4-Hydroxynonenal (4-HNE), a stable end-product of lipid peroxidation (LPO) can cause toxicity through apoptosis and necrosis. During current funded years, we have established that GSTA4-4 and RLIP76 are the major determinants of the intracellular concentrations of 4-HNE. Our preliminary studies show that 4-HNE binds to the death receptor Fas and induces Fas-mediated apoptosis through a novel pathway that is distinct from the canonical pathway in which Fas binds to FADD and recruits procaspase8 to assemble the death inducing signaling complex (DISC) for eventual execution of apoptosis. 4-HNE also binds to Daxx, a transcription repressor and causes its translocation from nucleus to cytoplasm to affect Fas-mediated apoptosis. In addition, 4-HNE promotes phosphorylation and translocation of p53 and activates its pro-apoptotic gene targets. Since exposure to oxidants causes LPO, we hypothesize that part of toxicity of oxidants is contributed by the pro-apoptotic effects of 4-HNE and that the over expression of GSTA4-4 (which catalyzes conjugation of 4-HNE to GSH) and RLIP76 (which mediates transport of GS- HNE conjugate) should attenuate the oxidant-induced toxicity by lowering the intracellular levels of 4-HNE. This hypothesis is supported by our preliminary studies showing that: a) Fas- and p53-mediated apoptosis is inhibited by the over-expression of GSTA4-4. b) Liposomal delivery of RLIP76 to RLIP76 (-/-) mice (which are more sensitive to oxidant toxicity) protects these mice from toxicity.
In Specific Aim 1 this hypothesis will be tested through proposed in-vitro studies using HepG2 cells transfected with Cyp2E1 and two model oxidant compounds, CCl4, and doxorubicin (DOX). In these studies we will also delineate the mechanisms of 4-HNE induced, Fas- mediated apoptosis and the regulatory role of Daxx and GSTs. Since our preliminary studies show that 4-HNE binds to Fas, Daxx, and p53, and affects their functions and/or cellular localization, studies are proposed in Specific Aim 2 to delineate the nature and significance of the interactions between 4-HNE and these proteins.
In Specific Aim 3, we hypothesize that RLIP76 (-/-) mice with impaired capacity to detoxify 4-HNE would be more sensitive to the toxicity of these agents and administration of RLIP76 proteoliposomes should attenuate this toxicity. This will be tested through proposed in vivo studies by comparing the toxicity of these oxidants in RLIP76 (-/-) and RLIP76 (+/+) mice, with and without treatment with RLIP76 proteolipsomes. In these studies we will also compare the 4-HNE levels and the extent of apoptosis in the liver of different groups of mice. Besides providing fundamental novel information on the mechanisms of oxidant toxicity and the role of 4-HNE in oxidative stress associated degenerative disorders, studies proposed in this application should also help to develop prevention strategies against the toxicity of drugs and xenobiotics which is a major clinical and environmental problem.
Proposed studies on the protective role of glutathione transferases and RLIP76 against oxidant toxicity should help in developing strategies for alleviating the toxicity of environmental oxidants and drugs and also for the prevention and treatment of oxidative stress-induced degenerative diseases.
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