Toxic lipid aldehydes such as 4-hydroxynonenal (HNE), formed during lipid peroxidation (LPO), are involved in the etiology of cataractogenesis and age related pathophysiology of ocular tissues. Glutathione (GSH) peroxidases (GPx) and Glutathoine S-transfereases (GSTs) provide defense against LPO. During funded years of current project we have shown that: 1) GST isozymes, GSTA1-1 and GSTA2-2 account for >65% of GPx activity towards lipid hydroperoxides and overexpression of these enzyme in lens epithelial (HLE-B3) or retinal pigment epithelial (RPE) cells attenuates LPO, and protects these cells from oxidant (e.g., H2O2, xanthine/xanthine oxidase (XO/XO, and naphthalene) induced-cytotoxicity, and apoptosis. 2) Overexpression of GSTA4-4 in cells prevents HNE-induced cytotoxicity and apoptosis by detoxifying HNE to form GSH-conjugate (GS-HNE). These findings suggest that GSTs play a major role in the defense against oxidative stress and can affect oxidative stress induced-signaling for apoptosis. To sustain GSTA4-4 mediated detoxification of HNE, GS-HNE must be transported from cells because its accumulation would inhibit GSTs. We have shown that the transport of GS-HNE from HLE-B3 cells is catalyzed primarily by RLIP76. We hypothesize that GSTA4-4 and RLIP76 constitute a major defense against oxidative stress by the HNE and excluding its metabolites (GS-HNE, GS-DHN and GS-HNA) from cells. Also, GSTA4-4 can regulate stress-induced signaling by modulating HNE concentration in cells. To test this hypothesis, we propose:
Specific Aim 1 - To investigate the contribution of GSTA4-4 in detoxification of HNE and its role in prevention of ROS induced opacification of lens. We will compare the cataractogenic effects of naphthalene, and high galactose diet in vivo in GSTA4-4 (-/-), GSTA4-4 transgenic mice (+/+ Lens++), and control mice. We will also compare naphthalene XA/XO, high sugar induced opacification of lens in culture from (-/-), (+/+ Lens++) and control mice.
In Specific Aim 2 - Similar studies as in Specific Aim 1 (using RLIP76 (-/-), RLIP76 (+/+ Lens++), and control mice) will be conduced to investigate the contribution of RLIP76 in defense against ROS and in prevention against oxidative stress-induced cataractogenes. Both GSTA4-4 and RLIP76 knock-out mice have been already developed by us.
In Specific Aim 3, we will examine the effect of the gradual depletion of intracellular HNE (by transiently transfecting HLE-B3 and RPE cells with GSTA4-4) on genes involved in adhesion, cell cycle control and proliferation. These studies would help in devising strategies to prevent/treat age-related degenerative ocular diseases such as cataract and ARMD.
