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O 17-20 MARCH, 2010 FESS PARKER’S DOUBLE TREE RESORT SANTA BARBARA, CALIFORNIA OXIDANTS AND ANTIOXIDANTS IN BIOLOGY TRANSLATIONAL REDOX SCIENCE 17-20 MARCH 2010 SANTA BARBARA, CALIFORNIA

CONFERENCE SCIENTIFIC PROGRAM ORGANIZERS ORGANIZERS ______

Chandan K. Sen Bruce N. Ames Dipak K. Das Enrique Cadenas César G. Fraga Dipak K. Das Thomas K. Hunt César G. Fraga Klaus Kraemer John Maguire Periannan Kuppusamy Lester Packer Lester Packer Junji Yodoi Chandan K. Sen Helmut Sies Roland Stocker Hideo Utsumi José Viña Junji Yodo

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CONTENTS

KEYNOTE ADDRESSES ...... 5

SESSION I WOUND HEALING...... 9

SESSION II REDOX SIGNALING AND INFLAMMATION...... 19

SESSION III C ARDIOVASCULAR ...... 29

SESSION IV TRANSLATIONAL SCIENCE BY MICRONUTRIENTS...... 35

SESSION V REDOX IMAGING...... 51

SESSION VI AGING ...... 55

POSTERS ...... 61

AUTHOR INDEX ...... 178

KEYNOTE ADDRESSES

Novel infectious agents in human carcinogenesis: State and perspectives New horizons in HIV/AIDS Harald zur Hausen Luc Montagnier Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany World Foundation AIDS Research and Prevention, UNESCO, Paris, France

During the past 30 years up to 21% of the global cancer inci- Combined antiretroviral therapy (ARV) keeps the virus multipli- dence has been linked to infectious events, involving specific viral, cation to a low level as evidenced by the disappearance of viral RNA bacterial and parasitic agents. Particularly the discovery of a role in the blood of HIV infected patients but does not completely elimi- of Hepatitis B virus in hepatocellular carcinomas and of high risk nate the virus: there is a viral reservoir which remains untouched even human papillomaviruses (HPV) in cervical, other anogenital and after long term treatment and regenerates viral multiplication if the oropharyngeal cancers triggered novel approaches in cancer pre- treatment is interrupted. vention by vaccination. A global application of these vaccines We have approached the identification of this reservoir by a new theoretically could reduce the cancer risk in females by 12-14%, in technology based on the detection of electromagnetic signals pro- males by 4-5%. Mechanisms of cell transformation by infectious duced by some viral and bacterial DNAs, in particular by HIV DNA agents will be analyzed. Even in cases where infectious agents act sequences. Thus, we have detected a reservoir of HIV DNA in the as direct carcinogens and where their persistence is necessary for blood of all patients treated by ARV, both in the plasma and in the the maintenance of the carcinogenic phenotype, additional modifi- erythrocyte fraction. cations of the host cell genome emerge as necessary events for ma- This will constitute a useful biomarker for complementary thera- peutics aimed at eradicating the virus infection such as immunother- lignant proliferation. Considerations will be presented to analyze apy and antioxidants. even cancers not yet linked to infectious events for a possible in- volvement of exogenous agents in their etiology. This involves in part cancers increased under immunosuppression, but also those with a reduced or not elevated incidence after immunosuppression. Malignant tumors arising either in the sequence of other infections will also be discussed or where those infections appear to exert protective functions. Finally potential synergistic effects of nutri- tional carcinogens with virus infections deserve further attention.

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SESSION I WOUND HEALING

Hyperbaric oxygen therapy and stem cell response in Homing to hypoxia: The role of oxygen tension in progenitor wound healing

cell trafficking to sites of injury Omaida C. Velazquez

The DeWitt Daughtry Family Department of Surgery, Leonard M. J. Glotzbach, H. Thangarajah, N. Vial, and G.C. Gurtner Miller School of Medicine, University of Miami, Miami, FL, USA

Department of Surgery, Stanford University, Stanford, CA, 94305 Wound healing occurs because of events in two compartments. Within the bone marrow, various signaling pathways trigger mobiliza- The isolation of circulating endothelial progenitor cells (EPCs) tion of bone marrow-derived progenitor cells (EPC) and other has altered our understanding of how new blood vessels form in stem/progenitor cells involved in the healing cascade. Within the wound, neovascularization occurs because of local chemical factors adult tissues (heart, brain, wounds) following injury. While an ex- that stimulate adjacent cells (angiogenesis) and because of recruited act characterization of the responsible cell(s) remains elusive, it is circulating EPC that differentiate into vascular channels (vasculogene- clear that a subset of the bone marrow is capable of selectively sis). Many chemo/cytokines trigger EPC release via induction of met- trafficking to areas of tissue damage and forming blood vessels de alloproteinase-9 (MMP-9) in bone marrow, and Nitric Oxide (.NO) has novo through a cascade of adhesion, migration, proliferation and been linked to this process. We have found that HBO2 will stimulate tubulization. We have identified the chemokine axis CXCR4/ EPC recruitment and augment healing of diabetic wounds in mice. Our . stromal cell-derived factor-1 (SDF-1) as a critical mediator of studies demonstrated that HBO2 increases synthesis of NO in bone ischemia-specific progenitor trafficking and have demonstrated marrow, increases the concentration of circulating stem cell factor, and that SDF-1 is transcriptionally regulated by tissue oxygen tension stimulates EPC mobilization. If NOS was inhibited, all of the HBO2- via hypoxia-inducible factor-1 (HIF-1). Since both hypoxia and mediated events were blocked. Therefore, HBO2 appears to stimulate EPC mobilization by a .NO dependent mechanism. We believe the se- SDF-1 are also observed in the bone marrow niche, hypoxia may . be a fundamental stimulus for progenitor cell trafficking and func- quence of events is as follows: HBO2
NOS
NO
nitrosylation tion. After homing to injured tissue, both bone marrow-derived of MMP9
cleavage of membrane-bound SCF
SCF prompts EPC proliferation and mobilization EPC released into peripheral circula- and adipose-derived mesenchymal stem cells respond to hypoxia
tion. However, EPC homing to areas in need of neovascularization by adopting a proangiogenic phenotype by proliferating, forming (such as a wound) does not appear to be specifically impacted by tubes and secreting vascular growth factors. Alterations in hypoxia HBO2. Homing signals such as Stromal Derived Growth Factor 1 alpha signaling may contribute to the pathophysiology of diseases with (SDF-1) appear to be more important for EPC homing. We have de- known defects in blood vessel growth, such as diabetes. It appears termined that (1) Diabetes results in decreased eNOS phosphorolation that hyperglycemia directly alters the molecular structure and func- within the bone marrow, thus reducing mobilization of endothelial tion of HIF-1 and its cofactors by methylglyoxylation of specific progenitor cells (EPCs), (2) Diabetes results in downregulation of arginine residues preventing assembly of transcriptional machin- SDF-1 within wounds, thus decreasing EPC homing to wounds, and ery. This results in both decreased angiogenesis and decreased re- (3) these two key diabetes-associated impairments in wound healing cruitment of progenitor cells to areas of ischemia in diabetes. This and neovascularization may be reversed by the combination of sys- suggests that the end manifestations of diabetes may be the result temic hyperoxia (to increase EPC mobilization) and wound SDF-1 of impaired hypoxia signaling following injury. injections (to increase EPC homing). Most recently, we have further determined that SDF-1 may mediate its EPC-homing effects via ef- Supported by NIH grants RO1-EB002265 and RO1-AG025016 fects on specific adhesion molecules.

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Impaired resolution of wound in diabetes Mathematical modeling of the role of oxygen in Sashwati Roy non-ischemic and ischemic wound healing Comprehensive Wound Center, Department of Surgery, The Ohio State University Medical Center, Columbus, OH Avner Friedman Acute inflammation resolves by mechanisms that are not fully understood. Persistence of the inflammatory response (chronic in- Mathematical Bioscience Institute, Ohio State University, flammation) can lead to scarring and loss of organ function. An ac- Columbus, OH, USA tively coordinated program of resolution initiates in the first few hours after an inflammatory response is triggered by tissue injury. It is well known that moderate hypoxia in a wound stimulates Resolution of inflammation is enabled if granulocytes are elimi- macrophages to produce growth factors, which improves healing. nated via efferocytosis and the tissue mononuclear cell population But moderate hyperoxia also improves healing since it enables (macrophages, m ) returns to baseline count and phenotypes. We cells to proliferate faster. However severe hypoxia impairs heal- observed that compared to non diabetic animals, dead cell clear- ing, as does extreme hyperoxia – which is toxic. Thus the role of ance activity (efferocytosis) was markedly impaired in wound oxygen in wound repair is quite sensitive. I will present a mathe- macrophages harvested from diabetic mice. Oxidants produced by matical model which focuses on the role that oxygen plays in cuta- NADPH oxidase are directly involved in the oxidation and exter- neous wound healing. The model includes the important cells and nalization of phosphatidyl serine (PS), a process that is critical for chemokines that are involved in the healing process, under both dead cell recognition. Compromised NADPH oxidase activity was non-ischemic and ischemic conditions. The predictions of the observed in macrophages harvested from diabetic wounds. Milk fat model are in excellent agreement with experimental results. The globule EGF factor 8 (MFG-E8; also known as lactadehrin) is se- model can be used as a tool for evaluation of different protocols of creted by activated m . MFG-E8 acts as a bridging molecule that oxygen treatment. is capable of binding to phosphatidyl serine on apoptotic cells as well as v3 or v5 integrin receptors on m . The presence of arginine residues renders this protein susceptible to glycation. Us- ing ELISA and Biacor assays we demonstrate that glyoxal, a prod- uct of glucose oxidation, can glycate MFG-E8. Once glycated the affinity of MFG-E8 for binding with PS markedly diminished. Diabetic wound tissue showed presence of glycated MFG-E8. These data suggest compromised NADPH oxidase activity in dia- betic wound cells result in diminished phosphatidylserine oxida- tion impairing the process of dead cell recognition by m . Glyca- tion of MFG-E8 diabetic macrophages represents another major mechanism that impairs dead cell recognition and clearance in dia- betic wounds. Such dysfunction in macrophage activity is in direct conflict with resolution of inflammation resulting in chronic in- flammation noted in diabetic wounds. Supported by RO1 DK 076566 to SR.

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Ischemic wound healing

Cell based therapies for peripheral vascular disease Thomas A. Mustoe

Douglas W. Losordo Department of Surgery, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Feinberg Cardiovascular Research Institute, Northwestern University School of Medicine, Northwestern Memorial Hospital, Chicago, IL USA Chronic wounds are a multibillion dollar health problem in the USA affecting millions of patients, falling into three major catego- As the population ages and the acute mortality from cardiovas- ries: pressure sores, diabetic foot ulcers, and venous leg ulcers. cular disease decreases, a large population of patients is emerging Local wound ischemia and ischemia reperfusion, (as well as the who have symptomatic chronic ischemic vascular disease, many of additive impact of aging, and bacterial biofilm) play a fundamen- whom remain severely symptomatic despite exhausting conven- tal role in the pathogenesis. We have utilized clinically relevant tional medical therapy and mechanical revascularization. In addi- and validated animal models to examine separately the impact of tion, mounting evidence suggests that microvascular insufficiency ischemia, and the altered response in the aged animal, and the ef- plays a significant role in the pathophysiology of ischemia. At the fects of ischemia reperfusion (oxidant injury). The skin unlike present time, there are no therapies that directly address the needs other organs, is subjected to repeated cycles of I-R, and the failure of this patient population. of an adaptive response to repeated cycles is notable. Classically, Pre-clinical and early clinical data indicate that a variety of therapeutic strategies for chronic wounds have focused on oxygen growth factors and stem/progenitor cells may be employed thera- delivery to offset the effects of ischemia, but future strategies will peutically for repair of ischemic tissue. include strategies to reduced oxidant injury. True breakthroughs Preclinical studies documented the potential therapeutic po- will almost certainly incorporate combination therapy, in recogni- tency of endothelial progenitor cells, both as cultured and freshly tion that chronic wounds have a multi factorial pathogenesis. isolated cells. Early phase clinical trials using a variety of ap- proaches have been completed providing data of feasibility, safety and bioactivity. Later phase trials are under way. Accordingly, the goal of ischemic tissue repair appears feasible and is being ap- proached in human clinical trials. The evolution of the strategy of ischemic tissue repair will re- quire an ongoing dialogue between clinicians, scientists, regulators and industry to take full advantage of advances in our understand- ing of the biology of these processes and their appropriate applica- tion to patients.

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Microcirculation in tissue injury and repair A novel role for caspase-8 in regulating the Mark G. Clemens cutaneous wound healing response Department of Biology, College of Liberal Arts and Sciences, Pedro Lee1, Stacey Lee1, and Colin Jamora1,2 University of North Carolina at Charlotte, Charlotte NC 28223

1 It is well known that vascular perfusion of tissues is essential to Division of Biological Sciences, Section of Cell and Developmental Biology, University of California, San Diego; 2Department of Medicine (Division of prevent injury as well as to allow repair following injury. Recent Dermatology), University of California, San Diego School of Medicine studies demonstrate that that effect on microvascular control of lo- cal inflammation associated with tissue injury contributes to mechanisms of both injury and repair. The effects of inflammation The wound healing response is a complex interplay of cells that on the integrity of the microcirculation are manifested in accumu- coordinate inflammation, stem cell proliferation and tissue remod- lation of inflammatory cells and in altered vasoreactivity as well as in the control of angiogenesis. All of these manifestations involve eling. The complex nature of this intercellular communication to altered function of the vascular endothelium with dysregulation of localize the wound healing response specifically to the site of the interactions between endothelin and gaseous vascular media- trauma and to reset itself once tissue repair is complete has been a tors, nitric oxide, carbon monoxide and hydrogen sulfide. All four challenging problem. We found that the protease caspase-8, which of these mediators are regulated by inflammation. During the in- is well established for facilitating receptor mediated apoptosis, jury phase, inflammatory stimuli alter vascular endothelial cell plays a novel role in regulating all phases of the wound healing re- signal transduction mechanisms resulting in impaired perfusion sponse. Using the conditional deletion of caspase-8 to mimic and oxygen supply with resulting tissue injury. During repair, these wound healing in the skin, we were able to decipher the crosstalk mechanisms also contribute to the regulation of existing vascula- occurring between epidermal keratinocytes, dermal fibroblasts, and ture and to angiogenesis. While nitric oxide mechanisms are leukocytes to regulate stem cell proliferation. Among the important widely recognized, the contributions of carbon monoxide and hy- constituents of this extracellular signaling is the cytokine interleu- drogen sulfide (H2S) are less well known. CO is made from the kin-1 (IL-1). Epidermal keratinocytes serve as a large reservoir metabolism of heme by heme oxygenase, the inducible form of for IL-1a and the reduction of caspase-8 leads to the formation of which (HO-1) is associated with inflammation. CO modulates the the NALP3 inflammasome, which mediates release of this cyto- function of heme proteins such as guanyl cyclase while the other kine into the extracellular milieu. Multiple responses are elicited metabolite of HO-1, bilirubin acts as an antioxidant. H2S is pro- by this cytokine depending upon the type of cell it encounters. duced by via the metabolism of cysteine by cystathionine gamma- lyase (CSE) which can also be induced by inflammation. H S has Further dissection of the reciprocal exchange of signals is also pro- 2 been shown to improve ulcer healing and to stimulate angiogene- viding insights into how the same processes that occur in the con- sis. While the primary action of H S is to activate ATP-activated text of tissue repair are usurped in inflammatory diseases. 2 potassium (KATP) channels in vascular smooth muscle cells, many of its actions appear to be independent of this mechanism. Taken together, current data strongly suggest that these three gaseous me- diators work in concert with each other and with vasoconstrictors such as endothelin to determine microvascular function during tis- sue injury and repair.

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SESSION II REDOX SIGNALING AND INFLAMMATION

Redox regulation of thioredoxin and glutaredoxin systems Inactivation of peroxiredoxin I by tyrosine phosphorylation at lipid rafts allows spatially controlled accumulation of H2O2 for Arne Holmgren, Rajib Sengupta, Yatao Du, intracellular signaling by growth factor or immune receptors Sergio Montano, and Jun Lu Sue Goo Rhee and Hyun Ae Woo Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 171 777 Stockholm, Sweden Department of Life Science, Division of Life and Pharmaceutical Sciences, Ewha Woman’s University, 11-1 Daehyun-dong, Seodaemun-ku, To balance the effects of ROS, thioredoxin and glutaredoxin Seoul 120-750, Korea systems act together with glutathione peroxidases and peroxiredox- ins and via disulfides ultimately also in redox signaling. Mammal- Despite its inherent toxicity, H2O2 is produced by mammalian ian Trx1 is an electron donor for ribonucleotide reductase (RNR). cells as a signaling molecule that oxidizes critical cysteine residues This enzyme comprises R1 + R2 in S-phase, which reduces NDPs of effectors such as protein tyrosine phosphatases in response to and supplies dNTPs for DNA replication. R1 + p53R2 in postmi- activation of cell surface receptors. It has remained unclear, how- totic cells generates dNTPs for DNA repair and mitochondrial ever, how H2O2 concentrations above the threshold required to DNA synthesis. Each dNTP made leaves an active site disulfide in modify effectors are achieved in the presence of the abundant de- the R1 subunit of RNR which is reduced by C-terminally located toxification enzymes peroxiredoxin (Prx) I and Prx II. We now cysteine (CXXC) residues, the target of Trx and Grx. With R1 + show that Prx I associated with lipid rafts is transiently phosphory- p53R2, Grx operating by a glutathionylation mechanism is cata- lated on tyrosine-194 and thereby inactivated both in cells stimu- lated via growth factor or immune receptors in vitro as well as in lytically more efficient (higher kcat/KM) than Trx, suggesting a ma- jor function of GSH and Grx in DNA repair and mitochondrial those at the margin of healing cutaneous wounds in mice. The lo- DNA-synthesis. Trx1 itself is subject to redox regulation by modi- calized inactivation of Prx I thus provides a means for allowing the fication of the three structural SH-groups as well as its involve- transient accumulation of H2O2 around lipid rafts, where signaling ment in NO-metabolism as the major S-denitrosylation mechanism components are concentrated, while preventing the toxic accumu- of protein S-nitrosothiols. Grxs catalyze GSH-dependent disulfide lation of H2O2 elsewhere. In contrast, Prx II was inactivated not by oxidoreductions and deglutationylation of proteins. Human Grx2a phosphorylation but rather by hyperoxidation of its catalytic cys- in mitochondria is also present as an inactive dimeric holoenzyme teine and only during sustained oxidative stress. containing an iron-sulfur cluster (2Fe2S) with GSH as a ligand. The 2Fe2S clusters of dithiol and monothiol glutaredoxins act to control apoptosis via the mitochondrial pathway and are involved in iron metabolism. Both Trx and Grx can be taken up in cells and have cytoprotective and antiapoptotic effects. A synthetic selenium drug, ebselen, is a very fast oxidant of reduced Trx and a direct substrate of mammalian TrxR and a promising drug for oxidative stress diseases or in treating cancer due to redox imbalance.

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Thioredoxin and TBP-2 in redox signaling

1 1,2 1 1 Immunoregulatory role of GIF/MIF, a redox-geared cytokine Zhe Chen , Eiji Yoshihara , Yoshiyuki Matsuo , Junji Yodoi 1 Department of Biological Responses, Institute for Virus Research, Katsuji Sugie1,2, Misa Kim-Saijo1 and Aoi Son2 Kyoto University; 2Graduate school of Biostudies, Kyoto University

Human Thioredoxin-1 (TRX) is a 12-kDa oxidoreductase con- 1La Jolla Institute for Allergy and Immunology and 2Kyoto University Institute taining a conserved dithiol-disulfide active site (-CPGC-). It was for Virus Research first identified from the supernatant of HTLV-I infected T cell line as adult T-cell leukemia-derived factor. Given the essential role in GIF/MIF (GIF, for brevity) is a 13-kDa polypeptide. Origi- the intracellular thiol-reducing system, TRX is known to regulate nally identified in T cell culture supernatants, the function of this multiple biological responses in a redox-dependent manner. We molecule has been controversial. The thioredoxin(TRX) motif in have identified several binding partners for TRX, including Thi- GIF alluded involvement of oxidoreductase activity in the cytokine oredoxin Binding Protein-2 (TBP-2/VDUP1/TXNIP), which binds biology. GIF-/- mice displayed an enhanced antibody response to to the reduced TRX inhibiting the biological activity of TRX in T-dependent antigens. The mutant mice showed more severe al- vivo. TBP-2 is a critical signal transducer in cell growth, lipid and lergic airway inflammation than wildtype mice. CD4 T cells of glucose metabolism, and immune responses. Several lines of evi- GIF-/- mice secreted elevated amounts of IL-4, the key cytokine dence indicate that TBP-2 is a crucial component of TRX- for IgE antibody formation and allergic inflammation. Therefore dependent redox regulation. It was suggested that ROS generated GIF suppresses IL-4 production and T-dependent immune re- in response to oxidative stresses promotes the formation of inter- molecular disulfide bridge between TRX and its targets, whereas sponses especially in allergy. Although GIF does not have the sig- TBP-2 overexpression abolishes the TRX function. The redox nal peptide sequence, it becomes secreted. GIF secreted from T regulation by TRX/TBP-2 system seems to be a ubiquitous phe- cells is cysteinylated at C60, whereas that in the cytosol is unmodi- nomenon in cellular responses, including mitogen-activated signal fied. The cysteinylation was essential for GIF to suppress IL-4 transduction (ERK, p38MAPK), ASK1-mediated cell apoptosis, production by CD4 T cells. Immunofluorescence studies revealed FoxO-mediated cellular longevity, and anti-inflammatory immune that cysteinylated GIF colocalized with ER and Golgi apparatus. responses. Conclusively, here we postulate a redox-regulated sig- GIF in T cell culture supernatants associated with TRX1, a poly- naling complex consisting of TRX or TRX family proteins, TBP-2, peptide with the capability to control inflammations. To determine and other putative signaling components (REDOXISOME), in re- the receptor for cysteinylated GIF, expression cloning was per- sponse to varieties of stresses. The core concept of redox regula- formed. One of the molecules identified was peroxiredoxin 1 tion is that under stress conditions, redoxisome is formed to acti- (Prdx-1). RNA interference experiments showed that knocking vate the stress signaling. The association/dissociation of redox- down of Prdx-1 diminished cellular binding of cysteinylated GIF. isome components induced by the environmental stresses account A previous study demonstrated that Prdx-1 bound GIF. Experi- for the complicated but precise regulation of redox signaling. ments are planned to determine whether cysteinylation is important 1. Teshigawara K, et al. Adult T leukemia cells produce a lymphokine that augments in- for this cytokine to bind Prdx-1 and whether Prdx-1 is involved in terleukin 2 receptor expression. J Mol Cell Immunol 1985;2:17-26. transmitting GIF signals at the cell surface. Altogether, the cyto- 2. Nishiyama A et al. Identification of thioredoxin-binding protein-2/vitamin D(3) up- regulated protein 1 as a negative regulator of thioredoxin function and expression. J kine GIF is subject to redox regulation when it becomes secreted, Biol Chem 1999;274:21645-21650. acquires the immunoregulatory activity and acts on target cells. 3. Dansen et al, Redox-sensitive cysteines bridge p300/CBP-mediated acetylation and FoxO4 activity. Nat. Chem. Biol 2009:5:664-672

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NADPH oxidases in the lung: beyond host defense Regeneration of infarcted myocardium with nutritionally

1 1 2 modified cardiac stem cells: Implication for redox signaling Albert van der Vliet , Derek Sham , Umadevi Wesley , David I. Kasahara1,3, and Milena Hristova1 Dipak K. Das Departments of 1Pathology and 2Microbiology and Molecular Genetics, College of Medicine, University of Vermont, Burlington, VT 05405, U.S.A., 3Department of Environmental Health, Harvard School of Public Health, Cardiovascular Research Center, University of Connecticut School of Medicine, Boston, MA 02115 Farmington, CT 06030-1110, USA In analogy to the deliberate production of reactive oxygen species (ROS) by NADPH oxidase in professional phagocytes as a critical component of A major hitch in the effectiveness of stem cell therapy is the death their antimicrobial activity, airway epithelial cells express homologs of of stem cells due to the oxidative environment present in the nor- NADPH oxidase, Dual Oxidase 1 and 2 (DUOX1/2), that can be activated by mal tissue. Reduction of oxidative stress or maintaining a reduced a variety of environmental stresses and bacterial stimuli, and contribute to maintenance of respiratory tract sterility and epithelial integrity. While sev- environment in the target tissue can enhance the stem cell survival eral studies have indicated that epithelial DUOX’s generate apical H2O2 as a and can enhance the cardiac regeneration after stem cell therapy. substrate for apical lactoperoxidase in the production of antimicrobial oxi- To study the efficiency of maintaining the reduced tissue environ- dants, recent studies in our laboratory have also demonstrated a critical role ment via pretreatment with natural antioxidant resveratrol in stem for DUOX1, the major epithelial DUOX isoform, in epithelial cell migration cell therapy. We pretreated male Sprague Dawley rats with res- and intracellular signaling pathways that mediate epithelial responses to a veratrol (2.5 mg/kg/day gavaged for 2 weeks). Left anterior de- variety of environmental or microbial insults by inducing various mediators of inflammation or tissue repair, such as matrix metalloproteinase-9, inter- scending coronary artery (LAD) occlusion followed by direct in- leukin-8 or mucins. These responses appear to involve a common mechanism jection of adult cardiac stem cells stably expressing EGFP on the that is initiated by cellular release of ATP to activate cell surface purinocep- border zone of the myocardium through survival surgery. The tors, a widely recognized signaling mechanism in airway epithelial responses prevalent of cardiac reduced environment was seen in resveratrol to mechanical injury or microbial infection. Similar recent findings of ATP treated rat hearts via significantly enhanced redox signaling ob- release and/or NADPH oxidase activation in stress responses in diverse or- served through the nuclear localization of nuclear factor-E2-related ganisms indicate their common role in biological response mechanisms to environmental stress throughout the animal and plant kingdoms. Activation factor-2 (Nrf2) and redox effector factor-1 (Ref-1) 7 days after of epithelial DUOX1 was found to promote the shedding of growth factors LAD occlusion. Significantly improved cardiac functional parame- and activation of the epidermal growth factor receptor (EGFR), whereas ters (left ventricular ejection fraction and fractional shortening) and DUOX1 also appears to stimulate the non-receptor tyrosine kinase c-Src, enhanced stem cell survival and proliferation (expression of cell which activates EGFR independent of ligand. Current studies are designed to proliferation marker Ki67) and differentiation of stem cells to- identify the proximal H2O2-dependent signaling events involved in EGFR wards the regeneration of the myocardium (expression of EGFP) activation, using redox proteomic strategies. Abnormal expression and/or activation of EGFR are important features of various lung diseases such as was evident 28 days after LAD occlusion in rats treated with res- asthma and lung cancer, and are associated with deregulated epithelial prolif- veratrol compared to control rats. Maintaining a reduced tissue en- eration, chronic epithelial wound responses, and mucus metaplasia. In this vironment by treatment with resveratrol in rats enhanced the car- regard, intriguing observations of enhanced epithelial DUOX1 expression by diac regeneration by adult cardiac stem cells via improved cell sur- Th2 cytokines and upregulation of lung DUOX1 in a mouse model of allergic vival, proliferation and differentiation leading to improved cardiac asthma may suggest a contributing role for DUOX1 in airway remodeling function. and mucus metaplasia as cardinal features of allergic asthma, and may impli- cate DUOX as an alternative potential therapeutic target.

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Mitochondria, oxidative stress and cell death Development of high field Overhauser-MRI and its application Sten Orrenius, Erik Norberg, Vladimir Gogvadze, to in vivo imaging and Boris Zhivotovsky

Institute of Environmental Medicine, Division of Toxicology, Hideo Utsumi1,2 Karolinska Institutet, Stockholm, Sweden

1Innovation Center for Medical Redox Navigation In addition to the long established role of the mitochondria in 2Department of Bio-functional Science, Faculty of Pharmaceutical Sciences energy metabolism, regulation of cell death has emerged as a sec- Kyushu University, Fukuoka 812-8582 ond major function of these organelles. This, in turn, seems to be e-mail: [email protected] intimately linked to their role as the major intracellular source of reactive oxygen species (ROS), which are mainly generated at Complex I and III of the respiratory chain. Excessive ROS produc- Proton Magnetic Resonance Imaging (MRI) has provided sig- tion can lead to the oxidation of macromolecules and has been im- nificant clinical utility in the diagnosis of diseases. Overhauser en- plicated in mtDNA mutations, ageing, and cell death. Although mi- hanced MRI (OMRI), which is a double resonance technique, crea- tochondrial dysfunction per se can result in ATP depletion and ne- tes highly-resolved images of free radical distributions in small a- crosis, these organelles are also involved in the regulation of apop- nimals by enhancing the water proton signal intensity via the O- totic cell death by mechanisms that have been conserved through verhauser Effect. We developed new-sequence for OMRI and suc- ceeded in simultaneous dual images by using nitroxyl radicals la- evolution. Hence, many toxic agents target the mitochondria and 14 15 cause the release into the cytosol of cytochrome c and other pro- beled with N and N nuclei and changing the external magnetic apoptotic proteins, which can trigger caspase activation and other field for ESR irradiation in OMRI (Utsumi et al. PNAS 2006), key events in apoptosis. Cytochrome c release occurs by a two-step which could visualize pharmacodynamics, redox reactions and re- process that is initiated by the dissociation of the hemoprotein from dox statue of inner and/or outer organs of individual animal.The its binding to cardiolipin in the inner mitochondrial membrane large difference of gyromagnetic ratio between electron and proton (IMM). Similarly, Apoptosis Inducing Factor (AIF) must also be spins restricted ESR excitation and the proton detection fields. We detached from the IMM, before it can be exported from the mito- have developed a prototype of OMRI scanner with circular- chondria into the nucleus. This occurs by proteolytic cleavage of transport-system, in which the sample object was transported the peptide chain that anchors it to the IMM. AIF cleavage is cata- between ESR to MR magnets circularly, which were operated at lyzed by mitochondrial calpain and preceded by oxidative modifi- 1.5 T (or 0.4 T) and 20 mT for MR detection and ESR excitation, cation of AIF triggered by mitochondrial ROS production. Subse- respectively. Phantom images of nitroxyl spin-probe were success- quent release of cytochrome c and AIF into the cytosol occurs via fully obtained with the OMRI system. The physical resolution of pores in the outer mitochondrial membrane formed by pro- the OMRI image for the phantom object was less than 0.2 mm. The apoptotic Bcl-2 family proteins, or by Ca2+/ROS-induced mito- developed OMRI system would have a significant advantage for chondrial permeability transition, although the latter mechanism imaging in vivo redox state,and also offer significant applicability might be more closely associated with necrotic cell death. Hence, it to simultaneous assessment of independent redox regulation in is apparent that mitochondrial ROS generation is critically in- gene-modified animals and disease models (JCBF 2009, PNAS volved in the control of both apoptotic and necrotic cell death and 2009). an important mediator of mitochondrial toxicity.

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Lipoic acid reverses the age-related loss of Nrf2-mediated antioxidant gene transcription

Tory M. Hagen

Linus Pauling Institute, Oregon State University, Corvallis, OR, USA

In order to define the benefits of micronutrients in improving elder health, we show that the dithiol antioxidant, (R)--lipoic acid (R-LA), improves hepatic antioxidant capacity, which otherwise declines with age. R-LA does not achieve this by acting as a die- tary antioxidant; rather, it reverses the age-related loss of endoge- nous antioxidants, especially glutathione (GSH). This has led to the discovery that nuclear levels of NF-E2 related-factor 2 (Nrf2), a transcription factor that regulates expression of GSH- synthesizing enzymes, markedly declines with age, but R-LA re- verses this loss. Our current focus is to understand why an age- related loss of Nrf2-mediated stress defenses occurs, and the mechanism(s) how R-LA acts to maintain this vital cellular de- fense system. Chromatin immunoprecipitation experiments for the Nrf2-mediated gene, glutamate-cysteine ligase, show that lower levels of Nrf2 bind to its Antioxidant Response Element (ARE) in aged versus young rat livers. We also have evidence showing that the active Nrf2/bZIP transcriptional complex is converted to a re- pressive motif during aging as shown by binding of the repressor, Bach1, and lower ARE-driven luciferase activity. Furthermore, Nrf2 occupies an alternate ARE site in livers of old rats, indicating an age-specific promoter switch. Our results show that the conver- sion of Nrf2 binding from its typical ARE site to an alternative one is not adequate to maintain basal expression of hepatic gcl in old rats, which provides a possible mechanism for the age-related loss of glutathione and other Phase II enzymes. However, R-LA im- proves Nrf2 action by increasing nuclear tenure of Nrf2 and also promotes its binding to the alternative ARE sequence. Thus, R-LA may be part of a unique class of dietary micronutrients that pro- mote “healthspan” by maintaining vital cellular defenses, which SESSION III otherwise decline with age. CARDIOVASCULAR

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Rescue of diabetes related impairment of myocardial Inducible nitric oxide-mediated nitroso-redox balance as a key angiogenesis: Potential and challenges modulator of tolerance to ischemia/reperfusion injury 1Samson Mathews Samuel, 1Mahesh Thirunavukkarasu, in diabetic patients 1Suresh Varma Penumathsa, 1Lijun Zhan, 2Gautam Maulik, and 1Nilanjana Maulik Hajime Otani 1Molecular Cardiology and Angiogenesis Laboratory, Department of Surgery, UCONN Health Center, Farmington, CT, USA, 2Department of Thoracic Sur- The Second Department of Internal Medicine, Kansai Medical University, 10-15 gery, Harvard Medical School, Boston, MA, USA Fumizono-cho, Moriguchi City, 570-8507 Japan

Maintaining a balanced redox status in the microenvironment might aid in alleviating diabetes mediated complications related to Although expression of inducible nitric oxide (iNOS) and oxi- impaired myocardial angiogenesis & ventricular remodeling. The dative stress are increased in the diabetic heart, the role of iNOS- cells defend themselves against oxidative stress induced damage mediated nitroso-redox balance in the pathophysiology of ische- using antioxidant proteins among which antiapoptotic & growth mia/reperfusion (I/R) injury has been poorly understood. Because stimulatory thioredoxin-1 (Trx1) plays a critical role. In the present iNOS-derived nitric oxide (NO) is a key player in cardioprotection study we have evaluated the reversal of diabetes mediated impair- against I/R injury in the preconditioned heart, it was hypothesized ment of angiogenesis in myocardial infarction (MI) model of strep- that inhibition of iNOS uncoupling enhances tolerance of the dia- tozotocin induced Type I diabetic rats by intramyocardial admini- betic heart to I/R injury through increased bioavailability of NO. stration of adenovirus encoding Trx1 (Ad.Trx1), after MI. The The present study demonstrated that iNOS-derived superoxide myocardial function was measured by echocardiography 30 days generation was reduced and nitric oxide bioavailability was in- after the intervention. The Ad.Trx1 administered group exhibited creased by treatment with NOS-cofactor, tetrahydrobiopterin reduced fibrosis, oxidative stress and cardiomyocyte and endothe- (BH4), before I/R in the heart isolated from the diabetic rat. This lial cell apoptosis as compared to diabetic MI group along with in- was associated with reduction of infarct size and improvement of creased capillary and arteriolar density. Western blot and immuno- left ventricular (LV) function after I/R. The cardioprotective effect histochemical analysis demonstrated myocardial overexpression of of BH4 was abrogated by treatment with a thiol reducing agent Trx1, HO-1, VEGF, p38MAPK and decreased p-JNK and p38MAPK in the Ad.Trx1 treated diabetic group. Alternatively, dithiothreitol (DTT) but not a NO-sensitive guanylyl cyclase in- we have observed significant reduction in the expression of VEGF hibitor ODQ, suggesting that iNOS-derived NO-mediated cardio- in SnPP (HO-1 enzyme inhibitor) treated non-diabetic and diabetic protection occurs through the protein S-nitrosylation but not the c- animals even after Ad.Trx1 therapy. Echocardiographic analysis af- GMP-dependent signaling pathway in the diabetic heart. Indeed, ter 4 weeks of MI revealed significant improvement in the myo- protein S-nitrosylation was increased by treatment with BH4 in the cardial functional parameters such as the ejection fraction, frac- diabetic heart and inhibited by DTT. These results suggest that tional shortening and E/A ratio in the Ad.Trx1 administered group iNOS-derived NO plays a crucial role in acquisition of tolerance to as compared to the diabetic MI group. For the first time our novel ischemia/reperfusion injury through the protein S-nitrosylation. approach of Trx1 gene therapy may prove to be a strategic thera- Therefore, inhibition of iNOS uncoupling and modulation of peutic modality in the treatment of diabetes related cardiac failure iNOS-mediated nitroso-redox balance may represent a novel by stabilizing the redox status in the myocardium, reducing cell therapeutic target for myocardial protection in diabetic patients. death, inducing neovessel formation & maturation & reducing ven- tricular remodeling after MI.

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Regulation of myocardial growth and death by oxidative stress Contribution of cholesterol oxidation products Junichi Sadoshima to the progression of the atherosclerotic lesion

Department of Cell Biology and Molecular Medicine, Cardiovascular Gabriella Leonarduzzi, Simona Gargiulo, Paola Gamba, Research Institute, New Jersey Medical School, NJ, USA Barbara Sottero, and Giuseppe Poli Oxidative stress plays an essential role in regulating growth and death of cardiac myocytes. Thioredoxin 1 (Trx1) has many protec- Department of Clinical and Biological Sciences, University of Turin, tive functions in the heart, including suppression of pathological San Luigi Hospital,, 10043 Orbassano(Turin), Italy hypertrophy. Trx1 facilitates the reduction of signaling molecules by cysteine thiol-disulfide exchange, thereby regulating their func- Cholesterol oxidation leads to the formation of a number of 27- tions. Trx1 upregulates DnaJb5, a heat shock protein 40, and forms carbon atoms compounds, termed oxysterols, which may either a multiple-protein complex with DnaJb5 and class II histone originate in the blood, cells and tissues, through both enzymatic deacetylases (HDACs), master negative regulators of cardiac hy- and not enzymatic reactions, or derive from the diet. These choles- pertrophy. Trx1 reduces critical cysteine residues in class II terol metabolites have been consistently demonstrated in vitro to HDACs, thereby inducing nuclear localization of class II HDACs be at least one or two orders of magnitude more reactive than and inhibiting pathological hypertrophy. The NADPH oxidase unoxidized cholesterol, showing remarkable pro-oxidant, pro- family is a group of membrane-associated proteins producing su- inflammatory, pro-apoptotic, and pro-fibrogenic effects. Moreover, peroxide in a wide variety of cell types. We have obtained evi- we recently observed that in vivo, namely in advanced atheroscle- dence that Nox4 plays an important role in mediating both death rotic lesions of human carotid, which are characterized by the and survival of heart cells in response to pressure overload and presence of several cells of the macrophage lineage, the more cho- ischemia/reperfusion in the heart. Nox4 plays an essential role in lesterol accumulates, the higher is the production of oxysterols. In mediating increases in oxidative stress in the failing heart by in- particular, oxysterols might significantly contribute to the vascular creasing superoxide production in mitochondria. In this presenta- changes due to atherosclerosis, as they appear involved in various tion, I will discuss the role of Trx1 and Nox4 in regulating growth key steps of this complex process, from endothelial cell dysfunc- and death of cardiac myocytes. tion and foam cell formation up to fibrotic degeneration of the arte- rial wall and vulnerable plaque rupture. As regards the way oxys- terols signal to the nucleus of the cells to exert their biochemical effects, a cholesterol oxide mixture compatible with that detectable in human hypercholesterolemic plasma and atherosclerotic lesions has shown the ability to trigger and sustain a molecular cascade in which the pivotal role is played by PKC and MEK/ERK phos- phorylation. Because of their very strong pro-inflammatory potential, cho- lesterol oxidation products could contribute to the progression of pathologies other than atherosclerosis, whose hypercholesterolemia represents a primary risk factor.

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SESSION IV TRANSLATIONAL SCIENCE BY MICRONUTRIENTS


Carotene dioxygenase-1 polymorphisms

Georg Lietz Colors with functions: Newcastle University, Human Nutrition Research Centre, School of Agriculture, Food Elucidating the biochemical and molecular basis of and Rural Development, Agriculture Building, Newcastle upon Tyne, NE1 7RU, UK carotenoid metabolism -carotene absorption and/or conversion into retinal is ex- Johannes von Lintig tremely variable among individuals. The proportion of low re- sponders to -carotene in well nourished individuals has been es-

Department of Pharmacology, School of Medicine, Case Western Reserve timated to be 45% [Wang et al, 2004]. Still, the mechanisms un- University, WB333, 10900 Euclid Ave. Cleveland, Ohio 44106-4965, USA
derlying this variability are not well understood, and could arise from genetic variations in genes involved in the absorption, trans- Carotenoids affect a rich variety of physiological functions port and/or conversion of carotenoids. Carotenoids are transported in nature and are beneficial for human health, serving as antioxi- across the intestinal epithelial layer via a facilitated process involv- dants in lipohilic environments and blue light filters in the macula ing the scavenger receptor-BI (SR-BI) [During et al, 2005]. A of human retina. These dietary compounds also serve as precursors number of polymorphisms in the SRBI gene have been found to significantly alter fasting plasma carotenoid concentrations [Borel of a unique set of apocarotenoid cleavage products, including reti- et al, 2007]. Once absorbed, pro-vitamin A carotenoids are con- noids (vitamin A and its derivatives). Although knowledge about verted to retinal via the -carotene 15,15’-monoxygenase retinoid biology has tremendously increased, the metabolism of (BCMO1). A first indication that genetic background might influ- their parent precursors remains poorly understood. Recently, mo- ence the conversion efficiency in humans comes from the discov- lecular players in carotenoid metabolism have been identified and ery of a very rare missense mutation in the BCMO1 gene, which is biochemically characterised. Moreover, mutations in their corre- associated with hypovitaminosis A and hypercarotenaemia [Lind- sponding genes impair carotenoid metabolism and induce various qvist et al, 2007]. However, this rare mutation cannot explain the pathologies in animal models. Polymorphisms in these genes alter high occurrence of the low responder phenotype. Screening of the carotenoid and retinoid homeostasis in humans as well. This lec- total open reading frame of the BCMO1 coding region lead to the ture summarizes our current knowledge about the molecu- identification of two common non-synonymous SNPs (R267S; lar/biochemical basis of carotenoid metabolism, and especially, the rs12934922 and A379V; rs7501331), with variant allele frequen- physiological role of carotenoids in retinoid-dependent physiologi- cies in a Caucasian population of 42% and 24%, respectively [Le- cal processes. ung et al, 2009]. Assessment of the responsiveness to a pharma- cological dose of -carotene in female volunteers confirmed that

carriers of both the A379V and R267S/A379V variant had a re- duced ability to convert -carotene [Leung et al, 2009]. Identifica- tion of novel functional SNPs in key enzyme of carotenoid me- tabolism indicates the potential importance of micronutrient-gene interactions. Furthermore, the effect of the genetic variants in BCMO1 is modulated by ethnicity, sex and BMI. Borel P, Moussa M, Reboul E et al. J Nutr; 137:2653-9 (2007). During A, Dawson HD, Harrison EH. Journal of Nutrition; 135:2305-2312 (2005). Leung W, Hessel S, Méplan C et al. Faseb J; 23, 1041–1053 (2009). Lindqvist A., Sharvill J., Sharvill D.E., and Andersson S., J. Nutr., 137, 2346-2350 (2007). Wang Z., Yin S., Zhao X., Russell R.M., and Tang B., Br. J. Nutr., 91, 121-131 (2004).

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-carotene 15,15’-monooxygenase 1 gene affects Determination of 9-cis -carotene and -carotene circulating levels of carotenoids in biological samples

Richard D. Semba,* Luigi Ferrucci, John R. B. Perry, Jian Qin, Kyung-Jin Yeum, Elizabeth J. Johnson, Amy Matteini, Markus Perola, Toshiko Tanaka, Kaisa Silander, * Norman I. Krinsky , Robert M. Russell, and Guangwen Tang Neil Rice, David Melzer, Anna Murray, Christie Cluett,

Linda P. Fried, Demetrius Albanes, Anna-Maria Corsi, Antonio Cherubini, Jack Guralnik, Stefania Bandinelli, Jean Mayer USDA-Human Nutrition Research Center on Aging,*Department of Andrew Singleton, Jarmo Virtamo, Jeremy Walston, and Biochemistry, School of Medicine, Tufts University, Boston, MA 02111

Timothy M. Frayling Determination of 9-cis -carotene (-C) and -carotene (-C) Johns Hopkins University School of Medicine* and thirteen other institutions in U.S., U.K., Italy, and Finland in biological samples may provide crucial information on biologi- cal activities of these carotenoids. However, these carotenoids are Introduction. Low circulating carotenoids have been associ- often co-eluted when analyzed using high performance liquid chro- ated with increased risk of cardiovascular disease, cancer, and matography (HPLC). There is a need to develop a method for 9- death, and with age-related declines of muscle strength and physi- cis -C and -C quantification. Both 9-cis -C and -C have peak cal performance. Whether genetic variation affects the circulating absorbance at 400 and 450 nm, whereas only 9-cis -C has peak levels of carotenoids has not been well studied. absorbance at 475 nm. Based on these spectral characters, we Methods. We conducted a genome-wide association study in developed an HPLC method to determine the amounts of 9-cis -C 1191 adults from the InCHIANTI study in Tuscany, Italy, to iden- and -C in biological sample by using peak absorbance ratios in tify novel common variants associated with circulating carotenoid these wavelengths. The absorbance of mixture of 9-cis -C and - levels. The effects were replicated in the Women’s Health and Ag- C was monitored at 475 and 400 nm. In which, the 9-cis -C was ing Studies (n = 615) and the Alpha-Tocopherol, Beta-Carotene quantified by using absorbance value at 475 nm; -C was then cal- Cancer Prevention Study (n = 2136). culated from the absorbance at 400 nm by subtracting the 9-cis -C Results. In meta-analyses involving all three studies, the G al- absorbance at 400 nm (calculated as 39% of the absorbance of 9- lele at rs6564851, near the -carotene 15,15’-monooxygenase 1 cis -C at 475 nm). This method was used to determine 9-cis -C (BCMO1) gene, was associated with higher circulating -carotene concentrations of American lactating women (n=12) in serum (7.1 -24 (P = 1.6 x 10 ) and -carotene (P = 0.0001) concentrations and ± 0.8 nM) and breast milk (1.1 ± 0.2 nM), and -C concentrations lower lycopene (P = 0.003), zeaxanthin (P = 1.3 x 10-5), and lutein of those lactating women in serum (54.2 ± 7.2 nM) and milk (8.3 ± (P = 7.3 x 10-15) concentrations, with effect sizes ranging from 1.8 nM). We also find that 9-cis -C concentrations of Korean 0.10-0.28 standard deviations per allele. No significant relation- women (n=16) in serum (15.6 ± 1.1 nM) and breast adipose tissue ship was found between this genetic variant and plasma retinol. (0.2 ± 0.1 nmol/g), and -C concentrations of those Korean women Conclusions. Variation in the -carotene 15,15’-monooxy- in serum (49.0 ± 3.9 nM) and adipose tissue (0.3 ± 0.1 nmol/g). genase 1 (BCMO1) gene, which catalyzes the first step in the con- Considering the significant amounts of 9-cis -C and -C in circu- version of provitamin A carotenoids to vitamin A in the small in- lation and tissues of humans from their dietary intakes, investiga- testine, is associated with plasma concentrations of carotenoids. tions on these carotenoids and their possible health benefits will be discussed.

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Control of oxidative phosphorylation by vitamin A illuminates a Public health aspects of -carotene as an important fundamental role in mitochondria energy homeostasis vitamin A source for humans

Rebeca Acin-Perez*, Beatrice Hoyos**, William S Blaner***, Hans K. Biesalski Giovanni Manfredi* and Ulrich Hammerling** Department of Biological Chemistry and Nutrition, University Hohenheim, *Department of Neurology and Neurobiology, Weill Medical College at Garbenstrasse 30, D 70597 Stuttgart Cornell, New York, NY. ** Immunology Program, Sloan-Kettering Institute for Cancer Research, New York, NY. *** Department of Human Nutrition, College of In developed countries major sources for preformed vitamin A Physicians and Surgeons, Columbia University, New York, NY (e.g., liver and liverproducts) are available but poorly consumed. Whereas many biological effects of vitamin A are attributable to As a consequence different groups (young females, pregnant its derivative, retinoic acid, its action in signal transduction does not women, elderly, obese people) with poor vitamin A intake and require prior metabolic conversion but is mediated by vitamin A (reti- subsequent low retinol but normal to high -carotene levels are de- nol) itself. Building on previous findings that retinol binds protein scribed. This raises the question whether the recommendation to kinase C (PKC) with high affinity we discovered an important regula- tory pathway in mitochondria, which depends on the presence of reti- consume a diet rich in -carotene to compensate a low intake of nol. This signal pathway is anchored on protein kinase C, is situated preformed vitamin A is indeed enough. Depending on the cleavage in the intermediate space, and transmits an activating signal to the py- ratio of -carotene to vitamin A, which migth be different in dif- ruvate dehydrogenase (PDH) complex located in the matrix. While in- ferent tissues, the importance of this provitamin as a source for vi- termediaries of this signal chain remain unidentified, the pyruvate de- tamin A is under discussion. To evaluate the state of the art regard- hydrogenase kinase, isoform 2 (PDK2), becomes inactivated by de- ing the role of -carotene as a source for vitamin A an international phosphorylation when PKC is active. Since PDK2 negatively regu- lates PDH, the reduced PDK2 activity translates into dephosphoryla- consensus meeting was organized on July 11th 2009. (Participants: tion of the E1 regulatory subunit of PDH, increasing acetyl-CoA pro- Tilman Grune, Jun Shi Chen, Georg Lietz, Andreu Palou, A. duction, oxygen consumption and ATP synthesis. The physiological Catharine Ross, Wilhelm Stahl, Guangweng Tang, David relevance of vitamin A for mitochondrial energy homeostasis became Thurnham, and Hans K. Biesalski). The experts discussed 17 ques- apparent in the LRAT null mouse strain rendered completely vitamin tions and reached an agreement formulated in a consensus answer. A deficient by dietary deprivation. This regimen resulted in markedly This consensus answer is based on published valid data, which reduced levels of oxygen consumption of their liver mitochondria, which were promptly normalized by the injection of vitamin A. The were carefully reviewed by the individual experts and are justified central role of PKC was established by targeted deletion of the prkcd by background statements. Ascertaining the impact of -carotene gene, whereas the need for binding retinol to the zinc-finger domains on the total intake of dietary vitamin A is complicated, as the effi- of PKC was deduced from the failure of complementing the prkcd ciency of conversion of -carotene to retinol is not a single ratio, gene ablation by a mutant form with an engineered deletion of the and different conversion factors have been used in various surveys retinol binding sites. While the upstream signal that activates PKC is and governmental recommendations. However, a role of -caro- still unknown, our evidence supports the hat retinol functions as a de-  vice to target oxidation to select cysteine residues of the zinc-finger tene in fulfilling the recommended intake for vitamin A is apparent domain where local unfolding is the crucial initiation event for en- from a variety of studies. Thus, besides, elucidating the various zyme activation. functions, the distribution and the uptake of -carotene, the con- Chiu, H-J, Fishman, D.A. and Hammerling, U. Vitamin A depletion casuses oxidative stress, sensus conference placed special emphasis on the provitamin A mitochondrial dysfunction and PARP-1-dependent energy deprivation. FASEB J 2008, 22:3878 Acin-Perez, R., Hoyos, B., Zhao, F., Vinogradov, V., Fischman, D.A., Harris, R.A., function of -carotene and the role of -carotene in the realization Leitegs, M., Nuttaporn, W., Blaner, W.S., Manfredi, G., and Hammerling, U. Control of oxi- dative phosphorylation by vitamin a illuminates a fundamental role in mitochondrial energy of the required/recommended total vitamin A intake, in both de- homoeostasis. FASEB J. 2010, 24: 627 veloped and developing countries.

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New classes of mitochondria-targeted antioxidants – Coenzyme Q in plasma membrane electron transport non-radical scavenging inhibitors of lipid peroxidation 1 1 2 Valerian E. Kagan Plácido Navas , Carlos Santos-Ocaña , Rafael de Cabo , and 1 University of Pittsburgh Emilio Siendones

Free radical reactions are implicated in the pathogenesis of major 1Centro Andaluz de Biología del Desarrollo, Universidad Pablo de chronic cardiovascular and neurodegenerative diseases, cancer, radia- Olavide-CSIC and Centre for Biomedical Research on Rare Diseases (CIBERER), ISCIII, E-41013 Sevilla, Spain tion injury and aging. It is assumed that oxygen radicals, particularly 2Laboratory of Experimental Gerontology, National Institute on Aging, NIH, – superoxide radicals (O2· ), hydrogen peroxide (H2O2) and hydroxyl Baltimore, MD 21224, USA radicals cause oxidative modification of biological molecules and ini- tiation of free radical chain reactions. Numerous attempts have been Coenzyme Q (CoQ) is an essential component of the mito- made to utilize free radical scavengers as therapeutic and/or preven- chondrial respiratory chain, functions on the activation of uncou- tive remedies. The initial optimism, however, faded over the years as pling proteins, the regulation of the permeability transition pore, as clinical trials showed relatively low efficiency of antioxidants in drug substrate for the dihydro-orotate dehydrogenase, and is substrate of discovery paradigms. Do peroxidation reactions develop as a random acyl-CoA dehydrogenase. CoQ is located in the inner mitochon- uncontrolled chain process in tissues normally or in disease condi- drial membrane, but also in serum lipoproteins, endo-membranes tions? There is a popular concept of chain breaking (sacrificial) wa- and the plasma membrane of eukaryotic cells. CoQ is a mediator in ter-soluble and lipid-soluble antioxidants such as vitamin C, vitamin a plasma membrane redox system (PMRS) involved in membrane E, ubiquinol, etc. Is antioxidant action their only or major function, antioxidant protection, in the maintenance of intracellular redox and do they act as “random” scavengers of “random” radicals? homeostasis, and regulation of cell signaling. NAD(P)H-dehydro- Recently, we discovered that during apoptosis, cytochrome c (cyt c), genases in PMRS include NADH-cytochrome b5 reductase en- switches its function from a shuttle of electrons between mitochon- coded by CB5R3 and NAD(P)H:quinone oxidoreductase 1 encoded drial complexes III and IV, to that of a peroxidase. We found that this by NQO1. CoQ-dependent PMRS blocks the triggering of the ce- is due to cyt c’s interactions with cardiolipin (CL). Trans-membrane ramide-dependent apoptotic program. PMRS is activated by vita- min E deficiency, calorie restriction, and mitDNA deficiency. The migration of CL from the inner to the outer mitochondrial membrane + - catalyzed by scramblase-3 early in apoptosis – is a pre-requisite for activity of PMRS also equilibrates the NAD /NADH ratio impor- its interaction with cyt c. The new catalytic function of cyt c is spe- tant in aging process. Yeast NQR1, homologous of mammal cific towards CL resulting in its oxidation to yield different mono-, CB5R3, regulates both chronological and replicative lifespan. This di- and tri- hydroperoxy- and hydroxy- molecular species. We will gene is induced by calorie restriction in parallel to the activation of respiration. Overexpression of NQR1 promotes oxygen consump- present data illustrating the principal differences in action of vitamin + E and vitamin C against random reactions of oxidative stress and en- tion, inhibits ethanol production, and maintains NAD /NADH bal- ance. We propose that PMRS regulates apoptosis, but also regu- zymatically-controlled reactions of lipid peroxidation. This will be + followed by demonstration of new possibilities to control lipid per- lates aging by connecting aerobic metabolism and NAD /NADH oxidation reactions by mechanism-based inhibitors of the peroxidase ratio. activity of cyt c/cardiolipin complexes. Finally, the data will be Supported by Spanish FIS Grant PI080500, NIH Grant 1R01AG28125-01A1, and the In- shown illustrating anti-apoptotic potential of these new classes of in- tramural Research Program of the NIA/NIH. hibitors of lipid peroxidation in vitro and in vivo.

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Coenzyme Q10 in health and disease ECTO-NOX, coenzyme Q , and superoxide production 10 Iain P. Hargreaves in aging National Hospital, London, UK

D. James Morré1,2, Dorothy M. Morré1,2, and Dale Kern3 A major difficulty encountered when assessing (coenzyme Q10) CoQ status in tissues is the lack of a commercially available non- 1NOX Technologies, Inc., 1291C Cumberland Avenue and 2Purdue University, 10 West Lafayette, IN 47906 and 3NuSkin International, 75 West Center Street, physiological internal standard. To address this problem we have Provo, UT 84601 synthesised a dipropoxy analogue of CoQ10, which has enabled the accurate determination of tissue CoQ10 status. A decrease in CoQ10 The aging related ECTO-NOX (arNOX) family of superoxide- status has been reported in a number of diseases, however, interest generating oxidases increases with age beginning at ca. 30 y. Shed has focused on the possible role CoQ10 deficiency may have in the forms are present in blood associated with serum LDL and in other pathophysiology of the rare adverse effects associated with hy- body fluids where they may contribute to action-at-a-distance phe- droxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in- nomena such as skin aging and coronary artery disease. arNOX hibitor (statin) treatment. Currently, there is insufficient evidence from human studies to link statin therapy unequivocally to pathol- proteins are membrane anchored with the catalytic N-termini di- ogically significant decreased tissue CoQ10 concentrations. To in- rected outward. The shed forms result from proteolytic cleavage vestigate involvement of mitochondrial respiratory chain (MRC) of ca. 30 kDa fragments. At least five different isoforms are indi- dysfunction in myotoxicity associated with statin treatment, MRC cated. A distinguishing feature is periodic generation of superox- activity and CoQ10 status was assessed in two patients experienc- ide anion estimated from superoxide dismutase-inhibited reduction ing myopathy following treatment with simvastatin (40mg/day) of ferricytochrome c. The electron donor for the shed forms is pro- and cyclosporin and simvastatin (40mg/day) and itraconazole. tein thiols. Competition for cytochrome c reduction by reduction Analysis of skeletal muscle biopsies revealed a decreased CoQ10 of CoQ10 by superoxide is not involved. Compared to H2O2 pro- status (77 and 132; reference range: 140 - 580 pmol/mg) and cyto- duction by mitochondria (ca. 0.1 nmoles/min/mg mitochondrial chrome oxidase (complex IV) activity (0.006 and 0.007 reference protein), human buffy coats or epidermal epithelia from subjects range: 0.014 – 0.034). To assess statin treatment in the absence of 55 to 65 y produce ca. 0.06 nmoles superoxide convertible into possible pharmacological interference primary astrocytes were cul- 7 tured with lovastatin (100 mM). Lovastatin treatment resulted in a H2O2 per 10 cells. For sera, ca. 0.3 nmoles/ml/min results in sev- eral millimoles on a daily basis in the proximity of circulating decrease in CoQ10 (97.9 ± 14.9; control: 202.9 ± 18.4 pmol/mg; p < 0.05), and complex IV activity (0.008 ± 0.001; control: 0.011 ± lipoproteins. Recent focus has identified safe and effective dietary 0.001; p< 0.05). The effect of rosuvastatin therapy was assessed on (e.g., CoQ10) and topical (AgeLoc, NuSkin Enterprises) means to blood mononuclear cell (BMC) CoQ10 status and mitochondrial inhibit arNOX activity. Both the cell-bound and shed arNOX are ATP synthesis in children with familial hypercholesterolemia. inhibited by CoQ10 but not by ubiquinol (CoQ10H2). As ubiqui- Samples were taken at baseline and after rosuvastatin treatment. nones with side chains shorter than that of CoQ8 do not inhibit, Twenty-nine patients were treated with 5, 10 or 20 mg rosuvastatin polyprenyl side chain involvement is indicated. Data for saliva for 29 weeks. After this time a significant 32% decrease in BMC and sera have demonstrated dose dependent, sustained but reversi- CoQ10 (p=0.024) was observed. BMC ATP synthesis was compa- ble 50% reductions in arNOX activity by oral administration of rable to baseline levels (p=0.60). In conclusion these results indi- CoQ10 in both male and female subjects. cate a possible association between statin treatment, decreased CoQ10 status and loss of complex IV activity.

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Chemoprevention by pomegranate and green tea Targeting inflammatory pathways “naturally” for prevention and therapy of cancer and other chronic diseases Susanne M. Henning, Piwen Wang, Angela Wong, Roberto Vicinanza, Lynn, S. Adams, William J. Aronson, and Bharat B. Aggarwal and Bokyung Sung David Heber

Center for Human Nutrition and Department of Urology, David Geffen School Cytokine Research Laboratory, Department of Experimental Therapeutics, The of Medicine, University of California Los Angeles, CA 90095 University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA Ellagitannins from pomegranate and polyphenols from green Chronic infections, obesity, alcohol, tobacco, radiation, envi- tea have limited bioavailability and are highly metabolized by ronmental pollutants, and high-calorie diet have been recognized colonic microflora. Ellagitannins are converted to gallagic acid as major risk factors for the most common types of cancer. All (GA), ellagic acid (EA), urolithin A (UA) and B (UB) and tea these risk factors are linked to cancer through oxygen radical- polyphenols to phenolic acids, which can be found in the circula- mediated inflammation. While acute inflammation that persists for tion and urine. During the absorption process polyphenols and their short-term mediates host defense against infections, chronic in- metabolites are glucuronidated, sulfated and methylated. These flammation that lasts for long-term can predispose the host to vari- metabolites exhibit biological activities. Recent findings show that ous chronic illnesses, including cancer. Linkage between cancer UB, GA, UA inhibit proliferation in aromatase overexpressing and inflammation is indicated by numerous lines of evidence; first, MCF-7-aro breast cancer cells via aromatase inhibition and estro- transcription factors NF-B and STAT3, two major pathways for gen receptor antagonism, and also inhibit inflammation via NFB. inflammation, are activated by most cancer risk factors; second, an Conjugation and methylation may alter their tissue distribution and inflammatory condition precedes most cancers; third, NF-B and biologic activities. Methylated and sulfated UB retained the aro- STAT3 are constitutively active in most cancers; fourth, hypoxia matase inhibitory activity. However, the methylated form of one of and acidic conditions found in solid tumors activate NF-B; fifth, the tea polyphenols, epigallocatechin gallate (EGCG), exhibited chemotherapeutic agents and gamma irradiation activate NF-B decreased anti-proliferative activity, decreased anti-inflammatory and lead to chemoresistance and radioresistance; sixth, most gene activity (NFB stimulation) and decreased stimulation of apoptosis products linked to inflammation, survival, proliferation, invasion, compared to the parent compound in human LNCaP prostate can- angiogenesis, and metastasis are regulated by NF-B and STAT3; cer cells. In a mouse and human tea intervention study we demon- seventh, suppression of NF-B and STAT3 inhibits the prolifera- strated that about 50% of EGCG was present in methylated form in tion and invasion of tumors; and eighth, most chemopreventive tissue. EGCG itself is a substrate for methyl transferases such as agents mediate their effects through inhibition of NF-B and catechol-O-methyl transferase (COMT) and 5-cytosine DNA STAT3 activation pathways. Thus suppression of these proin- methyl transferase I (DNMT1). In xenograft tumor tissue in SCID flammatory pathways may provide opportunities for both preven- mice drinking green tea both COMT and DNMT1 gene expression tion and treatment of cancer. We will discuss the potential of vari- was inhibited significantly. In cell culture experiments combining ous natural products in suppression of inflammatory pathways and EGCG with another methyl inhibitor such as quercetin decreased their role in prevention and therapy of cancer and other chronic EGCG methylation, decreased DNMT1 gene expression and in- diseases. creased EGCG uptake. In summary, to perform in vitro experi- ments with translational potential, the in vivo metabolism of phy- tochemicals will need to be considered carefully.

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Regulation of antioxidant gene expression by selected Resveratrol mimics caloric restriction and dietary polyphenols retards aging in the heart

Young-Joon Surh Tomas A. Prolla

WCU Department of Molecular Medicine and Biopharmaceutical Sciences, Departments of Genetics and Medical Genetics, Graduate School of Convergence Science and Technology and College of University of Wisconsin-Madison, Madison WI 53706, USA Pharmacy, Seoul National University, Seoul 151-742, South Korea Resveratrol in high doses has been shown to extend lifespan in The low risk of chronic diseases, such as coronary heart disease, some studies in invertebrates and to prevent early mortality in mice neurodegenerative disorders and certain cancers, observed in some fed a high-fat diet. We fed mice from middle age (14-months) to population groups results from consumption of relatively large old age (30-months) either a control diet, a low dose of resveratrol amounts of fruits and vegetables. Health benefits associated with (4.9 mg kg/day), or a calorie restricted (CR) diet and examined ge- functional foods and beverages containing a great array of bioac- nome-wide transcriptional profiles. We report a striking transcrip- tive phytochemicals or phytonutrients have been extensively inves- tional overlap of CR and resveratrol in the heart. Both dietary in- tigated and well-documented. As oxidative stress is implicated in a terventions inhibit gene expression profiles associated with cardiac wide array of human disorders, antioxidant phytochemicals have and skeletal muscle aging, and prevent age-related cardiac dys- also been frequently included as active ingredients of many func- function. A more recent analysis using transcriptional supermark- tional foods. The most important botanicals for use as nutracuetical ers of aging in multiple mouse strains shows that the anti-aging ef- formulations include tea, soy, pomegranate, grape seeds, pycno- fects of resveratrol are stronger in the heart, whereas lipoic acid genol, turmeric, ginkgo biloba, aloe, ginseng, etc. The majority of and Coenzyme Q10 have stronger effects in the brain. Our results bioactive chemical compounds contained in the dietary supple- suggest that transcriptional supermarkers of aging can be used to ments/nutraceuticals have antioxidant activities. Nuclear transcrip- identify and design tissue-specific CR mimetic anti-aging interven- tion factor erythroid 2p45 (NF-E2)-related factor 2 (Nrf2) plays a tions. crucial role in regulating induction of antioxidant or cytoprotective gene expression. Many bioactive substances derived from edible plants have been found to activate this particular redox-sensitive transcription factor, thereby potentiating cellular antioxidant or de- toxification capacity. Nutraceutical and functional food markets are rapidly growing, and there are ample outstanding opportunities of research for the development of botanicals as dietary supple- ments or nutraceuticals.

1. Surh, Y.-J. (2003) Cancer chemoprevention with dietary phytochemicals. Nature Re- views Cancer, 3: 768-780 2 . Surh, Y.-J., Kundu, J.-K., and Na, H.-K. (2008) Nrf2 as a master redox switch in turn- ing on the cellular signaling Involved in the induction of cytoprotective genes by some chemopreventive phytochemicals. Planta Med., 74: 1526-1539. 3. Surh, Y.-J., Dong, Z., Cadenas, E., and Packer, L. (2008) Dietary Modulation of Cell Signaling Pathways, CRC Press-Taylor & Francis

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SESSION IV REDOX IMAGING

Development of PET/SPECT probes and their application to Clinical (EPR) oximetry for improving diagnosis and in vivo molecular imaging treatment in ischemic diseases and tumors and wound healing

Hideo Saji Harold M. Swartz Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501 Japan Dartmouth Medical School, HB 7785, Hanover, NH 03755 “Molecular imaging” is a recently developed approach that is in vivo imaging of the spatial/temporal distribution of biologi- The technique of Electron Paramagnetic Resonance (EPR) cal/molecular biological processes (events) at the cellular/ molecular oximetry has now advanced to the stage where it is being used ex- level and has been contributing to the promotion of studies on the basis tensively in animal experiments and several clinical studies are in of life science, biological functions and causes of diseases, ge- process. This has required the development of special instrumenta- netic/regenerative medicine. In molecular imaging field, methods us- tion to safely and effectively accommodate human subjects. The ing radiation such as positron emission tomography (PET) and single potential advantages of EPR oximetry include that it measures the photon emission computed tomography (SPECT) play a major role. In actual pO2 directly in the tissues and these measurements can be tumors, rapid growth of cells and rapid expansion of the tumor mass made repeatedly from the same site. Most EPR oximetry ap- usually leaves the generation of new vasculature lagging behind and proaches require an initial minimally invasive step to place the results in the lack of oxygen delivery. This condition is under an un- oxygen sensitive materials at the sites of interest, but the subse- balanced redox status, oxidative levels in tumor cells being relatively quent measurements are completely non-invasive. The studies in higher. In such condition hypoxia-inducible factor-1 (HIF-1) is ex- human subjects underway at Dartmouth include pressed. HIF-1 is specifically degraded under normoxia, but stable un- 1. Measurements of oxygen in tumors to optimize therapy by der hypoxia. Thus, by utilizing the character of HIF-1, we attempted to enabling the therapy to be delivered at optimum times and/or develop PET/SPECT imaging agent for molecular imaging of cancer. altered to deal with significant hypoxia Since the degradation of HIF-1 occurs through protein hydroxylation 2. Measurements of oxygen in tissues at risk in peripheral vas- in the oxygen-dependent degradation domain (ODD) of HIF-1, it is cular disease to characterize the pathophysiological state and to measure response to therapy expected that probes containing the ODD and that are degraded in a 3. Acute and chronic measurements of oxygen in healing similar manner as HIF-1 can evaluate HIF-1 activity in vivo. Thus, wounds. we recently developed a protein (POS) in which the ODD is fused to In order to make the measurements with paramagnetic materi- the protein transduction domain (PTD) and a monomeric streptavidin als that are cleared for use in human subjects, most of the meas- (SAV). Furthermore, We synthesized a radiolabeled biotin derivative, urements are made using India ink as the oxygen sensitive mate- (3-123/125I-iodobenzoyl)norbiotinamide (123/125I-IBB) and conjugated rial. We are now developing an implantable resonator that should the chimeric protein—PTD-ODD-SAV (POS)—and 123/125I-IBB to 123/125 123/125 enable the measurements to be made with greater sensitivity and at produce I-IBB-POS ( I-IPOS). POS was degraded in an greater depths. Our technique uses multisite spectroscopy, which oxygen-dependent manner, and a clear image of the tumor was ob- can measure oxygen at several defined site simultaneously. The tained at 24 h after the injection of 123I-labeled-POS. Moreover, The 125 time resolution of the measurements can be seconds and the meas- intratumoral distribution of I-IPOS was heterogeneous and corre- urements have been repeated in human subjects from the same site sponded to the hypoxic areas. for more than four years.

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Imaging of tissue oxygenation: Novel probes and opportunities

Periannan Kuppusamy

Davis Heart and Lung Research Institute, The Ohio State University, Columbus, OH 43210, USA

Oxygen is a critical determinant in the prediction of treatment outcome of several disease including surgical interventions, cancer therapy, tissue graft, and cell therapy. There is a great need for methods capable of reliable noninvasive measurement and moni- toring of oxygen concentration in tissues. Although several meth- ods are utilized to measure oxygen concentration, a suitable tech- nique for noninvasive and repeated measurements of oxygen in the same tissue or cells on a temporal scale is warranted. While elec- trode techniques have evolved as the standard methods for meas- urement of oxygen, they generate analytical artifacts during assay procedures at the freshly probed sites. Near-infrared and nuclear magnetic resonance techniques are noninvasive methods. How- ever, they do not report the absolute values of oxygen concentra- tion, and lack the resolution of oxygen measurements. Electron paramagnetic resonance (EPR) oximetry enables reliable and accu- rate measurements of concentrations of molecular oxygen. EPR oximetry can measure directly and at the actual site of interest. The ability of EPR oximetry to make repeated measurements from lo- calized sites provides a very important capability that can enable critical aspects of a number of biomedical applications. We have developed innovative approaches using oxygen-sensing nano/ mi- crocrystalline probes to perform noninvasive cellular/tissue oxime- try/imaging in a variety of applications including myocardial ischemia/reperfusion injury, cellular cardiomyoplasty (cell ther- apy), organ transplantation, angiogenesis, cancer therapy, and wound healing. Current developments in in vivo EPR methodolo- gies enable a number of potential applications that could be very significant additions to clinical medicine. SESSION VI AGING

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Selective Autophagy on the cellular response to stress RasGrf1 deficiency delays aging in mice by mimicking caloric restriction Ana Maria Cuervo

José Viña, Consuelo Borrás1, Daniel Monleón2, Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY Raul López-Grueso1, Juan Gambini1, Leonardo Orlando3,

Federico V. Pallardó1, Eugenio Santos4, and Jaime Font de Mora3,5 Autophagy is the intracellular process that mediates the diges- 1Department of Physiology, School of Medicine, University of Valencia. tion of cellular components in lysosomes. The autophagic system 2Instituto de Investigación Sanitaria FIHCUV/Fundación de Investigación Hospital Clínico Universitario de Valencia. 3Laboratory of Cellular and fulfills two major functions in mammalian cells, serving both as an Molecular Biology, Centro de Investigación Príncipe, Valencia. 4Centro de alternative source of energy, when nutrients are scarce, and as an Investigación del Cáncer (USAL-CSIC), Univeristy of Salamanca. Spain efficient mechanism for the removal of any intracellular damage Lifespan is determined by environmental as well as genetic structure. Autophagic activity has been described to decline with factors. Caloric restriction extends longevity via regulatory pro- age in almost all organisms and tissues, as wells as in several age- teins that link metabolism and anti-aging effects. RASGRF1 is a related disorders. On light of the prevalent functions of this cata- Ras-guanine nucleotide exchange factor implicated in a variety of bolic process, cells with impaired autophagy are often energeti- physiological processes including learning and memory and glu- cally compromised and present severe problems in maintenance of cose homeostasis. Mice deficient for RasGrf1 display a remarkable cellular homeostasis and in the response to stress. Our group is in- increase in average and most importantly in maximal lifespan. This terested in the study of the changes with age in different auto- is not due to the role of RAS in cancer because tumor-free survival phagic pathways and on the consequences of those changes in the was also enhanced in these animals. Biomarkers of aging indicate aging organism. In addition, using both genetic manipulations and that RasGrf1 knockout mice (RasGrf1-/-) have a lower biological regulated diets, we have been able to prevent the decline in age than controls. Aged RasGrf1-/- displayed better motor coordi- autophagic activity in old rodents and analyze the beneficial effect nation than control mice. Protection against oxidative stress is pre- of this intervention both in cellular homeostasis and in their ener- served in old RasGrf1-/-. Furthermore, Sirtuin expression is in- getic balance. These models would help us evaluate the importance creased in RasGrf1-/- animals. Consistent with this, blood meta- of maintaining proper autophagic function until advanced ages, bolic profiles as revealed by metabolomic analysis resembled those and, on light of the observed cross-talk among autophagic path- observed in calorie-restricted animals. In addition, positron emis- ways, the effect that repairing one autophagic pathway may have sion tomography reveale that in vivo glucose consumption by the on the functioning of the others. heart is not altered by aging in the knockout model, indicating that RasGrf1 deficiency renders tissues resistant to aging. Thus, these observations link Ras signaling to the beneficial effects of caloric restriction on lifespan and suggest that RasGrf1 is an evolutionary conserved gene which could be targeted for the development of therapies with the potential to delay age-related processes.

Work was supported by grants SAF2008-00270; SAF2006-12470; BFU2007-65803/BFI from the Spanish Ministry of Education and Science; ISCIII2006-RED13-027 from the “Red Temática de investigación cooperativa en envejecimiento y fragilidad (RETICEF), and EU Funded COSTB35. D.M. is funded by the Ramon y Cajal Program of the MEC.

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Regulation of proteasome-mediated protein degradation Delaying age-related disease with micronutrients: triage theory during aging Bruce N. Ames Tilman Grune Children’s Hospital Oakland Research Institute Oakland, California, USA University of Hohenheim, Stuttgart, Germany An inexpensive intervention could delay the degenerative dis- Protein oxidation is a permanently ongoing process. Products eases accompanying aging, such as cancer, cardiovascular disease, of protein oxidation might disturb cellular metabolism and, there- cognitive decline, stroke, and immune dysfunction. Most of the fore, oxidized proteins should be degraded effectively. Therefore, world’s population, even in developed countries, has inadequate the bulk of oxidatively modified proteins is selectively recognized intake of one or more micronutrients (~40 essential vitamins, min- and degraded. This process is catalyzed by the proteasomal system erals, fatty acids and amino acids) that a varied and balanced diet and it is general accepted today, that the 20S proteasome is degrad- should provide. My triage theory (PNAS 103, 17589, 2006; AJCN ing the vast amount of oxidized proteins. On the other hand failure 90, 889, 2009) posits that, as a result of recurrent shortages of mi- of an adequate degradation of oxidized proteins is leading to vari- cronutrients during evolution, natural selection developed a meta- ous side effects, including formation of protein aggregates. Such bolic rebalancing response to shortage. The rebalancing favors protein aggregates are major players in the metabolic changes dur- micronutrient-dependent proteins needed for short-term survival ing aging of postmitotic cells. Protein aggregates accumulate dur- while starving those only required for long-term health. Triage ing aging in the lysosomal compartment. Located in the lysosomes theory predicts that the consequence of moderate shortages of even they might be involved in adverse metabolic pathways leading to a single micronutrient, though insufficient to cause overt clinical the apoptosis of senescence cells. Lysosomal accumulated protein symptoms, will impair functions essential for long-term health. aggregates can not be degraded by lysosomal cathepsins. The up- This impairment will result in insidious damage (e.g. increased take of protein aggregates from the cytosol into the endosomal- oxidative damage) that, over time, leads to the acceleration of age- lysosomal-compartment is mediated most likely by macroauto- associated diseases (e.g. increased cancer and brain dysfunction). phagy. Therefore, the understanding of the interaction of the cellu- As people with modest deficiencies have no overt clinical symp- lar protein degradation machinery, including proteasomes and toms, there has been little incentive to correct these deficiencies, lysosomes, are of increasing importance. though this could change if it can be shown that they are resulting in biochemical changes, e.g. chromosome breaks, that are markers of increased risk of age-related diseases, e.g. cancer. The consid- erable experimental and theoretical support for the triage idea will be discussed as will a strategy for determining the optimum level of each micronutrient in humans. The triage theory should help to put micronutrient nutrition on a firm foundation and lead to pre- ventive medicine for age-related diseases.

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Endothelial dysfunction in aged humans is related to oxidative stress and vascular inflammation. A practical approach from traslational research

Leocadio Rodriguez Mañas

Servicio de Geriatría, Hospital Universitario de Getafe; Carretera de Toledo Km. 12.5; 28905-Getafe, Madrid, Spain

Vascular diseases are the first cause of mortality and functional deterio- ration (including dependency) in Western Societies, reaching their highest incidences and prevalences in people older than 65 years old. Together to the classical (Hypertension, Smoking, Diabetes Mellitus, Hypercholesterolemia) and new cardiovascular risk factors (homocystein, CRP, markers of inflam- mation), ageing remains as the principal risk factor. Vascular endothelial dysfunction has been shown in many animal models of ageing and, since one decade, there are experimental data showing that endothelial dysfunction is occurring also during the human aging process. Endothelial dysfunction is now considered a crucial event in the development of many vasculopathies and vascular diseases, including ischemic heart disease, stroke and peripheral artery disease. Moreover, endothelial dysfunction has been shown to be a prognostic factor in cardiovascular diseases. We investigate the underlying mechanisms of human endothelial dysfunction accompanying the ageing process, particularly those related to oxidative stress and inflammation, in the vasculature of subjects between 13-91 years without cardiovascular disease or risk factors. In isolated mesenteric microvessels from these subjects, an age-dependent impairment of the endothelium-dependent relaxations to bradykinin (BK) was observed. In microvessels from subjects younger than 60 years old, BK-induced relaxation was mostly due (80%) to nitric oxide (NO) release while the rest was sensitive to cycloxygenase (COX) blockade. In microvessels from subjects older than 60 years old, this COX-derived vasodilatation was lost but a COX-derived vasoconstriction occurred, al- though the expression of both COX-1 and COX-2 was not altered. Evidence for age-related vascular oxidant and inflammatory environment was ob- served, which could be related to the development of endothelial dysfunc- tion. It must be underscored that these findings are remarkably similar to those observed in young people with diabetes or hypertension. These data raised some questions that we are now approaching: 1) If the effect of ageing on vascular endothelium in humans is very close to that produced by diabetes or hypertension, do these classical risk factors induce vascular disease in old people by adding new damage to endothelial dysfunction or there are other mechanisms involved? 2) These factors involved in producing endothelial dysfunction in selected populations, are also present in studies made on more representative cohorts of old people. POSTERS

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Effect of cigarette smoke on redox regulation in The effect of sprint training on oxidative stress chronic obstructive pulmonary disease and antioxidants

Amit R. Agarwal, Jerome V. Garcia, Harsh Sancheti, and Suhana Aiman1, Nor Anita MMN2 and Zaiton Z2 Enrique Cadenas 1Faculty of Sports Science and Recreation, Universiti Teknologi MARA, 40450 2 Department of Pharmacology and Pharmaceutical Sciences, University of Shah Alam, Selangor, Malaysia; Department of Physiology, Universiti Southern California, Los Angeles, California 90089, USA Kebangsaan Malaysia Medical Center, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia

Chronic obstructive pulmonary disease (COPD) is a persistent Numerous studies have shown that aerobic training reduces obstruction of airways in the lungs due to the narrowing of the pas- oxidative stress and improves antioxidant status. However, the in- sage ways. Cigarette smoking is responsible for approximately 80- formation on anaerobic training on oxidative stress and antioxidant 90% of COPD related deaths. Cigarette smoke contains over 4000 enzymes were scarce. The purpose of this study was to determine chemicals, including carcinogens, strong electrophiles and other the effect of sprint training on MDA and antioxidant enzyme ac- toxins. The current study was undertaken to determine the role of tivities. Sixteen male subjects participated in this study. They ran- cigarette smoke in the development of mitochondrial dysfunction domly assigned to training group and control group. Training due to oxidative stress. Rat lung epithelial cell cultures (RLE-6TN group engaged in sprint training for eight weeks (three times per cell line) were used to study the effects of cigarette smoke extract week). Venous blood samples were taken before and after training. (CSE), individual constituents of CSE like acrolein and nitric oxide Plasma malondialdehyde (MDA) was measured as oxidative stress on cell viability and redox status of the cell. We have adapted a maker. Antioxidant enzymes analyzed were superoxide dismutase method to prepare cigarette smoke extract in our laboratory by (SOD), glutathione peroxidase (GPX) and catalase (CAT). There smoking 4 cigarettes in 20 ml of serum free F-12 media. The was no significant difference in MDA was observed after the sprint smoke extract was characterized using Agilent 8453 UV-Visible training. Compared with the control group, SOD level remains un- Spectrophotometer with a consistent peak at 361 nm. We exposed changed following training. While there were significant im- cells to 0, 0.5%, 1%, 2.5%, and 5% of CSE for varying periods of provement of GPX and CAT activity after the training. In conclu- time. Our data indicate that treatment of RLE-6TN cells with doses sion, this study demonstrates that sprint training does not reduce less than 2.5% CSE for 4 h lead to an increase in the NADPH/ the oxidative stress but has enhanced antioxidant defences. NADP+ ratio with no change in the NADH/NAD+ ratio as meas- ured by HPLC. At 24 h, a decrease in both NADPH/NADP+ and NADH/NAD+ ratios, indicate of an initial compensatory response to CSE challenge.

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Zinc and the modulation of transcription factors STAT1 and STAT3 in fetal rat brain and neuronal cells Scavenging of superoxide in biological contexts: the role of ubiquinone, ubiquinol and SOD mimics Lucila Aimo, Suangsuda Supasai, Yo Omata,

Gerardo G. Mackenzie, and Patricia I. Oteiza Robert F. Anderson1, Andrej Maroz1, Sujata S. Shinde1, Departments of Nutrition and Environmental Toxicology, Robin A.J. Smith2, and Michael P. Murphy3 University of California Davis, Davis, CA 95616, USA

We recently observed that zinc is required for cell proliferation and 1Department of Chemistry, The University of Auckland, Private Bag 92019, prevention of neuronal apoptosis. The STAT pathway is involved in Auckland 1142, New Zealand, 2Department of Chemistry, Otago University, Box 3 the regulation of neurogenesis, neuronal differentiation and survival. 56, Dunedin 9054, New Zealand, Medical Research Council Dunn Human Nutrition Unit, Cambridge CB2 0XY, UK This work investigated the effects of low zinc availability on the modulation of transcription factors STAT 1 and STAT3 (STAT1/3) in Endogenous ubiquinones (UQ) are essential electron carriers in neuronal cell cultures and animal models of zinc deficiency. Marginal the mitochondrial electron transport chain, and the two-electron zinc diets (MZD) fed to rats throughout gestation caused a deregula- reduced ubiquinol form (UQH2) is a chain-breaking antioxidant tion of STAT1/3 modulation in gestation day 18.5 (GD 18.5) fetal which is active against lipid peroxidation within mitochondria. Su- brains. STAT activation (STAT-DNA binding) in total fetal brain .- peroxide, O2 , is the proximal radical produced during mitochon- homogenates was lower (24%) and higher (44%), respectively, in drial oxidative stress and its reactions with endogenous UQ/UQH2 MZD compared to control animals. In nuclear fractions from fetal and the exogenous UQ, such as Idebenone and MitoQuinone, de- brain, STAT-DNA binding was markedly lower for STAT1 (80%) and pend on the environment where interaction takes place. We have STAT3 (85%) in MZD compared to controls. In human neuroblastoma used pulse radiolysis to study such reactions in water, and in sol- IMR-32 cells, the incubation in zinc deficient media for 24 h, leads to vent systems mimicking the surface and core of the biological an increased total STAT1 and STAT3 activation. However, similarly .- membranes where UQ is preferentially located in vivo. While O2 to that observed in GD18.5 fetal brain, zinc deficiency caused a 50% is scavenged very rapidly by UQ in all solvents (108-109 M-1 s-1), reduction in nuclear STAT1- and STAT3-DNA binding. This was as- .- . 5 both O2 and the hydroperoxyl radical (HOO ) react slowly (<<10 -1 -1 sociated with a decreased transcription of STAT1/3-driven reporter M s ) with UQH2. In contrast, UQH2 react rapidly with other genes in zinc deficient cells compared to controls. To investigate the oxygen and carbon-centred radicals, indicating that the antioxidant relevance of STAT1 and STAT3 in IMR-32 cell proliferation and sur- role of UQH2 is mainly in preventing lipid peroxidation, not .- . vival, cells were incubated in the presence of inhibitors of STAT1/3 quenching O2 and HOO . In addition, both the mitochondria- (AG-490) or STAT3 (Stattic V) activation. Cell viability was signifi- targeted UQ, MitoQ, and the Mn(II) pentaazamacrocyclic, super- cantly lower after 24h incubation in the presence of the inhibitors oxide dismutase-mimic compound, mSOD1, are under study for (50% decrease at 20 uM AG490 and 1.3
M Stattic V). This was in their possible use as therapies to decrease oxidative damage in hu- part due to apoptotic cell death as evidenced by caspase-3 activation man pathologies. The appended positively-charged phosphonium and DNA fragmentation. In summary, zinc seems to be necessary for moiety induces ca. 1000-fold uptake of these compounds into mi- the physiological regulation of STAT1/3 in the nervous system. Zinc tochondria over the cytosol. Pulse radiolysis studies on the mecha- deficiency decreases nuclear STAT1/3 activation and dependent gene nisms of action of these compounds will also be discussed. transcription. An impaired STAT1/3 modulation could contribute to zinc deficiency-induced neuronal apoptosis. Supported by UC Davis and NIH (HD01743)

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Tissue engineering of peri-implant space with stem/progenitor Ferritin degradation and increase of labile iron pool is cells: a novel approach to improve implant-level oxygenation mediated by JNK1 in diallyl trisulfide treated prostate cancer cells PC-3 Mirela Anghelina, Omar I. Butt, Anna Bratasz, Leni Moldovan, 1Jedrzej Antosiewicz, 1Alicja Sielicka-Dudzin, Periannan Kuppusamy, and Nicanor I. Moldovan 1Andzelika Borkowska, 1Magorzata Halon,

Anna Herman-Antosiewicz, and Michal Wozniak Davis Heart and Lung Research Institute, Departments of Internal Medicine and 1Department of Bioenergetics and Physiology of Exercise, 2Department of Biomedical Engineering, Ohio State University, Columbus, OH, 43210 Medical Chemistry, 3Medical University of Gdansk, Department of Molecular Biology University of Gdansk Poland

Clinical use of implanted medical devices such as biosensors In our previous study, we demonstrated that JNK signaling path- and drug delivery devices is limited by the cellular foreign body way regulates ferritin degradation and labile iron pool in prostate can- reaction and consecutive fibrous encapsulation. Traditionally, at- cer cells treated with diallyl trisulfide (DATS). In the present work we tempts have been made to mitigate the impact of these processes extended this study to determine which of c-jun kinase is responsible by preventing their development, with little success so far. Based for ferritin degradation and what the role of iron in DATS-induced cell on our advances in the understanding of the coupling between an- death is. Moreover, we hypothesized that JNK1, Itch ligase axis will giogenesis and fibrogenesis in the subcutaneous space, we recently be responsible for ferritin degradation. PC-3 prostate cancer cells were proposed a different approach: to divert the normal 'wound- used in this study. In order to prove if kinases JNK participate in fer- healing' like process from fibrosis to a more stable neovasculariza- ritin degradation PC-3 cells were transfected with dominant negative tion, using stem/progenitor cells (SPC)-based tissue engineering. mutant of JNK1 or JNK2 and then treated with DATS. We observed Here we will illustrate this concept with our results obtained by us- that DATS did not induced ferritin degradation in cell transfected with ing mouse bone marrow derived c-kit+/sca-1+ SPC embedded in JNK1-DN. On the other hand DATS induced ferritin degradation in Matrigel, implanted in recipients near a nanoporous filter-limited cells transfected with JNK2-DN. Moreover DATS induced neither sensor, which continuously monitors device-level oxygen concen- ROS formation nor increase LIP in JNK1 transfected cells however trations by in vivo EPR oximetry. We show that the presence of such increase was observed in vector or in JNK2- transfected cells. In exogenous SPC significantly increases implant oxygenation for at order to established the role of iron in DATS induced cell death the least 10 weeks, coincident with morphological and immuno- cells were pretreated with iron chelators but neither DFO nor tujapicin histological markers of abundant neovascularization. We will also had any effect on cell death. In addition cell preloaded with iron com- show the current progress in our laboratory to develop novel engi- pound hemine had the same sensitivity to DATS. Moreover we ob- neered implant-tissue interfaces using SPC-containing biomateri- served that DATS increase activating phosphorylation of E3 ligaze als. Collectively, these advances create new bioengineering solu- Itch. The phosphorylation was blocked by JNK inhibitor SP600125. tions to the impact of foreign body reaction on the functioning of Cell transfected with JNK2-DN but not with JNK1-DN showed lower implants, including those dependent on oxygen availability. level of Itch phosphorylation. Consistently cells transfected with inac- tive form of Itch showed DATS induced ferritin degradation and ROS formation but were much more resistant to DATS induced cell death. In conclusion our data suggest that JNK1 regulates ferritin degradation LIP level and ROS formation in prostate cancer cell but it is not re- lated to Itch ligase activity.

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Effect of amifostine, a radiation-protecting drug, on oxygen TGRL lipolysis induced gene expression in concentration of normal tissue measured by EPR oximetry human aortic endothelial cells

Kazunori Anzai, Atsuko Matsumoto, Ken-ichiro Matsumoto, 1,2Hnin H. Aung, 3Michael Lame, 4K. Gohil, 2Dennis W. Wilson, Sushma Manda, Megumi Ueno, and Ikuo Nakanishi and 1John C. Rutledge

1 Research Center for Charged Particle Therapy, National Institute of Department of Internal Medicine, Division of Endocrinology, Clinical Nutrition, and Vascular Medicine, University of California, Davis, CA 95616; Radiological Sciences, Chiba 263-8555, Japan 2Department of Pathology, Microbiology and Immunology, University of California, Davis, CA 95616; 3Department of Molecular Biosciences, University of California, Davis, CA 95616; 4Division of Pulmonary and Critical Care For radiotherapy of cancer, oxygen concentration in the tumor Medicine, Department of Internal Medicine, University of California, Davis, CA tissues greatly affects the outcome of the therapy. The harmful ef- fects of radiation on normal tissues are also modified by the oxy- The endothelium plays an important role in the control of the gen concentration. Therefore, it is important to measure tissue vascular system. Elevation of triglyceride-rich lipoproteins oxygen concentrations in vivo. EPR oximetry is one of useful (TGRL) is a known cardiovascular risk factor. In the present study techniques for measuring oxygen concentration in vivo. In the pre- we investigated the effects of TGRL lipolysis on human aortic en- sent study, we examined the effect of amifostine, a powerful and dothelial cells (HAEC) focusing on intracellular pathways and clinically approved radiation-protecting drug, on the oxygen con- gene expression. Our previous studies indicate that TGRL lipolysis centration of a normal tissue by EPR oximetry using lithium octa- products in high concentrations injure HAEC. We hypothesized n-butoxy-substituted naphthalocyanine (LiNc-BuO) as a radical 1 that TGRL lipolysis products activate activating transcription fac- probe. LiNc-BuO was synthesized according to the literature . tor 3 through the Jun N-terminal kinase (JNK) signaling pathway The line width of LiNc-BuO probe in vitro linearly responded to and contribute to vascular inflammation in pathogenesis of athero- the oxygen concentration that was changed by using mixture of sclerosis affecting the expression of multiple pro-inflammatory cy- various concentrations of oxygen and nitrogen. The micro crystals tokines. To test this hypothesis, we incubated HAEC with either of LiNc-BuO was inoculated in the hind leg of mice and the EPR TGRL or TGRL lipolysis products for 3 hrs. RNA was isolated spectra of the LiNc-BuO were measured with a surface coil at- from HAEC after a 3 hr exposure to TGRL or TGRL lipolysis tached to an L-band EPR spectrometer. When amifostine, a radia- products and subjected to microarray analysis with subsequent tion-protecting drug, was administered intraperitoneally or intrave- verification using quantitative real-time PCR. Western blotting and nously to the mice, the line width of the EPR spectra of the probe immunofluorescence were used to study intracellular signaling decreased more than 60 min and then recovered to a normal level. pathways. Our results indicate that TGRL lIpolysis products initi- Since radiosensitivity of cells is dependent on the oxygen concen- ate a stress response in HAEC that has the molecular signature of tration and hypoxic cells are radio-resistant, the present findings inflammation with the induction of activating transcription factor 3 suggest that the lowering of oxygen concentration of normal tis- down stream of the JNK pathway. These results support the in- sues might contribute a part for the radiation-protection by ami- volvement of TGRL lipolysis products in endothelial dysfunction fostine. via induction of pro-inflammatory genes.

1 M. Afeworki et al., Free Radic. Biol. Med. 25, 72-78 (1998)

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Fra-2 mediates oxygen-sensitive induction of transforming MicroRNA biogenesis complex is dysregulated in growth factor  in cardiac fibroblasts ischemic cutaneous wounds

Ali Azad, Savita Khanna, Rebecca Schnitt, Sashwati Roy, J. Banerjee, S-R A. Hussain, S. Biswas, Y-C Chan, *J. Jiang, Hidenori Ichijo, and Chandan K. Sen *T. Schmittgen, S. Khanna, S. Roy, and C.K Sen

Davis Heart and Lung Research Institute, Department of Surgery, Comprehensive Wound Center, Department of Surgery, The Ohio State University Medical Center, Columbus, OH 43210, USA *College of Pharmacy. The Ohio State University, Columbus, OH 43210

In the ischemia-reperfused heart, transforming growth factor Background. Drosha, a RNA polymerase III, enzyme, and an asso- beta (TGF) proteins trigger the differentiation of cardiac fibro- ciated protein called DGCR8, are the major components of a multi- blasts (CF) to myofibroblasts contributing to fibrosis. Previously, subunit protein complex termed microprocessor complex. This com- we have reported that perceived hyperoxia (Circ Res 92:264-71), a plex plays a critical role in the biogenesis pathway, for processing mi- sub-lethal reoxygenation shock during IR, induces differentiation croRNA precursor RNAs, converting primary transcripts (pri-miRNA) of CF to myofibroblasts at the infarct site. In this study, we sought to pre-miRNAs. Using conditional epidermal specific deletion of to characterize the molecular mechanisms responsible for the O2- Dicer, an essential enzyme for the biogenesis of mature miRNA, we sensitive transcriptional induction of TGF in adult murine ven- have recently observed that impaired epithelialization is one of the ma- tricular CF and to test the significance of such findings in the in- jor causes of attenuated dermal wound healing. Objective. To deter- farcted myocardium in vivo using laser capture microdissection mine the role of miR biogenesis pathway in impairment of epitheliali- (LCM). We observed that among the AP_1 family of proteins spe- zation in ischemic wounds. Results. Among miR biogenesis complex, cifically Fra-2 mediates oxygen-induced TGF transcription in DGCR8 was significantly downregulated in ischemic wounds on day 3 adult cardiac fibroblasts. Spatially resolved infarct and non-infarct compared to non-ischemic wounds. In order to assess the role of tissues were collected at post-IR using LCM. All three isoforms of DGCR8 in the epithelialization process, we used RNA interference to TGF (TGF1, 2 and 3) were significantly induced in the myo- deplete DGCR8 in immortalized human keratinocytes. Knockdown of cyte-silent CF-rich peri-infarct heart tissue as well as in CF ex- DGCR8 invoked a pronounced reduction in keratinocyte migration and posed to hyperoxic insult during reperfusion following ischemia. proliferation. Furthermore, miRNA profiling showed that the expres- Reporter studies demonstrated that TGF transcription is hyper- sion of 19 miRNAs was downregulated relative to non-ischemic oxia inducible. Deletion of any one or both of the AP-1 binding wound while four miRNAs including miR-210 were upregulated. Re- sites in the TGF reporter construct resulted in loss of O2- cent studies show that aquaporin 3 (AQP3) serves as facilitator of sensitivity demonstrating that AP-1 confers O2-sensitivity to TGF keratinocyte migration and proliferation. We observed that compared transcription. Fra-2 and Ask-1 were identified as key mediators of to non-ischemic wounds, ischemic wounds showed reduced expression AP-1 dependent O2-sensitive TGF transcription. Knock down of of AQP3 during the early epithelialization phase of healing. Knock- Fra-2 significantly blunted expression of TGF1 in CF. Knock down of DGCR8 in human keratinocyte caused marked reduction in down of Ask-1 blunted hyperoxia-induced expression of Fra-2 the expression of AQP3. Studies are currently in progress to determine gene expression and nuclear localization. Collectively, these ob- mechanisms of AQP3 downregulation by miR biogenesis complex. servations point towards a central role of Ask-1 and Fra-2 in O2- Conclusions. This is the first study demonstrating that DGCR8, a inducible AP-1 activation and induction of TGF. Taken together, member of miR biogenesis complex, is downregulated in ischemic identification of Fra-2 as an O2-sensitive transcriptional regulator wounds. Such downregulation results in attenuated keratinocyte migra- of inducible TGF expression, positions Fra-2 as an important tion and proliferation conceivably via decreased AQP3 expression. player in reoxygenation-induced fibrosis. Supported by NIH Grant RO1-HL-073087 to CKS Supported by NIH RO1 GM-069589 and GM-077185.

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Dicer ablation in adult hearts induces oxidative stress, mitochondrial dysfunction and casues rapid loss of cardiac The cytotoxic effect of indigenously prepared function inorganic coordination complex

Jaideep Banerjee, Sashwati Roy, Surya C. Gnyawali, * *# † Savita Khanna, and Chandan K. Sen P. Banerjee , S. Banerjee , P. Majumder , S. Bhattacharya††, and S. Mazumder** Department of Surgery, The Ohio State University, Columbus, OH

Objective. Dicer, an RNAse III endonuclease, plays a key role *Department of Biochemistry, University of Calcutta, in miRNA biogenesis. The short-term effects of dicer deficiency †Department of Chemistry, Jadavpur University on the adult heart remain to be elucidated. We hypothesize that change in the level of crucial miRNAs upon heart-specific The cytotoxic effect of indigenously prepared inorganic coor- conditional dicer knock-out in adult animals result in rapid loss of cardiac function caused by mitochondrial dysfunction and oxida- dination complex was studied in cervical cancer cell line & ovarian tive stress. Methods. Double-transgenic (Myh6-cre/Esr1-Dicerfl/fl) cancer cell line & the IC 50 were found to be 75
M and 100
M mice were generated. At 8 weeks of age, vehicle or tamoxifen (20 respectively. The mechanism of cell killing is under study. Confo- mg/kg) was injected daily for five days. Heart function was deter- cal microscopic study of Chromosomal condensation by DAPI & mined using gated cardiac MRI (11.7T) and echocardiography. flow cytometric assay of ROS generation by DCFDA have been Myocardial miR profiling was performed using a bead array sys- studied at present for evaluating the mechanism of cytotoxicity of tem. Mitochondrial function was assessed by measurement of res- piratory control ratio (RCR) and membrane depolarization. Tissue inorganic coordination complex obtained from collaborator’s labo- redox status was assessed by EPR studies using nitroxide radical ratory. probe. Results. Significant (p<0.05, n=3) change in the levels of a number of miRNAs including miR-1, miR-133, miR-15b was ob- served. Decrease in ejection fraction, stroke volume and cardiac output was significant (p<0.05, n=5) in Dicer-/- compared to Dicer +/+ mice. Isolated mitochondria from Dicer-/- mice cardiac tissue showed significant (p<0.05, n=4) decrease in RCR. Predicted miR-15b targets regulate mitochondrial function. miR-15b over expressed HL-1 cells showed a loss (p<0.05, n=3) of mitochondrial membrane potential as assessed by JC-1 flow cytometry and a de- crease (p<0.05, n=4) in TMRM/PMPI ratio. Such compromise in mitochondrial function was associated with increased tissue oxida- tive stress in Dicer-/- mice, assessed by a significant increase in TBARS and GSSG/GSH ratio (p<0.05, n=4). EPR shows an in- crease (p<0.05, n=4) in the rate of decay of the nitroxide radical indicating a lower tissue oxygen status. Conclusion. This study provides first evidence demonstrating mitochondrial dysfunction and oxidative stress in dicer depleted adult mice heart which is fol- lowed by hypertrophy and acute loss of cardiac functions.

Acknowledgements NIH RO1-HL073087 to C.K.S.

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Enantioselective synthesis and antioxidant activity of Triptolide alleviates severe liver injury in mice 3-(3,4-dihydroxyphenyl)-glyceric acid – basic monomeric by attenuating the oxidative stress moiety of a biologically active polyether from Symphytum asperum and S. caucasicum

1 1 1 Vakhtang Barbakadze , Maia Merlani , Lela Amiranashvili , Xiaofeng Bao , Yan Lu , and Pingping Shen Lali Gogilashvili1, Karen Mulkijanyan1, Elina Yannakopoulou2, Kyriakos Papadopoulos2, and Bezhan Chankvetadze3 State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, 210093, PR China 1Kutateladze Institute of Pharmacochemistry, 36 P. Sarajishvili str., 0159 Tbilisi, Georgia; 2NCSR ‘Demokritos’, Institute of Physical Chemistry, Triptolide, a diterpene triepoxide, is one of the major compo- 15310 Athens, Greece; 3Department of Physical and Analytical Chemistry and nents of most functional extracts of Tripterygium wilfordii Hook Molecular Recognition and Separation Science Laboratory, School of Exact and Natural Sciences, Tbilisi State University, Chavchavadze Ave 3, 0179 Tbilisi, and is known to have various biological effects, including immu- Georgia nosuppressive, anti-inflammatory, anti-tumor and anti-fertility functions. We studied the inhibitory effect of triptolide on endo- The racemic and enantioselective synthesis of a novel glyceric toxemia (ETM)-induced liver damage in mice model. Hepatic acid derivative, namely, 2,3-dihydroxy-3-(3,4-dihydroxyphenyl)- damage was induced in C57BL/6 mice by lipopolysaccharide propionic acid was carried out and their antioxidant activities was (LPS) and D-galactosamine (D-GalN). Pretreatment with triptolide determined. The virtually pure enantiomers, (+)-(2R,3S)-2,3- decreased the ROS levels, mortality rate and liver injury after dihydroxy-3-(3,4-dihydroxyphenyl)-propionic acid and (–)-(2S,3 LPS/D-GalN injection. We utilized comprehensive proteomics to R)-2,3-dihydroxy-3-(3,4-dihydroxyphenyl)-propionic acid were identify alterations in liver protein expression during pretreatment synthesized for the first time via Sharpless asymmetric dihydroxy- with triptolide and pretreatment with N-acetylcysteine (NAC) after lation of trans-caffeic acid derivatives using the enantiocomple- mentary catalysts, [(DH LPS/D-GalN injection, Forty four proteins were found to be related 1,4-bis(9-O-dihydroquinine)-phthalazine Q) -PHAL] and 1,4-bis(9-O-dihydroquinidine)-phthalazine [(DH to oxidative stress, mitochondria, metabolism and signal transduc- 2 QD)2-PHAL], respectively. The determination of enantiomeric pu- tion. Furthermore; Both triptolide and NAC inhibited activation of rity of the novel chiral glyceric acid derivatives was performed by JNK and p38, phosphorylation of I B and activation of NF- B. high-performance liquid chromatographic (HPLC) techniques on These results demonstrated that triptolide inhibits the activation of the stage of their alkylated precursors. It was shown that the novel JNK and p38 by decreasing ROS levels, which in turn inhibits the racemic glyceric acid derivative as well as its enantiomeric pure hepatic injury. In addition, triptolide prolonged ERK activation, derivatives exhibit strong antioxidant activities against reactive indicating that ERK had a protective role in LPS/D-GalN-induced oxygen species, such as hypochlorite or free radicals such as liver damage and triptolide could inhibit the hepatic injury by pro- DPPH. No significant differences have been noted regarding the longing ERK activation. On the contrary, NAC had no effect on antioxidant activities of pure enantiomers and the racemic mono- ERK activity. These results show that triptolide could effectively mer. Their antioxidant activity is about 40-fold higher than that of cure severe liver injury by reducing the oxidative stress in the liver, the corresponding natural polyether and 3-fold higher of trans- thus suggesting its use as an inducer of antioxidative defense sys- caffeic acid itself. Such pronounced antioxidant properties give appropriate background for further deep investigation of the bio- tem for treating severe liver injury. logical activity of this phenolic compound, and it seems promising to use it either as food additive or medicinal preparation for treat- ment of oxidative disorder-related diseases.

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Allantoin- and pyrrolizidine alkaloids-free wound healing Dynamic regulation of signaling at regions of cell-cell contact by endoplasmic reticulum-bound protein-tyrosine phosphatase 1B compositions from Caucasian species of comfrey (Symphytum L.) Ahmed Bettaieb1, Ali Kinkhabwala2, Carsten Schultz3, 4 2 1 Vakhtang Barbakadze, Karen Mulkijanyan, Lali Gogilashvili, Benjamin Neel , Philippe Bastiaens , and Fawaz Haj Lela Amiranashvili, Maia Merlani, Zhana Novikova, 1 Department of Nutrition, University of California, Davis, CA 95616, U.S.A. [email protected], 2 Max Planck Institute of Molecular Physiology, Marine Sulakvelidze Otto-Hahn-Str. 11, 44227 Dortmund, Germany, 3 European Molecular Biology Laboratory, Heidelberg, Germany, 4 Ontario Cancer Institute, Toronto, Ontario, M5G 1L7, Canada Kutateladze Institute of Pharmacochemistry, 36 P. Sarajishvili str., 0159 Tbilisi, Georgia Tyrosine phosphorylation is critical for signal transduction and maintaining homeostasis. This process is mediated through the op- Extracts from the Caucasian species of comfrey – Symphytum posing actions of protein-tyrosine kinases and protein-tyrosine asperum and S.caucasicum have been used in folk medicine in the phosphatases (PTPs). Protein-tyrosine phosphatase 1B (PTP1B) is treatment of fractures and wounds. Extracts contain allantoin, claimed to be a cell proliferation-stimulating agent responsible for a widely expressed PTP that is localized on intracellular mem- the wound-healing properties of Symphytum, and hepatotoxic pyr- branes via a hydrophobic C-terminal targeting sequence. A role of rolizidine alkaloids which strongly restrict internal use of comfrey PTP1B in the regulation of many cellular functions has been sug- extracts. In the present study we obtained allantoin- and pyrroliz- gested, including growth factors, integrin and cadherin signaling. idine alkaloids-free composition containing crude polysaccharides PTP1B activity can be regulated through a variety of mechanisms and novel biopolymer from S. asperum and S.caucasicum roots – namely cellular localization, oxidation and sumoylation. Previ- poly[3-(3,4-dihydroxyphenyl)glyceric acid] (PDPGA), and at- tempted to appraise its pharmacological properties in vitro (an- ously, we and others showed that PTP1B dephosphorylates RTKs ticomplementary and antioxidant) assays and in vivo (mouse exci- such as the epidermal growth factor receptor (EGFR) and insulin sional wound and skin burn models). In contrast with polysaccha- receptor (IR) after receptor endocytosis, as they transit past the en- rides PDPGA exhibited marked antioxidant and anticomplemen- doplasmic reticulum (ER). However, other studies suggest that tary activity, Besides, ointment, containing 2.5% crude polysac- PTP1B can access some plasma membrane-bound substrates. To charides and PDPGA (C) was found to have pronounced wound delineate the mechanism of PTP1B interaction with such substra- healing properties, similar to 2.5% allantoin ointment (A) in exci- tes, we utilized advanced quantitative cellular imaging such as sional wound model. Secondly, we attempted to evaluate pharma- cological efficacy of ointment (C) in comparison with ointment photoactivation and fluorescence lifetime imaging microscopy in (A) in skin burn model. Ointment (C) appeared to be approxi- combination with substrate-trapping and mathematical modeling mately fourfold active than ointment (A) and twice as active as approaches. In addition, we developed activity sensors to image combined ointment (CA) containing 1.25% of high-molecular frac- regulation of PTP1B activation in live cells. We report that the ER tion from comfrey roots and 1.25% of allantoin when treatment of extends outwards to lie in close proximity to the PM only at appar- animals began immediately after burn induction. Significantly ently specialized regions of cell-cell contact, enabling PTP1B to lower (p < 0.01) values of burn area in (C)- than in (A)- and (CA)- dephosphorylate key substrates at these sites. Our studies show that treated animals were observed since day 2 till the end of the ex- periment. The obtained results allow to suggest that the wound ER-anchored PTP1B plays a dynamic role in regulating signaling healing effect of the investigated compositions results from syner- at regions of cell-cell contact and identify PM-proximal sub- gistic action of its constituents in the second phase of wound heal- regions of the ER as an important site for regulating cellular sig- ing, the inflammatory response. naling.

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Light distribution on the human retina over extended periods: Endothelial and inducible NO synthases expression in the Implications for photooxidative damage lungs of the developing mouse after prenatal administration of

vitamin A R.A. Bone and J.C. Gibert 1 1 1 Department of Physics, Florida International University, Miami, FL 33199 Raquel Carvalho , Maria de Lurdes Pinto , Ana Cláudia Coelho , Carlos Gonçalves2, and Vasco António Bairos2

Photoxidative damage has been implicated in the etiology of 1Department of Veterinary Sciences and Centre for Studies on Agricultural and age-related macular degeneration (AMD). The purpose of our Veterinary Sciences, University of Trás-os-Montes e Alto Douro, 5001-801 Vila study was to measure cumulative light distribution on the human Real, Portugal; 2 Institute of Histology and Embryology, Faculty of Medicine, University of Coimbra, 3004-504 Coimbra, Portugal retina over extended periods. Our hypothesis was that such distri- butions would tend to peak in the macula where AMD damage is Nitric oxide (NO) is a potent vasodilator shown to play a critical most pronounced. Using a head-mounted eye-tracker, we captured role in various functions. Recent studies relate the defective lung vas- the subject's field of view with a video camera (60 frames/s), su- cular development seen in the lungs of animals with respiratory dis- perimposed the gaze position on each frame, and continuously re- tress syndrome to eNOS deficiency. Inducible NOS is highly ex- corded pupil size. Of fifteen subjects (ages 18-65) who partici- pressed in late stages of lung fetal development. Vitamin A is crucial pated, five, who were familiar with the study, were assigned to a for normal cellular differentiation during fetal development. Our pre- control group; ten, who were naïve, formed a test group. In phase vious studies showed that maternal administration of vitamin A results 1, subjects viewed a repeating sequence of photographic images in an enhancement of lung fetal organogenesis, and an increase in vas- projected on a screen. In phase 2, they were seated in front of a cular endothelial growth factor (VEGF) pulmonary and plasma levels. monitor and performed arbitrary computer tasks, and in phase 3, A decrease of eNOS activity, associated to a decrease in VEGF ex- they moved freely around the lab and exterior of the building. The pression was reported by other authors. It has also been suggested that control group was specifically instructed to gaze at bright features NO up-regulates VEGF expression. In order to better understand the in the field of view and, in a second test, at dark features. The rest modulating action exerted by the vitamin A upon pulmonary eNOS of the participants were allowed to gaze freely. Based on the sub- and iNOS we have conducted an in vivo study in fetal and neonatal ject’s gaze position and pupil size, the relative cumulative light mice. The lungs were collected daily from the 15th gestational day till distribution over 5 to 15 min periods was calculated for a 20o(H) the 3th day of life and processed for routine immunohistochemistry. At 14o(V) area of the retina centered on the fovea. The data were the 16th day of gestation there is a markedly increase of eNOS presented as a plot of relative intensity of light versus retinal posi- expression, which does not occur in any other day under study. tion. For the control group, cumulative retinal light distributions Inversely, at this gestaional day, a decrease in iNOS expression is peaked and dipped in the fovea for the tests during which subjects observed. Prenatal administration of vitamin A did not alter signicantly were instructed to gaze at bright or dark features respectively in the eNOS or iNOS expression, however, there is a trend for an increase of field of view. The light distributions obtained from the test group eNOS expression in the lungs of animals subjected to vitamin A in phase 2, but not in phases 1 and 3, showed a tendency to peak in supplementation. Regarding eNOS expression it is important to notice the macula. In conclusion, our data so far do not confirm our hy- that at the 16th day of gestation, the lung undergoes marked vasculo- pothesis that the macula receives more light than the periphery un- genesis and angiogenesis, in which eNOS seems to play an essential der general viewing conditions. However, the specific task of role. The fact that vitamin A further stimulates eNOS expression working in front of a computer monitor exposed the retina to a without altering iNOS could be important to lung’s vascular develop- higher illuminance in the macular region. This could result in in- ment, suggesting a bennefical role of vitamin A in lung maturation by creased photooxidative damage with implications for development directly regulating angiogenic factors, such as VEGF and eNOS of AMD. expression.

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Evaluation of the in vivo photoprotective effect and the in vitro Antioxidant enzymes gender differences during mice fetal and antioxidant activity of topical formulations containing neonatal lung development quercetin 1 1 Raquel Carvalho, Maria de Lurdes Pinto , Ana Cláudia Coelho , Rubia Casagrande1,2; Sandra R. Georgetti1,2; Dario Santos2, Francisco Peixoto2, Carlos Gonçalves3, and Marcela M. Baracat1,2, Waldiceu A. Verri Jr3; Vasco António Bairos3 Fabiana T.M.C. Vicentini2, and Maria José Vieira Fonseca2

1Department of Veterinary Sciences and Centre for Studies on Agricultural and 1Department of Pharmaceutical Science, State University of Londrina, Lon- Veterinary Sciences, University of Trás-os-Montes e Alto Douro, 5001-801 Vila drina, PR; 2Department of Pharmaceutical Science, Faculty of Pharmaceutical Real, Portugal; 2Department of Biology, University of Trás-os-Montes e Alto Sciences of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP; Douro, 5001-801 Vila Real, Portugal; 3Institute of Histology and Embryology, 3Department of Pathology, State University of Londrina, Londrina, PR; Faculty of Medicine, University of Coimbra, 3004-504 Coimbra, Portugal The skin is continuously exposed to a combination of environ- Sexual dimorphism of the adult lung has recently been described in rodents. Female mice and rats have smaller, and per body mass, mental insults including UV radiation and high oxygen concentra- more alveoli and alveolar surface area than males of their respective tion, which constantly jeopardizes the integrity of cellular oxidi- species. These differences are regulated by estrogen receptors and zable structures of the skin. In this regard, antioxidants from natu- become apparent by the time of puberty. In humans, respiratory func- ral products such as quercetin (QC) present novel possibilities for tion of very prematurely born infants is influenced by sex, being the treatment and prevention of oxidative stress-mediated skin dis- characterized by a female advantage. Since the surfactant system and eases. Thus, the development of topical formulations added QC as the antioxidant enzyme system of the fetal lung have chronologically well as the correct evaluation of their in vitro and in vivo antioxi- similar developmental patterns and given the need of prolonged ex- dant activities would be very helpful to choose the most adequate posure to oxygen in male premature infants, we investigated if a sex one. Therefore, in the present study it was evaluated in vitro anti- difference would be present in antioxidant enzymes of the developing oxidant activities of different topical formulations (non-ionic mice lung. Mice lungs were collected daily from the 15th until the emulsion with high lipid content and anionic emulsion with low 3th day of life. Catalase, glutathione peroxidase, and superoxide dis- lipid content) added QC and the in vivo photoprotective effect of mutase were measured by the polarographic method and spectrome- these formulations. In vitro antioxidant activity was evaluated by try, respectively. Catalase and glutathione peroxidase are more abun- dant in the developing lung, whereas superoxide dismutase is unno- ability to inhibit lipid peroxidation, H-donor capability and scav- ticeable in all the days studied. Catalase and glutathione peroxidase enging hydroxyl radical effect. The skin oxidative stress was in- are higher near the time of birth and the first days of life. Surpris- duced in hairless mice by UVB irradiation and the protective effect ingly, a higher expression of catalase and glutathione peroxidase is of QC was determinate by cutaneous proteinase secretion/activity registered in males. Regarding catalase, the difference between males using sodium dodecyl sulphate polyacrylamide gel electrophoresis and females reached statistical significance at the 16th gestational substrate-embedded enzymography. Lipid peroxidation inhibition day, the day of birth and the 3rd day of life. Glutathione peroxidase and DPPH• scavenging assays could be successfully applied in th levels are significantly higher in the male’s lung at the 15 gesta- quality control for the antioxidant activity evaluation QC formula- nd tional day and at the 2 day of life. Whether these gender differences tions, nevertheless, the deoxyribose test was not suitable because in lung’s antixodant enzymes arise from a direct stimulation of their of excipient interference. The dose of 1.23 J/cm2 induced increase synthesis or by higher levels of reactive oxygen species in the male’s in the proteinase secretion/activity, which was inhibited by QC lung is yet to be investigated. However, gender differences in lung’s formulations. These results suggest QC as a promising drug for the antioxidant enzymes can cause significant changes in redox balance, treatment of photooxidative skin damages. which are frequently associated with lung dysfunction.

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The Tephrosia toxicaria extract inhibits lipid hydroperoxides formation Hypoxia-sensitive miR-200b regulates angiogenic response of endothelial cells by targeting Ets-1 Rubia Casagrande1,2, Vanessa V.M. Zimermann1, Renata M. Martinez1, Joice Sartorato Sanson1, César C. Andrei3, Yuk-Cheung Chan, Savita Khanna, Sashwati Roy, and Isabel C. Moreira4, Sandra R. Georgetti1,2, Marcela M. Baracat1,2 Chandan K. Sen

1Department of Pharmaceutical Science, State University of Londrina, Lon- Davis Heart & Lung Research Institute, Departments of Surgery, drina, PR; 2Department of Pharmaceutical Science, Faculty of Pharmaceutical The Ohio State University Medical Center, Columbus, Ohio, 43210

Sciences of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP; 3Department of Chemical, State University of Londrina, Londrina, PR; 4Federal Technological University of Parana, COALM/UTFPR, Londrina, PR MicroRNAs (miR) are small RNAs involving in epigenetic regulation of gene expression. MiR-200b plays a crucial role in The specie Tephrosia toxicaria is a source of flavonoids, which epithelial to mesenchymal transition in various cancer cells. are known to be antioxidants. However, there are few studies con- However, its role in other tumorigenic response such as angio- cerning the antioxidant mechanisms of T. toxicaria. Therefore, it genesis remains ambiguous. Here we report first evidence that was evaluated the whether the hexanic fraction (HF) and methano- endothelial miR-200b responds to hypoxic environment and lic fraction (MF) of T. toxicaria extract inhibits lipid peroxidation regulates angiogenesis via v-ets erythroblastosis virus E26 (lipid hydroperoxide formation) by a mechanism independent of oncogene homolog 1 (Ets-1). Exposure of human microvascular iron as well as the precision of the method. An emulsion of linoleic endothelial cells (HMECs) to hypoxia or stabilization of HIF1 acid 10 mM was prepared in tween 20. In 2 ml of emulsion it was suppressed miR-200b expression. Over-expression of miR-200b in added 20 μl of different concentrations of the samples (extract HMECs inhibited in vitro angiogenesis, which was accompanied fractions). Lipid hydroperoxides were determined before and after by down-regulation of matrix-metalloproteinase-1 (MMP-1) and the incubation of the emulsion with samples at 37ºC for 8 h and vascular endothelial growth factor receptor 2 (VEGFR2). In silico readings at 234 nm. The precision was evaluated during 3 days. studies revealed that the 3’ untranslated region of Ets-1, a crucial The inhibition of lipid hydroperoxides formation by T. toxicaria transcription factor in MMP-1 and VEGFR2 expression, contains was concentration dependent for both fractions. The linearity was two binding sites for miR-200b. Interestingly, miR-200b over- 2–40 and 2–20 μg/ml, and IC50 35,97 and 8,53 μg/ml for HF and expression significantly inhibited both mRNA and protein level of MF, respectively. The inter-day precisions presented the relative Ets-1. Knock-down of Ets-1 alone mimicked the impaired angio- standard deviation value near 10%. These results demonstrate that genic response that was observed in miR-200b overexpressed both fractions of the extract inhibited the lipid peroxidation and HMECs. This work provides first evidence demonstrating that hy- that the MF present greater antioxidant activity than the HF in this poxia-induced down-regulation of miR-200b de-repress Ets-1 ex- assay. These data suggest the use of T. toxicaria extracts are prom- pression in endothelial cells, thus further promoting angiogenesis ising sources of antioxidants against oxidative stress. under low oxygen environment.

Supported by NIH GM069589 and GM077185.

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ESR spectra of black and red hair and the effect of vitamin C (ascorbic acid) on the photoinduced free radicals Hypertension is associated with oxidative stress and in the hair exaggerated renal angiotensin II AT1 receptor and diminished dopamine D1 receptor function in old rats Eduard N. Chikvaidze1, Temur V. Gogoladze2, Alexander Miminoshvili3, and Inga Khachatryan1 Gaurav Chugh, Mustafa F. Lokhandwala, and Mohammad Asghar

1Department of Exact and Natural Sciences, Iv. Javakhishvili Tbilisi State Heart and Kidney Institute, College of Pharmacy, University of Houston, University, Tbilisi, Georgia, 2Tbilisi State Medical University, Tbilisi, Georgia, Houston, Texas 77204 3 Sukhumi State University, Tbilisi, Georgia The process of aging alters physiological functions including ESR spectra of melanin’s free radicals in black and red hair sodium homeostasis in the kidney and is associated with an in- have been investigated. It is shown that the ESR spectrum of black crease in blood pressure. Sodium homeostasis and thus blood pres- hair is slightly asymmetric singlet with g=2,003 and H=0,5 mTl. sure is maintained by the balance between angiotensin II type I The ESR spectrum of red hair is different from the spectrum of (AT1) receptor and dopamine D1 receptor function in the renal black hair and represents the superposition of the singlet of black proximal tubules. Although the underlying mechanism for age- hair and triplet. Under the action of visible light (blue with max = associated increase in blood pressure is not known, we hypothe- 450 nm, green with max = 510 nm, and red with max = 650 nm) sized that an imbalance in renal AT1 receptor and D1 receptor both in black and red hair appear photoinduced free radicals, which function during aging would favor elevated blood pressure. Renal is observed in the increase of the intensity of the ESR spectrum of hemodynamic, biochemical, molecular biology and western blot- those already existing in the hair. ting methodologies were used in the study. Aged (21-month) It should be noted that the ESR spectra of red hair from various Fischer 344 x Brown Norway rats had higher blood pressure com- donors is different. The antioxidant vitamin C has the different ef- pared to their younger (3-month) counterparts. The natriuretic ef- fects on the ESR spectra of hair. In particular, in the case with fects of AT1 receptor antagonist, candesartan, were potentiated black hair the concentration of photoinduced free radicals is re- whereas those of D1 receptor agonist, SKF 38393, were dimin- duced, whereas in red hair the disappearance of the triplet spec- ished in aged rats. The levels of mRNAs of AT1 receptor were in- trum is observed and at the same time the spectrum becomes a sin- creased and those of D1 receptor were decreased in the renal glet with the ESR parameters of black hair and the intensity of the proximal tubules of aged compared to younger rats. Markers of spectrum increases sharply. oxidative stress (protein carbonyls and 8-isoprostane) and stress- It is assumed, that antioxidants effective for black hair, may be activated transcription factors NF-B and Sp3 were up-regulated in ineffective for red hair, and vice versa. Therefore, in each specific the renal proximal tubules of aged rats. case is necessary to investigate separately the effectiveness of an These results document age-associated oxidative stress in the antioxidant. renal proximal tubules that involves NF-B and Sp3, and an exag- gerated AT1 receptor and diminished D1 receptor function. These age-related changes may provide an underlying mechanism for al- tered sodium homeostasis and higher blood pressure in aging. Financial support provided by NIH/NIA AG25056 and AG29904.

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gp91phox produced superoxide radical in classical activated Improved function of diabetic wound-site macrophages and microglia and contributes to the severity of traumatic brain accelerated wound closure in response to oral supplementation injury in mouse of a fermented papaya preparation Kenji Dohi1, Seiji Shioda2, Hirokazu Ohtaki2, Eric Collard and Sashwati Roy Tomoya Nakamachi2, Sachiko Yofu2, Kazue Satoh2, Yuko Mihara1, Kazuyuki Miyamoto1, Comprehensive Wound Center, Department of Surgery, Davis Heart and Lung Shohko Tsunawaki3, and Tohru Aruga1 Research Institute, The Ohio State University Medical Center, Columbus, OH 43210, USA 1 2 Department of Emergency and Critical Care Medicine, Department of Anatomy, Showa University School of Medicine, Tokyo, Japan Carica papaya Linn is widely known as a medicinal fruit. To 3Department of Infectious Diseases, National Research Institute for Child determine its role in wound healing, we investigated the effect of Health and Development, Tokyo, Japan standardized fermented papaya preparation (FPP) in adult obese diabetic (db/db) mice. Oral FPP (0.2g/kg for eight weeks) reduced Traumatic brain injury (TBI) is associated with reactive oxygen blood glucose elevations and improved lipid profile. FPP did not species (ROS), and one of the major enzymatic sources of superox- affect weight gain during the eight week supplementation period. ide anion production in the brain is NADPH oxidase. gp91phox FPP supplementation, prior to wounding, improved wound closure. (NOX2), one of the catalytic subunit of NADPH oxidase enzymes Reactive oxygen species support numerous aspects of wound heal- (NOX-es), plays as a key enzyme in various neuronal diseases. We ing, and NO generation is known to be compromised in diabetic hypothesized that gp91phox generates superoxide anion is a major wounds. Studies on viable macrophages isolated from the wound causative role in TBI. Using gp91phox knockout (gp91phox-/-) and site demonstrated that FPP supplementation enhanced respiratory wild-type (gp91phox +/+) mice (C57BL), unilateral controlled corti- burst function, as well as inducible NO production. Furthermore, cal impact traumatic brain injury was induced. The expressions and phox diabetic mice supplemented with FPP showed elevated CD68 and the roles of gp91 after TBI were investigated. We also studied phox CD31 expression at the wound site, suggesting effective recruit- the contributions of gp91 to superoxide anion generation after ment of monocytes and an enhanced pro-angiogenic response. TBI using in situ detection of oxidized HEt. In wild type mice, phox phox These data provide the first evidence that diabetic wound outcomes gp91 activated 24 h and 48 h after TBI. gp91 expressed may benefit from FPP supplementation by specifically influencing mainly in classically activated microglia like cells and weakly in the response of wound-site macrophages and subsequent angio- astrocyte and neuron after TBI. The contusion area and TUNEL phox-/- genic response. The challenge of wound healing in diabetes, and positive apoptotic like cells were suppressed in gp91 mice. - FPP’s long track-record of safe human consumption warrants test- TBI increased peroxynitrite (ONOO ) expression and superoxide - phox ing of FPP in a clinical setting. radical (O2 ) production (HEt stains cells) in gp91 immunoposi- phox tive cells. Gp91 containing NADPH oxidase expressed in clas- Supported by DK 076566 to SR and in part by a funding from Osato Research Institute, sically activated like microglia promotes ROS formation and has Japan an important roles of brain damages after TBI. Modulations of gp91phox-containing NADPH oxidase may provide a new therapeu- tic strategy to traumatic brain injury.

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Oxidant and antioxidant balances during brain Indiscriminate ("preventive") supplementation of vitamin E hypothermia therapy in patients with traumatic brain injury Yedidya Dotan, Ilya Pinchuk Moshe Leshno and Dov Lichtenberg

Kenji Dohi1, Kazue Satoh2, Kazuyuki Miyamoto1, Department of Physiology and Pharmacology, Sackler Faculty of Medicine, 1 2 1 Tel-Aviv University, Tel Aviv Israel 69978 Shunsuke Nakamura , Seiji Shioda and Tohru Aruga For many years, the prevailing concept was that LDL oxida- 1 Department of Emergency and Critical Care Medicine, School of Medicine, tion plays the central role in atherogenesis. As a consequence, sup- Showa University, Tokyo, Japan plementation of vitamin E, became very popular. Unfortunately, 2Department of Anatomy, School of medicine, Showa University, Tokyo, Japan the major randomized clinical trials yielded disappointing results Oxidative stress has important roles in traumatic brain injury and two independent meta-analyses concluded that vitamin E sup- (TBI). The control of free radical production is one of the targets in plementation increases mortality. This conclusion raised much the treatment for prevention from brain damage. Brain hypother- criticism, most of it relating to the choice of clinical trials included mia therapy (BHT) has neuroprotective effect and is thought to in the meta-analyses; the end point (only mortality) and the hetero- have the effect on the control of free radical production. On the geneity of the analyzed clinical trials with respect to both popula- other hand, the kinetic of free radical during BHT remains unclear. tion and treatment. The aim of this study is to investigate of the kinetic of free radical In an attempt to bring this controversy to an end, we applied a during BHT. Markov Model approach, which is free of most of the limitations Thirteen severe TBI patients are enrolled. 9 patients were of metaanalysis, to compare vitamin E supplemented cohorts with treated at 33°C
(BHT group). Other patients were controlled at non supplemented cohorts. The difference between the two virtual 35°C (normothermia group). Reactive oxygen metabolites (ROMs) cohorts was expressed in terms of the composite end point denoted were measured in jugular blood samples by the diacron reactive quality-adjusted life years (QALY). oxygen metabolites (d-ROM) test. Bio antioxidant potency (BAP) The main result was that the vitamin E-supplemented virtual was measured by the BAP test. cohort had 0.30 QALY (95% CI 0.21 to 0.39) less than the un- ROM level of BHT group at 33°C was lower than that of nor- treated virtual cohort. Based on the latter result, we conclude that mothermia group (35°C). ROM level during hypothermic phase indiscriminate supplementation of high dose vitamin E can not be was suppressed significantly in BHT group. On the other hand, recommended to the general public. We do not know the mecha- BAP level in BHT group was suppressed during hypothermic nisms responsible for the harmful effects of vitamin E, but recent phase. ROM levels were increased and BAP levels recovered studies show that the outcome of vitamin E supplementation may slowly along the rewarming. be negative on the cellular and molecular levels. We demonstrated that BHT suppressed free radical production Several recent studies, conducted with specific groups of pa- in clinical settings. However, BHT also led to suppression of anti- tients, demonstrated positive outcome. In other words, vitamin E is oxidant potential. These data supported that the imbalance of oxi- a “double-edge sword” that should not be consumed indiscrimi- dant and antioxidant balance exists in rewarming phase. Support- nately. Carefully-conducted randomized clinical trials with long ing therapies of antioxidant potential in rewarming phase in BHT follow-up and well-defined end points must be conducted to define may settle some troubles during rewarming phase in BHT. criteria capable of predicting who is likely to benefit from supple- mentation of vitamin E.

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Fisetin: Flavonoid and fighter against diabetic complications Topical application of vitamin E -tocotrienol in a porcine burn model promotes nerve regeneration and Jennifer Ehren1, N. Calcutt2, D. Schubert1, and P. Maher1 improves wound healing 1Cellular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, 2Department of Pathology, University of California at San Diego, J. Driggs, S. Biswas, S. Khanna, S. Roy and C.K. Sen La Jolla, CA

Comp. Wound Cntr., Dpt. of Surg., Davis Heart & Lung Res. Inst., An estimated 23.6 million Americans (8% of the population) Ohio State University Medical Center, Columbus, Ohio suffer from diabetes and an estimated 57 million Americans have prediabetes, a condition which increases the risk of developing Scar formation from a full thickness thermal injury (burn) re- Type 2 diabetes. Diabetic nephropathy (DN), a complication of sults in a rough scar devoid of rete ridge formation, poor vascula- diabetes, is a condition that often ends in kidney failure and has no ture and causes neuropathy due to nerve degeneration. We have re- known therapy. Fisetin, an orally active natural product, has anti- inflammatory properties and both direct and indirect antioxidant cently reported on neuroprotective properties of -tocotrienol activity. We examined the efficacy of fisetin on kidney function in (TCT) and now aim to test that neuroprotectivity in a porcine burn Akita mice, a diabetic animal model. Akita mice develop renal hy- model to demonstrate its efficacy. Domestic pigs (n=7) were anes- pertrophy, increased glomerular volume, and increased urine pro- thetized and clipped for dorsal treatment. One side of the dorsum tein secretion when compared to wild type (WT) mice. During the received 400 mg of control ointment via careful rubbing, while the early stages of DN, clusters of kidney blood vessels (glomeruli) other received 400 mg of TCT. The pigs were rubbed for four sub- undergo hypertrophy and a thickening of the basement membrane, sequent days, given a two day break then burned with 2.5 cm2 resulting in increased levels of albumin in the urine. Feeding Akita tungsten rods. HPLC analysis confirmed TCT was present on the mice fisetin prevented kidney weight increase seen in untreated treated side. TCT treated samples expressed a faster rate of leuko- Akita mice, yet had no marked change on WT mice kidney weight. cyte infiltration over 28 days, consistent with literature of dener- Similarly, urinary protein secretion decreased significantly in vated wounds slowing chemotaxis. TCT-treated wounds saw a 2.5 fisetin-fed Akita mice compared to untreated Akita mice, while no fold (p<0.03) increase in rete ridge formation. Accelerated in- significant change occurred in urinary protein secretion in fisetin- flammatory chemotaxis in TCT-treated samples significantly de- fed WT mice. In addition, fisetin decreased the serum inflamma- tion marker C-reactive protein (CRP) that is highly elevated in creased proliferating cells in the neodermis (p<0.03) and had also both human disease and diabetic mouse models. In contrast, fisetin shown increased angiogenesis (p<0.04) with clearly improved had no effect on increased blood glucose levels in Akita mice. morphology in the same timeframe. First evidence of TCT inhibit- Likewise, fisetin significantly improved several indices of Akita ing 12-Lox in vivo (70% decrease, p<0.01) was shown in d28 mice nerve function; large and small nerve fiber functions were mRNA levels via RT-PCR, indicating fewer lipid oxidive products tested by measuring nerve conduction velocity and paw withdrawal are present. By d65 the TCT-treated scars were expressing less to noxious heat, respectively. Diabetes-induced slowing of the mo- redness around the scar edge and better contour with the surround- tor nerve conduction velocity and decreased sensitivity to thermal ing skin. SEM micrographs demonstrate this relative smoothness stimuli were ameliorated by fisetin treatment. In summary, fisetin (10,000x). PGP9.5 staining shows nerve regeneration was four- restored levels of the aforementioned entities to those similar to fold in TCT-treated samples. This is the first report that shows WT mice. Fisetin shows significant promise as a treatment to ame- TCT promotes nerve regeneration and improves wound healing. liorate the consequences of diabetes.

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Mimetic peroxidase activities of uniformed platinum nanoparticles-apoferritin nanoreactor H2O2-induced mitochondrial fission in C2C12 myocytes

Jia Fan, Dongxue Xu, Li Xu, Jing-yan Wei, Yuliang Zhao, Xiying Fan, Rajaa Hussien, and George A. Brooks and Guangjun Nie* Exercise Physiology Laboratory, Department of Integrative Biology, University of California, Berkeley CA 94720-3140 CAS Key Laboratory for Biological Effects of Nanomaterials & Nanosafety, Na- tional Center for Nanoscience and Technology of China, Beijing, China; 11 Beiyijie, Zhongguancun, Beijing, China 100190 [email protected]; In skeletal muscle and many other cell types, mitochondria ex- Biomimetic enzymatic activities of nanostructures have been ist as an elaborate and dynamic network. The balance of continu- reported recently. Novel and superb catalytic activities have attrib- ous mitochondrial fission and fusion define the morphology of the uted to their unique size dimensions and enhanced surface area and mitochondrial reticulum. Environmental stimuli, such as oxidative the intrinsic properties of nanostructure compositions. We report stress, can influence fusion and fission rates, resulting in a trans- that apoferritin (apoFt) was used to template the synthesis of 1 to 2 formation of the network’s connectivity. Using confocal laser nm of biocompatible, non-toxic and stable Pt-nanoclusters (Pt- scanning microscopy of C2C12 mouse myocytes, we show that apoFt) with unique and intrinsic peroxidase activities. The Pt- acute exposure to the reactive oxygen species (ROS) hydrogen apoFt showed both catalase and HRP activities with distinctive en- peroxide (H2O2) induces a slow fragmentation of the mitochondrial zymatic properties, namely different response to pH and tempera- reticulum that is reversible over 24 hours. Although H2O2 rapidly ture. For the catalase activities, the Pt-apoFt showed significant in- decomposes in culture medium, the full extent of fragmentation crease in enzymatic activities with increasing pH and temperature, occurs 5-6 hours post-treatment, suggesting that H2O2 affects mi- two unique features not seen in both enzymes and other nanostruc- tochondrial morphology by modulating cellular physiology or acti- tures. For the HRP-like activities, physiological temperature and vating redox-sensitive signaling. Supraphysiological (>1 mM) slightly acidic conditions are for optimal HRP activities. These re- concentrations of H2O2 are cytotoxic, but lower concentrations sults firstly demonstrate that a Pt nanocluster possess differential (250 mM) sufficient to induce transient fragmentation do not lower unique enzymatic activities for different substrates under different cell viability. The extent of H2O2-induced mitochondrial fragmen- conditions. These nanostructures hold great promise for a wide tation correlates with decreases in inner mitochondrial membrane range of new applications in biomedicine and environmental sci- potential and maximal respiratory rate, suggesting a possible ence. mechanism. Because H2O2 is produced in contracting muscle, our results raise the possibility that ROS generation may contribute to exercise-induced changes in mitochondrial morphology in vivo.

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Anthocyanin metabolites inhibit viability of Caco-2 cells Genistein decreases CYP1A1 via inhibiting the bindings of the Sarah C. Forester1, Andrew L. Waterhouse1, and arylhydrocarbon receptor nuclear translocator and estrogen Patricia I. Oteiza2 receptor alpha to the xenobiotic response element

Department of Viticulture and Enology1, and Departments of Nutrition and 1 1 Environmental Toxicology2 University of California, Davis Erik B. Froyen and Francene M. Steinberg

Anthocyanins are a class of polyphenols abundant in the skins 1Department of Nutrition, University of California, Davis, Davis, CA 95616 of red grapes. Gut microflora metabolize anthocyanins to primar- ily phenolic acids and aldehydes. These metabolites may explain The consumption of soy protein and the associated isoflavones the relationship between anthocyanin consumption and reduced in- (genistein and daidzein) have been linked with a decreased risk for cidence of colon cancer. Previously, gallic acid, 3-O-methyl-gallic certain cancers. One proposed mechanism for cancer prevention is acid, and 2,4,6-trihydroxybenzaldehyde were found to decrease through decreasing the phase I cytochrome P450 1A1 (CYP1A1) Caco-2 cell viability more than other anthocyanin metabolites. To enzyme. This study evaluated the hypotheses that dietary soy investigate the underlying mechanisms, we have now investigated the capacity of these three anthocyanin meta-bolites to modulate isoflavones, genistein and daidzein, will 1) decrease hepatic the cell cycle, and to induce apoptotic cell death. Caco-2 cells CYP1A1 enzyme activity and protein in male and female Swiss were incubated for 24-72 h in the presence of 10 to 100 μM gallic Webster mice; 2) decrease mRNA in mouse liver cells (Hepa- acid, 3-O-methylgallic acid, 2,4,6-trihydroxy-benzaldehyde, and 1c1c7); and 3) inhibit the bindings of the arylhydrocarbon receptor malvidin-3-glucoside. Malvidin-3-glucoside did not affect cell vi- (AhR), AhR nuclear translocator (ARNT), and estrogen receptor ability within in the range of concentrations tested. The aldehyde alpha (ER) to the CYP1A1 xenobiotic response element (XRE). only reduced cell viability at the highest treatment concentration The mice were fed for 1, 3, 5, or 7 d of one of four treatments: con- (100 μM). Both gallic acid and 3-O-methylgallic acid induced a trol (casein AIN-93G), or control supplemented with 1500 mg/kg time-dependent decrease in cell viability. After 72 h incubation, diet of flavone (positive control), genistein, or daidzein (all agly- the three metabolites caused an arrest in the G0/G1 phase of the cell cones). To investigate the molecular mechanisms associated with cycle, which would lead to a decrease in cell proliferation. Apopto- CYP1A1 modulation, Hepa-1c1c7 cells were treated with control sis was monitored as caspase-3 activation and DNA fragmentation. [0.1% (v:v) DMSO], 1 mol/L -naphthoflavone, or 5 mol/L ge- The three metabolites, at a concentration of 50 μM, activated nistein, daidzein, or the daidzein metabolite equol, followed by caspase-3 (evaluated as PARP cleavage and enzyme activity). ® However, only gallic acid and 3-O-methylgallic acid increased the TaqMan and chromatin immunoprecipitation assays. Genistein levels of mono- and oligonucleosomes within the cytoplasm and decreased liver CYP1A1 activity and protein in the male and fe- caused apoptotic nuclear morphologic changes (Hoechst staining). male mice. No significant differences in mRNA were detected in 3-O-methylgallic acid and gallic acid led to a decrease (67.8 % and the cells following treatment with isoflavones. In support of the 47.0 %, respectively) in NF- B-DNA binding. In conclusion, the animal results, the cells treated with genistein, but not daidzein and decreased Caco-2 cell viability caused by 3-O-methyl-gallic acid equol, exhibited decreases in ARNT and ER bindings to the and gallic acid can occur as a cones-quence of both, the induction CYP1A1 XRE. In conclusion, these data provide novel molecular of apoptosis and inhibition of cell proliferation. The inhibition of mechanisms associated with the CYP1A1 decreases in mice treated transcription factor NF- B, which promotes cell proliferation and with genistein under acute and basal conditions. survival, can underlie the observed effects.

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Systems biology of human aging -- Network model 2009 PGE2-mediated induction of oncostatin M production in human chronic wounds John D. Furber1 and Pat Langley2

1Legendary Pharmaceuticals, Gainesville, FL 32604; 2School of Computing and Kasturi Ganesh1, Savita Khanna1, Urmila Gnyawali1, Informatics, Arizona State University, Tempe, AZ 85287 1 2 Narashimham L. Parinandi , Krishna Rao Maddipati , 1 1 1 The many observable signs of human senescence have been Gayle M. Gordillo , Chandan K. Sen , and Sashwati Roy hypothesized by various researchers to result from several primary 1Comprehensive Wound Center, The Ohio State University, Columbus, OH, causes. Close inspection of the biochemical and physiological USA, 2Wayne State University, Detroit, MI, USA pathways associated with age-related changes and with the hy- pothesized causes reveals several parallel cascades of events that Cytokine screening studies in our laboratory recognized oncostatin involve several important interactions and feedback loops. We pre- M (OSM), an IL-6 family cytokine produced by activated macro- sent a network diagram to aid in conceptualizing the many proc- phages, as being highly abundant in human chronic wound fluid. Pros- esses and interactions among them, including promising interven- taglandin E2 (PGE2) is generated by the sequential metabolism of ara- tion points for therapy development. This diagram is maintained on chidonic acid by cyclo-oxygenase and prostaglandin E synthase. LC- the Web as a reference for researchers and students. Content is up- MS studies indicated presence of high levels of PGE2 in human dated as new information comes to light (www.LegendaryPharma. chronic wound fluid. We hypothesized that PGE2 present in wound com/chartbg.html). In addition, several researchers have proposed fluid induces OSM production by macrophages. Adult chronic wound to adapt the network model's contents into an interactive website (defined as wound present > 4 weeks) patients (25-80 years old) un- with links to references and background materials. A symposium dergoing VAC® (negative pressure) therapies of their wounds at the to promote this development was held at Arizona State University, Comprehensive Wound Center (CWC) were recruited (IRB approved) December 2008; abstracts are at http://circas.asu.edu/ sympo- for the study. VAC dressing (sponges) were collected. In addition, sia/aging/. A second symposium was held 8-9 Dec 2009 at the Na- paired peripheral blood samples were collected from each patient. tional Institute on Aging in Baltimore, Maryland. Wound fluid and inflammatory cells were derived from the VAC This network model includes both intracellular and extracellu- dressing by lavaging the wound dressing with saline solution. High lar processes. It ranges in scale from the molecular to the whole- levels of PGE2 and OSM (p<0.01; n=19) were observed in chronic body level. Important pathways include: wound fluids as compared to plasma from the same subjects. Wound • Extracellular proteins become damaged. derived macrophages, but not pair-matched blood monocyte derived • Lysosomes accumulate reactive, crosslinked lipofuscin. macrophages, produced high levels of OSM. To characterize the • Mitochondrial DNA mutates. mechanism of OSM production in wound fluid, differentiated cultured • Lamin-A progerin in nucleus. human macrophages were treated with PGE2 for 24h. PGE2 potently • Nuclear envelope pore proteins oxidized. induced OSM (protein and mRNA). This induction was completely • Nuclear mutations, telomere shortening, chromosome breaks, blocked using a receptor tyrosine kinase (RTK) inhibitor. Characteri- chromatin alterations and epigenetic DNA adducts change gene zation studies indicated that PGE2 strongly induces the phosphoryla- expression. tion of a RTK, Axl, in macrophages. Wound macrophage biology is • Oxidized aggregates in cytoplasm. uniquely different from biology of peripheral blood monocyte derived • Proteasomes. macrophages. In wound macrophages, PGE2 activates OSM via an • Redox poise increases. Axl dependent mechanism. OSM is an inducer of MMP-9 which is • Inflammatory cascades. abundant in chronic wounds and may complicate healing outcomes. • Neuroendocrine and immune systems. Supported by NIH RO1 DK-076566 to SR. • ER stress.

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Evaluation of physical-chemical and antioxidant stability and Oral tocotrienol is transported to human organs and in vivo efficacy of formulations added soybean extract particularly helpful for patients waitlisted for liver transplantation

Sandra R. Georgetti1,2, Rubia Casagrande1,2, Marcela M. Baracat1,2, Urmila Gnyawali, Viren Patel, Gayle M. Gordillo, Savita Khanna, Waldiceu A. Verri Jr3, Fabiana T.M.C. Vicentini2, Cameron Rink, Bassel Shneker, Sashwati Roy, Kasturi Ganesh, and Maria José Vieira Fonseca2 J. Layne Moore, Atom Sarkar, Benjamin Sun, David Feldman, Robert Kirkpatrick, Elmahdi Elkhammas, Emily Klatte, 1Department of Pharmaceutical Science, State University of Londrina, Michel Miller, Michael Firstenberg and Chandan K. Sen Londrina, PR; 2Department of Pharmaceutical Science, Faculty of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Ribeirao Davis Heart and Lung Research Institute, Department of Surgery, 3 The Ohio State University Medical Center Preto, SP; Department of Pathology, State University of Londrina, Londrina, PR Recent works in our laboratory has established alpha-tocotrienol as The skin is constantly challenged by oxidative stress. There- the most potent neuroprotective form of natural vitamin E effective fore, topical administration of antioxidants provides an efficient against stroke. In this study we tested the delivery of mixed to- manner to enrich the endogeneous cutaneous protection system. cotrienols (TCT), taken orally, to major human organs. TCT was de- However, novel formulations must be stable and active in vivo. tected in all organs collected (brain, adipose, skin, cardiac muscle, car- diac valve, liver, and skin) from patients. Results from mixed TCT Therefore, the present study was designed to evaluate both physi- supplemented humans were compared with those from a control group cal-chemical and antioxidant activity stability of different formula- who took tocopherols (TCP). TCT supplementation increased the liver tions containing SE as well as in vivo protection against TPA- and adipose tissue levels of , , and  - TCT significantly. In the car- induced oxidative stress in the skin of hairless mice (estimation of diac muscle, levels of  and -TCT increased significantly. TCT sup- H2O2 and lipid peroxidation). A non-ionic emulsion with high lipid plementation significantly increased , , and  - TCT levels in whole content (F1) and anionic emulsion with low lipid content (F2) con- blood and skin when compared to pre-supplementation (time 0) base- line values. When observing our liver transplantation patients we taining or not the SE were stored at 4C, 30C/60% relative humid-  came across a striking finding. The model for end-stage liver disease ity (RH), and 40 C/70% RH for 6 months. At pre-determined times (MELD) is used to prioritize cirrhotic liver patients who are waiting (initial, 1, 2, 3 and 6 months) samples were collected for the for liver transplantation. When we fitted MELD score to a linear model evaluation of physical (pH, centrifugation and globule size), and both pre- and post-supplementation, 50% (7) of patients on TCT sup- functional (inhibition of lipid peroxidation) stability. Both formula- plementation (n=14) showed a negative sloping (reduction) MELD tions were stable in all parameters evaluated during the 6 months. score post supplementation. When we compared that to the tocopherol supplemented group, only 1 patient out of 5 (20%) showed a negative The topical treatment of hairless mice with F1 or F2 containing SE sloping (reduction) MELD score. Of the TCT supplemented liver pa- abolished the TPA-induced generation of H2O2 and lipid peroxida- tients 5 out 7 (71%) patients with cirrhosis due to viral hepatitis (hepa- tion (MDA formation). Therefore, present data suggest that formu- titis B, 1/1 and C, 4/6) had a reduction in MELD score. However, all lations containing SE are promising therapeutic approaches to in- the patients with a diagnosis of cryptogenetic cirrhosis (n=3) who were hibit oxidative damages of the skin. supplemented with TCT had an increase in MELD score. When all pa- tients were considered those who were supplemented with TCT had a statistically significantly lower MELD score slope than those supple- mented with TCP. Thus, TCT is transported to all vital human tissues studied including the brain. Furthermore, tocotrienol supplementation may lower MELD score in patients waiting for liver transplantation providing them with a longer time to wait until an appropriate liver is available for transplantation. [Supported by NINDS NS42617 to CKS].

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Development of a multi-modal in vivo system to image wound tissue oxygen and perfusion Tocopherol Transfer Protein (TTP) modulates cigarette smoke (CS) sensitive lung transcriptome 1-2 1-3 1-3 1 Surya Gnyawali , Jeff Xu , Jiwei Huang , Urmila Gnyawali , 1-4 1-3 1-2 Kun Huang , Ronald Xu , and Chandan K. Sen Kishorchandra Gohil, Saji Oommen, Vihas T. Vasu, Hnin Hnin Aung, and Carroll E. Cross 1Comprehensive Wound Center, 2Davis Heart and Lung Research Institute, 3 4 Department of Biomedical Engineering, Department of Biomedical Department of Internal Medicine, Division of Pulmonary and Critical Care Informatics, The Ohio State University, Columbus, OH 43210
Medicine, University of California, Davis, CA 95616

Assessment of tissue oxygenation and perfusion is important Oxidant and inflammatory processes are implicated in CS- for evaluating wound healing/regression and guide followed by induced lung diseases. Our primary hypothesis is that lung’s intrin- therapeutic processes. However, many existing techniques and sic antioxidant status affects the expression of multiple genes that clinical practices are subjective to background bias, tissue hetero- determine physio-pathological phenotypes of lungs. -Tocopherol geneity, and inter-patient variations. We sought to develop a multi- (AT) is suggested to be a potent antioxidant. It is sequestered in modal imaging system for in vivo, non-invasive, real-time quantita- lungs and may be a component of lung’s antioxidant network. tive assessment of tissue oxygen and perfusion. Our imaging sys- Mice and patients deficient in the TTP gene have very low sys- tem integrated a broadband light source, a high resolution CCD camera, a highly sensitive infrared camera and a liquid crystal tun- temic AT inspite of feeding a normal diet. In this study, 5 month able filter. A user-friendly software interface was developed to old male TTP-deficient (KO) mice and their congenic normal (WT) mice, n = 5/group, were exposed to either air or CS (60 mg control all components systematically. Image registration and re- 3 construction algorithms were developed for reliable reconstruction particulates/m ) for 3 and 10 days. Post-exposure lung tissue was of tissue oxygenation without interferences of blood concentration, dissected, RNA extracted, 10 mg from each lung pooled, group tissue scattering, pigmentation and background absorption. Ther- wise, and subjected to GeneChip (430A 2.0) analysis. Differential mal images and hyperspectral visual/near infrared images were co- analysis of the transcriptomes (~15,000 mRNAs) identified CS- registered. Validity of the algorithm was tested on a tissue- sensitive genes that were modulated by AT-TTP deficiency. AT- simulating heterogeneous phantom. The tissue perfusion was re- TTP deficiency potentiated CS-induced cyp1a1 and cyp1b1 expres- constructed from the measurements of the dynamic changes of sion; two genes driven by aromatic hydrocarbon receptor (AhR). hemoglobin concentration and temperature distribution subjected There was selective potentiation of CS-induced Nrf2-driven gene to vascular occlusion and reperfusion. Image fusion technique that battery by AT-TTP deficiency. Lymphocyte and leukocyte specific co-registered tissue morphology, oxygenation and perfusion maps genes were the largest gene-clusters modulated by AT-TTP defi- was displayed in real-time. Clinical feasibility test for quantitative imaging of tissue oxygenation and perfusion was also performed ciency. These experiments have uncovered a large network of on healthy human subjects. Results show that the imaging system lung-tissue genes that are modulated by AT-TTP deficiency. Since is powerful to detect tissue oxygenation and perfusion in real-time lung-tissue does not express TTP, the data suggest that lung AT is with minimal bias introduced by tissue heterogeneity and back- the primary modulator of these gene-network. Furthermore, the ef- ground absorption. Images are independent of human skin color. fects on lymphoid and myeloid specific genes uncovered in this This imaging system, initially targeted to quantitative assessment study may indicate a role for AT in bone-marrow-lung-tissue ho- of chronic wounds, will help in translational research and clinics. meostatic adaptations under physiological conditions and, when Supported by NHLBI R01 GM-077185 mice, and possibly humans, are exposed to environmental toxins.

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Hemangioma growth is nox-4 dependent and inhibited by oral feeding of blueberry extract Concordant changes of activity and the level of mRNA of two superoxide dismutase isoforms in rat blood cells taking 1,2 1,2 2,3 vitamin E Gayle M. Gordillo , Huiqing Fang , Sashwati Roy , 2,3 and Chandan K. Sen Maliheh Hajiani1, Aboualfazl Golestani1, Mehdi Froozandeh2, 3 1 1 1 2 Ali Akbar Owji , Shahnaz Khaghani , Naghmeh Ghannadian , and Division of Plastic Surgery, Laboratory of Molecular Medicine Davis Heart 1* 3 Parvin Pasalar Lung Research Institute, Division of General Surgery, The Ohio State University, Columbus, Ohio 43210 1Department of Biochemistry, Azad University of Jahrom; 2Depatment of Biotechnology, Terabit Moddares University, Tehran, Iran; 3Department of Hemangiomas are endothelial cell tumors that are the most Biochemistry, Shiraz University of Medical Sciences, Shiraz, Iran common soft tissue tumor in infants occurring in up to 10% of Caucasian infants. Using an established murine model, we have Vitamin E is the most important lipid soluble antioxidant. More shown that injection of endothelial cells (EOMA) results in endo- recently, it has been proposed as a gene regulator, and its effects have been observed at the level of gene expression, mRNA stabil- thelial cell tumor (HE) formation that requires the expression of ity, protein translation and protein stability. Aim: To investigate the oxidant inducible monocyte chemoattractant protein-1 (MCP-1). effects of vitamin E on activity and expression of the most impor- We sought to identify the source of oxidants stimulating MCP-1 tant endogenous antioxidant enzyme; superoxide dismutase (SOD), expression in EOMA cells. NADPH oxidase is the primary source in rats. Materials and Methods: 28 male Sprauge-Dawly rats were of oxidant production in endothelial cells. Real-time PCR was used divided into four groups; the control group and three dosing groups to screen EOMA cells for all isoforms of gp91, the catalytic receiving daily 50 μl intraperitoneal injections of 0, 10, 30, and subunit of NADPH oxidase, and nox-4 was identified as the pre- 100 mg/kg of -tochopherol acetate dissolved in liquid paraffin for dominant isoform. Immunohistochemistry studies showed nox-4 6 weeks. Quantitative real time RT-PCR, western blotting and en- expression was strikingly elevated in human HE validating nox-4 zyme assay were used to assess the level of Cu/Zn- SOD and Mn- as a therapeutic target. Post-transcriptional gene silencing of nox-4 SOD mRNA, protein, and the enzyme activity in blood cells re- using RNA interference techniques in EOMA cells resulted in de- spectively at weeks 0, 2, 4 and 6 following vit E administrations. creased oxidant production, decreased angiogenic capacity on Ma- Total antioxidant capacity was also assessed in plasma for all the trigel and decreased HE size in vivo. We then sought to determine mentioned groups at same time intervals. Results: Mn-SOD activ- ity showed significant increase (P<0.002) in 100 mg/kg dosing whether antioxidant delivery using a nutritional intervention such group after 4 and 6 weeks with no significant increase in its mRNA as blueberry extract (BBE) was effective in treating HE. BBE (150 or protein levels. Cu/Zn-SOD activity and the level of its mRNA ug/ml) was shown to inhibit c-Jun N-terminal kinase activation, and protein showed no significant changes during the study. Total AP-1 and NF-kB DNA binding as well as MCP-1 expression in antioxidant status showed significant increase in 100 mg/kg dosing EOMA cells. Oral gavage feeding of BBE (20 mg/kg) to mice with group after 2, 4, and 6 weeks during the study. Conclusion: only HE resulted in significantly decreased HE size and prolonged sur- chronic administration of higher doses of -tocopherol in rats is vival. These results indicate that HE formation is driven by nox-4 associated with the increase activity of Mn-SOD. Since neither derived oxidant production and that treatment with BBE may be an mRNA nor protein levels of the enzyme showed any significant effective alternative intervention. changes, it seems the enzyme is controlled at the level of its activ- ity. Cu/Zn-SOD seems to be constitutive with constant activity GMG supported by K08 GM066964 during the study in all examined groups.

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Age-dependent increase in cholesterol efflux, clearance and Dose-dependent modulation of systemic lipid peroxidation and catabolism in the aging intact 3xTG mouse model of activity of antioxidant enzymes by vitamin E in the rat Alzheimer's disease

Maliheh Hajiani, Abolfazl Golsetani, Ahmad Shariftabrizi, Ryan T. Hamilton§, Amanda J. Compadre§, Julian Lemus§, Roghieh Rastegar, Seyedmehdi Payabvash, Amirali Hasanzadeh, Esosa Agbonwaneten§, Jia Yao§, Ronald W. Irwin§, and Ahmad Reza Dehpoor, and Parvin Pasalar Roberta Díaz Brinton§

§ 1Department of Basic Sciences,Azad University of Jahrom; 2Department of Department of Pharmacology and Pharmaceutical Sciences, School of  Biochemistry , Tehran University of Medical Sciences; 3Department of Pharmacy, and Department of Neuroscience, Pharmacology , Tehran University of Medical Sciences University of Southern California, Los Angeles, CA 90089

The objective of this work was to examine the time-dependent Dysregulation of cholesterol homeostasis is an antecedent to pro-oxidant versus antioxidant effect of various doses of vitamin E the development of Alzheimer's pathology. Elevated neuronal used commonly in experimental studies. Erythrocyte activity of membrane cholesterol induces an aging-dependent production of superoxide dismutase (SOD), glutathione peroxidase (GPX), cata- amyloid beta that leads to amyloid plaque formation in vivo. This lase (CAT) and plasma lipid peroxidation levels were investigated study proposes that cholesterol trafficking, efflux and catabolism is following biweekly intramuscular administration of 100, 300, 600 increased in the triple transgenic AD (3xTG-AD) model. To test mg/kg of vitamin E at a baseline timepoint, and 2, 4, 6 weeks after this hypothesis entorhinal cortex sections (C2) were analyzed by initiating treatment.Vitamin E had an antioxidant effect when ad- western blot in intact female non-transgenic (non-TG) and 3xTG- ministered at low doses over short time period, and increased the AD mice. 3xTG-AD mice had elevated levels of 24-S-hydroxylase antioxidant enzymes. At higher doses and over long time periods, (CYP46) and ATP binding cassette protein 1 (ABCA1) at all ages it increased the level of lipid peroxidation, and attenuated the ac- while non-TG mice had an age-dependent increase in both. Apol- tivity of antioxidant enzymes. These results suggest that time de- ipoprotein E and F increased age-dependently in the 3xTG-AD pendent variations in vitamin E effects should be considered in de- while it was unchanged in the non-TG. Cholesterol uptake by sign and interpretation of experimental antioxidant studies, as well apolipoprotein E-receptor appeared to be unchanged at all ages in as during clinical trials both mice whereas low density lipoprotein-receptor decreased age dependently in both. Lysosomal trafficking of cholesterol by Niemman Pick C1 was decreased age dependently in both. Mito- chondrial cholesterol trafficking proteins steroid activated receptor and peripheral benzodiazopine receptor had a dose dependent in- crease with age in the 3xTG-AD while was unchanged in the non- TG. These findings suggest that cholesterol and lipid efflux, clear- ance and catabolism pathways are upregulated while uptake and lysosomal trafficking are decreased suggesting that cholesterol steady states appear to be associated with the mitochondrial and extracellular compartments in the 3xTG-AD mice.

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Protein unfolded low density lipoprotein inhibits Age-dependent increases in AMPK, JNK, and GSK3 mitochondrial bioenergetics: Implication for the atherogenic and role for caloric restriction: phenotype of coronary artery disease Implication for mitochondrial energy substrate control

Ryan T. Hamilton, Juliana Hwang-Levine, Howard N. Hodis, and Ryan T. Hamilton, Fei Yin, and Enrique Cadenas Enrique Cadenas Department of Pharmacology and Pharmaceutical Sciences, School of Department of Pharmacology and Pharmaceutical Sciences, School of Phar- Pharmacy, University of Southern California, Los Angeles, CA 90089, USA macy,
Atherosclerosis Research Unit Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA Aging-dependent hypometabolism precedes the develop-

ment of Alzheimer’s disease by a decade and is mitochondrial- Elevated low-density lipoprotein (LDL) is a risk factor for the dependent. Several canonical signaling pathways lead to this hy- development of atherosclerosis and the unfolded-LDL subfraction pometabolic phenotype including age-dependent activation of JNK in vivo (LDL–) is intimately associated with the development of and GSK3 phosphorylation while activation of AMPK may pro- cardiovascular disease. JNK-2 is implicated in the formation of vide a protective mechanism and they all converge on mitochon- foam cells and atherosclerotic lesions and interaction of oxLDL drial function. It is known that both GSK3 and JNK phosphory- with CD36 activates JNK-2 phosphorylation. These findings sup- lation increase with age in Fischer rats and that both pathways in- port a protein unfolded LDL-dependent activation of CD36 and hibit age-dependently PDH activity. These observations supported SR-A, the induction of JNK-2 and a subsequent mitochondrial en- the hypothesis that the interplay between JNK and GSK3 signal- ergy-redox regulation. We found that protein unfolded LDL de- ing with AMPK may provide mechanistic insight into Alzheimer’s creased ATP production in BAEC by 61 ± 23%, mitochondrial pathology and that caloric restriction a known modulator of life respiratory capacity by 31 ± 8%, and maximal anaerobic metabo- span may shift the balance in this paradigm. It was observed that lism by 25 ± 10% while increased proton leak by 36 ± 12% and there was an age-dependent increase in AMPK protein expression basal anaerobic metabolism by 140 ± 46%. ATP synthase and as well as its phosphorylation but its activation to total AMPK lev- PDH activity were decreased by protein unfolded LDL (42 ± 4% els were unchanged. Short-term caloric restriction at 6 months of and 47 ± 13%, respectively) and p-JNK-2-treated mitochondria age increased AMPK phosphorylation while it decreased AMPK had concomitant decreases in PDH (52 ± 5%) and ATP synthase phosphorylation at 14 months of age and p-AMPK/AMPK was un- activity (67 ± 19%) while JNK inhibitor blocked this loss in activ- changed at all ages. These findings suggest that AMPK may be ity (2± 10% and 11 ± 3%). Protein unfolded LDL decreased the mechanistically involved in neuroprotective functions of age- ATP to ADP ratio by 61 ± 10% and glutathione by 52 ± 13% and dependent hypometabolism and that caloric restriction may func- pre-treatment with JNK inhibitor ablated this decrease in tion protectively at younger age than older age. ATP/ADP by 1 ± 5% and GSH by 16 ± 15%. These findings dem- onstrate a novel mechanism by which protein unfolded LDL (LDL–) impairs mitochondrial bioenergetics as mediated by p- JNK-2 and implicates a necrotic phenotype (low GSH/GSSG and low ATP) and the importance of LDL– in the development of a cardiovascular disease phenotype.

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Effects of high-dose B-complex vitamins plus calcium, Ameliorative effect of photoilluminated riboflavin onto the magnesium and zinc (Berocca®) on subjective mood and cisplatin-induced oxidative stress in vivo

cognitive performance in healthy males 1 2 1 1 Iftekhar Hassan , Iyad Ali , Sandesh Chibber , Imrana Naseem * 1 1 1 Crystal F. Haskell , David O. Kennedy , Anthony Watson , 1 1 2 2 Department of Bichemistry, Faculty of Life Sciences, Aligarh Muslim Rachel Veasey , Jens Lüdemann , Volker Baroth , University, Aligarh, U.P., India; 2Department of Biochemistry and Genetics, Stephen Beveridge2, and Silvia Maggini2 Faculty of Medicine, An-Najah National University, Nablus, Palestine. 2Presenting author 1Brain Performance and Nutrition Research Centre, Northumbria University, UK; 2Bayer Consumer Care Ltd, Basel, Switzerland Cisplatin is a widely used anticancer drug against various can- cers and solid tumors. It is documented that it generates reactive A significant proportion of the general population report supple- oxygen species leading to its major side effects like nephrotoxicity menting their diet with one or more vitamins or minerals, with stress, and hepatotoxicity which limits its clinical use for long time. Ribo- fatigue or improvement of cognitive performance the most commonly flavin is an essential vitamin found in all forms of life. Being a reported reasons for doing so. In this respect, very few studies have well known photosensitizer, it generates reactive oxygen species assessed the relationship between supplementation with vita- (ROS) upon photo-illumination at high concentration which are mins/minerals and psychological functioning in healthy cohorts of known to cause the oxidative damage to cellular biomolecules viz. non-elderly adults. In this randomized, placebo-controlled, double- proteins, lipids and DNA. This property of riboflavin is utilized in blind, parallel groups trial the effect of a high-dose B-complex vitamin photodynamic therapy (PDT) and ribophototherapy for the treat- and mineral supplement (Berocca®) was assessed in 215 males aged ment of various diseases including cancer. We have tried to show 30 to 55 years, who were in full time employment. Participants at- the ameliorative effect of riboflavin onto cisplatin induced oxida- tended the laboratory prior to, and on the last day of a 33-day treat- tive stress in mice. We treated mice with riboflavin and cisplatin ment period. During both visits participants completed the Profile of separately and also in the two combination of cisplatin and ribofla- Mood States (POMS), Perceived Stress Scale (PSS) and General vin in the ratio of 1:0.5 and 1:1 under photoilluminated condition. After 8 doses of the treatment for a month, the assay of major anti- Health Questionnaire (GHQ-12). Cognitive performance and task re- oxidant enzymes (superoxide dismutase, catalase, glutathione re- lated modulation of mood/fatigue were assessed with the 60 minute ductase, alkaline phosphatase) and antioxidant proteins (reduced Cognitive Demand Battery (CDB). In addition, on the final day, par- glutathione, sulfhydryl groups) and glutathione –S-transferase ticipants completed the Stroop task for 40 minutes whilst engaged in along with the estimation of liver and kidney function markers inclined treadmill walking and subsequent executive function was as- were carried-out of their kidney, liver and serum. Beside these, the sessed. Results show that participants in the vitamin/mineral group had extent of lipid peroxidation and protein oxidation were also studied significant improvements in their ratings on the PSS, GHQ-12 and the in the samples. The level of antioxidant enzymes and the proteins ‘vigor’ subscale of the POMS. They also performed better on one (of were significantly compromised in the cisplatin treated group three) CDB battery tasks (Serial 3s subtractions) and rated themselves along with higher extent of lipid peroxidation and protein oxida- as less ‘mentally tired’ both pre- and post- completion of the Cognitive tion as compared to the control. The pattern was similar but not so Demand Battery. In conclusion, these findings suggest that healthy pronounced in the riboflavin treated group. The groups under the members of the general population may benefit from augmented levels treatment with combination of both cisplatin and riboflavin showed of vitamins/minerals via direct dietary supplementation. Specifically that all the parameters tended towards the normal levels in a dose dependent manner with increasing dose of riboflavin. Hence, it can supplementation led to improved ratings of stress, mental health, and be hypothesized that riboflavin shows ameliorative effect onto the vigor, and improved cognitive performance during intense mental cisplatin –induced oxidative stress in vivo. processing.

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Multiple pathophysiological roles of mitochondrial fission and 57% average increase in equine serum nitric oxide levels fusion upon nanophotodynamic effect of fullerene-induced mitochondrial oxidative stress following oral administration of multi-nutrient nitric oxide precursor Cheng-En Hsieh1 and Mei-Jie Jou2

1 School of Medicine, Chang Gung University, Kwei-Shan, Tao-Yuan, Taiwan Gerald K. Huff, Tim Baum (Avid Labs), 2 Department of Physiology and Pharmacology, Chang Gung University, and Tamara Sofi (THE Labs) Kwei-Shan, Tao-Yuan, Taiwan Subjects evaluated and treated in Las Vegas, Nevada by Gerald K. Mitochondrial fission and fusion machinery possesses vital Huff, DVM, 2315 North Decatur, Las Vegas, CA 89108. Testing per- function in maintaining the mitochondrial integrity and network, formed by Avid Labs, 100 Memorial Drive, Greer, South Carolina bioelectrical and chemical connectivity, turnover of mitochondria, 29650. Formulation performed by THE Labs, 27128 Paseo Espada, and segregation and protection of mitochondrial DNA (mtDNA). Substantial evidence indicates that extensive mitochondrial fission San Juan Capistrano, CA 92675 occurs during oxidative insult, a key reason for various diseases Equine laminitis causes chronic lameness or death in countless and molecular aging, yet the detailed cellular functions and effects horses every year. Laminitis causes inflammation to the laminae, lead- of such morphological response remain unclear. We investigated ing to rotation or sinking of the coffin bone. If the hoof does not re- pathophysiological roles of mitochondrial fission and fusion ma- align, pain will continue and the horse may be euthanized. USDA chinery upon mitochondria-targeted reactive oxygen species grant funded Veterinary researchers Moore and Eades (Louisiana State (mROS) generation by the photodynamic effect (PDE) of a new University, school of Veterinary Medicine) believe the initiating factor class of water-soluble nanophotosensitizer carbon 60, tris-malonic in the onset of laminitis is an imbalance in blood flow to laminae acid carboxyfullerene (C3), coupled with 543 nm laser in wild- caused by decreased production of nitric oxide (NO) (which normally type, fission enhanced and fusion enhanced 143B osteosarcoma relaxes blood vessels and increases blood flow) and increased release cells. Mitochondrial level of dynamic alterations including mor- of endothelin-1 (which causes blood vessel contraction and subsequent phology, oxidative strength, lipid peroxidation,  m, cardiolipin decreases in blood flow). This imbalance ultimately leads to decreased (CL), and mCa2+ were time-lapsed imaged using laser scanning laminar blood flow, laminar swelling, tissue death, and subsequent confocal microscopy coupled with various fluorescent probes in- cluding MitoTracker Green, 2',7'-dichlorofluorescin diacetate separation of the laminae. Studies suggest equine ulcer formation may (DCF), C11-BODIPY581/591, tetramethyl rhodamine methyl ester be caused by a similar NO deficiency pathology. In this equine study (TMRM), 10-N-nonyl acridine orange (NAO), and rhod-2. Our re- 23 horses were tested for serum NO levels (samples drawn from the sults revealed that fission of mitochondria reduced significantly jugular vein) using highly reproducible Elisa Assays in order to estab- mROS propagation for less lipid peroxidation and  m loss lish baselines. Of these 23 subjects, 10 were tested for serum NO lev- whereas fusion of mitochondria facilitated mitochondrial destruc- els before and one half hour after being orally administered NitrOxide, tion. Whereas, fission of mitochondria accelerated and fusion de- a medical food containing amino acids (to include L-arginine), B vi- 2+ creased  m depolarization, CL peroxidation, mCa overload, and tamins, network antioxidants, flavanoids, trace minerals, and herbs. eventual cell death upon lethal doses of mitochondrial oxidative Subjects experienced an average 57% increase of NO production over insults. Thus, rearrangement of mitochondrial network via fission baseline in serum NO levels. Study graph showing individual subject or fusion may provide potential diverse protection against or aug- levels is available. mentation towards mitochondrial oxidative stress associated apop- Med-Pharmex Animal Health funded this study and generously granted permission to share tosis. study results within the context of scientific conferences. Further studies are being planned and funded.

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Gender differences of LDL receptor production in response to statin treatment Differential regulation of brain mitochondrial function by clinically relevant progestins 1,2 1 Juliana Hwang-Levine , Enrique Cadenas , and Ronald W. Irwin, Jia Yao, S. S. Ahmed, Ryan T. Hamilton, 2 Howard N. Hodis Enrique Cadenas, and Roberta Díaz Brinton

1 2 Pharmacology & Pharmaceutical Sciences, School of Pharmacy, Department of Pharmacology and Pharmaceutical Sciences, Atherosclerosis University of Southern California, Los Angeles, CA 90089 Research Unit, Schools of Pharmacy and Medicine, University of Southern California Mitochondrial dysfunction and reduction of the cerebral meta- bolic rate for glucose precede neurodegenerative diseases includ- Low-density lipoprotein receptor (LDLr) is a cell surface re- ing Alzheimer’s disease. Previously, we identified brain mitochon- ceptor that recognizes apoB and mediates endocytosis of choles- drial proteomic changes associated with acute estradiol (E2) re- terol ester-rich LDL. An LDLr precursor is synthesized at a mo- placement that indicated enhanced brain mitochondrial efficiency lecular weight of 120 kD; N- and O-linked sugars are added to the as evidenced by increased respiratory control ratio, elevated cyto- LDLr in the endoplasmic reticulum and Golgi apparatus resulting chrome-c oxidase activity and expression while simultaneously re- in an increase of LDLr weight to 160kD. Mature 160 kD LDLr is ducing free radical generation in brain (Nilsen, Irwin, et al., J. Neu- rosci 2007). We extended these findings to demonstrate E2 and transported to the cell surface to become the active binding site for progesterone (P4) regulate oxidative metabolism in brain mito- the LDL particle removing cholesterol from circulation. This study chondria (Irwin, et al., Endocrinology 2008). P4 is neuroprotective was conducted to determine whether LDLr production differs be- whereas the synthetic progestin, medroxyprogesterone acetate tween genders. Male and female human aortic endothelial cells (MPA), most widely prescribed for the treatment of menopausal (HAEC) were cultured according to the manufacturer protocol. symptoms is not neuroprotective (Nilsen & Brinton, Endocrinol- Cells were replated for 24 h in phenol red-free media prior to sim- ogy 2002). In this current study we used a functional biochemical vastatin (Sim) treatment. LDLr protein levels were determined by approach to identify the mechanism responsible for interactions be- western blot analyses in cell lysates and normalized to -actin. tween estrogen and progestins. We investigated brain mitochon- Female HAEC exhibit a greater 160 kD/120 kD LDLr protein ratio drial function of ovariectomized (OVX) 5-month female rats (n = than male HAEC when cultured in normal media. Treatment with 5) following 24h treatment with MPA, E2, E2 + MPA, vehicle, and 5 mM Sim increased both the 160 kD and 120 kD proteins in male ShamOVX. MPA alone and MPA + E2 resulted in diminished mi- and female HAEC with increase in the 160 kD protein greater in tochondrial respiration and ATP generation. Treatment changes in male HAEC than female HAEC. These data indicate for the first mitochondrial proteins relative to controls were determined via time that expression and production of LDLr in HAEC treated with immunoblot and enzyme activity assay for tissue lysates of hippo- statin varies between genders. With statin treatment, there is a sub- campus, cortex, cerebellum, and purified whole brain mitochon- dria. Progestin-mediated alterations in oxygen utilization were stantial increase in the 160 kD/120 kD LDLr protein ratio in male measured via the Seahorse XF24 metabolic analyzer in primary HAEC as compared with female HAEC indicating that male cells cultured neurons and isolated brain mitochondria. The set of mito- are more responsive to statin. These results may in part explain the chondrial proteins altered by interactions between E2 and MPA in- differential effect of lipid-lowering therapies on cardiovascular cluded ATP synthase, PDH, COXIV, MnSOD, and Prdx. We are outcomes between genders. Further exploration of these findings currently investigating several clinically relevant progestins in or- may lead to improve cardiovascular prevention for women. der to distinguish the negative from the positive neural effects of hormone therapy.

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Effects of multivitamin supplementation (Supradyn®) on Red wine polyphenols may influence human space memory perceived fatigue and cognitive functions during extended

multi-tasking assessment F. Jagla, B. Cimrova, S. Budac, M. Jergelova, S. Bendzala, and O.Pechanova 1 1 1 David O. Kennedy , Crystal F Haskell , Bernadette Robertson , 1 1 2 2 Institute of Normal and Pathological Physiology, Slovak Academy of Sciences, Joanne Gee , Rebecca Jones , Jens Lüdemann , Volker Baroth , 2 2 Bratislava, Slovakia Volker Spitzer , and Silvia Maggini

Background: The nitric oxide (NO) release within the brain is 1Brain Performance and Nutrition Research Centre, Northumbria University, UK; 2Bayer Consumer Care Ltd, Basel, Switzerland necessary for long–term potentiation, the electrophysiological events related to synaptic plasticity and learning. The NO synthase Fatigue is one of the generally recognized symptoms of micro- (NOS) inhibitors disrupt the performance of animals in learning nutrient deficiencies. Despite the general consensus that fatigue is tasks while the NO donors display an antiamnestic action. It seems a highly prevalent problem, and can be associated with vita- that NO may be involved in post-training memory processes in min/mineral deficiencies, to date no research has directly investi- rats. It was the reason to analyze the influence of a polyphenolic TM gated the effect of multi-vitamin/mineral supplementation on lev- compound, the NO synthase activator - Provinols , upon the hu- els of fatigue and cognitive functions. In this placebo controlled, man space memory functions. double-blind, randomized, controlled trial the effect of a commer- Methods: The analysis of the accuracy of visually evoked sac- cially available multi-vitamin/mineral (Supradyn®) was assessed cadic eye movements and of saccades driven by memory informa- in a cohort of 220 females aged 25 to 50 years, who reported sub- tion about the eye landing position was used in healthy volunteers. jective fatigue and who were in full time employment or childcare. The saccades elicited by the visual targets were followed by the Participants attended the laboratory prior to, and on the last day of memory-guided saccades. The whole procedure was repeated 2 the 9 week treatment period. During both visits mood was assessed hours after, following the Provinols™ administration (4 mg/kg of with questionnaires. Cognitive performance and task related modu- body weight). According to the clock face the saccadic landing po- lation of mood/fatigue were also assessed during two discrete 20 sitions were randomly selected at 1, 2, 4, 5, 7, 8, 10 and 11 hour. minute periods during which participants completed a four module The EEG spectral powers related to the eye movements were ana- version of the Multi-Tasking Framework, with mood assessments lyzed as well. before and after completion. The results showed that those in the Results and Discussion: The Provinols™ positively affected vitamin/mineral group exhibited an attenuation of the negative ef- the accuracy of both the saccades. The EEG spectral powers point fects on mood/fatigue of extended task completion. Multi-tasking to the cortex activation by Provinols™. The memory-guided sac- for this group was also improved overall in terms of accuracy, and cade task comprises perception, memorization and execution of a on two of the modules (Mental Arithmetic and Stroop) in terms of saccade. Perception is under control of the network which subserve both faster and more accurate responses. An analysis conducted in also the attention mechanisms. Memorization is controlled by the a subsection of the cohort (N = 102) demonstrated significant re- dorsolateral prefrontal cortex and programming and generation of a ductions in blood levels of homocysteine following the vita- MGS involve the working memory. It is suggested that Provi- mins/mineral supplement. These findings suggest that healthy nols™ may affect attentional mechanisms as well. members of the general population that report fatigue may benefit Partly supported by VEGA grant No. 2/0160/08 and APVV - 0538-07. from augmented levels of vitamins/minerals via direct dietary sup- plementation.

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Arachidonic acid pathway as a key target for neuroprotection by nanomolar -tocotrienol

Savita Khanna1, Narasimham L. Parinandi2, Sainath R. Kotha2, Expression level of peroxiredoxin isoforms Sashwati Roy1, Cameron Rink1, Douglas Bibus3, and in response to cervical carcinogenesis Chandan K. Sen1 1 1 1 1 1 Kiyoon Kim , Miran Yu , Seulhee Han , Inkyung Oh , Dorothy M. Davis Heart and Lung Research Institute, Department of Surgery, 2 1 1 2Department of Internal Medicine, The Ohio State University Medical Center, Young-Jun Choi , Sungsoo Kim , Kyungsik Yoon , Columbus, OH 43210 and 3The Holman Center for Lipid Research and Lipid Minhyung Jung2 and Wonchae Choe1 Technologies and The Center for Spirituality and Healing, University of Minnesota, Austin, MN 55912 1)Department of Biochemistry and Molecular biology, Medical Science and Previous studies from our laboratory have identified - Engineering Research Center for Bioreaction to Reactive Oxygen Species, Biomedical Science Institute, School of Medicine, Kyunghee University, Seoul tocotrienol (TCT) as a natural form of vitamin E with potent neu- 130-701; 2Department of Obstetrics and Gynecology, School of Medicine, roprotective properties. At nM concentrations, TCT protects Kyung Hee University, Seoul 130-701, Korea against a variety of insults e.g. glutamate via inhibition of a 12-Lox catalyzed arachidonic acid (AA) pathway of neurodegeneration. Despite considerable progress in understanding the function of Phospholipase A2 (PLA2) activity mobilizes AA from membrane peroxiredoxin (Prx) in cancer, its expression patterns have not been phospholipids (PLs). The goals of the current study were to test extensively studied in response to cervical carcinogenesis. We whether: (i) PLA2 is sensitive to glutamate challenge; (ii) PLA2 is evaluated the expression of Prx isoforms in normal tissue, cervical implicated in glutamate-induced cytotoxicity; and (iii) TCT regu- intraepithelial neoplasia (CIN1, CIN2, and CIN3), and cervical lates glutamate-induced PLA2 function. HT4 neural cells were cancer. We found strong pattern of increased Prx II and III immu- studied in culture. Glutamate (10 mM), at 30 min of treatment, in- nostaining with increasing severity of the lesion. No difference in duced the release of [3H] AA from cells that was significantly at- staining intensity by grade of lesion was observed for Prx I, and tenuated by calcium chelators (EGTA and BAPTA), cPLA2- IV. Therefore, we conclude that Prx II and III are upregulated in specific inhibitor, AACOCF3, and TCT (250 nM). Glutamate also response to the development of cervical cancer. caused the elevation of free polyunsaturated fatty acid (AA and docosahexaenoic acid) levels and disappearance of PL-esterified AA in HT4 cells. These responses were significantly attenuated by TCT. Moreover, glutamate induced a time-dependent (0-30 min) translocation and enhanced serine phosphorylation of cPLA2 in a TCT-inhibitible manner. cPLA2-specific inhibitor (AACOCF3), transient knock-down of cPLA2 expression by cPLA2 siRNA trans- fection, as well as TCT significantly protected against the gluta- mate-induced cytotoxicity in HT4 cells. Results of this study repre- sent the first evidence demonstrating that glutamate-induces cPLA2 activation and that PLA2 is directly implicated in glutamate- induced cytotoxicity. Importantly, glutamate inducible PLA2 is sensitive to nM TCT. Thus, TCT may limit membrane AA mobili- zation in response to glutamate insult. Support: NIH Grant NS42617

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Supplementation of a Selenocystine derivative prevents Long-term treatment with schisandrin B ameliorates the im- the ionizing radiation induced oxidative stress in mice paired mitochondrial antioxidant status in various tissues of chronic ethanol-intoxicated rats A. Kunwar and K.I. Priyadarsini Philip Y. Lam, Po Yee Chiu, Hoi Yan Leung, Na Chen, Radiation and Photochemistry Division, Bhabha Atomic Research Centre, Pou Kwan Leong, and Kam Ming Ko Trombay, Mumbai-400085, India Department of Biochemistry, The Hong Kong University of Science & Radioprotectors are employed to minimize the damaging ef- Technology, Clear Water Bay, Hong Kong SAR, China fects of ionizing radiation to normal cells during radiotherapy. In present study we report the in vivo radioprotection effecicay of a Chronic alcohol consumption can lead to multiple organ dys- water-soluble synthetic derivative of selenocystine, 3,3'-Diseleno- function. In the present study, a rat model of chronic ethanol in- dipropionic acid (DSePA). The radio protective effects of DSePA toxication was utilized to investigate the effect of schisandrin B was examined in Swiss albino mice, after administration at a dos- (Sch B, a dibenzocyclooctadiene compound isolated from Fructus age of 2 mg/kg body weight (i.p.), for 5 days prior to whole body Schisandrae) on ethanol-induced oxidative damage in the brain, exposure to sub and supra lethal doses of -radiation. DSePA in- heart, liver and kidney. Chronic ethanol consumption induced gen- hibited radiation induced hepatic lipid peroxidation, protein car- eralized oxidative tissue damage, as indicated by increases in bonylation, loss of hepatic function and deformation in hepatic ar- plasma sorbitol dehydrogenase (SDH), alanine aminotransferase chitecture. It also protected against the depletion of endogenous (ALT) activities and reactive oxygen metabolites (ROMs) level, as antioxidants viz., glutathione (GSH), GPx, superoxide dismutase well as mitochondrial malondialdehyde (mtMDA) production in (SOD), and catalase in the hepatic tissue of irradiated mice. Protec- various tissues of rats. The ethanol-induced oxidative damage was tion towards GI tract and hematopoietic system was confirmed by associated with impairment in mitochondrial antioxidant status and the restoration of radiation induced reduction in villi height, num- activation of heat shock response. Among all tissues examined, the ber of crypt cells and spleen cellularity. The mRNA expression liver was most susceptible to ethanol-induced oxidative damage, as analysis revealed that DSePA treatment caused augmentation of evidenced by a significant increase in mtMDA production and de- GADD45 and inhibition of p21 levels in spleen and the hepatic crease in mitochondrial ATP generation capacity (ATP-GC). tissue. Additionally, it also reversed the radiation induced altera- Long-term Sch B treatment was found to enhance mitochondrial tions in expression of pro-apoptotic BAX and anti-apoptotic Bcl-2 antioxidant and functional status in a tissue non-specific manner. genes, which may be favoring towards survival. In line with these The ethanol-induced oxidative stress was ameliorated by Sch B observations, DSePA improved the 30-day survival of the irradi- treatment, as indicated by the reversal of altered plasma SDH, ALT ated mice by 30%. Based on all these studies it is concluded that activities and ROMs level, as well as mtMDA production and heat DSePA exhibits potent radioprotective effects against whole body shock protein 25/70 expression in various tissues of chronic etha- -radiation and the probable mechanisms of action involves main- nol-intoxicated rats. The results suggest that long-term Sch B tenance of antioxidant enzymes, prophylactic action and inhibition treatment may increase the resistance of mitochondria to oxidative of apoptosis. stress and thereby ameliorate the impaired mitochondrial antioxi- dant status in chronic ethanol-intoxicated rats.

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Effect of benzyl isothiocyanates on the toxicity of chemothera- peutic drugs in cancer cell lines and normal A link between mtOGG1-mediated respiratory defect human lymphocytes and mitochondrial ROS

1,3 1,3 1,3 1 1 1 Young-Kyoung Lee , June-Hyung Kim , You-Mie Kim , Younghyun Lee ,Yang Jee Kim , Young Joo Choi , 2,3 1,3 1 1 1 1 Soo-Han Yoon and Gyesoon Yoon Hae Dong Woo , Joong Won Lee , Tae Kyung Ha ,Yeonju Choi , 1 1 1Department of Biochemistry & Molecular Biology and 2Department of Sunyeong Lee , and Hai Won Chung 3 Neurosurgery, School of Medicine and Department of Molecular Science & 1 Technology (BK21), The Graduate School, Ajou University, Suwon, 443-721, School of Public Health and Institute of Health and Environmental Sciences, Korea. E-mail: [email protected] Seoul National University, Seoul, South Korea Mitochondrial genome is a 16kb-long and double-stranded circular Natural isothiocyanates (ITCs) isolated from cruciferous vege- molecule. It encodes 13 proteins essential for respiratory chain reac- tables are known to be effective chemopreventive agents. Sul- tion, but known to be highly prone to oxidative damage because of its foraphane(SFN) and Benzyl isothiocyanate(BITC) are naturally unprotected structure and its environment close to continuous reactive occurring members of the isothiocyanate family. In this study, we oxygen species (ROS) production. Diverse mtDNA mutations and de- evaluated the effects of ITCs(BITC or SFN) on anticancer drugs letions have recently been postulated in carcinogenesis. However, (Cisplatin or 5-Fluorouracil)-induced cytotoxicity in HL-60 cells there is no direct evidence whether and how mtDNA damage contrib- and normal human lymphocytes. HL-60 cells were treated with utes to cancer development. Here, we focused on elucidating the po- ITCs (BITC or SFN) for 3 h followed by treatment with che- tential involvement of mtDNA damage in hepatocellular carcinoma motherapeutic drugs(3μM Cisplatin or 10μM 5-Fluorouracil). Pre- (HCC). Firstly, we assessed mitochondrial function of SNU HCC treatment of BITC caused a significant decrease of cell viability cells. Expression levels of respiratory complexes and O2 consumption and an increase of DNA migration. It also increased apoptotic cell rates were largely decreased whereas ROS production was signifi- death compared with either agent alone. However, SFN-mediated cantly increased in most SNU HCC cells compared to Chang cell, an effects were not observed. Cellular exposure to BITC in combina- immortalized human normal hepatocyte. Secondly, we examined dele- tion with anticancer drugs increased reactive oxygen species tion of mtDNA from SNU cells with Southern blotting. Deleted forms (ROS) generation and decreased intracellular glutathione content. of mtDNA were not detected clearly but there were obviously distinct Pretreatment with N-acetyl cysteine effectively inhibited BITC/ digestive patterns by restriction enzyme between Chang and SNU anticancer drugs-induced apoptosis. In human lymphocytes, ITCs cells. Surprisingly, when we monitored expression level of mitochon- did not enhance the cytotoxic effects of anticancer drugs and re- drial 8-oxoguanine DNA glycosylase (mtOGG1), one of the DNA re- duced anticancer drugs-induced DNA damage. This implies that a pair enzymes localized in mitochondria, mtOGG1 expressions were treatment of BITC/anticancer drugs could be useful to increase the clearly down-regulated in the SNU cells of which mitochondrial func- efficacy with avoiding possible side effects following conventional tions were defective unlike ncOGG1, isotype of mtOGG1 located in anticancer drugs. In conclusion, these findings suggest that BITC nuclear. Similar results were also found in human HCC tissues. Fi- may be effective as a therapeutic agent to treat leukemia. nally, we observed that O2 consumption rate was decreased by mtOGG1 knockdown using siRNA dose-dependently in Chang cells. Taken together, our results suggest that mtDNA damage induced by down-regulated mtOGG1 may contribute to tumor development via mitochondrial dysfunction and mitochondrial ROS.

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The antioxidant response element (ARE) has a major role in the defense against oxidative stress and cancer PI3K regulation of cellular bioenergetics Joseph Levy, Michael Danilenko, and Yoav Sharoni Chen Li, Bangyan Stiles, and Enrique Cadenas Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University and Soroka Medical Center, Beer-Sheva, Israel Department of Pharmacology and Pharmaceutical Sciences, School of In recent years, evidence has accumulated indicating that the can- Pharmacy, University of Southern California, Los Angeles, CA, USA cer preventive action of various dietary ingredients such as caro- tenoids, polyphenols and isothiocyanates is, at least in part, due to the PI3K regulation of cellular bioenergetics was examined in a induction of phase II enzymes. Expression of these enzymes is regu- liver-specific PTEN knockout mouse model that exhibits a robust lated by the antioxidant response element (ARE) and the transcription PI3K/Akt signaling. Bioenergetics (glycolysis and mitochondrial factor Nrf2. Dietary inducers of Nrf2 are diversified in their chemical respiration) of cultured hepatocytes was measured by extracellular flux analysis and data showed that hepatocytes cultured from structure but are usually electrophiles which react with Keap1 to dis- -/- rupt its inhibitory activity on Nrf2. It is interesting that hydrophobic PTEN knockout mice (PTEN ) exhibited higher anaerobic and carotenoids such as lycopene which activate Nrf2 lack any electro- aerobic metabolism. Treatment of cultured hepatocytes with the philic group and thus are unlikely to interact directly with Keap1. In- PI3K inhibitor LY290004 led to a decrease in mitochondrial respi- deed, we have demonstrated that intact carotenoids do not directly ac- ration suggesting the involvement of the PI3K/Akt pathway in the tivate the ARE transcription system and that carotenoid oxidation mitochondrial energy-transducing process. Mitochondria isolated from PTEN-/- mouse liver had a higher respiratory control ratio products are the active mediators in this stimulation. Furthermore, the -/- activity of the carotenoid derivatives depends on the relative position than those of wild type mice. PTEN hepatocytes showed in- creased phosphorylation of Akt (Ser473), GSK3 (Ser9) and of the methyl group to the terminal aldehyde which determines the re- 21 activity of the conjugated double bond in reactions such as Michael GSK3 (Ser ), increased association of phospho Akt and de- addition to SH groups in Keap1.We recently found that Nrf2 is in- creased association of unphosphorylated GSK3/ isoforms with volved in other anti-cancer effects of phyto-nutrients in hormone de- mitochondria. Pyruvate dehydrogenase activity was higher in PTEN-/- hepatocytes. The increased mitochondrial bioenergetics in pendent cancers. Various phyto-nutrients inhibit estrogen signaling, -/- the major risk factor in breast and endometrial cancer. Using overex- PTEN mice may be partially due to PI3K regulation at (a) cyto- pression of Nrf2 and siRNA for this gene, we demonstrated that Nrf2 solic level, through the phosphorylation of Akt and GSK3/ is involved in the phytonutrient-induced inhibition of the estrogenic thereby favoring the translocation of Akt while inhibiting the trans- activity. Although the effect of estrogens in breast and endometrial location of GSK3/ to mitochondria and the subsequent modula- cancer is harmful, it is beneficial for bone formation. Thus, we inves- tion of mitochondrial protein phosphorylation and activity; and (b) tigated the effect of the phyto-nutrients on estrogenic activity in os- transcriptional level, through the regulation of NRFs and PGC1 which modulate the transcription of mitochondrial genes. It may be teoblasts. Surprisingly, we found that the same dietary compounds, which inhibit estrogenic activity in cancer cells, did not inhibit and summarized that PI3K activation enhances mitochondrial bioener- even stimulated the expression of estrogen-induced genes in os- getics through enhancing substrate availability (glycolysis) and the teoblast like cells. The results suggest that the Nrf2 transcription fac- regulation of mitochondrial function at the cytosolic and transcrip- tor is involved in these differential activities of plant-derived phytonu- tional levels. trients in bone and cancer cells.

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New strategy for high-sensitivity RSNOs assay based on (-)-Epicatechin reduces L-NAME induced hypertension and catalysis of gold nanoparticles oxidant production in rats Yang Liu1, Hong-ying Jia1, Li-bo Du1, Xue-ji Zhang2, Corina Litterio1, Paula D. Prince1, Patricia I. Oteiza2,3, Ying-lin Sha3, Lu Han1, and Qiu Tian1 César G. Fraga1,2, and Mónica Galleano1 1Institute of Chemistry, The Chinese Academy of Sciences, Beijing 100190, 2 1 China.; Department of Chemistry, University of South Florida, 4202 East Physical Chemistry-PRALIB, Pharmacy and Biochemistry, University 3 2 Fowler Avenue, CHE-205A, Tampa, Florida 33620-5250 USA; Single Molecule Buenos Aires-CONICET, Buenos Aires, Argentina; Departments of and Nano-biomedicine Laboratory, Department of Biophysics, Peking Nutrition and 3Environmental Toxicology, University of California, University School of Basic Medical Sciences, Biochemistry Building 204#, Xue- Davis, USA yuan Road 38#, Beijing 100083, China

Flavanoids are naturally occurring plant compounds, which S-nitrosothiol (RSNOs) by the nitric oxide (NO)-dependent S- biological effects could explain some of the cardiovascular bene- nitrosation of thiol-containing proteins and peptides has multiple fits linked to the consumption of fruit and vegetables. Dietary in- biological functions including platelet deactivation, immunosu- tervention studies in humans and animals indicate that flavanol- pression, neurotransmission, and apoptosis. Most of the functions rich foods, i.e., wine, tea, and chocolate may exert blood pressure are attributed to NO release during the RSNOs decomposition. (BP) lowering effects, and other cardiovascular benefits. However Therefore, the quantification of S-nitrosothiols is essential to un- the biochemical mechanisms mediating those benefits are still un- derstanding the NO-mediated biological events. This study aims to der debate. To advance on the understanding the effects of promote a highly sensitive assay for RSNOs by catalysis of gold flavanols on BP, we used a model of induced hypertension in rats nanoparticles in biological media. by treatment with the NO-synthase inhibitor L-NAME. The addi- Gold nanoparticles have been shown to quantitively catalyze tion of (-)-epicatechin (EC) in the diet (0.2-4.0 mg/g diet) signifi- NO releasing whenever they come into contact with RSNOs and cantly modulated the L-NAME-dependent BP increase in a dose- the approximate limit of detection of the assay is in the picomole to dependent manner. The decrease in BP was associated with the fetomole range that rely on the ligand exchange reaction on the presence of EC in plasma, the decrease in markers of oxidative surface of the nanoparticles. The process is ascribed to the forma- damage, i.e. malondialdehyde, nitrated proteins, and oxidized glu- tion of the Au-thiolate on the surface of gold nanoparticles and the tathione, and an increase in plasma NO. These results are suggest- simultaneous cleavage of the S-N bond with release of nitric oxide. Furthermore, the quantitive detedtion of RSNOs is free from inter- ing that by lowering cell oxidants EC is maintaining an optimal - - NO steady-state level. These conditions finally result in the main- ference of various endogenous substances, such as NO2 , NO2 and tenance of normal BP values. GSH, which may co-exist under biological conditions.

This work was partially supported by NIH AT2966; UBACyT (B801 and B802, and Support by NSFC(No. 90813021 & 20875093) ANPCyT PICT 994. MG and CGF are members of the CIC, CONICET, Argentina Jia HY, Liu Y, Zhang XJ, et al. Potential Oxidative Stress of Gold Nanoparticles by In- duced-NO Releasing in Serum, J Amer Chem Soc 131(2009)40-41

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NSAIDs and acidic environment induce gastric mucosal Generation of reactive oxygen species by heating cellular mitochondrial dysfunction aqueous solution containing oxygen
H. Matsui, K. Hiyama, T. Kaneko, Y.N. Nagano, Ken-ichiro Matsumoto1, Minako Nyui1, Masato Kamibayashi1, O. Shimokawa, and A. Hirayama Toshihiko Ozawa1,2, Ikuo Nakanishi1, and Kazunori Anzai1

Graduate School of Comprehensive Human Sciences, 1Radiation Modifier Research Team, Heavy-Ion Radiobiology Research Group, University of Tsukuba, Japan Research Center for Charged Particle Therapy, National Institute of 2
Radiological Sciences, Chiba-shi, Chiba 263-8555, Japan, Department of Analytical Pharmacology, Yokohama College of Pharmacy, Yokohama-shi, Backgrounds & Aims: We have previously reported that both Kanagawa 245-0066, Japan gastric cellular exposure to acidic environment and indomethacin treatments induced lipid peroxidation and cellular apoptosis (Na- Interest for hyperthermia clinical cancer treatment has been in- gano, 2005). However, possible mechanisms of these phenomena creased recently, especially in combination use with existing still remain unknown. In the present study, we elucidate whether treatments, i.e. chemotherapy, radiotherapy and immunotherapy to both indomethacin treatment and extracellular acidic environment increase efficiency of those treatments. Mechanism for hyperther- cause mitochondrial dysfunction or not. Moreover, we investigate mal cell killing or sensitization to other stresses/treatments has the effect of rebamipide on these cellular injuries. been unclear. Relation of reactive oxygen species (ROS) to the ef- Methods; To examine the relations between extracellular pH fect of hyperthermia has been widely alleged and/or reported. Ni- and mitochondrial function, cellular oxygen consumptions in vari- troxyl radicals have been conventionally used as chemical redox ous pH media (1~7.4) were measured with an oxygen electrode. probe in vitro and in vivo experiments in fields of electron par- Mitochondrial membrane potential was measured with a fluores- amagnetic resonance (EPR) spectroscopy. In this presentation, cent probe JC-1 and a spectrofluorometer. Intercellular pH was ex- temperature-dependent free radical reactions were investigated us- amined with a fluorescent probe BCECF-AM and a ing nitroxyl radicals as redox probes. Gluthathione (GSH)- spectrofluorometer. We measured intracellular reactive oxygen dependent EPR signal loss of nitroxyl radicals occurred tempera- species (ROS) with electron paramagnetic resonance (EPR) using ture-dependently. Details of temperature-dependent reaction of ni- spin trapping reagents such as CYPMPO. We also treated cells troxyl radicals and GSH were investigated. Heating a solution of a with both indomethacin and rebamipide in various pH, and hydroxylamine showed EPR signal recovery. Heating a spin trap- measured mitochondrial function. ping agent showed temperature-dependent increase of EPR signal Results: Both the indomethacin treatment and the low pH con- due to the hydroxyl radical adduct. Most of reactions were sup- dition inhibited RGM1 oxygen consumption, and decreased the pressed by bubbling N2 gas. However, heating carbamoyl- mitochondria membrane potential. The intercellular pH decrease PROXYL with GSH in the N2 gas atmosphere showed temporally below pH7.0 induced ROS generation. This ROS was decided as enhanced signal decay. Results showed a possibility that heating an •- mitochondrial super oxide anion. Moreover, rebamipide inhibited aqueous solution containing oxygen can generate O2 . both acidic environment and indomethacin induced cellular injury. Conclusion: Extracellular low pH conditions, the same as in- domethacin treatments were likely to induce cellular injuries via super oxide anion generation under mitochondrial dysfunction.

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High-resolution redox imaging of intact cells by rxYFP, Cellular migration induced by nitric oxide involves the a ratiometric oxidation- sensitive fluorescent protein participation of the GTP-binding proteins Ras and Rac

Giuseppe Maulucci1, Marina Mele2, Valentina Labate2, H.P. Monteiro1, R. Eller-Borges1, M. Curcio1, M.S. Moraes1, Marco De Spirito1, Tommaso Galeotti2, and Giovambattista Pani2 and W.L. Batista2

1Institute of Physics,LABCEMI Microscopy Core Facility and 2Institute of 1Department of Biochemistry – UNIFESP – Campus São Paulo, SP – Brazil. General Pathology, Unit of Redox Signaling, Catholic University Medical 2Department of Biological Sciences – UNIFESP – School, Largo F. Vito #1, 00168 Rome, Italy Campus Diadema, SP – Brazil

Plasmid-encoded oxidation sensitive fluorescent probes prom- The small GTP-binding proteins Ras and Rac are molecular ise to pave new avenues for realt time imaging of redox signaling in switches exchanging GDP for GTP and converting external signals live cells. We have recently shown that rxYFP, a redox-sensitive in response to a variety of stimuli. Ras and Rac play important variant of the Yellow Fluorescent Protein, can be used ratiometri- roles in cell proliferation, cell differentiation, and cell migration. cally to construct high resolution redox maps of live cells. Confocal Particularly in the case of Rac, the protein is directly involved in analysis of cell fluorescence at two excitation wavelenghts allows in the reorganization and changes in the cytoskeleton during cell mo- fact to monitor, voxel by voxel, the distribution of the probe be- tility. It is known that nitric oxide (NO) is involved in cell migra- tween its reduced and oxidised states, while normalizing fror probe tion, however only few studies addressed the relationships between concentration and photobleaching. By taking advantage of this NO and Rac in signaling processes associated with cell migration. property, we were able to confirm that, in human and murine ma- On the other hand, it is well established that NO stimulates the Ras lignant cells, the nucleus is significantly more reduced than the cy- – ERK1/2 MAP kinases signaling pathwaya. In addition, NO also tosol; additionally, simple deconvolution of redox images con- stimulates the interactions between Ras and the PI3-Kinase signal- structed with untargeted rxYFP allowed to identify hypereduced ing pathwayb. In this communication, we describe the participation perinuclear spots corresponding to mitochondria, as further con- of NO in the activation of Rac in rabbit aortic endothelial cells firmed by the use of a mitochondrially targeted form of the probe. (RAEC). RAEC cells were stimulated with bradykinin, which In 2D scans, the cell border of adherent cells consistently appears promotes endogenous production of NO. We showed that endoge- to be significantly more oxidised than the inner cytosol, while in nous production of NO stimulated Rac and promoted cell migra- 3D cell reconstruction oxidation is polarized towards the bottom of tion over a time period of 24 h. The use of specific inhibitors of the cell; finally, in a spontaneously migrating cell, the leading front PI3-Kinase and of Ras, resulted in inhibition of NO-stimulated Rac appears to me more oxidised than the trailing edge. This set of ob- activation. These results were corroborated by using RAEC cells servations suggests, in keeping with our previous biochemical stud- permanently transfected with the negative dominant mutant of Ras. ies, that integrin signaling and cytoskeleton rearrangement are asso- In conclusion, the NO-mediated activation of Rac involves the di- ciated with local pro-oxidant activity, with relevant implications rect participation of PI-3 Kinase and Ras. for cell invasion and metastasis. Taken together these examples un- a FRBM 35: 381-396, 2003. derscore the power of rxYFP for the real time, high resolution bJBC 273: 29923-29928, 1998. analysis of redox signaling in cultured cells. Financial Support: We acknowledge the financial support of FAPESP (09/ 52730-7) and CNPq

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NAC modulates homoaggregation, viability and Age related oxidase (arNOX) implicated in aging-related proliferation of CD40-activated human B cells oxidative damage to skin proteins Philippe Nadeau1 and Sonia Néron1

Dorothy M. Morré1,2, D. James Morré1,2, Christiaan Meadows1, 1Héma-Québec, Recherche et Développement, Québec, Canada, G1V 5C3 3 4 Zoe D. Draelos , and Dale Kern CD40 ligation by CD154 (CD40 ligand) is essential to B-cell

1 activation in the germinal center. We have developed a co-culture NOX Technologies, Inc., 1291C Cumberland Avenue, West Lafayette, IN 47906, 2Purdue University, West Lafayette, IN 47907, 32444 N. Main Street, model in which proliferation and Ig secretion by purified human High Point, NC 27262 and 4Dale Kern, NuSkin International, 75 W. Center blood B cells can be regulated by the intensity of the CD40-CD154 Street, Provo, UT 84601 interaction. In this system, CD154+ fibroblasts and cytokines are used as surrogates for activated T-cells. Recent studies on tonsillar Little is known about the sources of reactive oxygen species in B cells, as well as human and mouse B-cell lines, showed that skin that result in protein oxidation and cross-linking of potential variations in REDOX potential accompany CD40-mediated B-cell importance to skin aging. Our work has identified arNOX a cell regulation. Indeed, CD40-induced Reactive Oxygen Species (ROS) surface-located hydroquinone oxidase inhibited by coenzyme Q10 modulate signalling pathways involved, for example, in cell sur- that generates superoxide. arNOX appears at about age 30 and then vival, differentiation and Ig secretion. One objective of our study increases with increasing age to about age 65. The protein is shed was to determine the role of REDOX homeostasis in CD40- into body fluids including sera, urine, perspiration and saliva. mediated B–cell activation. As a first step, we have tested whether arNOX activity may be measured spectrophotometrically using re- B-cell homeostasis could be modulated by decreasing REDOX po- duction of ferricytochrome c as the method of quantitation. tential. N-acetylcysteine (NAC), a thiol molecule with antioxidant arNOX-catalyzed oxidation of protein bound tyrosines was meas- properties, was added at 10 and 20 mM to B cells stimulated with a ured using a fluorescence-labeled tyrosine analog, tyramine, which high ratio of CD154+ cells. NAC treatment induced strong homo- when coupled to proteins carrying a tyrosyl radical, rendered the aggregation of B cells, increased cellular viability, while decreas- proteins fluorescent. The assay demonstrated the potential for ing proliferation. Besides, analysis of CD45+CD19+Ax405- cells by arNOX-induced oxidative damage (dityrosine formation) to human flow cytometry indicated that NAC-treated B cells decreased in collagen and elastin and to surface proteins of human fibroblasts size and granularity and exhibited a lower relative fluorescence for and frozen sections from epidermal punch biopsies. Additionally, 5-(and 6-)chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate autofluoresence indicative of Advanced Glycation Endproducts (CM-H2DCF-DA), a ROS detection reagent. Using Fluorescent correlated with the arNOX activity of epidermal biopsy specimens Cell Barcoding (FCB), we observed that various signalling path- consistent with a role for arNOX as a major source of oxidative ways were indeed positively or negatively modulated in NAC- damage and cross-linking of skin proteins amenable to topical treated B cells. Overall, our results suggest that variations in RE- (AgeLoc) arNOX-specific inhibitor combinations. DOX potential modulate viability and proliferation of CD40- activated B cells, likely via activation/inhibition of distinct signal- ling pathways. Whether REDOX potential could be altered by the strength of the CD40 signal, and the precise mechanisms by which ROS modulate cultured B cells, remain to be further investigated.

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Design of pH-sensitive polymeric micelle possessing reduced forms of TEMPO for imaging of ROS. Anti-oxidative stress Intramolecular base-accelerated radical-scavenging reaction nanoparticles for novel theranostics by vitamin E derivatives bearing a pyridine moiety Yukio Nagasaki 1 2 1 3 Graduate School of Pure and Applied Sciences, Graduate School of Ikuo Nakanishi , Haruko Yakumaru , Minako Nyui , Kei Ohkubo , Comprehensive Human Sciences, Tsukuba Research Center for Interdisciplinary Ken-ichiro Matsumoto1, Kiyoshi Fukuahra4, Haruhiro Okuda4, Materials Science, Tsukuba Advanced Research Alliance, University of Tsukuba 3 1,5 1 and Satellite Laboratory of International Center for Materials Shunichi Fukuzumi , Toshihiko Ozawa , Kazunori Anzai , and Nanoarchitechtonics, Ten-noudai 1-1-1, Tsukuba, Ibaraki 305-8573, Japan Nobuo Ikota2,6

Reactive oxygen species (ROS) are known to play versatile roles 1Research Center for Charged Particle Therapy, National Institute of on the occasion of many important events. However, excessive pro- Radiological Sciences, Chiba 263-8555, Japan, 2Research Center for Radiation duction of ROS causes significant adverse effect to living body. Such Emergency Medicine, National Institute of Radiological Sciences, Chiba 263- 8555, Japan, 3Department of Material and Life Science, Graduate School of oxidative stresses must be controlled appropriately. For example, arte- 4 rial re-canalizations achieved by thrombolysis and intravascular inter- Engineering, Osaka University, Osaka 565-0871, Japan, Division of Organic Chemistry, National Institute of Health Sciences, Tokyo 158-8501, Japan, vention are main treatment strategies to restore blood supply in 5Department of Health Pharmacy, Yokohama College of Pharmacy, Yokohama ischemic stroke and heart attack. However, reperfusion has a dilemma 245-0066, Japan, and 6School of Pharmacy, Shujitsu University, Okayama 703- of ischemia-reperfusion injury caused by ROS, which is produced after 8516, Japan a long ischemic period and can extend a damaged area. Therefore, pro- tection of organs affected by ROS has been perused not to cause the Fine-tuning of the radical-scavenging activity of antioxidants is larger damaged area than that associated with the arterial occlusion. It of considerable importance with regard to the development of ef- is known that stable nitroxyl radical such as 2,2,6,6-tetramethylpipe- fective chemopreventive agents against oxidative stress and asso- ridin 1-oxyls (TEMPO) reacts effectively with ROS compounds. ciated diseases. We have reported that the scavenging reaction of However, these compounds have not been applied clinically because DPPH• (2,2-diphenyl-1-picrylhydrazyl radical) by 2,2,5,7,8-penta- they were inactivated due to the acute reduction by anti-oxidant sys- methylchroman-6-ol (PMC), a vitamin E model, proceeds via a tems such as catalase, glutathione peroxidase in vivo. In order to over- proton-coupled electron transfer in deaerated methanol (MeOH) come poor bioavailability and biocompatibility, we developed a novel and that pyridines significantly accelerate this reaction by stabiliz- core-shell type nanoparticle containing nitroxyl radicals, amino- ing a radical cation intermediate derived from PMC.1 In this study, TEMPO, and named radical-containing-nanoparticle (RNP). RNP we synthesized three vitamin E derivatives bearing a pyridine moi- formed a micelle possessing amino-TEMPO in the core under the physiological conditions, and demonstrated high performances of the ety with a linkage of different spacers. All the vitamin E deriva- tives thus obtained efficiently scavenged DPPH• in deaerated prolonged blood circulation time by the compartmentalization of • TEMPO into the micelle and pH-sensitivity to help TEMPO radicals MeOH. The second-order rate constant for DPPH -scavenging by act as antioxidant due to the collapse of nanoparticle in low pH condi- one of the vitamin E derivatives determined by the stopped-flow tion such as ischemic-tissue in vivo. The preparation, physicochemical technique is about 3-fold larger than that by PMC. The DPPH•- and biological characterization and anti-oxidant properties against scavenging activity is found to be depending on the length of the ischemia-reperfusion injury will be summarized in this paper. spacers.

The author would like to express his sincere appreciation to Professors H.Matsui, A.Matsumura, Nakanishi, I. et al. Org. Biomol. Chem. 2005, 3, 626. K.Suzuki, Drs. T.Mamiya, Mr.Marushima, (Medical School of University of Tsukuba), A.Hirayama, (Tsukuba Tech. University), Dr.H.Tsurushima (AIST), Dr.K.Toh, D.Miyamoto and Mr.T.Yoshitomi (TIMS, University of Tsukuba) for their collaboration.

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Beneficial effects of vitamin E on the mitochondrial Squalene sensitizes anti-cancer therapy through the inhibition dysfunction in hippocampus and frontal cortex of aged rats of DNA damage checkpoint activity by Wip1

1 1 1 1 1 2 Hiroshi Nishida , Naoto Tatewaki , Yuki Nakajima , Ana Navarro , Manuel J. Bandez , José M. López-Cepero , Kayoko Nakayama1, Nobuo Ikekawa2, and Tetsuya Konishi1 1 3 4 Carmen Gómez , Alejandro D. Boveris , Enrique Cadenas , and 1 3 Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan. Alberto Boveris 2Niigata Bio Research Center, Niigata, Japan

1Department of Biochemistry and Molecular Biology, and 2Department of Cell ATM (Ataxia Telangiectasia Mutated) protein kinase plays a cru- Biology and Histology, Faculty of Medicine, University of Cádiz, 11003 Cádiz, cial role in cellular DNA damage responses. The inhibition of ATM Spain; 3School of Pharmacy and Biochemistry, University of Buenos Aires, leads to an abolishment of one such signal pathway termed as “check- C1113AAD Buenos Aires, Argentina; and 4Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern points”. It is expected that the discovery of checkpoint inhibitor will be California, Los Angeles, CA 90089-9121. ([email protected]) an effective assistance of anti-cancer therapy. Recent study reported that Wip1, a magnesium dependent protein phosphatase, de- High doses of vitamin E improved brain mitochondrial func- phosphorylates serine 1981 (Ser1981) of ATM during the DNA dam- tion and neuromuscular and exploratory performances and in- age checkpoint response. On the other hand, Squalene (SQ) has been creased median life span in aging mice (Navarro et al., Am J thought to assist anti-cancer therapies such as chemo and/or radiation Physiol 289: R1392-R1399, 2005). Age-dependent mitochondrial therapy though the detailed mechanism is unknown. Here, we report the inhibitory effect of SQ on ATM-dependent checkpoint signaling dysfunction is more marked in rat hippocampus and frontal cortex pathway. SQ itself did not affect on cell cycle distribution of A549 that in whole brain (Navarro et al., Am J Physiol 294: R501-R509, human lung adenocarcinoma cells. The cell viability was decreased by 2008). Such observations prompted us to test vitamin E in the mi- SQ treatment after UV (25, 50 and 100 J/m2) or gamma-irradiation tochondrial function of rat hippocampus and frontal cortex upon (IR; 2 and 6 Gy) in SQ dose-dependent manner (10, 30, 100 mM). SQ aging. Dietary supplementation with vitamin E (2.0 or 5.0 g/kg of treatment disrupted G2/M checkpoint in DNA damaged cells analyzed food) from 9 to 12 mo of age was effective in preventing by 93 % using phosphor-Histone H3 antibody (Ser10). IR-induced phosphory- the age-dependent decrease in hippocampus and frontal cortex res- lations of ATM effectors such as p53 (Ser15) and Chk1 (Ser317) were piration from 4 to 12 mo of age. Vitamin E also restored by 95 % remarkably inhibited by the treatment of SQ in cells. However, In vitro the selective impairment of complexes I and IV activities. The two kinase activities of ATM and AT and Rad3 related (ATR) toward effects, inactivation by aging and restoration by vitamin E, were PHAS-I were not inhibited by SQ. Of importance, SQ increased intra- observed in mtNOS activities, biochemical and functional, in hip- cellular Wip1 protein expression in both of unperturbed and perturbed pocampal and frontal cortex mitochondria. Vitamin E also pre- cells, and suppressed ATM activation in IR treated cells. The enhanced vented the increase in oxidation products (TBARS and protein car- expression of Wip1 by SQ treatment lasted for 12 h, and was con- firmed by Wip1-siRNA experiment. Consistent with potential inhibi- bonyls) and in H O production in hippocampal and frontal cortex 2 2 tion of ATM by SQ, xenograft study demonstrated that the tumor mitochondria. Hippocampal mitochondrial mass was moderately growth of MKN45 gastric cancer cell in vivo was obviously inhibited decreased, by 19 %, upon aging, an impairment that was restored by SQ treatment in X-ray exposed or doxorubicine treated mice, sig- by vitamin E. nificantly. The expression of Wip1 in tumors were also increased by oral administration of SQ. Taken together these findings, SQ inhibits ATM protein kinase activity in cells following DNA damage through the intracellular induction of Wip1 expression, and was confirmed by xenograft study with radio/chemo-therapies.

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Luteolin exerts the anti-gut inflammatory activity on dextran sulfate sodium induced colitis in mice Zinc and ERK regulation in neurons and in the developing rat brain Yosuke Nishitani1, Yuzo Koda2, and Masashi Mizuno2,3

1Organization of Advanced Science and Technology, Kobe University, Kobe, Johnathan R. Nuttall, Yin Htet, and Patricia I. Oteiza 2 3 Japan; Faculty of Agriculture, Kobe University, Kobe, Japan; Department of Agrobioscience, Graduate School of Agricultural Science, Kobe University, Departments of Nutrition and Environmental Toxicology, Kobe, Japan University of California, Davis, Davis, CA 95616, USA Luteolin is a flavonoid present in significant amounts in vegeta- bles including celery and carrots, which has anti-inflammatory ac- We previously observed that severe and marginal zinc nutrition tivities both in vivo and in vitro. However, the impact of luteolin on during gestation can disrupt brain development and affect associ- intestinal inflammation has not yet been fully elucidated. In this ated cell signals, including the extracellular signal regulated study, we evaluated the anti-gut inflammatory activity of luteolin us- kinases (ERK). ERK1/2 have central roles in modulating neuro- ing in vivo and in vitro gut inflammation models. Colitis was in- genesis and neuronal survival, and their inhibition could underlie duced in C57BL/6 mice by the administration of 2% dextran sulfate the adverse effects of developmental zinc deficiency. We currently sodium (DSS) to drinking water. Luteolin (5, 20, 50 mg/kg) was ad- investigated the underlying mechanisms leading to ERK1/2 inhibi- ministered intragastrically daily, one week prior to commencement tion when zinc decreases. Zinc deficiency caused a marked de- of DSS treatment. Oral administration of 20 or 50 mg/kg of luteolin crease in ERK1/2 phosphorylation at Thr202 and Tyr204 in prolif- significantly ameliorated the shortening of colon length (6.5 ± 0.2 erating IMR-32 neuroblastoma cells and differentiated cortical cm and 6.4 ± 0.2 cm, respectively), compared with untreated DSS- neurons. Marginal zinc deficiency imposed to rats throughout ges- induced colitis mice (5.6 ± 0.2 cm). Similar results were obtained tation led to low ERK1/2 activation in the offspring brain cortex at from histologic analysis of the colon sections in DSS-induce colitis postnatal day 2 (P2). Accordingly, a decrease in cellular zinc im- mice. In addition, the treatment with 20 or 50 mg/kg of luteolin had a paired the activation of ERK1/2 downstream targets (nuclear tendency to inhibit the aberrant mRNA expression in inflamed tissue MSK-1). Upstream events in the ERK1/2 pathway, c-Raf (Ser338) through notable suppression of TNF-, IL-6, IL-1, and IFN- and MEK1/2 (Ser217/Ser221) phosphorylation, were not affected mRNA expression. In an in vitro gut inflammation model with the by zinc deficiency in neuronal cells and P2 rat brain cortex. These co-culture system, consisting Caco-2 cells (apical side) and findings suggest that ERK1/2 inactivation in zinc deficiency could RAW264.7 cells stimulated by LPS (basolateral side), treatment with be due to altered activities of downstream phosphatases. PP2A, is luteolin (100 μM) from apical chamber significantly inhibited TNF- a serine phosphatase regulated by zinc and that plays a key role in  production from RAW264.7 and IL-8 mRNA expression in Caco- the inactivation of ERK2. While total phosphatase activity was not 2. HPLC analysis, post stimulation by LPS, revealed the presence of affected by zinc deficiency, PP2A activity increased with the time luteolin (4 μM) and its glucuronate conjugate (10 μM) in the baso- of exposure to zinc deficient media in IMR-32 cells. In summary, lateral chamber. Furthermore, TNF- production by LPS-stimulated low zinc availability decreases ERK1/2 phosphorylation in neu- RAW264.7 was significantly inhibited by the direct treatment of 4 ronal cells in culture and in the developing rat brain, which is in μM luteolin to RAW264.7. In summary, these findings seem to sug- part due to an increased PP2A activation. ERK1/2 inactivation gest that oral administration of luteolin exhibits the anti- could explain the adverse effects of zinc deficiency on neurogene- inflammatory activity in the gut and the agent which exerts the activ- sis, neuronal survival, and ultimately on brain development. ity may be luteolin itself and not its metabolite. Supported by UC Davis and NIH (HD01743)

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The oxidative potential of hyperbaric oxygen treatment: Rapid diffusion of hydrogen protects the retina: A summary of recent experimental work focused on its Administration to the eye of hydrogen-containing saline in exposure time and pressure-related oxidative effects retinal ischemia-reperfusion injury 1 1 1 1 1 2 3 Sukru Oter , Ahmet Korkmaz , Turgut Topal , Serdar Sadir , Hideaki Oharazawa , Tsutomu Igarashi , Takashi Yokota , 1 1 2 1 2 4 Mehmet Ozler , Bulent Uysal , Recai Ogur , Hiroaki Fujii , Hisaharu Suzuki , Mitsuru Machida , 3 3 2 5 4 Kemal Simsek , and Hakan Ay Hiroshi Takahashi , Shigeo Ohta , and Ikuroh Ohsawa 1 2 3 1Department of Ophthalmology, Musashikosugi Hospital, Nippon Medical Departments of Physiology, Public Health and Undersea & Hyperbaric School, Kanagawa, Japan, 2Department of Ophthalmology, Nippon Medical Medicine; Gulhane Military Medical Academy; Ankara, Turkey School, Tokyo, Japan, 3Department of Molecular Biology, 4The Center of 5 Molecular Hydrogen Medicine and Department of Biochemistry and Cell Biol- The risk for oxygen toxicity during hyperbaric oxygen (HBO) ogy, Institute of Development and Aging Sciences, Nippon Medical School, Ka- nagawa, Japan exposure is a known fact. Our previous experimental works have been focused the pressure- and exposure time-related correlation of Introduction: Retinal ischemia-reperfusion (I/R) injury by transient HBO. The persistence of oxidative stress markers following HBO elevation of intraocular (IOP) is known to induce neuronal damage was also examined. A total of 162 Sprague-Dawley rats were used through the generation of reactive oxygen species. Previous studies for these experiments. The clinically approved maximal pres- indicate that molecular hydrogen (H2) is an efficient antioxidant gas sure/duration range, namely 3 ATA/2 hours, was chosen as the that selectively reduces the hydroxyl radical (•OH) and suppresses highest limits. After HBO exposure, the major target organs of hy- oxidative stress-induced injury in several organs. This study was con- peroxic oxidative stress, i.e., the lung, brain and blood, were taken ducted to explore the neuroprotective effect of H2-loaded eye drops on for biochemical assays. Malondialdehyde (MDA) as indicator of retinal I/R injury. Methods: Retinal ischemia was induced in rats by lipid peroxidation, and antioxidant enzymes superoxide dismutase raising IOP for 60 minutes. H2-loaded eye drops were prepared by dis- (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) were solving H2 gas into a saline to saturated level and administered to the the main measured parameters in these studies. In brain tissues, ni- ocular surface continuously during the ischemia and/or reperfusion pe- trite plus nitrate values (NOX), end products of nitric oxide degra- riods. One day after I/R injury, apoptotic cells in the retina were quan- dation, were also examined. MDA and SOD levels of blood, brain tified and oxdative stress was evaluated by markers such as 4- and lung tissue as well as lung CAT activity revealed clear pres- hydroxynonenal and 8-hyroxy-2-deoxyguanosine. Seven days after I/R sure-related rises. Blood, brain and lung tissue MDA and SOD lev- injury, retinal damage was quantified by measuring the thickness of els also indicated a relation with HBO exposure time. NOX values the retina. Result: When H2-loaded eye drops were continuously ad- of brain tissue were also found to be increased at maximal expo- ministered, H2 concentration in the vitreous body immediately in- sure levels. The measured oxidative stress markers usually re- creased and I/R-induced •OH level decreased. The drops reduced the turned to their baseline levels within 90 min after HBO exposure. number of retinal apoptotic and oxidative stress marker-positive cells, In conclusion, within its approved limits for therapeutic use, the and prevented retinal thinning with an accompanying activation of safety of HBO was proven by these results. The parallel increase of Müller glia, astrocytes and microglia. The drops improved the recov- antioxidant enzymes and the relative short persistence of oxidation ery of retinal thickness by > 70%. Conclusions: Our results suggest products support this conclusion. that H2-loaded eye drops will be a highly useful neuroprotective and anti-oxidative therapeutic treatment for acute retinal I/R injury.

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Beneficial effects of spirulina on non-alcoholic steatohepatitis Melatonin as an ideal protective agent against model rats by anti-oxidative and anti-inflammatory activities hyperbaric oxygen induced oxidative reactions Wing Pak1, Fusako Takayama1, Mitsumasa Mankura1, 1 1 2 1 1 1 1 Toru Egashira , Hiromu Kawasaki , Yasumasa Kodo , Sukru Oter , Bulent Uysal , Mehmet Ozler , Turgut Topal , 1 1 1 1 2 3 1 Manaka Mine , Shigeru Okada , and Akitane Mori Serdar Sadir , Kemal Simsek , Recai Ogur , and Ahmet Korkmaz 1Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2 1 2 3 Okayama University, Japan; Spirulina Bio-Lab. Co., Ltd. Departments of Physiology, Undersea & Hyperbaric Medicine, and Public Health; Gulhane Military Medical Academy; Ankara, Turkey Nonalcoholic steatohepatitis (NASH) is a chronic liver disease characterized by necroinflammatory activity, hepatocyte balloon- Exposure to hyperbaric oxygen (HBO) leads to an increase of ing with Mallory’s hyaline, and occasional fibrosis. The “two-hit dissolved oxygen in the blood and delivery to the tissues. There- theory” is widely accepted as the mechanism of NASH pathogene- fore it has been successfully used for the treatment of a variety of sis. Recently, Spirulina (SP), a blue-green algae, is drawn more at- clinical conditions, especially, related to hypoxia. However, the tention, because of its anti-oxidative, nutritional and medicinal oxygen toxicity risk of HBO has also been of scientific interest. properties. The aim of this study is to investigate the SP effect on Recently, our research team set a series of studies examining the NASH and to elucidate the mechanism. Methods: Fee radical scav- use of melatonin as a protecting antioxidant against this stressing • • 1 – enging activities of SP against O2 , HO , O2 and ONOO were ex- condition. In these experiments the effectiveness of exogenously amined by electron spin resonance method and fluorophotometric administered and endogenously secreted melatonin was tested in measurement. 8-Hydroxy 2’-deoxyguanosine was quantified by acutely exposed and chronically administered HBO treatments. A our ELISA assay. Animal experiment was using NASH model total of 247 Sprague-Dawley rats were used for the experimental (PCT/JP 2007/52477) were induced fatty liver in rats by feeding sets. Acute one session HBO was administered at 3 ATA for 2 h. them a choline deficient high fat diets (CDHF), then treated in ad- Chronic HBO exposure was set as daily 60 min sessions at 2.5 dition with oxidative stress (OS). Animal experiment was per- ATA for 10 days. Exogenous melatonin was injected i.p. prior to formed with 5 groups; Control (with MF chow diets), CDHF, HBO sessions at the doses of 10 mg/kg in the one session and 5 NASH, NASH+2SP and NASH+6SP. After experimental period, mg/kg in the chronic exposure groups. After HBO exposure blood, blood and liver were collected from rats under anesthesia, to de- lung and brain tissues were harvested for assays. Oxidative stress termine OS related injuries and Western blotting analysis such as and antioxidant system status were examined. A clear increasing NF- B. Results: SP was shown to have anti-oxidative activities. effect of HBO on almost all oxidative parameters was seen in all The hepatobiliary enzyme leakages and liver fibrosis were exhib- experiments and the antioxidant enzymes presented also higher ac- ited in NASH rats. The decrease of plasma SOD like activity, and tivities. All experiments indicated a limiting effect of exogenously increases of plasma myeloperoxidase activity, serum iron level, injected as well as endogenously secreted melatonin on HBO- and the reactive oxygen species from the liver mitochondrial me- induced oxidative action. The present experimental results warrant tabolism, and the NF- B nuclear import were also demonstrated in melatonin as an effective agent against HBO-induced oxidative NASH rats. The SP administration significantly abated these stress, but have to be supported by clinical trials in order to be changes. Conclusion: The present study demonstrated SP could adopted into clinical conditions. prevent the NASH progression through anti-oxidative and anti- inflammatory activities.

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Glutathione disulfide as a cell death signal Regulation of mevalonate pathway and Ras signalling by lycopene in prostatic cancer cells Han-A Park, Savita Khanna, Cameron Rink, Surya Gnyawali, Natalia Kubicki, Sashwati Roy, and Chandan K. Sen

Paola Palozza, Rossella Simone, Assunta Catalano, and Department of Surgery, Laboratory of Molecular Medicine, Davis Heart & Maria Colangelo Lung Research Institute, The Ohio State University Medical Center, Columbus, OH, 43210 Institute of General Pathology, Catholic University School of Medicine, 00168 Rome, Italy. E-mail: [email protected] Reduced glutathione (GSH) is a low molecular weight intracellular thiol in all aerobic cells. Under conditions of oxidative Cancer cells have abnormal cholesterol biosynthetic pathways stress, large amounts of GSH are rapidly oxidized to GSSG. Cell and farnesylation is a key process in oncogene activation. Tomato death is often associated with high GSSG levels. Such results are lycopene has been suggested to prevent the risk of prostate cancer, interpreted as evidence for oxidative stress without addressing any although the exact molecular mechanism(s) is still unknown. Since functional significance of GSSG in the death process. We hypothe- the carotenoid share similar synthetic pathways with cholesterol, sized that under specific conditions, GSSG functionally partici- we tested the hypothesis that it may exert its antitumor effects pates in signaling for cell death. through changes in mevalonate pathway and in Ras activation. In- Mechanisms that trigger the oxidation of GSH to GSSG in a cubation of the Ras-activated prostatic carcinoma LNCaP cells, cell such as exposure to ROS or to ROS-generating cytokines also with a 24-h lycopene treatment (2.5-10 μM) dose-dependently re- induce numerous other cellular responses. Thus, it is challenging to duced intracellular total cholesterol by decreasing 3-hydroxy-3- dissect which of those responses actually contributed to the death methylglutaryl-coenzyme A (HMG-CoA) reductase expression and process. To address this complication, we raised cellular GSSG by inactivating Ras, as evidenced by its translocation from cell content by microinjection. Control cells were injected with either GSH or vehicle. GSSG, but not GSH, caused cell death at physio- membranes to cytosol. Concomitantly, lycopene reduced the Ras- logically relevant concentrations. GSSG-induced cell death was dependent activation of NF-kB via inhibition of ROS production protected in the presence of the 12-lipoxygenase(Lox) inhibitors. and decreased phosphorylation of JNK, ERK1/2 and p38. These Previous work from our laboratory has identified 12-Lox as a key effects were accompanied by an arrest of cell cycle progression executioner of neural cell death. Results of this study indicate that and by apoptosis induction, as evidenced by a decrease in cyclin GSSG induces 12-Lox dependent cell death. Furthermore, GSSG- D1 and pAKT levels, and by an increase in p21, p27 and p53 lev- dependent glutathionylation of 12-Lox was identified a critical els and in Bax/Bcl-2 ratio. The addition of mevalonate prevented player in neural cell death. We tested the hypothesis by using glu- the growth-inhibitory effects of lycopene as well as its increase in taredoxin, a deglutathionylating enzyme, and noted that glutare- Ras cytoplasmatic accumulation and the subsequent changes in doxin transfected cells were protected against glutamate challenge. NF-kB. The ability of lycopene in inhibiting HMG-CoA reductase To test the significance of our findings, GSSG was stereotaxically expression and cell growth and in inactivating Ras was also found injected to the brain in vivo and MRI was performed to quantify in prostatic PC-3 cancer cells. These findings provide a novel tissue lesion. Strategies directed at improving or arresting of intra- mechanistic insight into the preventive effects of lycopene in hu- cellular GSSG clearance may be effective in minimizing oxidative stress-related tissue injury or potentiating the killing of tumor cells, man prostate cancer. respectively. This work unfolds a new paradigm that is likely to extend beyond the specific cell type studied and shed light on fun- damental mechanisms underlying cell death.

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The time-dependent effect of ProvinolsTM on brain Different mechanisms of melatonin and N-acetylcysteine effects in adult spontaneously hypertensive rats NO synthase activity in experimental hypertension O. Pechanova1, J. Zicha2, L. Paulis1, S. Vranková1, 2 2 O. Pechanova, L. Jendeková, and S. Vranková Z. Dobesová , and J. Kunes 1Institute of Normal and Pathological Physiology, Slovak Academy of Sciences, Institute of Normal and Pathological Physiology, Slovak Academy of Sciences, Bratislava, Slovak Republic, Institute of Physiology, Academy of Sciences of Bratislava, Slovak Republic Czech Republic, Prague, Czech Republic

The imbalance in nitric oxide (NO) and reactive oxygen spe- Red wine polyphenols have been reported to possess beneficial cies production is often found in both experimental and human hy- properties for preventing cardiovascular diseases but their neuro- pertension. The aim of our study was to determine possible effects protective effects during chronic L-NAME treatment have not been of N-acetyl-5-methoxytryptamine (melatonin, 10 mg/kg/day) and elucidated. The aim of this study was to analyze a time course of N-acetylcysteine (1.5 g/kg/day) in adult spontaneously hyperten- TM Provinols effects on brain NO synthase activity and oxidative sive rats (SHR) with established hypertension. After a six-week- damage in L-NAME-induced hypertension. Male Wistar rats, 12 treatment, blood pressure was measured and NO synthase (NOS) weeks old, were divided into six groups: control groups, groups activity, concentration of conjugated dienes, protein expression of treated with N(G)-nitro-L-arginine methyl ester (L-NAME, 40 endothelial NOS, inducible NOS and nuclear factor-kappaB (NF- mg/kg/day) for 4 or 7 weeks and groups receiving ProvinolsTM (40 kappaB) in the left ventricle were determined. Both treatments im- mg/kg/day) plus L-NAME for 4 or 7 weeks. At the end of the proved the NO pathway by means of enhanced NOS activity and treatment, marker of membrane oxidative damage - conjugated di- reduced reactive oxygen species level as indicated by decreased enes (CD) in the brain and NO synthase activity in the cerebral conjugated diene concentrations and lowered NF-kappaB expres- cortex, cerebellum and brainstem were determined. L-NAME sion. N-acetylcysteine (but not melatonin) also increased the endo- thelial NOS protein expression. However, only melatonin was able treatment for 4 or 7 weeks led to the increase in blood pressure, to reduce blood pressure significantly. Subsequent in vitro study elevation of CD concentration and decrease of NO synthase activ- TM revealed that both N-acetylcysteine and melatonin lowered the tone ity in the brain parts investigated. Provinols partially prevented of phenylephrine-precontracted femoral artery via NO-dependent blood pressure rise and elevation of CD concentration. Comparing relaxation. Nevertheless, melatonin-induced relaxation also in- TM to the L-NAME treated group, Provinols increased NO synthase volved NO-independent component which was preserved even af- activity after 4 weeks of treatment. However, the prolonged Provi- ter the blockade of soluble guanylate cyclase by oxadiazolo[4,3- nolsTM treatment for 7 weeks had no effect on NO synthase activity a]quinoxalin-1-one. In conclusion, both N-acetylcysteine and decreased by L-NAME treatment. In conclusion, ProvinolsTM par- melatonin were able to improve the NO/reactive oxygen species tially prevents L-NAME induced hypertension via the different balance in adult SHR, but blood pressure was significantly lowered mechanisms depending on the duration of treatment. Prevention of by melatonin only. This implies that a partial restoration of oxidative damage in the brain with modulating effect on NO syn- NO/reactive oxygen species balance achieved by the antioxidants thase activity is suggested. such as N-acetylcysteine has no therapeutic effect in adult rats with established hypertension. The observed antihypertensive effect of The study was supported by APVV-0538-07 and VEGA 2/0178/09. melatonin is thus mediated by additional mechanisms independent of NO pathway. The study was supported by APVV-0538-07 and VEGA 2/0178/09.

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Antioxidant properties and protective effects of raw, Vitamin A and ,-carotene oxidative reactive secondary boiled and fried garlic on DNA damage in sequence chain-growth model explaining antioxidant-related hypercholesterolemic hamsters mortality rate increase Yara S. Queiroz1, Marcelo L. Ribeiro2, Demetrius P. Arçari2, Richard C. Petersen Marcela P. Monteiro1, Geni R. Sampaio1, and Elizabeth A.F.S. Torres1* University of Alabama at Birmingham

1Department of Nutrition - School of Public Health - University of São Paulo The ,-carotene antioxidant analogue for vitamin A was iden- (USP); 2Clinical Pharmacology and Gastroenterology Unit - São Francisco tified in a smoker study with 29,133 men to form increased risks University Medical School, Bragança Paulista, S.P. Av. Dr. Arnaldo 715 São Paulo - S.P. - Brazil - 01246-904 for lung cancer in addition to total mortality related to numerous other varied pathologies such as coronary heart disease, stroke, hy- Garlic (Allium sativum L.) is rich in several bioactive com- pertension and cardiomyopathy JAMA 290:476-485; 2003. Conju- pounds that can act as free radical scavengers. Oxidative stress gated isoprene carotenoids that act as antioxidants have previously plays an important role in the pathogenesis of numerous chronic been considered as a source for C=C double-bond addition reac- age-related free radical-induced diseases. The aim of this study tions by free radicals with the large -system. So, for free-radical was to evaluate the antioxidant activity of garlic as well as the abil- pathogenesis, a chain-growth cardiovascular disease (CVD) poly- ity to influence DNA in hypercholesterolemic hamsters. Fifty ani- mer model was tested. 100 gelatin capsules containing a low- mals were divided into five diet groups: control, hypercholesterol viscosity liquid of vitamin A 8000IU/capsule or ,-carotene (HC), raw garlic/HC, boiled garlic/HC and fried garlic/HC. The 25,000IU vitamin A/capsule, Nature Made, Mission Hills, CA, experimental diets were supplemented with 5% lyophilized garlic were incised and squeezed into separate containment wells 4.0mm for 30 days. The antioxidant capacity was measured using oxygen deep and 9.0mm diameters. Each antioxidant was combined with a radical absorbance capacity (ORAC) and the DNA damage in- Fenton-metal redox couple consisting of cobalt naphthenate and duced was investigated by the comet assay. ORAC measured in dibenzoyl peroxide, Sigma Aldrich, St. Louis, MO, to test expected liver improved by 9%, 17% and 10% for raw garlic/HC, boiled viscosity increases. When 4% cobalt naphthenate and 3% diben- garlic/HC and fried garlic/HC in relation to control, respectively. zoyl peroxide were added slowly over a 3 day period to both vita- Our results demonstrate that garlic was able to decrease DNA min A and ,-carotene, viscosity suddenly increased to a point damage after intervention in liver cells, were significantly lower in where both low-viscosity antioxidants began to gel into a sticky hypercholesterolemic animals treated with boiled garlic (TM = solid for a CVD atherosclerosis model. All liquid flow stopped af- 5.31 ± 0.71) and raw garlic (TM = 7.57 ± 1.14) than those the hy- ter 4 days for vitamin A and 9 days for ,-carotene. Over time a percholesterol group (TM = 8.69 ± 0.88). The data presented here cancer-related model for polymerization cure shrinkage estimated show that the forms usually consumed garlic is not genotoxic in at approximately 50vol% occurred where both liquid-oil antioxi- liver. The observed protection may be related to the antioxidant ac- dants gelled and adhered to the containment substrate surface. Both tivity of the garlic bioactive compounds. polymers also interlaced as ruffled ribbon-like networks glued to the wells underneath. Further, 0.5mm films exposed to oxygen air polymerized with shrinkage as harder material for a reperfusion in- jury model.

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The Janus face of supplemental oxygen in ischemic stroke: Cyclic adenosine monophosphate (cAMP) mediates the New insight on therapeutic potential and identifying the anti-inflammatory effects of LA and vitamin D

oxygen-sensitive transcriptome Sonemany Salinthone1,2, Vijayshree Yadav1,2, Dennis N. Bourdette1,2, and Daniel W. Carr1,3 Cameron Rink1, Sashwati Roy1, Mahmood Khan2, Pavan Ananth1, 2 1 1 1Portland Veterans Affairs Medical Center, Portland, OR 97239; 2Department Periannan Kuppusamy , Chandan K. Sen , and Savita Khanna of Neurology, Oregon Health & Sciences University, Portland, OR 97239; 3Department of Endocrinology, Oregon Health & Sciences University, Portland, 1Department of Surgery, 2Department of Internal Medicine, OR 97239 The Ohio State University Medical Center, Columbus, OH 43210 Abnormal regulation of the inflammatory response is an impor- To date, the significance of targeting acute ischemic stroke (AIS)- tant component of many diseases such as diabetes, Alzheimer’s caused focal hypoxia remains unclear. Despite the support of case re- disease and multiple sclerosis (MS). Lipoic acid (LA) and vitamin ports and small animal research, three clinical pilot studies that probed D are anti-inflammatory agents that are being investigated as alter- the efficacy of hyperbaric oxygen (HBO) to treat AIS reported insig- native/complementary therapeutics for these conditions. LA is a nificant, or potentially harmful, outcomes. We hypothesize that con- sulfur containing fatty acid that has been shown to decrease paraly- flicting outcomes in literature stem from a limited window of benefit sis, demyelination and axonal injury in the animal model of MS, for supplemental oxygen to treat AIS. The objectives of this study experimental autoimmune encephalomyelitis (EAE). Vitamin D is were two-fold: (1) to define the therapeutic window of opportunity for a prohormone that has also been shown to be effective in amelio- supplemental oxygen by employing normobaric (NBO) and HBO at rating clinical manifestations of EAE. However, the mechanisms the onset of ischemia (iNBO, iHBO) or at reperfusion (rNBO, rHBO) of action of LA and vitamin D are not completely understood. In in a rodent model of middle cerebral artery occlusion (MCAO); and this study, we are examining the role of cAMP in mediating the ef- (2) to employ high density transcriptome screening towards uncover- fects of LA and vitamin D. cAMP is a small molecule second ing oxygen-sensitive molecular mechanisms implicated in AIS. The messenger that acts as a potent immunomodulator and is synthe- sized upon activation of G-protein coupled receptors, such as the efficacy for supplemental oxygen to correct AIS-affected tissue pO2 was evaluated using EPR oxymetry. Stroke lesion volume was quanti- histamine, adenosine and beta adrenergic receptors. We found that fied using 4.7T MRI at 48h along with histochemical assessment of LA inhibits IL-2, IL-6 and IL-17 production while enhancing IL-10 oxidative stress and neurodegeneration. Unbiased query of oxygen re- synthesis. Using pharmacological inhibitors, we determined that sponsive gene networks in stroke-affected tissue was performed using LA stimulates cAMP production via activation of the histamine GeneChip™. Verification of gene candidates was achieved by real- and adenosine, but not the beta adrenergic receptors. In addition, time PCR from laser captured neurons in stroke affected brain tissue. oral administration of LA to MS patients resulted in increased cAMP levels. Average cAMP levels in 28 MS subjects were NBO was sufficient to correct penumbral tissue pO2 during AIS. iNBO and iHBO attenuated, while rNBO and rHBO exacerbated, AIS- 27.9% higher four hours (13.195 ± 0.779) after ingestion of LA associated lesion volume, oxidative stress and neurodegeneration. Di- compared to baseline (9.513 ± 1.271). We also discovered that vi- rected microarray analysis revealed key oxygen-sensitive gene net- tamin D treatment stimulates cAMP production and inhibits IL-6 works related to chemokine signaling and neutrophil recruitment. AIS secretion in T cell enriched PBMCs. Collectively, the data provide presents a temporal window of opportunity to minimize brain injury evidence that LA and vitamin D may be effective as treatment al- using supplemental oxygen therapy. Findings provide key information ternatives in inflammatory diseases by inhibiting the production of relevant to the successful design of clinical trials aimed at testing the pro-inflammatory mediators via activation of the cAMP signaling effects of supplemental oxygen in stroke affected patients. Supported by pathway. The information also adds to our understanding of the NIH CTSA Pilot Award UL1 RR025755 bioactivity of LA and vitamin D.

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Fermented garlic, a novel candidate food for the The modulation of the redox status of isolated brain prevention of diabetic nephropathy mitochondria by energy linked substrates: Quantification by high performance liquid chromatography Emiko Sato, Masahiro Kohno, and Yoshimi Niwano

Harsh Sancheti§, Jerome V. Garcia§, Li-Peng Yap§, New Industry Creation Hatchery Center (NICHe), Tohoku University, Japan Derick Han‡, and Enrique Cadenas§ Our previous study showed that spontaneous fermentation of § Department‡ of Pharmacology & Pharmaceutical Sciences, School of garlic under a fixed condition potentiates its fundamental antioxi- Pharmacy, Research Center for Liver Diseases, Keck School of Medicine, dative properties. Scavenging activities against O -· and H O of University of Southern California, Los Angeles, CA 90089-9121, USA 2 2 2 80% EtOH extract of the fermented garlic were increased 13-folds The balance between oxidized and reduced species, i.e., glu- and more than 10-folds respectively, as compared with those of the tathione (GSH/GSSG) and pyridine dinucleotides (NADH/NAD, control garlic extract. Polyphenol content and potent scavengers against H O of the extract were also increased in the fermented NADPH/NADP), determine the cellular redox status. Oxidative 2 2 garlic. Furthermore, we have shown that the fermented garlic has stress and altered redox status are widely considered as major an ability to scavenge ·OH, indicating that the fermented garlic components of aging and age-related diseases. The isolation of mi- possesses desirable antioxidative properties in terms of scavenging tochondria from organs is a widely used tool in studying mito- activities against reactive oxygen species (ROS). Diabetic neph- chondrial biology. However, Inherent in the long isolation process, ropathy is one of the three major diabetic complications, and re- are alterations in mitochondrial redox status. Previous work from quires dialysis for the treatment. It is well known that the concen- our laboratory has shown that different isolation methods can alter tration of circulating methylglyoxal (MG), a uremic toxin, is the redox status with respect to the glutathione pool. Substrate sup- higher in the patients with diabetic nephropathy than in healthy plementation of isolated mitochondria resulted in higher buffering people. Recently, it has been reported that free radials are gener- capacity against H2O2 challenges, in part due to increased GSH ated via a non-enzymatic reaction between MG and H2O2 in vitro. levels. The aim of the present study is to extend our previous work Thus we postulate that the free radical species is one of the triggers and monitor changes in the pyridine dinucleotide pool. Upon sub- of oxidative injury in the kidney, which may in turn lead to dia- strate supplementation, changes in the pyridine dinucleotide pool betic nephropathy. We have tried to develop a functional food for were quantified using HPLC and changes in membrane potential the prevention of diabetic nephropathy by applying the fermented have been monitored using fluorescent dye Rhodamine 123. Our garlic. In this study, we report that the extract of the fermented gar- data shows that substrate supplementation shifts the mitochondrial lic have an ability to decrease the free radicals generated via a re- redox status towards reduced state. These data are in agreement action between MG and H2O2. Our results suggest that it is possi- with our previous work and shows changes in redox status of pyri- ble to develop functional food for the prevention of diabetic neph- dine dinucleotides when supplemented with mitochondrial energy ropathy by applying the extract of fermented garlic, which can de- substrates. crease the free radicals suggested as putative triggers of oxidative injury in the kidney.

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Dietary -tocopherol and plasma -tocopherol are correlated with the urinary metabolite -CMBHC but not -CEHC in TGF 1 induces mitochondrial ROS through GSK3 caucasians and African Americans inactivation-mediated respiratory defects

1 1 2 Martha G. Sensel , Mary C. Cambou , Weiqing Liu , 1,3 1,3 1,3 3 3 1 Yong-Hak Seo , Hyun-Jung Jung , Hae-Ok Byun , Katie M. Lebold , Maret Traber , and Lenore Arab You-Mie Kim1,3, Soo-Han Yoon2,3, and Gyesoon Yoon1,3 1David Geffen School of Medicine and 2Department of Biomathematics, 3 University of California, Los Angeles, CA 90095; Linus Pauling Institute, 1Department of Biochemistry & Molecular Biology and 2Department of Oregon State University, Corvallis, OR 97331 Neurosurgery, School of Medicine and 3Department of Molecular Science & Technology (BK21), The Graduate School, Ajou University, Suwon, 443-721, Clarity on the relationships between food source and supplemental Korea. E-mail: [email protected] vitamin E intake with urinary tocopherol (toc) metabolites are needed to discern the utility of the latter as biomarkers in human studies. We examined relationships between dietary intake, plasma (pl.) levels, and urinary metabolites of -toc in 262 African American (AA) and Cau- We previously reported that transforming growth factor 1 casian (CAU) subjects from Los Angeles. Dietary intakes of -toc (TGF 1) induces senescence through persistent mitochondrial were 10.6 (11.2)* and 13.3 (13.7) mg/day for AAs and CAUs, respec- ROS generation through respiratory defects. In the present study, tively. Plasma -toc levels were 10. 7 (3.6) and 11.2 (3.2)
g/mL for we investigated the molecular mechanism of how mitochondrial AAs and CAUs, respectively. Creatinine-adjusted urinary levels of the respiration is damaged by TGF 1. During the TGF 1-induced se- major metabolite -CEHC and its precursor, -CMBHC, were 2.59 (3.23) and 0.37 (0.49)
mol/g creatinine, respectively in AAs and nescent process, no significant changes of respiratory protein ex- 3.20 (2.95) and 0.49 (0.71)
mol/g creatinine, respectively, in CAUs. pressions were observed. However, TGF 1 immediately triggered Pearson correlation analysis of log-transformed data showed signifi- phosphorylation on negative regulatory sites of both GSK3 and  cant correlations between dietary -toc and lipid-adjusted pl. -toc (r within 15 min and the phosphorylation levels were continuously = 0.39l; p < 0.0001), dietary -toc and urinary -CMBHC (r = 0.36; maintained, well corresponding to the intracellular ROS profile. p < 0.0001); and lipid-adjusted pl. -toc and urinary -CMBHC The GSK3 inactivation was upstream event of the ROS genera- (r = 0.31; p < 0.0001). Correlations for dietary or pl. -toc with urinary -CEHC were not significant. In the subset of 154 subjects who did tion, independent of AKT activation. Interestingly, a conventional not consume supplemental vitamin E, correlations were reduced, but GSK3 inhibitor, SB415286, directly inhibited not only cellular showed the same tendency (dietary vs. lipid-adjusted pl. -toc, r = 0.16 respiration without alteration of respiratory protein expression in and p = 0.006; dietary -toc vs. urinary -CMBHC, r = 0.28 and both Mv1Lu and Chang cells. In addition, the GSK3 inhibition was p = 0.0009; lipid-adjusted pl. -toc, vs. urinary -CMBHC: r = 0.20 enough to induce senescence, accompanying prolonged ROS pro- and p = 0.012). Compared to CAUs, AAs had stronger correlations of duction. These results were further confirmed by knockdown of urinary -CMBHC with both dietary -toc (p = 0.008 vs. p = 0.05) and pl. -toc (p = 0.05 vs. p = 0.17). Although correlations with uri- GSK3 with siRNAs. Our present results suggest that GSK3 in- nary -CEHC were not significant in either AAS or the overall group activation is involved in the TGF 1-induced respiratory defects, of non-supplement users, CAUs showed an r = 0.280 and p = 0.025 for thereby continuously elevating cellular ROS level and inducing se- pl. -toc and urinary -CEHC. Further, urinary -CEHC was highly nescence. correlated with urinary -CMBHC (r = 0.528; p < 0.001). The obser- vation that dietary intake and pl. levels of lipid-adjusted -toc are cor- related with urinary levels of the intermediary -toc metabolite -CMBCH, but generally not with the terminal metabolite -CECH is perplexing, but suggests that -CMBCH might serve as a biomarker for -toc intake. *all descriptive data are expressed as mean (SD)

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Multi-nutrient approach to nitric oxide production Investigation of the effects of long-term hyperbaric more efficient than L-arginine alone oxygenation on the oxidative and antioxidant parameters of Tamara Sofi, Joseph Juliano, and Michael Ricciardi rats’ lung tissue THE Labs, Research & Development Division, 27128 Paseo Espada, Kemal Simsek1, Hakan Ay1, Sukru Oter2, Mehmet Ozler2, San Juan Capistrano, CA 92675 3 2 2 4 Ergun Ucar , Bulent Uysal , Turgut Topal , Ozgur Yesilyurt , Nitric Oxide (NO) synthesized from L-arginine by NO syn- 1 and Senol Yildiz thase has a large variety of physiological actions in the cardiovascu- lar, periphery, immune, and nervous system. It is believed to be the Departments of 1Undersea & Hyperbaric Medicine, 2Physiology, 3Pulmonary Medicine and 4Pharmacology; Gulhane Military Medical Academy; Ankara, body’s nutrient delivery system and is considered therapeutic Turkey when produced for 30 minutes or more. Via trial and error, various multi-nutrient applications were tested until increased NO produc- Despite to its common clinical use and known benefits, hyper- tion crossed the 30 minute threshold using less than 1 gram l- baric oxygen (HBO) is also reported to enhance the production of arginine per dose. In this clinical study 50 human subjects ranging reactive oxygen species and therefore can cause oxidative stress in from 40 to 80 years of age, diagnosed with NO deficiency condi- several tissues. Previous studies had shown that HBO induced oxi- tions to include hypertension and diabetes, were divided into 3 dative stress is directly proportional to both its exposure pressure groups receiving either (a) placebo, (b) L-arginine alone, or (c) and duration. Nevertheless, these studies were usually performed Hemoxide, a multi-nutrient formula containing L-arginine and other with single-session HBO exposures. On the other hand, the clinical nutrient cofactors to include choline bitartrate. Loading dose was 3 use of HBO commonly depends on a long-term exposure period. In capsules per day, 2 times per day, for 3 days. NO levels were this study, it was aimed to enlighten the oxidative effect of HBO in tested via skin surface temperature changes and Siemens’ NO the lung tissue of rats which were exposed to HBO from 5 up to 40 breathalyzer. Subjects receiving placebo had no spike in NO pro- sessions. A total of 60 male Sprague-Dawley rats were divided into duction from baseline. Subjects receiving l-arginine alone experi- 6 study groups exposed to 5, 10, 15, 20, 30 and 40 daily 2.8 enced an average 8% spike over baseline in NO production within ATA/90 min HBO sessions (n = 8 for each). Animals were sacri- the first 10 minutes and then gradually declined to flat line at the 20 ficed 24 h after the last HBO sessions. An additional control group minute point. Subjects receiving Hemoxide experienced increased was set for obtaining normal data (n = 12). Lung malonyldialde- NO levels for 1-4 hours reaching the same initial 10 minute 8% in- hyde (MDA) and carbonylated protein (PCO) levels were deter- crease as L-arginine subjects, but then peaked at a 33% average in- mined as measures of oxidative stress, along with the activities of crease over baseline at the 20 minute point (where L-arginine sub- the antioxidant enzymes superoxide dismutase (SOD) and glu- jects flat lined). Levels in the Hemoxide subjects did not drop be- tathione peroxidase. For the first 15 sessions, none of the measured low 20% increase over baseline until the 65-70 minute point, parameters represented any changes. But in the 20, 30 and 40- gradually declining to 8% over baseline (where l-arginine subjects session groups MDA, PCO and SOD were found to be signifi- peaked) at the 110 minute point. Study results were submitted to cantly increased indicating that oxidative stress is established in the FDA which made this application a medical food. Since this rats’ lung tissue: Thus, the standard prophylactic use of antioxi- study, THE labs has translated the acetylcholine, dopamine, and dants during HBO therapy seems to be reasonable, in particular for serotonin neurotransmitter delivery systems. patients with longer exposure procedures.

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Novel boron carrier based on core cross-linked micelles Protective effects of astaxanthin against singlet oxygen-induced composed of PEG-b-PLA copolymer damage in human dermal fibroblasts in vitro with polymerizable boron cluster Kumi Tominaga, Nobuko Hongo, Mariko Karato, and

Shogo Sumitani1, Motoi Oishi1-3, and Yukio Nagasaki*1-5 Eiji Yamashita

1 Life Science Division, Fuji Chemical Industry Co. Ltd., Toyama, Japan Graduate School of Pure and Applied Sciences, University of Tsukuba, 2Tsukuba Research Center for Interdisciplinary Materials Science, 3Center for Tsukuba Advanced Research Alliance, 4Graduate School of Comprehensive Astaxanthin is widely distributed in nature especially in the Human Sciences, 5Satellite Laboratory of International Center for Materials marine organisms such as crustaceans and fish. We recently com- Nanoarchitechtonics pared the singlet oxygen quenching ability of astaxanthin in sol-

vents with that of 26 common hydrophilic and lipophilic antioxi- Boron neutron capture therapy has attracted much attention as dants, including vitamins, polyphenols, and certain drugs, under the selective cancer therapy using 10B compounds, which effi- the same conditions. The result showed that the activity of astaxan- ciently generate the a-particles and 7Li nuclei within ten mm thin was approximately 6,000 times, 800 times, 560 times, and 75 through the nuclear reaction of 10B atom with low-energy thermal times greater than that of vitamin C, coenzyme Q , catechin, and neutrons. The success of BNCT is dependent on the delivery sys- 10 alpha-lipoic acid, respectively, which indicates that astaxanthin has tems to accumulate sufficient quantity of 10B to tumor tissues. the strongest singlet oxygen-quenching ability among these com- Herein, we designed and prepared core cross-linked micelles (CL 1) pounds . Here we studied the protective effects of astaxanthin micelles) composed of poly(ethylene glycol)-block-poly(lactide) against singlet oxygen induced damage in human dermal fibro- copolymer bearing a methacryloyl group at PLA end using polym- blasts in vitro comparing to 7 common antioxidants (-carotene, erizable boron cluster. This micelle enables to prolong the blood lutein, -tocopherol, coenzyme Q10, -lipoic acid, vitamin C, and circulation time and deliver the 10B compounds to the tumor tissues catechin). Human dermal fibroblasts were treated with 10 μM of without leakage of 10B compounds as well as disassociation of the each antioxidant for 24 h. And after singlet oxygen exposure, and micelle in the blood stream because of the existence of cross- the human dermal fibroblasts were incubated for 24 h and the cell linked core through the covalent bonds. viability was determined by the MTT assay. Only astaxanthin ex- The average diameter of the CL micelle was 85 nm determined hibited the protective effect in this condition. Even at 3 μM the ef- by dynamic light scattering measurements. To clarify biodistribu- fect was identical and an almost 100% protection. Bedsides, a tion of both micelles, 125I-labeled CL and non cross-linked (NCL) dose-dependent protective effect was observed for -tocopherol micelles were injected into BALB/c mice bearing tumor via tail alone among the tested antioxidants at up to 1,000 μM. A relation- vein. The CL micelle showed prolonged blood circulation time (t 1/2 ship between the protective effect of astaxanthin and collagen pro- = 16.5 h) compared with NCL micelle (t = 11.8 h). In addition, 1/2 duction ability in the cells and a difference of action mechanisms accumulation of CL micelle into tumor tissue was higher than
of the protective effect between astaxanthin and -tocopherol will that of NCL micelles. These results clearly indicate that the stabil- be discussed. The anti-wrinkling effect of astaxanthin, which was ity of the CL micelle remarkably increased due to the covalently reported in clinical and animal studies, may be attributed to its pro- cross-linked bonds between polymer and boron cluster. tective effect against singlet oxygen-induced damage. Y. Nishida et al.: Carotenoid Science 11, 16 (2007).

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ESR detection for alkyl-oxy radical and superoxide Complex I syndrome in isolated rabbit heart subjected to radical decay by natural antioxidants ischemia-reperfusion

Mitsuko Ukai 1Laura B. Valdez, 1Tamara Zaobornyj, 1Silvina S. Bombicino, 1Darío E. Iglesias,2Martin Donato, 2Verónica D’Annunzio, Department of Education, Hokkaido University of Education, 2Ricardo Gelpi, and 1Alberto Boveris Hakodate 040-8567, Japan 1Laboratory of Free Radical Biology, School of Pharmacy and Biochemistry, Antioxidants play a role as efficient scavengers for radicals. 2Laboratory of Cardiovascular Physiopathology, Department of Pathology, Oxidation processes are crucial for food storage. Oxidation is the School of Medicine; University of Buenos Aires, Buenos Aires, Argentina major causes of chemical spoilage of food. They are rancidity and/or deterioration of the nutritional quality, color, flavor and tex- The effects of ischemia-reperfusion on left ventricle mitochon- ture. The total antioxidant capacity is an important factor for the drial function were studied in isolated rabbit heart using the Lan- evaluation of the food quality. Basic methods to measure antioxi- gendorff technique with 15 min of stabilization, 15 min of ische- dant capacity in foods have been reviewed. Three assays should be mia and 5 and 30 min of reperfusion. Tissue O2 consumption, mi- recommended for the standardization, oxygen radical absorbance tochondrial state 3 respiration with malate-glutamate, and complex capacity (ORAC), the Folin - Ciocalteu method and the Trolox (6- I activity were similarly decreased, by 20-25% and 40-45%, after 5 hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) equiva- and 30 min reperfusion, respectively. Hydrogen peroxide produc- lent antioxidant capacity (TEAC). Methods of analysis of water tion after 30 min of reperfusion was 80% higher than the H2O2 soluble and lipid soluble antioxidant capacity in foods were ex- production in control mitochondria. Mitochondrial NO production tended by the modifications of the ORAC procedure. These meth- decreased 35% in ischemic hearts with a recovery to control values ods have been compiled into the USDA food database of antioxi- during reperfusion. The pattern of mtNOS biochemical activity dant capacity of foods. The spin trapping abilities against alkyl-oxy was in agreement with the changes in mtNOS functional activity in and superoxide radicals were studied using a new reagent CYP- malate/glutamate-supported O2 consumption. The mitochondrial MPO. For careful detection of spin adducts, we used borosilicate level of protein carbonyls (40%), TBARS (50%) and nitrotyrosine ESR flat cells. CYPMPO is a colorless crystalline and very soluble (3-fold) were markedly increased after the process of ischemia- into aqueous solutions. We examined plant specimens from teas, reperfusion. This experimental model shows an early reduction of vegetables and fruits. CYPMPO successfully trapped alkyl-oxy as mitochondrial NO production during acute hypoxia, which may re- well as superoxide radicals in the plant extracts. Upon illumination lease NO-mediated cytochrome oxidase inhibition. In reoxygena- tion, mtNOS activity is up-triggered, increasing mitochondrial NO, of Hg - Xe arc lamp light onto phosphate buffer solution contain- - - ing AAPH and CYPMPO, we generated the alkyl-oxy radicals in O2 and ONOO productions. During reperfusion, mitochondria de- the very pure form. Similarly, superoxide radicals were generated velop to a condition named “complex I syndrome” in which enzy- by HPX, DTPA, CYPMPO and XOD. The spin adducts by CYP- matic complex I inactivation is associated with protein nitration MPO were sensitive and very stable. The trapping activity for al- and oxidative damage to proteins and phospholipids. kyl-oxy radical was measured by the GSH equivalent, and that for the superoxide radical by the SOD equivalent. We concluded that CYPMPO has a high capability for radical trapping and is useful.

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Quercetin reduces inflammatory pain by inhibiting Regulation of cell signals by procyanidins at the cytokine production and oxidative stress intestinal epithelium

1 2,3 4 Sandra V. Verstraeten1, Matieu Da Silva2, Grayson K. Jaeggers2, Waldiceu A. Verri, Jr , Daniel A. Valério , Sandra R. Georgetti , 2,3 2,4 Danilo A. Magro2, Thiago M. Cunha2, Fabiana T.M.C. Vicentini3, Patricia I. Oteiza , and César G. Fraga 2 3 2 Silvio M. Vieira , Maria J.V. Fonseca , Sergio H. Ferreira , 1 2 4 Department of Biolological Chemistry-IQUIFIB-IIMHNO, School of Pharmacy Fernando Q. Cunha , and Rubia Casagrande and Biochemistry, UBA-CONICET, Argentina; Departments of 1Nutrition and 3Environmental Toxicology, University of California, Davis, CA, USA; and 4 1Departamento de Ciências Patológicas - Centro de Ciências Biológicas, Physical Chemistry-PRALIB, School of Pharmacy and Biochemistry, Universidade Estadual de Londrina; 2Department of Pharmacology, FMRP, UBA-CONICET, Argentina USP, Ribeirão Preto; 3Department of Pharmaceutical Sciences, USP, Ribeirão Preto; 4Departamento de Ciências Farmacêuticas - Centro de Ciências de Based on the findings that large procyanidins (LP) protect in- Saúde, Universidade Estadual de Londrina testinal cells from different pro-inflammatory stimuli, we have now tested the hypothesis that LP interact with intestinal epithelial Quercetin is a flavonoid with antioxidant and antinociceptive cells and through these interactions prevent pro-inflammatory effects. However, the mechanisms involved in its antinociceptive events initiated at lipid rafts, e.g. calcium mobilization, oxidant effect are not fully elucidated. Cytokines and reactive oxygen spe- formation, and activation of select cell signal. We used both, cies have been implicated in the cascade of events resulting in liposomes (as model of membranes with different lipid composi- inflammatory pain. Therefore, we evaluated the antinociceptive tion) and Caco-2 cells (as model of intestinal epithelium), to inves- mechanism of quercetin focusing on the role of cytokines and oxi- tigate the effects of a fraction enriched in LP isolated from cocoa. dative stress. The treatment with quercetin dose- (30-300 mg/Kg) LP increased the resistance to Triton-X 100-mediated disruption of dependently inhibited the second phase of formalin test, acetic acid glycolipid-enriched liposomes. In Caco-2 cells, LP inhibited NF- and phenyl-p-benzoquinone overt pain-like behavior, and car- B activation initiated by different pro-stimulatory compounds at a rageenin-induced mechanical hypernociception. Quercetin also in- different extent. The the highest inhibitory effects were observed hibited the hypernociception induced by cytokines (TNF - 43% for agents that initiate NF- B -activation at lipid rafts, i.e. tumor and CXCL1 - 38%), but not of inflammatory mediators that di- necrosis factor alpha and deoxycholate. Furthermore, LP inhibited rectly sensitize the nociceptor such as PGE2 and dopamine. Quer- deoxycholate-induced calcium mobilization, oxidant production cetin did not affect carrageenin- or cytokine (TNF and CXCL1)- and the activation of mitogen-activated kinases (MAPKs). In sum- induced leukocyte recruitment, which can be a source of hyperal- mary, LP can interact with synthetic and biological membranes and gesic mediators. On the other hand, quercetin reduced carrageenin- protect them from different pro-inflammatory stimuli. The ob- induced IL-1 production as well as carrageenin-induced decrease tained results suggest that LP has could have selective interactions of reduced glutathione (GSH) levels. Concluding, these results with particular areas of the membrane, preventing oxidant and sig- suggest that quercetin exerts its analgesic effect by inhibiting pro- naling events initiated at lipid rafts. nociceptive cytokine production and the oxidative imbalance me- diation of inflammatory pain. Supported by grants from CHNR-State of California Vitamin Price Fixing Consumer Set- tlement Fund and Mars Inc., and UBACyT B801.

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Glucose 6-phospate dehydrogenase deficiency Labile iron-mediated cerulein-induced oxidative stress in accelerated Coronavirus 229E infection due to rat acinar AR42J cells impaired inflammatory response

1Michal Wozniak, 3Maciej Sledzinski, 2Andzelika Borkowska, Yi-Hsuan Wu#, Shin-Ru Lin#, Mei-Ling Cheng#+, 2Alicja Sielicka-Dudzin, 2Magorzata Halon, and Chuen-Mao Yang*, and Daniel Tsun-Yee Chiu*#+ 2Jedrzej Antosiewicz # Graduate Institute of Medical Biotechnology, Chang Gung University, 259, st + 1 2 Wen-hwa 1 Rd, Kwei-san, Tao-yuan, Taiwan; Department of Clinical Department of Medical Chemistry, Bioenergetics and Physiology of Exercise, Pathology, Chang Gung Memorial Hospital, Kwei-san, Tao-yuan, Taiwan 3General and Endocrine Surgery and Transplantation, Medical University of Gdask, Poland Glucose 6-phosphate dehydrogenase (G6PD), the key regula- Reactive oxygen species (ROS) have been implicated in the tory enzyme in the pentose phosphate pathway, provides reducing pathogenesis of acute pancreatitis (AP). However, the mechanism power to all cells in the form of NADPH to meet the cellular needs of ROS formation in the pancreas remains poorly defined. We ob- for reductive biosynthesis and maintenance of the cellular redox served that during acute pancreatitis (AP), the iron storage protein status. We have previously demonstrated that cellular susceptibility ferritin in the rat pancreas undergoes degradation accompanied by toward viral infection is modulated by G6PD status, yet the de- an increased formation of protein carbonyls. In order to get an in- tailed mechanism remains elusive. One possibility is that cellular sight into the mechanism of ROS formation, we also performed redox status modulated by G6PD may involve in fine-tuning of experiments on the pancreatic acinar AR42J cells stimulated by ce- immune response against viral infection. To test this hypothesis, rulein. In such cells we observed increased labile iron pool (LIP) we used G6PD-knockdown lung epithelial A549 cells as host and that is accompanied by a decrease in the cellular L ferritin level determined the viral production upon coronavirus infection in the and an increase in the ROS formation. The changes in ferritin level present of TNF- as an infectious model system. Upon 15 ng/ml of were inversely correlated with the ROS formation. Moreover, we TNF- treatment, G6PD-knockdown A549 cells generated less su- observed that in cells transfected with plasmid encoding inactive peroxide compared to control during earlier time period (<4 hours). mutant of JNK1 cerulein did not induce ferritin degradation. In In addition, G6PD-knockdown A549 cells also displayed reduced conclusion, our data suggest that LIP significantly participates in level of phosphorylated p38 and ERK. Moreover, the proinflam- the cerulein induced ROS formation and that both ferritin degrada- matory protein Cox-2 and its downstream product PGE2 were sig- tion and iron dependent ROS formation is controlled by JNK1. nificantly attenuated in G6PD-knockdown A549 cells following TNF- treatment. Interestingly, our data also demonstrated a down-regulated expression of viral gene (nucleocapsid) in G6PD- knockdown cells under increased inflammation condition as com- pared to G6PD knockdown cell not treated with TNF-. Take to- gether, these results support our hypothesis that altered redox status due to G6PD knockdown may involve in modulating cellular immune response against viral infection.

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2-Deoxyglucose diet induces ketosis, enhances NO stress mediated by neural NO synthase (nNOS), mitochondrial function and reduces Alzheimer’s like pathology a potential accelerator of melanoma progression? in the triple transgenic Alzheimer’s mouse model

Sun Yang and Frank L. Meyskens Jia Yao, Ronald Irwin, Shuhua Chen, Eric Hernandez, Chao Family Comprehensive Cancer Center, and Roberta Díaz Brinton University of California Irvine, Orange, CA 92868 Department of Pharmacology and Pharmaceutical Sciences, Our laboratory has been extensively involved in the study of ab- School of Pharmacy, University of Southern California, Los Angeles, CA 90033 normal redox status and redox-sensitive signalings such as APE/Ref- 1, AP-1 and NF- B in the past decade for the experimental therapeu- Previously, we have shown that mitochondrial bioenergetic tics of human melanoma. Our goal is to identify key factors underly- deficits precede Alzheimer’s disease (AD) pathology in the triple ing melanoma development. It has been well-documented that UV transgenic AD (3xTg-AD) mouse model(Yao et al., 2009). Both radiation exposure especially sunburn at a young age is particularly basic science and clinical studies suggested a change in brain linked to melanoma incidence. As an important environmental car- metabolic profile prior to the onset or diagnosis of AD. In the cur- cinogen, UV radiation not only generates ROS, but also produces a rent study, we sought to 1) further investigate brain metabolic large amount of nitric oxide (NO) in human skin. Utilizing a NO- change with age and AD progression in the 3xTg-AD mouse donor DETA to mimic NO stress, we demonstrated that melanoma model; 2) to investigate the impact of 2-deoxyglucose (2-DG), a proliferation and invasion potential were significantly stimulated by common compound to induce ketogenesis, on AD pathology. At 3 DETA treatment, associated with inductions of many proteins in- month female 3xTg-AD mice had higher SCOT expression relative volved in cell growth (c-Jun, JunD), anti-apoptosis (Bcl-2, APE/Ref- to non-transgeic (nonTg) female mice, suggesting an early shift to 1) and metastasis signalings (MMP-1, Snail). Notably, long-term ex- ketogenic phenotype to compensate the decrease in brain glucose posure of DETA/NO stress resulted in over-growth of primary normal metabolism in these mice. The expression of SCOT decreased with human melanocytes with formation of foci in in vitro culture, indicat- age in the 3xTg-AD mice. At 12 months, both SCOT expression ing gain of additional vertical growth potential. As melanocytes are and activity were significantly lower in the 3xTg-AD mice relative to nonTg. To investigate the impact of 2-DG on AD like pathol- originated from neural crest, we proposed that neural NO synthase (nNOS) might play an important role in generating NO and mediating ogy, both nonTg and 3xTg-AD female at 6 month were fed with NO stress in human melanoma. First, both our in vitro cell culture either regular diet (AIN-93G) or diet containing 0.04% 2-DG for 3 (immunoblotting) and in vivo human biopsy (immunohistochemistry) weeks. Serum ketone levels as well as hippomcapal SCOT levels studies demonstrated marked elevation of nNOS expressions in mela- were increased with 2-DG in both nonTg and 3xTg-AD mice. In noma. More interestingly, induction of nNOS was evident with UVB addition, compared to the control group, 2-DG increased the ex- radiation and bFGF incubation. Knockdown of nNOS with siRNA ef- pression of enzymes involved in fatty acid metabolism as well as ficiently reduced the DETA-induced melanoma proliferation and in- oxidative phosphorylation. In 3xTg-AD mice, 2-DG diet also re- vasion potential, with reductions of c-Jun, Bcl-2 and MMP-1. A duced AD like pathology. The reduction in amyloid pathology is novel synthesized nNOS inhibitor JI-11 was utilized to inhibit nNOS likely due to up-regulation of alpha secretase pathway. All to- activity; our data showed that co-treatment of JI-11(1μM) signifi- gether, data suggest that in 3xTg-AD mice there is a shift towards cantly attenuated the alterations induced by UVB radiation and ketogenic phenotype early in AD progression, suggesting the acti- DETA treatments. Based on our studies, we propose that targeting vation of a compensatory pathway to the decrease in glucose me- nNOS with application of specific synthetic inhibitors to diminish tabolism. A relative short-term 2-DG treatment induces ketogene- NO stress represents an innovative and promising strategy for mela- sis in both genotypes, increases fatty acid oxidation and reduces noma prevention. AD like pathology. The long-term impact of 2-DG remains to be further investigated.

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. NO modulation of glutathonylation of proteins: Regulation of insulin signaling by lipoic acid: Implications for aging and neurodegeneration Therapeutic implications for neurodegeneration

¶ Li-Peng Yap, Jerome V. Garcia, Derick Han and Enrique Cadenas Li-Peng Yap, Jerome V. Garcia, Derick Han¶ and Enrique Cadenas

¶ Pharmacology & Pharmaceutical Sciences, School of Pharmacy and Research Pharmacology & Pharmaceutical Sciences, School of Pharmacy and ¶Research Center for Liver Diseases, Keck School of Medicine, University of Southern Center for Liver Diseases, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA California, Los Angeles, CA 90089, USA

The interaction between redox and energy changes establishes Aberrant insulin signaling – entailing decreased Akt (also a regulatory mechanism that controls cellular energy levels in re- known as protein kinase B (PKB) and increased glycogen synthase sponse to redox changes (i.e., increased generation of H2O2 and kinase (GSK) activities– may account for (a) the early energy . NO) through specific post-translational modifications of cytosolic changes due to decreased glucose metabolism and mitochondrial and mitochondrial proteins. The role of these protein modifications generation of energy, and (b) the perpetuation of a cycle that leads in aging and age-related neurodegeneration is established by an to the later neuropathological hallmarks of Alzheimer’s disease: . age-dependent increase in the levels of NO –a consequence of in- A plaque and NFT formation. Hence, the modulation of insulin crease expression of neuronal nitric oxide synthase and inflamma- signaling bears therapeutic potential for the early and late stages of tion– leading to nitration, S-nitrosation, and S-glutathionylation of AD. The use of insulin itself in Alzheimer’s patients has shown in- specific proteins. Acute exposure of primary cortical neurons and creased cognitive function but with mixed results concerning A . astrocytes to a NO flux, mirroring neuroinflammation, led to S- accumulation, as both insulin and A are proteolytically degraded glutathionylation of proteins in a dose- dependent manner due to by the same enzyme (insulin-degrading enzyme). Therefore, com- oxidation of the cellular redox environment. The significance of a pounds that elicit an “insulin-like” effect may be effective in the higher redox buffering capacity was reflected in the extent of intra- use in AD through the modulation of intracellular insulin signaling cellular formation of S-glutathionylated proteins. Increasing con- without competition at the IDE level. centrations of GSNO and GSSG formation as a consequence of Our studies using small animal imaging (microPET) show a . NO exposure correlated with S-glutathionylation of proteins. Glu- decrease in glucose metabolism in a triple transgenic mouse model tathonylation of GAPDH, a key glycolytic enzyme, led to signifi- (APPSWE + PS1M164V + TauP301L) that develop both the pathological cant inhibition of its activity. and cognitive changes reflected in AD in humans. Decrease in Glutathionylation of GAPDH and inhibition of its activity were glucose metabolism in these mice occurs with increasing age and also observed in a triple transgenic model of Alzheimer’s disease precedes substantial plaque and tangle formation as well as cogni- as a function of age. Metabolic changes observed in Alzheimer’s tive changes. Our studies show that feeding mice lipoic acid in- disease –decreased pyruvate dehydrogenase activity and mito- duces insulin-like effects, demonstrated by increase glucose me- chondrial respiration– and redox changes preceded the occurrence tabolism as well as modulation of insulin signaling in neurons of histopathology and accompanied cognitive deficits and were through modulation of the phosphorylation status of Akt and compounded by inactivation of cytosolic GAPDH upon S- GSK3 in vivo and in vitro. glutathionylation.

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Novel EPR imaging of fluctuating oxygenation associated with a defect of vascular integrity in Signal transduction pathways mediate H2O2-induced necrosis transplanted tumors in primary cultured hepatocytes

Hironobu Yasui1,2, Shingo Matsumoto2, Maria D. Ybanez and Derick Han Nallathamby Devasahayam2, Sankaran Subramanian2, James B. Mitchell2, and Murali C. Krishna2 Research Center for Liver Diseases, University of Southern California, 2011 Zonal Avenue, LA, CA 90089-9121, USA

1Laboratory of Radiation Biology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, In this study, the signaling pathways that regulate necrosis in- 2 Sapporo, Japan. Radiation Biology Branch, Center for Cancer Research, duced by H O in primary cultured hepatocytes were examined. National Cancer Institute, Bethesda, MD 20892, USA 2 2 H2O2 treated to hepatocytes is consumed within minutes, but hepa- Fluctuation in blood flow is one of tumor phenotype, which tocytes undergo necrosis several hours later. H2O2 treatment in- develops heterogeneity of oxygen and subsequently invokes lack duces a “lag phase” where signaling transduction pathways includ- of cancer treatment efficacy. Both to improve therapeutic planning ing glycogen synthase kinase-3 (GSK-3) and protein kinase C effectively against solid tumor and to make precise prognosis in (PKC) are activated. GSK-3 inhibitor or silencing GSK-3 was cancer treatment, noninvasive dynamic imaging of spatial and effective in reducing necrosis caused by H2O2 (~ 50- 80%) in pri- mary cultured hepatocyes. This suggests GSK-3 plays an essen- temporal pO2 profile density is needed. Here, we report for the first time to our knowledge that rapid imaging with pulsed electron par- tial role in mediating H2O2-induced necrosis. PKC inhibitor treat- amagnetic resonance imaging (EPRI) capacitated direct monitoring ment also protected against H2O2-induced necrosis. PKC inhibitor of oxygen fluctuation within minutes in natural state of murine treatment to primary hepatopcytes, however, increased activity of squamous cell carcinoma (SCCVII) and human colorectal carci- Akt. GSK-3 is downstream target of Akt, and PKC inhibitor noma (HT29) tumors with reliable resolution. Special resonator treatment consequently resulted in increased phosphorylation of applicable for both EPRI and magnetic resonance imaging (MRI) GSK-3 (serine 9, which inactivates GSK-3), likely due through increase in Akt activity. This suggests PKC is a negative regulator provided pO2 maps with anatomical guidance and microvessel density without positional movement. Oxygen images every 3 min of Akt and also suggests part of the protective mechanism of PKC in two different tumor-bearing mice model disclosed tumor-size- inhibitor against H2O2-induced necrosis may be mediated through and tumor-type-dependent variation of fluctuating oxygenation. inactivation of GSK-3 in primary cultured hepatocytes. Taken to- Immunohistochemical analysis for CD31 and SMA revealed that gether our data demonstrates that signaling pathways involving the fluctuation of oxygenation was correlated with pericyte density GSK-3 and PKC mediate H2O2-induced necrosis, suggesting strongly rather than vascular density in tumor. These results sug- H2O2 induces a “programmed necrosis” in primary cultured hepa- tocytes. gested that EPR oxymetric imaging combined with MRI can be a potent method to detect fluctuating oxygenation in solid tumor non-invasively.

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The mitochondrial energy-redox axis in aging and Design of pH-sensitive polymeric micelle possessing caloric restriction: reduced forms of TEMPO for imaging of ROS Potential role of nicotinamide nucleotide transhydrogenase Toru Yoshitomi1, Takashi Mamiya2, Hirofumi Matsui2,3, Fei Yin, Harsh Sancheti, and Enrique Cadenas Aki Hirayama3,6, and Yukio Nagasaki1-5

1Graduate School of Pure and Applied Sciences 2Graduate School of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, 3 4 5 University of Southern California, Los Angeles, CA, USA Comprehensive Human Sciences TARA TIMS, University of Tsukuba MANA, NIMS, Tennoudai 1-1-1, Tsukuba, Ibaraki, Japan 6 Tsukuba University of Technology, Kasuga 4-12-7, Tsukuba, Ibaraki, Japan As cellular powerhouses and established cellular sources of H2O2, mitochondria play a central role in the aging progress. The Recently, a non-invasive imaging of reactive oxygen species steady-state level of mitochondrial H2O2 is determined at its ap- (ROS) has attracted attentions as a new method of evaluating dis- proximation equilibrium by the rate of H2O2 generation (electron ease and response to clinical treatment. For example, inflammation leak during NADH oxidation in the respiratory chain) and of H2O2 sites are known to be acidic pH, in which ROS are excessively gen- removal (NADPH-dependent, GSH and thioredoxin supported erated. A variety of low-molecular-weight electron paramagnetic processes in the mitochondrial matrix). Generation of mitochon- resonance (EPR) imaging probes such as 1-hydroxy-2,2,6,6-tetra- drial NADPH is largely dependent on the inner-membrane Nicoti- methylpiperidinyloxy (TEMPO-H) have been developed for the namide Nucleotide Transhydrogenase (NNT), which catalyzes the + detection of ROS. However, such probes are hard to utilize in vivo reduction of NADP to NADPH utlizing the proton gradient as the due to their preferential renal clearance. To solve this issue, we driving force and NADH as the electron donor. Thus, NNT repre- have developed pH-sensitive polymeric micelle possessing reduced sents a critical link between mitochondrial metabolic function (en- forms of TEMPO (RNP(R)) using self-assembling of amphiphilic ergy component) and redox homeostasis (redox component) by block copolymers composed of a hydrophilic poly(ethyleneglycol) coupling NADPH generation to the TCA cycle and active respira- segment and a hydrophobic poly(styrene-TEMPO-H) segment tion; a mitochondrial energy-redox axis is hereby defined. Our re- containing amino groups. Due to a protonation of the amino groups sults demonstrate that aging in Fischer 344 rats is accompanied by in the hydrophobic core, RNP(R) disintegrates in response to (a) impaired energy metabolism; (b) shift of redox state; (c) de- acidic pH. When RNP(R) was mixed with horseradish peroxidase creased NNT activity, and (d) increased H2O2 levels. Furthermore, (HRP)/ hydrogen peroxide (H2O2) couple as a model of in vivo oxi- these energy and redox changes are attenuated by short-term ca- dants, the EPR spectra of RNP(R) were obtained. The EPR signal loric restriction in senescent animals but not in young animals. of RNP(R) under acidic condition (pH=5.6) increased much faster SiRNA to NNT in PC12 cells elicited changes in both energy and than that under neutral condition (pH=7.4), though the enzymatic redox status that validate the role of NNT in the energy-redox axis, activity of HRP is almost the same under the present experimental mainly a substantial shift toward anaerobic glycolysis. Data from condition. These results suggest that the disintegration of RNP(R) the PC12 cell model and an NNT-knockout mouse model accelerates the oxidation reaction in acidic region, indicating that strengthen the importance of the interdependent mitochondrial en- RNP(R) is anticipated as EPR imaging probe for imaging of ROS at ergy-redox axis in aging and provides evidence for a regulatory low pH regions. role of NNT.

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Anti-senescence effect of natural antioxidants green tea Cytosolic ferritin degradation is prerequisite for polyphenols and quinic acid in C. Elegans mitochondrial ferritin-induced iron mobilization L.Z. Zhang1, X.Z. Zhao-Wilson2, and B.L. Zhao1

1 Yinghui Zhang, Marc Mikhael, Dongxue Xu, Yiye Li, State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China. 2BioMarker Shan Soe-Lin, Bo Ning, Yuliang Zhao, Prem Ponka, and Pharmaceuticals, Inc. 5941 Optical Court, San Jose, CA 95138, USA Guangjun Nie*

Natural antioxidants play important roles in anti-aging process. We CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety studied the anti-senescence effect of natural antioxidants, green tea National Center for Nanoscience and Technology of China, Beijing 100190 polyphenols and quinic acid in C. Elegans and found they have differ- ent effects and pass through different pathways. We found that Epigal- locatechin gallate (EGCG) extended C. elegans longevity under Ferritin sequesters and stores iron, consequently protecting stresses. Under heat stress (35°C), EGCG improved the mean longev- cells against iron-mediated free radical damage. However, the ity by 13.1% at 0.1mg/ml. Under oxidative stress, EGCG could im- mechanism of iron exit from the ferritin cage and its reutilization prove the mean longevity of C. elegans by 172.9% at 0.1mg/ml. How- are largely unknown. In a previous study we found that mitochon- ever, EGCG could not extend the lifespan of C. elegans under normal drial ferritin (MtFt) expression led to a decrease in cytosolic fer- culture conditions. Further studies demonstrated that the significant longevity-extending effects of EGCG on C. elegans could be attributed ritin. Here we showed that treatment with lysosomal inhibitors to the in vitro and in vivo free radical scavenging effects of EGCG, and largely blocked ferritin loss in both MtFt-expressing and wild type the up-regulative effects of EGCG on stress-resistance-related proteins cells. Moreover, ferritin in cells treated with lysosomal inhibitors including SOD-3 and HSP-16.2 in mutant C. elegans with SOD- was found to store more iron than did ferritin in untreated cells. 3::GFP and HSP-16.2::GFP expression. Considering that the death rate The prevention of cytosolic ferritin degradation in MtFt-expressing of a population is closely related to the mortality caused by external cells significantly blocked iron mobilization from the protein cage stress, it could be concluded that the survival-enhancing effects of EGCG on C. elegans under stresse are very important for anti-aging induced by MtFt expression. These studies also showed that block- research. Quinic acid (QA) from plants can extend the life span of C. age of ferritin loss by leupeptin resulted in decreased ferritin syn- elegans in normal conditions. We found that QA could extend C. ele- thesis and prolonged ferritin stability, potentially resulting in di- gans lifespan by 11.4% under normal culturing conditions, 17.8% un- minished iron availability. Lastly, we found that proteasomes were der thermal stress, and 29.7% under oxidative stress. Both sir-2.1 and responsible for ferritin degradation in cells pretreated with ferric daf-16 are required for QA to extend worm lifespan, which suggests ammonium citrate. Thus, the current studies suggest that ferritin that QA causes worms to live longer in a signaling pathway involving sir-2.1 and daf-16. In the QA-treated worms, the downstream DAF-16- degradation precedes the release of iron in MtFt-expressing cells; targeted genes, sod-3 and hsp-16.2, were consequently up-modulated. that MtFt-induced cytosolic ferritin decrease is partially prevent- However, hsp-16.2 was not indispensable for the lifespan-extending able by lysosomal protease inhibitors; and that both lysosomal and effect of QA suggesting that HSP-16.2 was only one of the effectors proteasomal pathways may be involved in ferritin degradation. for QA. Meanwhile, QA exhibited an ability to keep the ROS at a lower level in worms by scavenging free radicals. The ability of QA to extend worm lifespan combined with the association of QA with sir- 2.1, daf-16, sod-3, hsp-16.2 and ROS demonstrates that QA possesses great potential in anti-aging. Supported by a grant from the National Natural Science Foundation of China (30370361).

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The role of mitochondrial ferritin on hydrogen peroxide Protective effect of green tea polyphenols against induced SH-SY5Y cell damage 6-OHDA-induced apoptosis through ROS-NO pathway in Parkinson’s disease models Nan Zhang1, Xiang-Lin Duan1, Zhen-Hua Shi1, Zhen Li1, 3 2 1* Baolu Zhao Guang-Jun Nie , Bao-Lu Zhao , Yan-Zhong Chang State key Laboratory of Brain and Cognitive Sciences, Institute of Biophysics, 1Laboratory of Molecular Iron Metabolism, College of Life Science, Hebei Academia Sinica, Beijing 100101, China Normal University, Shijiazhuang 050016, Hebei Province, China (*[email protected]) To investigate the protective effect of green tea polyphenols 2State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, against 6-OHDA induced apoptosis in Parkinson’s disease, we de- Academia Sinica, Beijing, 100101, China veloped a method to simultaneously detect NO and ROS in bio- 3CAS Key Laboratory for Biological Effects of Nanomaterials & Nanosafety, Na- tional Center for Nanoscience and Technology of China, Beijing, 100190, logical system using electron spin resonance (ESR) spin trapping China technique. In the models of 6-OHDA induced SH-SY5Y cell and 6-OHDA injected rat neuron, the cell apoptosis was detected by Mitochondrial ferritin (MtFt) is a newly identified mitochon- MTT, flow cytometric analysis using Annexin V and PI and dria protein encoded by an intronless gene on chromosome 5q23.1 ELISE methods. ROS and NO free radicals were detected by ESR in humans. Previous studies suggested that overexpression of MtFt technique. The NOS was detected by RT-PCR and Western blot caused a redistribution of iron from cytosol to mitochondria thus assays. The protein bound 3-nitro-tyrosine (3-NT) was measured high levels of MtFt result in an iron deficient phenotype in cytosol, by competitive ELISE assay. More cell survived and less cell suf- it expression also inhibited the in vivo tumor growth due to cytoso- fered apoptosis in the culture cells and substantia nigra treated by green tea polyphenols. ROS and NO free radical generation, lic iron deprivation, and MtFt over-expression in HeLa cells in- thiobarturic acid reacted substances (TBARS) content, ni- creases resistance to oxidative stress. In addition the study of trite/nitrate concentration and protein bound 3-NT were decreased MtFt may be useful for revealing mitochondria iron metabolism and antioxidant abilities increased by the treatment of green tea and finding possible therapeutic applications in neurological disor- polyphenols. The protein levels and activity of NOS were de- ders involving increased iron deposition. creased by the treatment of green tea polyphenols. Conclusions: In this study, we examined that the role of MtFt on H2O2 in- These results suggest that green tea polyphenols protected cell duced cell damage using the MtFt overexpressed SH-SY5Y cells. apoptosis induced by 6-OHDA through ROS-NO pathway in vitro Our results showed that overexpression of MtFt restrained the in- and in vivo.

crease of ROS and the decrease of mitochondrial membrane poten- Nie,G..J., Jin,C-F., Zhao,B-L. Distinct effects of tea catechins on 6- tial, maintained the level of anti-apoptotic protein Bcl-2 expres- hydroxydopamine-induced apoptosis in PC12 cells. Arch. Biochem. Bio- sion, inhibited the activation of pro-apoptotic protein caspase3, phys. 397,84-90,2002 Nie, G.J., Cao, Y-L., Zhao, B-L. Protective effects of green tea polyphenols and thus inhibited the apoptosis of SH-SY5Y cells induced by H2O2. their major component, (-)-epigallocatechin-3-gallate (EGCG), on 6- Interestingly, when Vector-SY5Y cells were treated with H2O2 for hydroxyldopamine-induced apoptosis in PC12 cells. Redox Report, 7,170- 24 h, the significantly upregulated the levels of ferritin, DMT1 177, 2002 S-H Guo, E Bezard, Baolu Zhao. Protective effect of green tea polyphenols on without IRE and downregulated the levels of TfR, DMT1 with IRE the SH-SY5Y cells against 6-OHDA induced apoptosis through ROS-NO compared to the control groups; while no significant changes were pathway. Free Rad Biol Med 39: 682-695, 2005. found in MtFt-SY5Y cells under the same treatment. The mecha- Shuhong Guo, Jingqi Yan, Erwan Bezard, Tangbin Yang, Xianqiang Yang, Baolu Zhao。Protective effects of green tea polyphenols in the 6-OHDA rat nism maybe was that overexpression of MtFt decreased the free model of Parkinson’s disease through inhibition of ROS-NO path- iron level by regulating the iron metabolism related proteins, and way
Biological Psychiatry 621353-1362 2007

then inhibited the fenton reaction, decreased the production of This work was supported by a grant of National Natural Scientific Foundation ROS, thus inhibited the cell apoptosis induced by H2O2. (30170239) and 973 grant (2006CB500700).

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Effects of aqueous extracts from Vitis coignetiae Pulliat leaves on non-alcoholic steatohepatitis model rat Anti-oxidation and lifespan-extension activities of CordyMax in oxidative stress and aging models Chengzhu Zhao 1, Fusako Takayama1, Toru Egashira1, 1,2 3 3 3 Mitsumasa Mankura1, Keiji Ueki2, Azusa Hasegawa1, Jia-Shi Zhu , Yan Zhang , Jieying Yang , Ninzhi Tan , 3 Hiromu Kawasaki1, Shigeru Okada1, and Akitane Mori1 and Chunsheng Zhao

1 1Pharmanex Research Institute, Provo, UT 84601, 2Pharmanex Beijing Clinical Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 3 Okayama University. 2Hiruzen winery Co., Ltd Pharmacology Center: 2 Xinkang Road, Beijing 100088, China; School of Pharmacy, Shihezi University, Shihezi City, Xinjiang 832000, China

Background: Vitis coignetiae Pulliat (Yamabudo) is used as Cordyceps sinensis is traditionally believed as an anti-aging health juice and wine based on the abundant polyphenols and an- herb in China. We have reported the effects of CordyMax (CM), a thocyanins in its fruit. However, the similar benefits of leaves of mycelia fermentation product of C. sinensis, in glucose-lipid- this plant were not been well demonstrated. This study investigated energy metabolisms, anti-fatigue and endurance enhancement. In the therapeutic effects of aqueous extracts from Vitis coignetiae this study we examined the effect of CM in antioxidant and Pulliat leaves (VCPL) on an animal model of nonalcoholic steato- lifespan extension in mice. The antioxidant activity was tested in hepatitis (NASH) at the progressive processes. mice (6 months old) that received 60 days of vehicle or CM (0.5, Methods: NASH model (PCT/JP 2007/52477) were prepared 1.0, or 1.5 g/kg) and a single dose of 11 Gry 60Co gamma-radiation by loading oxidative stress following fatty liver formation in rats on Day 60. Compared to controls, CM increased plasma total thiol- by feeding choline deficient high fat diets (CDHF). After the pro- groups, GSH and GSH-peroxidase, and liver CAT, SOD and GSH- gress to steatohepatitis, VCPL administration to NASH rats was reductase (p < 0.05). CM reduced liver protein carbonyl-groups performed for 3 weeks. Animal experiment was performed in 5 and 8-OHdG (p < 0.05). For examining the lifespan-extension ef- groups; Control (with MF chow diets), CDHF, NASH, NASH + fect of CM, 250 mice of 12 months of age (both sexes) were re- 100VCPL and NASH+300VCPL. After the experimental period, ceived either vehicle or CM (0.5, 1.0, or 1.5 g/kg) mixed with the blood and liver were collected from anesthetized animals for the forage. Calorie intake was adjusted to match the levels for controls samples to determine the extent of oxidative stress injury and the twice per week. Compared to controls, the 75% survival time was overall effects of VCPL , biochemically and histologically. extended 94-108 days in the CM dosage groups, the 50% survival Results: The electron spin resonance measurements assay time extended 10-66 days, the 25% survival time extended 29-44 demonstrated the strong anti-oxidative activities of VCPL. In days and the 12.5% survival time extended 7-50 days (86 wks so NASH, hepatobiliary enzyme leakages and liver fibrosis were ex- far; treatment continues). The Kaplan-Meier Survivor analysis re- hibited. There were demonstrated OS in liver mitochondria and in- vealed the extended lifespan and the reduced risks of death by CM creases in nuclear NF- B. VCPL administration after the steatohe- (p < 0.05). In conclusion, CM therapy significantly improves the patitis, abated these changes. body’s antioxidant capacity and extends the lifespan in mice, sup- Conclusion: The strong anti-oxidation activities of VCPL may porting the traditional belief on the anti-aging function of CM in be beneficial in ameliorating NASH. As the OS enhances the NF- humans. B nuclear import which promotes the induction of inflammatory proteins.

176 177 A Beveridge, S...... 108 Acin-Perez, R...... 40 Bhattacharya, S...... 73 Adams, L.S...... 47 Bibus, D...... 116 Agarwal, A.R...... 62 Biesalski, H.K...... 41 Agbonwaneten, E...... 105 Biswas, S...... 71,90 Aggarwal, B.B...... 46 Blaner, W.S...... 40 Ahmed, S.S...... 113 Bombicino, S.S...... 159 Aiman, S...... 63 Bone, R.A...... 78 Aimo, L...... 64 Borkowska, A...... 67,162 Albanes, D...... 38 Borrás, C...... 56 Ali, I...... 109 Bourdette, D.N...... 149 Ames, B.N...... 59 Boveris, A...... 134,159 Amiranashvili, L...... 75,76 Boveris, A.D...... 134 Ananth, P...... 148 Bratasz, A...... 66 Anderson, R.F...... 65 Brinton, R.D...... 105,113,165 Andrei, C.C...... 82 Brooks, G.A...... 93 Anghelina, M...... 66 Budac, S...... 114 Anita, Nor ...... 63 Butt, O.I...... 66 Antosiewicz, J...... 67,162 Byun, H.-O...... 153 Anzai, K...... 68,127,133 Arab, L...... 152 C Arçari, D.P...... 147 Cadenas, E. .62,106,107,112,113, Aronson, W.J...... 47 ...... 123,134,150,166,167,170 Aruga, T...... 87,88 Calcutt, N...... 91 Asghar, M...... 85 Cambou, M.C...... 152 Aung, H.H...... 69,101 Carr, D.W...... 149 Ay, H...... 139,154 Carvalho, R...... 79,80 Azad, A...... 70 Casagrande, R...... 81,82,98,160 Catalano, A...... 142 B Chan, Y.-C...... 71,83 Bairos, V.A...... 79,80 Chang, Y.-Z...... 174 Bandez, M.J...... 134 Chankvetadze, B...... 75 Bandinelli, S...... 38 Chen, N...... 119 Banerjee, J...... 71,72 Chen, S...... 165 Banerjee, P...... 73 Chen, Z...... 22 Banerjee, S...... 73 Cheng, M.-L...... 163 Bao, X...... 74 Cherubini, A...... 38 Baracat, M.M...... 81,82,98 Chibber, S...... 109 Barbakadze, V...... 75,76 Chikvaidze, E.N...... 84 Baroth, V...... 108,115 Chiu, D.T.-Y...... 163 Bastiaens, P...... 77 Chiu, P.Y...... 119 Batista, W.L...... 129 Choe, W...... 117 Baum, T...... 111 Choi, Y...... 120 AUTHOR INDEX Bendzala, S...... 114 Choi, Y.-J...... 117,120 Bettaieb, A...... 77 Chugh, G...... 85

Chung, H.W...... 120 Fang, H...... 102 Grune, T...... 58 Iglesias, D.E...... 159 Cimrova, B...... 114 Feldman, D...... 99 Guralnik, J...... 38 Ikekawa, N...... 135 Clemens, M.G...... 17 Ferreira, S.H...... 160 Gurtner, G.C...... 10 Ikota, N...... 133 Cluett, C...... 38 Ferrucci, L...... 38 Irwin, R.W...... 105,113,165 Coelho, A.C...... 79,80 Firstenberg, M...... 99 H Colangelo, M...... 142 Fonsecca, M.J.V...... 160 Ha, T.K...... 120 J Collard, E...... 86 Font de Mora, J...... 56 Hagen, T.M...... 28 Jaeggers, G.K...... 161 Compadre, A.J...... 105 Forester, S.C...... 94 Haj, F...... 77 Jagla, F...... 114 Corsi, A.-M...... 38 Fraga, C.G...... 124,161 Hajiani, M...... 103,104 Jamora, C...... 16 Cross, C.E...... 101 Frayling, T.M...... 38 Halon, M...... 67,162 Jendeková, L...... 144 Cuervo, A.M...... 57 Fried, L.P...... 38 Hamilton, R.T...105,106,107,113 Jergelova, M...... 114 Cunha, F.Q...... 160 Friedman, A...... 13 Hammerling, U...... 40 Jia, H.-Y...... 125 Cunha, T.M...... 160 Froozandeh, M...... 103 Han, D...... 150,166,167,169 Jian, J...... 71 Curcio, M...... 129 Froyen, E.B...... 95 Han, L...... 125 Johnson, E.J...... 39 Fujii, H...... 138 Han, S...... 117 Jones, R...... 115 D Fukuahra, K...... 133 Hargreaves, I...... 45 Jou, Mei-Jie...... 110 D’Annunzio, V...... 159 Fukuzumi, S...... 133 Hasanzadeh, A...... 104 Juliano, J...... 155 Da Silva, M...... 161 Furber, J.D...... 96 Hasegawa, A...... 176 Jung, H.-J...... 153 Danilenko, M...... 122 Haskell, C.F...... 108,115 Jung, M...... 117 Das, D.K...... 25 G Hassan, I...... 109 de Cabo, R...... 43 Galeotti, T...... 128 Heber, D...... 47 K De Spirito, M...... 128 Galleano, M...... 124 Henning, S.M...... 47 Kagan, V...... 42 Dehpoor, A.R...... 104 Gamba, P...... 33 Herman-Antosiewicz, A...... 67 Kamibayashi, M...... 127 Devasahayam, N...... 168 Gambini, J...... 56 Hernandez, E...... 165 Kaneko, T...... 126 Dobesová, Z...... 145 Ganesh, K...... 97,99 Hirayama, A...... 126,171 Karato, M...... 157 Dohi, K...... 87,88 Garcia, J.V...... 62,150,166,167 Hiyama, K...... 126 Kasahara, D.I...... 24 Donato, M...... 159 Gargiulo, S...... 33 Hodis, H.N...... 106,112 Kawasaki, H...... 141,176 Dotan, Y...... 89 Gee, J...... 115 Holmgren, A...... 20 Kennedy, D.O...... 108,115 Draelos, Z.D...... 130 Gelpi, R...... 159 Hongo, N...... 157 Kern, D...... 44,130 Driggs, J...... 90 Georgetti, S.R...... 81,82,98,160 Hoyos, B...... 40 Khachatryan, I...... 84 Du, L.-B...... 125 Ghannadian, N...... 103 Hristova, M...... 24 Khaghani, S...... 103 Du, Y...... 20 Gibert, J.C...... 78 Hsieh, C.-E...... 110 Khan, M...... 148 Duan, X.-L...... 174 Glotzbach, J...... 10 Htet, Y...... 137 Khanna, S...... 70-72,83,90,97,99 Gnyawali, S...... 72,100,143 Huang, J...... 100 ...... 116,143,148 E Gnyawali, U...... 97,99,100 Huang, K...... 100 Kim, J.-H...... 121 Egashira, T...... 141,176 Gogilashvili, L...... 75,76 Huff, G.K...... 111 Kim, K...... 117 Ehren, J...... 91 Gogoladze, T.V...... 84 Hussain, S.-R.A ...... 71 Kim, S...... 117 Elkhammas, E...... 99 Gogvadze, V...... 26 Hussien, R...... 93 Kim, Y.J...... 120 Eller-Borges, R...... 129 Gohil, K...... 69,101 Hwang-Levine, J...... 106,112 Kim, Y.-M...... 121,153 Golestani, A...... 103,104 Kim-Saijo, M...... 23 F Gómez, C...... 134 I Kinkhabwala, A...... 77 Fan, J...... 92 Gonçalvez, C...... 79,80 Ichijo, H...... 70 Kirkpatrick, R...... 99 Fan, X...... 93 Gordillo, G.M...... 97,99,102 Igarashi, T...... 138 Klatte, E...... 99

180 181

Ko, K.M...... 119 Losordo, D.W...... 14 Moldovan, N.I...... 66 Koda, Y...... 136 Lu, J...... 20 Monleón, D...... 56 Kodo, Y...... 141 Lu, Y...... 74 Montagnier, L...... 7 Oh, I...... 117 Kohno, M...... 151 Lüdeman, J...... 108,115 Montano, S...... 20 Oharazawa, H...... 138 Konishi, T...... 135 Monteiro, H.P...... 129 Ohkubo, K...... 133 Korkmaz, A...... 139,140 M Monteiro, M.P...... 147 Ohsawa, I...... 138 Kotha, S.R...... 116 Machida, M...... 138 Moore, J.L...... 99 Ohta, S...... 138 Krinsky, N.I...... 39 Mackenzie, G.C...... 64 Moraes, M.S...... 129 Ohtaki, H...... 87 Krishna, M.C...... 168 Maddipati, K.R...... 97 Moreira, I.C...... 82 Oishi, M...... 156 Kubicki, N...... 143 Maggini, S...... 108,115 Mori, A...... 141,176 Okada, S...... 141,176 Kunes, J...... 145 Magro, D.A...... 160 Morré, D.M...... 44,130 Okuda, H...... 133 Kunwar, A...... 118 Maher, P...... 91 Morré, D.J...... 44,130 Omata, Y...... 64 Kuppusamy, P...... 54,66,148 Majumder, P...... 73 Mulkijanyan, K...... 75,76 Oommen, S...... 101 Mamiya, T...... 171 Murphy, M.P...... 65 Orlando, L...... 56 L Manda, S...... 68 Murray, A...... 38 Orrenius, S...... 26 Labate, V...... 128 Manfredi, G...... 40 Mustoe, T.A...... 15 Otani, H...... 31 Lam, P.Y...... 119 Mankura, M...... 141,176 Oteiza, P.I...... 64,94,124,137,161 Lame, M...... 69 Maroz, A...... 65 N Oter, S...... 139,140,154 Langley, P...... 96 Martinez, R.M...... 82 Nadeau, P...... 131 Owji, A.A...... 103 Lebold, K.M...... 152 Matsui, H...... 126,171 Nagano, Y.N...... 126 Ozawa, T...... 127,133 Lee, J.W...... 120 Matsumoto, A...... 68 Nagasaki, Y...... 132,156,171 Ozler, M...... 139,140,154 Lee, P...... 16 Matsumoto, K.-I...... 68,127,133 Nakajima, Y...... 135 Lee, S...... 16,120 Matsumoto, S...... 168 Nakamachi, T...... 87 P Lee, Y...... 120 Matsuo, Y...... 22 Nakamura, S...... 88 Pak, W...... 141 Lee, Y.-K...... 121 Matteini, A...... 38 Nakanishi, I...... 68,127,133 Pallardó, F.V...... 56 Leonarduzzi, G...... 33 Maulik, G...... 30 Nakayama, K...... 135 Palozza, P...... 142 Leong, P.K...... 119 Maulik, N...... 30 Naseem, I...... 109 Pani, G...... 128 Leshno, I.P.M...... 89 Maulucci, G...... 128 Navarro, A...... 134 Papadopoulos, K...... 75 Leumus, J...... 105 Mazumder, S...... 73 Navas, P...... 43 Parinandi, N.L...... 97,116 Leung, H.Y...... 119 Meadows, C...... 130 Neel, B...... 77 Park, A.-A...... 143 Levy, J...... 122 Mele, M...... 128 Néron, S...... 131 Pasalar, P...... 103,104 Li, C...... 123 Melzer, D...... 38 Nie, G...... 92,173,174 Patel, V...... 99 Li, Y...... 173 Merlani, M...... 75,76 Ning, B...... 173 Paulis, L...... 145 Li, Z...... 174 Meyskens, F.L...... 164 Nishida, H...... 135 Payabvash, S...... 104 Lichtenberg, D...... 89 Mihara, Y...... 87 Nishitani, Y...... 136 Pechanova, O...... 114,144,145 Lietz, G...... 37 Mikhael, M...... 173 Niwano, Y...... 151 Peixoto, F...... 80 Lin, S.-R...... 163 Miller, M...... 99 Norberg, E...... 26 Penumathsa, S.V...... 30 Litterio, C...... 124 Miminoshvili, A...... 84 Novikova, Z...... 767 Perola, M...... 38 Liu, W...... 152 Mine, Manaka...... 141 Nuttall, J.R...... 137 Perry, J.R.B...... 38 Liu, Y...... 125 Mitchell, J.B...... 168 Nyui, M...... 127,133 Petersen, R.C...... 146 Lokhandwala, M.F...... 85 Miyamoto, K...... 87,88 Pinto, M.L...... 79,80 López-Cepero, J.M...... 134 Mizuno, M...... 136 O Poli, G...... 33 López-Grueso, R...... 56 Moldovan, L...... 66 Ogur, R...... 139,140 Ponka, P...... 173

182 183

Prince, P.D...... 124 Sen, C.K.. 70-72,83,90,97,99,100 T W Priyadarsani, K.I...... 118 ...... 102,116,143,148 Takahashi, H...... 138 Walston, J...... 38 Prolla, T.A...... 49 Sengupta, R...... 20 Takayama, F...... 141,176 Wang, P...... 47 Sensel, M.G...... 152 Tan, N...... 177 Waterhouse, A.L...... 94 Q Seo, Y.-H...... 153 Tanaka, T...... 38 Watson, A...... 108 Qin, J...... 39 Sha, Y.-l...... 125 Tang, G...... 39 Wei, J.-Y...... 92 Queiroz, Y.S...... 147 Sham, D...... 24 Tatewaki, N...... 135 Wesley, U...... 24 Shariftabrizi, A...... 104 Thangarajah, H...... 10 Wilson, D.W...... 69 R Sharoni, Y...... 122 Thirunavukkarasu, M...... 30 Wong, A...... 47 Rastegar, R...... 104 Shen, P...... 74 Tian, Q...... 125 Woo, H.A...... 21 Rhee, S.G...... 21 Shi, Z.-H...... 174 Tominaga, K...... 157 Woo, H.D...... 120 Ribeiro, M.L...... 147 Shimokawa, O...... 126 Topal, T...... 139,140,154 Wozniak, M...... 67,162 Ricciardi, M...... 155 Shinde, S.S...... 65 Torres, E.A.F.S...... 147 Wu, Y.-H...... 163 Rice, N...... 38 Shioda, S...... 87,88 Traber, M...... 152 Rink, C...... 99,116,143,148 Shneker, B...... 99 Tsunawaki, S...... 87 X Robertson, B...... 115 Sielicka-Dudzin, A...... 67,162 Xu, D...... 92,173 Rodriguez Mañas, L...... 60 Siendones, E...... 43 U Xu, J...... 100 Roy, S...... 12,70,71,72,83,86,90 Silander, K...... 38 Ucar, E...... 154 Xu, R...... 100 ...... 97,99,102,116,143,148 Simone, R...... 142 Ueki, K...... 176 Russell, R.M...... 39 Simsek, K...... 139,140,154 Ueno, M...... 68 Y Rutledge, J.C...... 69 Singleton, A...... 38 Ukai, M...... 158 Yadav, V...... 149 Sledzinski, M...... 162 Utsumi, H...... 27 Yakumaru, H...... 133 S Smith, R.A.J...... 65 Uysal, B...... 139,140,154 Yamashita, E...... 157 Sadir, S...... 139,140 Soe-Lin, S...... 173 Yang, C.-M...... 163 Sadoshima, J...... 32 Sofi, T...... 111,155 V Yang, J...... 177 Saji, H...... 52 Son, A...... 23 Valdez, L.B...... 159 Yang, S...... 164 Salinthone, S...... 149 Sottero, B...... 33 Valério, D.A...... 160 Yannakopouylou, E...... 75 Sampaio, G.R...... 147 Spitzer, V...... 115 van der Vliet, A...... 24 Yao, J...... 105,113,165 Samuel, S.M...... 30 Steinberg, F.M...... 95 Vasu, V.T...... 101 Yap, L.-P...... 150,166,167 Sancheti, H...... 62,150,170 Stiles, B...... 123 Veasey, R...... 108 Yasui, H...... 168 Sanson, J.S...... 82 Subramanian, S...... 168 Velazquez, O.C...... 11 Ybanez, M...... 169 Santos, D...... 80 Sugie, K...... 23 Verri, W.A...... 81,98,160 Yesilyurt, O...... 154 Santos, E...... 56 Sulakvelidze, M...... 76 Verstraeten, S.V...... 161 Yeum, K.-J...... 39 Santos-Ocaña, C...... 43 Sumitani, S...... 156 Vial, N...... 10 Yildiz, S...... 154 Sarkar, A...... 99 Sun, B...... 99 Vicentini, F.T.M.C...... 81,98,160 Yin, F...... 107,170 Sato, E...... 151 Sung, B...... 46 Vicinanza, R...... 47 Yodoi, J...... 22 Satoh, K...... 87,88 Supasai, S...... 64 Vieira Fonseca, M.J...... 81,98 Yofu, S...... 87 Schmittgen, T...... 71 Surh, Y.-J...... 48 Vieira, S.M...... 160 Yokota, T...... 138 Schnitt, R...... 70 Suzuki, H...... 138 Viña, J...... 55 Yoon, G...... 121,153 Schubert, D...... 91 Swartz, H.M...... 53 Virtamo, J...... 38 Yoon, K...... 117 Schultz, C...... 77 Von Lintig, J...... 36 Yoon, S.-H...... 121,153 Semba, R.D...... 38 Vranková, S...... 144,145 Yoshihara, E...... 22

184 185 SPONSORS

Yoshitomi, T...... 171 Zhang, Y...... 173,177 DSM Nutritional Products Yu, M...... 117 Zhao, B.L...... 172,174,175 Sight & Life Foundation Zhao, C...... 176,177 Jarrow Formulas Z Zhao, Y...... 92,173 Osato Research Institute Zaiton, Z...... 63 Zhao-Wilson, X.Z...... 172 Linus Pauling Institute / Oxygen Club of California Conference Endowment Fund ______Zaobornyj, T...... 159 Zhivotovsky, B...... 26 Amino Up Chemical Co. Zhan, L...... 30 Zhu, J.-S...... 177 Zhang, L.Z...... 172 Zicha, J...... 145 Bayer Consumer Care AG Zhang, N...... 174 Zimermann, V.V.M...... 82 Glenn Foundation for Medical Research Zhang, X.-j...... 125 zur Hausen, H...... 6 GWR Medical Inc.

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