Reactive oxygen species (ROS) causing oxidative stress to the ocular lens have been implicated in cataracts of various etiologies. However, the mechanism of how the ROS propagate the injury is not well established. Based upon our demonstration that under oxidative stress, a large amount of lipid-derived aldehydes (LDAs), especially 4-hydroxynonenal, is formed in the ocular lens, we hypothesize that LDAs act as toxic messengers that mediate the injurious cataractogenic effects of ROS. We propose that LDAs extend oxidative injury in the lens by causing modifications to membrane proteins (including gap junction and channel proteins), resulting in altered membrane fluidity and calcium homeostasis, leading to apoptosis and cataract. Since LDAs are extremely reactive, exact measurement of their in vivo concentration has been problematic. To quantitate LDAs formed under oxidative stress in rat lens, and assess whether scavenging of LDAs prevents the formation of protein-LDA adducts we will use a cell-permeable tetrapeptide (N-acetyl-Asn-Tyr-Thr-Cys-NH2; NYTC) which readily binds LDAs. The 125I-NYTC-LDA conjugates will be separated by HPLC, quantified by radioactivity determination and characterized by ESI-MS (Aim 1). To understand the mechanism(s) of LDA-mediated injury, we will quantify and identify protein-LDA adducts in cataractous lenses, and will correlate the extent of protein modification to alterations in membrane fluidity, calcium homeostasis and induction of apoptosis and assess whether scavenging of LDAs by NYTC prevents the injury (Aim 2). We have recently shown that LDAs are cataractogenic. However, the metabolic pathways through which the lens detoxifies LDAs, and how these toxicants cause cataract, are not known. Therefore, we will identify the major metabolites of LDAs and assess the relative significance of individual metabolic pathways for LDAs in the ocular lens. We will further investigate whether the metabolism of LDAs in the lens is altered with age and/or hyperglycemia (Aim 3). We will also augment the metabolism of LDAs in a human lens epithelial cell line (HLEC) by overexpressing a relevant glutathione-S-transferase isozyme (which conjugates the LDAs to GSH), and aldehyde dehydrogenase (which catalyzes LDA oxidation), and ask whether such augmentation abrogates oxidative injury (Aim 4). Completion of our proposal will clearly establish the role of oxidative stress in the age related cataractogenesis, especially the mechanism through which ROS-induced injury is propagated. The use of NYTC should provide the basis for novel and more targeted therapeutic strategies for ameliorating cataractogenesis.
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