The PI had previously proposed to investigate the physiological roles of glutathione (GSH) S-transferase and GSH-peroxidases in protection mechanisms against the deleterious effects of lipid peroxidation in ocular tissues. During this funded period, he discovered that several GSH isozymes belonging to a novel group of GSTs are differentially expressed in human and bovine ocular tissues. GST isozymes of this novel group, provisionally designated as GSTs 5.8, express high activities towards 4-hydroxyalkenals and fatty expoxides. In addition, these isozymes express GSH-peroxidase activity towards phospholipid hydroperoxides. High activities of novel GST 5.8 isozymes toward these toxic products of lipid peroxidation suggest an important role of these isozymes in protective mechanisms against lipid peroxidation in ocular tissues. The PI's preliminary studies have also shown that micromolar concentrations of 4-hydroxynonenal (4-HNE) in culture medium produce opacification in lenses in organ culture, and the induction of GST 5.8 in lens epithelium by t-butylated hydroxytoluene (BHT) attenuates 4-HNE mediated opacification of lenses. In this proposal, the PI will test the hypothesis that a novel group of GST isozymes (GSTs 5.8) which are differentially expressed in ocular tissues are specifically involved in detoxification of the toxic products of lipid peroxidation and that their induction provides added protection against the toxicants formed during lipid peroxidation. He proposes to purify GST 5.8 isozymes from human, bovine, and rat ocular tissues and determine their primary structures through amino acid sequencing, and study their kinetic parameters towards 4-hydroxyalkenals, lipid hydroperoxides and epoxides. In vivo concentrations of these toxic products of lipid peroxidation will also be determined to co-relate their kinetic parameters towards GSTs 5.8 in order to elucidate the physiological roles of GSTs 5.8. The cDNA of GSTs 5.8 will be cloned, sequenced and the genetic interrelationships among various ocular GSTs 5.8 will be determined and their interrelationships will be elucidated. Using immunohistochemical and in situ hybridization techniques, their localization in ocular tissues will be established. Since the inclusion of 4-HNE/hydroperoxides in culture medium induces opacity in lenses in organ cultures, we will optimize conditions for 4-HNE/hydroperoxide mediated opacification of lenses in culture. Since his preliminary studies have indicated that BHT in culture medium attenuates 4-HNE mediated cataractogenesis, the PI will conduct experiments to co-relate the protective effect of BHT to the induction of specific GST isozymes through analyses of GST isozymes in control and BHT treated lenses and estimation of the GSH-conjugates of lipid peroxidation products. These studies will establish the protective role of GSTs 5.8 against oxidant/lipid peroxidation mediated cataractogenesis and help in devising strategies for prevention of cataractogenesis and other pathophysiologies in ocular tissues caused by lipid peroxidation.
