CONTROL OF CELL DIVISION IN THE OCULAR LENS

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The long objective is to understand changes in the lens epithelium which accompany cataract formation and aging. Cataract surgery is the most frequently performed operation for persons over 60, and senile cataract is the world's leading cause of blindness. Oxidative insult may cause cataracts. Hydrogen peroxide, an oxidant present in aqueous humor, is elevated in the aqueous of humans with cataracts. Immediate goals are to determine the morphological and biochemical events responsible for H2O2-induced injury in young and old rabbit and mouse lens epithelial cells and in human cells from cataractous and non-cataractous individuals. The long-term effect of H2O2 will be evaluated on these cells and on rabbit lenses in vivo and in organ culture. Neither the type of insult induced by H2O2 nor the long-term effects of H2O2 on lenses or on lens epithelial cells is known. This project addresses these unknowns. TEM, SEM as well as probes to specific cytoskeletal proteins will be used to evaluate the morphological response of cells to sublethal doses of H2O2. Specific aims are to: 1) determine if H2O2 induces single-strand DNA breaks and if the number of breaks correlates with the inhibition of mitosis noted in lens cells exposed to H2O2; 2) determine the susceptibility of various phases of the cell cycle to H2O2 insult; 3) determine if H2O2 damage is due to a defect in energy metabolism. We will ascertain the effect of H2O2 on levels of ATP, NAD, GSG, GSSG, on the activities of glyceraldehyde-3- phosphate dehydrogenase, hexokinase, and Na-K-ATPase, on glucose uptake and lactate production, and on the level of specific intermediates of the glycolytic sequence. Changes in phosphate metabolites and pH will be measured in living cells by high resolution NMR spectroscopy; 4) investigate the long-term effect of H2O2 injection into the anterior chamber of young and old rabbits, and determine the level of H2O2 that damages the epithelium and whether this leads to cataract formation; 5) determine the effect of H2O2 on macromolecular synthesis and if H2O2 induces the synthesis of any "new proteins" conferring resistance to H2O2; 6) compare the response of lens cells to H2O2 and hyper baric oxygen; and 7) investigate the effect of age on insulin receptors and tyrosine kinase activity, and determine if insulin or IGF stimulates the phosphorylation of specific proteins. The mitogenicity of FGF, insulin and IGF will be evaluated in cultured cells and in rabbit lenses in organ culture.