we have demonstrated that serum amyloid a (SAA) activates the NLRP3 inflammasome in an ATP-ROS dependent manner. the hypothesis of this study was that an increase in mitochondrial ROS production enhances NLRP3 S-glutathionylation in SAA-stimulated macrophages. Mouse peritoneal exudate macrophages were treated with antimycin A, a complex III inhibitor, or FCCP, an oxidative phosphorylation uncoupler, after 8h of SAA stimulation and the cells were analyzed after 16h of concomitant treatment. Intracellular and extracellular NLRP3 inflammasome components were analyzed by western blotting, IL-1β secretion was measured by ELISA, and NLRP3 S-glutathionylation was analyzed by immunoprecipitation followed by western blotting. SAA alone induced mild S-glutathionylation of NLRP3, which was substantially enhanced by antimycin a and FCCP. Both antimycin a and FCCP augmented the association between NLRP3 and caspase1. Surprisingly, only FCCP was able to increase IL-1β secretion in SAA-stimulated macrophages. Our data demonstrate that NLRP3 is a target for S-glutathionylation induced by changes in mitochondrial redox status, which may impact NLRP3 inflammasome assembly and activity. Financial support: VLC-CoBRE pilot project. SUNY-Presidential Research Award.

doi:10.1016/j.freeradbiomed.2011.10.372

249 Contribution of Leukocytes to Microvascular Oxidative and Nitrosative Stresses in Endothelial Dysfunction Saptarshi Kar1, and Mahendra Kavdia1 1Wayne State University Endothelial oxidative stress causes biochemical changes within the endothelium due to the complex chemical interactions between the free radicals NO, O2

•– and peroxynitrite, which are not completely understood. the consequences of the biochemical changes within the endothelium include a reduction in NO production, increase in peroxynitrite formation and increase in expression of adhesion molecules in the endothelium. in this study, a biotransport model for the free radicals was developed in the microcirculation with a small number of leukocytes positioned along the luminal surface of the endothelium. the model was simulated for different levels of endothelial oxidative stress with the leukocytes considered inactive and activate depending on the case simulated. the simulation results predict the NO, O2

•– and peroxynitrite concentration profiles within the blood vessel and the leukocytes. the key results for this study were a) Activation of the leukocytes causes significant increase in O2

•– and peroxynitrite concentration in the lumen, endothelium and smooth muscle region compared to normal physiology, b) Transition from normal physiology to endothelial oxidative stress causes a 270% increase in peroxynitrite concentration and a 411% increase in O2

•– within the leukocytes and c) in comparison to normal physiology, activation of leukocytes increases its O2

•– and peroxynitrite concentration by 6900 and 3018%, respectively. the results from this study show that increase in O2

•– and peroxynitrite concentration within the leukocytes help in their priming (through peroxynitrite) and activation (secondary stimuli through O2

•–). Furthermore, the increased luminal O2

•– and peroxynitrite concentration provide an explanation to the previous experimental findings that leukocyte rolling/adhesion and activation are independent events. Supported by NIH R01 HL084337.

doi:10.1016/j.freeradbiomed.2011.10.373

250 Regular Exercise Ameliorates Redox and Inflammatory Signaling in Human Peripheral Blood Mononuclear Cells Si-Young Kim1 1Sejong University There has been considerable accumulation of evidence supporting that exhaustive exercise causes oxidative stress, which may lead to various disorders as well as inflammation. However, it is considered that regular exercise can attenuate oxidative stress and inflammation. in the present study, we investigated the effects of one-bout exhaustive exercise and long-term regular exercise on oxidative stress and inflammatory responses in human peripheral blood mononuclear cells (PBMCs). in the first experiment, twenty volunteers who participated in the guided exercise program were subjected to progressive exercise until they were exhausted on the treadmill followed by resting conditions. Isolated human PBMCs were collected immediately following exercise and after 1-h recovery. One-bout exhaustive exercise induced expression of glutamate-cysteine ligase catalytic subunit (GCLC), interleukin 1β (IL-1β), and cyclooxgenase 2 (COX-2) as well as nuclear factor kappa B (NF-κB) DNA binding activity and phosphorylation of both IKKα and IκBα in PBMCs. Initial induction of the above oxidative signaling and pro-inflammatory response fully returned to the basal level during 1 h of recovery. in a follow-up experiment, twenty men who participated in the exercise training of different two intensities (40% and 80% of VO2max) for 4 times per week for 12 weeks were randomly allocated to low- and high-intensity exercise groups (n=10; each group). After 12 weeks, the expression of IL-1β, p-IKKα, and p-IκBα in the high-intensity exercise group was significantly decreased in comparison with control and low-intensity exercise groups. On the other hand, there were not significant differences in NF-κB DNA binding activity and COX-2 expression among all groups, and the levels of glutathione and GCLC in the high-intensity exercise group were statistically higher than those in the control group. Taking all these findings into account, although the one-bout exhaustive exercise transiently induced NF-κB signaling and subsequent inflammatory response, regular exercise may potentiate cellular antioxidant and anti-inflammatory capabilities.

doi:10.1016/j.freeradbiomed.2011.10.374 251 Mitochondrial ROS Modulate Gene Expression Induced by Inflammatory Mediators Andrey Kozlov1, J. Catharina Duvigneau2, Ingeborg Kehrer1, Jamile Paier-Pourani1, Andrea Muellebner2, Lothar Lauterboeck2, and Heinz Redl1 1L. Boltzmann Inst. Exper. Clin. Traumatol., 2University of Veterinary Medicine Systemic inflammatory response induced by LPS results in a number of pathologic events including increased generation of mitochondrial O2

•– and an up-regulation of genes related to inflammation (IL-6, iNOS) and to ER stress (GRP78, XBP1) occurring in liver and other organs. the aim of this study was to elucidate whether or not ROS rising from mitochondrial O2

•– modulate the expression of these genes in hepatocyte culture exposed to inflammatory mediators (IM). Using laser scan microscopy we detected low levels of ROS in mitochondria (mROS) but not in cytoplasm of control hepatocytes (using fluorescent dyes CH2Xros and DCF-DA, respectively). Treatment of hepatocytes with IM elevated both mROS and cytoplasmic ROS. Expression of IL-6 and iNOS (determined by qPCR)

SFRBM 2011 S105

occurred prior to the increase in ROS levels, suggesting that mROS do not participate in the initial induction of IL-6 and iNOS expression. To investigate whether mROS are able to modulate mRNA levels, we analyzed the effects of Mito-TEMPO, a mitochondria targeted antioxidant. Mito-TEMPO decreased specifically mROS levels, and simultaneously decreased the levels of IL-6 and iNOS, but upregulated the levels of markers of ER stress. These data suggest that mROS interfere differentially with the signalling cascades leading both to up- and down-regulation of genes at mRNA level.

doi:10.1016/j.freeradbiomed.2011.10.375

252 Are Biodiesel (B20) Emissions Less Harmful than Petrodiesel (B0)? Muyao Li1, Matthew E Poynter1, Brian C Palmer1, Erin Parker1, Brooke T Mossman2, Britt a Holmen3, and Naomi K Fukagawa1 1Department of Medicine, College of Medicine, 2Department of Pathology, College of Medicine, 3School of Engineering, University of Vermont Combustion of biodiesel fuels is commonly perceived to be less harmful than petrodiesel fuel. However, the biological effects of biodiesel exhaust and the potential impact on human health remain uncertain. We compared the effects of exhaust particles generated by a Volkswagen light-duty diesel engine fueled by a blend of 20% soy biodiesel and 80% petrodiesel (B20) to those generated from combustion of 100 % petrodiesel (B0). Particles were added for 24 hr at 10 and 20 µg/ml in vitro to cell lines representing human lung epithelial cells (BEAS-2B) and macrophages (THP-1). in addition, C57BL/6 mice were exposed in vivo to the same particles by oropharyngeal aspiration (~ 80 µg/dose) for 3 consecutive days. Particles derived from both types of fuel had minimal cytotoxicity in the two cell lines. However, cytokine concentrations in the medium of B20-treated cells and in bronchoalveolar lavage fluid (BALF) of animals exposed to B20 were significantly higher than after treatment with B0 (e.g. G-CSF in THP-1 cells and IL-8 in BEAS-2B cells, as well as G-CSF, IP-10, IL-6 in BALF and lung tissues of the mice). Furthermore, production of Toll-like receptor (TLR4) by THP-1 cells and activation of ERK1/2 in BEAS-2B cells was indicative of pro-inflammatory responses after B20 exposure although differences were not found in lung tissue. in cells, B20 did not induce more oxidative stress than B0 after 24 hours exposure, but in vivo there was a trend towards increased protein oxidation in lung tissue and lower total glutathione content in blood after B20 treatment. These findings suggest a potential risk for adverse health outcomes after B20 exhaust exposure. Indeed, these data show that the addition of 20% soy biodiesel resulted in the formation of smaller ultrafine particles compared to the baseline B0 fuel, with added fatty acid methyl esters, carboxylic acids and aldehydes only detected in the B20 particle samples. Further analysis of the ultrafine particles, even soluble components, may shed light on the likely components in biofuel emissions that lead to a more inflammatory profile.

doi:10.1016/j.freeradbiomed.2011.10.376

253 Role of NADPH Oxidase and Renin-Angiotensin System in Intermittent Hypoxia-Induced Inflammation in Rat Adrenal Medulla Yu Liu1, and Man Lung Fung1 1Department of Physiology, the University of Hong Kong We have shown that intermittent hypoxia (IH) associated with recurrent apnea induces oxidative stress and inflammation in rat adrenal medulla. However the pathogenic mechanism is not clear at present. We hypothesized that the expression of NADPH oxidase (NOX) induced by renin-angiotensin system (RAS) plays a role in the tissue inflammation during chronic IH. Adult SD rats were exposed to air [normoxic (Nx) control] or IH treatment (8 hrs/day) which mimicked a severe recurrent sleep apneic condition for 14 days. Injections of apocynin, an inhibitor of NOX, (25 mg/kg i.p.) or vehicle were performed before the IH treatment every day. the mRNA levels of NOX subunits p22-phox, NOX-2 and NOX-4 and the protein expressions of IL-6, TNF-α and COX-2 were examined by RT-PCR and ELISA. the protein expressions of NOX-4 and RAS components (AGT, AT1 and AT2) were examined by Western blot. Our results showed that the protein expression of IL-6, TNF-α and COX-2 were significantly higher in the IH group than that of the Nx and apocynin-treated hypoxic (AIH) group. the mRNA levels of p22-phox, NOX-2 and NOX-4 were also increased markedly in the IH group, when compared with other two groups. in addition, IH treatment significantly induced the protein expression of NOX-4. Furthermore, the protein expressions of AGT, AT2 and AT2 were increased in the IH group, indicating that the up-regulation of NOX may be induced by the increased RAS expression. in conclusion, we showed that NOX plays a pathogenic role in the IH-induced local inflammation in rat adrenal medulla.

doi:10.1016/j.freeradbiomed.2011.10.377 254 Reactive Oxygen Species and the Macroalgal Wound Response Ruth E. McDowell1, Charles D. Amsler1, James B. McClintock1, and Bill J. Baker2 1University of Alabama at Birmingham, 2University of South Florida The vertebrate immune system uses reactive oxygen species (ROS) produced by cells involved in the innate immune response to defend against invading pathogens, notably bacteria. As such, ROS are an important feature of the vertebrate inflammatory wound response and likely have been for millions of years. ROS are analogously involved in defense against pathogens in some of the algae, which represent eukaryotic lineages separated from animals by over 1 billion years. Although studying the conserved features of wounding and immunity in these distantly related organisms can provide new insights into vertebrate innate immunity, the production of reactive species in macroalgae (seaweeds) upon wounding is uncharacterized. Here we show that the release of strong oxidants is common after macroalgal wounding where we studied along the Western Antarctic Peninsula. 80% of species studied released strong oxidants within 1 minute of wounding, and cellular production of strong oxidants remained common over an hour post-wounding. Despite the fact that hydrogen peroxide is a major contributor to the oxidants detected from wounded microalgae and terrestrial (vascular) plants, peroxide does not appear to be a common component of the Antarctic macroalgal wound response. the characteristics of the wound response in the species studied are diverse and vary in magnitude, duration, and the identity of oxidants produced,

SFRBM 2011S106