This project has as its general objective to gain insight into the functions of GSH in the prevention of cataracts. Particular attention is being paid to a mouse cataract model in which L-buthionine-S, R-sulfoximine (L-BSO), an inhibitor of GSH biosynthesis, induces large corticonuclear cataracts when administered in a series of 6-8 injections over a 2-day period, starting approximately at one week of age (standard mouse cataract). The swiftness and reproducibility of this cataract entail initiation of opacification typically on Day 3 of the experiments, shortly after GSH reaches near-zero levels, with disturbance of the lens bow and anterior cortical fiber swelling, followed by lysis of all mature fibers in less than 24 hrs. The biochemical correlates of this process include proteolysis, influx of Na+ and loss of k+, influx of Ca2+ and rapid loss of protein from the lens. It may be generated by observed disturbances in the central lens epithelium that imply compromised energy metabolism and lipid peroxidation. It is believed that this model presents a unique opportunity to study the role of GSH in preventing exudative changes leading to epithelial cell pathology, fiber cell membrane damage and cortical cataracts. Future studies will seek to determine the sequence of biochemical changes preceding gross pathology and possibly coinciding with ultrastructural changes. 2D electrophoresis of proteins will be directed mainly to obtaining information on changes in cytoskeletal and membrane polypeptides, which reside in water-insoluble fractions. Ag staining and radioactive tagging of SH groups will be used to increase sensitivity of detection. The changes observed will be quantified by densitometry. Other early biochemical changes to be observed will include: disulfide formation, levels of ATP and ATPase activities, changes in Na+, K+, Ca+ and their uptake by incubated lenses, mitochondrial and glycolytic activities, malondialdehyde. GSH ester and ascorbate will be used to prevent the cataract and test which biochemical events are essential for its development. Attempts will be made to generate the BSO cataract in vitro after preliminary depletion of GSH with the drug in vivo, in order to enable manipulation of the system when opacification is imminent (e.g., by GSH repletion, variation of Ca2+, protease inhibitors). Shorter incubations will be used to study cation fluxes and phosphorylation of proteins, using radioactive tracers. Alternative BSO cataract models in mouse and rat may be studied if they satisfy specific needs (e.g., large lenses, slower or less serve cataracts). However, the main pursuit of this project in the near future will be to reveal the sequence of changes in lens biochemistry which results in the standard mouse BSO cataract, a uniquely rapid and synchronous in vivo experimental cataract correlated with near total GSH depletion.
