Thérèse Rouleau1 1Université de Montréal, Canada Introduction: Hypoalveolarization is a main characteristic of bronchopulmonary dysplasia (BPD) that affects 50% of infants born < 28 weeks of gestation. Animal studies report that ascorbylperoxide (AscOOH) generated in parenteral nutrition (PN) devoid of light protection induces oxidized redox potential and loss of alveoli following an exaggerated apoptosis. Because AscOOH is a substrate of glutathione peroxidase, we hypothesized that improving of the in vivo glutathione level can limit BPD development. The aim was to assess in newborn animals that addition of glutathione in PN prevents the deleterious effects of AscOOH. Methods: Guinea pigs (n = 40) aged of 3 days were divided in 6 groups to receive the following intravenous solutions through a catheter in jugular vein. Sham: animals fed regular chow, catheter was closed; PN(-L): animals fed exclusively with parenteral nutrition (dextrose, amino acids, lipids, vitamins), PN was light-protected by opaque materials; PN(+L): PN without photo-

PN(+L)+GS after 4 days

of continuous infusion, lungs were collected and prepared for determinations of redox potential (mV) calculated from GSH and GSSG concentrations (capillary electrophoresis), activated caspase-3 (apoptosis) (Western blot) and alveolarization (average number of intercepts between a 1 mm line and histological structures from 4 fields / animal). ANOVA, p<0.05. Results: Plasma glutathione in PN(+L)+GSSG was higher than in PN(+L) group (0.5 0.2 vs. 14.2 0.4 M, p<0.01). Results from PN(-L) were not different than those from Sham. The presence of GSSG has corrected (p<0.05) the modifications induced by PN(+L) or AscOOH (see Table).

Mean S.E.M. Redox Apoptosis

Alveolarization Sham -212 2 0.18 0.0

2 34 2

PN(-L) -211 3 0.19 0.04

32 1 PN(+L) -

204 1* 0.29 0.03*

28 1* PN(+L)+GSSG -

211 3* 0.16 0.02*

32 1* AscOOH -

205 0.31 0.0

27

AscOOH+GSSG -213

0.21 0.0

31 *: p<0.05 in PN(+L) 0.05 in AscOOH GSSG. Conclusion: Addition of GSSG in PN could prevent deleterious effect of ascorbylperoxide on lung development of preterm infants by correcting their well-known low glutathione level.

400 Attenuation of GSTP1 Decreases Fas S-Glutathionylation-Mediated Lung Epithelial Cell Apoptosis and Fibrotic Remodeling David McMillan1, Karolyn Lahue1, James Nolin1, Sidra Hoffman1, Sarah Abdalla1, David Chapman1, Jos van der Velden1, Vikas Anathy1, and Yvonne Janssen-Heininger1 1University of Vermont, USA Pulmonary fibrosis is a debilitating lung disease characterized by

excessive epithelial cell death, fibroblast activation, and collagen production. The death receptor Fas induces lung epithelial cell death through caspases 8 and 3, and our group has previously demonstrated that apoptosis occurs following S-glutathionylation of Fas (Fas-SSG) at cysteine 294 in these cells [1]. S-glutathionylation, the conjugation of glutathione to reactive cysteines, is catalyzed in part by glutathione S-transferase pi 1 (GSTP1). To date, little is known about the role of GSTP1 in pulmonary fibrosis. We hypothesized that GSTP1 is a critical mediator of Fas-SSG-mediated apoptosis in lung epithelial cells and fibrosis. GSTP1 inhibition and knockout were analyzed in the

of lung fibrosis. To block GSTP1, the inhibitor TLK199 was administered oropharyngeally to mice starting 14 days post-delivery of

nd 28. GSTP-

/- mice and their wild-type littermates were also euthanized on days 14 and 28 post- of collagen content, fibrosis markers, caspase activity and Fas-SSG. Bleomycin- -induced collagen production was significantly attenuated following both GSTP1 inhibition and knockout at 14 and 28 days. Moreover, fibrosis markers including Col1A1, Col5A1, FN1, and FSP1 were also attenuated in GSTP-/- and TLK199-treated mice. Bleomycin-induced Fas-SSG was also decreased in the lungs of TLK199-treated mice. Our results demonstrate that GSTP1-catalyzed S-glutathionylation may be an important driver of lung fibrosis and that Fas is an important target in this process. Our findings also point to the therapeutic potential of TLK199 for fibroproliferative lung diseases based on the observed reversal of existing collagen increases. [1] Anathy, et al. J Cell Biol, 2009 This research was funded by NIH grants R01HL079331 and 2T32HL076122-11A1

401 Oxygen and Parenteral Nutrition, Two Main Oxidants, in the Neonatal Intensive Care: it All Adds Up Ibrahim S. Mohamed1, Wesam Elremaly1, Thérèse Rouleau1, and Jean-Claude Lavoie1 1Université de Montréal, Canada Introduction: Bronchopulmonary dysplasia (BPD) affects near 50% of infants born < 29 weeks of gestation. Oxidative stress is suspected. The hypothesis was that the two main sources of oxidant load, which are oxygen supplement (FiO2>21%) and peroxides contaminating parenteral nutrition (PN), have additive effects on oxidative stress and BPD. The objective was to assess the effect of early exposure to O2 and PN on oxidative stress at 36 weeks post-menstrual age (PMA) and on BPD in preterm infants. Study design: A prospective observational study including 116 infants < 29 weeks of gestation. Baseline clinical characteristics, FiO2 on day 7, duration of PN and clinical outcomes data were collected. In 39 infants, whole blood GSH and GSSG at 36 weeks PMA (at time of BPD diagnosis) were measured to calculate the redo t-test, Chi-square, ANOVA analyses were used as appropriate, P<0.05 was considered significant. Results: FiO2 higher level of GSSG and oxidized redox potential.

FiO2<25% FiO2 PN>14 d GSHa 7.6 (0.5) 7.4 (0.6) 7.5 (1.2) 7.5 (0.4) GSSGa 0.18

(0.02) 0.29 (0.04)*

0.13 (0.02)

0.26 (0.03)*

Redoxb -198 (2) -191 (2)* -203 (5) -193 (2)*

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doi: 10.1016/j.freeradbiomed.2014.10.049

Mean (s.e.m.); aGSH, GSSG are in nmol/mg of protein, bRedox potential in mV. *=P<0.05 Infants with PN duration > 14 days and FiO2 oxidized redox potential (-190.7±2.3 mV) than infants with PN duration > 14 days but FiO2< 25% (-196.5±2.2 mV) and those

2< 25% were most reduced (-203.6±6.1 mV), suggesting an additive effect, ANOVA (P = 0.03). The effect of both FiO2 and PN on BPD was:

PN > 14days Total FiO2 < 25% 5/27 (19%) 21/27 (78%) 54 FiO2 3/7 (43%) 35/35 (100%) 42 Total 33 62 95

Conclusion: Early O2 supplement and PN have additive effects that induce prolonged oxidative stress and more BPD. Strategies targeting judicious use of O2 and either decreasing the duration or developing a safer formulation of PN can be targeted to decrease BPD.

402 Glutaredoxin 1 Controls the Extent of Collagen Deposition and TGF Signaling in a House Dust Mite Mouse Model of Allergic Airway Disease James D Nolin1, Sidra M Hoffman1, Karolyn G Lahue1, David H McMillan1, Jos L van der Velden1, and Yvonne MW Janssen-Heininger1 1University of Vermont, USA Asthma is a multi-faceted disease accompanied by loss of epithelial integrity leading to remodeling of the airways. Alterations in the redox environment, including glutathione, have been reported in asthma. Glutathione can be conjugated to protein cysteines to control protein function in a process known as glutathionylation. The thioltransferase, glutaredoxin 1 (Grx1), deglutathionylates proteins under physiological conditions, restoring sulfhydryl groups. Using the ovalbumin model of allergic airways disease, Grx1 deficient mice were shown to have faster resolution of disease compared to WT mice [1]. In the present study, we determined the role of Grx1 in a house dust mite (HDM) model of allergic airway disease, using three complimentary approaches: mice globally lacking Grx1 (Glrx1-/-), an airway epithelial specific Grx1 over expressing mouse (Glrx1 Tg), and WT mice in which either WT or a catalytically inactive mutant form (Mut) of recombinant Grx1 (rGrx1) protein was administered. In response to HDM, Glrx1-/- mice demonstrated enhanced protein S-glutathionylation, airway hyperresponsiveness (AHR), as well as elevated collagen deposition. Increases in phosphorylated Smad3 (pSmad3) as well as nuclear Smad4, both markers of

he lungs of Glrx1-/- mice. In contrast, Glrx1 Tg mice showed decreased AHR, dampened collagen production following HDM exposure. Administration of WT rGrx1 into airways following HDM challenge resulted in decreases in collagen deposition, while Mut rGrx1 had no impact on HDM-induced collagen production. Together, these findings demonstrate the importance of Grx1 and HDM-induced fibrotic

features associated with severe asthma. [1] Hoffman, A. J. Physiol. 2012 Funding: R01 HL060014, T32 ES007122-30

403 Regulation of Aberrant Expression of EC-SOD in Human IPAH by Epigenetic Mechanisms Crystal Woods1, Sujatha Venkataraman1, Robert Stearman1, Gary Mouradian1, Leah Villegas1, Russell Bowler2, Timothy McKinsey1, Bradley Ferguson1, Kaori Ihida-Stansbury3, Mark Geraci1, Kurt Stenmark1, Frederick Domann4, and Eva Nozik-Grayck1 1University of Colorado Anschutz Medical Center, USA, 2National Jewish Health, USA, 3University of Pennyslvania, USA, 4University of Iowa, USA Epigenetic mechanisms, including DNA methylation and histone acetylation, regulate gene expression in idiopathic pulmonary arterial hypertension (IPAH). Extracellular superoxide dismutase (EC-SOD) is a key vascular antioxidant enzyme that may be down-regulated in IPAH. Epigenetic mechanisms regulate cell-specific differences in the expression of lung EC-SOD and loss of EC-SOD in lung cancer, though the contribution of EC-SOD promoter methylation or deacetylation to low EC-SOD expression in IPAH is unknown. We hypothesized that EC-SOD gene is epigenetically silenced by aberrant DNA methylation and/or histone deacetylation, resulting in decreased expression of EC-SOD in patients with IPAH. We used lung tissue and PASMC provided by the Pulmonary Hypertension Breakthrough Initiative from subjects with IPAH at transplantation and failed donors (FD). Lung EC-SOD mRNA expression was decreased by 40% in IPAH vs FD (n=16, p<0.05). EC-SOD mRNA and protein in PASMC was also decreased in IPAH vs FD (n=4-6). In genomic lung DNA, % methylation at 18 CpG sites in the EC-SOD promoter was tested by bisulfite sequencing. The methylation status of the EC-SOD promoter was highly variable and not significantly different between FD and IPAH. Furthermore, EC-SOD mRNA was unchanged in IPAH PASMC after treatment with 5-azaC, suggesting DNA methylation was not responsible for low EC-SOD. In contrast, 24 hour treatment with TSA, a histone deacetylase activity (HDAC) inhibitor, increased EC-SOD mRNA 2-fold in IPAH PASMC, while treatment in FD PASMC had no effect. IPAH PASMC proliferate faster than FD PASMC. Using the xCELLigence assay (Roche), the doubling time increased in IPAH PASMC treated with TSA, while TSA did not significantly change doubling time in FD PASMC. TSA also decreased migration more in IPAH vs FD PASMC (n=2-3). Though HDAC isoform expression was variable between IPAH and FD PASMC, HDAC1 and 2a activity was increased in all 6 IPAH PASMC compared to 4 of the 6 FD PASMC. These data suggest that the decrease in EC-SOD gene expression in PAH is not likely regulated by DNA methylation but may be regulated by loss of histone acetylation due to enhanced HDAC activity.

404 Chlorinated Fatty Acids Are Biomarkers and Potential Mediators of Chlorine Gas Toxicity Joo-Yeun Oh1, Jaideep Honavar1, Stephen Doran1, Gloria Benavides1, Victor M. Darley-Usmar1, Sadis Matalon1, David Ford2, and Rakesh P. Patel1 1Center for Free Radical Biology, University of Alabama at Birmingham, USA, 2Center for Cardiovascular Reseach, Saint Louis University, USA Accidental or intentional exposure to chlorine gas results in significant toxicity that comprises during- and post-exposure phase. The latter occurs over hours to weeks resulting in acute lung injury, reactive airways, pulmonary fibrosis and pulmonary and systemic vascular dysfunction, that is characterized by inflammation and dysfunction in endothelial nitric oxide synthase

S165SFRBM 2014

doi: 10.1016/j.freeradbiomed.2014.10.050

doi: 10.1016/j.freeradbiomed.2014.10.051

doi: 10.1016/j.freeradbiomed.2014.10.052