Further, we found that in the MCT-treated rats there was an increase in covalently modified EGFR dimers formed via a dityrosine crosslink resulting in constitutive EGFR activation. the dityrosine cross-link appears to be caused by an increase in hydrogen peroxide (H2O2) secondary to a decrease in catalase activity. Mechanistically, we found that an active metabolite of MCT, monocrotaline pyrrole (MCTP), directly attenuated the activity of human recombinant catalase in vitro. We also found that co-culture of pulmonary endothelial cells (EC) pre-exposed to MCTP with pulmonary smooth muscle cells (SMC) induced a significant accumulation of H2O2 in SMC and resulted in an increase in the proliferation rate of the SMC. This increase in proliferation correlated with enhanced levels of covalent EGFR dimers and EGFR autophosphorylation and these events were blocked by pretreatment with either PEG-catalase or the EGFR inhibitor, AG1478. Finally, we found that treatment with the EGFR inhibitor, gefinitib, significantly attenuated EGFR signaling and prevented the development of PH in MCT-exposed rats. Thus, we have identified a new mechanism whereby oxidative stress stimulates ligand-independent EGFR dimerization and EGFR signaling which contributes to the progression of PH.

doi:10.1016/j.freeradbiomed.2011.10.359 277 Altered Lung Glutathione Redox Status in Glutathione Reductase Knockout Mice: Implications for Alveolar Epithelial Cell Differentiation Mary Robbins1, Morgan Locy1, Cynthia Hill1, Jason Hansen2, Lynette Rogers1, and Trent Tipple1 1The Research Institute at Nationwide Children's Hospital, 2Emory University Altered intracellular glutathione (GSH) redox potential contributes to the regulation of cellular proliferation and differentiation. in the alveolar epithelium, type 2 cells are progenitors for type 1 cells, the primary cells through which gas exchange occurs. We have previously demonstrated that adult GSH reductase (GR) knockout mice (GR-KO) have altered lung GSH redox states. the present studies tested the hypothesis that newborn GR-KO mice have a more oxidized redox potential in the lung and altered numbers of type 1 alveolar epithelial cells (AEC). Total GSH levels were determined by reverse-phase HPLC on lung samples from E19-D14 newborn wild-type (WT) and GR-KO mice. Western Blot analyses were performed on whole lung homogenates from E19, D14, D28 and D70 WT and GR-KO mice for type I marker T1α and type 2 marker surfactant protein C (SP-C). β-actin was used as a loading control. Data (mean±SEM, n=3) were analyzed by 2-way ANOVA followed by Bonferroni post hoc with significance noted at P<0.05. Our data indicate that GSH and GSSG concentrations in newborn GR-KO mice were 50-70% lower than in age-matched WT mice. These differences contributed to a more oxidizing intracellular GSH redox potential that was age-dependent, with a +10 mV difference on E19 (-220 vs -210 mV GR-KO) and a +30 mV difference on D14 (-220 WT vs -190 mV GR-KO). Independent effects of genotype and day of life on lung T1α expression and an interaction between day of life and genotype were detected. T1α protein levels were 46% lower in GR-KO mice than in WT controls at E19 (0.25±0.01 vs 0.46±0.09) and 60% lower than in WT controls at D70 (0.70±0.19 vs 1.73±0.20). No effects of day of life or genotype on lung SP-C expression were detected. GR-KO mice have compromised lung antioxidant defenses characterized by less total GSH and a more oxidized GSH redox potential compared to WT controls. Differences in expressions of T1α but not SP-C suggest that GR-KO mice have persistent deficits in type 1 AEC abundance. We speculate that differences in lung redox status in GR-KO mice directly contribute to altered lung cell phenotype. GR-KO mice are an ideal model in which to study the effects of altered intracellular

redox status on lung cellular differentiation.

doi:10.1016/j.freeradbiomed.2011.10.360 278 Mouse Strain-Dependent Susceptibility To Quantum Dot Induced Lung Inflammation and Oxidative Stress David K Scoville1, Collin C White1, Dianne Botta1, Lisa McConnachie1, Megan Zadworny1, Xiaoge Hu2, Xiaohu Gao2, and Terrance J Kavanagh1 1University of Washington - Dept. of Environmental and Occupational Health Sciences, 2University of Washington - Dept. of Bioengineering Quantum dots (QDs) are nanoparticles typically composed of a CdSe core, a ZnS shell, and an assortment of polymer coatings specific to the application. the fluorescent properties of QDs (resistant to photobleaching; narrow spectral emission) make them useful in biomedical imaging. However, due to their small size and heavy metal composition, there is concern over possible toxicity of QDs. in addition to eliciting an inflammatory response, macrophage phagocytosis of QDs could result in degradation and release of ionic Cd, potentially leading to oxidative stress. Using 8 genetically inbred parental mouse strains from the Collaborative Cross (CC) we have investigated differential susceptibility to lung inflammation and oxidative stress after oropharyngeal aspiration of amphiphilic polymer-coated QDs, with the ultimate goal of identifying susceptibility genes and pathways using systems genetics. Inflammatory markers included total protein content in bronchoalveolar lavage fluid (BALF), and the relative percentage of neutrophils present in the BALF cellular fraction as assessed by flow cytometry. We used the ratio of % neutrophils in BALF from QD treated mice vs. saline treated mice as a measure of sensitivity to QD-induced lung inflammation among these 8 strains. There was significant strain-dependant variation in sensitivity toward QD induced inflammation (A/J > NZO/H1LtJ > NOD/ShiLtJ = WSB/EiJ = CAST/EiJ > C57BL/6J = 129S1/SvImJ = PWK/PhJ), as defined by % neutrophils in the BALF. No significant increase in BALF protein levels for any strain was noted, suggesting no acute cytotoxicity at the dose used (6 ug Cd equivalents/kg BW). Interestingly, basal lung glutathione levels were higher in a resistant strain (C57BL/6) than in two relatively sensitive strains (A/J, p=0.032 and NZO/H1LtJ, p=0.0081). These results provide phenotypic data that will be useful for mapping potential quantitative trait loci (QTLs) associated with QD-induced lung inflammation and oxidative stress in recombinant inbred mouse lines from the CC. Analysis of such QTLs could lead to insights regarding the molecular mechanisms responsible for QD toxicity and ultimately provide guidance on how to modify the composition of QDs to reduce their toxicity.

doi:10.1016/j.freeradbiomed.2011.10.361 279 Acute Ozone Exposures Result in Site-Specific Airway Injury and Mitochondrial Damage Katherine L Tuggle1, Jessica Fetterman1, David Westbrook1, Scott Ballinger1, Edward M Postlethwait1, and Michelle V Fanucchi1 1University of Alabama at Birmingham Rationale: Half of the US population is exposed to unhealthy levels of ozone, an ambient air pollutant associated with decreased lung function and exacerbation of chronic medical conditions. Animal studies have shown that acute ozone exposure causes pulmonary injury that includes site-specific epithelial

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