%0 Journal Article %~ PubMed %A Freewan, Mohammed %A Rees, Martin D %A Sempertegui Plaza, Tito S %A Glaros, Elias %A Lim, Yean J %A Wang, Xiao Suo %A Yeung, Amanda W S %A Witting, Paul K %A Terentis, Andrew C %A Thomas, Shane R %T Human Indoleamine 2,3-Dioxygenase is a Catalyst of Physiological Heme Peroxidase Reactions - Implications for the Inhibition of Dioxygenase Activity by Hydrogen Peroxide. %B Journal of Biological Chemistry %D 2013 %C United States %I American Society for Biochemistry and Molecular Bi %V 288 %N 3 %P 1548-1567 %@ 0021-9258 %X %Z FOR Codes: 30406 %0 Journal Article %~ PubMed %A Shanu, Anu %A Groebler, Ludwig %A Kim, Hyun-Bo %A Wood, Sarah %A Weekly, Claire M %A Aitken, Jade B %A Harris, Hugh H %A Witting, Paul K %T Selenium inhibits renal oxidation and inflammation but not acute kidney injury in an animal model of rhabdomyolysis. %B Antioxidants & Redox Signaling %D 2013 %C United States %I Mary Ann Liebert, Inc. Publishers %V 18 %N 7 %P 756-769 %@ 1557-7716 %X Acute kidney injury (AKI) is a manifestation of rhabdomyolysis (RM). Extracellular myoglobin accumulating in the kidney after RM promotes oxidative damage, which is implicated in AKI. Aim: Test whether selenium (Se) supplementation diminishes AKI and improves renal function Results: Dietary selenite increased Se in the renal cortex as demonstrated by X-ray fluorescence microscopy. Experimental RM-stimulated AKI as judged by increased urinary protein/creatinine, clusterin and kidney injury molecule-1 (KIM-1), decreased creatinine clearance (CCr) and increased plasma urea and damage to renal tubules. Concentrations of cholesterylester (hydro)peroxides and F2-isoprostanes increased in plasma and renal tissues after RM while aortic and renal cyclic guanidine monophosphate (cGMP; marker of nitric oxide (NO) bioavailability) decreased. Renal superoxide dismutase-1, phospho-P65, TNF?? gene, MCP-1 protein and the 3-chloro-tyrosine/tyrosine ratio (Cl-Tyr/Tyr; marker of neutrophil activation) all increased after RM. Dietary Se significantly decreased renal lipid oxidation, phospho-P65, TNF?? gene expression, MCP-1 and Cl-Tyr/Tyr, improved NO bioavailability in aorta but not in the renal microvasculature and inhibited proteinuria. However, CCr, plasma urea and creatinine, urinary clusterin and histopathological assessment of AKI remained unchanged. Except for the Se++ group, renal angiotensin-receptor-1/2 gene/protein expression increased after RM with parallel increases in MEK1/2 inhibitor-sensitive MAPkinase(ERK) activity. Innovation: We employed synchrotron radiation to identify Se distribution in kidneys in addition to assessing reno-protection after RM. Conclusion: Se-treatment has some potential as a therapeutic for AKI as it inhibits oxidative damage and inflammation and decreases proteinuria albeit histopathological changes to the kidney and some plasma and urinary markers of AKI remain unaffected after RM. %Z FOR Codes: 110312 60111 %0 Journal Article %~ PubMed %A Ding, Alice %A Kalaignanasundaram, Priyadharshani %A Ricardo, Sharon D %A Abdelkader, Amany %A Witting, Paul K %A Broughton, Brad R S %A Kim, Hyun Bo %A Wyse, Benjamin F %A Phillips, Jacqueline K %A Evans, Roger G %T Chronic treatment with tempol does not significantly ameliorate renal tissue hypoxia or disease progression in a rodent model of polycystic kidney disease. %B Clinical and Experimental Pharmacology & Physiology %D 2012 %C Australia %I Wiley-Blackwell Publishing Asia %V 39 %N 11 %P 917-929 %@ 1440-1681 %X %Z FOR Codes: 110107 %0 Journal Article %~ PubMed %A Groebler, Ludwig K %A Wang, Xiao Suo %A Kim, Hyun Bo %A Shanu, Anu %A Hossain, Farjaneh %A McMahon, Aisling C %A Witting, Paul K %T Cosupplementation with a synthetic, lipid-soluble polyphenol and vitamin C inhibits oxidative damage and improves vascular function yet does not inhibit acute renal injury in an animal model of rhabdomyolysis. %B Free Radical Biology & Medicine %D 2012 %C United States %I Elsevier Inc. %V 52 %N 9 %P 1918-1928 %@ 0891-5849 %X We investigated whether cosupplementation with synthetic tetra-tert-butyl bisphenol (BP) and vitamin C (Vit C) ameliorated oxidative stress and acute kidney injury (AKI) in an animal model of acute rhabdomyolysis (RM). Rats were divided into groups: Sham and Control (normal chow), and BP (receiving 0.12% w/w BP in the diet; 4weeks) with or without Vit C (100mg/kg ascorbate in PBS ip at 72, 48, and 24h before RM induction). All animals (except the Sham) were treated with 50% v/v glycerol/PBS (6mL/kg injected into the hind leg) to induce RM. After 24h, urine, plasma, kidneys, and aortae were harvested. Lipid oxidation (assessed as cholesteryl ester hydroperoxides and hydroxides and F(2)-isoprostanes accumulation) increased in the kidney and plasma and this was coupled with decreased aortic levels of cyclic guanylylmonophosphate (cGMP). In renal tissues, RM stimulated glutathione peroxidase (GPx)-4, superoxide dismutase (SOD)-1/2 and nuclear factor kappa-beta (NF??) gene expression and promoted AKI as judged by formation of tubular casts, damaged epithelia, and increased urinary levels of total protein, kidney-injury molecule-1 (KIM-1), and clusterin. Supplementation with BP±Vit C inhibited the two indices of lipid oxidation, down-regulated GPx-4, SOD1/2, and NF-?? gene responses and restored aortic cGMP, yet renal dysfunction and altered kidney morphology persisted. By contrast, supplementation with Vit C alone inhibited oxidative stress and diminished cast formation and proteinuria, while other plasma and urinary markers of AKI remained elevated. These data indicate that lipid- and water-soluble antioxidants may differ in terms of their therapeutic impact on RM-induced renal dysfunction. %Z FOR Codes: 110106 60199 %0 Journal Article %~ PubMed %A Aitken, Jade B %A Lay, Peter A %A Duong, T T Hong %A Aran, Roshanak %A Witting, Paul K %A Harris, Hugh H %A Lai, Barry %A Vogt, Stefan %A Giles, Gregory I %T Synchrotron radiation induced X-ray emission studies of the antioxidant mechanism of the organoselenium drug ebselen. %B Journal of Biological Inorganic Chemistry %D 2012 %C Germany %I Springer %V 17 %N 4 %P 589-598 %@ 1432-1327 %X Synchrotron radiation induced X-ray emission (SRIXE) spectroscopy was used to map the cellular uptake of the organoselenium-based antioxidant drug ebselen using differentiated ND15 cells as a neuronal model. The cellular SRIXE spectra, acquired using a hard X-ray microprobe beam (12.8-keV), showed a large enhancement of fluorescence at the K(?) line for Se (11.2-keV) following treatment with ebselen (10 ?M) at time periods from 60 to 240 min. Drug uptake was quantified and ebselen was shown to induce time-dependent changes in cellular elemental content that were characteristic of oxidative stress with the efflux of K, Cl, and Ca species. The SRIXE cellular Se distribution map revealed that ebselen was predominantly localized to a discreet region of the cell which, by comparison with the K and P elemental maps, is postulated to correspond to the endoplasmic reticulum. On the basis of these findings, it is hypothesized that a major outcome of ebselen redox catalysis is the induction of cellular stress. A mechanism of action of ebselen is proposed that involves the cell responding to drug-induced stress by increasing the expression of antioxidant genes. This hypothesis is supported by the observation that ebselen also regulated the homeostasis of the transition metals Mn, Cu, Fe, and Zn, with increases in transition metal uptake paralleling known induction times for the expression of antioxidant metalloenzymes. %Z FOR Codes: 30401 30299 110902 %0 Journal Article %~ PubMed %A Groebler, Ludwig K %A Liu, Joe %A Shanu, Anu %A Codd, Rachel %A Witting, Paul K %T Comparing the potential renal protective activity of desferrioxamine B and the novel chelator desferrioxamine B-N-(3-hydroxyadamant-1-yl)carboxamide in a cell model of myoglobinuria. %B The Biochemical Journal %D 2011 %C United States %I Portland Press Ltd. %V 435 %N 3 %P 669-677 %@ 1470-8728 %X Accumulating Mb (myoglobin) in the kidney following severe burns promotes oxidative damage and inflammation, which leads to acute renal failure. The potential for haem-iron to induce oxidative damage has prompted testing of iron chelators [e.g. DFOB (desferrioxamine B)] as renal protective agents. We compared the ability of DFOB and a DFOB-derivative {DFOB-AdAOH [DFOB-N-(3-hydroxyadamant-1-yl)carboxamide]} to protect renal epithelial cells from Mb insult. Loading kidney-tubule epithelial cells with dihydrorhodamine-123 before exposure to 100????M Mb increased rhodamine-123 fluorescence relative to controls (absence of Mb), indicating increased oxidative stress. Extracellular Mb elicited a reorganization of the transferrin receptor as assessed by monitoring labelled transferrin uptake with flow cytometry and inverted fluorescence microscopy. Mb stimulated HO-1 (haem oxygenase-1), TNF?? (tumour necrosis factor ??), and both ICAM (intercellular adhesion molecule) and VCAM (vascular cell adhesion molecule) gene expression and inhibited epithelial monolayer permeability. Pre-treatment with DFOB or DFOB-AdAOH decreased Mb-mediated rhodamine-123 fluorescence, HO-1, ICAM and TNF?? gene expression and restored monolayer permeability. MCP-1 (monocyte chemotactic protein 1) secretion increased in cells exposed to Mb-insult and this was abrogated by DFOB or DFOB-AdAOH. Cells treated with DFOB or DFOB-AdAOH alone showed no change in permeability, MCP-1 secretion or HO-1, TNF??, ICAM or VCAM gene expression. Similarly to DFOB, incubation of DFOB-AdAOH with Mb plus H2O2 yielded nitroxide radicals as detected by EPR spectroscopy, indicating a potential antioxidant activity in addition to metal chelation; Fe(III)-loaded DFOB-AdAOH showed no nitroxide radical formation. Overall, the chelators inhibited Mb-induced oxidative stress and inflammation and improved epithelial cell function. DFOB-AdAOH showed similar activity to DFOB, indicating that this novel low-toxicity chelator may protect the kidney after severe burns. %Z FOR Codes: 30401 %0 Journal Article %~ PubMed %A Weekley, Claire M %A Aitken, Jade B %A Vogt, Stefan %A Finney, Lydia A %A Paterson, David J %A de Jonge, Martin D %A Howard, Daryl L %A Witting, Paul K %A Musgrave, Ian F %A Harris, Hugh H %T Metabolism of Selenite in Human Lung Cancer Cells: X-Ray Absorption and Fluorescence Studies. %B Journal of the American Chemical Society %D 2011 %C United States %I American Chemical Society %V 133 %N 45 %P 18272-9 %@ 0002-7863 %X Selenite is an inorganic form of selenium that has a cytotoxic effect against several human cancer cell lines: one or more selenite metabolites are considered to be responsible for its toxicity. X-ray absorption spectroscopy was used to monitor Se speciation in A549 human lung cancer cells incubated with selenite over 72 h. As anticipated, selenodiglutathione and elemental Se both comprised a large proportion of Se in the cells between 4 and 72 h after treatment, which is in accordance with the reductive metabolism of selenite in the presence of glutathione and glutathione reductase/NADPH system. Selenocystine was also present in the cells but was only detected as a significant component between 24 and 48 h concomitant with a decrease in the proportion of selenocysteine and the viability of the cells. The change in speciation from the selenol, selenocysteine, to the diselenide, selenocystine, is indicative of a change in the redox status of the cells to a more oxidizing environment, likely brought about by metabolites of selenite. X-ray fluorescence microscopy of single cells treated with selenite for 24 h revealed a punctate distribution of Se in the cytoplasm. The accumulation of Se was associated with a greater than 2-fold increase in Cu, which was colocalized with Se. Selenium K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy revealed Se-Se and Se-S bonding, but not Se-Cu bonding, despite the spatial association of Se and Cu. Microprobe X-ray absorption near-edge structure spectroscopy (??-XANES) showed that the highly localized Se species was mostly elemental Se. %Z FOR Codes: 30401 %0 Journal Article %~ PubMed %A Dong, Lan-Feng %A Jameson, Victoria J A %A Tilly, David %A Prochazka, Lubomir %A Rohlena, Jakub %A Valis, Karel %A Truksa, Jaroslav %A Zobalova, Renata %A Mahdavian, Elahe %A Kluckova, Katarina %A Stantic, Marina %A Stursa, Jan %A Freeman, Ruth %A Witting, Paul K %A Norberg, Erik %A Goodwin, Jacob %A Salvatore, Brian A %A Novotna, Jana %A Turanek, Jaroslav %A Ledvina, Miroslav %A Hozak, Pavel %A Zhivotovsky, Boris %A Coster, Mark J %A Ralph, Stephen J %A Smith, Robin A J %A Neuzil, Jiri %T Mitochondrial targeting of α-tocopheryl succinate enhances its pro-apoptotic efficacy: A new paradigm for effective cancer therapy. %B Free radical biology & medicine %D 2011 %C United States %I Elsevier Inc. %V 50 %N 11 %P 1546-55 %@ 0891-5849 %X Mitochondria are emerging as intriguing targets for anti-cancer agents. We tested here a novel approach, whereby the mitochondrially targeted delivery of anti-cancer drugs is enhanced by the addition of a triphenylphosphonium group (TPP(+)). A mitochondrially targeted analog of vitamin E succinate (MitoVES), modified by tagging the parental compound with TPP(+), induced considerably more robust apoptosis in cancer cells with a 1-2 log gain in anti-cancer activity compared to the unmodified counterpart, while maintaining selectivity for malignant cells. This is because MitoVES associates with mitochondria and causes fast generation of reactive oxygen species that then trigger mitochondria-dependent apoptosis, involving transcriptional modulation of the Bcl-2 family proteins. MitoVES proved superior in suppression of experimental tumors compared to the untargeted analog. We propose that mitochondrially targeted delivery of anti-cancer agents offers a new paradigm for increasing the efficacy of compounds with anti-cancer activity. %Z FOR Codes: 111201 %0 Journal Article %~ PubMed %A Dong, Lan-Feng %A Jameson, Victoria J A %A Tilly, David %A Cerny, Jiri %A Mahdavian, Elahe %A Marín-Hernández, Alvaro %A Hernández-Esquivel, Luz %A Rodríguez-Enríquez, Sara %A Stursa, Jan %A Witting, Paul K %A Stantic, Bela %A Rohlena, Jakub %A Truksa, Jaroslav %A Kluckova, Katarina %A Dyason, Jeffrey C %A Ledvina, Miroslav %A Salvatore, Brian A %A Moreno-Sánchez, Rafael %A Coster, Mark J %A Ralph, Stephen J %A Smith, Robin A J %A Neuzil, Jiri %T Mitochondrial targeting of vitamin E succinate enhances its pro-apoptotic and anti-cancer activity via mitochondrial complex II. %B Journal of Biological Chemistry %D 2011 %C United States %I American Society for Biochemistry and Molecular Bi %V 286 %N 5 %P 3717-3128 %@ 1083-351X %X Mitochondrial complex II (CII) has been recently identified as a novel target for anti-cancer drugs. Mitochondrially targeted vitamin E succinate (MitoVES) is modified so that it is preferentially localized to mitochondria, greatly enhancing its pro-apoptotic and anti-cancer activity. Using genetically manipulated cells, MitoVES caused apoptosis and generation of reactive oxygen species (ROS) in CII-proficient malignant cells but not their CII-dysfunctional counterparts. MitoVES inhibited the succinate dehydrogenase (SDH) activity of CII with IC(50) of 80 ?M, whereas the electron transfer from CII to CIII was inhibited with IC(50) of 1.5 ?M. The agent had no effect either on the enzymatic activity of CI or on electron transfer from CI to CIII. Over 24 h, MitoVES caused stabilization of the oxygen-dependent destruction domain of HIF1? fused to GFP, indicating promotion of the state of pseudohypoxia. Molecular modeling predicted the succinyl group anchored into the proximal CII ubiquinone (UbQ)-binding site and successively reduced interaction energies for serially shorter phytyl chain homologs of MitoVES correlated with their lower effects on apoptosis induction, ROS generation, and SDH activity. Mutation of the UbQ-binding Ser(68) within the proximal site of the CII SDHC subunit (S68A or S68L) suppressed both ROS generation and apoptosis induction by MitoVES. In vivo studies indicated that MitoVES also acts by causing pseudohypoxia in the context of tumor suppression. We propose that mitochondrial targeting of VES with an 11-carbon chain localizes the agent into an ideal position across the interface of the mitochondrial inner membrane and matrix, optimizing its biological effects as an anti-cancer drug. %Z FOR Codes: 111201 %0 Journal Article %~ PubMed %A Kim, Hyun Bo %A Shanu, Anu %A Wood, Sarah %A Parry, Sarah N %A Collet, Michael %A McMahon, Aisling %A Witting, Paul K %T Phenolic antioxidants tert-butyl-bisphenol and vitamin E decrease oxidative stress and enhance vascular function in an animal model of rhabdomyolysis yet do not improve acute renal dysfunction. %B Free radical research %D 2011 %C Switzerland %I Informa Healthcare %V 45 %N 9 %P 1000-12 %@ 1029-2470 %X Rhabdomyolysis (RM) caused by severe burn releases extracellular myoglobin (Mb) that accumulates in the kidney. Extracellular Mb is a pro-oxidant. This study tested whether supplementation with tert-butyl-bisphenol (BP) or vitamin E (Vit E, as ??-tocopherol) at 0.12% w/w in the diet inhibits acute renal failure (ARF) in an animal model of RM. After RM-induction in rats, creatinine clearance decreased (p < 0.01), proteinuria increased (p < 0.001) and renal-tubule damage was detected. Accompanying ARF, biomarkers of oxidative stress (lipid oxidation and hemeoxygenase-1 (HO-1) gene and protein activity) increased in the kidney (p < 0.05). Supplemented BP or Vit E decreased lipid oxidation (p < 0.05) and HO-1 gene/activity and restored aortic cyclic guanylyl monophosphate in control animals (p < 0.001), yet ARF was unaffected. Antioxidant supplementation inhibited oxidative stress, yet was unable to ameliorate ARF in this animal model indicating that oxidative stress in kidney and vascular cells may not be causally related to renal dysfunction elicited by RM. %Z FOR Codes: 111501 %0 Journal Article %~ PubMed %A Witting, Paul K %A Song, Changjie %A Hsu, Kenneth %A Hua, Susan %A Parry, Sarah N %A Aran, Roshanak %A Geczy, Carolyn %A Freedman, Saul Benedict %T The acute-phase protein serum amyloid A induces endothelial dysfunction that is inhibited by high-density lipoprotein. %B Free radical biology & medicine %D 2011 %C United States %I Elsevier Inc. %V 51 %N 7 %P 1390-8 %@ 0891-5849 %X The acute-phase protein serum amyloid A (SAA) is elevated during inflammation and may be deposited in atheroma where it promotes atherosclerosis. We investigated the proatherogenic effects of SAA on the vascular endothelium and their regulation by high-density lipoprotein (HDL). Exposure of human aortic endothelial cells (HAEC) to SAA (0.25-25??g/ml) decreased nitric oxide ((???)NO) synthesis/bioavailability, although the endothelial NO synthase monomer-to-dimer ratio was unaffected. SAA (10??g/ml) stimulated a Ca(2+) influx linked to apocynin-sensitive superoxide radical anion (O(2)(???-)) production. Gene expression for arginase-1, nuclear factor ??B (NF-??B), interleukin-8, and tissue factor (TF) increased within 4h of SAA stimulation. Enzymatically active Arg-1/2 was detected in HAEC cultured with SAA for 24h. Therefore, in addition to modulating (???)NO bioavailability by stimulating O(2)(???-) production in the endothelium, SAA modulated vascular l-Arg bioavailability. SAA also diminished relaxation of preconstricted aortic rings induced by acetylcholine, and added superoxide dismutase restored the vascular response. Preincubation of HAEC with HDL (100 or 200, but not 50, ??g/ml) before (not after) SAA treatment ameliorated the Ca(2+) influx and O(2)(???-) production; decreased TF, NF-??B, and Arg-1 gene expression; and preserved overall vascular function. Thus, SAA may promote endothelial dysfunction by modulating (???)NO and l-Arg bioavailability, and HDL pretreatment may be protective. The relative HDL to SAA concentrations may regulate the proatherogenic properties of SAA on the vascular endothelium. %Z FOR Codes: 110201 30401 110704 %0 Journal Article %~ PubMed %A Cho, Jin-Gun %A Witting, Paul K %A Verma, Manisha %A Wu, Ben J %A Shanu, Anu %A Kairaitis, Kristina %A Amis, Terence C %A Wheatley, John R %T Tissue vibration induces carotid artery endothelial dysfunction: a mechanism linking snoring and carotid atherosclerosis? %B Sleep %D 2011 %C United States %I The American Academy of Sleep Medicine %V 34 %N 6 %P 751-757 %@ 0161-8105 %X We have previously identified heavy snoring as an independent risk factor for carotid atherosclerosis. In order to explore the hypothesis that snoring-associated vibration of the carotid artery induces endothelial dysfunction (an established atherogenic precursor), we utilized an animal model to examine direct effects of peri-carotid tissue vibration on carotid artery endothelial function and structure. %Z FOR Codes: 110203 110201 %0 Journal Article %~ PubMed %A Morrison, Daniel E %A Issa, Fatiah %A Bhadbhade, Mohan %A Groebler, Ludwig %A Witting, Paul K %A Kassiou, Michael %A Rutledge, Peter J %A Rendina, Louis M %T Boronated phosphonium salts containing arylboronic acid, closo-carborane, or nido-carborane: synthesis, X-ray diffraction, in vitro cytotoxicity, and cellular uptake. %B Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry %D 2010 %C Germany %I Springer %V 15 %N %P 1305-18 %@ 1432-1327 %X The preparation of boronated triaryl and tetraaryl phosphonium salts of the type [PPh(3)CH(2)R]Br [R is 4-boronophenyl (1), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-yl)phenyl (2), 3-boronophenyl (3), 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-yl)phenyl (4), 2-boronophenyl (5), 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-yl)phenyl (6), and closo-1,2-carboran-1-yl (7)] is described. These compounds were prepared by the reaction of triphenylphosphine with benzylic bromides or 1-bromomethyl-closo-1,2-carborane in acetonitrile solution at 85 ??C. The zwitterionic nido-7,8-carborane derivative PPh(3)CH(2)C(2)B(9)H(11) (8) was prepared by treatment of 7 with cesium fluoride in refluxing ethanol. All compounds were fully characterized by multinuclear ((1)H, (11)B, (13)C, and (31)P) 1D- and 2D-NMR spectroscopy, electrospray ionization mass spectrometry, and elemental analysis, and single-crystal X-ray structures were determined for compounds 1, 3, 7, and 8. The cytotoxicities and boron uptake of selected derivatives were investigated in vitro using human glioblastoma (T98G) and canine kidney tubule (MDCK II) cells. The zwitterionic species 8 was found to be the least cytotoxic agent while also delivering the greatest amount of boron to the T98G cells, peaking at 9.15 ?? 2.65 ??g B/mg protein. %Z FOR Codes: 30401 30201 %0 Journal Article %~ PubMed %A Liu, Joe %A Obando, Daniel %A Schipanski, Liam G %A Groebler, Ludwig K %A Witting, Paul K %A Kalinowski, Danuta S %A Richardson, Des R %A Codd, Rachel %T Conjugates of Desferrioxamine B (DFOB) with Derivatives of Adamantane or with Orally Available Chelators as Potential Agents for Treating Iron Overload. %B Journal of medicinal chemistry %D 2010 %C United States %I American Chemical Society %V 53 %N 3 %P 1370-82 %@ 0022-2623 %X Desferrioxamine B (DFOB) conjugates with adamantane-1-carboxylic acid, 3-hydroxyadamantane-1-carboxylic acid, 3,5-dimethyladamantane-1-carboxylic acid, adamantane-1-acetic acid, 4-methylphenoxyacetic acid, 3-hydroxy-2-methyl-4-oxo-1-pyridineacetic acid (N-acetic acid derivative of deferiprone), or 4-[3,5-bis(2-hydroxyphenyl)-1,2,4-triazol-1-yl]benzoic acid (deferasirox) were prepared and the integrity of Fe(III) binding of the compounds was established from electrospray ionization mass spectrometry and RP-HPLC measurements. The extent of intracellular (59)Fe mobilized by the DFOB-3,5-dimethyladamantane-1-carboxylic acid adduct was 3-fold greater than DFOB alone, and the IC(50) value of this adduct was 6- or 15-fold greater than DFOB in two different cell types. The relationship between logP and (59)Fe mobilization for the DFOB conjugates showed that maximal mobilization of intracellular (59)Fe occurred at a logP value approximately 2.3. This parameter, rather than the affinity for Fe(III), appears to influence the extent of intracellular (59)Fe mobilization. The low toxicity-high Fe mobilization efficacy of selected adamantane-based DFOB conjugates underscores the potential of these compounds to treat iron overload disease in patients with transfusional-dependent disorders such as beta-thalassemia. %Z FOR Codes: 30405 30404 110103 %0 Journal Article %~ PubMed %A Niknami, Marzieh %A Vignarajan, Soma %A Yao, Mu %A Hua, Sheng %A Witting, Paul K %A Kita, Yoshihiro %A Shimizu, Takao %A Sved, Paul %A Patel, Manish I %A Dong, Qihan %T Decrease in expression or activity of cytosolic phospholipase A(2)alpha increases cyclooxygenase-1 action: A cross-talk between key enzymes in arachidonic acid pathway in prostate cancer cells. %B Biochimica et biophysica acta %D 2010 %C Netherlands %I Elsevier BV %V 1801 %N 7 %P 731-7 %@ 0006-3002 %X The eicosanoid pathway is activated in many types of cancers including prostate. Eicosanoids are synthesized from intracellular arachidonic acid (AA), which is released from membrane glycerophospholipids mainly by the action of cytosolic phospholipase A(2)alpha (cPLA(2)alpha). Thus, targeting cPLA(2)alpha has been proposed as a treatment option. The aim of this study was to determine the effect of cPLA(2)alpha inhibition on cyclooxygenase (COX) expression and PGE(2) production. Inhibition of cPLA(2)alpha expression by siRNA or activity by Efipladib in prostate cancer cell lines (PC3 and LNCaP) led to an increase in COX-1 protein and PGE(2) levels in a dose-dependent manner from 24 to 72 h. The COX-2 response was less evident. Efipladib treatment increased COX-1 promoter transcriptional activity without changing the rate of COX-1 protein degradation. Treatment with Efipladib also led to a decrease in most LOX products (HETEs) as measured by LC/MS/MS. Replenishing 5- and 12-HETEs abolished Efipladib-induced COX-1 and PGE(2) levels. Decreasing 5- and 12-HETE production, as a result of treating cells with inhibitors MK886 and Baicalein, respectively, mimicked the effect of Efipladib on COX-1 and PGE(2) levels. Hence, the mechanism underlying the cPLA(2)alpha inhibition-induced COX-1 is likely due to a decrease in LOX products, which may exert a negative feedback on COX-1 gene expression in prostate cancer cells. Considering that PGE(2) is a potent promoter of cancer cell proliferation and survival, understanding the mechanism coupling cPLA(2)alpha with COX-1 is of potential clinical significance. %Z FOR Codes: 111201 110104 %0 Journal Article %~ PubMed %A Wu, Ben J %A Yan, Ling %A Charlton, Francesca %A Witting, Paul %A Barter, Philip J %A Rye, Kerry-Anne %T Evidence that niacin inhibits acute vascular inflammation and improves endothelial dysfunction independent of changes in plasma lipids. %B Arteriosclerosis, Thrombosis, and Vascular Biology %D 2010 %C United States %I Lippincott Williams & Wilkins %V 30 %N 5 %P 968-975 %@ 1524-4636 %X OBJECTIVE: To determine if niacin can confer cardiovascular benefit by inhibiting vascular inflammation and improving endothelial function independent of changes in plasma lipid and lipoprotein levels. METHODS AND RESULTS: New Zealand white rabbits received normal chow or chow supplemented with 0.6% or 1.2% (wt/wt) niacin. This regimen had no effect on plasma cholesterol, triglyceride, or high-density lipoprotein levels. Acute vascular inflammation and endothelial dysfunction were induced in the animals with a periarterial carotid collar. At the 24-hour postcollar implantation, the endothelial expression of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and monocyte chemotactic protein-1 was markedly decreased in the niacin-supplemented animals compared with controls. Niacin also inhibited intima-media neutrophil recruitment and myeloperoxidase accumulation, enhanced endothelial-dependent vasorelaxation and cyclic guanosine monophosphate production, increased vascular reduced glutathione content, and protected against hypochlorous acid-induced endothelial dysfunction and tumor necrosis factor alpha-induced vascular inflammation. CONCLUSION: Previous human intervention studies have demonstrated that niacin inhibits coronary artery disease. This benefit is thought to be because of its ability to reduce low-density lipoprotein and plasma triglyceride levels and increase high-density lipoprotein levels. The present study showed that niacin inhibits vascular inflammation and protects against endothelial dysfunction independent of these changes in plasma lipid levels. %Z FOR Codes: 1102 %0 Journal Article %~ PubMed %A Wang, Yutang %A Liu, Hanzhong %A McKenzie, Gavin %A Witting, Paul K %A Stasch, Johannes-Peter %A Hahn, Michael %A Changsirivathanathamrong, Dechaboon %A Wu, Ben J %A Ball, Helen J %A Thomas, Shane R %A Kapoor, Vimal %A Celermajer, David S %A Mellor, Andrew L %A Keaney, John F %A Hunt, Nicholas H %A Stocker, Roland %T Kynurenine is an endothelium-derived relaxing factor produced during inflammation. %B Nature Medicine %D 2010 %C United States %I Nature Publishing Group %V 16 %N 3 %P 279-285 %@ 1078-8956 %X Control of blood vessel tone is central to vascular homeostasis. Here we show that metabolism of tryptophan to kynurenine by indoleamine 2,3-dioxygenase (Ido) expressed in endothelial cells contributes to arterial vessel relaxation and the control of blood pressure. Infection of mice with malarial parasites (Plasmodium berghei) or induction of endotoxemia in mice led to endothelial expression of Ido, decreased plasma tryptophan concentration, increased kynurenine concentration and hypotension. Pharmacological inhibition of Ido increased blood pressure in systemically inflamed mice but not in mice deficient in Ido or interferon-gamma, which is required for Ido induction. Both tryptophan and kynurenine dilated preconstricted porcine coronary arteries; the dilating effect of tryptophan required the presence of active Ido and an intact endothelium, whereas the effect of kynurenine was endothelium independent. The arterial relaxation induced by kynurenine was mediated by activation of the adenylate and soluble guanylate cyclase pathways. Kynurenine administration decreased blood pressure in a dose-dependent manner in spontaneously hypertensive rats. Our results identify tryptophan metabolism by Ido as a new pathway contributing to the regulation of vascular tone. %Z FOR Codes: 110201 %0 Journal Article %~ PubMed %A Antao, Shane Tony %A Duong, T T Hong %A Aran, Roshanak %A Witting, Paul K %T Neuroglobin over-expression in cultured human neuronal cells protects against hydrogen peroxide insult via activating phosphoinositide-3 kinase and opening the mitochondrial KATP channel. %B Antioxidants & redox signaling %D 2010 %C United States %I Mary Ann Liebert, Inc. Publishers %V 13 %N 6 %P 769-81 %@ 1557-7716 %X Cultured neurons tolerate low H(2)O(2) concentrations (< or =50 microM) through the activity of constitutive antioxidant response elements (ARE). At H(2)O(2) levels (> or =100 microM), neurons increase expression of the gene encoding for inducible hemoxygenase-1 while superoxide dismutase-2 and catalase remain unchanged. Despite this adaptive response, the endogenous antioxidant systems are overwhelmed, leading to decreased viability. Elevating the neuronal cell content of human neuroglobin (Ngb) prior to insult with 100 or 200 microM H(2)O(2) enhanced cell viability and this resulted in a significant decrease in oxidative stress and an increase in the intracellular ATP concentration, whereas in parental cells exposed to the same H(2)O(2)-insult, oxidative stress and ATP increased and decreased, respectively. The mechanism for this increase in ATP involves sustained activation of the mito-K(ATP) channel and an increase in phosphoinositide-3 kinase (PI3K)-mediated phosphorylation of Akt. Pharmacological inhibitors directed toward PI3K (wortmannin and LY294002), or the mito-K(ATP) channel (glybenclamide) inhibited the H(2)O(2)-mediated increase in ATP in cells overexpressing human Ngb and consequently cell viability decreased. Neuroglobin''s ability to bolster the intracellular pool of ATP in response to added H(2)O(2) is central to the preservation of cytoskeletal integrity and cell viability. %Z FOR Codes: 110999 110299 110106 %0 Journal Article %~ PubMed %A Carter, Elizabeth A %A Rayner, Benjamin S %A McLeod, Andrew I %A Wu, Lindsay E %A Marshall, Craig P %A Levina, Aviva %A Aitken, Jade B %A Witting, Paul K %A Lai, Barry %A Cai, Zhonghou %A Vogt, Stefan %A Lee, Yao-Chang %A Chen, Ching-Iue %A Tobin, Mark J %A Harris, Hugh H %A Lay, Peter A %T Silicon nitride as a versatile growth substrate for microspectroscopic imaging and mapping of individual cells. %B Molecular bioSystems %D 2010 %C United Kingdom %I Royal Society of Chemistry %V 6 %N 7 %P 1316-22 %@ 1742-2051 %X Herein is described a general sampling protocol that includes culture, differentiation and fixing of cells in their preferred morphology on the one sample substrate (Si(3)N(4)) to enable subsequent diverse modern microspectroscopic analyses. The protocol enables unprecedented correlated and complementary information on the intracellular biochemistry of metabolic processes, diseases and their treatment, which offers the opportunity to revolutionize our understanding of cell and tissue biology at a molecular level. The culture of adherent cells onto inexpensive Si(3)N(4) membranes allows microspectroscopic analyses across the electromagnetic spectrum, from hard X-ray fluorescence (both XRF and XANES), through to visible and fluorescence light microscopies, and infrared microspectroscopy without substrate interference. Adherent mammalian cell lines (3T3-L1 adipocytes and H9c2 cardiac myocytes) illustrate the in vitro application of these protocols. The cells adhered strongly to Si(3)N(4) membranes and visually displayed normal proliferative and phenotypic growth; more importantly, rapid alcohol fixation of cells did not affect their structural integrity for subsequent analyses. %Z FOR Codes: 100402 60101 30201 %0 Journal Article %~ PubMed %A Szuchman-Sapir, Andrea J %A Pattison, David I %A Davies, Michael J %A Witting, Paul K %T Site-specific hypochlorous acid-induced oxidation of recombinant human myoglobin affects specific amino acid residues and the rate of cytochrome b(5)-mediated heme reduction. %B Free radical biology & medicine %D 2010 %C United States %I Elsevier Inc. %V 48 %N 1 %P 35-46 %@ 0891-5849 %X Myeloperoxidase catalyzes the reaction of chloride ions with H(2)O(2) to yield hypochlorous acid (HOCl), which can damage proteins. Human myoglobin (HMb) differs from other Mbs by the presence of a cysteine residue at position 110 (Cys110). This study has (i) compared wild-type and a Cys110Ala variant of HMb to assess the influence of Cys110 on HOCl-induced amino acid modification and (ii) determined whether HOCl oxidation of HMb affects the rate of ferric heme reduction by cytochrome b(5). For wild-type HMb (HOCl:Mb ratio of 5:1 mol:mol), Cys110 was preferentially oxidized to a homodimeric or cysteic acid product-sulfenic/sulfinic acids were not detected. At a HOCl:Mb ratio 10:1 mol:mol, methionine (Met) oxidation was detected, and this was enhanced in the Cys110Ala variant. Tryptophan (Trp) oxidation was detected only in the Cys110Ala variant at the highest HOCl dose tested, with oxidation susceptibility following the order Cys>Met>Trp. Tyrosine chlorination was evident only in reactions between HOCl and the Cys110Ala variant and at a longer incubation time (24 h), consistent with the formation via chlorine-transfer reactions from preformed chloramines. HOCl-mediated oxidation of wild-type HMb resulted in a dose-dependent decrease in the observed rate constant for ferric heme reduction (approx two-fold at HOCl:Mb of 10:1 mol:mol). These data indicate that Cys110 influences the oxidation of HMb by HOCl and that oxidation of Cys, Met, and Trp residues is associated with a decrease in the one-electron reduction of ferric HMb by other proteins; such heme-Fe(3+) reduction is critical to the maintenance of function as an oxygen storage protein in tissues. %Z FOR Codes: 604 %0 Journal Article %~ PubMed %A Shanu, Anu %A Parry, Sarah N %A Wood, Sarah %A Rodas, Elicia %A Witting, Paul K %T The synthetic polyphenol tert-butyl-bisphenol inhibits myoglobin-induced dysfunction in cultured kidney epithelial cells. %B Free radical research %D 2010 %C United Kingdom %I Informa Healthcare %V 44 %N 8 %P 843-53 %@ 1029-2470 %X Abstract Rhabdomyolysis caused by severe burn releases extracellular myoglobin (Mb) that accumulates in the kidney and urine (maximum [Mb] approximately 50 microM) (termed myoglobinuria). Extracellular Mb can be a pro-oxidant. This study cultured Madin-Darby-canine-kidney-Type-II (MDCK II) cells in the presence of Mb and tested whether supplementation with a synthetic tert-butyl-polyphenol (tert-butyl-bisphenol; t-BP) protects these renal cells from dysfunction. In the absence of t-BP, cells exposed to 0-100 microM Mb for 24 h showed a dose-dependent decrease in ATP and the total thiol (TSH) redox status without loss of viability. Gene expression of superoxide dismutases-1/2, haemoxygenase-1 and tumour necrosis factor increased and receptor-mediated endocytosis of transferrin and monolayer permeability decreased significantly. Supplementation with t-BP before Mb-insult maintained ATP and the TSH redox status, diminished antioxidant/pro-inflammatory gene responses, enhanced monolayer permissiveness and restored transferrin uptake. Overall, bolstering the total antioxidant capacity of the kidney may protect against oxidative stress induced by experimental myoglobinuria. %Z FOR Codes: 110199 %0 Journal Article %~ PubMed %A Hua, Susan %A Song, Changjie %A Geczy, Carolyn L %A Freedman, S Ben %A Witting, Paul K %T A role for acute-phase serum amyloid A and high-density lipoprotein in oxidative stress, endothelial dysfunction and atherosclerosis. %B Redox Report %D 2009 %C United Kingdom, Aust %I Maney Publishing %V 14 %N 5 %P 187-196 %@ 1743-2928 %X The acute-phase protein serum amyloid A (SAA) is a clinically useful marker of inflammation and associates strongly with increased risk of cardiovascular events. Chronically elevated SAA concentrations may contribute to physiological processes that lead to atherosclerosis, including endothelial dysfunction, an early and predictive event in the development of cardiovascular disease. Accumulating data suggest that SAA can be a direct mediator in the development and progression of atherogenesis and atherothrombosis. SAA may affect key events underlying acute coronary syndromes, including cholesterol transport, contribute to endothelial dysfunction, promote thrombosis, and enhance leukocyte trafficking and activation. This review summarizes the evidence supporting a role for SAA as a potential regulator of inflammation and endothelial dysfunction, which underlie the adverse outcomes that complicate coronary artery disease. The findings suggest that novel therapeutic strategies to reduce SAA levels and/or oppose the actions of SAA may have beneficial effects in patients with coronary artery disease. %Z FOR Codes: 1102 1107 601 %0 Journal Article %~ PubMed %A Whiteman, Ineka T %A Gervasio, Othon L %A Cullen, Karen M %A Guillemin, Gilles J %A Jeong, Erica V %A Witting, Paul K %A Antao, Shane T %A Minamide, Laurie S %A Bamburg, James R %A Goldsbury, Claire %T Activated actin-depolymerizing factor/cofilin sequesters phosphorylated microtubule-associated protein during the assembly of alzheimer-like neuritic cytoskeletal striations. %B The Journal of neuroscience %D 2009 %C United States %I Society for Neuroscience %V 29 %N 41 %P 12994-13005 %@ 1529-2401 %X In Alzheimer''s disease (AD), rod-like cofilin aggregates (cofilin-actin rods) and thread-like inclusions containing phosphorylated microtubule-associated protein (pMAP) tau form in the brain (neuropil threads), and the extent of their presence correlates with cognitive decline and disease progression. The assembly mechanism of these respective pathological lesions and the relationship between them is poorly understood, yet vital to understanding the causes of sporadic AD. We demonstrate that, during mitochondrial inhibition, activated actin-depolymerizing factor (ADF)/cofilin assemble into rods along processes of cultured primary neurons that recruit pMAP/tau and mimic neuropil threads. Fluorescence resonance energy transfer analysis revealed colocalization of cofilin-GFP (green fluorescent protein) and pMAP in rods, suggesting their close proximity within a cytoskeletal inclusion complex. The relationship between pMAP and cofilin-actin rods was further investigated using actin-modifying drugs and small interfering RNA knockdown of ADF/cofilin in primary neurons. The results suggest that activation of ADF/cofilin and generation of cofilin-actin rods is required for the subsequent recruitment of pMAP into the inclusions. Additionally, we were able to induce the formation of pMAP-positive ADF/cofilin rods by exposing cells to exogenous amyloid-beta (Abeta) peptides. These results reveal a common pathway for pMAP and cofilin accumulation in neuronal processes. The requirement of activated ADF/cofilin for the sequestration of pMAP suggests that neuropil thread structures in the AD brain may be initiated by elevated cofilin activation and F-actin bundling that can be caused by oxidative stress, mitochondrial dysfunction, or Abeta peptides, all suspected initiators of synaptic loss and neurodegeneration in AD. %Z FOR Codes: 110999 %0 Journal Article %~ PubMed %A Reichelt, Melissa E %A Shanau, Ana %A Willems, Laura %A Witting, Paul K %A Ellis, Natasha A %A Blackburn, Michael R %A Headrick, John P %T Endogenous adenosine selectively modulates oxidant stress via the A1 receptor in ischemic hearts. %B Antioxidants & redox signaling %D 2009 %C United States %I Mary Ann Liebert, Inc. Publishers %V 11 %N 11 %P 2641-50 %@ 1557-7716 %X We tested the impact of A1 adenosine receptor (AR) deletion on injury and oxidant damage in mouse hearts subjected to 25-min ischemia/45-min reperfusion (I/R). Wild-type hearts recovered approximately 50% of contractile function and released 8.2 +/- 0.7 IU/g of lactate dehydrogenase (LDH). A1AR deletion worsened dysfunction and LDH efflux (15.2 +/- 2.6 IU/g). Tissue cholesterol and native cholesteryl esters were unchanged, whereas cholesteryl ester-derived lipid hydroperoxides and hydroxides (CE-O(O)H; a marker of lipid oxidation) increased threefold, and alpha-tocopherylquinone [alpha-TQ; oxidation product of alpha-tocopherol (alpha-TOH)] increased sixfold. Elevations in alpha-TQ were augmented by two- to threefold by A1AR deletion, whereas CE-O(O)H was unaltered. A(1)AR deletion also decreased glutathione redox status ([GSH]/[GSSG + GSH]) and enhanced expression of the antioxidant response element heme oxygenase-1 (HO-1) during I/R: fourfold elevations in HO-1 mRNA and activity were doubled by A1AR deletion. Broad-spectrum AR agonism (10 microM 2-chloroadenosine; 2-CAD) countered effects of A1AR deletion on oxidant damage, HO-1, and tissue injury, indicating that additional ARs (A(2A), A(2B), and/or A3) can mediate similar actions. These data reveal that local adenosine engages A1ARs during I/R to limit oxidant damage and enhance outcome selectively. Control of alpha-TOH/alpha-TQ levels may contribute to A1AR-dependent cardioprotection. %Z FOR Codes: 110201 %0 Journal Article %~ PubMed %A Niknami, Marzieh %A Patel, Manish %A Witting, Paul K %A Dong, Qihan %T Molecules in focus: Cytosolic phospholipase A(2)-alpha. %B The international journal of biochemistry & cell biology %D 2009 %C United Kingdom %I Elsevier %V 41 %N 5 %P 994-7 %@ 1357-2725 %X Cytosolic phospholipase A(2)-alpha (cPLA(2)-alpha) cleaves its preferred substrate, arachidonic acid, at the sn-2 position of membrane glycerophospholipids. Stimulation of cells with agents that mobilize intracellular calcium and/or promote the phosphorylation of cPLA(2)-alpha leads to (i) translocation of the enzyme from cytosol to endoplasmic reticulum, Golgi apparatus and perinuclear membranes-where it associates with the arachidonic acid in close proximity to downstream eicosanoid-producing enzymes; and (ii) the change in configuration induced by phosphorylation increases the phospholipid binding affinity and arachidonic acid release. As a mediator of growth factors, cytokines, chemokines, and hormones that modulate survival and growth in various cell types, cPLA(2)-alpha has attracted considerable attention as a potential therapeutic target in control of inflammation and cancer. The importance of the enzyme may have been underestimated by the relatively normal phenotype in the enzyme knockout animals. A clear phenotype has emerged when these knockout animals are used as models of various diseases. %Z FOR Codes: 111201 %0 Journal Article %~ PubMed %A Duong, Thi Thuy Hong %A Witting, Paul Kenneth %A Antao, Shane Tony %A Parry, Sarah Nicole %A Kennerson, Marina %A Lai, Barry %A Vogt, Stefan %A Lay, Peter Andrew %A Harris, Hugh Hamlyn %T Multiple protective activities of neuroglobin in cultured neuronal cells exposed to hypoxia re-oxygenation injury. %B Journal of neurochemistry %D 2009 %C United Kingdom %I Wiley-Blackwell %V 108 %N 5 %P 1143-1154 %@ 1471-4159 %X Oxidative stress is associated with the pathology of acute and chronic neurodegenerative disease. We have cloned a human neuroglobin (Nb) construct and over-expressed this protein in cultured human neuronal cells to assess whether Nb ameliorates the cellular response to experimental hypoxia-reoxygenation (H/R) injury. Parental cells transfected with a blank (pDEST40) vector responded to H/R injury with a significant decrease in cellular ATP at 5 and 24 h after insult. This was coupled with increases in the cytosolic Ca(2+), and the transition metals iron (Fe), copper (Cu), and zinc (Zn) within the cell body, as monitored simultaneously using X-ray fluorescence microprobe imaging. Parental cell viability decreased over the same time period with a approximately 4 to 5-fold increase in cell death (maximum approximately 25%) matched by an increase in caspase 3/7 activation (peaking at a 15-fold increase after 24 h) and condensation of beta-actin along axonal processes. Over-expression of Nb inhibited ATP loss and except for significant decreases in the sulfur (S), chlorine (Cl), potassium (K) and Ca(2+) contents, maintained cellular ion homeostasis after H/R insult. This resulted in increased cell viability, significantly diminished caspase activation and maintenance of the beta-actin cytoskeletal structure and receptor-mediated endocytosis. These data indicate that bolstering the cellular content of Nb inhibits neuronal cell dysfunction promoted by H/R insult through multiple protective actions including: (i) maintenance of cellular bioenergetics; (ii) inhibition of Ca(2+) influx; (iii) a reduction in cellular uptake of Fe, Cu and Zn at the expense of S, Cl and K; and (iv) an enhancement of cell viability through inhibiting necrosis and apoptosis. %Z FOR Codes: 1109 %0 Journal Article %~ PubMed %A Rayner, Benjamin S %A Hua, Susan %A Sabaretnam, Tharani %A Witting, Paul K %T Nitric oxide stimulates myoglobin gene and protein expression in vascular smooth muscle. %B Biochemical Journal %D 2009 %C United Kingdom %I Portland Press Ltd. %V 423 %N 2 %P 169-177 %@ 1470-8728 %X Mb (myoglobin) is a haemoprotein present in cardiac, skeletal and smooth muscle and is primarily responsible for the storage and ''facilitated transfer'' of molecular oxygen from the cell membrane to mitochondria. Also, Mb plays a role in regulating *NO (nitric oxide) homoeostasis through (i) binding *NO (Mb-NO complex); (ii) oxidation of *NO to nitrate; and (iii) formation of vasoactive S-nitroso-Mb [Rayner, B.S., Wu, B.-J., Raftery, M., Stocker, R. and Witting, P.K. (2005) J. Biol. Chem. 280, 9985-9993]. Pathological *NO concentrations affect mitochondrial function and decrease cell viability through inducing apoptosis. Treatment of cultured rat VSMCs (vascular smooth muscle cells) with cumulative doses (0.1, 1 or 10 microM) of *NO from the donors diethylamineNONOate or spermineNONOate (N-[2-aminoethyl]-N-[2-hydroxy-3-nitrosohydrazine]-1,2-ethelenediamine) yielded a time-dependent increase in Mb gene expression. Concomitant transcriptional activation increased the concentration of Mb within cultured rat or primary human VSMCs as judged by Western blot analysis and indirect immunofluorescence microscopy. Cell viability did not decrease in these cells at the *NO doses tested. Importantly, sub-culturing isolated rat aortic segments for 7 days in the presence of L-arginine at 37 degrees C stimulated *NO production with a parallel increase in Mb in the underlying VSMCs. Overall, exposure of VSMCs (either in cell culture or intact vessels) to pathological *NO promotes an up-regulation of the Mb gene and protein, suggesting a feedback relationship between *NO and Mb that regulates the concentration of the potent cell signalling molecule in the vessel wall, similar to the role haemoglobin plays in the vessel lumen. %Z FOR Codes: 60108 %0 Journal Article %~ PubMed %A Niknami, Marzieh %A Dong, Qihan %A Witting, Paul %T Pitfalls in the use of arachidonic acid oxidation products to assign lipoxygenase activity in cancer cells. %B Free radical research %D 2009 %C United Kingdom %I Informa Healthcare %V 43 %N 10 %P 951-6 %@ 1029-2470 %X Arachidonic acid (AA) reaction with cyclooxygenase (COX) and lipoxygenases (LOX) yield eicosanoids that can mediate prostate cancer proliferation and enhance both tumour vascularization and metastasis. Increasingly measurement of eicosanoids with liquid chromatography is employed to implicate LOX activity in different biological systems and in particular link LOX activity to the progression of cancer in experimental models. This study demonstrates that simply identifying patterns of eicosanoid regio-isomerism is insufficient to designate LOX activity in prostate cancer cells and the analysis must include complete stereochemical assignment of the various isomers in order to validate the assignment of LOX activity. %Z FOR Codes: 111299 %0 Journal Article %~ PubMed %A Song, Changjie %A Hsu, Kenneth %A Yamen, Eric %A Yan, Weixing %A Fock, Jianyi %A Witting, Paul K %A Geczy, Carolyn L %A Freedman, S Ben %T Serum amyloid A induction of cytokines in monocytes/macrophages and lymphocytes. %B Atherosclerosis %D 2009 %C Ireland %I Elsevier Ireland Ltd %V 207 %N 2 %P 374-83 %@ 0021-9150 %X Serum amyloid A (SAA) is a biomarker of inflammation. Elevated blood levels in cardiovascular disease and local deposition in atheroma implies a role of SAA as a mediator rather than just a marker of inflammation. This study explored SAA-induced cytokine production and secretion by mononuclear cells. %Z FOR Codes: 801 %0 Journal Article %~ PubMed %A Song, Changjie %A Shen, Ying %A Yamen, Eric %A Hsu, Kenneth %A Yan, Weixing %A Witting, Paul K %A Geczy, Carolyn L %A Freedman, S Ben %T Serum amyloid A may potentiate prothrombotic and proinflammatory events in acute coronary syndromes. %B Atherosclerosis %D 2009 %C Ireland %I Elsevier Ireland Ltd %V 202 %N 2 %P 596-604 %@ 0021-9150 %X Elevated serum amyloid A (SAA) levels, like C-reactive protein (CRP), predict coronary events. Both induce monocyte tissue factor (TF), and peripheral blood mononuclear cells (PBMC) from patients with coronary artery disease (CAD) express higher TF in response to CRP. This study examined SAA induction of TF and tumour necrosis factor-alpha (TNF) in PBMC from patients with CAD and in monocytoid THP-1 cells. %Z FOR Codes: 110201 110106 110704 %0 Journal Article %~ PubMed %A Dong, Lan-Feng %A Freeman, Ruth %A Liu, Ji %A Zobalova, Renata %A Marin-Hernandez, Alvaro %A Stantic, Marina %A Rohlena, Jakub %A Valis, Karel %A Rodriguez-Enriquez, Sara %A Butcher, Bevan %A Goodwin, Jacob %A Brunk, Ulf T %A Witting, Paul K %A Moreno-Sanchez, Rafael %A Scheffler, Immo E %A Ralph, Stephen J %A Neuzil, Jiri %T Suppression of tumor growth in vivo by the mitocan alpha-tocopheryl succinate requires respiratory complex II. %B Clinical Cancer Research %D 2009 %C United States %I American Association for Cancer Research %V 15 %N 5 %P 1593-1600 %@ 1078-0432 %X PURPOSE: Vitamin E analogues are potent novel anticancer drugs. The purpose of this study was to elucidate the cellular target by which these agents, represented by alpha-tocopoheryl succinate (alpha-TOS), suppress tumors in vivo, with the focus on the mitochondrial complex II (CII). EXPERIMENTAL DESIGN: Chinese hamster lung fibroblasts with functional, dysfunctional, and reconstituted CII were transformed using H-Ras. The cells were then used to form xenografts in immunocompromized mice, and response of the cells and the tumors to alpha-TOS was studied. RESULTS: The CII-functional and CII-reconstituted cells, unlike their CII-dysfunctional counterparts, responded to alpha-TOS by reactive oxygen species generation and apoptosis execution. Tumors derived from these cell lines reciprocated their responses to alpha-TOS. Thus, growth of CII-functional and CII-reconstituted tumors was strongly suppressed by the agent, and this was accompanied by high level of apoptosis induction in the tumor cells. On the other hand, alpha-TOS did not inhibit the CII-dysfunctional tumors. CONCLUSIONS: We document in this report a novel paradigm, according to which the mitochondrial CII, which rarely mutates in human neoplasias, is a plausible target for anticancer drugs from the group of vitamin E analogues, providing support for their testing in clinical trials. %Z FOR Codes: 30405 %0 Journal Article %~ PubMed %A Sabaretnam, Tharani %A Harris, Matthew J %A Kockx, Maaike %A Witting, Paul K %A Le Couteur, David G %A Kritharides, Leonard %T The Effects Of Hydrogen Peroxide And Apolipoprotein E Isoforms On Apolipoprotein E Trafficking In Hepg2 Cells. %B Clinical and experimental pharmacology & physiology %D 2009 %C Australia %I Wiley-Blackwell Publishing Asia %V 36 %N 12 %P e96-102 %@ 1440-1681 %X 1. The major source of apolipoprotein E (apoE) is the liver. In the present study, the effects of oxidative stress and apoE isoforms on apoE distribution and trafficking were established using the HepG2 liver tumour cell line. 2. Hydrogen peroxide (0, 25, 250 and 1000 micromol/L) was associated with rapid and concentration-dependent redistribution of apoE into the early endosomal compartment. This redistribution was achieved with a much lower concentration (25 micromol/L) than that needed to induce changes in intracellular apoE mRNA expression, apoE protein levels and markers of oxidative stress (250-1000 micromol/L). 3. Live cell imaging of apoE3-green fluorescent protein revealed a significant decrease in traffic velocity in response to oxidative stress. 4. The E4 isoform was associated with reduced trafficking velocity compared with the E3 isoform under basal conditions. 5. The results indicate that oxidative stress and apoE isoforms influence apoE trafficking and distribution within HepG2 cells. Altered apoE hepatocyte trafficking may provide a mechanistic link between oxidative stress, ageing and some diseases in older people. %Z FOR Codes: 601 %0 Journal Article %A Nikinami, M %A Patel, M %A Witting, Paul %A Dong, Q %T Aberrant activation of arachidonic acid and eicosanoid pathways-targets for treating prostate cancer %B Recent Patents on Endocrine, Metabolic & Immune Drug Discovery %D 2008 %C United Kingdom %I Bentham Science Publishers Ltd %V 2 %N %P 9-15 %@ 1872-2148 %X %Z FOR Codes: 110306 %0 Journal Article %~ PubMed %A Dong, L-F %A Low, P %A Dyason, J C %A Wang, X-F %A Prochazka, L %A Witting, P K %A Freeman, R %A Swettenham, E %A Valis, K %A Liu, J %A Zobalova, R %A Turanek, J %A Spitz, D R %A Domann, F E %A Scheffler, I E %A Ralph, S J %A Neuzil, J %T Alpha-tocopheryl succinate induces apoptosis by targeting ubiquinone-binding sites in mitochondrial respiratory complex II. %B Oncogene %D 2008 %C United Kingdom %I Nature Publishing Group %V 27 %N 31 %P 4324-4335 %@ 1476-5594 %X Alpha-tocopheryl succinate (alpha-TOS) is a selective inducer of apoptosis in cancer cells, which involves the accumulation of reactive oxygen species (ROS). The molecular target of alpha-TOS has not been identified. Here, we show that alpha-TOS inhibits succinate dehydrogenase (SDH) activity of complex II (CII) by interacting with the proximal and distal ubiquinone (UbQ)-binding site (Q(P) and Q(D), respectively). This is based on biochemical analyses and molecular modelling, revealing similar or stronger interaction energy of alpha-TOS compared to that of UbQ for the Q(P) and Q(D) sites, respectively. CybL-mutant cells with dysfunctional CII failed to accumulate ROS and underwent apoptosis in the presence of alpha-TOS. Similar resistance was observed when CybL was knocked down with siRNA. Reconstitution of functional CII rendered CybL-mutant cells susceptible to alpha-TOS. We propose that alpha-TOS displaces UbQ in CII causing electrons generated by SDH to recombine with molecular oxygen to yield ROS. Our data highlight CII, a known tumour suppressor, as a novel target for cancer therapy. %Z FOR Codes: 111203 %0 Journal Article %~ PubMed %A Szuchman-Sapir, Andrea %A Pattison, David I %A Ellis, Natasha A %A Hawkins, Clare L %A Davies, Michael J %A Witting, Paul K %T Hypochlorous acid oxidizes methionine and tryptophan residues in myoglobin. %B Free radical biology & medicine %D 2008 %C Orlando, FL, USA %I Elsevier %V 45 %N 6 %P 789-98 %@ 0891-5849 %X After acute myocardial infarction (AMI), infiltrating proinflammatory cells generate two-electron oxidants such as hypochlorous acid (HOCl). Myoglobin (Mb) is present at approximately 0.3 mM in cardiomyocytes and, therefore, represents a significant target for oxidation. Exposure of horse Mb (50 microM) to reagent HOCl (0-500 microM) or activated human neutrophils (4-40x10(6) cells/ml) yielded oxidized Mb (Mb(ox)) as judged by amino acid analysis and peptide mass mapping. HOCl/Mb ratios of 1-5 mol/mol gave Mb(ox) with up to four additional oxygen atoms. Hydrolysis of Mb(ox) followed by amino acid analysis indicated that methionine (Met) and tryptophan (Trp) residues were modified by HOCl. Peptide mass mapping revealed that Met55 was oxidized at a lower HOCl/Mb ratio than Met131 and this preceded Trp7/14 modification (susceptibility Met55>Met131>Trp7>Trp14). Incubation of Mb with activated neutrophils and physiological chloride anion yielded Mb(ox) with a composition similar to that determined with HOCl/Mb ratios <2 mol/mol, with oxidation of Met, but not Trp, detected. These data indicate that Mb undergoes site-specific oxidation depending on the HOCl/protein ratio. As Mb is released from necrotic cardiomyocytes into the vasculature after AMI, HOCl-modified Mb may be a useful surrogate marker to gauge the extent of myocardial inflammation. %Z FOR Codes: 110106 %0 Journal Article %~ PubMed %A Parry, Sarah N %A Ellis, Natasha %A Li, Zhe %A Maitz, Peter %A Witting, Paul K %T Myoglobin induces oxidative stress and decreases endocytosis and monolayer permissiveness in cultured kidney epithelial cells without affecting viability. %B Kidney & blood pressure research %D 2008 %C Switzerland %I S. Karger AG %V 31 %N 1 %P 16-28 %@ 1423-0143 %X BACKGROUND: Muscle degradation caused by severe burn releases myoglobin (Mb), which accumulates in the kidney (termed myoglobinuria). Mb is a pro-oxidant. AIM: To demonstrate that Mb promotes oxidative stress and dysfunction in cultured Madin-Darby canine kidney type II (MDCK II) cells. METHODS: The glutathione redox ratio was used to monitor oxidative stress. Regulation of antioxidant response genes was determined with RT-PCR. Propidium iodide and annexin V staining were markers of necrosis and apoptosis, respectively. Mitochondrial function was assessed by monitoring mitochondrial depolarisation. Endocytosis was determined with immune fluorescence microscopy, and monolayer permeability was monitored with labelled inulin. RESULTS: Kidney epithelial cells exposed to (0-100 muM) Mb showed a dose-dependent decrease in the glutathione redox ratio indicative of enhanced oxidative stress. In parallel, the expression of antioxidant genes for superoxide dismutase (SOD)-1/2, inducible haemoxygenase (HO-1) and catalase (CAT) increased in MDCK II cells, coupled with increases in corresponding activity. Notably, apoptosis and necrosis remained unaffected. However, transferrin endocytosis and monolayer permeability decreased significantly, while clathrin distribution and mitochondrial function were unaffected. CONCLUSION: Low concentrations of Mb promote oxidative stress in kidney epithelial cells that manifest as subtle changes to function without decreasing viability. Whether this impairs kidney function in burns patients is not clear. %Z FOR Codes: 110302 %0 Journal Article %~ PubMed %A Witting, P K %A Zeng, B %A Wong, M %A McMahon, A C %A Rayner, B S %A Sapir, A J %A Lowe, H C %A Freedman, S B %A Brieger, D B %T Polymorphonuclear leukocyte phagocytic function increases in plasminogen knockout mice. %B Thrombosis research %D 2008 %C United Kingdom %I Pergamon %V 122 %N 5 %P 674-82 %@ 0049-3848 %X Mice lacking plasminogen (PG-/-) require alternative pathways of fibrinolysis for survival. This may depend on polymorphonuclear leukocytes (PMN) that can clear soluble and insoluble fibrin(ogen) through PG-independent processes. Our objective was to demonstrate that PMNs from PG-/- mice exhibit increased Mac-1 dependent phagocytic activity, which may explain their increased fibrin(ogen)lytic activity compared with wild type (PG+/+) mice. %Z FOR Codes: 110201 1108 %0 Journal Article %~ PubMed %A Thomas, Shane R %A Witting, Paul K %A Drummond, Grant R %T Redox control of endothelial function and dysfunction: molecular mechanisms and therapeutic opportunities. %B Antioxidants & Redox Signaling %D 2008 %C United States %I Mary Ann Liebert, Inc. Publishers %V 10 %N 10 %P 1713-1765 %@ 1557-7716 %X The endothelium is essential for the maintenance of vascular homeostasis. Central to this role is the production of endothelium-derived nitric oxide (EDNO), synthesized by the endothelial isoform of nitric oxide synthase (eNOS). Endothelial dysfunction, manifested as impaired EDNO bioactivity, is an important early event in the development of various vascular diseases, including hypertension, diabetes, and atherosclerosis. The degree of impairment of EDNO bioactivity is a determinant of future vascular complications. Accordingly, growing interest exists in defining the pathologic mechanisms involved. Considerable evidence supports a causal role for the enhanced production of reactive oxygen species (ROS) by vascular cells. ROS directly inactivate EDNO, act as cell-signaling molecules, and promote protein dysfunction, events that contribute to the initiation and progression of endothelial dysfunction. Increasing data indicate that strategies designed to limit vascular ROS production can restore endothelial function in humans with vascular complications. The purpose of this review is to outline the various ways in which ROS can influence endothelial function and dysfunction, describe the redox mechanisms involved, and discuss approaches for preventing endothelial dysfunction that may highlight future therapeutic opportunities in the treatment of cardiovascular disease. %Z FOR Codes: 110201 %0 Journal Article %~ PubMed %A Duong, T T Hong %A Antao, Shane %A Ellis, Natasha A %A Myers, Simon J %A Witting, Paul K %T Supplementation with a synthetic polyphenol limits oxidative stress and enhances neuronal cell viability in response to hypoxia-re-oxygenation injury. %B Brain research %D 2008 %C Netherlands %I Elsevier BV %V 1219 %N 0 %P 8-18 %@ 0006-8993 %X Oxidative stress is associated with the pathology of acute and chronic neurodegenerative disease. Cultured human neuronal cells exposed to experimental hypoxia-re-oxygenation (H/R) injury responded with an increased production of reactive oxygen species (ROS) and a significant decrease in intracellular ATP. Expression of genes encoding for hypoxia-inducible factor 1-alpha (HIF1-alpha), inducible haemoxygenase-1 (HO-1), glucose transporter-1 (Glut-1), the oxygen-sensor neuroglobin (Nb) and Cu,Zn-superoxide dismutase (SOD1), catalase (CAT) and glutathione peroxidase-1 (Gpx-1) increased significantly in response to the insult. Enhanced expression of HO-1, SOD1 and CAT correlated with an increase in the corresponding protein activity. Despite the cellular response to bolster antioxidant capacity, apoptosis and necrosis increased following H/R injury. In contrast, ROS accumulation, the endogenous gene response and cell death was limited in neuronal cells pre-incubated with 50 or 100, but not 10 microM of the phenolic antioxidant 3,3'',5,5''-tetra-t-butyl-biphenyl-4,4''-diol (BP) prior to H/R injury. These data indicate that the early endogenous gene response to H/R injury is unable to inhibit neuronal dysfunction and that increasing cellular antioxidant capacity with a synthetic polyphenol (>10 microM) is potentially neuro-protective. %Z FOR Codes: 110302 %0 Journal Article %~ PubMed %A Witting, Paul K %A Rayner, Benjamin S %A Wu, Beng-Jing %A Ellis, Natasha A %A Stocker, Roland %T Hydrogen peroxide promotes endothelial dysfunction by stimulating multiple sources of superoxide anion radical production and decreasing nitric oxide bioavailability. %B Cellular Physiology and Biochemistry %D 2007 %C Switzerland %I S. Karger AG %V 20 %N 5 %P 255-268 %@ 1015-8987 %X Hydrogen peroxide (H(2)O(2)) is an oxidant implicated in cell signalling and various pathologies, yet relatively little is known about its impact on endothelial cell function. Herein we studied the functional and biochemical changes in aortic vessels and cultured porcine aortic endothelial cells (PAEC) exposed to H(2)O(2). Exposure of aortic rings to 25 or 50 muM, but not 10 muM, H(2)O(2) for 60 min prior to constriction significantly decreased subsequent relaxation in response to acetylcholine (ACh), but not the nitric oxide ((.)NO) donor sodium nitroprusside. Treatment of PAEC with 50 muM H(2)O(2) significantly decreased ACh-induced accumulation of (.)NO, as measured with a (.)NO-selective electrode, yet such treatment increased nitric oxide synthase activity approximately 3-fold, as assessed by conversion of L-arginine to L-citrulline. Decreased (.)NO bioavailability was reflected in decreased cellular cGMP content, associated with increased superoxide anion radical (O(2)(-.)), and overcome by addition of polyethylene glycol superoxide dismutase. Increased cellular O(2)(-.) production was inhibited by allopurinol, diphenyliodonium and rotenone in an additive manner. The results show that exposure of endothelial cells to H(2)O(2) decreases the bioavailability of agonist-induced (.)NO as a result of increased production of O(2)(-.) likely derived from xanthine oxidase, NADPH-oxidasse and mitochondria. These processes could contribute to H(2)O(2)-induced vascular dysfunction that may be relevant under conditions of oxidative stress such as inflammation. Copyright (c) 2007 S. Karger AG, Basel. %Z FOR Codes: 111601 %0 Journal Article %~ PubMed %A Neuzil, Jiri %A Widén, Cecilia %A Gellert, Nina %A Swettenham, Emma %A Zobalova, Renata %A Dong, Lan-Feng %A Wang, Xiu-Fang %A Lidebjer, Caroline %A Dalen, Helge %A Headrick, John P %A Witting, Paul K %T Mitochondria transmit apoptosis signalling in cardiomyocyte-like cells and isolated hearts exposed to experimental ischemia-reperfusion injury. %B Redox report : communications in free radical research %D 2007 %C United Kingdom %I Maney Publishing %V 12 %N 3 %P 148-162 %@ 1743-2928 %X Ischemia-reperfusion (I/R) is a condition leading to serious complications due to death of cardiac myocytes. We used the cardiomyocyte-like cell line H9c2 to study the mechanism underlying cell damage. Exposure of the cells to simulated I/R lead to their apoptosis. Over-expression of Bcl-2 and Bcl-x(L) protected the cells from apoptosis while over-expression of Bax sensitized them to programmed cell death induction. Mitochondria-targeted coenzyme Q (mitoQ) and superoxide dismutase both inhibited accumulation of reactive oxygen species (ROS) and apoptosis induction. Notably, mtDNA-deficient cells responded to I/R by decreased ROS generation and apoptosis. Using both in situ and in vivo approaches, it was found that apoptosis occurred during reperfusion following ischemia, and recovery was enhanced when hearts from mice were supplemented with mitoQ. In conclusion, I/R results in apoptosis in cultured cardiac myocytes and heart tissue largely via generation of mitochondria-derived superoxide, with ensuing apoptosis during the reperfusion phase. %Z FOR Codes: 110302 %0 Book Section %A Neuzil, J %A Witting, Paul %A Kontush, A %A Headrick, J P %T Role of vitamins E and C in nuclear factor-kB and nitric oxide signaling %B Encyclopedia of Vitamin E %D 2007 %C United States %I CABI %V %N %P 0 %@ 9781845930752 %E Preedy, V R %E Watson, R %X %Z FOR Codes: 111199 %0 Journal Article %~ PubMed %A Dong, Lan-Feng %A Swettenham, Emma %A Eliasson, Johanna %A Wang, Xiu-Fang %A Gold, Mikhal %A Medunic, Yasmine %A Stantic, Marina %A Low, Pauline %A Prochazka, Lubomir %A Witting, Paul K %A Turanek, Jaroslav %A Akporiaye, Emmanuel T %A Ralph, Stephen J %A Neuzil, Jiri %T Vitamin E analogues inhibit angiogenesis by selective induction of apoptosis in proliferating endothelial cells: the role of oxidative stress. %B Cancer research %D 2007 %C United States %I Wb Saunders Company %V 67 %N 24 %P 11906-11913 %@ 1538-7445 %X "Mitocans" from the vitamin E group of selective anticancer drugs, alpha-tocopheryl succinate (alpha-TOS) and its ether analogue alpha-TEA, triggered apoptosis in proliferating but not arrested endothelial cells. Angiogenic endothelial cells exposed to the vitamin E analogues, unlike their arrested counterparts, readily accumulated reactive oxygen species (ROS) by interfering with the mitochondrial redox chain and activating the intrinsic apoptotic pathway. The vitamin E analogues inhibited angiogenesis in vitro as assessed using the "wound-healing" and "tube-forming" models. Endothelial cells deficient in mitochondrial DNA (mtDNA) were resistant to the vitamin E analogues, both in ROS accumulation and apoptosis induction, maintaining their angiogenic potential. alpha-TOS inhibited angiogenesis in a mouse cancer model, as documented by ultrasound imaging. We conclude that vitamin E analogues selectively kill angiogenic endothelial cells, suppressing tumor growth, which has intriguing clinical implications. %Z FOR Codes: 111299 %0 Journal Article %~ PubMed %A Wu, Ben J %A Kathir, Krishna %A Witting, Paul K %A Beck, Konstanze %A Choy, Katherine %A Li, Cheng %A Croft, Kevin D %A Mori, Trevor A %A Tanous, David %A Adams, Mark R %A Lau, Antony K %A Stocker, Roland %T Antioxidants protect from atherosclerosis by a heme oxygenase-1 pathway that is independent of free radical scavenging. %B The Journal of experimental medicine %D 2006 %C United States %I Rockefeller University Press %V 203 %N 4 %P 1117-27 %@ 0022-1007 %X Oxidative stress is implicated in atherogenesis, yet most clinical trials with antioxidants, particularly vitamin E, have failed to protect against atherosclerotic diseases. A striking exception is probucol, which retards atherosclerosis in carotid arteries and restenosis of coronary arteries after angioplasty. Because probucol has in vitro cellular-protective effects independent of inhibiting lipid oxidation, we investigated the mode of action of probucol in vivo. We used three models of vascular disease: apolipoprotein E-deficient mice, a model of atherosclerosis; rabbit aortic balloon injury, a model of restenosis; and carotid injury in obese Zucker rats, a model of type 2 diabetes. Unexpectedly, we observed that the phenol moieties of probucol were insufficient, whereas its sulphur atoms were required for protection. Probucol and its sulphur-containing metabolite, but not a sulphur-free phenolic analogue, protected via cell-specific effects on inhibiting macrophage accumulation, stimulating reendothelialization, and inhibiting vascular smooth muscle cell proliferation. These processes were mediated via induction of heme oxygenase-1 (HO-1), an activity not shared by vitamin E. Our findings identify HO-1 as the molecular target of probucol. They indicate 2-electron rather than radical (1-electron) oxidants as important contributors to atherogenesis, and point to novel lead compounds for therapeutic intervention against atherosclerotic diseases. %Z FOR Codes: 110104 %0 Journal Article %~ PubMed %A Witting, Paul K %A Liao, Wen-Qiang %A Mathew, Z %A Neuzil, Jiri %T Expression of human myoglobin in H9c2 cells enhances toxicity to added hydrogen peroxide. %B Biochemical and biophysical research communications %D 2006 %C United States %I Academic Press %V 348 %N 2 %P 485-93 %@ 0006-291X %X Hydrogen peroxide (H2O2) is implicated in cardiac myocyte (CM) damage during myocardial ischemia-reperfusion (IR) injury. Myoglobin (Mb) is present in CM at significant concentrations and reacts with H2O2 to yield one- and two-electron oxidants that may promote myocardial injury. Paradoxically, hearts from mice lacking Mb are more susceptible to H2O2-induced dysfunction than the corresponding controls [U. Flogel, A. Godecke, L.O. Klotz, J. Schrader, Role of myoglobin in the anti-oxidant defense of the heart, FASEB J. 18 (2004) 1156-1158]. We have overexpressed wild-type or Y103F variant of human Mb in cultured CMs to test whether Mb protects against H2O2 insult. Contrary to expectation, cells expressing WT or the Y103F Mb show increased mitochondrial dysfunction and apoptosis, and decreased ATP in response to H2O2 that follows the order native < Y103F Mb < WT human Mb consistent with the increasing pro-oxidant activity for these proteins. These data indicate that (i) Mb promotes oxidative damage to cultured CM and (ii) Mb may be a useful target for the design of inhibitors of myocardial IR injury. %Z FOR Codes: 110106 %0 Journal Article %A McMahon, Aisling %A Duong, Thi Thuy Hong %A Brieger, David %A Witting, Paul %T Is there potential for antioxidants to enhance thrombolysis therapy in patients with ischemic stroke? %B Future Cardiology %D 2006 %C United Kingdom %I Future Medicine Ltd. %V 2 %N %P 659-665 %@ 1479-6678 %X %Z FOR Codes: 110101 %0 Journal Article %~ PubMed %A de Haan, Judy B %A Witting, Paul K %A Stefanovic, Nada %A Pete, Josefa %A Daskalakis, Michael %A Kola, Ismail %A Stocker, Roland %A Smolich, Joseph J %T Lack of the antioxidant glutathione peroxidase-1 does not increase atherosclerosis in C57BL/J6 mice fed a high-fat diet. %B Journal of lipid research %D 2006 %C United States %I American Society for Biochemistry and Molecular B %V 47 %N 6 %P 1157-67 %@ 0022-2275 %X Oxidative stress is thought to contribute to the initiation and progression of atherosclerosis. As glutathione peroxidase-1 (Gpx1) is an antioxidant enzyme that detoxifies lipid hydroperoxides, we tested the impact of Gpx1 deficiency on atherosclerotic processes and antioxidant enzyme expression in mice fed a high-fat diet (HFD). After 12 weeks of HFD, atherosclerotic lesions at the aortic sinus were of similar size in control and Gpx1-deficient mice. However, after 20 weeks of HFD, lesion size increased further in control but not in Gpx1-deficient mice, even though plasma and aortic wall markers of oxidative damage did not differ between groups. In control mice, the expression of Gpx1 increased and that of Gpx3 decreased at the aortic sinus after 20 weeks of HFD, with no change in the expression of Gpx2, Gpx4, catalase, peroxiredoxin-6, glutaredoxin-1 and -2, or thioredoxin-1 and -2. By comparison, in Gpx1-deficient mice, the expression of antioxidant genes was unaltered except for a decrease in glutaredoxin-1 and an increase in glutaredoxin-2. These changes were associated with increased expression of the proinflammatory marker monocyte chemoattractant protein-1 in control mice but not in Gpx1-deficient mice. In summary, a specific deficiency in Gpx1 was not accompanied by an increase in markers of oxidative damage or increased atherosclerosis in a murine model of HFD-induced atherogenesis. %Z FOR Codes: %0 Journal Article %~ PubMed %A Hack, Benjamin %A Witting, Paul K %A Rayner, Benjamin S %A Stocker, Roland %A Headrick, John P %T Oxidant stress and damage in post-ischemic mouse hearts: effects of adenosine. %B Molecular and cellular biochemistry %D 2006 %C United States %I Springer New York LLC %V 287 %N 1-2 %P 165-75 %@ 0300-8177 %X Despite the general understanding that ischemia-reperfusion (I/R) promotes oxidant stress, specific contributions of oxidant stress or damage to myocardial I/R injury remain poorly defined. Moreover, whether endogenous ''cardioprotectants'' such as adenosine act via limiting this oxidant injury is unclear. Herein we characterized effects of 20 min ischemia and 45 min reperfusion on cardiovascular function, oxidative stress and damage in isolated perfused mouse hearts (with glucose or pyruvate as substrate), and examined whether 10 muM adenosine modified these processes. In glucose-perfused hearts post-ischemic contractile function was markedly impaired (< 50% of pre-ischemia), cell damage assessed by lactate dehydrogenase (LDH) release was increased (12 +/- 2 IU/g vs. 0.2 +/- 0.1 IU/g in normoxic hearts), endothelial-dependent dilation in response to ADP was impaired while endothelial-independent dilation in response to nitroprusside was unaltered. Myocardial oxidative stress increased significantly, based on decreased glutathione redox status ([GSSG]/[GSG + GSSH] = 7.8 +/- 0.3% vs. 1.3 +/- 0.1% in normoxic hearts). Tissue cholesterol, native cholesteryl esters (CE) and the lipid-soluble antioxidant alpha-tocopherol (alpha-TOH, the most biologically active form of vitamin E) were unaffected by I/R, whereas markers of primary lipid peroxidation (CE-derived lipid hydroperoxides and hydroxides; CE-O(O)H) increased significantly (14 +/- 2 vs. 2 +/- 1 pmol/mg in normoxic hearts). Myocardial alpha -tocopherylquinone (alpha-TQ; an oxidation product of alpha -TOH) also increased (10.3 +/- 1.0 vs. 1.7 +/- 0.2 pmol/mg in normoxic hearts). Adenosine treatment improved functional recovery and vascular function, and limited LDH efflux. These effects were associated with an anti-oxidant effect of adenosine, as judged by inhibition of I/R-mediated changes in glutathione redox status (by 60%), alpha-TQ (80%) and CE-O(O)H (100%). Provision of 10 mM pyruvate as sole substrate (to by-pass glycolysis) modestly reduced I/R injury and changes in glutathione redox status and alpha-TQ, but not CE-O(O)H. Adenosine exerted further protection and anti-oxidant actions in these hearts. Functional recoveries and LDH efflux correlated inversely with oxidative stress and alpha -TQ (but not CE-O(O)H) levels. Collectively, our data reveal selective oxidative events in post-ischemic murine hearts, which are effectively limited by adenosine (independent of substrate). Correlation of post-ischemic cardiovascular outcomes with specific oxidative events (glutathione redox state, alpha-TQ) supports an important anti-oxidant component to adenosinergic protection. %Z FOR Codes: %0 Journal Article %~ PubMed %A Rayner, Ben S %A Duong, T T Hong %A Myers, Simon J %A Witting, Paul K %T Protective effect of a synthetic anti-oxidant on neuronal cell apoptosis resulting from experimental hypoxia re-oxygenation injury. %B Journal of neurochemistry %D 2006 %C United Kingdom %I Blackwell Publishing Ltd. %V 97 %N 1 %P 211-21 %@ 0022-3042 %X Oxidative stress is associated with the pathology of acute and chronic neurodegenerative disease. Cultured neuronal cells exposed to hypoxia-reoxygenation (H/R) injury, as a model for stroke, yield a burst of reactive oxygen species (ROS) as measured with electron paramagnetic resonance (EPR) spectroscopy in combination with spin trapping. Added superoxide dismutase inhibited spin-adduct formation verifying that superoxide radical anion was formed in neuronal cells following H/R injury. The intracellular ADP/ATP ratio increased rapidly over the first 5 h following injury and this was due primarily to the decreased cellular pools of ATP, consistent with the notion that H/R promotes mitochondrial dysfunction leading to decreased ATP reserve and increased ROS formation. As an early response to the enhanced oxidative stress, genes encoding for hypoxia-inducible factor 1-alpha (HIF1-alpha), inducible haemoxygenase-1 (HO-1), and the oxygen-sensor neuroglobin increased significantly. Up-regulation of the HO-1 gene was paralleled by increased HO protein expression and activity. Despite this cellular response, apoptosis increased significantly following H/R injury indicating that the endogenous anti-oxidant defenses were unable to protect the cells. In contrast, addition of a phenolic anti-oxidant, bisphenol (BP), prior to H/R injury, inhibited ROS production and gene regulation and significantly decreased neuronal cell apoptosis. Added BP was converted stoichiometrically to the corresponding diphenoquinone indicating the synthetic anti-oxidant effectively decreased oxidative stress through a radical scavenging mechanism. Together, these data indicate that BP has the potential to act as a neuro-protective drug. %Z FOR Codes: %0 Journal Article %~ PubMed %A Bhindi, Ravinay %A Witting, Paul K %A McMahon, Aisling C %A Khachigian, Levon M %A Lowe, Harry C %T Rat models of myocardial infarction. Pathogenetic insights and clinical relevance. %B Thrombosis and haemostasis %D 2006 %C Germany %I Schattauer GmbH %V 96 %N 5 %P 602-10 %@ 0340-6245 %X Animal models of cardiovascular pathology contribute towards understanding and treatment of a broad range of conditions. Specifically in the context of acute myocardial infarction (AMI), rat models have been commonly used in studies of pathogenesis, investigation and novel therapies, although there has often been difficulty in translating experimental findings to clinical benefit. However, recent years have seen two important changes to our clinical approaches to AMI. First, there is increasing recognition that the pathophysiology of human AMI is a process occurring at many levels, not just within the epicardial coronary artery, but also within the microvasculature and the myocardium. Second, contemporary treatments are shifting away from thrombolytic dissolution of epicardial coronary thrombus to direct mechanical approaches using angioplasty and stents. These changes in our understanding of AMI have implications for the relevance of these animal models. The following discussion therefore reviews and examines the current rat models of AMI, places them in a clinical context, discusses their advantages and limitations, and outlines likely future developments, providing an overview of the place of these important models of AMI. %Z FOR Codes: %0 Journal Article %A McMahon, A C %A Duong, T T H %A Brieger, D %A Witting, Paul %T Scope for antioxidants to enhance thrombolytic therapies in stroke patients %B Future Cardiology %D 2006 %C United Kingdom %I Future Medicine Ltd. %V 2 %N %P 659-665 %@ 1479-6678 %X %Z FOR Codes: 110201 %0 Journal Article %~ PubMed %A Witting, Paul %A Harris, Hugh %A Rayner, Benjamin %A Aitken, Jade %A Dillon, Carolyn %A Stocker, Roland %A Lai, Barry %A Cai, Zhonghou %A Lay, Peter %T The Endothelium-Derived Hyperpolarizing Factor, H(2)O(2), Promotes Metal-Ion Efflux in Aortic Endothelial Cells: Elemental Mapping by a Hard X-ray Microprobe. %B Biochemistry %D 2006 %C United States %I American Chemical Society %V 45 %N 41 %P 12500-12509 %@ 0006-2960 %X Hydrogen peroxide (H(2)O(2)) is a physiologic oxidant implicated in vascular cell signaling, although little is known about the biochemical consequences of its reaction with endothelial cells. Submicrometer-resolution hard X-ray elemental mapping of cultured porcine aortic endothelial cells (PAEC) has provided data on the global changes for intracellular elemental density within PAEC and indicates an efflux of metal ions and phosphorus from the cytoplasm after H(2)O(2) treatment. The synchrotron-radiation-induced X-ray emission experiments (SRIXE) show that H(2)O(2)-treated cells are irregularly shaped and exhibit blebbing indicative of increased permeability due to the damaged membrane. The SRIXE results suggest that H(2)O(2)-induced damage is largely restricted to the cell membrane as judged by the changes to membrane and cytoplasmic components rather than the cell nucleus. The SRIXE data also provide a mechanism for cell detoxification as the metal-ion efflux resulting from the initial H(2)O(2)-mediated changes to cell membrane potentially limits intracellular metal-mediated redox processes through Fenton-like chemistry. They may also explain the increased levels of these ions in atherosclerotic plaques, regardless of whether they are involved in plaque formation. Finally, the SRIXE data support the notion that cultured endothelial cells exposed to H(2)O(2) respond with enhanced cellular metal-ion efflux into the extracellular space. %Z FOR Codes: %0 Journal Article %~ Isi %A Rayner, B. %A Harris, H. %A Carter, E. %A Vogt, S. %A Cai, Z. %A Lai, B. %A Chin, C. %A Lee, Y. %A Lay, P. %A Witting, P. %T The use of synchrotron radiation to measure ion flux and cellular protein and lipid changes within cardiac ischemia reperfusion injury. %B Atherosclerosis Supplements %D 2006 %C Ireland %I Elsevier Ireland Ltd %V 7 %N 3 %P 587-587 %@ 1567-5688 %X %Z FOR Codes: