346 4-HNE Significantly Alters L-FABP Structural and Functional Dynamics Rebecca L Smathers1, Philip Reigan1, Kristofer S Fritz1, James J Galligan1, Colin T Shearn1, and Dennis R Petersen1 1University of Colorado Anschutz Medical Campus Lipid peroxidation (LPO) is implicated in the pathogenesis of various diseases involved with chronic oxidative stress – including diabetes, Alzheimer's, Parkinson’s, Non-Alcoholic Fatty Liver Disease and Alcoholic Liver Disease (ALD). Reactive aldehydes generated from LPO, such as 4-hydroxynonenal (4-HNE), often result in protein modification through adduction of protein side chains. the specificity of these modifications is protein-dependent, frequently posing detriment to conformational structure, stability and function. Utilizing a proteomic wide scan for 4–HNE modified proteins, liver fatty acid binding protein (L-FABP) was found in hepatic cytosolic fractions from both rats and mice chronically fed an ethanol-containing diet. Sites of modification were identified by MALDI-TOF/TOF mass spectrometry in both the lipid-bound (holo) and unbound (apo) states of recombinant mouse L-FABP in vitro. Two adducts (Lys57 andCys69) were found on the periphery of apo-L-FABP, while six peripheral adducts (Lys6, Lys31, His 43, Lys46, Lys57 and Cys69) were found on holo-L-FABP. Molecular modeling, in conjunction with binding and stability assays, has provided novel insights into the consequences of L-FABP adduction by 4-HNE. These modifications were found to significantly reduce the area, shape and structural integrity of the binding pocket and ligand portals. Furthermore, a fourth, solvent accessible, binding portal for ligand entry/exit in holo L-FABP was identified post-adduction. the dynamics of these modeling simulations is supported with in vitro data, showing decreased stability of adducted protein in the apo state and decreased capacity and affinity for multiple fatty acids. Collectively, these data demonstrate the consequences of L-FABP adduction by 4-HNE in silico and in vitro – revealing insight into the complex molecular dynamics involved in protein modification via reactive aldehydes. Supported by R37 NIH/AA009300 (DRP) and NIH/AAA F31 AA18898-03 (RLS). doi:10.1016/j.freeradbiomed.2011.10.298

347 Effect of Hydrogen Sulfide On Cellular Bioenergetics Asaf Stein1, Zhengkuan Mao1, Angela Betancourt1, and Shannon Bailey1 1University of Alabama at Birmingham Hydrogen sulfide (H2S) controls biological processes associated with cardiovascular regulation, hepatic function, inflammation, and mitochondrial bioenergetics. H2S, like nitric oxide, can inhibit mitochondrial respiration by binding to cytochrome c oxidase (CcO). Moreover, at lower concentrations, H2S can induce a reversible hypometabolic state in non-hibernating rodents. the ability to reversibly slow metabolism on a cellular level highlights the therapeutic potential of H2S in treatment of ischemia-reperfusion and for protection of organs used in transplantation. To better understand the effect of H2S on mitochondrial function, cellular bioenergetics was measured in Huh7 cells (liver hepatoma cells) and primary rat hepatocytes after exposure to sodium hydrosulfide (NaHS). Cellular bioenergetics was assessed using the XF24 Analyzer (Seahorse Biosciences) in cells treated with the ‘sulfide donor’ NaHS. Incubation with NaHS decreased cellular O2 consumption rate (OCR) in both liver cell types presumably through H2S generation and subsequent inhibition of CcO. Inhibition of OCR exhibited some dose-dependence in Huh7 cells, with higher [NaHS] resulting in larger decreases in OCR. Similar results were found with primary hepatocytes. At higher [NaHS] (500 µM), both basal and maximal respiration (FCCP-stimulated) was inhibited. Respiration remained depressed in cells 6 hr post-NaHS treatment. These data support the concept

that physiological effects of H2S may be mediated through modulation of mitochondrial function. Time course studies are warranted to better understand the molecular effects of H2S on cellular bioenergetics. Continued study of H2S-induced hypometabolism at the cellular level will yield knowledge necessary for moving H2S-based therapies from the bench to the bedside.

doi:10.1016/j.freeradbiomed.2011.10.299 348 Examination of the Effects of Gamma-Irradiation of Cigarettes On the Induction of Oxidative Stress by Cigarette Smoke Particulate Matter. Mark Taylor1, Michelle Lawton1, Tony Carr1, Natalia Cockcroft1, Emma Bishop1, and Ian M Fearon1 1British American Tobacco, Southampton, U.K. Introduction: a number of patents have suggested the use of γ-irradiation of either tobacco or manufactured cigarettes as a method of reducing the toxicity and disease potential of cigarette smoke. in this study we have investigated the use of post-production γ-irradiation of cigarettes as a potential harm reduction technique by exposing in vitro models of smoking-related diseases and disease processes to cigarette smoke extracts derived from γ-irradiated cigarettes. Methods: Cultured lung epithelial (H292) and THP-1 monocytic cells were exposed to particulate matter (PM) from γ-irradiated (10 kGy) and non-irradiated 3R4F reference cigarettes. After exposure, the levels of pro-inflammatory cytokines secreted into the culture media were assessed. in H292 cells we also examined the levels intracellular reduced glutathione (GSH) using the GSH-GloTM assay (Promega). Machine-smoked toxicant yields were measured using standard analytical techniques. Results: PM caused a dose-dependent decrease in GSH levels alongside dose-dependent increases in the levels of MCP-1 and IL-8 in H292 cells. PM also caused an increase in IL-6 production by THP-1 cells. Each of these effects was significantly reduced when the PM was derived from γ-irradiated cigarettes. These effects were not associated with changes in smoke toxicant yields in the irradiated cigarettes. Conclusion: Cigarette smoke PM-induced oxidative stress and the production of inflammatory proteins was reduced by γ-irradiation of cigarettes. Further studies are required to elucidate the mechanisms underlying these altered responses.

doi:10.1016/j.freeradbiomed.2011.10.300 349 Oxidative Stress during Storage of Packed Red Blood Cells Amen Florencia1, Florencia Tomasina1, Ana Denicola1,2, Cristina Touriño3, and Leonor Thomson1,2 1Facultad de Ciencias, 2and Center for Free Radical and Biomedical Research, 3Facultad de Medicina, Universidad de la República During storage, red blood cells (RBC) undergo a series of structural and functional alterations collectively known as storage lesions. These include the loss of discoid morphology, membrane changes with increased phosphatidyl serine exposure, vesicle formation, and eventually lysis. Stored units become more acidotic, and higher concentrations of free hemoglobin in the suspension liquid, together with the presence of bioactive lipids

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