The objective of the proposed work is to evaluate oxidative stress as a factor in the development of human senile cataract. Emphasis will be placed on studying the mechanism of formation of nuclear cataract, a major cause of loss of lens transparency in the aging human. The hypothesis to be tested is that molecular O2 and UVA light can each contribute to cataract formation in regions of the lens where antioxidant levels are low. Although a number of lens and non-lens studies have implicated UVA radiation as being biologically toxic, the role of this major component of sunlight in cataractogenesis is currently unclear. To investigate oxidative mechanisms in the lens, the PI will use two in vivo guinea pig models involving treatment of the animals with hyperbaric 02 (HBO) and/or UVA light. Each model produces an increase in the level of lens nuclear light scattering combined with damage to nuclear fiber cell membranes. The PI and his collaborators will employ a variety of techniques including SDS-PAGE, HPLC, laser methods in vivo and in vitro, mass spectrometry and infrared spectroscopy to investigate oxidative effects on lens proteins and membrane lipids, and to identify crystallins that have precipitated in the lens because of in vivo oxidative stress. The studies are designed to determine the relative contributions of lens membrane damage and crystallin modifications to the observed loss of nuclear transparency, and to identify reactive species of oxygen that may be involved. In addition, possible HBO-induced myopia in the guinea pig lens will be investigated, and a mouse/vitamin E-deficient/HBO model will be used to search for a link between poor nutrition occurring early in life and subsequent nuclear cataract. In vitro HBO and UVA methods will complement in vivo studies as a means of elucidating oxidative, as well as protective, mechanisms. In the in vitro work, roles for alpha-crystallin and protein-thiol mixed disulfides in preventing crystallin precipitation in 02-stressed lenses will be evaluated, and the mechanism of UVA blockage of pentose shunt activity in H202-challenged cultured lenses will be determined. Quantitative measurement of UVA-induced generation of H202, superoxide anion and singlet oxygen during in vitro irradiation of guinea pig lens proteins will aid in elucidating in vivo mechanisms. Molecular methods will be employed to investigate possible up-regulation of particular lens antioxidant enzymes for the two in vivo models, and for evaluating antioxidant polyphenol compounds as possible anticataract agents. Overall, the studies will provide valuable information on protecting the lens against 02- and UVA-induced damage, and on guarding against the formation of maturity-onset cataract.