The investigators have demonstrated that a number of glutathione (GSH) S-transferase (GST) isoenzymes are present in lens and other ocular tissues.GSTs conjugate toxic electrophilic xenobiotics to GSH and express GSH-peroxidase II activity towards lipid hydroperoxides. They have demonstrated that GSTs can conjugate endogenously generated toxic intermediates formed during lipid peroxidation to GSH. Thus GSTs can disrupt the autocatalytic chain of lipid peroxidation by conjugating lipid epoxides and alpha-beta unsaturated carbonyls (such as hydroxyalkenonals) to GSH and, by reducing lipid hydroperoxides. Since lipid peroxidation may be one of the major contributory factors in cataractogenesis, the """"""""induction"""""""" of GSTs and their substrate GSH could delay cataractogenesis by enhancing the protection of lens against lipid peroxidation. To examine this hypothesis, the investigators have developed an in vitro lens organ culture system and have demonstrated that inclusion of 1-10 Um t-butylated hydroxytoluene (BHT) in lens culture media enhances intracellular levels of GSH, GST and GSH-Px almost two fold by increased de novo synthesis. Using this system, they will establish the optimal conditions of induction of GSH, GST and GSH-Px by BHT and other dietary antioxidants and investigate the effects of antioxidants on individual GST isoenzymes and all the three forms of GSH-Px activities (GSH-Px I, GSH Px II, and the novel GSH Px which uses the peroxides of phospholipids as substrates). They will isolate, characterize, and quantitate GST and GSH-Px isoforms in lenses with and without """"""""induction"""""""" using the techniques of HPLC, FPLC, chromatofocusing, and immunoaffinity chromatography. The investigators have established expertise for these techniques and have required antibodies. The effect of induction of GSH, GST and GSH-Px on lipid peroxidation will be determined by isolating and quantitating the intermediates of lipid peroxidation and their GSH-conjugates by TLC, HPLC, FPLC and by characterizing them through mass, NMR spectroscopy, chemical and enzymatic analysis. They will also evaluate the protective effect of the induction of GSH/GST/GSH-Px system by BHT on naphthalene induced cataractogenesis in brown Norway rats in vivo as well as using in vitro lens cultures. Various GST isoenzymes of ocular tissues, particularly those having higher activity toward toxic intermediates of lipid peroxidation will be characterized. In addition, the GSH-Px isoform which specifically expresses activity towards peroxidized phospholipids (e.g. dilinoeyl phosphatidyl choline-peroxide; DPC-peroxide) will be purified and characterized. These studies will establish the role of GSH, GSTs and related enzymes in the defense against lipid peroxidation and exogenous toxicants and will provide insight into the ways and means by which these detoxification mechanisms could be manipulated to the advantage of lens of delaying/preventing cataractogenesis.
