Trapping of Protein-Centered Radicals with a Nitrone Spin Trap Prevents EndotoxinInduced Experimental Acute Respiratory Distress Syndrome Mouse Model

511 Trapping of Protein­Centered Radicals with a  Nitrone Spin Trap Prevents Endotoxin­Induced  Experimental Acute Respiratory Distress  Syndrome Mouse Model  Sandra E Gomez-Mejiba1, Maria S Gimenez2, Zili Zhai1, And Dario C Ramirez1 1 2 OMRF, Oklahoma City, IMIBIO-SI, UNSL, San Luis, Argentina Inflammatory reactions can be triggered by end-oxidation products of proteins and might be effectively prevented by blocking protein oxidation with nitrone spin traps such as 5,5dimethyl-1-pyrroline N-oxide (DMPO). In the lung, inflammation is induced by air pollutants such as the bacterial endotoxin lipopolysaccharide (LPS). Here we tested the effect of blocking free radical reactions in the lung with DMPO on inflammation triggered by LPS in an acute respiratory distress syndrome mouse model. We used male C57BL/6J mice with acute intratracheal instillation of either 50 μl of vehicle (saline) or LPS (50, 125, 250, 500 μg/mouse), with fifty μl of DMPO (0.05, 0.25, 0.5, 2.5 nmol/mouse) or vehicle intratracheally instilled 1 h before and 23 h after the instillation of LPS. Twenty four h after the LPS instillation, the bronchoalvelolar lavage fluid (BALF), blood and lung tissue were collected. LPS, DMPO or a combination of both at the doses and follow-up time used did not cause death of animals; however, mice instilled with LPS became cachexic and asthenic. Interestingly, DMPO treatment greatly improved these conditions. Cytospin preparation and differential cytology of the BALF showed a cell population mainly composed of neutrophils which were reduced in a dose-dependent manner by DMPO. LPS increased markers of oxidative stress (carbonyls, nitrotyrosine and chlorotyrosine), inflammation (TNF-α, IL-1β, nitrite/nitrate, and IL-6) and tissue damage (lactic dehydrogenase) in the BALF supernatant, serum and lung tissue homogenates; these changes were prevented by DMPO. Histology of the lung tissue of mice instilled with LPS and DMPO showed increased protein-DMPO nitrone adduct formation along with reduced ICAM-1 expression and TNF-α in the lung parenchyma. Protein nitrone adducts also increased in BALF supernatant and serum, indicating that because of its pharmacokinetic properties DMPO might act locally and systemically to trap protein-centered radicals or that proteins tagged with DMPO in the lung can reach systemic circulation. These results suggest that DMPO blocks inflammation caused by endotoxin by trapping protein-centered radicals, suppressing ICAM-1/chemokine expression, or blocking further inflammatory effects of oxidized proteins. Delivery of DMPO into the lung might be a potential preventive therapy against the systemic effects of endotoxin in the lung. Supported by NIEHS 5R00ES015415-04. doi:10.1016/j.freeradbiomed.2010.10.524

512 Tuning of the Pro­oxidant and Antioxidant Activity  of Trolox Through the Controlled Release from  Biodegradable Poly(trolox ester) Polymers  Paritosh Wattamwar1, Sarita S Hardas1, D. Allan Butterfield1, Kimberly W Anderson1, and Thomas Dziubla1 1 University of Kentucky Concentration dependant pro-oxidant and antioxidant effect of [1] antioxidant molecules has been known in literature . This two way effect of antioxidants can be used in tissue engineering applications to control the oxidative stress level in the cells and thereby modulate cell response. If the rate of release of antioxidants from the surface of a material can be controlled, then the antioxidant/pro-oxidant response of the cell to the materials can be tuned. Polymers composed of antioxidants linked through hydrolysable bonds provide a means of controlling release of

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these antioxidants. Trolox, a water-soluble analogue of Vitamin E, is one such antioxidant that has shown a concentration dependant pro-oxidant effect. In our previous work, we have synthesized a polymer of trolox, poly(trolox ester), which upon biodegradation results in release of active antioxidant trolox. In this work, we evaluated the concentration dependant effect of free trolox as compared with antioxidant polymer poly(trolox ester) on oxidative stress level in human umbilical vein endothelial cells (HUVECs) . Slow release of trolox as a degradation product of Poly(trolox ester) resulted in the suppression of oxidative stress in the cells as compared to the pro-oxidant effects observed by addition of free trolox. 1.Tafazoli, S., J.S. Wright, and P.J. O'Brien, Prooxidant and antioxidant activity of vitamin E analogues and troglitazone. Chem Res Toxicol, 2005. 18(10): p. 1567-74. doi:10.1016/j.freeradbiomed.2010.10.525

513 Isotope­Reinforced Polyunsaturated Fatty Acids  Protect Yeast Cells from Oxidative Stress  Shauna A. Hill1, Kathleen Hirano1, Vadim V. Shmanai2, Beth N. Marbois1, Dragoslav Vidovic3, Andrei V. Bekish4, Bradley Kay1, Vincent Tse1, Jonathan Fine1, Catherine F. Clarke1, and Mikhail S Shchepinov5 1 2 University of California, Los Angeles, National Academy of 3 4 Sciences of Belarus, Oxford University Belarussian State 5 University, Minsk. Belarus, Retrotope, Inc The facile abstraction of bis-allylic hydrogens from polyunsaturated fatty acids (PUFAs) is the hallmark chemistry responsible for initiation and propagation of autoxidation reactions. PUFA autoxidation has deleterious effects on the biological system and has been linked to age-related degenerative diseases and associated with apoptosis. The products of these autoxidation reactions can form cross-links to other membrane components, damage proteins and nucleic acid. We report that substitution of hydrogen atoms with deuterium atoms uniquely at the bis-allylic sites are much more resistant to autoxidation reactions, due to the isotope effect. This is shown using coenzyme Q-deficient Saccharomyces cerevisiae coq mutants with defects in biosynthesis of coenzyme Q (Q). Q functions in respiratory energy metabolism and also functions as a lipid-soluble chain terminating antioxidant. Although yeast cannot synthesize PUFAs, they are able to incorporate exogenously supplied PUFAs. Yeast coq mutants incubated in the presence of the PUFAs αlinolenic or linoleic acid exhibit 99% loss of colony formation after four hours, demonstrating a profound loss of viability. In contrast, coq mutants treated with monounsaturated oleic acid or with one of the deuterated PUFAs:11,11-D2-Linoleic or 11,11,14,14-D4-αLinolenic retain viability similar to wild-type yeast. Deuterated PUFAs also confer protection to wild-type yeast subjected to heat stress. In addition, PUFA-treated yeast coq mutants show greater levels of oxidative damage as compared to treatment with deuterated PUFAs. These results indicate that isotope-reinforced PUFAs are stabilized compared to standard PUFAs, and they protect coq mutants and wild-type yeast cells against the toxic effects of lipid autoxidation products. These findings suggest new approaches to controlling ROS-inflicted cellular damage and oxidative stress. doi:10.1016/j.freeradbiomed.2010.10.526

SFRBM/SFRRI 2010