The ocular tissues are constantly exposed to toxic substances present in the environment and those produced within the tissues. Glutathione (GSH) plays an important role in protecting the ocular tissues from the oxidative stress due to endogenous oxidants, as well as from the environmental pollutants, and xenobiotics. One of the major defense systems against the toxic oxidants and xenobiotics involves GSH S-transferases (GST). GST represent a family of multifunctional enzymes which catalyze the conjugation of the electrophilic xenobiotics and their metabolites to GSH. These conjugates can then be metabolized to water soluble, non-toxic mercapturic acids by the enzymes of mercapturic acid pathway. GST can also non-catalytically bind the toxic xenobiotics, thereby reduce their toxic effects by removing them from circulation. Multiple forms of GST isoenzyme are present in the ocular tissues, some of which can also reduce the lipid hydroperoxides through their GSH peroxidase II activity. The studies are aimed at establishing the role of GST in the detoxification mechanisms of lens and other ocular tissues. The multiple isoenzymes of GST present in bovine, human, and rat lens, retina, and cornea will be purified using affinity chromatography, isoelectric focusing, and HPLC. The isoenzymes and their individual subunits isolated by in vitro hybridization will then be characterized for their structural, catalytic (conjugating and hydroperoxide reducing activity), immunological, and non-catalytic binding properties. Since we have demonstrated that dietary antioxidants t-butylated hydroxytoluene (BHT) and t-butylated hydroxyanisole (BHA), induce GST in rat lens, a detailed study of the effect of these antioxidants on the induction of individual isoenzymes and subunits of GST and also on the enzymes of mixed function oxygenase system in ocular tissues will be performed. The study of the differential induction of subunits will define the role of individual subunits of GST in the conjugation of xenobiotics, reduction of lipid hydroperoxides, and chemical removal of xenobiotics by non-catalytic binding. The enzymes of mercapturic acid pathway, particularly the cysteinyl glycinase and N-acetyltransferase and those of mixed function oxygenase system will also be characterized. The studies will provide a better understanding of the mechanisms by which the ocular tissues defend themselves from the chemical and oxidative stress.
