Endogenous Metabolites Modulate Macromolecular Free Radicals Derived from Aromatic Amine Drugs in Neutrophil-Like Myeloid Leukemia Cells

33 Modulation of Myeloperoxidase Activity by SelfGenerated Hypochlorous Acid 1

Dhiman Maitra , Carlos E. Souza1, Faten Shaeib1, Rasha M. Abdulridha1, Ibrahim Abdulhamid2, Ghassan M. Saed1, Michael P. Diamond1, Subramaniam Pennathur3, and Husam M. Abu-soud1 1 2 Wayne State University, Children's Hospital of Michigan, 3 University of Michigan Myeloperoxidase (MPO) is a hemoprotein that generates hypochlorous acid (HOCl) from chloride (ClǦ) and hydrogen peroxide (H2O2). Here, we investigated whether the HOCl formed during steady-state catalysis was able to destroy the MPO heme moiety and function as a major source of free iron. UV-visible spectra and H2O2-specific electrode measurements recorded during steady-state HOCl synthesis by MPO showed that the degree of MPO heme destruction increased by multiple additions of 10 μM (plasma levels) of H2O2 allowing the enzyme to function only at a fraction of its maximum activity (5-10%). MPO heme destruction occurred only in the presence of ClǦ. Stopped-flow measurements revealed that the HOCl-mediated MPO heme destruction was complex and occurred through transient ferric species whose formation and decay kinetics indicated it can participate in heme destruction along with subsequent free iron release. MPO heme depletion was confirmed by identifying a number of heme degradation products utilizing HPLC analyses, while the buildup of free iron was confirmed colorimetrically utilizing the ferrozine assay. Melatonin, a potent MPO inhibitor known to switch the MPO catalytic activity from peroxidation to catalase-like activity, prevented HOCl-mediated feedback heme destruction. Taking together, with our recent finding that MPO and free iron are elevated in ovarian cancer, we can postulate that the source of free iron in ovarian cancer is due to HOCl-mediated feedback destruction of MPO.

doi:10.1016/j.freeradbiomed.2012.10.060

34 Fluorescent Heme Degradation Products in Sickle Cell Disease: Does HOCl Play a Role in Sickle Cell Disease 1

Dhiman Maitra , Jaeman Byun2, Ghassan M. Saed1, Michael P. Diamond1, Subramaniam Pennathur2, Ibrahim Abdulhamid3, and Husam M. Abu-soud1 1 2 3 Wayne State University, University of Michigan, Children's Hospital of Michigan Sickle cell disease (SCD) is a debilitating genetic condition which leads to decreased red blood cell (RBC) lifespan, increased hemolysis and iron overload. Recently we have shown that hypochlorous acid (HOCl), a potent oxidant generated by myeloperoxidase (MPO), can oxidatively cleave the heme moiety in free heme as well as purified hemoglobin. It has also been reported that MPO levels are significantly elevated in the plasma of SCD patients and the plasma concentration of MPO showed a significant inverse correlation with the hemoglobin concentration. Based on our recent discovery on the role of HOCl on the destruction of hemoglobin heme moiety and the evidence of the presence of high plasma levels of MPO in SCD patients we hypothesized that hemoglobin heme destruction in SCD is being caused by MPO generated HOCl. To test our hypothesis HPLC and mass spectrometric analysis was performed with RBC isolated from SCD patients. HPLC analysis showed the presence of fluorescent heme degradation products in the RBC of SCD patients, levels of which increased ~ 10 fold when the RBCs were treated with a molar excess of HOCl. This indicated that these

products were indeed generated by the action of HOCl on the heme moiety. Seven different heme fragmentation products were tentatively identified from their molecular weight by LC-MS analysis. These fragments are tri-, di- or mono-pyrrole derivatives of the parent heme tetrapyrrole ring. Some of the fragments, namely m/z 437 and m/z 473, also showed methyl-esterification in the terminal carboxylic group. Our results indicate that MPO and HOCl may play a causative role in SCD, and thus inhibiting MPO might be an interesting therapeutic target for ameliorating the pathological conditions associated with SCD.

doi:10.1016/j.freeradbiomed.2012.10.061

35 Salt as an Oxidative Stress in Gastric Mucosal Cells 1

Hirofumi Matsui , Yumiko Nagano1, Masato Tamura1, Ichinosuke Hyodo1, Hiroko Indo2, Hideyuki Majima2, and Aki Hirayama3 1 2 3 University of Tsukuba, University of Kagoshima, Tsukuba University of Technology Sodium chloride (NaCl) has been reported to involve acute gastric mucosal lesions. Moreover, NaCl rich food has been reported to be a carcinogenic factor for the stomach. However, there are few reports to clear the pathogenesis for NaCl-induced gastric lesions and neoplasms. Recently, we have reported that gastric acid is an oxidative stress which involves cellular apoptosis via mitochondrial ROS production (Matsui, J Gastro. 2011). ROS have been reported to play a very important role for the gastric lesions formation and carcinogenesis. NaCl-derived hyperosmotic condition may inhibit mitochondrial functions to produce ROS. To elucidate it, we investigated the relations between NaCl and ROS production in gastric epithelial cells. Methods: A gastric epithelial cell-line RGM-1 and RGM-MnSOD which is a MnSOD over-expressing stable clone were used. These cells were exposed hypertonic conditions with NaCl. Cellular cytotoxicity was investigated by TC-1 method. EPR spectra were detected with a reagent CYPMPO. Result: NaCl treatment involved supeoxide anion production. The hyperosmotic condition involved not only necrosis but also apoptosis. MnSOD overexpression restrained the ROS production and cell death. Conclusions: NaCl is an oxidative stress for gastric epithelial cells.

doi:10.1016/j.freeradbiomed.2012.10.062

36 Endogenous Metabolites Modulate Macromolecular Free Radicals Derived from Aromatic Amine Drugs in Neutrophil-Like Myeloid Leukemia Cells 1,2

Karim Michail and Arno G. Siraki1 1 2 University of Alberta, Alexandria University, Egypt Our lab has previously demonstrated that aromatic amine drugs and xenobiotics induce the formation of free radicals on protein i and lipid macromolecules . These findings open possibilities that primary N-centered cation radicals originating from the peroxidation of aniline-based drugs/xenobiotics might also cooxidize endogenous small molecule metabolites which could produce a futile free radical cycle of reactions. In a series of experiments using electron paramagnetic resonance (EPR) spectrometry, we identified preliminary structural characteristics for a number of molecules which acted as co-oxidants in the ii peroxidase metabolism of anilines . Herein, we show that N,Ndimethylglycine (DMG), a product of homocysteine metabolism, is

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readily co-oxidized into secondary C-centered radicals by aromatic amine drugs and xenobiotics triggered by human neutrophil myeloperoxidase (MPO) and catalytic amounts of H2O2. Furthermore, in the presence of DMG, we show that oxygen consumption (determined by oximetry) is significantly enhanced; whereas the yield of azobenzene (determined by GC/MS), an aniline dimeric end-product, is remarkably attenuated. On the other hand, glycine, sarcosine (methylglycine), and betaine (trimethylglycine) did not mimic DMG in its unique behavior. In human promyelocytic leukemia cells (HL-60), DMG, but not sarcosine or betaine, dose-dependently enhanced the formation of protein-centered MPO radicals as evidenced by immuno-spin trapping and western blotting analysis. Interestingly, significant induction of MPO radicals was achieved by levels of DMG close to those observed in hyperhomocysteinemia. We have also found a correlation between DMG-enhanced protein radical formation and caspase activation and glutathione depletion. We postulate that the co-oxidation of small molecule metabolites, such as DMG into C-centered free radicals in the course of the extra-hepatic peroxidative metabolism of anilines could modulate the haematotoxicity of the latter compounds via initiating and/or promoting oxidative modification of protein targets. Although we have shown that a direct chemical interaction between DMG and aniline free radical metabolites is possible, there could be intracellular signaling or biochemical mechanisms which account for the enhancement of protein free radical formation. References: 1 Chem. Res. Toxicol. 23, 2010 (880-87) and 24, 2011(1031-39) 2 Unpublished data

doi:10.1016/j.freeradbiomed.2012.10.063

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CBA-based assay exhibits multiple advantages, including: a) better sensitivity; b) no interference by metal chelators; c) welldefined stoichiometry; d) possibility of real-time monitoring of TyrOOH formation. In summary, we developed a new, improved assay for detection and quantification of tyrosine-derived hydroperoxides which can be used in model studies of oxidative protein damage.

doi:10.1016/j.freeradbiomed.2012.10.064

38 Oxidant and Antioxidant Activities of Mammalian Catalase 1

Madhur M. Goyal1, Rashee Mittal , and Anjan Basak1 1 Jawaharlal Nehru Medical College, India Catalase is well known antioxidant enzyme which protects cells from the toxic effects of hydrogen peroxide. It catalyses the dissociation of hydrogen peroxide directly into H2O and O2. In a few reports, mammalian catalase has been identified as the source of ROS, having peroxidase and oxidase activity. Our results show that apart from the known catalytic action, bovine liver catalase can generate Hydroxyl Radicals (HRs) in experimental conditions. With present data catalase seems unique in combining favorable and unfavorable properties. It is remarkable how, on the one hand, enzyme is essential for survival due to its antioxidant nature and yet, on the other hand, by producing free radicals it cause possible damage to living tissues. Present study emphasize on the need of further research to explore the exact role of catalase in human health and diseases.

doi:10.1016/j.freeradbiomed.2012.10.065

A Novel Assay for Real-Time Monitoring of Tyrosine Hydroperoxide Using Coumarin Boronic Acid (CBA)

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1,2

Radoslaw Michalski , Jacek Zielonka1, Joy Joseph1, and Balaraman Kalyanaraman1 1 2 Medical College of Wisconsin, Lodz University of Technology, Poland Protein hydroperoxides are marker products of oxidative modification of proteins. However, there is no rapid and reliable assay for measuring protein hydroperoxides. Tyrosyl hydroperoxide (TyrOOH) is produced when tyrosyl radical reacts with superoxide radical anion. This can be the case in many physiological situations, especially when tyrosyl radical is formed within a protein, and prevented from dimerization. Also, free tyrosyl hydroperoxide is a metastable oxidant and therefore can diffuse far from its site of formation. Formation of TyrOOH may lead to modification of the structure of proteins, as well as inactivation of enzymes, oxidation of other biological targets (glutathione, ascorbate), DNA damage etc. Here we present the assay for tyrosine hydroperoxide based on oxidation of coumarin boronic acid (CBA) to its fluorescent derivative 7hydroxycoumarin (COH). .

HO B

HO

O

O

CBA (non-fluorescent)

TyrOOH

HO

O

O

COH (blue fluorescent)

The rate constant of the reaction of CBA with TyrOOH was -1 -1 determined to be 8.3 ± 0.3 M s at pH 7.4. The reaction can be monitored in real-time, and can be used to detect in-situ generated TyrOOH. Comparing to the commonly used reagent for hydroperoxides detection, FOX (FeSO4/Xylenol Orange), the

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The Links between Mitochondrial Superoxide and Endothelial Phenotype 1

Ning Pan , Yukio Shimasaki1, and John F. Keaney1 1 University of Massachusetts Medical School Endothelial dysfunction has been associated with the pathogenesis of many vascular diseases such as atherosclerosis and hypertension. It is likely that multiple mechanisms contribute to endothelial dysfunction, however reactive oxygen species Ɣ (ROS), including O2 Ǧ, production appears particularly important. Mitochondria are well known as a major source of ROS within most cells under physiological and pathological conditions. Ɣ Among the determinants of mitochondrial O2 Ǧ, we chose uncoupling protein 2 (UCP2) as a modulator. Our previous data indicated that depletion of UCP2 increased endothelial Ɣ mitochondrial O2 Ǧ and decreased both cell proliferation and migration. Our recent data showed that this growth defect could be rescued by over-expression of mitochondrial superoxide dismutase (SOD2), confirming an important role of mitochondrial Ɣ O2 Ǧ in the endothelium. To investigate the mechanism, we examined two major pathways that regulate the cells growth, AKT and extracellular signal-regulated protein kinase (ERK) pathways. As part of the Akt pathway, eNOS produces NO• to modulate endothelial function. By applying a nitroxide synthase inhibitor LNAME, we showed that NO• is not a key player in explaining the phenotype. Our signaling analysis also showed that Akt pathway is not primarily responsible for the phenotype. In contrast to Akt, ERK1/2 is hyper-activated as a result of UCP2 deficiency in endothelial cells, including the ones from humans. Cell cycle

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