Lipid peroxidation (LPO) caused by oxidative stress is involved in the mechanisms of cataractogenesis and ocular toxicity in general. LPO products, phospholipid hydroperoxides (PL-OOH) and 4-hydroxynonenal (4-HNE) cause cataracts in animal models, and their concentrations are increased in human senile cataracts. Glutathione (GSH) and its associated enzymatic mechanisms provide protection against the toxic manifestations of LPO primarily through free radical scavenging and glutathione peroxidase (GPx) activity. The alpha class glutathione S-transferases (GSTs) constitute the bulk of GST protein in ocular tissues. We hypothesize that the alpha class GST isozymes, hGSTA1-1 and hGSTA2-2 which reduce PL-OOH through their Se-independent GPx activity, and the isozymes, hGST5.8, and hGSTA4-4 which detoxify 4-HNE through its conjugation to GSH, act as the major antioxidant enzymes and protect ocular tissues from the toxic manifestations of LPO. This hypothesis stems from our preliminary studies showing that hGSTA1-1 and hGSTA2-2 can utilize PL-OOH as substrates, while the major Se-dependent enzyme, GPx 1, can not. GSTs contribute more than 60 percent of the total GPx activity toward PL-OOH in liver and cultured human lens epithelial (HLE) cells, and HLE cell transfected with hGSTA1-1 or A2-2 acquire resistance to H202 and naphthalene induced LPO, cytotoxicity, and apoptosis. Likewise, HL-60 cells transfected with mGSTA4-4 acquire resistance to the cytotoxic, and apoptotic effects of 4-HNE, and pro-oxidants which induce LPO. The following Aims are proposed to validate this hypothesis: 1. To quantitate GPx activity of hGSTA1-1, hGSTA2-2 toward PL-OOH in ocular tissues and cultured cells in relation to Se-dependent GPxs. To examine if these isozymes can reduce membrane PL-OOH in situ in ocular tissues and cells in culture. To examine if HLE and RPE cells transfected with hGSTA1-1, hGSTA2-2 are protected from H202 and naphthalene mediated LPO, cytotoxicity and apoptosis. 2. To clone 4-HNE metabolizing isozyme hGST5.8 and evaluate the relative roles of hGST5.8 and hGSTA4-4 (another 4-HNE metabolizing human GST we have cloned) in metabolism of 4-HNE in ocular tissues. To transfect HLE and RPE cells with these enzymes and to examine if their overexpression protects against cytotoxicity, and apoptosis caused by 4-HNE and oxidants such as H202 and naphthalene which promote LPO. 3. To examine if mGSTA4-4 knock out (-/-) mice lacking the ability to metabolize 4-HNE are more susceptible to naphthalene cataract and toxicity of LPO products during iron overload. These studies will help in understanding the role of LPO in cataractogenesis.
