and, in addition, radioisotopes of copper and iron will bc used to identify transition metal complexes which may be present in the aqueous humor and lens. Finally, the oxidative mechanism of X-ray cataract will be explored with the use of a lipid soluble spin-trapping agent and the hydroxyl radical scavenger dimethylthiourea.
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