445 Urinary Biomarkers of Oxidative Damage are Predominantly Determined by Non­genetic Factors Kasper Broedbaek1,2, Rasmus Ribel-Madsen3, Trine Henriksen1,2, Allan Weimann1,2, Morten Petersen1,2, Jon Andersen1,2, Shoaib Afzal1,2, Brian Hjelvang1,2, Allan Vaag3, Pernille Poulsen3,4, L Jackson Roberts 2nd5, and Henrik E Poulsen1,2 1Rigshospitalet, Copenhagen, Denmark, 2Bispebjerg Hospital, Copenhagen, Denmark, 3Steno Diabetes Center, Gentofte, Denmark, 4Novo Nordisk A/S, Soeborg, Denmark, 5 Vanderbilt University Medical Center Oxidative damage is thought to be important in both cancers and in many non-cancerous conditions. To what extend this damage is determined by genetic and environmental factors is unknown. The aim of this study was to examine the contribution of genetic versus environmental factors to oxidative damage by performing a classical twin study. The study population included 198 elderly twins (aged 62–83 years), 46 monozygotic (MZ) and 53 dizygotic (DZ) twin pairs. Urine samples were collected for measurement of biomarkers of oxidative damage (8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 8-oxo-7,8-dihydroguanosine (8-oxoGuo) and 2,3-dinor-5,6-dihydro-15-F2t-IsoP (F2-IsoP-M)). Classical twin analyses including intraclass correlation coefficients and heritability estimates were used to determine the relative contributions of genes and environment to the variation in urinary markers of oxidative damage. There were no statistically significant differences in intraclass correlations between MZ and DZ twins neither for 8-oxodG (rMZ = 0.55, rDZ = 0.47; P = 0.43), F2-IsoP-M (rMZ = 0.33, rDZ = 0.22; P = 0.42) nor 8-oxoGuo (rMZ = 0.45, rDZ = 0.58; P = 0.21). The heritability estimates for 8-oxodG, F2-IsoP-M and 8-oxoGuo excretion were h2 = 0.17, h2 = 0.22 and h2~ 0, respectively. Furthermore, significant positive correlations between 8-oxodG, F2-IsoP-M and 8-oxoGuo were shown. In conclusion, we demonstrated that in a large population of elderly Danish twins, the rate of oxidative damage is predominantly determined by potentially modifiable non-genetic factors.

446 Using GTP as a Biomarker of Oxidative Stress to Determine Synergistic Interactions between Iron and Paraquat  Fernando Cardozo-Pelaez1, and Traci Brown1 1University of Montana Iron (Fe) and paraquat (PQ) act synergistically causing cellular damage via reactive oxygen species (ROS). A two-electron oxidation of guanine in DNA, via ROS, results in the formation of 8-hydroxy-2′-deoxyguanosine (oxo8dG), which is the major oxidation product of guanine. Guanine is the most readily oxidized base and oxo8dG one of the most frequently studied oxidized DNA base products. Changes in the level of oxo8dG have attracted considerable interest as a biomarker of oxidative stress. In the other hand, Guanosine 5′-triphosphate (GTP), required for RNA synthesis and several other normal cellular functions, is more readily available than guanine in DNA with cytosolic concentrations in the high micromolar range. This suggests that under conditions of oxidative stress, more oxidized GTP (oxo8GTP) than oxo8dG in DNA would be produced in cells; thus, serving as a better assessment of oxidative damage. Using a PQ/Fe exposure paradigm we tested whether guanine oxidation in the GTP pool will serve as a better biomarker of oxidative stress than oxo8dG levels in DNA. Human neuroblastoma SH-SY5Y cells were exposed to either PQ (500 μM), Fe (500 μM), or a combination of both (500 μM each) for a 4 hr period. After exposure, levels of oxo8dG in cellular DNA from all the exposure groups were not significantly different from levels in unexposed

cells. The levels of oxo8GTP were elevated, as compared to unexposed cells, only in cells exposed to the Fe/PQ combination. This data supports the notion that PQ and Fe work synergistically to generate ROS and identifies oxo8GTP as a better biomarker for oxidative stress. This work was supported by NIA grant 5R01AG031184.

447 Mitochondrial Aconitase: Selective Target of Reactive Species with Metabolic Impact? Fiorella Scandroglio1,2, Verónica Tórtora1,2, Rafael Radi1,2, and Laura Castro1,2 1Biochemistry Dept. Facultad de Medicina, 2Center for Free Radical and Biomedical Research, Uruguay Superoxide radical and reactive species derived from its reaction with •NO (ONOO– and CO3

•–) rapidly react with the Fe-S cluster of the Krebs cycle enzyme aconitase, yielding an inactive protein in vitro. The selective reactivity of reactive species toward aconitase is further supported by proteomic analysis of mitochondria from animal models of sepsis, diabetes and aging which revealed aconitase inactivation and nitration in vivo. Using isolated mitochondria from different rat tissues with varied metabolic profiles, we aimed to evaluate the flux control coefficient elicited by aconitase over the Krebs cycle and the respiratory chain reactions. We determined aconitase specific activitiy in rat kidney (103 ± 20 mU/mg), liver (25 ± 6 mU/mg), heart (255± 43 mU/mg) and brain (37 ± 5 mU/mg). Titrations with fluorocitrate, showed tissue variations with IC50 of 17, 4, 12, and 3 μM in kidney, liver heart and brain, respectively. Of the tissues examined, the flux control coefficient exhibited the highest value in brain (0.98 ± 0.03). Before significant changes in oxygen consumption were observed, aconitase inhibition threshold were determined to be ~44, ~24, <5% for kidney, liver and brain respectively. These results indicate that aconitase exerts a high level of control over respiration (particularly in brain) and support the hypothesis that inactivation of aconitase may provide a control mechanism to prevent O2

•– formation by the respiratory chain. Supported by ANII FCE_398

448 Free Radical Metabolism by Cytochrome P450­2E1 and NADPH Oxidase Activation Forms Protein Radicals and Tyrosine Nitration in Obesity­Associated Non­alcoholic Fatty Liver Disease Saurabh Chatterjee1, Douglas Ganini DaSilva2, Jinjie Jiang1, Marcelo G Bonini3, Fabian Leinisch1, Maria Kadiiska1, and Ronald P Mason1 1NIEHS, 2Universidade Federal de São Paulo, UNIFESP, São Paulo, 3University of Illinois at Chicago Obesity is treated as a low-grade inflammatory trigger and is often associated with non-alcoholic fatty liver disease where free radical metabolism plays a significant role. We hypothesize that free radical metabolism through cytochrome p450 isoforms (mainly CYP2E1) and NADPH oxidase activation forms protein radicals and post translational protein tyrosine nitration in diet-induced models of obesity. We utilize the administration of a low dose of carbon tetrachloride (CCl4)(0.8mM/kg) that is well tolerated in diet- restricted lean controls in terms of liver pathology as a model to generate metabolic oxidative stress in diet-induced obese (DIO) mice. Results indicated DIO mice with increased metabolic oxidative stress had significant increase in protein-DMPO (spin trap) adducts in liver homogenates and in CD-68 specific Kupffer cells, indicating protein radical generation. Immunospecific staining for 4-hydroxynonenal, a lipid peroxidation product, was

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also observed in the sinusoidal cells as early as 6h after CCl4 administration. Administration of CYP2E1 inhibitor diallylsulphide inhibited 4-HNE formation, decreased serum ALT levels and improved liver lesions in DIO mice treated with CCL4, ascertaining the role of this enzyme in inducing metabolic oxidative stress. To investigate the mechanisms of protein radical formation in Kupffer cells, mice were administered apocynin, an inhibitor of translocation of the p47phox subunit. Results indicated that serum ALT levels, DMPO-nitrone adduct formation and protein tyrosine nitration were inhibited by apocynin. Also NADPH oxidase activation was observed in CCl4-treated DIO mice as assessed by western blot analysis of the gp91phox subunit in the immunoprecipitate of p47phox protein. p47phox knockout mice did not show elevations in protein nitrone adducts, protein tyrosine nitration or signs of hepatocellular necrosis. These studies thus indicate the participation of NADPH oxidase as a potential source of protein radical formation with obesity-induced non-alcoholic fatty liver disease.

449 Ohr (Organic hydroperoxide resistance protein) Possesses a Previously Undescribed Activity: Lipoyl­dependent Peroxidase José Renato Rosa Cussiol1, Thiago Geronimo Pires Alegria1, Luke Ignatius Szweda2, and Luis Eduardo Soares Netto1 1Universidade de São Paulo, 2Oklahoma Medical Research Foundation Ohr belongs to a family of proteins which share a unique α/β fold named Ohr/OsmC. These proteins are exclusively present in bacteria being most of them pathogenic of many plants and animals. Previously, several Ohr/OsmC proteins were characterized as cys-based thiol peroxidases because most of them have the ability to react with organic hydroperoxides through a conserved and reactive cysteine residue of its polypeptide backbone. Ohr/OsmC peroxidase activity requires reduction of the enzyme’s disulfide group formed upon catalytic reduction of the organic hydroperoxide. However, the enzyme’s physiological reducing agent has not yet been identified. Here, we showed for the first time that Ohr from Xylella fastidiosa has the ability to reduce peroxides sustained by enzymes from the intermediary metabolism. Lipoylated enzymes present in the bacterial extracts of Xylella fastidiosa interacted physically and functionally with Ohr, whereas thioredoxin and glutathione systems failed to support Ohr peroxidase activity. Furthermore, we were able to reconstitute in vitro three lipoyl-dependent systems as Ohr physiological reducing systems. We also showed that OsmC from Escherichia coli, an orthologue of Ohr, is specifically reduced by lipoyl-dependent systems. These results represent the first evidence that a Cys-based thiol peroxidase is directly reduced by lipoylated enzymes. Moreover, we demonstrated that YhfA from Escherichia coli, another protein that belongs to Ohr/OsmC family, does not possess peroxidase activity. This protein lacks a conserved arginine residue in the active site, responsible to lower the pKa of the peroxidatic cysteine residue. These results suggest that not all members from Ohr/OsmC family are thiol peroxidases, probably displaying other biological functions in the bacterium.

450 Riboflavin­mediated Photo­oxidation of Tyrosine Residues in Milk Proteins Depends on the Protein Structure Trine Kastrup Dalsgaard1, Jacob Holm Nielsen1, and Michael J. Davies2 1Aarhus University, Denmark 2The Heart Research Institute, Sydney, Australia Oxidative damage to proteins is a major problem in the food industry resulting in changes in palatability, nutritional content, and structure. Photo-oxidative damage is of major importance to milk and milk products as a result of the presence of the sensitizer riboflavin in these materials. Visible light exposure may therefore generate excited state and radical species that may play a role in milk deterioration. In this study, the formation and lifetime of radicals generated on three different milk proteins (BSA, β-lactoglobulin, β-casein) with different secondary structures were examined, with the aim of investigating the effects on protein structure on riboflavin-mediated protein photo-oxidation. ESR studies have provided evidence for the formation of long-lived protein radicals on exposure to visible light in the presence of riboflavin (5μg/mL). Studies with iodinated proteins indicate that these are tyrosine-derived phenoxyl radicals. The accumulation of tyrosyl radicals was lower with β-casein than with β-lactoglobulin or BSA. At the same time the concentration of dityrosine (detected by HPLC) was higher with β-casein than BSA or β-lactoglobulin. In contrast, accumulation of the alternative tyrosine oxidation product, DOPA, was highest in BSA, followed by β-lactoglobulin and β-casein. The flexible structure of β-casein appears to favor radical-radical termination of tyrosyl radicals to give dityrosine resulting in a lower steady-state level of protein radicals. In BSA and β-lactoglobulin, the tyrosyl radicals dimerize less rapidly due to the lower flexibility of the globular structure of these proteins, thereby resulting in the formation of higher concentrations of DOPA. As dityrosine is involved in protein-protein cross-linking, these data are consistent with a larger extent of oxidative cross-linking of β-casein than of the other proteins.

451 Immunological Detection of NFK in Photo­oxidized Lens Alpha­crystallin Marilyn Ehrenshaft1, Baozhang Zhao1, Usha P. Andley2, Ronald P. Mason1, and Joan E. Roberts3 1NIEHS/NIH, 2Washington Univ. School Med., 3Fordham Univ. The crystalline proteins (alpha, beta and gamma crystallin) comprise approximately 90% of the water soluble proteins in the eye lens, and are responsible for maintaining lens transparency and allowing the lens to focus light undistorted onto the retina. Alpha crystallins are the major lens crystallins, and act as both structural proteins and as chaperones to protect all lens proteins from damage leading to lens deterioration. Because crystallin proteins do not turn over in the lens, the accumulated damage they incur can lead to cataracts, which are the leading cause of blindness in the world. Porphyrins are endogenous biomolecules that are also used as therapeutic agents. Accumulation of these photosensitizing compounds in the eye can exacerbate lens aging through increased levels of singlet oxygen, resulting in polymerization of lens proteins and structural alterations to some of the protein amino acid residues. Oxidation of tryptophan residues, for example, into kynurenine and N-formylkynurenine (NFK) causes irreversible discoloration of the normally transparent lens, leading to development of cataracts. Because NFK is itself a singlet oxygen-generating photosensitizer, the presence of NFK in lens proteins serves to exacerbate lens deterioration. In this work we have used polyclonal anti-NFK

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