Studies on Experimental Models

Oxidative Stress in Applied Basic Research and Clinical Practice

Editor-in-Chief Donald Armstrong

For other titles published in this series, go to www.springer.com/series/8145

Note from the Editor-in-Chief All books in this series illustrate point-of-care testing and critically evaluate the potential of antioxidant supplementation in various medical disorders associated with oxidative stress. Future volumes will be updated as warranted by emerging new technology, or from studies reporting clinical trials. Donald Armstrong Editor-in-Chief

Samar Basu  •  Lars Wiklund Editors

Studies on Experimental Models

Editors Samar Basu Professor of Biochemistry and Medical Inflammation Director of Chaire d’Excellence Laboratorie de Biochimie Biologie Moléculaire et Nutrition Faculté de Pharmacie Université d’Auvergne 28 Place Henri-Dunant BP 38 63001 Clermont-Ferrand France and Head, Oxidative stress and Inflammation Department of Public Health and Caring Sciences Faculty of Medicine Uppsala University SE-751 85 Uppsala Sweden [email protected] [email protected]

Lars Wiklund Professor of Anesthesiology and Intensive Care Medicine Department of Surgical Sciences/ Anesthesiology and Intensive Care Medicine Faculty of Medicine Uppsala University SE-751 85 Uppsala Sweden [email protected]

ISBN 978-1-60761-955-0 e-ISBN 978-1-60761-956-7 DOI 10.1007/978-1-60761-956-7 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2011923976 © Springer Science+Business Media, LLC 2011 All rights reserved. This work may not be translated or copied in whole or in part without the written ­permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com)

Preface

The notion that reactive oxygen species are involved in diseases originated in the 1930s when scientists realized that radiation injury may lead to formation of free radicals and in turn initiate cancer and other pathologies. Even though research in free radicals has been stretched exponentially in the last three decades, there are still major questions as to what extent free radicals are involved in health and various diseases. In recent years, there is a growing recognition that free radicals, and thereby oxidative stress, is involved in atherosclerosis, cardiovascular diseases, cancer, ischemia–reperfusion injury, radiation injury and neurological diseases, etc. In addition, free radicals are perhaps involved in both aging and redox process. Despite the great amount of research being carried out in the field, there are still uncertainties about the overall mechanisms behind free radical-related pathologies, as well as how free radicals may play a fundamental role in protecting cells through redox signaling. With these concepts in mind, there is a wide-spread consensus today that the use of antioxidants as a therapeutic approach may counteract free radical-mediated pathologies. However, the role of antioxidants in normal physiology and redox signaling is still in its infancy. Since oxidative stress is related to various diseases and pathologies, scientists are eager to study the disease in humans. However, it is not always ethical to study all the aspects of the disease in humans; consequently, it is mandatory to study the disease process and the mechanisms behind it through experimental models. The models generally involve animals, in  vitro/cell culture studies, and even in primates and humans to a certain extent. This book contains mainly data on the experimental models or review of such models of oxidative stress in various diseases, and is divided into six parts that present a sketch of models in humans, animals and in vitro methods. Part I deals with diabetes, which includes the role of oxidative stress and antioxidant therapies in experimental models of diabetes, diabetes complications, micronutrient intake and its relevance to atherosclerosis and insulin resistance. Part II deals with stroke, including arachidonic acid metabolism and the role of alpha-tocotrienol as a therapeutic agent, assessment of oxidative stress, heat-shock proteins and doxorubicininduced oxidative stress in the heart, as well as biomarkers of oxidative stress in cardiovascular diseases. Part III deals with neurology, which includes MPTP and

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oxidative stress, oxidative stress in Alzheimer’s disease, retinal disturbances in patients and animals models with Huntington’s, Parkinson’s and Alzheimer’s disease, stress gene regulation in Alzheimer’s blood cells, Gpx4 knockout mice and transgenic mice in aging, experimental models of myocardial, and cerebral ischemia. Part IV deals with ocular diseases, including neovascular models of the rabbit eye and purinergic signaling in volume regulation of glial cells in rat retina. Part V is concerned with toxicological/environmental aspects that contain Helicobacter Pylori-induced oxidative stress and inflammation, experimental models for ionizing radiation research, cigarette smoke-induced oxidative stress in preclinical models, smokers and patients with airways disease, exhaled breath condensate biomarkers in airway inflammation in COPD, induction of oxidative stress by iron/ascorbate in isolated mitochondria and by UV irradiation in human skin, carbon tetrachlorideinduced oxidative stress, lipid metabolites after exposure of toxicants, oxidative stress in porcine endotoxemia and exercise as a model to study oxidative stress. The final part concerns in vitro/cell culture models that contain mitochondrial oxidative stress, protection against oxidant-induced neuronal cell injury, oxidative DNA biomarkers, oxidative stress in cell culture, animals and humans, role of cAMP and G protein signaling in cardiovascular dysfunction, cellular and chemical assays of antioxidants, and arsenic-induced oxidative stress. We hope that this book will contribute to the advancement of oxidative stress research using appropriate animal models and will serve as a valuable reference for basic and clinical scientists. Editor Co-Editor

Samar Basu Lars Wiklund

Contents

Part I  Diabetes Role of Oxidative Stress and Targeted Antioxidant Therapies in Experimental Models of Diabetic Complications..................................... Judy B. de Haan, Karin A. Jandeleit-Dahm, and Terri J. Allen

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Experimental Models of Oxidative Stress Related to Cardiovascular Diseases and Diabetes...................................................... Maria D. Mesa, Concepcion M. Aguilera, and Angel Gil

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Part II  Cardiovascular Arachidonic Acid Metabolism and Lipid Peroxidation in Stroke: Alpha-Tocotrienol as a Unique Therapeutic Agent...................................... Cameron Rink, Savita Khanna, and Chandan K. Sen Assessment of Oxidative Stress in the Brain of Spontaneously Hypertensive Rat and Stroke-Prone Spontaneously Hypertensive Rat Using by Electron Spin Resonance Spectroscopy.................................. Fumihiko Yoshino, Kyo Kobayashi, and Masaichi-Chang-il Lee

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Small Heat Shock Proteins and Doxorubicin-Induced Oxidative Stress in the Heart.......................................................................... 105 Karthikeyan Krishnamurthy, Ragu Kanagasabai, Lawrence J. Druhan, and Govindasamy Ilangovan Oxidative Stress in Cardiovascular Disease: Potential Biomarkers and Their Measurements........................................... 131 Subhendu Mukherjee and Dipak K. Das In vivo Imaging of Antioxidant Effects on NF-k B Activity in Reporter Mice................................................................................ 157 Ingvild Paur, Harald Carlsen, and Rune Blomhoff vii

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Part III  Neurology MPTP and Oxidative Stress: It’s Complicated!............................................ 187 V. Jackson-Lewis, M.A. Tocilescu, R. DeVries, D.M. Alessi, and S. Przedborski Oxidative Stress in Alzheimer’s Disease: A Critical Appraisal of the Causes and the Consequences.......................... 211 Jaewon Chang, Sandra Siedlak, Paula Moreira, Akihiko Nunomura, Rudy J. Castellani, Mark A. Smith, Xiongwei Zhu, George Perry, and Gemma Casadesus Retinal Disturbances in Patients and Animal Models with Huntington’s, Parkinson’s and Alzheimer’s Disease............................ 221 C. Santano, M. Pérez de Lara, and J. Pintor Stress Gene Deregulation in Alzheimer Peripheral Blood Mononuclear Cells........................................................................................... 251 Olivier C. Maes, Howard M. Chertkow, Eugenia Wang, and Hyman M. Schipper The Use of Gpx4 Knockout Mice and Transgenic Mice to Study the Roles of Lipid Peroxidation in Diseases and Aging................................ 265 Qitao Ran and Hanyu Liang An Experimental Model of Myocardial and Cerebral Global Ischemia and Reperfusion............................................................................... 279 Lars Wiklund and Samar Basu Part IV  Ocular Diseases Neovascular Models of the Rabbit Eye Induced By Hydroperoxide........... 303 Toshihiko Ueda, Takako Nakanishi, Kazushi Tamai, Shinichi Iwai, and Donald Armstrong Purinergic Signaling Involved in the Volume Regulation of Glial Cells in the Rat Retina: Alteration in Experimental Diabetes..................... 319 Andreas Bringmann Part V  Toxicology/Environmental Helicobacter pylori-Induced Oxidative Stress and Inflammation................ 343 Hyeyoung Kim and Young-Joon Surh

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Experimental Models for Ionizing Radiation Research............................... 371 Kristin Fabre, William DeGraff, John A. Cook, Murali C. Krishna, and James B. Mitchell Experimental Models to Study Cigarette Smoke-Induced Oxidative Stress In Vitro and In Vivo in Preclinical Models, and in Smokers and Patients with Airways Disease................................................................. 399 Hongwei Yao and Irfan Rahman Exhaled Breath Condensate Biomarkers of Airway Inflammation and Oxidative Stress in COPD........................................................................ 421 Paolo Montuschi Induction of Oxidative Stress by Iron/Ascorbate in Isolated Mitochondria and by UV Irradiation in Human Skin................................. 441 Ingrid Wiswedel, Wolfgang Augustin, Sven Quist, Harald Gollnick, and Andreas Gardemann Carbon Tetrachloride-Induced Hepatotoxicity: A Classic Model of Lipid Peroxidation and Oxidative Stress...................... 467 Samar Basu Enhanced Urinary Excretion of Lipid Metabolites Following Exposure to Structurally Diverse Toxicants: A Unique Experimental Model for the Assessment of Oxidative Stress............................................... 481 Francis C. Lau, Manashi Bagchi, Shirley Zafra-Stone, and Debasis Bagchi Oxidative Stress in Animal Models with Special Reference to Experimental Porcine Endotoxemia.......................................................... 497 Miklós Lipcsey, Mats Eriksson, and Samar Basu Models and Approaches for the Study of Reactive Oxygen Species Generation and Activities in Contracting Skeletal Muscle.......................... 511 Malcolm J. Jackson Exercise as a Model to Study Interactions Between Oxidative Stress and Inflammation.................................................................................. 521 Christina Yfanti, Søren Nielsen, Camilla Scheele, and Bente Klarlund Pedersen Exercise as a Model to Study Oxidative Stress.............................................. 531 Mari Carmen Gomez-Cabrera, Fabian Sanchis-Gomar, Vladimir Essau Martinez-Bello, Sandra Ibanez-Sania, Ana Lucia Nascimento, Li Li Ji, and Jose Vina

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Part VI  In Vitro/Tissue Culture Models of Mitochondrial Oxidative Stress.................................................... 545 Enrique Cadenas and Alberto Boveris Protection of Oxidant-Induced Neuronal Cells Injury by a Unique Cruciferous Nutraceutical............................................................................... 563 Zhenquan Jia, Soumya Saha, Hong Zhu, Yunbo Li, and Hara P. Misra Oxidatively Generated Damage to DNA and Biomarkers........................... 579 Jean Cadet, Thierry Douki, and Jean-Luc Ravanat Measuring Oxidative Stress in Cell Cultures, Animals and Humans: Analysis and Validation of Oxidatively Damaged DNA............................... 605 Hanna L. Karlsson and Lennart Möller The Roles of cAMP and G Protein Signaling in Oxidative Stress-Induced Cardiovascular Dysfunction................................................. 621 Soumya Saha, Zhenquan Jia, Dongmin Liu, and Hara P. Misra Cellular and Chemical Assays for Discovery of Novel Antioxidants in Marine Organisms....................................................................................... 637 Tim Hofer, Tonje Engevik Eriksen, Espen Hansen, Ingrid Varmedal, Ida-Johanne Jensen, Jeanette Hammer-Andersen, and Ragnar Ludvig Olsen Arsenic-Induced Oxidative Stress: Evidence on In Vitro Models of Cardiovascular, Diabetes Mellitus Type 2 and Neurodegenerative Disorders........................................................................................................... 659 Rubén Ruíz-Ramos, Patricia Ostrosky-Wegman, and Mariano E. Cebrián Index . ............................................................................................................... 681

Contributors

Concepcion M. Aguilera Department of Biochemistry and Molecular Biology II, Institute of Nutrition and Food Technology “José Mataix”, Centre of Biomedical Research, University of Granada, Campus de la Salud, Armilla, Granada, Spain Terri J. Allen Diabetic Complications Laboratory, JDRF Diabetes Division, Baker IDI Heart and Diabetes Institute, Melbourne, Australia D.M. Alessi Graduate student, Integrated Program of Cellular, Molecular and Biophysical studies at Columbia University, USA Donald Armstrong Department of Ophthalmology, University of Florida College of Medicine, Gainesville, Florida, USA Wolfgang Augustin Department of Pathological Biochemistry, Otto-von-Guericke University, Magdeburg, Germany Debasis Bagchi InterHealth Research Center, Benicia, CA, USA; Pharmacological and Pharmaceutical Sciences, University of Houston College of Pharmacy, Houston, TX, USA Manashi Bagchi InterHealth Research Center, Benicia, CA, USA Samar Basu Laboratorie de Biochimie, Biologie Moléculaire et Nutrition Faculté de Pharmacie, Université d’Auvergne 28, Clermont-Ferrand, France; Oxidative stress and Inflammation, Department of Public Health and Caring Sciences, Faculty of Medicine, Uppsala University, Uppsala, Sweden

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Contributors

Rune Blomhoff Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway Alberto Boveris Physical Chemistry, School of Pharmacy & Biochemistry, University of Buenos Aires, Buenos Aires, Argentina Andreas Bringmann Department of Ophthalmology and Eye Hospital, University of Leipzig, Leipzig, Germany Enrique Cadenas Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA, USA Jean Cadet Laboratoire “Lésions des Acides Nucléiques”, SCIB-UMR-E n°3 (CEA/UJF), Institut Nanosciences et Cryogénie, CEA/Grenoble, 38054 Grenoble Cedex 9, France Harald Carlsen Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway Gemma Casadesus Department of Neuroscience, Case Western Reserve University, Cleveland, OH, USA Rudy J. Castellani Department of Pathology, University of Maryland, Baltimore, MD, USA Mariano E. Cebrián Departamento de Toxicología, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), Ave. Instituto Politecnico National, Mexico, DF, Mexico Jaewon Chang Department of Neuroscience, Case Western Reserve University, Cleveland, OH, USA Howard M. Chertkow Bloomfield Centre for Research in Aging, Lady Davis Institute for Medical Research, Montréal, Québec, Canada H3T 1E2; Departments of Neurology, Neurosurgery and Medicine (Geriatrics), McGill University, Montréal, Québec, Canada H3G 146 John A. Cook Radiation Biology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA

Contributors

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Dipak K. Das Cardiovascular Research Center, University of Connecticut Health Center, School of Medicine, Farmington, CT, USA William DeGraff Radiation Biology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA Judy B. de Haan Diabetic Complications Laboratory, JDRF Diabetes Division, Baker IDI Heart and Diabetes Institute, Melbourne, Australia R. DeVries Departments of Neurology, Center for Motor Neuron Biology and Disease, Columbia University, New York, NY, USA Thierry Douki Laboratoire “Lésions des Acides Nucléiques”, SCIB-UMR-E n°3 (CEA/UJF), Institut Nanosciences et Cryogénie, CEA/Grenoble, 38054 Grenoble Cedex 9, France Lawrence J. Druhan Division of Cardiovascular Medicine, Davis Heart and Lung Research Institute, Ohio State University, Columbus, OH, USA Tonje Engevik Eriksen MabCent-SFI, University of Tromsø, Tromsø, Norway Mats Eriksson Department of Anaesthesia and Intensive Care, Uppsala University, Uppsala, Sweden Kristin Fabre Radiation Biology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA Andreas Gardemann Department of Pathological Biochemistry, Otto-von-Guericke University, Magdeburg, Germany Angel Gil Department of Biochemistry and Molecular Biology II, Institute of Nutrition and Food Technology “José Mataix”, Centre of Biomedical Research, University of Granada, Campus de la Salud, Armilla, Granada, Spain Harald Gollnick Department of Dermatology and Venereology, Otto-von-Guericke University, Magdeburg, Germany Mari Carmen Gomez-Cabrera Department of Physiology, Faculty of Medicine, University of Valencia, Fundacion Investigacion Hospital Clinico Universitario/INCLIVA, Spain

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Contributors

Jeanette Hammer-Andersen MabCent-SFI, University of Tromsø, Tromsø, Norway Espen Hansen MabCent-SFI, University of Tromsø, Tromsø, Norway Tim Hofer MabCent-SFI, University of Tromsø, Tromsø, Norway Sandra Ibanez-Sania Department of Physiology, Faculty of Medicine, University of Valencia, Fundacion Investigacion Hospital Clinico Universitario/INCLIVA, Spain Govindasamy Ilangovan Division of Cardiovascular Medicine, Davis Heart and Lung Research Institute, Ohio State University, Columbus, OH, USA Shinichi Iwai Departments of Pharmacology and Ophthalmology, Showa University School of Medicine, Tokyo, Japan Malcolm J. Jackson School of Clinical Sciences, University of Liverpool, Liverpool, UK V. Jackson-Lewis Departments of Neurology, Center for Motor Neuron Biology and Disease, Columbia University, New York, NY, USA Karin A. Jandeleit-Dahm Diabetic Complications Laboratory, JDRF Diabetes Division, Baker IDI Heart and Diabetes Institute, Melbourne, Australia Ida-Johanne Jensen MabCent-SFI, University of Tromsø, Tromsø, Norway Li Li Ji The Biodynamics Laboratory, Department of Kinesiology, University of Wisconsin at Madison, WI, USA Zhenquan Jia Edward Via Virginia College of Osteopathic Medicine, Blacksburg, VA, USA Ragu Kanagasabai Division of Cardiovascular Medicine, Davis Heart and Lung Research Institute, Ohio State University, Columbus, OH, USA Hanna L. Karlsson Unit for Analytical Toxicology, Department of Biosciences and Nutrition, Novum, Karolinska Institutet, Huddinge, Stockholm, Sweden

Contributors

Savita Khanna Department of Surgery, The Ohio State University Medical Center, Columbus, OH, USA Hyeyoung Kim Department of Food and Nutrition, Brain Korea 21 Project, College of Human Ecology, Yonsei University, Seoul, South Korea Kyo Kobayashi Department of Clinical Care Medicine, Division of Pharmacology, Kanagawa Dental College, Yokosuka, Japan Murali C. Krishna Radiation Biology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA Karthikeyan Krishnamurthy Division of Cardiovascular Medicine, Davis Heart and Lung Research Institute, The Ohio State University, Columbus, OH, USA Francis C. Lau InterHealth Research Center, Benicia, CA, USA Masaichi-Chang-il Lee Department of Clinical Care Medicine, Division of Pharmacology, Kanagawa Dental College, Yokosuka, Japan Yunbo Li Edward Via Virginia College of Osteopathic Medicine, Blacksburg, VA, USA Hanyu Liang Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA Miklós Lipcsey Department of Anaesthesia and Intensive Care, Uppsala University Hospital, Uppsala, Sweden Dongmin Liu Department of Human Food, Nutrition and Exercise, Virginia Tech, Blacksburg, VA, USA Olivier C. Maes Bloomfield Centre for Research in Aging, Lady Davis Institute for Medical Research, Montréal, Québec, Canada Vladimir Essau Martinez-Bello Department of Physiology, Faculty of Medicine, University of Valencia, Fundacion Investigacion Hospital Clinico Universitario/INCLIVA, Spain

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Contributors

Maria D. Mesa Department of Biochemistry and Molecular Biology II, Institute of Nutrition and Food Technology “José Mataix”, Centre of Biomedical Research, University of Granada, Campus de la Salud, Armilla, Granada, Spain Hara P. Misra Edward Via Virginia College of Osteopathic Medicine, Blacksburg, VA, USA James B. Mitchell Radiation Biology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA Lennart Möller Unit for Analytical Toxicology, Department of Biosciences and Nutrition, Novum, Karolinska Institutet, Huddinge, Stockholm, Sweden Paolo Montuschi Department of Pharmacology, Faculty of Medicine, Catholic University of the Sacred Heart, Rome, Italy Paula Moreira Center for Neuroscience and Cell Biology of Coimbra, University of Coimbra, Coimbra, Portugal Subhendu Mukherjee Cardiovascular Research Center, University of Connecticut Health Center, School of Medicine, Farmington, CT, USA Takako Nakanishi Departments of Ophthalmology, Showa University School of Medicine, Tokyo, Japan Ana Lucia Nascimento Department of Physiology, Faculty of Medicine, University of Valencia, Fundacion Investigacion Hospital Clinico Universitario/INCLIVA, Spain S. Nielsen The Department of Infectious Diseases, Rigshospitalet, The Centre of Inflammation and Metabolism, University of Copenhagen Faculty of Health Sciences, Copenhagen, Denmark Akihiko Nunomura Department of Neuropsychiatry, University of Yamanashi, Yamanashi, Japan Ragnar Ludvig Olsen MabCent-SFI, University of Tromsø, Tromsø, Norway Patricia Ostrosky-Wegman Departamento de Medicina Genómica y Toxicología Ambiental, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Circuito Escolar, Cd. Universitaria, Mexico, DF, Mexico

Contributors

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Ingvild Paur Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway Bente K. Pedersen The Department of Infectious Diseases, Rigshospitalet, The Centre of Inflammation and Metabolism, University of Copenhagen, Faculty of Health Sciences, Copenhagen, Denmark George Perry Department of Neuroscience, Case Western Reserve University, Cleveland, OH, USA; Department of Biology, Center for Neuroscience and Cell Biology of Coimbra, University of Coimbra, Coimbra, Portugal; UTSA Neurosciences Institute, University of Texas at San Antonio, San Antonio, TX, USA M. Pérez de Lara Departamento de Bioquímica y Biología Molecular, Escuela de Optica UCM, Madrid, Spain J. Pintor Departamento de Bioquímica y Biología Molecular, Escuela de Optica UCM, Madrid, Spain S. Przedborski Departments of Neurology, Center for Motor Neuron Biology and Disease, Columbia University, New York, NY, USA Sven Quist Department of Dermatology and Venereology, Otto-von-Guericke University, Magdeburg, Germany Irfan Rahman Department of Environmental Medicine, Lung Biology and Disease Program, University of Rochester Medical Center, Rochester, NY, USA Qitao Ran Department of Cellular and Structural Biology; Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio; South Texas Veterans Health Care System, San Antonio, TX, USA; Jean-Luc Ravanat Laboratoire “Lésions des Acides Nucléiques,” SCIB-UMR-E n°3 (CEA/UJF), Institut Nanosciences et Cryogénie, CEA/Grenoble, 38054 Grenoble Cedex 9, France Cameron Rink Department of Surgery, The Ohio State University Medical Center, Columbus, OH, USA

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Contributors

Rubén Ruíz-Ramos Departamento de Toxicología, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), Mexico, DF, Mexico Soumya Saha Edward Via Virginia College of Osteopathic Medicine, Blacksburg, VA, USA; Department of Human Food, Nutrition and Exercise, Virginia Tech, Blacksburg, VA, USA C. Santano Departamento de Bioquímica y Biología Molecular, Escuela de Optica UCM, Madrid, Spain Fabian Sanchis-Gomar Department of Physiology, Faculty of Medicine, University of Valencia, Fundacion Investigacion Hospital Clinico Universitario/INCLIVA, Spain C. Scheele The Department of Infectious Diseases, Rigshospitalet, The Centre of Inflammation and Metabolism, University of Copenhagen, Faculty of Health Sciences, Copenhagen, Denmark Hyman M. Schipper Bloomfield Centre for Research in Aging, Lady Davis Institute for Medical Research, Montréal, Québec, Canada; Departments of Neurology, Neurosurgery and Medicine (Geriatrics), McGill University, Montréal, Québec, Canada Chandan K. Sen Department of Surgery, The Ohio State University Medical Center, Columbus, OH, USA Sandra Siedlak Department of Pathology, Case Western Reserve University, Cleveland, OH, USA Mark A. Smith Department of Pathology, Case Western Reserve University, Cleveland, OH, USA Young-Joon Surh Department of Molecular Medicine and Biopharmaceutics, Graduate School of Convergence Sciences and Technology, Seoul National University, Seoul, South Korea Kazushi Tamai Department of Ophthalmology, Nagoya City University School of Medicine, Nagoya, Japan M.A. Tocilescu Department of Neurology, Center for Motor Neuron Biology and Disease, Departments of Pathology and Cell Biology, Columbia University, New York, NY, USA

Contributors

Toshihiko Ueda Department of Ophthalmology, Showa University School of Medicine, Tokyo, Japan Ingrid Varmedal MabCent-SFI, University of Tromsø, Tromsø, Norway Jose Vina Department of Physiology, Faculty of Medicine, University of Valencia, Fundacion Investigacion Hospital Clinico Universitario/INCLIVA, Spain C. Vives-Bauza Department of Neurology, Center for Motor Neuron Biology and Disease, Columbia University, New York, NY, USA Eugenia Wang Gheens Center on Aging, and Department of Biochemistry and Molecular Biology, School of Medicine, University of Louisville, Louisville, KY, USA Lars Wiklund Department of Surgical Sciences/Anaesthesiology and Intensive Care Medicine, Uppsala University, 751 85 Uppsala, Sweden Ingrid Wiswedel Department of Pathological Biochemistry, Otto-von-Guericke University, Magdeburg, Germany Hongwei Yao Department of Environmental Medicine, Lung Biology and Disease Program, University of Rochester Medical Center, Rochester, NY, USA Christina Yfanti The Department of Infectious Diseases, Rigshospitalet, The Centre of Inflammation and Metabolism, University of Copenhagen, Faculty of Health Sciences, Copenhagen, Denmark Fumihiko Yoshino Department of Clinical Care Medicine, Division of Pharmacology, Kanagawa Dental College, Yokosuka, Japan Shirley Zafra-Stone InterHealth Research Center, Benicia, CA, USA Hong Zhu Edward Via Virginia College of Osteopathic Medicine, Blacksburg, VA, USA Xiongwei Zhu Department of Pathology, Case Western Reserve University, Cleveland, OH, USA

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Part I

Diabetes

Role of Oxidative Stress and Targeted Antioxidant Therapies in Experimental Models of Diabetic Complications Judy B. de Haan, Karin A. Jandeleit-Dahm, and Terri J. Allen

Abstract  Diabetic patients, whether of type 1 or type 2 origin, are at greater risk of developing complications of the vasculature than non-diabetic patients. Macrovascular complications such as diabetes-associated atherosclerosis lead to accelerated and often more advanced lesions than seen in the general population. Microvascular complications such as nephropathy, retinopathy and neuropathy as well as diabetic cardiomyopathy are further complications associated with the diabetic milieu. Understanding the mechanisms leading to and accelerating these complications is a major research initiative of many laboratories. To facilitate these studies, the design and use of appropriate animal models has been central to the study of these diabetic complications. A new and emerging concept underpinning many of these end-organ complications is oxidative stress, particularly of mitochondrial origin, which is understood to play a critical role in the initiation and progression of these diabetic complications. Thus the development of experimental models that specifically delineate the cause and role of ROS in diabetic complications is now becoming a major research area. This chapter focuses on some of the latest oxidative stress-driven experimental models of diabetic complications. Use of the ApoE/GPx1 double-knockout mouse has revealed the importance of antioxidant defense in limiting accelerated diabetes-associated atherosclerosis and diabetic nephropathy, while RAGE knockout mice have shown that oxidative stress is inextricably linked with pathophysiological cell signaling, particularly through RAGE. The use of NOX knockout mice is shedding light on the contribution of the NADPH oxidases to the ROS milieu as well as the contribution of the various isoforms (NOX 1, 2 and 4) to the individual diabetic complications. Furthermore, these models are helping to understand the types of ROS involved and their cellular location, which may help in the specific targeting of these ROS to reduce ROSmediated pathogenesis. For example, antioxidants that target mitochondrial ROS (location) or ROS such as hydrogen peroxide (specificity) may offer an alternate T.J. Allen (*) Diabetic Complications Laboratory, JDRF Diabetes Division, Baker IDI Heart and Diabetes Institute, St Kilda Road Central, Melbourne, VIC 8008, Australia e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_1, © Springer Science+Business Media, LLC 2011

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approach to reduce diabetes-driven oxidative stress. It is only via manipulation of experimental models of diabetes-driven oxidative stress that the contribution of the various ROS will be revealed, and only then that effective treatment regimens can be designed to lessen the effect of oxidative stress on diabetic complications. Keywords  Antioxidant defense • Diabetic complications • Ebselen • Experimental models • Glutathione peroxidase • Oxidative stress

1 Introduction Diabetes mellitus is a metabolic disorder that is characterized by chronic hyperglycemia with disturbances in carbohydrate, fat and protein metabolism and occurs as a result of defects in insulin secretion and/or action [1]. Whether diabetes occurs as a result of type 1, the early-onset and predominantly insulin-dependent form, or type 2, the late-onset form that is associated with metabolic syndrome, obesity and insulin-resistance, individuals with diabetes are at greater risk of developing diabetes-associated complications [2, 3]. The predominant complications include cardiovascular disease, nephropathy, retinopathy, neuropathy and a specific impairment in the heart muscle leading to cardiomyopathy [4]. It is now well accepted that most diabetic complications arise from chronic hyperglycemic damage to the vascular system [5]. Vascular disease can be separated into that affecting the macrovasculature resulting in atherosclerosis of major vessels and/or stroke, and microvascular complications resulting in retinopathy, nephropathy and neuropathy. Diabetic cardiomyopathy is understood to occur as a result of abnormal myocardial metabolism in diabetes, rather than as a result of micro- or macro-vascular disease [6–8]. While many studies address the causative mechanisms of the individual ­diabetes-driven complication, it is now becoming increasingly apparent that oxidative stress is an important underpinning phenomenon that assists with the progression towards more severe and often fatal complications. Evidence from numerous studies suggests an important causal role for increased reactive oxygen species (ROS), particularly mitochondrial ROS, in the pathogenesis of the major complications associated with diabetes [9]. Several pathways, including the polyol pathway [10], increases in advanced glycation end products (AGEs) [11], activation of ­protein kinase C [12] and increases in hexosamine flux, have been identified where hyperglycemia triggers increased ROS production that in turn may initiate, progress or amplify end-organ damage in diabetes. One postulate suggests that all of these pathways are activated as a result of glucose-induced overproduction of superoxide by the mitochondrial electron transport chain [2]. Indeed it has been estimated that 1–2% of all electrons passing through the respiratory chain contribute to the ­formation of superoxide [13, 14], with the rate of production varying greatly depending on the environment and/or disease state [14]. Other potential sources of ROS production include nitric oxide synthases (NOS), nicotinamide adenine

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dinucleotide phosphate (NADPH) oxidases (NOX), xanthine oxidase, lipoxygenase and cytochrome P450 mono-oxygenases [13, 15]. Particular attention has focused on the members of the NOX/DUOX family of NADPH oxidases since these enzymes mediate physiological functions such as host defense, cell signaling, and thyroid hormone biosynthesis through the generation of ROS, including superoxide anion and hydrogen peroxide [16]. However, it is becoming increasingly apparent that alterations in diabetes-driven cellular ROS arise not only as a consequence of overproduction of ROS, but also as a result of ineffective removal by antioxidant defenses [17, 18]. The design and use of appropriate animal models to study diabetic complications has been examined in several reviews covering mouse models of type 1 and type 2 diabetes as well as related phenotypes such as obesity and insulin resistance [5, 19–22]. However, the development of experimental models that specifically delineate the cause and role of ROS in diabetic complications is now becoming a major research area, enabling both an understanding of the mechanisms of ROSmediated damage as well as facilitating the design of targeted therapeutic approaches to limit diabetes-mediated ROS action. This chapter will focus on some of the latest oxidative stress-induced experimental models of diabetic complications with particular emphasis on cardiovascular disease, nephropathy, and diabetic cardiomyopathy.

2 Diabetes-Associated Atherosclerosis Diabetes mellitus is a major pro-atherosclerosis risk factor, with a two- to fourfold higher incidence of cardiovascular disease in diabetic patients than in the general population [23]. Other risk factors include hyperglycemia, dyslipidemia, hypertension and obesity. However, these risk factors only partly explain the more advanced lesions [24] and increased incidence of cardiovascular disease observed in these patients. Understanding the underlying mechanisms that accelerate diabetes-­ associated atherosclerosis remains an important research area and various pathways have been implicated that include the biochemical process of advanced glycation [25] and the receptor for AGEs, RAGE [26]. In addition, various proteins that have been implicated in the atherosclerotic process per se have been shown to be upregulated in the diabetic condition. These include vascular cell adhesion molecule-1 (VCAM-1), monocyte chemoattractant protein-1 (MCP-1) and connective tissue growth factor (CTGF) [27]. Strong evidence now suggests that ROS derived from the hyperglycemia-driven increase in mitochondrial electron transport chain activity [9], glucose autoxidation [28] and enzymes such as NAD(P)H oxidase play a causal role in mediating many of the pro-atherogenic changes observed [29]. Indeed, ROS are known to upregulate a number of pro-atherogenic processes such as monocyte infiltration, platelet activation [30], smooth muscle cell migration [31], cell adhesion [32], release of CTGF [33] and increased production of AGEs [28]. Specifically, the accumulation

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of oxidized low-density lipoproteins (oxLDL) in vessel walls is an early initiator of atherosclerotic events [34]. In turn, oxLDL is atherogenic for several reasons, namely (1) it is chemotactic for circulating monocytes, the cellular precursors of arterial macrophages, (2) it is responsible for inhibiting the migration of ­macrophages from the aortic wall, thus trapping them inside the lesion and (3) it is directly cytotoxic to endothelial cells, thus aiding in the erosion of the endothelial surface and promoting thrombosis [35–37]. Furthermore, macrophages preferentially take up oxLDL, thereby promoting atherosclerosis [38], while the oxidative bursts of macrophages and neutrophils recruited to inflammatory sites further ­compound the oxidative insult [37]. Importantly, a heightened state of oxidative stress has been observed in diabetic patients [39]. Several laboratories, including ours, have focused on the role of antioxidant defense in regulating the flow of ROS during the atherogenic process. Initial studies focused on the levels of the various antioxidant enzymes known to play a role in ROS removal. One study showed that migrating smooth muscle cells and macrophages in atheromatous plaques express these enzymes intensively [40], while a different study assessed antioxidant levels in ApoE−/− mice prior to and during visible aortic lesion formation [41]. In that study, there was a coordinated increase in a wide range of antioxidant enzymes prior to visible atherogenic changes, while the expression of many antioxidant enzymes decreased during the period of lesion formation. This led these authors to suggest that the induction of antioxidant ­activities partially prevents the progression of atherogenesis, while the subsequent decline in antioxidant capacity may contribute to lesion formation. It is now increasingly recognized that knowledge of antioxidant enzyme involvement in ­limiting oxidative processes may delineate where potential therapeutic targets exist to reduce non-diabetes and diabetes-associated atherosclerosis. Indeed, overexpression of the antioxidant enzyme catalase, which removes hydrogen peroxide, reduced the severity of lesions in ApoE-deficient mice [42]. Recent attention has focused on the most abundant isoform of the glutathione peroxidase (GPx) family, glutathione peroxidase-1 (GPx1), based on a number of important clinical observations that strongly support a major role for GPx1 in limiting atherosclerosis. Blakenberg et  al. [43] first described a patient cohort where blood GPx1 activity was the strongest predictor of cardiovascular disease risk, with an inverse association between GPx1 activity and cardiovascular events. Based on this data, these authors suggested assessment of GPx1 for prognostic value in addition to that of traditional risk factors. Furthermore, they suggested that increasing GPx1 activity might lower the risk of cardiovascular events. Schnabel et  al. [44] demonstrated that plasma homocysteine (HCys) was related to future cardiovascular events in a patient cohort with coronary artery disease and that this occurred mainly in patients with low erythrocyte GPx1 activity. These authors proposed that HCys elicits its cardiovascular effect by directly affecting GPx1 activity. Winter et al. [45] found a significant association between a polymorphism in the human GPx1 gene and the risk of coronary artery disease, while Hamanishi et al. [46] found additional GPx1 polymorphisms within a diabetic population that correlated with reduced GPx1 activity and an increased risk of atherosclerosis. Recently, Nemoto et al. [47]

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reported that the presence of a Pro197Leu substitution within the GPx-1 gene may play a crucial role in determining genetic susceptibility to coronary arteriosclerosis in type 2 diabetic patients since this polymorphism was associated with increased coronary artery calcification as assessed by multi-slice computed tomography. Although in vitro and clinical data are highly supportive of a role for ROS in underpinning diabetes-associated atherosclerosis, it is only through interrogation of appropriate animal models that a true picture of the role of ROS and antioxidant defense is revealed.

2.1 Experimental Models of Diabetes-Associated Atherosclerosis with an Emphasis on Oxidative Stress 2.1.1 The GPx1 Knockout Mouse Glutathione Peroxidase-1 and Its Role in the Antioxidant Pathway Glutathione peroxidase-1 is a ubiquitously expressed antioxidant enzyme present in the cytosol and mitochondria of all living cells. One of its major functions is the second-step detoxification of hydrogen peroxide within the antioxidant pathway. A build-up of hydrogen peroxide and its subsequent non-enzymatic conversion to noxious hydroxyl radicals is prevented by the rapid interaction of GPx1 with its substrate, H2O2, and its co-factor, reduced glutathione (GSH). GPx1 is also involved in the removal of lipid peroxides [48] and it acts as a peroxynitrite reductase in the reduction of potentially damaging peroxynitrite radicals [49] (Fig.  1). In the absence of this antioxidant enzyme, a build-up of ROS ensues that are known to damage DNA, proteins and lipids [49]. GPx1 knockout (−/−) mice, generated in our laboratory [50] and by others [51, 52], have become an excellent research tool with which to establish a role for ROS in the progression and promotion of oxidant stress-mediated pathogenesis. Furthermore they have allowed us to draw meaningful conclusions about the protective role of this isoform of the GPx family of antioxidant enzymes, since standard assays do not discriminate between the different isoforms. In addition, most studies investigating the role of the GPxs do so by limiting selenium intake, which results in non-specific reductions in selenium-dependent enzymes [53], including all the selenium-dependent isoforms of GPx. The GPx1 knockout model also facilitates the distinction between the contributions of Gpx1, catalase (a peroxisomal H2O2 metabolizing enzyme) and thioredoxin peroxidase in the peroxidation of H2O2 to water. Our initial studies using this experimental model of oxidative stress showed an important role for GPx1 in the protection against ischemic-reperfusion mediated stroke [54]. In this instance, Gpx1−/− mice that were subjected to focal cerebral ischemia for 2  h via occlusion of the mid-cerebral artery showed significantly ­elevated lipid hydroperoxide levels compared with stroked control brains as well as

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NADPH Oxidase Xanthine Oxidase Cyclo-oxygenase Mitochondrial leakage dysfunctional eNOS

ROS

(O2.-)

Cu/Zn-SOD Mn-SOD

H2O2

1st step

+ O2

2 GSH

NO

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Fenton Reaction

(peroxynitrite)

2 GSH

(transition metals)

.OH

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GSSG

H2O

(hydroxyl radical)

NO2(nitrite)

LH L•

LOOH (lipid peroxide)

LOH + H2O (lipid alcohol)

2 GSH GSSG

Fig.  1  Removal of ROS by GPx1. The generation of superoxide radicals (−O2·) is greatly enhanced in the diabetic milieu via enzymes such as NADPH oxidase. −O2· is neutralized to water via a two-step process involving superoxide dismutase (SOD) in a first step, and glutathione peroxidase-1 (GPx1) or catalase in a second step. An imbalance in this pathway favors the build-up of hydrogen peroxide (H2O2). Fenton-type reactions occur when H2O2 or −O2· interact with transition metals such as iron (Fe2+), resulting in the production of noxious hydroxyl radicals (OH). These radicals initiate peroxidative damage to lipids, forming lipid hydroperoxides (LOOH). The functional importance of GPx1 rests in its ability to remove both hydrogen peroxide and lipid peroxides and neutralize these to water and lipid alcohol (LOH) respectively. In addition, GPx1 removes peroxynitrite radicals that form as a result of the interaction of −O2· with nitric oxide (NO). Two reduced glutathione (GSH) are consumed each time GPx1 reduces ROS, generating oxidized glutathione (GSSG)

accelerated caspase-3 activation and apoptosis of neuronal cells. Similar results were obtained in GPx1−/− mice subjected to cold-induced cerebral damage as a model of head trauma [55]. Given the strong evidence for a role for ROS in the initiation and progression of atherosclerosis, we felt that the GPx1−/− mouse model was ideally suited to studying the consequences of a lack of GPx1 on pro-atherogenic processes associated with diabetes. However, in order to understand the role of GPx1 in diabetes-associated atherosclerosis, it was important to first consider the contribution of GPx1 to proatherogenic processes not limited to the diabetic milieu. An underlying assumption would then be that the diabetic milieu, with its highly pro-oxidant environment, would facilitate even greater responses than those seen in a non-diabetic environment.

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 Px1−/− Mice Fed High Fat Diets as a Model to Study G Pro-atherogenic Mechanisms Our initial studies were performed in control mice and Gpx1−/− mice fed high fat diets (15% fat and 1% cholesterol) for 20 weeks on a C57Bl/J6 background [56]. In these animals, our biochemical analysis confirmed increased uptake of cholesterol into the vasculature of control and Gpx1−/− mice. Despite increases in nonenzymatic antioxidants after HFD-feeding (a-TOH in both plasma and vasculature, and total CoQ levels in vasculature), a rise in a-tocopheryl quinone (a-TQ), a well established marker of oxidative stress, suggested enhanced oxidative events within the vasculature of Gpx1−/− mice. However, Gpx1−/− mice failed to show an increase in aortic root lesions, nor were peroxidative events increased in plasma or aortic wall lipids compared with that seen in control animals. These results suggested that increased oxidative events within vasculature did not translate into increased lipid peroxidative damage and did not influence lipid deposition within the aortic sinus region in this strain of mice. However, the importance of GPx1 in the protection against atherosclerosis became apparent to us [57] and others [58] when the lack of GPx1 was coupled with a lack of apolipoprotein E (ApoE) in ApoE/GPx1 double-knockout (dKO) mice. ApoE−/− mice are now recommended as the murine model in which to study atherosclerosis, since these mice develop more extensive and more pathophysiologically relevant lesions throughout the aortic tree [59] that are not restricted to the aortic root as observed in high-fat-fed, non-genetically altered mice [60–62]. This allows for a more robust analysis of pathophysiologically relevant factors affecting atheroscleosis throughout the aortic tree. Furthermore, ApoE−/− mice develop atherosclerosis over a relatively short period of time as a consequence of their impaired clearance of plasma lipoproteins [63]. It should however be highlighted that LDL-R knockout mice are also a useful model in which to study pro-atherogenic processes [64], particularly since diabetes does not have as great a dyslipidemic effect in this strain of mice [19]. ApoE/GPx1 Double-Knockout Mouse Model After the establishment of our ApoE/GPx1 dKO colony (on a C57Bl/J6 background), we initially investigated the effect of a lack of GPx1 on atherosclerosis during aging. Our data showed increased lesion formation in the aortic arch and sinus region of 6- and 12-month-old female ApoE/GPx1 dKO mice fed a regular diet (4% fat) compared with age- and sex-matched ApoE−/− controls (Fig. 2a, b). In addition, Torzewski et al. [58] provided evidence that a lack of GPx1 accelerated atherosclerosis in their ApoE/GPx1 dKO mice after high fat feeding. In their study, atherosclerotic lesions were significantly increased in female ApoE/GPx1 dKO mice placed on a Western-type diet for 24 weeks. Moreover, their lesions showed increased cellularity, with an increase in macrophage content in early lesions and an increase in smooth muscle cells in advanced lesions. Furthermore, a deficiency

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a

b 400000

dKO

Aortic sinus lesions (µm2)

ApoE−/−

*

300000 200000 100000

Ap Ap oE oE −/ −/ − −G Px 1− /−

0

Fig.  2  Aortic sinus lesions, detected after staining with Oil Red O, of 6-month-old female ­ poE-deficient and ApoE/GPx1 double-knockout (dKO) mice are shown in (a). Quantitation of A lesions (b) show that sinus lesions are significantly increased in ApoE/GPx1 double-knockout mice compared with age and sex-matched ApoE-deficient controls. n = 10 mice/group. *P < 0.05 vs. ApoE−/− aortas

of GPx1 led to an increase in ROS within the aortic wall, lower levels of bioactive nitric oxide and increased nitrotyrosine, a marker of peroxynitrite production, clearly demonstrating that oxidative stress is increased in this pro-atherogenic model. ApoE/GPx1 dKO peritoneal macrophages also showed increased in  vitro proliferation in response to macrophage-colony-stimulating factor [58]. Collectively, these results, together with our results in aging mice, suggest that a deficiency of GPx1 accelerates and modifies atherosclerotic lesion progression in apolipoprotein E-deficient mice.

 iabetic ApoE/GPx1 dKO Mice as a Model of Accelerated D Diabetes-Associated Atherosclerosis Based on recent clinical studies that suggest a major protective role for GPx1 in diabetes-associated atherosclerosis [43, 44, 46], we induced diabetes in our ApoE/ GPx1 dKO mice to determine whether this is merely an association or whether GPx1 has a direct effect on diabetes-associated atherosclerosis. ApoE-deficient and ApoE/GPx1 dKO mice were rendered diabetic using the diabetogenic agent streptozotocin (STZ). STZ destroys the pancreatic b-islet cells, thus providing a robust model of type 1 diabetes. Furthermore, the National Institutes of Health (NIH), in collaboration with the Juvenile Diabetes Research Foundation, established the Animal Models of Diabetic Complications Consortium (AMDCC),

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which recently reported that the STZ-diabetic ApoE−/− mouse, despite some ­limitations, is currently considered the preferred model for studying macrovascular disease in diabetes [19]. Aortic lesion formation and atherogenic pathways were assessed after 10 and 20 weeks of diabetes. In our study, we showed that atherosclerotic lesions within the aortic sinus region, as well as lesions within the arch, thoracic, and abdominal region, were significantly increased in diabetic ApoE/GPx1 dKO aortas compared with diabetic ApoE−/− aortas [57]. This increase in aortic lesion deposition was accompanied by increases in macrophages, alpha-smooth muscle actin, RAGE and various proinflammatory (VCAM-1 and MCP-1) and profibrotic markers such as vascular endothelial growth factor (VEGF) and CTGF [57]. Quantitative reversetranscription polymerase chain reaction analysis showed increased expression of RAGE, VCAM-1, VEGF, and CTGF. Nitrotyrosine levels were significantly increased in diabetic ApoE/GPx1 dKO mouse aortas. These findings were observed despite upregulation of other antioxidants. Our results clearly showed that lack of functional GPx1 accelerates diabetes-associated atherosclerosis via upregulation of proinflammatory and profibrotic pathways in ApoE−/− mice and establishes GPx1 as an important antiatherogenic therapeutic target in patients with or at risk of ­diabetic macrovascular disease [57]. 2.1.2 The NOX Knockout Mouse Under physiological conditions, NADPH oxidases have a very low constitutive activity but are increased in response to various stimuli, including the diabetogenic milieu [29, 65–67]. NADPH oxidases appear to be especially important in redox signaling in that they are specifically activated by diverse agonists and, in turn, regulate the activation of protein kinases, transcription factors and other biological molecules. The NADPH oxidases, NOX1, NOX2, NOX4 and NOX5 have been localized to vascular tissue; however, the expression and subcellular localization of each vary [68]. NOX2 and NOX4 are the major source of ROS in endothelial cells and are implicated in vasodilator dysfunction and in the modulation of redox-­ sensitive signaling pathways that influence adhesion molecule expression, cell permeability, growth and migration [69]. Vascular smooth muscle cells, which have NADPH oxidase activity, lack NOX2 [70]. In this instance, activity is largely dependent upon NOX4 expression [71]. Initial studies in mice into the function of the NOX isoforms showed that both NOX2 and NOX4 expression increase at an early time point (4–8 weeks after initiation of diabetes with STZ) in the progression towards more developed lesions in diabetic ApoE−/− mice, suggestive of a role for NOX2 and NOX4-containing NADPH oxidases in the initiation of diabetes-­ associated atherosclerosis [72]. In addition, recent data in vascular cells suggests a distinct role for NOX1 and NOX4 in the production of superoxide and hydrogen peroxide respectively, which may partially account for the limited pharmacological effect of antioxidant treatments with superoxide scavengers that do not remove hydrogen peroxide [73].

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However, the role and relative importance of the NOX isoforms in vascular injury, including their role in diabetes, remains unclear. To address this question more specifically, NOX knockout mice including the knockout of the NOX1 and NOX2 isoforms have been generated [70, 74]. Various studies including NOX2 and p47phox KO mice on the ApoE background have lead to conflicting conclusions regarding the relative roles of NOX isoforms in mediating atherogenesis. One study by Kirk et  al. [75] reported that lack of the phagocytic NOX2 isoform failed to inhibit atherosclerosis in either ApoE−/− mice or NOX2−/− mice fed high-fat diets, leading these authors to conclude that superoxide generated by phagocytic NOX enzymes does not promote atherosclerosis in mice with either diet-induced or genetic forms of hypercholesterolemia. It should, however, be noted that in this study, lesions were assessed only within the aortic sinus, a region known to be influenced by factors such as turbulent blood flow, in contrast to the rest of the aorta, and therefore may not necessarily be a true reflection of the susceptibility of the aorta to atherogenic changes [76]. A second study also reported that knockout of p47phox (a cytosolic protein required for NADPH oxidase functionality [77]) had no effect on aortic sinus lesion progression in ApoE−/− mice, nor did it impact on blood pressure levels [78]. In contrast, two further studies in ApoE−/− mice support a role for NADPH oxidase-mediated pro-atherogenic processes. In the first study, p47phox KO mice had significantly less lesions throughout the aortic tree than ApoE(−/−) mice, with no differences in lesions within the sinus region, regardless of whether mice were fed standard food or a high-fat diet [79], while a second study, using ApoE/NOX2 dKO mice showed that the absence of NOX2 resulted in a 50% reduction in lesions from the arch to the iliac bifurcation in NOX2(−/y)/ ApoE(−/−) mice fed a high-fat diet compared with high-fat-diet-fed ApoE(−/−) mice, while NOX2 knockout did not impact on lesion development within the aortic sinus region [80]. These findings were also associated with a marked decrease in aortic ROS production and an increase in NO bioavailability in NOX2(−/y)/ ApoE(−/−) mice [80]. The latter two studies highlight the importance of not limiting the sampling of lesions to the sinus region, and this approach will be particularly important for future studies using these NOX knockout mice in a diabetic context where it has been reported by several laboratories that diabetic ApoE−/− mice develop even more diffuse and accelerated lesions throughout the aortic tree [27, 57, 59]. Studies using the NOX−/− mice should therefore reveal the contribution of the various NOX isoforms to diabetes-associated pathogenesis, and in ­particular to diabetes-associated-atherosclerosis. Recently, another isoform, NOX5, has also been reported; however even less is known about it. This calcium-dependent isoform is only present in humans, but has been linked to oxidative stress and human coronary artery disease [81, 82] and may mediate PDGF-induced proliferation in human aortic endothelial cells via a pathway that includes JAK/STAT activation [83]. Regulation of the NADPH oxidases is a major focus of several laboratories, since modulators of the NOX enzymes offer yet another way to control ROS ­production. In this light, a recent study has identified a novel NADPH oxidase ­regulatory protein, Poldip2, that associates with p22phox, NOX1 and NOX4 and

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colocalizes with p22phox at sites of NOX4 localization. Poldip2 was shown to increase NOX4 enzymatic activity threefold and to positively regulate basal ROS production in vascular smooth muscle cells. These authors suggest that Poldip2 may be a novel therapeutic target for vascular pathologies with a significant ­vascular smooth muscle cell migratory component, such as restenosis and ­atherosclerosis [84]. 2.1.3 The RAGE Knockout Mouse Model The impact of the AGE/RAGE axis on oxidative stress is increasingly being ­realized in experimental models investigating a role for RAGE in diabetic vascular complications [85, 86]. Hyperglycemia increases non-enzymatic glycation, characterized by the binding of glucose or by-products to amino groups of proteins. This reaction leads to the formation of complex AGEs that alter the structure and ­function of proteins. Glycated proteins such as AGEs activate membrane receptors such as RAGE and induce intracellular oxidative stress and a pro-inflammatory state [87, 88]. Activation of RAGE in the diabetic vasculature is considered to be a key mediator of atherogenesis. Initial studies using ApoE/RAGE dKO mice established a role for RAGE in the progression of atherosclerosis in mice fed a regular food diet. RAGE expression was shown to increase with the progression of lesions, with expression detected specifically within endothelial cells and vascular smooth muscle cells [59]. In ­addition, ApoE/RAGE dKO mice showed significantly reduced lesions within the aortic sinus region. Similarly, reductions in aortic lesions were also detected in a second murine model expressing dominant negative RAGE on an ApoE−/− background, where RAGE was specifically reduced within endothelial cells, highlighting the importance of endothelial RAGE to atherogenesis. These studies also revealed significant reductions in the levels of VCAM-1 in ApoE/RAGE dKO mice, implying a role for RAGE in mediating vascular inflammation. Reductions in RAGE also led to reduced oxLDL-mediated upregulation of VCAM-1 and matrix metalloproteinase-2 (MMP-2) in RAGE-deficient endothelial cells. Furthermore, endothelial rings derived from ApoE/RAGE dKO mice displayed significantly enhanced endothelium-dependent relaxation to acetylcholine compared with ApoE−/− mice implying a role for RAGE in mediating endothelial dysfunction [59]. These authors suggested that unifying mechanisms exist whereby increased endothelial RAGE and its ligands mediate vascular, oxidative and inflammatory stresses that culminate in atherosclerosis in the vessel wall. A recent study further supports the importance of AGEs, through interaction with RAGE, in mediating atherogenesis via oxidative mechanisms. Lesions were significantly reduced after 12-weeks of high-fat feeding of low-density lipoprotein receptor-deficient (LDL-R)/RAGE dKO mice [89]. Importantly, these reductions were associated with concomitant reductions in oxidative stress in the vessel wall. Furthermore, macrophages derived from these RAGE-deficient mice showed reduced oxLDL-induced activation of mitogen-activated protein (MAP)

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kinases and reductions in oxidative stress markers. In addition, the inflammatory markers ICAM-1 and VCAM-1 were reduced in LDL-R/RAGE dKO aortas. These authors speculate that oxLDL may be a ligand of RAGE in the hyperlipidemic state and that RAGE inactivation inhibits atherosclerosis by reducing oxLDL-induced pro-inflammatory responses and oxidative stress associated with the hyperlipidemic condition. Using a different mouse model, the AGER1 transgenic mouse, according to recent data by Torreggiani et al. [90], again demonstrated a link between AGEs and oxidative stress. In their experimental model they show that AGER1 overexpression reduces basal levels of AGEs and intracellular oxidative stress (assessed by antioxidant GSH status in different tissues). These mice also showed enhanced resistance to diet-induced hyperglycemia and oxidative stress, and were protected against injury-induced arterial intimal hyperplasia, oxidative stress and inflammation. Recent data from our laboratory has shown an important role for RAGE in ­diabetes-associated atherosclerosis [91]. Using ApoE/RAGE dKO mice made ­diabetic with STZ, we have shown reduced lesions in these mice compared with ­diabetic ApoE−/− controls. These beneficial effects on the vasculature were associated with reductions in leukocyte recruitment and decreased expression of proinflammatory mediators, including the nuclear factor-kappaB subunit p65, VCAM-1, and MCP-1. Importantly, RAGE knockout had a profound effect on reducing oxidative stress, as reflected by reduced staining for nitrotyrosine and reduced expression of the various NADPH oxidase subunits, NOX2, p47phox, and rac-1. Furthermore, RAGE ligands, including S100A8/A9, high mobility group box  1 (HMGB1), and the advanced glycation end product carboxymethyllysine were reduced in plaques from diabetic ApoE/RAGE dKO mice. Our study, using ApoE/RAGE dKO mice, ­provides direct evidence for RAGE playing a central role in the development of diabetes-associated atherosclerosis. Our findings strengthen the use of this model for investigating the role of RAGE in diabetes-associatedatherosclerosis and emphasize the potential use of strategies that target RAGE in the prevention and treatment of diabetic macrovascular complications.

3 Diabetic Nephropathy Diabetic nephropathy (DN) is now the most common cause of chronic kidney ­disease (CKD) and accounts for 40–50% of renal cases [92]. Approximately onethird of diabetic patients develop end-stage kidney disease necessitating renal replacement therapy within 25 years of disease onset [92]. Long-term prospective and interventional studies have shown that the risk of developing kidney disease is directly related to the exposure to elevated blood sugar levels. Therefore, therapies aimed at reducing the toxic effects of high glucose may be beneficial in reducing DN in diabetic patients. Indeed, recent clinical studies have shown that

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intensive glucose control and blockade of the renin–angiotensin system reduce the risk and progression of DN [93, 94]. However, such therapies have not ­eliminated DN in diabetic patients. Therefore the development of other novel therapeutics that specifically target DN may be helpful for most patients with diabetes. Accumulating evidence suggests an important role for oxidative stress in DN [95]. Oxidative stress has been linked with high glucose. Indeed, the increased oxidative stress as a consequence of high glucose elicits vascular inflammation and alters gene expression of growth factors and cytokines within the kidney [96]. In particular, high glucose induces intracellular ROS directly via auto-oxidation and glucose metabolism, and indirectly through the formation of AGEs that then bind to RAGE [97]. ROS have been shown to mimic the stimulatory effects of high glucose and upregulate transforming growth factor-beta 1 (TGF-b1), plasminogen activator inhibitor-1 (PAI-1), and extracellular matrix (ECM) proteins in glomerular mesangial cells, leading to mesangial expansion [98]. ROS also activate other signaling molecules such as MAP kinases and protein kinase C, as well as transcription factors such as nuclear factor-kappa B, activator protein-1 and specificity protein-1, leading to the upregulation of cytokines, growth factors and ECM proteins [99]. Furthermore, oxidative stress may arise as a result of reduced removal by antioxidant enzymes. Clearly, strategies that limit oxidative stress may prevent or lessen the development and progression of DN. Diabetic patients have been reported to have a reduced capacity to remove oxidative stress as a result of lowered antioxidant function. Under normal physiological conditions, the kidney expresses abundant amounts of the major antioxidants, superoxide dismutase-1 (SOD1), GPx1 and catalase. However, under pathophysiological conditions, these enzymes decrease, leaving the kidney vulnerable to the increased ROS known to accompany diabetes [100]. The importance of the GPx family of enzymes in limiting DN has been shown in STZtreated diabetic rats where a dietary deficiency of the essential trace element selenium (selenium as an integral part of the active site of the GPx enzymes) led to decreased GPx activity and enhanced DN [101]. There are several GPx isoforms present in the kidney; however, GPx1 accounts for >96% of the GPx activity [50]. Protection against oxidative stress is therefore most likely as a result of the function of the GPx1 isoform. However, to date, no study has directly linked GPx1 to the protection against DN. Our initial studies using diabetic C57Bl/J6 GPx1−/− mice surprisingly failed to show accelerated DN [102]. Thus, the significance of a lack of GPx1 may not have been properly revealed since lipid levels are unaffected in this diabetic model. Indeed, elevated lipids may be critical in accelerating DN since clinical observations suggest that hyperlipidemia is an important contributory factor to the progression of diabetic renal disease [103, 104]. Furthermore, Lassila et al. [105] have shown accelerated nephropathy in diabetic ApoE−/− mice. Therefore, mice with both an ApoE and a Gpx1 deficiency represent a more advanced model in which to study the consequences of a lack of GPx1.

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3.1 Diabetic ApoE/GPx1−/− dKO Mouse as a Model of DN In addition to the accelerated diabetes-associated atherosclerosis detailed above, we have also detected a significant increase in characteristic markers of diabetic nephropathy in ApoE/GPx1 dKO mice rendered diabetic with STZ. This occurred against a background of elevated lipids, highlighting the involvement of both ­lipids and oxidative stress in the progression of diabetic nephropathy. Furthermore, these results emphasize the importance of using the correct experimental model to address questions of oxidative stress in diabetic nephropathy, since our previous studies in diabetic GPx1−/− mice on a C57Bl/J6 background without increased lipid involvement, failed to show significant differences [102]. In our studies using diabetic ApoE/GPx1 dKO mice, we demonstrate increased albuminuria that is associated with pathological changes that include mesangial expansion and ­upregulation of pro-fibrotic (collagen I and III, fibronectin and TGF-b) and proinflammatory mediators (VCAM-1 and MCP-1) in the diabetic ApoE/GPx1 dKO kidneys. In doing so, we establish a role for GPx1 in limiting and/or preventing diabetic nephropathy in the physiologically relevant milieu of increased lipids known to accompany diabetes [104, 105]. It is also noteworthy that lack of GPx1 caused an upregulation of collagen I and III but not type IV collagen in the diabetic kidney. Type I and III collagen are mainly expressed in the interstitial region of the kidney with some involvement in glomeruli under certain pathological situations, while type IV collagen plays a significant role in glomerular pathology [106, 107]. It is therefore likely that GPx1 plays a role in the protection of the interstitium against extracellular matrix deposition. GPx1 may therefore be of particular importance in the protection of the kidney interstitium against diabetesmediated changes, as it is now recognized that tubulointerstitial changes are a prominent feature of DN and are closely linked to the progression and ultimate decline in glomerular filtration rate [108].

3.2 Experimental Models of NADPH Oxidase-Mediated Oxidative Stress in DN It is now well established that NADPH oxidase is the main source of ROS in DN [29, 67, 69, 109]. NADPH oxidases have a distinct cellular localization in the kidney [110]. Reactive oxygen species are produced by fibroblasts, endothelial cells, ­vascular smooth muscle cells, mesangial cells, tubular cells and podocytes. NOX-1 and -4, as well as all the components that comprise the phagocytic NADPH oxidase including NOX2, are expressed in the kidney. Expression of these NOX isoforms is seen in renal vessels, glomeruli and podocytes as well as cells of the thick ascending limb of the loop of Henle, macula densa, distal tubules, collecting ducts and cortical interstitial fibroblasts [110]. Furthermore, enhanced NADPH oxidase activity has been associated with oxidative damage to DNA in diabetic glomeruli [111, 112].

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A recent study by Gorin et al. [67] has shown a role for NOX4 in DN. In this model of STZ-induced diabetes in the Sprague-Dawley rat, antisense nucleotide treatment to NOX4, administered for 2  weeks after STZ injection, reduced renal extracellular matrix accumulation and renal hypertrophy via inhibition of Akt/­ protein kinase B and ERK1/2. However, the mechanism(s) by which diabetes and high glucose activate the NADPH oxidases remain(s) speculative. It may involve a direct effect on a specific NOX isoform such as NOX4, or via other mediators such as Ang II and/or TGF-b. Thus, the renin–angiotensin system may contribute to the upregulation of NADPH oxidase-mediated ROS production in DN. Indeed, the angiotensin-converting enzyme inhibitor as well as an Ang II type 1 (AT1) receptor blocker were able to inhibit the increase in ROS generation and reduce NAD(P)H oxidase subunit p47phox protein expression in rats with type 1 diabetes [113]. As mentioned previously, NOX1 and 2 knockout mice are now available to delineate the precise contribution of the various NOX isoforms to the development of DN and are expected to highlight the significance of NADPH oxidase as a ­possible new therapeutic target in the reduction and/or prevention of DN [70].

4 Diabetic Cardiomyopathy Extensive clinical and experimental evidence has substantiated the existence of a specific diabetes-associated cardiac disease, characterized by structural, functional and metabolic changes of the heart, in the absence of vascular complications [4]. These cardiomyopathic changes cannot be explained solely by poor coronary perfusion. Structural changes include left ventricular hypertrophy (LVH) and/or cardiac interstitial fibrosis [114, 115]. In particular, increased muscle mass indicative of myocardial hypertrophy, as well as cavity dilation and depressed ventricular performance consistent with impaired cardiac function, have been reported in diabetic subjects [115]. Large clinical studies of diabetic patients support this notion, e.g., the Framingham Heart Study, which demonstrated an association between diabetes and increased left ventricular (LV) wall thickness and increased heart mass [116]. These clinical findings have been confirmed in experimental models where an increase in wall thickness and LV dimensions were observed in diabetic rats and these changes were accompanied by cardiac dysfunction [114, 117]. Moreover, LV-weight to body-weight ratio, a marker of LVH, was higher in diabetic rats compared with non-diabetic control rats [114]. Histopathological changes include diffuse myocardial fibrosis [118] with changes in the myocardial extracellular matrix [119]. In particular, accumulation of myocardial collagen types I, III and VI have been reported. The increase in type I and III collagen may in turn contribute to altered cardiac hemodynamics due to increased stiffness and loss of elasticity. Furthermore, the increase in type III and VI collagen may impede the exchange of cellular substrates, which has been suggested to increase oxidative stress [119]. Oxidative stress has been implicated in the progression toward overt diabetic heart failure and has recently been reviewed by Mellor et  al. [120]. Evidence

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implicating ROS in diabetic cardiomyopathy include increased cardiac lipid ­peroxidation [121, 122] and elevated oxidized glutathione in diabetic heart tissue [7]. Recently superoxide production was shown to increase in the diabetic Zucker obese rat via a mechanism that includes glucose-6-phosphate dehydrogenase (G6PD)-derived NADPH [123]. Strong evidence in support of a role for ROS in the development of cardiomyopathy is also evident under non-diabetic conditions. Indeed, the chronic release of ROS has been linked to the development of LVH and progression to heart failure [13], impaired calcium handling and the stimulation of myocytes to produce more ROS [124]. Furthermore, abnormal activation of NADPH oxidase in response to angiotensin II, noradrenaline and tumor necrosis factor-a contributes to cardiac myocyte hypertrophy [13]. In addition, fibrosis and collagen deposition involved in the remodeling of the failing myocardium are dependent on ROS release [13]. Intervention studies using various antioxidants have provided further evidence in support of a role for ROS in cardiomyopathy. For example, vitamin E therapy delayed the development of heart failure, improved the redox state and reduced lipid peroxidation in a guinea pig model of ascending aortic constriction [125]. In addition, in animals with vitamin E deficiency, myopathy of the heart muscle is frequently observed [125]. Strong evidence for the protective role of antioxidants in diabetic cardiomyopathy comes from diabetic OVE26 mice specifically overexpressing the potent antioxidant protein metallothionein in their cardiomyocytes [124, 126]. These mice, which normally exhibit extensive cardiomyopathy, showed reduced cardiac damage and improved cardiomyocyte survival. Furthermore, mice overexpressing the antioxidant enzyme GPx1, albeit in a non-diabetic model, ­display improved LV function through the inhibition of LV remodeling, as well as reduced cardiomyocyte apoptosis and interstitial fibrosis compared with wild-type mice [127]. Two further studies have investigated the functional significance of GPx1 in the protection of the myocardium during ischemia/reperfusion injury. Mice lacking GPx1 were shown to be more susceptible to ischemia/reperfusion injury [52] while conversely, mice overexpressing GPx1, were protected from such injury [128]. Similarly, overexpressing the antioxidant enzyme catalase preserved normal cardiac morphology and prevented contractile defects in diabetic OVE26 mice [129], suggesting an important role for peroxide-reducing antioxidants in the protection against diabetic cardiomyopathy. In addition, MnSOD has been shown to play an important role in maintaining normal heart function. MnSOD-deficient mice die from cardiomyopathy, neurodegeneration and metabolic acidosis within the first 10 days of life [130]. Interestingly, Cu/ZnSOD knockout mice exhibit a less severe phenotype than that of MnSOD-deficient mice, where Cu/ZnSOD knockout mice are essentially normal but more susceptible to neuronal injury [131]. It is presently thought that myocyte apoptosis is a major contributory component to the progression of diabetic cardiomyopathy [13, 132, 133]. Cell death is an important cause of various myocardial abnormalities – in particular, loss of contractile tissue, compensatory hypertrophy of myocardial cells and fibrosis [133]. However the mechanisms leading to diabetic myocardial apoptosis remain unclear [133]. ROS are powerful mediators of apoptosis [13, 132]. Indeed, Cai et al. [133]

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showed that treatment with high glucose caused an increase in ROS production as  well as increased apoptosis of cardiac myoblasts and suggested that the ­hyperglycemia-induced apoptosis occurred via ROS-mediated mechanisms [133]. Furthermore, treatment of these cardiomyocytes with superoxide or H2O2 induced apoptosis via different apoptotic pathways, implying that there are several redoxsensitive apoptotic mechanisms in cardiac myocytes. Mitochondrially derived ROS have been proposed to contribute to diabetic cardiomyopathy. Evidence for this was shown in a recent study by Song et  al. [134] who used the OVE26 mouse model of severe type 1 diabetes to measure myocyte contractility and the source of ROS generation via a mitochondrial specific ROS detection system. Initial contractility was impaired in their diabetic myocytes and ROS production was elevated after exposure to either high glucose or angiotensin II (AngII). In particular, superoxide, detected via the mitochondrial sensor MitoSOX Red, confirmed that mitochondria are a major source of ROS in these diabetic myocytes. These authors speculate that the AngII-induced increase in mitochondrial ROS occurs via a pathway that includes cytoplasmic superoxide derived from NADPH oxidase. This assumption is based on the fact that AngII acts on the AT1 cell surface receptor and not on mitochondria, implying that a pathway exists linking the cardiomyocyte AngII receptor to mitochondrial generation of ROS. Such a pathway has also been proposed by Zhang et al. [135] who suggest that cytoplasmic superoxide, derived from NADPH oxidase, induces the opening of mitochondrial KATP channels, thereby increasing mitochondrial ROS production. In the diabetic heart, NADPH oxidase is a likely mediator of this effect since it is present in the heart [136], it is upregulated by diabetes [137, 138] and, furthermore, hyperglycemia [138] and AngII [135] are known inducers of NADPH oxidase-mediated ROS. This mechanism of cytosolic ROS generation leading to increased mitochondrial ROS may also occur in other diabetic complications such as diabetic nephropathy. Evidence comes from recent data of Coughlan et al. [139] who show that AGEs lead to increases in cytosolic ROS, which facilitate the production of mitochondrial superoxide. Inhibiting cytosolic ROS production with apocynin or lowering AGE concentration with the AGE-crosslink breaker alagebrium prevented the production of mitochondrial ROS. Furthermore, RAGE deficiency prevented diabetes-induced increases in renal mitochondrial superoxide and renal cortical apoptosis in RAGE knockout mice [139]. Our laboratory has investigated whether lack of the antioxidant enzyme GPx1 affects known markers of diabetic cardiomyopathy in C57Bl/J6 GPx1−/− mice made diabetic with STZ (Fig.  3a). In our model, elevated levels of H2O2, fatty acid hydroperoxides and peroxynitrite occur as a consequence of the lack of their removal by the second step antioxidant enzyme GPx1. Thus, Gpx1 knockout mice are a useful model to address the role of GPx1 and the involvement of these particular ROS in diabetic cardiomyopathy. In this model, we detected significantly increased picrosirius red staining after 3 months of diabetes in diabetic GPx1−/− hearts compared with diabetic controls (Fig. 3b), suggesting that fibrosis is enhanced by the lack of GPx1. After 6 months of diabetes, we observed a significant increase in the gene expression of the cardiac hypertrophic peptide, atrial natriuretic peptide (ANP) (Fig. 3c),

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Fig. 3  Diabetic GPx1 knockout mice hearts display features of accelerated diabetic cardiomyopathy. Despite achieving equivalent blood glucose levels of approximately 20 mM as shown in 3a, diabetic GPx1 knockout hearts show significantly increased percent fibrosis as shown in 3b (*P < 0.05; assessed by picrosirius staining after 3 months of diabetes); significant increases in the hypertrophic marker ANP as shown in 3c (*P < 0.05); and a significant increase in heart/body weight ratio after 6 months of diabetes as shown in 3d (**P < 0.01) compared with diabetic ApoE-deficient control hearts. n = 6–9 mice/group except for ANP analysis where 4–7 mice were used/group

together with an increase in the heart/body weight ratio (Fig.  3d) ­compared with diabetic control hearts, suggesting that lack of GPx1 may contribute to diabetic hypertrophy. We have also shown that gene expression of the pro-fibrotic mediators TGF-b, CTGF, collagen I, III and IV and fibronectin, is significantly elevated in the hearts of ApoE/GPx1−/− mice, suggesting a link between hypercholesterolemia, oxidative stress and cardiac fibrosis (Fig. 4). Importantly, GPx1 activity is reported to decline with developing diabetic cardiomyopathy [140].

5 Targeted Antioxidant Therapies Although results from basic research suggest a protective role for vitamins in the prevention of cardiovascular disease, evidence from clinical trials indicate no overall benefit for major cardiovascular events [141]. In some trials the use of

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Fig.  4  Quantitative RT-PCR gene expression analysis of pro-fibrotic markers in 6-month-old male ApoE-deficient and ApoE/GPx1 double-knockout mice hearts. Lack of GPx1 significantly increased expression of the pro-fibrotic mediators, TGF-b (**P < 0.01) and CTGF (*P < 0.05), as well as the fibrosis genes, fibronectin (**P < 0.01) and collagen 1 (**P < 0.01), 3 (**P < 0.01) and 4 (***P < 0.001). n = 9 for ApoE−/− group; n = 14 for dKO

vitamins increased cardiovascular mortality. For example, the HOPE trial showed an increase in the incidence of congestive heart failure (CHF) in patients treated with vitamin E [142], while the use of vitamin E is also contra-indicated in patients with left ventricular dysfunction [143]. Failure of vitamins in clinical applications may arise due to the lack of specificity in targeting the correct ROS and/or their cellular location. Indeed, vitamins E and C that target radical (or one electron) oxidants may not have adequately removed critical ROS and ROSgenerating pathways in major clinical trials, thus explaining their equivocal results [144]. It is becoming increasingly clear that enzymatically generated non-radical (or two electron) oxidants such as H2O2 and peroxynitrite are more important, since these oxidants modulate redox-sensitive thiol and methionine residues in proteins. Protection of these often critical residues in enzymes and/or structural proteins prevents inactivation and/or destruction of vital protein function. Bolstering the function of antioxidants such as GPx1 that target the removal of H2O2 and peroxynitrite could be a potentially more important therapeutic strategy than the administration of vitamins, such as vitamin E and C. Indeed, our studies

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with ebselen, a GPx1 mimetic, highlight the effectiveness of this approach in reducing diabetes-associated atherosclerosis in our experimental models [145].

5.1 The GPx1-Mimetic Ebselen Ebselen (2-phenly-1, 2-benzisoselenazol-3[2H]-one) is a synthetic, lipid-soluble, non-toxic seleno-organic compound [146] with anti-inflammatory and antioxidant activities [147]. In particular, because of its glutathione peroxidase mimetic activity, ebselen has been widely reported to act against membrane hydroperoxides, including hydrogen peroxide and lipid peroxides [148–150]. In this context, ebselen has been shown to inhibit hydrogen peroxide-induced cell death in various cell types, including HepG2 [151], human HL-60 [152], PC12 cells [153] and HUVEC [154]. In addition, ebselen scavenges other ROS, including peroxyl radical and peroxynitrite [146]. Ebselen also directly inhibits several enzymes involved in inflammatory processes. These include 5-lipoxygenases, NOS, NADPH oxidase, protein kinase C and ATPase [146]. This is of particular relevance to pathologies such as atherosclerosis where inflammatory involvement is often thought of as etiological.

5.2 Ebselen in an Experimental Model of Diabetes-Associated Atherosclerosis Studies from our laboratory support a role for targeted antioxidant defense against diabetes-associated atherosclerosis [145]. Based on the knowledge that inflammatory responses are elevated in diabetes-associated atherosclerosis and the strong antiinflammatory effects of ebselen, we postulated that ebselen would reduce oxidative stress, pro-inflammatory mediators and pro-atherogenic pathways in diabetic ApoE−/− mice. Indeed, previous studies supported a role for ebselen in reducing various oxidative-stress mediated pathologies in experimental models, including cerebral infarction [155], the protection of the endothelium in stroke-prone hypertensive rats [156], and cardiac dysfunction in a murine model of chronic iron overload [157]. Of dual relevance, ebselen reduced oxidative damage of proteins and partially restored endothelial dysfunction in Zucker diabetic rats [158]. Furthermore, ebselen decreased arterial lesions in a superoxide-driven non-inflammatory transgenic murine model consistent with a potential role for ebselen in reducing atherosclerosis [159]. Other evidence in support of a role for ebselen in reducing oxidant-mediated pathogenesis came from limited patient studies where ebselen improved acute ischemic stroke outcome [155] and delayed neurological deficits after aneurysmal subarachnoid hemorrhage [160], suggesting that ebselen may be a promising neuroprotective agent [155]. However, prior to our study in ApoE−/− mice [145], no study had directly evaluated the effect of ebselen on diabetes-associated atherosclerosis.

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In our study, 8-week-old male C57Bl/J6 ApoE−/− mice were rendered diabetic with STZ (two injections of 100  mg/kg body weight) and divided into ebselengavaged and non-gavaged groups. Ebselen was gavaged twice daily at 10  mg/kg body weight starting at 10 weeks of age. Animals were maintained for 20 weeks of diabetes for gene expression, immunohistochemical analyses, en face aortic lesion analysis as well as an assessment of lesions within the aortic sinus region. Our study showed that administration of ebselen to diabetic ApoE-deficient mice attenuated lesion formation in most regions of the aorta (the arch, thoracic and abdominal aortic regions). The reduced lesion formation was accompanied by a decrease in oxidative stress as reflected by reduced nitrotyrosine levels as well as a reduction in the NOX2 subunit of the NADPH oxidases. Our results support the observations of Brodsky et  al. [158] where ebselen decreased nitrotyrosine in Zucker diabetic rats, albeit a model not associated with atherosclerosis. Our data therefore highlights the importance of ebselen in its role as a peroxynitrite reductase and strengthens the notion that ebselen functions in a similar fashion to GPx1 [49]. Furthermore, the cellularity associated with a pro-atherosclerotic phenotype (a-smooth muscle cells and macrophages) was decreased by ebselen in association with a reduction in expression of the pro-atherosclerotic mediators RAGE and VEGF in the aorta. Interestingly, our study showed that ebselen elicits its anti-atherogenic effects in a site-specific manner since ebselen did not affect lesions within the aortic sinus. Our results support a growing list of studies where site-specific effects of modulators of atherosclerosis have been observed [76]. For example, in a study by Witting et al. [161], the lipid-lowering antioxidant probucol decreased lesion formation in most aortic regions but resulted in increased lesion formation within the aortic sinus. Indeed, it has been postulated that parts of the aorta behave differently due to local hemodynamic factors such as low shear stress, turbulence, oscillating flow and inherent properties of the vessel wall [76, 161]. However, importantly, our data showing a reduction in atherosclerotic plaque in most regions of the aorta after 20 weeks of diabetes, supports the notion of Blankenberg et al. [43] that bolstering GPx-like activity reduces atherosclerosis, and is in agreement with our findings in diabetic ApoE/GPx1 dKO mice where lack of GPx1 greatly accelerated plaque deposition in the aorta of ApoE−/−GPx1−/− mice [57]. Our results therefore strongly suggest that ebselen is an effective anti-atherogenic agent against diabetic macrovascular disease.

5.3 A Mechanistic Understanding of the Anti-atherogenic Action of Ebselen We have undertaken a series of in  vitro analyses in human aortic endothelial cells (HAECs) to further understand the anti-atherogenic actions of ebselen that we observed in our diabetic experimental models [145]. In these experiments, HAECs were pre-treated with 0.03 mM ebselen prior to exposure to 100 mM H2O2 for 30 min.

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Our in  vitro studies in HAECs suggest that one of the key mechanisms whereby ebselen confers its anti-atherogenic effects is via modulation of the transcription factor NF-қB. Pre-treatment with ebselen reduced the H2O2-mediated increase in IқBkinase (IKK) complex phosphorylation on critical activatory residues. Since IKK is a key regulator of NF-қB activation [162], it is predicted that by reducing IKK phosphorylation, ebselen anchors NF-қB in the cytoplasm, thereby preventing the activation of pro-inflammatory genes. It is therefore likely that ebselen affects downstream cellular targets regulated by NF-қB. To explore this notion further, we also investigated the effects of ebselen on NOX2 expression in HAECs, since NOX2 is known to be regulated by NF-қB [163] and in our ­diabetic ApoE−/− animal model, ebselen was associated with decreased NOX2 gene and protein expression. Our in vitro data showed reduced NOX2 protein levels after ebselen treatment and supports the idea that ebselen affects downstream targets of NF-қB. We also explored the consequences of ebselen pre-treatment on the cytokine TNF-a, since TNF-a is an important diabetes-associated pro-inflammatory mediator and is involved in the activation of NF-қB [164]. Our in vitro data showed that the H2O2-induced upregulation of TNF-a was reduced by ebselen. Furthermore, recent data by Yuen et al. [165] has shown that the pro-inflammatory cytokine IL-6 mediates its insulin-regulatory effect in HAECs via enhanced TNF-a production. Our results in HAECs support growing evidence that ebselen inhibits TNF-a-induced proinflammatory responses in endothelial cells [153] and other cell types [166, 167]. Finally, our in vitro analysis has also shown that ebselen blocks the H2O2-mediated phosphorylation of c-Jun-N-terminal kinase (JNK), a kinase involved in the activation of the transcription factor AP-1. This is important, given that several laboratories suggest a role for JNK in TNFa-mediated endothelial cell activation [168] and in particular through interactions of AP-1 with NF-қB [169], and because others have suggested that the effect of ebselen on JNK may be ­cell-type-specific [154, 170]. Collectively, our results with ebselen have implications not only for inflammatory genes known to be regulated by these pathways [1], but also on the pro-atherosclerotic pathway itself, since inflammatory events are integrally linked with atherosclerosis. Although our studies show the potential benefit of ebselen in the protection against diabetes-associated atherosclerosis, novel GPx-Like compounds, synthesized for their greater solubility and efficacy than ebselen are now available, and are at present a major focus of our laboratory to assess pre-clinically their anti-atherogenic potential. Indeed, this approach and the screening of more efficient ebselen analogs have shown their greater protection against mitochondrial oxidative ­damage and cell death in fibroblasts derived from Friedreich ataxia patients [171]. In these studies, ebselen analogs were several-hundred-fold more effective at ­preventing cell death than ebselen.

5.4 Mitochondrially Targeted Antioxidants Large amounts of free radicals are produced by the mitochondria during oxidative respiration and are known to play an important role in the physiology of a cell. However, mitochondrially derived ROS also contribute to the pathophysiology of a

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cell and have been implicated in pathologies that include ischemia-reperfusion injury, diabetes, atherosclerosis, endothelial dysfunction and cardiovascular ­diseases. To effectively remove mitochondrially derived ROS, strategies for the targeted delivery of antioxidants to mitochondria are being developed [172–174]. Indeed, another explanation for the failure of antioxidants to produce beneficial effects in clinical trials is that they do not accumulate preferentially in the ­mitochondria. Recent advances in mitochondrially targeted antioxidants have ­concentrated on those that specifically target the matrix-facing surface of the inner mitochondrial membrane and therefore protect against mitochondrial oxidative damage [175]. In particular, antioxidants conjugated with a lipophilic triphenylphosphonium cation such as mitoquinone, mitovitamin E and mitophenyltertbutyline accumulate in the mitochondrial matrix at concentrations several-fold greater than cytosolic non-mitochondrially targeted antioxidants because of the high negative membrane potential of the inner mitochondrial membrane [176, 177]. Indeed, several studies in mice fed mitochondrially targeted antioxidant compounds for several weeks show accumulation of these compounds in the mitochondria of ­various tissues including brain, heart, liver and kidney [178]. In vitro experiments using both mitoquinone and mitovitamin E have shown promising reductions in peroxide-mediated oxidant stress and apoptosis while maintaining proteasomal function in bovine aortic endothelial cells, suggesting a potential role for mitochondrially targeted antioxidants in the protection against oxidative stress-mediated endothelial cell dysfunction [173]. Targeting of the antioxidant enzymes and their mimetics to the inner ­mitochondrial space may be another approach to boost antioxidant defense in this compartment. This technique has recently been employed to reduce cardiomyopathy in transgenic mice expressing mitochondrially targeted catalase. In their study, Kohler et  al. [179] were able to attenuate cardiac mitochondrial oxidative stress and left ­ventricular dysfunction in transgenic mice expressing mitochondrially targeted catalase after antiretroviral-induced cardiomyopathy. In addition, a mitochondrially targeted form of the GPx1-mimetic ebselen, mitoperoxidase, has been developed, and shown to decrease glucose or H2O2-mediated apoptosis in cell lines [180]. However, mitoperoxidase was shown to be only slightly more effective in the ­protection against H2O2 and glucose-mediated apoptosis than ebselen in in  vitro assays [180]. The effectiveness of reducing diabetes-associated complications such as atherosclerosis and nephropathy with these mitochondrially targeted antioxidants still needs rigorous testing in experimental models of diabetes before clinical studies are conducted to evaluate the effectiveness of mitochondrially targeted antioxidants.

5.5 NOX Inhibitors It is now becoming increasingly clear that increased NADPH oxidase activity in tissues vulnerable to hyperglycemia takes place downstream of the advanced glycation end products and protein kinase C pathways, two of the primary mechanisms

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involved in the pathogenesis of diabetic complications [181]. It has been proposed that compounds that suppress NADPH oxidase activity may offer therapeutic ­benefits for diabetic complications, in particular diabetic nephropathy where NOX involvement is increasingly being appreciated [96]. In addition, it has recently been shown that upregulation of NOX is associated with upregulation of the endothelin pathway in the pathology of DN and that the dual endothelin receptor antagonist, CPU0213, attenuates DN by suppressing NOX in male Sprague-Dawley rats made diabetic with STZ [182]. Furthermore, it has also been suggested that inhibition of Nox1 may be an efficient strategy to suppress neointimal formation in the prevention of vascular complications associated with diabetes [183]. In light of these observations, several small-molecule and peptide inhibitors of the NOX enzymes have been developed and shown to have promise in experimental studies, but issues of specificity, potency, and toxicity militate against any of the existing published compounds as candidates for drug development [184]. Apocynin, a proven NADPH oxidase inhibitor, has been used widely in animal models of oxidative tissue injury and shown to improve vascular and renal complications. More recently, Nam et al. [185] evaluated the effects of apocynin on diabetic nephropathy in a type 2 diabetic rat model. In their model, using aged Otsuka Long Evans Tokushima Fatty (OLETF) rats, they demonstrate improvements in glomerular and mesangial expansion, significantly decreased glomerular VEGF expression and reductions in the oxidative stress markers, urinary 8-OHdG and MDA, in the apocynin-treated OLETF rats. However, apocynin is considered to be non-selective in its mode of action as evidence suggests that it also targets other enzymes such as Rho-kinase [186]. Additional inhibitors of NAD(P)H oxidase include diphenylene iodonium (DPI), 4-(2-amino-ethyl)-benzenesulfonyl fluoride (AEBSF) and the PI3K inhibitor wortmannin [187]. The usefulness of these compounds as therapeutic strategies is limited due to their effects on multiple enzymes and other potential targets, and in the case of AEBSF, being an irreversible serine protease inhibitor [188]. Other approaches have included the novel tat peptide inhibitors [184] and the Vas2870 compound [189]. One of the most specific inhibitors developed so far is gp91(ds-tat), an 18-amino acid peptide that interferes with NADPH oxidase assembly and activation [190]. In particular, this peptide functions by mimicking the binding region of NOX2, and possibly NOX1, that interacts with p47phox. In doing so, it inhibits subunit assembly, resulting in the specific inhibition of superoxide production from NADPH oxidase and not from other oxidases such as xanthine oxidase. However, there are limitations with this approach – most notably, the mode of administration of the peptide that at present can only be via intravenous injection due to its limited bioavailability [184]. gp91(ds-tat) has shown promise in reducing vascular ROS associated with AngII-mediated hypertension in mice [190] as well as reducing endothelial dysfunction and vascular ROS in the Dahl salt-sensitive rat model [191]. VAS2870, discovered via a high-throughput screening assay specific for NADPH oxidase activity [189], attenuates PDGF-dependent smooth muscle cell chemotaxis in  vitro via a mechanism that includes the complete abolition of NAD(P)H oxidase activation and ROS production [189]. Only limited in vitro and

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some in  vivo data is available for these novel compounds and further testing in experimental models is necessary to determine if these approaches represent ­feasible therapeutic strategies for diabetic complications. In addition, a more preferable strategy in the design of NOX-inhibitors would be to develop agents with isoform specificity, as it may not be beneficial to block all NOX isoforms in specific disease states such as diabetes, considering the important signaling role provided by some of the NOX isoforms and the role of NOX2 in the innate immune response, although broadly active NOX inhibitors may prove to be useful in some settings.

6 Conclusions This review has highlighted the use of several experimental models to assess the contribution of ROS to diabetic complications such as diabetes-associated atherosclerosis, diabetic cardiomyopathy and diabetic nephropathy. The deleterious ­consequences of excess ROS, such as hydrogen and lipid peroxides as well as peroxynitrite, is being revealed through the use of the ApoE/GPx1 double-knockout mouse. In addition, this model emphasizes the importance of antioxidant defense in limiting these particular ROS. Indeed, lack of GPx1 contributes to accelerated diabetes-associated atherosclerosis, diabetic nephropathy, and may also impact on the functioning of the diabetic heart. Furthermore, use of RAGE knockout mice has shown that oxidative stress is inextricably linked with pathophysiological cell ­signaling, particularly signaling through RAGE with its concomitant increase in diabetes-mediated pro-atherogenic effects. Particular attention has focused on the utility of NOX-knockout mice, since the contribution of the NADPH oxidases to the ROS milieu is considered paramount, given that the NOX enzymes are an important upstream source of ROS. The availability of the NOX1 and NOX2 knockouts will shed valuable information on the contribution of these isoforms to individual diabetic complications. Specific targeting of ROS is one mechanism to reduce ROS and it is becoming increasingly clear that knowledge of the type of ROS involved will increase the likelihood of achieving this aim. For example, experimental models have shown that the specific targeting of ROS such as hydrogen and lipid peroxides as well as peroxynitrite may be more beneficial than the blanket antioxidant approach taken in large clinical trials and may explain the ­failure of vitamin trials in the past. Specifically, the use of ebselen, a glutathione peroxidase mimetic, has shown promise against diabetes-associated atherosclerosis and diabetic nephropathy in experimental models. Antioxidants that target ­mitochondrial ROS may offer an alternative approach to reduce diabetes-driven oxidative stress since it is becoming increasingly clear that mitochondria play a vital role in diabetes-mediated ROS production. Furthermore, targeting the source of ROS by inhibiting ROS production using, for example, NOX inhibitors, is yet another approach being explored by several laboratories. It is only via manipulation of experimental models of diabetes-driven oxidative stress that the contribution of

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the various ROS will be revealed, and only then can effective treatment regimens be designed to reduce ROS and lessen the effect of oxidative stress on diabetic complications. Acknowledgments  T.J.A. and K.J-D. are supported by an Australian National Health and Medical Research Council (NH&MRC) Senior Research Fellowship. All three authors acknowledge support from the Australian NH&MRC and the Australian National Heart Foundation.

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Experimental Models of Oxidative Stress Related to Cardiovascular Diseases and Diabetes Maria D. Mesa, Concepcion M. Aguilera, and Angel Gil

Abstract  In this chapter we summarize the commonly used animal models employed in the study of cardiovascular diseases and diabetes, two of the most prevalent oxidative stress-induced diseases. A number of animal models of atherosclerosis support the notion that reactive oxygen and nitrogen species have a causal role in atherosclerosis and other vascular diseases. Experimental atherosclerosis is induced by specific lipidrich diets or in genetically modified strains that cause hyperlipidemia and cardiovascular diseases. Rabbits are the animals most used for the study of atherosclerosis due to their facility to generate atherosclerosis with defined fat-enriched diets. Indeed, a myocardial infarction-prone derived from Watanabe LDL receptor-deficient rabbit strain is also used. Since mice are highly resistant to atherosclerosis, only genetically modified mice, such as the knock-out mouse for apo E, LDL receptor or both, have been used. In addition, reactive species also contribute to the pathogenesis of b-cell destruction in type 1 diabetes and b-dysfunction on type 2 diabetes. Alloxan induces the formation of reactive oxygen species and provokes b-cell death in type 1 and 2 diabetes mellitus, while streptozotocin involves DNA alkylation and fragmentation. Zucker rats are the most important animal models of genetic obesity and metabolic syndrome. Finally, genetically engineered diabetic models include transgenic and knock-out mice. Keywords  Atherosclerosis • Animal models • Cardiovascular diseases • Diabetes • Oxidative stress

A. Gil (*) Department of Biochemistry and Molecular Biology II, Institute of Nutrition and Food Technology “José Mataix”, Centre of Biomedical Research, University of Granada, Campus de la Salud, 18100 Armilla, Granada, Spain e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_2, © Springer Science+Business Media, LLC 2011

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1 Introduction Oxidative stress has been implicated in a wide variety of degenerative processes diseases and syndromes, due to a weakening of the antioxidant defense or excess production of radicals that can overwhelm the scavenging capacity of cellular ­antioxidant systems. Some of these oxidation-linked diseases or disorders can be ­exacerbated, perhaps even initiated, by numerous environmental pro-oxidants and/ or pro-oxidant drugs and foods [1]. Animal experimentation is essential for ­understanding the molecular basis, pathogenesis, actions of therapeutic agents and genetic or environmental influences that increase the risks of disease. The species of animal used is determined by several factors. In general, smaller animals are preferred because they are more manageable and experiments are cheaper. One of the guiding principles of animal research is to use the lowest possible animal and, nowadays, permission would not be granted for using larger animals unless a similar experiment could not be performed on a rodent. However, a major criticism of using rodents is that they may not adequately reflect the human situation and this occasionally justifies the use of larger animals [2]. In this chapter we summarize the commonly used animal models employed in the study of cardiovascular diseases and diabetes, two of the most prevalent oxidative stress-induced diseases. Due to the vastness of the topic, we will review the most important animal models used, their advantages, disadvantages and limitations. In addition, we will present data related to the evaluation of oxidative stress and some antioxidants in different models of those diseases.

2 Animal Models of Cardiovascular Diseases A great number of cardiovascular diseases have their origins in the development of atherosclerosis. Oxygen radicals, and other reactive oxygen species (ROS), may play an important role in the pathophysiology of atherosclerosis, stroke, and other ­cardiovascular diseases [3]. Inflammatory cells and inflammatory mediators, such as cytokines, and matrix metalloproteinases (MMP) play an important role in the plaque rupture [4, 5]. On the basis of pathological features, the human stable ­coronary plaque is usually enriched in collagen fibers and contains more smooth muscle cells, few macrophages/foam cells and lipid accumulation. In contrast, vulnerable plaques have a large lipid or necrotic core (mainly cholesterol clefts), a thin fibrous cap and many inflammatory cells at the so-called “shoulder” region [6]. Better ­understanding of mechanisms that provoke increases in oxidative stress in different cardiovascular diseases may lead to more effective antioxidant prevention or ­treatment of them. Various animal models of atherosclerosis support the notion that ROS released from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, myeloperoxidase (MPO), xanthine oxidase (XO), lipoxygenase (LO), nitric oxide synthase (NOS) and enhance ROS production from dysfunctional mitochondrial respiratory

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chain have a causal role in atherosclerosis and other vascular diseases. Human investigations also support the oxidative stress hypothesis of atherogenesis. This is further supported by the observed impairment of vascular function and enhanced atherogenesis in animal models that have deficiencies in antioxidant enzymes [7]. The use of suitable animal models is essential for investigation of the ­mechanisms underlying cardiac events and development of the therapeutic ­strategies; however, ideal animal models that can mimic human coronary ­atherosclerosis and subsequent acute myocardial infarction are still lacking. The perfect animal model should display coronary lesions resembling those seen in human atherosclerosis. No suitable large animal model of high-risk (vulnerable) plaque exists. Lack of such a model has ­hampered studies designed to validate imaging technologies and to scrutinize the effects of therapeutic interventions in atherosclerotic arteries [8]. Furthermore, the major problem in current animal models of cardiovascular disease is that the ­distribution and composition of ­lipoproteins is very different from humans [9]. There are a number of experimental animal models used in the study of ­atherosclerosis (Table 1). Atherosclerosis is induced by specific lipid-rich diets, or in genetically modified strains that develop hyperlipidemias and cardiovascular diseases. Within animal models, monkeys and pigs generate pathological arterial lesions similar to those in humans [8] where intimal diffuse and eccentrically enlargement as a consequence of mechanical stress and blood flow against arterial wall, mainly in iliac and secondary bifurcation are found. However, the difficult handling and the high price impede the use of these animals and therefore, smaller animals are preferred.

Table 1  Animal experimental models of cardiovascular disease Dietary-induced atherosclerosis Genetically unmodified animals High intake of cholesterol and saturated fat: rabbit, guinea pig, chicken, pig, monkey Genetically modified animals New Zealand rabbit expressing apo A-I, overexpressing lecitin-cholesterol-acyl-transferase, human hepatic lipase or human apo B-100 Watanabe heritable hyperlipidemic rabbits (WHHLMI) expressing human Lipoprotein (a) Transgenic knock-out and knock-in animals C57BL/6 mouse Apo E knock-out (apo E−/−) LDL receptor knock-out (LDL−/−) Double apo E and LDL receptor knock-out Knock-in expressing human apo E, apo E2k or apo E3-Leiden Platelet function deficient murine strains ATP-binding cassette transporter A1 mutated murine strains (ABCA1)

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2.1 Rabbit Dietary-Induced Atherosclerosis Rabbits are the animals most used for the study of atherosclerosis due to their facility to generate atherosclerosis with specific diets, especially cholesterol-rich diets. Among all strains of rabbits, the New Zealand rabbit, with a body weight of between 2 and 5 kg, is the most commonly used in the laboratory [10]. In addition, gender differences provide an interesting field of observation, since female animals are less prone to diet-induced atherosclerosis than male animals [11]. Different amounts of cholesterol included alone or in combination with other dietary fats and the duration of the treatments may induce atheromatous lesions at different degrees. Atheromatous plaques have been developed with 0.5% of cholesterol during 3 weeks [12] or 12 weeks [13], 1% of cholesterol during 10 weeks [14] or 12 weeks [15] and 2% of cholesterol during 4 weeks [16]. Other authors have used cholesterol in combination with saturated fats. Juzwiak et al. [17] developed atherosclerosis by feeding rabbits with 0.5 g/kg of cholesterol plus 0.5 g/kg of saturated fat for 12 weeks. Our research group has used 1.5% of cholesterol plus 3% of lard and has demonstrated that the evolution of the lesion degree is ­time-dependent (Fig. 1). We have observed that after 10 days of this atherogenic diet, rabbits had small deposits of lipid-full macrophages in the aortic arch. These deposits of foam cells became larger after 20 days of treatment, at the time that new mild lesions appeared in the thoracic and abdominal aorta. Experiments of 30 days led to large fatty streaks (macrophage infiltrates) in all aorta sections [18]. Particularly, the highest grade of lesion was found in the aortic arch, followed by the thoracic aorta and, finally, by the abdominal aorta; indeed, the higher cholesterol accumulation

Fig. 1  Evolution of fatty streak in rabbits fed 1.5% cholesterol and 3% lard-rich diet for 10, 20 and 30 days

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and a lower monounsaturated to ­polyunsaturated fatty acids ratio (MUFA/PUFA), a good marker of lipid oxidation susceptibility, were found in the aortic arch compared with the thoracic and ­abdominal aortas [18]. Finally, after 50 days of feeding animals with the ­atherogenic diet, aorta lesions became typical fibroatheromatous plaques, containing higher levels of cholesterol [19]. We have demonstrated that this atherogenic diet caused an increase in plasma and lipoprotein concentrations of cholesterol, phospholipids and triacylglycerols and enhanced LDL oxidative damage after 30 days [20] or 50 days [21]. In ­addition, plasma retinol and the essential co-antioxidant coenzyme Q10 were lost, and their concentrations were drastically reduced within the first 10 days of dietary intervention. A concomitant increase in peroxidation products was also found in these animals [18]. As a consequence of diet-induced oxidative damage, other tissues were also affected. Specifically, the in vitro susceptibility to oxidation of erythrocyte and liver microsome and mitochondria membranes were increased after 30 days of dietary intervention [22, 23], and liver membrane mitochondria hydroperoxides were increased, while the content of CoQ10 dropped after 50 days [24]. In addition, the hepatic mitochondria lipid profile and the enzymatic and nonenzymatic antioxidant defense systems were also altered [25]; these alterations make this ­atherosclerotic experimental model an adequate model of liver steatosis [26]. We have employed this animal model for the assessment of dietary impact on cardiovascular disease. Indeed, we studied the effect of different dietary sources of fat, as well as, a hydroalcoholic extract of Curcuma longa, exhibiting antioxidant properties, for the treatment or prevention of atherosclerosis. Our first aim was to determine the effect of feeding different diets enriched in different oils: sunflower oil, virgin olive oil, refined olive oil and fish oil for a period of 30 days, on ­atherosclerotic rabbits that had been on the cholesterol- and lard-rich diet for 50 days. In this way, we demonstrated the beneficial role of virgin olive oil and fish oil on atherosclerosis. The replacement of the atherogenic diet by other unsaturated fat-enriched diets using sunflower, refined olive or virgin olive oils recovered LDL susceptibility to oxidation to values close to those in healthy animals, while fish oil increased LDL oxidation. However, both virgin olive, and, to a lesser extent, fish oil, stopped the progression of the atherosclerotic lesions [19]. In addition, virgin olive, sunflower and fish oil enhanced the hepatic antioxidant defense ­system, and decreased the content of hydroperoxides [25]. Secondly, we ­demonstrated that oral administration of a hydroalcoholic extract of C. longa extract reduces oxidative stress and attenuates the development of fatty streaks in rabbits fed a high cholesterol diet [18]. Specifically, we have observed that ­supplementation with C. longa extract decreases the susceptibility of LDL to lipid peroxidation and diminishes LDL lipids in a dose-dependent manner [20]. In ­addition, the extract reduced the susceptibility to oxidation of erythrocyte and liver microsome membranes in vitro and thus may contribute to the prevention of ­atherosclerotic effects caused by the diet rich in cholesterol and lard [23]. In 1980, Watanabe developed an LDL receptor-deficient rabbit strain (Watanabe heritable hyperlipidemic-WHHL), derived from the New Zealand strain, as a model of human familiar hypercholesterolemia [27]. These original WHHL rabbits showed very remarkable atherosclerosis plaques in aortas starting from the

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age of 3 months; however, the lesion of coronary arteries are rather limited in these ­animals [28]. Furthermore, cellular and extracellular matrix compositions of ­coronary plaques are largely different from atherosclerotic lesions of the aorta and other arteries in WHHL rabbits [29]. Later, a novel myocardial infarctionprone WHHLMI colony was selected, in which the cumulative incidence of spontaneous myocardial infarction at the age of 30 months has increased from 23 to 97% [30]. In fact, coronary atherosclerosis can be detected at the age of 2 months and the lesions are mainly composed of macrophage-derived foam cells. With aging, the coronary plaques are expanded and cause severe lumen stenosis. The histological features of macrophage-rich fatty streaks at the early stage are gradually ­transformed into a typical fibrous plaque, fibroatheromatous plaque or unstable plaque. These coronary lesions have several histological features ­resembling those of human ­coronary plaques [31]. For example, coronary plaques, indeed, contain a large lipid core covered by thin fibrous caps ­accompanied by an accumulation of macrophages and foam cells. Some plaques are associated with intraplaque hemorrhage or ­calcification. Furthermore, these macrophages in the lesions express high levels of MMP-1, MMP-2, MMP-9 and MMP-12, membrane type-1 MMP, tissue factor and interleukin-1, suggesting that these coronary lesions mimic typical human ­vulnerable plaques. However, although these coronary plaques bear many ­histological similarities to those in humans and myocardial infarction occurs in almost all WHHLMI rabbits, ­pathological examinations failed to reveal coronary plaque rupture as seen in humans [6]. For these reasons, WHHLMI rabbits may become an important means for elucidating the possible mechanisms of plaque rupture by exposing the plaques to additional risk factors beyond hyperlipidemia. It is worthwhile to point out the insight gained from myocardial infarction-prone WHHLMI rabbits for the study of human acute coronary syndromes [6].

2.2 Murine Models of Atherosclerosis Mice are highly resistant to atherosclerosis. The only exception in mice is the C57BL/6 strain that develop atherosclerosis after the intake of 15% fat, 1.25% cholesterol and 0.5% cholic acid [32]. However, this ­unphysiological and inflammatory diet induces very small lesions at 4–5 months of age and it does not resemble the pathology of human atherosclerosis [33]. A transplantation-based mouse model of atherosclerosis regression has been developed by allowing plaques to form in a model of human atherosclerosis, the apo E-deficient mouse, and then placing these plaques into recipient mice with a normolipidemic plasma environment. Under these conditions, the depletion of foam cells occurs. Interestingly, the disappearance of foam cells was primarily due to migration in a CCR7-dependent manner to regional and systemic lymph nodes after 3 days in the normolipidemic (regression) environment. Further studies using this transplant model demonstrated that liver X receptor and HDL are other factors likely to be

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involved in plaque regression. Through the use of this transplant model, the process of uncovering the pathways regulating atherosclerosis regression has begun, which will ultimately lead to the identification of new therapeutic targets [34].

2.3 Genetically Modified Animals The challenge lies in bridging the gap between genetically friendly small animal models and human-like bigger animal models. Sequencing of the mouse and human genome, development of a single nucleotide polymorphism (SNP) database and in silico quantitative trait loci (QTL) linkage analysis may enhance the understanding of atherosclerosis and help develop new therapeutic targets [35]. The animal model most commonly used in the study of atherosclerosis is the apo E knock-out (apo E−/−) mouse, which has been used to study the role of different genes after genetic manipulation. These animals do not express apo E and develop hyperlipidemia, hypercholesterolemia, hypertriaclyglycerolemia, low levels of HDL, and atherosclerosis after feeding on either a normal or a fat-rich diet; the severity of the disease depends on the strain of the animals [36]. Another ­characteristic found in lesions of these apo E−/− is the presence of oxidation ­specific epitopes [37]. The sequential events involved in lesion formation have been described to be similar to those found in larger animal models and human ­atherosclerosis [38]. However, evidence of plaque rupture followed by formation of occlusive thrombi or thromboembolism has not been detected in these mice as had been described in human atherosclerotic lesions [36]. A selective injury of plaques in apo E−/− animals produced platelet adhesion to subendothelial tissues and thrombus formation containing lipid debris in foam cells and fibrin. This simple procedure, in which direct plaque injury is allowed to ­interact with flowing blood and cellular components, could be extended to ­investigate the relative contribution of various elements to thrombogenesis and ­cellular proliferation [39]. The complexity of lesions in the apo E−/−, together with the benefits of using the mouse as a model of human disease, makes it a desirable system in which to study both environmental and genetic determinants of atherosclerosis [33]. LDL receptor deficiency found in knock-out mouse (LDL−/−) slightly increases plasma cholesterol (LDL and VLDL cholesterol) that does not generate ­atherosclerosis. However, these animals are very susceptible to dietary changes that modified atherosclerotic phenotype and, subsequently, feeding then a fat-rich diet enhances hypercholesterolemia and leads to atherosclerosis [40]. Specifically, the lesion pathology in this model is not as well characterized as in the apo E−/− model, but it seems that the lesions can progress beyond the foamcell fatty-streak stage to the fibro-proliferative intermediate stage [33]. In addition, the doubleknockout model apo E−/− and LDL−/− develops severe hyperlipidemia and atherosclerosis, and in this case is also accelerated with fatty diets [40]. Another murine model of atherosclerosis is based on the genetic replacement of a specific gene (knock-in). Knock-in mice expressing human apo E2 show a

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plasma lipoprotein profile similar to type III hyperlipidemic subjects. These individuals have lower VLDL clearance, and thus develop atherosclerosis in spite of a normal diet [41]. In addition to knock-out and knock-in mouse models, there are a number of transgenic animal models. Most of them consist on the introduction of the human gene of apo E or a mutated form of the gene, such as apo E2k or apo EE3-Leiden [42–44]. A number of apo E isoforms have been expressed in mouse for the study of their influence on the development of atherosclerosis [45]. These models have good potential since they respond very well to dietetic and pharmacological factors. On the other hand, murine strain defective in platelet function and the development of in  vivo models for the determination of atherosclerotic physiology and pathophysiology have allowed new tools for the identification of the individual role of platelet proteins in the adhesion, activation, secretion and procoagulant activity [46]. The mutations ABCA1, ATP-binding cassette transporter A1, result in Tangier disease, in which cholesterol esters are accumulated in tissues and plasmatic levels of HDL are very low, increasing the risk of cardiovascular disease. These transporters play a crucial role in the cellular phospholipids and cholesterol traffic. Mice and chicken knock-out models for this transporter develop symptoms of Tangier disease. Thus, transgenic animals with the human gene restore cholesterol metabolism in these mutants [47].

2.4 Other Experimental Models of Cardiovascular Diseases The development of atherosclerosis is also possible by inducing an intraluminar mechanical damage in the aorta. It generates an endothelial proliferative response similar to the pathophysiology of the atherosclerotic process [48]. Fernandez and Volek [9] have shown that guinea pigs may be considered to be a suitable model for the study of the influence of dietary restriction on lipoprotein metabolism, since, as in humans, most of their cholesterol is associated with LDL and they have the cholesterol ester transfer protein and the lipoprotein lipase. Lacunar ischemic stroke accounts for 25% of all ischemic strokes, but the exact etiology is unknown. Numerous pathophysiological features have been proposed, including atheroma and endothelial dysfunction. Bailey et al. [49] have recently revised the relevance of all available potential animal models of lacunar stroke. Some animal models produce small subcortical infarcts, but few mimic the human small vessel pathology. Models of small vessel disease could help to improve the understanding of human lacunar disease, particularly to clarify factors associated with the small vessel morphological changes preceding brain damage [49]. The study of mechanisms of action underlying the use of electrical neuromodulation for angina and myocardial ischemia may illuminate heart–brain interactions that influence these conditions. Dejongste et al. [50] have discussed the experimental models to unravel the heart–brain interactions involved in the use of electrical neuromodulation for ischemic disease.

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Finally, other animal models have been developed for the study of cardiovascular disease. Granada et al. [8] have reviewed some of the most promising porcine models, focusing on their applicability in the development and validation of ­coronary imaging technologies and interventional devices. Models simultaneously showing hyperlipidemia, inflammation and associated complications of diabetes and ­hypertension will serve the purpose better as they mimic the actual clinical ­condition [35]. All these animal models, as many others, also have advantages and disadvantages, mainly for the evaluation of nutritional and pharmacological treatments.

3 Experimental Models of Diabetes The increasing worldwide incidence of diabetes in adults constitutes a global ­public health burden. b-Cells are particularly weakly protected from free radical toxicity [51]. Reactive species contribute differently to the pathogenesis of b-cell destruction in type 1 diabetes, and b-dysfunction on type 2 diabetes [52]. Indeed, reactive ­nitrogen species (RNS) have been also shown to participate in type 2 diabetes [53]. Type 1 diabetes mellitus is characterized by a specific destruction of the pancreatic b-cells, commonly associated with immune-mediated damage, which finally progress to absolute insulinopenia. It has been reported that oxidative stress plays an important role in the pathogenesis of this disease [54]. In type 1 diabetes, proinflammatory cytokines induce different signaling cascades, affecting the rate of ROS and RNS generation and provoking an apoptotic program of b-cell death [55]. On the other hand, type 2 diabetes represents a heterogeneous group of disorders characterized by insulin resistance and impaired insulin secretion and defined by a raised fasting or postchallenge blood glucose. In this case, the causes of b-cell dysfunction is not fully understood, but glucolipotoxicity has been considered to be an important contributing factor [52, 56, 57]. Experimental models for the study of obesity, diabetes, hyperlipidemia and other metabolic complications would accelerate the ongoing process of finding a cure for these diseases. Surgical and toxin-mediated pancreatic damage are valuable tools in the study of the consequences of diabetic complications. In addition, genetically modified diabetic animals are also used for the study of this disease. One of the most straightforward ways of studying diabetes in animals is to remove the pancreas, either partially or totally. Firstly, this technique was applied in dogs, but smaller animals are preferred; rats and mice are the most commonly used [2]. These authors have reviewed the current status of the most commonly used rodent diabetic models developed spontaneously, through genetic engineering or artificial manipulation. In addition to these models, the Psammomys obesus, rhesus monkeys and many other species have been also used for this purpose [58–60]. The most important models of type 1 diabetes (i.e., streptozotocin or alloxan-induced diabetes) as well as type 2 diabetes (i.e., Goto-Kakizaki – GK-rats, obese Zucker rats and others genetically modified mouse models) are detailed below.

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3.1 Streptozotocin and Alloxan Murine Diabetes Models Streptozotocin and alloxan are the most prominent diabetogenic chemicals in ­diabetes research. Both are cytotoxic glucose analogs and can selectively damage the insulin producing pancreatic b-cells, resulting in hyperglycemia via different pathways [61]. These molecules are readily transported into pancreatic b-cells by GLUT-2, in which they cause alteration of glucose metabolism and b-cell toxicity, resulting in insulin deficiency [61]. Injection of alloxan and streptozotocin ­principally induce similar blood glucose and plasma insulin responses and cause an insulin-dependent type 1-like diabetes syndrome by different mechanisms [62]. Alloxan is a pyrimidine derivative that induces diabetes in animals as a result of two distinct pathological mechanisms leading to the specific necrosis of the ­pancreatic b-cells. Specifically, it acts through the inhibition of glucose-induced insulin secretion, by inhibiting glucokinase, and because of its ability to induce ROS formation [62]. Alloxan has been the agent with the greater impact upon the understanding of ROS mediated mechanisms of b-cell death in type 1 and 2 diabetes mellitus. In general, multiple doses of alloxan led to significantly higher mortality than did a single dose. Federiuk et al. [63] have tried to induce diabetes in rats by administrating different doses of alloxan. These authors have observed firstly, that when animals were given 120  mg/kg of alloxan intravenously (i.v.), about 70% died within the first 2 days. Secondly, some animals received multiple moderate-to-high doses of alloxan intraperitoneally (i.p.), with a first dose of 100–150 mg/kg followed by a second administration of the same dose 48 or more hours later. Of those animals, 96% developed diabetes: 72% developed type 1 and 28% developed type 2 diabetes mellitus. However, this alloxan regimen led to a mortality of 55% within the 1st week. Other animals received only a single moderate (100–150 mg/kg, i.p.). Among them, 80% developed type 1 and 20% developed type 2 diabetes. Finally, they limited alloxan administration to a single high dose of 200 mg/kg, i.p., causing diabetes in 80%, 87.5% of which was type 1, and 12.5% of which was type 2 [63]. On the other hand, streptozotocin is a nitrosourea derivative isolated from Streptomyces achromogenes with broad-spectrum antibiotic and antineoplastic activity. It inhibits insulin secretion and causes a state of insulin-dependent diabetes mellitus due to its specific chemical properties, namely its DNA alkylating activity that along a defined chain of events results in the fragmentation of DNA. Indeed, protein glycosylation may be an additional damaging factor contributing to the ­functional defects of the b-cells after exposure to streptozotocin [62]. Due to its chemical properties, in particular greater stability, streptozotocin is the agent of choice for reproducible induction of a diabetic metabolic state in experimental animals. There are a number of reliable protocols for establishing streptozotocin diabetes in mice [61]. Multiple low doses of streptozotocin, e.g., 40 or 55  mg/kg on 5 ­consecutive days, induce a suboptimal injury of pancreatic b-cells and progression of diabetes relies, in part, on a secondary autoimmune insulinitis, as in human type 1 diabetes. These protocols have been used extensively to study the immunological

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pathways that lead to insulinitis and b-cell death. However, the degree of diabetic complications will depend on the activity of streptozotocin and the susceptibility of the mouse strain to streptozotocin-induced pancreatic injury [64]. A single high dose of streptozotocin or two doses given on consecutive days (>200 mg/kg or 2 × 100 mg/kg, respectively) can produce diabetes in rodents, probably as a result of direct toxic effects. Increasing the streptozotocin dosage causes greater cytotoxicity and more rapid destruction of pancreatic b-cells, resulting in a higher incidence and severity of diabetes. However, high dosages have nonspecific cytotoxicity, making it difficult to interpret findings [61]. Chen and Wang [65] have revised the different varieties of streptozotocin-treated rodents.

3.2 Neonatal Streptozotocin-Induced Diabetes Administration of streptozotocin to neonatal rats (2 days postpartum) produces a type 2 diabetes mellitus model in adulthood. At the 4th week, glycemia is normal or mildly elevated due to b-cell regeneration. However, hyperglycemia develops progressively with age, and by the 12th week, marked impairment of glucosestimulated insulin release could be observed [66].

3.3 Streptozotocin-Spontaneously Hypertensive Rat Two-day-old spontaneously hypertensive rats (SHRs) injected (i.p.) with streptozotocin develop a hyperglycemic syndrome, associated with other biochemical, morphological and hemodynamic properties that, to some extent, involve insulin resistance combined with hypertension, which replicates the natural history of patients with type 2 diabetes [67]. Plasma glucose levels in streptozotocin-SHRs increase in a dose-dependent manner at the 12th week. During long-term observations, hyperglycemia persisted until the 28th week but later gradually ameliorated, accompanied by continued hypertension as measured at the 52nd week [68, 69].

3.4 Fat-Fed/Streptozotocin-Induced Diabetic Rodents Fat-fed/streptozotocin diabetic rodents, developed by combination of diet-induced insulin resistance and relatively-low-dose streptozotocin, provide a novel animal model for type 2 diabetes. It simulates the human metabolic syndrome and is suitable for experimental in vivo evaluation of antidiabetic agents [65]. In C57BL/6J mice, insulin resistance could be induced by diets enriched in either fructose or fat, and hyperglycemia is introduced by injecting a dose of streptozotocin that does not cause

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diabetes in chow-fed mice. Initially, insulin concentration increases, but then decrease to levels still higher than those in chow-fed mice following streptozotocin injection. Accompanied by this decrease in insulin levels after streptozotocin injection, fat- or fructose-fed C57BL/6J mice become significantly hyperglycaemic [70]. Another rodent model employs 7-week-old, nonobese, out-bred male Sprague– Dawley rats, fed a 40% fat-enriched diet for 2 weeks before injection with streptozotocin (50 mg/kg, i.v.). Before the injection, fat-fed rats have glucose concentrations similar to chow-fed counterparts and insulin resistance. Streptozotocin injection elevates glucose, insulin, free fatty acids and triacylglycerol concentrations. In addition, fat-fed/streptozotocin rats are not insulin deficient compared to normal chow-fed rats but display hyperglycemia associated with reduced insulin-stimulated adipocyte glucose clearance [71].

3.5 Zucker Diabetic Fatty Rats The inbred line of Zucker diabetic fatty rats was established in 1985 and developed to a genetic model in 1991. These animals present a mutation in the leptin receptor, which is the molecular base of their characteristic phenotype [65]. Male Zucker rats homozygous for nonfunctional leptin receptors (fa/fa) develop obesity, ­hyperlipidemia and hyperglycemia, but rats with homozygous dominant (+/+) and heterozygous (fa/+) genotypes remain lean and normoglycemic. Insulin resistance occurs in young fa/fa rats followed by evolution of an insulin secretory defect that triggers hyperglycemia [72]. Thus, the Zucker male rat has become an experimental model for type 2 diabetes, with a predictable progression from prediabetic to ­diabetic state [65]. Indeed, obese Zucker rats are the best known and most widely used animal model of genetic obesity and, at present, the more representative rat strain to study the metabolic syndrome, since they present changes similar to those seen in human metabolic syndrome [73]. A direct or indirect consequence of the lack of a leptin receptor-mediated counterregulation is that obese Zucker rats display markedly elevated circulating leptin levels compared with their lean counterparts [74]. Old classical orexigenic peptides such as neuropeptide Y (NPY), galanin, orexins and melanin-concentrating hormone are upregulated in obese Zucker rats [75]. This strain is characterized by an increased expression of ghrelin both at the peripheral and central levels, which could participate in the development of extra weight in the obese Zucker rats [76]. Subsequently, Zucker rats develop severe obesity associated with hyperphagia, defective nonshivering thermogenesis and preferential deposition of energy in adipose tissue. In addition, obese Zucker rats present a range of endocrinological abnormalities, dyslipidaemia, mild glucose intolerance, hyperinsulinemia, and insulin resistance, similar to those of human metabolic syndrome [73]. Hyperglycemia initially appears at about 7 weeks of age, and all obese male rats are fully diabetic by 12 weeks. Between 7 and 10 weeks, serum insulin levels are high but subsequently drop as pancreatic b-cells cease to respond to glucose stimuli [77]. In this diabetic model, GLUT-4 in the adipose

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tissue, heart and skeletal muscle is reduced by 25–55% [78]. Hyperinsulinemia is detectable at 3 weeks and persists throughout the animals’ lives, the islets of Langerhans’ hypertrophy moderately and increase in number. In addition, the animals present renal damage [79]. The increase in plasma triacylglycerols concentrations exhibited by obese Zucker rats is due to the accumulation of VLDL, and the increase in cholesterol to the increase in cholesterol in the VLDL and HDL fractions [80]. These rat glucose levels are in reality normal or only slightly higher than normal. Therefore, these animals are not the best model to study the effective treatments to control alterations of glucose homeostasis [73]. Nevertheless, some researchers have succeeded in identifying several vascular changes characteristic of diabetes in these rats [81]. In fact, Zucker rats may constitute an experimental model in which hypertension, partially justified by endothelial damage, was specifically associated with the genotype for obesity [82]. This endothelial damage caused by increased oxidative stress may interact with nitric oxide function [83]. Obesity is associated with a state of chronic inflammation, and, in this context, TNF-a, a proinflammatory cytokine, is overexpressed and likely mediates insulin resistance in Zucker rats [84]. Apart from the insulin resistance, Zucker rats have inadequate b-cell compensation. Studies demonstrate that the Zucker rat carries a genetic defect in b-cell transcription that is inherited independently of the leptin receptor mutation and insulin resistance. The genetic reduction in b-cell gene transcription in homozygous animals probably contributes to the development of diabetes in the background of insulin resistance [85]. In prediabetic Zucker rats, there is no change in insulin mRNA levels; however, gene-expression patterns in the islets of diabetic Zucker rats show marked alterations, including a decrease in insulin mRNA levels associated with reduced islet insulin secretion [86]. In Zucker rats, hyperphagia leads to hyperinsulinemia, which upregulates transcription factors that stimulate lipogenesis. This causes ectopic deposition of triacylglycerols in nonadipocytes, thereby providing fatty acid substrate for pathological nonoxidative metabolism, such as ceramide synthesis, leading to lipoapoptosis of b-cells [65]. Studies show that free fatty acid-induced suppression of insulin output in prediabetic Zucker rats is mediated by nitric oxide (NO), since free fatty acids induce a fourfold rise in NO, by the upregulation of mRNA of inducible NO synthase (iNOS), and reduced insulin output in cultured prediabetic Zucker islets [87]. These modifications result in an increase in the nitrogen-free radical peroxynitrite. Indeed, the generation of ROS may interact with NO function, leading to endothelial dysfunction [83]. For all these reasons, Zucker rats are generally applied to studies of diabetes with obesity and cardiovascular complications due to the dyslipidemia background [82].

3.6 Genetically Engineered Diabetic Mice Genetically engineered diabetic models include transgenic and knock-out mice developed for the study of the pathophysiological consequences of specific alterations in a

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single gene or in a set of candidate diabetogenes, especially the ­expression of key actors in insulin signaling, action or secretion [88]. These models allow investigations of gene interactions and environment influences in the development of type 2 diabetes, providing new insights of the pathogenesis of the disease [65]. For example, the nonobese diabetic (NOD) mouse and bio-breeding (BB) rats resemble type 1 diabetes, while a number of type 2 diabetic rodent models are more frequently used and the most important are highlighted forward. 3.6.1 Otsuka Long-Evans Tokushima Fatty Rats These diabetic rats are characterized by polyuria, polydipsia and mild obesity. Clinical and pathological features in Otsuka Long-Evans Tokushima Fatty Rats (OLETF) rats resemble those of human type 2 diabetes, especially the diabetic nephropathy [89]. OLETF rat obesity is secondary to the hyperphagia and may be the outcome of two regulatory disruptions. One depends upon a peripheral pathway secondary to the loss of sensitivity to cholecystokinin A (CCK-A), related to the lack in the expression of the receptor mRNA in the pancreas, small intestine and brain, which consequently leads to satiety signal delay. The other is related to a central pathway critical to overall energy balance influenced by the increased ­dorsomedial hypothalamus NPY [90]. Indeed, OLETF rat is an animal model of obesity with visceral fat accumulation, specifically in islets, and hyperlipidemia, reminiscent of the characteristics of human type 2 diabetes [91]. This characteristic allows the use of this experimental model for the study of supplementation with functional lipid on metabolic syndrome alterations [92]. The OLETF rat is a ­valuable model for characterizing actions of CCK in energy balance and weight control and has provided novel insights into interactions between exercise and food intake [93]. Together these data support the use of the OLETF rat as a model for examining obesity development and for investigating how interventions at critical developmental points in time can alter genetic influences on food intake and body weight [94]. In addition, one of the characteristics of these animals is the development of diabetic nephropathy [89]. It seems to be a consequence of the hyperglycemic-derived ROS that causes lipoperoxidation in glomeruli [95]. In fact, a NADPH oxidase inhibitor has been described to reduce oxidative stress and improve diabetic nephropathy by reducing proteinuria in OLETF rats [96]. 3.6.2 Goto-Kakizaki Rats The GK rat is a nonobese and normolipidemic rodent model of mild type 2 diabetes with early hyperglycemia, hyperinsulinemia and insulin resistance. In contrast to many other rodent models of noninsulin-dependent diabetes, GK rats do not exhibit hyperlipidemia or obesity. The neonatal b-cell mass deficit is considered to be the primary defect leading to basal hyperglycemia, which is detectable around

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3.5 weeks of age. After 8 weeks, hyperglycemia deteriorates and glucose-stimulated insulin release by the islets is more severely impaired [65]. Specific characteristics make this model an appropriate tool for identification of the mechanisms that increase the risk of abnormal b-cell mass/function and of type 2 diabetes. Careful comparison of the alterations so far detected in the type 2 diabetes human b-cell population and those found in the diabetic GK b-cell population, put a number of striking commonalities into the foreground. None of the available rodent models of spontaneous type 2 diabetes has revealed such close appropriateness for modeling the human diabetic b-cell. Although the GK b-cell is not a blueprint for the diseased b-cell in humans, there are, however, sufficiently valuable similarities to justify more efforts to understand the etiopathogenesis of type 2 diabetes in this rat model [97]. As in humans, GK rats develop renal lesions, structural changes in peripheral nerves and retinal damage [2]. Researches in GK rats have provided evidence about signals derived from glucose metabolism (e.g., changes in intracellular calcium, cyclic nucleotides, generation of biologically active lipids and increase in intracellular adenine and guanine nucleotides) and about implication of protein dephosphorylation as the major regulatory steps involved in glucose-stimulated insulin secretion [98]. GK rat islets have increased mRNA expression of markers of early islet endothelial cell activation and also some defense mechanisms, specifically interleukin-1. Indeed, counteracting islet endothelial cell inflammation and oxidative stress might be one way to prevent b-cell dysfunction in type 2 diabetes [99]. Hyperglycemia in the GK rat is associated with the development of agedependent renal structural changes, such as segmental glomerular sclerosis and tubule-interstitial fibrosis that are similar to those described in patients with prolonged type 2 diabetes who have not developed renal disease, although albuminuria or progressive nephropathy does not result [100]. Identification of the mediators involved in the above processes and in particular of the conditions that will determine progression of subclinical morphological changes to nephro­pathy and renal failure will likely result in future novel therapeutic approaches to diabetic nephropathy. Diabetic retinopathy is a consequence of oxidative stress and advanced glycation end-product-derived apoptosis. GK rat appears to be a suitable model for experimental studies on initial or latent-phase diabetic retinopathy, but also for the assessment of the antiapoptotic beneficial effect of antioxidants (a combination of vitamins C and E) or inhibitors of advanced glycation [101]. The major inconvenience of this model is that there are some strains with different phenotypes that may exhibit differential characteristics. For this reason it is very important to take this into account when results are discussed. 3.6.3 db/db (C57BL/KsJ-db/db) Mice The diabetes db gene mutation occurred spontaneously in the leptin-receptordeficient C57BL/KsJ strain of mice from Bar Harbor, ME, USA [102]. In diabetic

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mutant db/db mice, progressive impairment of glucose-induced insulin response is ­observable with increasing age. While plasma levels of insulin and glucagon in db/db mice, which peaked at 7 weeks of age, do not reflect the state of hyperglycemia, the glucagon/insulin ratio (G/I) is roughly in parallel with the development of hyper­glycemia [103]. This experimental model has been used for the investigation of antiglycemic agents, acting via GLP-1, and drugs improving insulin resistance [65]. Recently, Mariappan et al. [104] have found that NF-kappaB activity in db/db mice is associated with increased oxidative stress and cardiac dysfunction in type 2 diabetes. This study demonstrates that downregulation of increased ROS may restore cardiac function. Finally, the db/db mouse is the best model of experimental diabetic nephropathy. These animals display substantial glomerular pathology, including mesangial matrix expansion, modest albuminuria, and a decline in creatinine clearance after 5 months of age [105]. Experimental data point to a role of augmented insulin ­receptor activity in nephropathy of type 2 diabetes [106]. 3.6.4 ob/ob (C57BL/6J-ob/ob) Mice In the C57BL/6J-ob/ob mouse developed at the Jackson Laboratory in Bar Harbor, ME, USA [107] the ob gene was transferred from the stock of origin onto the B/6 genomic background and is located on chromosome 6. This is a model of severe obesity, insulin resistance, and diabetes caused by leptin deficiency [65]. Despite the degree of obesity and hyperinsulinemia, C57BL/6J ob/ob mice had milder hyperglycemia and, surprisingly, normal circulating adiponectin levels despite stillprominent signs of insulin resistance, mainly in the liver. C57BL/6J ob/ob mice had also a rapid clearance of circulating triacylglycerols and severe hepatic steatosis [108]. NPY, a neuromodulator implicated in the control of energy balance, is overproduced in the hypothalamus of ob/ob mice and has been associated with obesity [109]. These animals have 70% higher triacylglycerol concentrations ­compared to ob/+ mice. These observations indicate that obesity-induced type 2 diabetes in mice increases the secretion of triacylglycerol-rich lipoproteins [110]. This animal model has been employed for the study of implications of GLP-1 [111], GIP [112], PPARg [113] and PTP1B [114] in diabetes and associated obesity. Sonta et al. [115] have suggested that vascular NADPH oxidase may be a major source of increased oxidative stress in diabetes and obesity in ob/ob mice, while Boudina et al. [116] have indicated that reduced mitochondrial oxidative capacity may contribute to cardiac dysfunction in ob/ob mice. In conclusion, there are numerous experimental models of oxidative stress related to cardiovascular disease and diabetes that mimic most of the metabolic and molecular alterations associated with those diseases, including the impairment of the oxidative status. However, the ideal models are still lacking and further ­investigations are needed to find new models that closely resemble human oxidative stress-mediated diseases.

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Part II

Cardiovascular

Arachidonic Acid Metabolism and Lipid Peroxidation in Stroke: Alpha-Tocotrienol as a Unique Therapeutic Agent Cameron Rink, Savita Khanna, and Chandan K. Sen

Abstract  Under normal physiological conditions, the human brain has one of the highest metabolic profiles of all organs, using 25% of glucose and 20% of all oxygen consumed by the body. When challenged by metabolic disruption as in ischemic or hemorrhagic stroke, brain tissue that is enriched with arachidonic acid (22:6n − 3 polyunsaturated fatty acid) is highly susceptible to oxidative stress. The consequence of increased generation of radical species in stroke-affected brain tissue is the uncontrolled oxidative metabolism of arachidonic acid, generating a host of secondary products that are culpable neuromodulators of the cell death cascade. In this chapter, preclinical models of ischemic and hemorrhagic stroke injury are explored. Subsequently, the arachidonic acid cascade is examined as a common pathological contributor of oxidative stress in both aforementioned stroke subtypes. Finally, the unique neuroprotective properties of the natural vitamin E alpha-tocotrienol are discussed as a potent intervention of the stroke-induced arachidonic acid cascade. Keywords  Alpha-tocotrienol • Arachidonic acid • Ischemia • Lipid peroxidation • ROS • Stroke • Vitamin E

1 Introduction Worldwide, 15,000,000 people suffer stroke every year [1]. Of these, stroke claims the lives of 5,000,000, while another 5,000,000 are left permanently disabled [1]. In the United States alone, stroke is the third leading cause of death and the leading cause of serious long-term disability with ~795,000 Americans afflicted by a new or recurring stroke event each year [2]. Stroke is broadly defined by a cerebrovascular disruption of blood supply to brain tissue which is either ischemic or hemorrhagic

C.K. Sen (*) Department of Surgery, The Ohio State University Medical Center, Columbus, OH 43210, USA e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_3, © Springer Science+Business Media, LLC 2011

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in origin. Stroke pathology of ischemic origin accounts for 87% of all strokes subtypes presented clinically, while intracerebral and subarachnoid hemorrhagic constitute the remainder (10% and 3% respectively) [2]. Despite substantial progress in the past decade in medical imaging and clinical diagnosis of stroke, potential stroke therapeutics have failed to translate from the laboratory to the clinic. Intravenous tissue plasminogen activator (tPA) remains the only U.S. FDA-approved pharmacological treatment available for acute ischemic stroke therapy. However, with a limited therapeutic window of £3 h from the onset of symptoms [3, 4], fewer than 3% of ischemic stroke admissions receive tPA treatment [5]. No therapeutic recourse for hemorrhagic stroke has met FDA approval and prospective neuroprotective agents have a long history of failure in clinical trials [6–8]. Emblematic of the limited availability for therapeutic intervention is a 20% mortality rate for stroke patients during the acute phase. Long-term prognosis of stroke survivors is poor as well – 40% do not recover independence with selfcare and 25% are unable to walk independently [9]. Given a growing life expectancy and aging population in developed nations, stroke incidence is projected to rise for decades to come. Indeed, while the high incidence, mortality, and morbidity associated with stroke contribute to substantial public awareness and scientific interest across many fields of study, little therapeutic progress has been achieved. An underlying hallmark of stroke pathology is the generation of reactive oxygen species (ROS) that induce oxidative damage to cellular proteins, lipids and DNA – a process collectively referred to as oxidative stress. The consequence of oxidative stress in stroke-affected brain tissue is an accumulation of dysfunctional proteins, lipid peroxidation products, and damaged nuclear or mitochondrial DNA, which culminate in cell death. Importantly, oxidative stress is well established in stroke pathologies of both ischemic and hemorrhagic origin; however, the etiological differences of stroke subtypes contribute to different pathological patterns of oxidative stress. This chapter outlines the etiology and oxidative stress patterns associated with both ischemic and hemorrhagic stroke. Methodological approaches as well as strengths and weaknesses associated with models of both stroke subtypes are explored. The oxidative metabolism of arachidonic acid is examined in great detail as a common pathological contributor of oxidative stress in both ischemic and hemorrhagic stroke. Finally, therapeutic management of oxidative stress in stroke is discussed in light of the historic failure of neuroprotective agents in stroke clinical trials. Alpha-tocotrienol, a lesser characterized natural vitamin E isomer, is discussed as a promising therapeutic agent in the intervention of the arachidonic acid cascade.

2 Ischemic Stroke (87% of all stroke cases) The etymology of the word ischemia is derived from the Greek words ischaimos which means “to restrain” and haima meaning “blood.” In simple terms, ischemic stroke occurs when an artery of the brain is blocked. The brain depends on continuous blood flow to deliver oxygen, nutrients (i.e., oxygen, glucose), and to remove carbon

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dioxide and cellular waste. When brain tissue is unable to maintain homeostasis and subsequent trafficking of essential lipids, proteins, nutrients and waste, the terminal result is energetic failure (ATP depletion) and cell death. Ischemic stroke has several etiological origins. The most common is due to the narrowing of the arteries in the neck or head. This is most often caused by atherosclerosis – a chronic disease process directly influenced by diet. High fat diets leading to elevated LDL cholesterol and triglyceride levels are significant risk factors for atherogenesis. If cerebrovascular arteries become too narrow, blood cells collect and form blood clots. These blood clots can block the artery where they are formed (thrombosis), or can dislodge and become trapped in smaller or more distant arteries of the brain (embolism). A thrombotic stroke occurs when diseased or damaged cerebral arteries become blocked by the formation of a blood clot within the brain. Clinically referred to as focal cerebral thrombosis or cerebral infarction, this classification of stroke is responsible for 50% of all clinically presented stroke cases. Cerebral thrombosis can also be divided into two additional categories that correlate to the location of the blockage within the brain: large-vessel thrombosis and small-vessel thrombosis. Large-vessel thrombosis is the term used when the blockage is in one of the brain’s larger blood-supplying arteries such as the carotid or middle cerebral artery, while small-vessel thrombosis involves one (or more) of the brain’s smaller, yet deeper penetrating arteries (Φ : 0.2–15 mm). This latter type of stroke is also referred to as a “lacunar” stroke event. An embolic stroke is also caused by cerebrovascular occlusion, but in this case the clot (or embolus) is formed in peripheral arteries outside of the cerebrovascular system. Often from the heart, an embolus will travel the bloodstream until it becomes lodged in the brain and cannot travel any further. This naturally restricts the flow of blood to the brain and results in almost immediate physical and neurological deficits. While thrombotic and embolic events are the most common cause of ischemic stroke, there are many other pathological events, including traumatic injury to the blood vessels of the neck, or disorders of blood clotting, which also induce ischemic stroke of the brain.

2.1 Patterns of Oxidative Stress in Ischemic Stroke The brain is an energy-demanding organ. Despite representing only 2% of total body weight, the human brain receives 15% of cardiac output and uses 20% of the oxygen consumed by the body [10, 11]. As hypermetabolic brain tissue requires a disproportionate share of oxygen and energy substrate for oxidative metabolism, the disruption of cerebral blood flow poses an immediate and significant threat to homeostatic function. Foremost, ischemic stroke induces energetic failure (ATP deficiency) due to the lack of substrate (both oxygen and glucose) available to maintain cellular respiration. Within 15 min of ischemic stroke onset, ATP levels of stroke-affected brain tissue decrease by one-third. As a result, ATP-dependent ion pumps (i.e., Na+/K+ ATPase) fail to maintain transmembrane ion gradients resulting

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in Ca2+ influx through voltage-sensitive Ca2+ channels. Ischemia-induced energetic failure in brain tissue also results in excessive extracellular glutamate release. Consequently, glutamate-mediated over-stimulation of ionotropic NMDA receptors continues to drive intracellular Ca2+ uptake. The subsequent rapid accumulation of intracellular Ca2+ results in activation of lipases (i.e., phospholipase A2) that contribute to membrane degradation and lipid peroxidation in stroke-affected brain tissue enriched with polyunsaturated fatty acids (PUFAs). Moreover, the intracellular Ca2+ influx also induces Ca2+ and calmodulin codependent activation of neuronal and endothelial nitric oxide synthase (NOS). NOS generates nitric oxide (NO) gas, a signaling molecule with well-characterized vasodilatory effects. The potential for NO to improve ischemic stroke-affected cerebral blood flow via vasodilation is clear; however, increasing evidence suggests that NO also contributes to oxidative damage during ischemic stroke. With one unpaired electron, NO reacts with most free radicals at near diffusion-limited rates [11]. Reaction of NO with superoxide radical (O2−) leads to the production of highly reactive peroxynitrite (ONOO−). As much as 2% of oxygen consumed by mitochondria is converted to superoxide anion under normal respiratory conditions [12, 13]. In a pathological setting of ischemic stroke, generation of superoxide is greatly enhanced and in combination with elevated NO levels contributes to a massive increase in peroxynitrite formation [14, 15]. In addition to evidence supporting peroxynitrite-mediated lipid peroxidation, peroxynitrite also exerts cytotoxic effects directly on mitochondria by inhibiting respiration at complexes I, II, III and V [16, 17].

2.2 Experimental Models of Ischemic Stroke Animal models of ischemic stroke have long focused on middle cerebral artery (MCA) occlusion on the basis of clinical data supporting as high as 75% of all ischemic stroke cases occur in the MCA territory [18]. Today, the intraluminal suture model of middle cerebral artery occlusion (MCAO) in rodents is the most frequently employed preclinical model of ischemic stroke (Fig.  1) [19]. First reported by Koizumi et al. in 1986 and later modified by Longa et al. in 1989, this model introduced a minimally-invasive small animal technique of acute focal ischemia and reperfusion [20, 21]. Several variations of techniques and approaches to induce MCAO in rodents have been published since the model’s inception; however, none have been as widely accepted [22–24]. Advantages of this model include the ability to conduct experiments using transgenic mouse lines, and fewer physical facility and surgical barriers as compared to large animal preclinical models. Despite the popularity of the intraluminal suture method, there are significant limitations to the approach that have been extensively documented in the literature. These shortcomings contribute to a relatively high degree of variability in lesion volume and location across and within studies [19, 25–30]. Foremost, real-time placement of the occluder in the MCA cannot be visualized. Consequently, overshooting or undershooting the MCA origin is a common occurrence that contributes

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Fig.  1  Intraluminal suture method of middle cerebral artery occlusion (MCAO) in rodents. (a) 1.0–1.5 cm ventral midline incision exposes the common carotid artery (CCA), internal carotid artery (ICA) and external carotrid artery (ECA). (b) The surgical field of view is limited to the bifurcation of the CCA into the ECA and ICA. The ICA supplies aspects of the Circle of Willis, including the MCA and anterior cerebral artery (ACA). The MCA perfuses the cerebral cortex. (c) The distal ECA is tied off to introduce a nylon monofilament suture (4-0 rat, 6-0 mouse) with a blunted tip. The suture is advanced from the ECA into the ICA until the MCA origin (18 mm rat, 8 mm mouse). MCA occlusion is verified by >70% drop in cortical blood flow as measured by laser Doppler flowmetry. The ischemic area at risk is overlaid in yellow. Following 90  min of occlusion, the suture is removed, restoring blood flow to model reperfusion

to variability in lesion volume outcomes. Laser Doppler flowmetry improves the accuracy of occluder placement; however, it provides relative cerebral perfusion at a single point-measurement, thereby making it difficult to assess whether the targeted MCA territory is fully occluded. Further contributing to the variability of stroke lesion volume in the intraluminal thread model is the potential for premature reperfusion of the MCA territory. This phenomenon has been documented in as high as 25% of experimental animals [19]. Finally, with the intraluminal suture tip occluding the proximal MCA, the remaining suture in the internal carotid artery may unintentionally induce subcortical ischemic stroke lesions as well [31]. Large animal preclinical models hold several key advantages when compared to rodent stroke models. First, the size and anatomical feature set of a large animal brain more closely mimics that of humans as compared to rodent models. Canines, for example, have a highly evolved gyrencephalic neocortex with a white to gray matter ratio that closely approximates humans [32, 33]. Second, large animal vascular architecture accommodates an array of endovascular devices and interventional radiologic techniques permitting a minimally invasive approach to inducing stroke with real-time visualization of occlusion. Leveraging these advantages, a recently developed canine model of endovascular MCA occlusion and reperfusion reports a minimally invasive approach with high reproducibility [34]. This approach benefits from real-time visualization of MCAO using c-arm fluoroscopy, and clinically relevant high-resolution MR imaging (Fig. 2). Alternatives to endovascular MCAO

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Fig.  2  A minimally invasive endovascular approach to MCAO in a large animal setting. The canine Circle of Willis is accessed via microwire, guided by c-arm fluoroscopy originating from a femoral artery puncture. (a) The M1 segment of the right middle cerebral artery (RMCA) is occluded with an embolic coil. (b) Contrast injection of the RICA under c-arm fluoroscopy demonstrates the coil-occluded segment of the RMCA as compared to the LMCA. (c) Two seconds later, contrast agent has perfused into smaller capillary vessels. The LMCA territory blushes normally while the ischemic RMCA territory is not patent, defining the stroke area at risk. The occluder is left in place for 60  min to produce acute focal ischemia of the RMCA territory. Following 60 min, the embolic coil is retrieved to model reperfusion. (d) At 24 h, magnetic resonance imaging (MRI) using a clinical 3T scanner reveals the stroke-affected lesion volume from which infarct volume is calculated. MRI slices are stacked and rendered in an oblique 3D view to enhance visualization of the stroke-induced lesion (red). Oblique cross-sections (e and f ) reveal the size of the cortical lesion (red) in a single MRI slice as compared to the remaining unaffected tissue (green)

in large animal systems include transorbital MCA occlusion via vessel clamp [35–39]. While variants of such models have reported highly reproducible infarct volumes, they necessitate an invasive approach including removal of the orbital globe, transection of the optic nerve and opthalmic artery, and craniectomy of the posteromedial orbit [37]. Thus far, the effects of the orbital wound and traumainduced inflammatory response have not been resolved separately from the strokeinduced pathology. The craniectomy approach to model stroke in a preclinical setting is also associated with severe head trauma, changes in intracranial pressure, and cerebrospinal fluid loss [40, 41]. Because in humans stroke is not necessarily associated with head trauma, the trauma aspect of invasive surgery may be viewed as a major confounding factor.

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In general, large animal models of ischemic stroke also pose additional ethical, veterinary and housing considerations that obligate larger fiscal and personnel requirements when compared to rodent stroke models [42, 43]. Consequently, the selection of experimental stroke model and animal system used should account for the study objective. Small animal (rodent) models remain powerful tools for studying biological mechanisms of ischemic stroke due to the availability of transgenic lines and a wide array of molecular biology tools (i.e., rodent antibodies). In contrast, large animal stroke models are essential for generating proof-of-concept experiments for potential stroke therapeutics prior to moving to a clinical setting.

3 Hemorrhagic Stroke (13% of all stroke cases) Intracerebral hemorrhage (ICH) occurs when a vulnerable blood vessel within the brain bursts, allowing blood to leak inside the brain. The sudden increase in pressure within the brain can cause damage to the brain cells surrounding the blood. If the amount of blood increases rapidly, the sudden buildup in pressure can lead to unconsciousness or death. ICH usually occurs in selective parts of the brain, including the basal ganglia, cerebellum, brainstem, or cortex. The most common cause of ICH is high blood pressure (hypertension). Since high blood pressure itself is asymptomatic, many people at risk for ICH are not aware that they have high blood pressure, or that it needs to be treated. Less common causes of ICH include trauma, infection, tumors, blood clotting deficiencies, and abnormalities in blood vessels. Subarachnoid hemorrhage (SAH) occurs when a blood vessel just outside the brain ruptures and the area between the arachnoid membrane and the pia mater rapidly fills with blood. A patient with SAH may have a sudden, intense headache (referred to as a thunderclap headache), neck pain, and nausea or vomiting. SAH may arise spontaneously or due to trauma with the sudden buildup of pressure outside the brain causing rapid loss of consciousness or death. SAH is most often caused by cerebral aneurysms – small areas of rounded or irregular swellings in the arteries. Where the swelling is most severe, the blood vessel wall becomes weak and prone to rupture.

3.1 Patterns of Oxidative Stress in Hemorrhagic Stroke In addition to a disruption of cerebral perfusion which by itself imposes similar patterns of oxidative stress as compared to ischemic stroke, hemorrhagic stroke also incorporates patterns of oxidative stress associated with iron-mediated free radical injury. Specifically, the rupture of cerebral blood vessels and subsequent red blood cell lysis results in release of heme in hemorrhagic stroke-affected tissue. Heme is metabolized by heme oxygenase (HO) in the brain into iron, carbon monoxide, and biliverdin [44]. Following heme degradation by HO, free iron concentration can

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reach as high as 10  mmol/L, which induces significant brain edema and directly contributes to free radical formation and lipid peroxidation [45]. Free iron greatly enhances hydroxyl radical production and subsequent lipid peroxidation via the Fenton reaction [46, 47]. Furthermore, as iron deposition in brain tissue increases with age, iron-mediated free radical formation, lipid peroxidation and neurotoxicity become increasingly relevant in hemorrhagic stroke pathology [48]. This is particularly noteworthy given the elevated risk of a cerebral stroke event with age, and the risk for intracranial bleeding in ischemic stroke cases – a phenomenon termed “hemorrhagic transformation” that occurs in as many as 70% of all ischemic stroke patients [49, 50]. Finally, carbon monoxide produced by HO-mediated heme metabolism also amplifies generation of reactive oxygen species in brain mitochondria [51], thereby further contributing to elevated oxidative stress under hemorrhagic stroke conditions.

3.2 Experimental Models of Hemorrhagic Stroke Traditionally, preclinical models of ICH and SAH have focused on a stereotaxic approach to recreate the human condition of hemorrhagic stroke either through clostridial collagenase-induced hemorrhage or autologous blood injection [52]. Both experimental approaches present distinct advantages and disadvantages and have been applied across a broad range of species, from rodents to primates. Collagenases catalyze the hydrolysis of basal lamina (collagen type IV), and by direct injection into neocortex, provide site and dose-dependent control of the hemorrhagic volume via deterioration of cerebral capillaries [53, 54]. A key advantage of this model is the induction of reproducible size and shape of the hematoma [55]. However, as compared to the autologous blood infusion model, clostridial collagenaseinduced hemorrhage triggers an exacerbated inflammatory response, due to the bacterial origin of collagenase [53]. As the inflammatory cascade plays a critical role in the evolution and resolution of ICH and SAH pathology [56, 57], enhanced inflammation due to the use of clostridial collagenase represents a substantive shortcoming of this approach. Autologous blood injection creates reproducible size and specific site hematomas that do not leak into cellular compartments outside of the injection location, i.e., intraventricular, subarachnoid or subdural space [55]. An overt disadvantage of this approach, however, is that it does not emulate vessel rupture as the etiology of the hemorrhagic stroke event. Furthermore, duration of injection and time to reach the desired hematoma volume is significantly shorter than the events leading to spontaneous ICH and SAH in humans [55, 58]. Finally, a mechanical method of SAH has been proposed as an extension of the intraluminal thread model of MCAO in which the monofilament occluder is intentionally passed beyond the MCA origin to puncture the intracranial ICA bifurcation [55, 59, 60]. In this model, SAH intensity has been shown to correlate with filament size [59]. This endovascular approach of SAH improves upon previously described collagenase and autologous clot approaches by negating the effects of stereotaxic-, needle-, and

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injection-associated trauma. However, the site of SAH hemorrhage is limited due to the restricted endovascular approach via the ICA.

4 Brain Oxygen, Lipid Metabolism, and Oxidative Stress Regardless of stroke mechanism, brain tissue is highly susceptible to oxidative stress as it (1) consumes an inordinate amount of oxygen to meet high metabolic demands; (2) contains high concentrations of polyunsaturated fatty acids (PUFAs) that are vulnerable to lipid peroxidation; and (3) has lower antioxidant capacity as compared to other organ systems. Neurons are particularly vulnerable to oxidative damage in stroke-affected brain tissue due to lower levels of the endogenous antioxidant glutathione as compared to resident glial cells [61]. The consequence of increased generation of free radical species in stroke-affected brain tissue enriched with PUFAs is an accumulation of lipid peroxidation products that have proven to be culpable neuromodulators of the cell death cascade. Brain tissue is highly enriched with the n − 6 PUFA arachidonic acid (AA, 20:4n − 6) and the n − 3 PUFA docosahexaenoic acid (DHA, 22:6n − 3), which are major components of phospholipid membranes. Together, AA and DHA account for ~20% of all fatty acids in the mammalian brain [62]. Both AA and DHA are nutritionally essential to brain function and structure during early development [63–68], and influence membrane fluidity, signal transduction, and gene transcription throughout life [67, 69–71]. Neither AA or DHA are synthesized de novo, but are obtained from the diet and circulated to the brain directly in the plasma [67], or elongated from n − 6 and n − 3 PUFA linoleic (18:2n − 6) and alpha-linolenic acid (18:3n − 3) precursors in the liver [72]. A number of pathological conditions in the human brain are associated with disturbed PUFA metabolism of AA [73, 74], including stroke.

4.1 Arachidonic Acid Metabolism in Brain In resting cells, AA is stored within phospholipid membranes, esterified to glycerol. A receptor-dependent event, requiring a transducing G-protein coupled receptor, initiates phospholipid hydrolysis and releases AA into the intracellular space. Three enzymes mediate this deacylation reaction: phospholipase A2 (PLA2), phospholipase C (PLC), and phospholipase D (PLD), each with different sites of attack on the phospholipid backbone. PLA2 catalyzes the hydrolysis of phospholipids at the sn-2 position, releasing AA in a single-step reaction. By contrast, PLC and PLD do not release free AA directly. Rather, they generate lipid products containing arachidonate (diacylglycerol and phosphatidic acid, respectively), which can be released subsequently by diacylglycerol- and monoacyl­ glycerol-lipases.

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4.2 Phospholipase A2 Phospholipase A2 (PLA2) isozymes encompass a diverse family of at least 15 different isozyme groups [75] classified into five distinct categories (1) secreted small molecular weight sPLA2, (2) larger cytosolic calcium-dependent cPLA2, (3) calciumindependent iPLA2, (4) platelet-activating factor acetylhydrolases (PAFA), and (5) lysosomal PLA2 isozymes. The entire PLA2 family is characterized by a common function – the enzymatic hydrolysis of the sn-2 ester bond of glycerophospholipids – thereby producing a free fatty acid (i.e., AA) and lysophospholipid (i.e., lysophosphatidylcholine, LPC). Currently, only sPLA2 and cPLA2 have welldefined roles in stroke-mediated AA metabolism [76] and are therefore the focus of discussion here. Under conditions of stroke in which reactive oxygen and nitrogen species are abundant, there occurs a rapid accumulation of free fatty acids, due to increases in intracellular Ca2+ and activation of PLA2s [77–86]. The sPLA2s are characterized by the requirement of histidine in their active site, Ca2+ for catalysis and the presence of six conserved disulfide bonds [75]. Under ischemic stroke conditions, sPLA2 mRNA and protein expression is significantly upregulated [85, 87] and activity is induced by inflammatory cytokine tumor necrosis factor-alpha (TNF-a) [88]. The cPLA2s are the only PLA2s that demonstrate a preference for AA in the sn-2 position of phospholipids [89]. Localized predominantly in gray matter [90], they lack the disulfide bonding network of sPLA2s and function through the action of a serine/aspartic acid dyad [75]. Following ischemic stroke, cPLA2 subunit mRNA and protein expression is elevated [79, 83]. Stroke-induced intracellular Ca2+ accumulation mediates cPLA2 subunit translocation to the membrane phospholipid bilayer [91] and activity is induced by phosphorylation of serine residue 505 by mitogen-activated protein kinase [92–94]. In addition to AA, glycerophospholipid metabolism by PLA2s following stroke also elevates LPC levels in blood, cerebrospinal fluid, and brain tissue following ischemic and hemorrhagic stroke [86, 95–97]. LPCs act as potent mediators of inflammation in the brain following stroke via stimulated release of interleukin 1b [98] and subsequent activation of microglia [99, 100]. Once released, free AA has three potential fates: reincorporation into phospholipids, diffusion outside the cell, and metabolism. In a pathological setting, such as ischemic and hemorrhagic stroke, free AA accumulates and undergoes uncontrolled oxidative metabolism by both enzymatic and nonenzymatic processes (Fig.  3). This uncontrolled metabolism, referred to as the “arachidonic acid cascade,” includes the formation of prostaglandins, leukotrienes, thromboxanes, isoprostanes and nonenzymatic lipid peroxidation products [101]. The arachidonic cascade amplifies the overall production of free radicals, both reactive oxygen and nitrogen species, and subsequently oxidative damage to lipids, proteins and nucleic acids. Taken together, the release of AA by PLA2 and downstream meta­ bolism plays a significant role in oxidative tissue injury following ischemic and hemorrhagic stroke.

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AA in cell membrane (phosphatidylcholine)

AA release (i.e. cPLA2)

TCT

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enzymatic oxidative lipid metabolism

initiating radicals

i.e. Fe2+, LOO◊-, LO◊-,◊OH, ONOO-

. lipid radical (LO -)

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Fig. 3  Oxidative metabolism of arachidonic acid following ischemic stroke. Following release from the lipid membrane bi-layer by phospholipase A2 (PLA2), arachidonic acid (AA) undergoes oxidative metabolism under enzymatic or non-enzymatic mechanisms. Abbreviations: MDA malondialdehyde, 4HNE 4-hydroxynonenal, PGG2 prostaglandin G2, PGH2 prostaglandin H2, PGD2 prostaglandin D2, PGI2 prostaglandin I2, TXA2 thromboxane A2, PGF2a prostaglandin F2a, 5-LOX 5-lipoxygenase, 12-LOX 12-lipoxygenase, 15-LOX 15-lipoxygenase, 5-HPETE 5-hydroperoxyeicosatetraenoic acid, 12-HPETE 12-hydroperoxyeicosatetraenoic acid, 15-HPETE 15-hydroperoxyeicosatetraenoic acid, LTA4 leukotriene A4. Independent of antioxidant function common to all vitamin E isoforms, alpha-tocotrienol (TCT) is a potent inhibitor of PLA2 and 12-LOX at nanomolar concentrations

4.3 Nonenzymatic Oxidative Lipid Metabolism Beyond the initial damage to lipid membranes, reaction of free radical species with double bonds of PUFAs produce alkyl radicals, which in turn react with molecular oxygen to form a peroxyl radical (ROO˙). Peroxyl radicals can abstract hydrogen from adjacent PUFAs to produce a lipid hydroperoxide (ROOH) and a second alkyl radical, thereby propagating a chain reaction of lipid oxidation [102]. Lipid peroxides degrade and give rise to a,b-unsaturated aldehydes that include 4-hydroxynonenal (4HNE)m malondialdehyde (MDA), and acrolein [103–106]. These aldehydes covalently bind to proteins through reaction with thiol groups and alter their function. They also react with amino groups to form cyclic adducts. The best characterized of the lipid peroxide aldehydes is 4HNE, a nine-carbon a,b-unsaturated aldehyde that at low concentration modulates cellular signaling in brain tissue. Under pathological conditions such as stroke, 4HNE-mediated protein carbonylation induces a number of deleterious effects in cells including inhibition of nucleic acid synthesis, elevation of intracellular calcium, and inhibition

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of mitochondrial respiration [107]. The localized concentration of 4HNE may increase to as high as 4.5  mM within the phospholipid bilayer, causing protein cross-linking of essential membrane transporters (i.e., glucose and glutamate transporters, sodium/potassium ATPases) [108–110]. The modification of adenine nucleotide translocation by 4HNE also suppresses ADT and ATP transport through the inner mitochondrial phospholipid membrane. Taken together, these actions significantly attenuate the energy-producing capacity of mitochondria in tissue already devastated by stroke-induced energetic failure. The C-3 position of 4HNE is highly reactive, undergoing Michael addition reaction with cellular thiol. This leads to the formation of adducts with endogenous antioxidant glutathione, and ultimately efflux from the cell via glutathione conjugate transporter RLIP76 [111]. 4HNE-mediated inactivation of thioredoxin and thioredoxin reductase through modification of cysteine and selenocysteine residues at the active site has also been linked to dysregulation of cellular redox status [112]. Reactive species attack on lipid hydroperoxides also results in the formation of isoprostanes via b-cleavage of the peroxyl acid and subsequent molecular rearrangement. Isoprostanes have D-, E- and F-ringed structures similar to cyclooxygenasegenerated prostaglandins, except that their hydrocarbon chains are in the cis position in relation to the pentane ring as opposed to the trans position observed in prostaglandins. While measurement of isoprostanes is employed as a “gold standard” to quantify cumulative oxidative stress from neurological stress, their bioactivity remains poorly characterized [113, 114]. One particular isoprostanes metabolite, 15-A2t-IsoP, has been shown to induce neurodegeneration in cultured neurons via mitochondrial ROS production, glutathione depletion, 12-lipoxygenase activation, and caspase cleavage [113]. Furthermore, 15-A2t-IsoP has recently been shown to potentiate hypoxia-induced neuronal cell death, suggesting a greater role in stroke pathology [114]. Finally, the F2-isoprostane acts as a potent vasoconstrictor of brain capillaries by inducing COX-mediated synthesis of thromboxane in endothelial cells [115]. In a stroke setting, vasoconstriction and platelet aggregation by thromboxane is counterproductive to resolving arteriothrombosis.

4.4 Enzymatic Oxidative Lipid Metabolism Cyclooxygenase, lipoxygenase, and epoxygenase enzymes are pivotal players in the generation of oxygenated derivatives of AA in the pathological setting of stroke. Cyclooxygenases convert AA to prostaglandins and thromboxanes, lipoxygenases catalyze the metabolism of AA into leukotrienes and lipoxins, and epoxygenase activity produces epoxyeicosatrienoic acids. The functional role of these enzymes and downstream lipid-derived products is largely dependent upon environment and cellular localization. In a stroke setting, several products of these pathways act within neurons to modulate the activities of ion channels, protein kinases, ion pumps, and neurotransmitter uptake systems. The newly formed eicosanoids may also exit the cell of origin and act at a distance, by binding to G-protein-coupled

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receptors present on nearby neurons or glial cells. Finally, the actions of the eicosanoids may be terminated by diffusion, uptake into phospholipids, or enzymatic degradation. 4.4.1 Cyclooxygenase The cyclooxygenase (COX) family of isozymes is composed of heme-containing bifunctional enzymes with two catalytic sites. The first active site adds two oxygen molecules to AA to form the hydroxy endoperoxide prostaglandin G2 (PGG2). Subsequently, PGG2 translocates to a second active site for peroxidative reduction to prostaglandin H2 (PGH2). As there is no channel within the COX monomer to shuttle PGG2 to the active reducing site, PGG2 may be reduced on the same or a neighboring enzyme [116]. Importantly, the peroxidase activity of COX converts PGG2 to PGH2 by removal of oxygen, which may thereby serve as a source of oxygen radicals [117, 118]. It has recently been demonstrated that COX-dependent neuronal death is linked to superoxide anion generation [119]. Three cyclooxygenase isoforms (COX-1, COX-2, and COX-3) exist in brain tissue [120]. COX-1 and COX-2 are both constitutively expressed in neural tissue [121, 122]. COX-1 is traditionally characterized as a “housekeeping” enzyme in homeostatic production of prostaglandins for activities that include maintenance of glycerophospholipid levels and membrane remodeling [123, 124]. Unlike COX-1, however, COX-2 expression and activity is induced in response to pathogenic stimuli including ischemic and hemorrhagic stroke [125–129]. Suggestive evidence supports COX-2 isozyme coupling with upstream phospholipase and downstream synthase activity for the production of lipid signaling eicosanoids. Both cPLA2 and COX-2 knockout mice share phenotypic similarities and decreased susceptibility to ischemia/reperfusion brain injury [130–132], supporting a functional coupling between cPLA2 and COX-2 enzymes [132–134]. While COX-1 and COX-2 are homodimers, sharing 60% homology in cDNA and amino acids, subtle yet significant differences in substrate binding and catalytic sites confer unique properties. COX-1 contains valine residues at positions 434 and 523, while COX-2 possesses isoleucine. This small difference results in a larger and more flexible active site in COX-2 as compared to COX-1, thus accounting for greater eicosanoid production by COX-2 at low AA concentration [101, 135, 136]. COX-3 is a lesser characterized splice variant of COX-1, highly abundant in astrocytes, endothelial cells, and pericytes, though absent in neurons [137]. Acetaminophen is a potent and selective inhibitor of COX-3 activity [138]. The COX-mediated metabolism of AA to PGH2 is a precursor to five primary bioactive prostanoids – PGD2, PGE2, PGF2a, thromboxane (TXA2) and prostacyclin (PGI2). The highest levels of extracellular protanoid release in an ischemic stroke setting occurs during reperfusion [139]. The prostanoids bind to G proteincoupled receptors that differ in their effects on cAMP and/or phosphoinositol turnover, and intracellular Ca2+ mobilization [140]. For example, PGE2 has been shown to elicit both neuroprotective and neurotoxic effects following cerebral

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ischemia that are dependent on receptor class binding. PGE2 activation of the E-prostanoid 1 receptor (EP1) disrupts neuronal calcium homeostasis by impairing sodium/calcium exchange, further agonizing Ca2+ activation of phospholipase and the arachidonic acid cascade. Pharmacological and gene inhibition of EP1 receptor has been demonstrated to reduce stroke-induced brain injury, oxygen–glucose deprivation, and neurotoxicity [141]. Conversely, PGE2 signaling via EP2 receptor is neuroprotective in cerebral ischemia and dependent on activation of the cAMP– PKA pathway [142]. Such paradoxical outcomes suggest an additional level of complexity in the prostanoid response to ischemic injury that warrants additional investigation. 4.4.2 Lipoxygenases The first human lipoxygenase (LOX) activity was described in platelets via the transformation of AA to a prostaglandin-like endoperoxide [143]. Today, the LOX enzyme family includes four members that are classified on the basis of the carbon position in which they oxidize arachidonic acid: 5-, 8-, 12-, and 15-LOX. Despite variances in amino acid sequences and tissue distribution among family members, the active site of each isozyme is highly conserved [144]. The structure of LOX at the active site is composed of an N-terminal b-barrel domain and a C-terminal domain containing a hydrophobic substrate-binding site [145]. A nonheme iron atom is coordinated by three histidine residues and the carboxy-terminal isoleucine. The oxidation of ferrous iron (Fe2+) to ferric iron (Fe3+) activates the enzyme that is then capable of excising a hydrogen atom from a hydrocarbon at one of the four double bonds. This abstraction generates a radical metabolite of AA that rapidly reorganizes its double bonds to take on a more stable conformation. Next, insertion of molecular oxygen generates a hydroperoxide radical that is reduced to the hydroperoxide anion by the simultaneous oxidation of iron to the ferric state [145]. A proton is then accepted to form a highly reactive fatty acid hydroperoxide, hydroperoxyeicosatetraenoic acid (HPETE). These hydroperoxide derivatives of AA are short-lived and readily metabolized into more stable compounds, including hydroxyeicosatetraenoic acids (HETEs) and leukotrienes. While each LOX isoform carries out the same general reaction, namely, hydrogen abstraction from arachidonic acid, each has a unique gene structure, amino acid sequence and tissue distribution profile. Only three forms of LOX are present in brain tissue, 5-, 12-, and 15-LOX [101]. Of these, 12-LOX is the most abundant isoform found in the brain [146], with significant mRNA expression in rat cortical neurons, astrocytes, and oligodendrocytes [147]. The LOX-mediated metabolites of AA serve as second messengers following stroke by modulating inflammation, apoptosis, and synaptic activity. Hydroperoxy- (HPETE) and hydroxy- (HETE) eicosatetraenoic acids.  While shortlived, HPETEs are potent neurotoxins. Highly reactive oxygen radicals are produced during the conversion of HPETEs to HETEs [148], contributing to the overall burden of oxidative stress following stroke. Under conditions of glutathione (GSH)

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depletion, as in acute focal stroke, 12-LOX derived 12-HPETE triggers nitric oxide (NO)-induced neural cell death [149]. Meanwhile, inhibitors of cPLA2 and 12-LOX have been demonstrated to prevent neurotoxicity in  vitro [150]. Recent evidence supports findings that the hydroxy- (HETE) derivatives of LOX-mediated metabolism possess potent biological activity as well. 12-HETE has been shown to increase mitochondrial NO production, induce cytochrome C release, and subsequently cause mitochondrial dysfunction [151]. 5-, 12- and 15-HETE are potent mediators of increased vascular permeability [152]. Elevated levels of HETEs in a setting of ischemic and hemorrhagic stroke may therefore contribute to the documented phenomenon of blood brain barrier breakdown and stroke-induced edema [153]. Leukotrienes.  The LOX catalyzed dehydration reaction generates an epoxide intermediate, leukotriene A4 (LTA4) [101]. LTA4 is a highly unstable intermediate that is readily hydrolyzed to LTB4 or conjugated with glutathione by cysteinyl leukotriene C4 synthase to produce leukotriene C4 (LTC4), leukotriene D4 (LTD4), and leukotriene E4 (LTE4) [154]. Together, LTC4, LTD4 and LTE4 are collectively referred to as cysteinyl-leukotrienes (Cys-LTs). Total leukotriene levels accumulate in the mammalian brain and cerebrospinal fluid during and after cerebral ischemia [155, 156]. LTB4 is characterized as a powerful chemotactic agent that induces adhesion of proinflammatory leukocytes to the endothelium, stimulates phagocytosis, and activates neutrophils and other leukocytes in a paracrine manner [157–160]. The Cys-LTs are potent vasoconstrictors of both venous and arterial smooth muscle [161, 162], and are also reported to produce vascular leak and vasogenic edema [163]. Furthermore, pharmacological inhibition of Cys-LT receptors attenuates pathological outcomes in a rodent model of focal cerebral ischemia, suggesting roles for Cys-LTs in mediating ischemic injury [159, 164]. Like LTB4, Cys-LTs also operate as potent activators and chemoattractants for leukocytes, particularly eosinophils and monocytes, thereby indirectly propagating inflammation and oxidative stress [159]. 4.4.3 Cytochrome P450 Cytochrome P450 epoxygenases (cP450) are heme-containing enzymes that use molecular oxygen to oxidize PUFAs by transfer of one atom of oxygen to their substrate and the other to water. Brain cP450 epoxygenases metabolize AA by inserting a single oxygen atom into an unactivated hydrocarbon at one of the four double bonds, thereby producing four regioisomers: 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs). Compared to other organs, expression of cP450 in brain tissue is quite low, accounting for only 1–10% of levels observed in the liver [165, 166]. Relative expression of cP450 in brain tissue is found to be highest in astrocytes localized near blood vessels, as well as the adjacent vascular endothelium [167]. The activity of cP450 epoxygenases localized in and around the cerebrovasculature has been shown to modulate angiogenesis via capillary endothelial migration and tube formation [168, 169]. In a pathological setting, such as ischemic stroke, EETs also serve as

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potent vasodilators of cerebral arterioles by increasing K+ currents in cerebral arteriole smooth muscle cells [170]. Elevated extracellular glutamate, as released by neurons and astrocytes under conditions of acute ischemic stroke, increases the formation of EETs in cultured astrocytes [171]. Importantly, glutamate alone has no effect on isolated cerebral blood vessels [172, 173]. However, when injected into rat brain tissue via cranial window, glutamate induces significant dilation of pial arterioles [171]. A cP450 inhibitor, miconazole, abrogates this effect [171, 174], suggesting that glutamate stimulated activity of cP450 enhances EET formation and vasodilation. In addition to their vasodilatory effects, beneficial roles of EETs have been demonstrated within the vasculature that are independent of smooth muscle cell relaxation [175]. Specifically, it has been shown that EETs possess potent antiinflammatory effects by inhibiting cytokine-induced endothelial cell adhesion molecule expression and preventing leukocyte adhesion to the vascular wall [176–178]. More recently, EETs have been shown to protect neurons [179] and astrocytes [180] against ischemic cell death induced in vitro by oxygen–glucose deprivation. Like other eicosanoids, the biological activity of EETs is expected to occur via cell surface receptors. To date, however, the identification of putative EET receptors and mechanisms of protection remain to be adequately characterized [175]. Once metabolized by cP450, AA-derived EETs are subject to several metabolic fates, including incorporation back into membrane phospholipids, and hydration by soluble epoxide hydrolase (sEH) [181] to di-hydroxyeicosatrienoic acids (DHET). No essential function of DHETs has yet to be identified [182]. Strategies to inhibit sEH activity in order to prolong the neuroprotective action of EETs are currently of therapeutic interest. Inhibitors of sEH have historically been developed as antihypertensive agents, but recent data indicate they also decrease vascular smooth muscle proliferation, prevent cardiac hypertrophy, and improve renal hymodynamics [182].

5 Management of Oxidative Stress in Stroke An accepted adage among stroke research scientists and clinicians alike is “time is brain” [183]; meaning that as time passes from the onset of stroke, interventional therapies are less likely to translate into functional recovery of stroke-affected tissue. When one considers the unique physiological and biochemical properties of hypermetabolic brain tissue, enriched with PUFAs that are highly susceptible to oxidative metabolism, the underlying truth of this maxim becomes evident. Preclinical models of stroke have revealed substantive evidence of elevated oxidants as early as 20 min following the onset of stroke [184, 185], and uncontrolled oxidative metabolism of AA as early as 1 h following hemorrhagic and ischemic stroke [186, 187]. In the case of ischemic stroke, a significant increase in reactive oxygen species and AA metabolism accompany reperfusion of stroke-affected tissue as well [188]. The rapid and overwhelming imbalance of pro-oxidants over

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antioxidants in stroke injury leads to a pathological setting that is highly resistant to late-phase (>6  h) therapeutic intervention. Potential neuroprotective agents administered after the onset of stroke that aim to salvage ischemic tissue, limit infarct size, and resolve stroke-mediated oxidative stress and inflammation have failed in clinical study. Of 114 neuroprotective agents tested during the twentieth century, not one was proven successful in clinical trials [189]. As clinicians and scientists recognize the limited therapeutic window for managing oxidative stress in stroke, research emphasis is shifting from late phase treatment modalities toward acute phase (<6 h) and prophylactic treatment regimens.

5.1 Therapeutic Window of Opportunity for Hyperbaric Oxygen Therapy in Acute Ischemic Stroke Recent studies to test the therapeutic window of opportunity for hyperbaric oxygen (HBO) in acute ischemic stroke support this fundamental paradigm shift [190–192]. HBO (100% O2 greater than atmospheric pressure) has long been proposed as a commonly available treatment option for stroke patients to correct ischemiainduced hypoxia in brain tissue. The three clinical pilot studies to probe the efficacy of HBO to treat acute ischemic stroke, however, reported mixed and potentially harmful outcomes with HBO treatments that overlapped thrombolytic therapy or were applied as late as 2 weeks after the onset of stroke [193–195]. Indeed, one recent preclinical investigation has uncovered specific application phases during ischemia and following reperfusion in which HBO-mediates both protective and harmful outcomes [192]. Specifically, HBO during stroke-induced ischemia was found to sufficiently correct brain hypoxia while significantly attenuating markers of oxidative stress (i.e., 4HNE) and pathological outcomes [192]. Conversely, HBO at the onset of ischemic stroke reperfusion, when ROS are known to be elevated [196], exacerbated oxidative stress while worsening stroke outcomes. Others have reported a limited window between 3 h and 6 h acute ischemic stroke reperfusion in which HBO is beneficial, but harmful at 12 h and beyond [190, 191]. Given the distinct and limited window of HBO therapy, the challenge now comes in translating these findings to an efficient and effective clinical response. Improved diagnostic processes to rapidly and repeatedly monitor cerebral perfusion of acute ischemic stroke patients would benefit the clinical prescription of HBO therapy.

5.2 Neuroprotective Properties of Natural Vitamin E, Alpha-Tocotrienol A promising prophylactic therapy for management of oxidative stress in the acute phase of stroke stems from a lesser-characterized form of natural vitamin E,

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alpha-tocotrienol (TCT). To date, alpha-tocopherol (TOC) remains the best characterized of the eight naturally occurring vitamin E isomers, as it is the most bio-available form with an established transport mechanism. On a concentration basis, however, the neuroprotective effects of nanomolar TCT represent the most potent biological function of all natural vitamin E isomers [197]. While all eight family members possess antioxidant efficacy, recent studies of the biological functions of vitamin E indicate that TCT possesses unique antioxidant independent function not shared by other family members [197]. Structurally, TCT differs from TOC only by the presence of three trans double-bonds in the hydrocarbon tail. This seemingly small structural difference accounts for significant neuroprotective action by TCT, but not shared by TOC. The major TCT-sensitive signaling pathways that are known to be involved in ischemic stroke-induced neurodegeneration include cPLA2 and 12-LOX. Activation of cPLA2 has been recognized to play a major role in stroke-mediated neurotoxicity [130]. As previously described, cPLA2 activity under conditions of ischemic stroke plays a critical role in liberating AA from membrane phospholipids. Free AA subsequently undergoes enzymatic and nonenzymatic oxidative metabolism to produce lipid-derived mediators of inflammation and oxidative stress that contribute to stroke pathology [130]. Nanomolar TCT has recently been reported to attenuate both activation and translocation of cPLA2 under conditions of glutamatemediated toxicity in neural cells [198]. Once cleaved from the sn-2 position of glycerophospholipids, AA may be metabolized by 12-LOX to proinflammatory HPETEs, HETEs, and leukotrienes. 12-LOX deficient mice are highly resistant to ischemic stroke injury [199], and TCT has also been shown to be a potent 12-LOX inhibitor in neural cells subjected to glutamate-mediated neurotoxicity [200]. Furthermore, prophylactic supplementation of TCT in stroke-prone spontaneously hypertensive rats significantly attenuated ischemic stroke lesion volume [199]. In the case of both PLA2 and 12-LOX, the TCT-dependent mechanism of neuroprotection in stroke has been shown to occur at nanomolar concentrations. Importantly, this concentration of TCT is readily achievable in brain tissue via oral supplementation [201, 202]. Taken together, these outcomes support significant neuroprotective potential for TCT in management of oxidative stress in the context of ischemic stroke. As a naturally occurring vitamin with a long history of safe consumption and no documented adverse side effects, TCT represents a safe and inexpensive prophylactic for at-risk stroke patients.

6 Conclusion In summary, this chapter outlines several mechanisms of oxidative damage in ischemic and hemorrhagic stroke as related to the arachidonic acid cascade. The use of in vivo preclinical stroke models, such as those described, has significantly contributed to the knowledge base of oxidative arachidonic acid metabolism and the pathogenic role of oxidative stress in stroke-mediated brain injury.

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The unique metabolic properties and lipid composition of brain tissue support a cellular environment that is highly susceptible to rapid and lethal oxidative damage following stroke. The clinical failure of therapeutic agents targeting neuroprotection in late phase stroke recovery suggests that once set in motion, the arachidonic acid cascade is highly resistant to pharmacological intervention. Further confounding the problem is substantial evidence supporting multiple oxidative pathways of AA-metabolism that contribute to brain stroke injury and oxidative stress. Therefore, research efforts focused on prophylactic and acute phase interventions that target multiple AA metabolism pathways hold significant promise in management of oxidative stress following stroke. One such intervention is prophylactic TCT supplementation. TCT is a natural, lipid-soluble antioxidant that accumulates in brain tissue following oral supplementation [201, 202]. This lesser characterized vitamin E isomer also possesses unique antioxidant independent properties that attenuate the arachidonic acid cascade at multiple levels [198–200], including PLA2 and 12-LOX (Fig. 3). For these reasons, prophylactic TCT supplementation may be of significant benefit to high-risk stroke patients, such as those diagnosed with atrial fibrilation, hypertension, or a transient ischemic attack (TIA). Acknowledgment  Supported by NIH NS42617 to CKS.

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Assessment of Oxidative Stress in the Brain of Spontaneously Hypertensive Rat and Stroke-Prone Spontaneously Hypertensive Rat Using by Electron Spin Resonance Spectroscopy Fumihiko Yoshino, Kyo Kobayashi, and Masaichi-Chang-il Lee

Abstract  It is well accepted that reactive oxygen species (ROS)-induced oxidative stress is a potential contributor to the pathogenesis of ischemia–reperfusion injury. The vascular system may be the first target of ROS generated during the pathological processes of hypertension and stroke; ROS also have important roles in the pathogenesis of vascular incompetence. The blood–brain barrier-permeable nitroxyl spin probe, 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl, has the potential to be useful in understanding redox status under conditions of oxidative stress induced by ROS in the rodent brain, using the in  vivo electron spin resonance (ESR) technique. These methods have also been applied to the spontaneously hypertensive rat (SHR) and stroke-prone SHR (SHRSP) models of essential hypertension and stroke, respectively. We have previously evaluated the degree of oxidative stress in the brains of SHRSP or SHR, as compared to normal (control) Wistar-Kyoto rat. This chapter focuses on biomedical applications of ESR, especially, the L-band ESR technique, using the rodent disease model. These ESR methods may constitute a useful tool for assessment of oxidative stress in the rodent brain of hypertension and stroke models such as the SHR and SHRSP; in addition, these ESR techniques have the potential to contribute to the development of neuroprotective drugs for treatment of hypertension and stroke in humans, in the near future. Keywords  Brain • ESR imaging • In vivo L-band • Nitroxyl spin probe • Oxidative stress • Spontaneously hypertensive rat • Stroke-prone spontaneously hypertensive rat

Masaichi-Chang-il Lee (*) Department of Clinical Care Medicine, Division of Pharmacology, Kanagawa Dental College, Yokosuka 238-8580, Japan e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_4, © Springer Science+Business Media, LLC 2011

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1 Introduction It is well known that free radicals, including reactive oxygen species (ROS) such as superoxide (O2•–) or hydroxyl radical (HO•), contribute to the development of several age-related diseases by causing oxidative stress [1]. Free radical reactions and oxidative stress induced by ROS are in most cases held in check by antioxidant defense mechanisms; however, where an excessive amount of ROS are produced or defense mechanisms are impaired, oxidative stress leading to events such as lipid peroxidation may occur [1–3]. One well-accepted hypothesis for the mechanism of hypertension is that oxidative stress caused by excess O2•– scavenging endothelial nitric oxide (NO, a vascular smooth muscle relaxant), could contribute to increased vascular smooth muscle contraction and hence the elevated total peripheral resistance. These phenomena would appear to be important in contributing to several pathological conditions in the central nervous system (CNS), including stroke [4–6]. Therefore, ROS-induced oxidative stress has been implicated as a potential contributor to the pathogenesis of the ischemia–reperfusion CNS injury characteristic of hypertension and stroke. Electron spin resonance (ESR) has been recognized as one of the most powerful techniques for the detection of ROS in the form of free radicals in biological tissues and cells. We have developed an ESR-based technique to detect free radical reactions induced by ROS in biological systems in  vitro and in  vivo [7–13]. Regarding in vivo ESR applications, nitroxyl radicals are very useful as exogenous spin probes to measure free radical distribution, oxygen concentration, and redox metabolism in biological systems. It has been suggested that the blood–brain barrier (BBB)-permeable molecule 3-methoxycarbonyl-2,2,5, 5-tetramethylpyrrolidine-1-oxyl (MC-PROXYL) is a suitable spin probe for the study of free radical reactions induced by ROS in the brains of small animal models using in vivo ESR detection [7–9, 12–18]. Using a combination of these ESR methods collectively focused on animal models of disease, recent results show that oxidative stress plays a key role in the vascular injury that occurs in hypertension and stroke. The spontaneously hypertensive rat (SHR) or strokeprone SHR (SHRSP), models of essential hypertension or stroke respectively, exhibit several characteristics of the increased oxidative stress induced by ROS [19–25]. This chapter will focus on oxidative stress induced by ROS that have a critical role in the pathogenesis of vascular disease in the brain, in hypertension, or stroke, using the rodent model. Thus, we demonstrate in this chapter that results obtained from our laboratory by means of in vivo L-band ESR techniques using MC-PROXYL, could serve as a useful basis for measuring oxidative stress induced by ROS in the brain of a model of hypertension such as the SHR or a model of stroke such as the SHRSP. Further, ESR techniques may be applicable to the assessment of antioxidant properties of drugs used for clinical treatment of human hypertension or stroke following their investigation in SHR or SHRSP.

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2 L-Band ESR/Nitroxyl Spin Probe Methods Nitroxyl radicals are very useful as exogenous spin probes for measurement of free radical distribution in biological systems [7–9, 18, 26–30], especially stable radicals such as nitroxyl radical 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) or 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (hydroxyTEMPO). These are often used as spin probes for in  vivo ESR, operating in the lower frequency microwave bands (£1  GHz), the so-called L-band, where the dielectric losses of biological systems are lower [29, 31]. A nitroxyl radical has been reported to lose its paramagnetism when exposed to a reducing agent in biological systems [32, 33]. The signal decay rate of the nitroxyl radical gives evidence of oxidative stress induced by ROS and changes of redox status in biological systems [7–9, 18, 30, 34]. Significant advances in the field of ESR imaging in recent years have now made it possible to visualize the distribution and metabolism of free radicals and the degree of tissue oxygenation in  vivo [35–37]. During the last decade, significant advances in L-band, and in vivo ESR techniques have provided useful information on oxidative stress in biological systems [30, 33, 38, 39]. In vivo ESR spectroscopy and ESR imaging are useful for investigation of the redox status of living organisms noninvasively. ESR imaging is particularly useful for determining the in vivo spatial distribution of free radicals in animals. It has been suggested that BBB-permeable MC-PROXYL is a suitable spin probe for the study of free radical reactions in the brain by in vivo ESR [7, 14, 16–18]. Since the permeability of the BBB to a compound is dependent on its lipophilicity and molecular weight, the partition coefficients between 1-octanol and water of various nitroxyl compounds have been measured [14]. The partition coefficient values of carbamoylPROXYL (0.87) and hydroxy-TEMPO (4.83) were consistent with those reported by Fuchs et  al. [15]. MC-PROXYL was more lipophilic (partition coefficient = 8.70) than carbamoyl-PROXYL and other spin probes, implying high permeability of the BBB to this compound. Our study also demonstrated that relatively large amounts of MC­-PROXYL were distributed to the brain compared to the BBBimpermeable carbamoyl-PROXYL. This was observed using ESR imaging for 3D or ESR-computed tomography (CT) of the head regions of mice (Fig. 1) [7, 18].

Fig. 1  Typical spatial 3D images obtained at 3 min after treatment with carbamoyl-PROXYL (a), and MC-PROXYL (b) in the head region of a live mouse [18]. To assign the area imaged, representative ESR images were superimposed on a photograph of the mouse studied

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Results of the studies mentioned above indicate that the in vivo ESR/nitroxyl spin probe techniques employed herein could be a powerful tool to selectively detect free radicals and to monitor free radical reactions in  vivo. Quantitative ESR analysis using MC-PROXYL has the potential to be useful for understanding redox status under conditions of oxidative stress in the rodent brain [7, 18].

3 SHR and SHRSP The development of genetic models for research on hypertension and stroke (SHR and SHRSP), has contributed to the ability to predict and prevent these conditions [40–42]. Cerebral ischemia and reperfusion cause delayed neuronal death in rodents such as Mongolian gerbils and SHRSP, both of which have been used as experimental stroke models [25]. The SHR, a model of essential hypertension, has several characteristics of increased oxidative stress [43, 44]. There is significant in vitro evidence indicating that O2•– contributes to increased systemic vascular tone in SHR [20, 45]. It has also been shown by in vivo fluorescence microscopy that blood vessels of SHR generate excessive amounts of O2•– [19]. Severe hypertension and cerebrovascular diseases develop in SHRSP [21]. ROS generation occurs after reperfusion and this free radical-induced oxidative stress can greatly damage neurons in the SHRSP [46]. Oxidative stress induced by excessive generation of O2•– in SHRSP tissues leads to increased hypertensive responses that may be relevant to the pathophysiology of stroke [8, 18, 23, 47]. This would therefore be an important mechanism of oxidative stress for production of excessive ROS, mainly O2•–, in animal models such as the SHR or SHRSP.

4 Measurement of Oxidative Stress in SHR and SHRSP Brain Using ESR In vivo L-band ESR and L-band ESR imaging methods have also been employed in brains from normotensive rats (Wistar-Kyoto rat, WKY), SHR, and SHRSP using MC-PROXYL. We had previously confirmed that the decay rate of MC-PROXYL in the brains of SHRSP, a hypertensive reference strain [8, 18] in which cerebral ischemia–reperfusion is known to occur, was due to oxidative stress. In these studies, ESR imaging and noninvasive L-band ESR showed that the decay rate of the MC-PROXYL ESR signal was more rapid in SHRSP brains than in the WKY brain (Fig. 2) [8, 9, 12, 18]. We performed pharmacokinetic profiles of MC-PROXYL ESR signals in the SHR and SHRSP brain. These can be divided into phase I and phase II, according to a twocompartment model of distribution, using in vivo L-band ESR (Fig. 3) [8, 18]. Nitroxyl spin probes are administrated systemically and are eliminated principally by a combination of excretion via the kidney and reduction to the hydroxyl amines [8, 18, 48].

Assessment of Oxidative Stress in the Brain of Spontaneously 0.12 0.10 0.08 0.06

K1

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0.05 0.04 0.03

0.04

0.02

0.02

0.01

0

0

Fig. 2  L-band ESR signal decay rate constant of MC-PROXYL in the head region of live WKY, SHR, and SHRSP after i.v. injection of MC-PROXYL [8]. Rats were treated with MC-PROXYL (140 mmol/l, 10 ml/kg) via the tail vein, and ESR spectra were measured at the head region of live rats. K1 and K2 indicate the decay rate constant (min−1) in phase I and phase II as shown in Fig. 3. Each column represents the mean ± SD (n = 3–6). *p < 0.05 versus the corresponding value for WKY. †p < 0.05 versus the corresponding value for SHR

The decay of the nitroxyl spin probe, MC-PROXYL, which is reflected in ESR signal intensity in the head region, appears to follow the usual pharmacokinetic pattern for excretion of drugs: those that tend to stay in the vascular system are excreted very rapidly (phase I) due to reduction by ascorbate, while lipophilic nitroxyl spin probes, such as MC-PROXYL, show much slower excretion via the kidney (phase II) (Fig. 3) [48]. The decay rate constants for MC-PROXYL in phase I (K1) and phase II (K2) in the brain were faster in SHR and SHRSP than in WKY. Furthermore, the phase I decay rates (K1) in SHRSP were more rapid than in SHR, while no significant difference was observed in phase II decay rates (K2) between SHRSP and SHR (Figs. 2 and 3) [8]. These results suggest that the higher oxidative stress in SHRSP brain occurred in the cerebral vascular system rather than the tissue responsible for degrading the MC-PROXYL ESR signal in the brain. Taken together, our data indicated that the level of oxidative stress in the cerebrovascular system in rodent brain was in the following descending order: SHRSP > SHR > WKY (Figs. 2 and 3), which is the same descending order as for blood pressure in these animals. Thus, the pathophysiology of hypertension, and therefore stroke, could involve increases in the level of oxidative stress by ROS generation in the cerebrovascular system.

5 Clinical Significance of the Measurement of Oxidative Stress in an Animal Model of Stroke Using ESR The most convincing evidence that oxidative stress induced by ROS generation is involved in brain disease such as stroke is the repeated observation that compounds that inhibit lipid peroxidation, or scavenge ROS, can prevent post-traumatic

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time (min) Fig. 3  A typical L-band ESR signal decay of MC-PROXYL in the head region of live WKY (a), SHR (b), and SHRSP (c) after i.v. injection of MC-PROXYL [8]. Rats were treated with MC-PROXYL (140 mmol/l, 10 ml/kg) via the tail vein, and ESR spectra were measured at the head region of live rats. The logarithmic signal intensity of the second peak of the ESR spectrum of the nitroxyl spin probes was plotted against time. Linearity was observed in phase I and phase II of the corresponding semilogarithmic plots, respectively

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pathophysiological changes and promote functional recovery and survival in experimental studies. Nevertheless, the significance of ROS and lipid peroxidation ultimately depends on whether it can be demonstrated that early application of an effective ROS scavenger can promote survival and neurological recovery after CNS injury and stroke in humans [4]. It is well known that ROS scavengers may decrease edema and tissue damage in stroke [49, 50]. Thus, the neuroprotective properties of antistroke agents could be associated with their antioxidant properties, indicating ROS scavenging activity. However, there is very little direct evidence of their antioxidant properties, specifically due to a lack of techniques for detecting ROS and for the assessment of oxidative stress levels in vivo, particularly in a suitable stroke animal model. Therefore, we need further studies using ESR techniques, both in vitro and in vivo, aimed at characterizing antioxidant properties of neuroprotective agents. Since we have evaluated the degree of oxidative stress in the brain of SHR and SHRSP as mentioned above, this means that we have now effectively established novel in vivo ESR methods for assessment of antioxidant properties of drugs or foods using SHR or SHRSP. Propofol anesthesia has been associated with lower intracranial pressure and cerebral swelling than volatile anesthesia in brain tumor patients undergoing craniotomy [51]. The potential neuroprotective effect of propofol may be mediated by its antioxidant properties which have been shown to play a role in apoptosis, ischaemia-reperfusion injury and inflammatory-induced neuronal damage [51, 52], due to reduction of lipid peroxidation via the generation of ROS [52–54]. We evaluated high oxidative stress in the brain of SHRSPs using in  vivo L-band ESR [8, 18]. Consequently, it is clear from our SHRSP investigations, that ESR techniques are highly useful in the assessment of the antioxidant properties of propofol mediumchain triglyceride/long-chain triglyceride (MCT/LCT) for clinical use. Indeed, we confirmed that oxidative stress in the brain of SHRSPs was higher compared to that in WKYs anesthetized with pentobarbital (Fig. 4a). When propofol MCT/LCT was used as an anesthetic, rather than pentobarbital, the level of oxidative stress in SHRSP then became similar to that seen in WKY (Fig. 4b), suggesting that propofol MCT/ LCT reduced SHRSP-induced oxidative stress in the brain. Consequently, propofol MCT/LCT could be particularly useful in adjusting levels of anesthesia in cases of ROS-induced brain disease. A better knowledge of clinical and laboratory markers of functional outcome would result in a more individualized and possibly improved approach to management of the patient with stroke. It is well accepted that infarct size as detected in neuroimaging studies constitutes a strong predictor of clinical outcome. However, CT scan or brain magnetic resonance imaging (MRI) information concerning the extent of a cerebral infarction is usually available too late after clinical onset to be of help in the decision-making phase. The predictive value of neuroimaging is then limited to the long-term phase of stroke. As a result, prediction of stroke outcome basically relies on clinical findings. As previously mentioned, we have now developed an in vivo ESR imaging system with reconstructed 3D images or high-quality ESR-CT images by using MC-PROXYL in a rodent model (Figs. 1 and 5). It should be noted that such a clinical indicator of acute stroke provides little information in

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Fig. 4  Effects of propofol MCT/LCT on SHRSP-induced oxidative stress in the brain [12]. Rats were anesthetized with pentobarbital (30 mg/kg, i.v.) (a) and propofol MCT/LCT (20 mg/kg, i.v.) (b) L-band ESR was used to determine the signal decay of MC-PROXYL in the head region of live WKYs and SHRSPs after i.v. injection of MC-PROXYL. Rats were treated with MC-PROXYL (140 mM, 5 ml/kg) via the tail vein, and ESR spectra were measured within the head region of live rats. The logarithmic signal intensity of the second peak within the ESR spectrum of the nitroxyl spin probes was plotted against time. Inset: regression line fitted to a typical plot of L-band ESR data. Data are presented as mean ± SD of WKYs (n = 5) or SHRSPs (n = 5) with pentobarbital and with propofol MCT/LCT. *Significance p < 0.05 from the corresponding control value of the WKYs

relation to the prevention of stroke. Further advances in the instrumentation used for ESR imaging and in the development of optimized nontoxic spin probes will make ESR technology one of the most novel diagnostic tools for acute stroke or clinical predictors for prevention of stroke in the future. We have already indicated that our ESR assessment can characterize the degree of oxidative stress in the SHR model of hypertension and the SHRSP model of stroke (Fig. 6) [8]. It is likely that the level of oxidative stress using ESR assessment may give useful information on pathological developments in the progress from hypertension to stroke.

6 Conclusion After brain injury by ischemic or hemorrhagic stroke or trauma, the production of ROS may increase in the vascular system. The pathophysiology of hypertension or stroke is associated with excess of ROS generation in the vascular system, and

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Fig. 5  (A) A typical slice pattern of 3D ESR-CT images (xy-plane) obtained at 3 min after the treatment with MC-PROXYL in the head region of living mouse [9]. To assign the anatomic position, representative ESR-CT images in slices on the x–y plane (0, the midsagittal section) derived from the corresponding images in panel (B) are a picture of superimposed by mouse studied. The ESR-CT images of MC-PROXYL were used as gray scale pixels

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Fig. 6  2D ESR projection images (y–z plane) of MC-PROXYL distribution in isolated brains of WKY (a), SHR (b), and SHRSP (c) [8]. ESR was measured at 5, 11, 17, and 23  min after i.v. treatment with MC-PROXYL (isolated 30  s after the treatment). As indicated by the attached color scale (16 colors, white and 100 being the maximum ESR signal), ESR images were reproduced in 16 colors and signals lower than 10% of the maximal signal intensity detected in all slices were regarded as noise

results in induction of various pathological cascades of cerebrovascular damage. In conclusion, we have demonstrated that ESR methods using MC-PROXYL will be useful for understanding redox status under conditions of oxidative stress in the SHR or SHRSP brain. We have used ESR imaging and noninvasive L-band ESR to characterize the higher degrees of oxidative stress in the SHRSP or SHR than in the WKY brain, and the lower extent of such stress in the SHR than in the SHRSP brain. Indeed, we may be able to confirm propofol MCT/LCT as neuroprotective anesthesia against human stroke after screening for antioxidant properties in stroke models such as SHRSP. Thus, our ESR biomedical application suggests that it could be used to assess antioxidant effects on oxidative stress in the brain using the SHR and SHRSP animal models of disease. After screening drugs or foods for antioxidant activity using ESR assessment, it will be possible to find and develop novel drugs with such properties for the prevention of hypretension or stroke in the near future. We hope that further advances in the instrumentation used for ESR imaging and the development of optimized nontoxic spin probes will make this technology even more promising for novel clinical prediction or noninvasive diagnosis of human hypertension or stroke.

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Small Heat Shock Proteins and Doxorubicin-Induced Oxidative Stress in the Heart Karthikeyan Krishnamurthy, Ragu Kanagasabai, Lawrence J. Druhan, and Govindasamy Ilangovan

Abstract  Doxorubicin (Dox) and its derivatives are used as chemotherapeutics, either alone or in combination with other agents. Dilated cardiomyopathy and congestive heart failure due to cardiotoxicity continues to be the most serious side effect, imposing severe limitations in the use of these agents despite the arrival of new classes of Dox-derivatives and new formulations. In this chapter we summarize the recent understanding of the mechanism of Dox-induced cardiotoxicity and its relevance to the stress-inducible proteins, with special emphasis on the small heat shock proteins such as Hsp27, Hsp20, etc. The heat shock proteins are expressed as a response to the oxidative stress in the heart due to the redox reactions of these drugs and the generation of reactive oxygen species (ROS). On the other hand, ROS are also known to induce various MAP kinases and phosphorylate and activate the stress-responding transcription factors, including the heat shock factors (HSF). Activation of HSF-1 leads to the induction of a series of heat shock proteins, depending upon the type of exerted stress. Recent studies have confirmed that Dox-induced oxidative stress indeed leads to HSF-1 activation to induction of heat shock proteins, especially small Hsps in the heart. The Dox-induced small Hsps have been found to be involved in cell signaling and can be either cardioprotective or detrimental. Additionally, a few transgenic animal models have shown that selective overexpression of these proteins can be cardioprotective against Dox. These results establish the fact that proper regulation of the function of small Hsps could eliminate cardiotoxicity and serve as a potential therapeutic target to protect the heart from Dox-induced toxicity. Keywords  Adriamycin • Doxorubicin • Heart failure • Heat shock factor-1 • Oxidative stress • Small heat shock proteins

G. Ilangovan (*) Division of Cardiovascular Medicine, Davis Heart and Lung Research Institute, Ohio State University, 460 West 12th Ave, 43210 Columbus, OH, USA e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_5, © Springer Science+Business Media, LLC 2011

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1 Introduction Doxorubicin (Dox, Adriamycin) is one of the original anthracyclines isolated in the early 1960s from the pigment-producing bacterium Streptomyces peucetius. To this day, adriamycin and its derivatives remain some of the most effective anticancer drugs [1, 2]. Adriamycin is a potent and broad spectrum antineoplastic agent used in the treatment of several cancers, such as Hodgkin’s lymphoma, aggressive nonHodgkin’s lymphomas, acute lymphoblastic leukemia, metastatic breast carcinoma, ovarian carcinoma, lung carcinoma, and sarcoma. It has continued to be considered a first-line antineoplastic drug for more than 30 years [3]. However, this drug has proven to be a double-edged sword because it also causes a serious side effect, namely, cardiotoxicity leading to cardiomyopathy and congestive heart failure (CHF). The risk of developing irreversible cardiomyopathy due to Dox treatment may be increased by several factors. These include the degree of anthracycline exposure (high-dose anthracycline infusion or higher cumulative doses are responsible for more frequent cardiac problems), age (the elderly and very young patients are at greater risk), a history of cardiac disease, previous cancer therapies (e.g., mediastinal radiation therapy), and the concurrent use of chemotherapy regimens that include paclitaxel or trastuzumab [4, 5]. The mortality rate of anthracyclinerelated CHF is greater than 50% within 2 years from diagnosis in patients with New York Heart Association class III/IV [6, 7]. In order to overcome this unwanted side effect, new developments such as new derivatives with less toxicity to the heart, new formulations in the form of encapsulation into delivery vehicles (such as microemulsions), new targeted delivery modalities to selectively deliver the drug to the desired site (avoiding the accumulation in the heart etc.) [8], have emerged. However, there has been no significant improvement towards avoiding cardiotoxicity and complete elimination of this side effect has not been achieved. Fortunately, the mechanisms of killing tumor cells (intended effect) and the cause of cardiomyopathy (unwanted effect) by Dox and its analogs are found to be different, indicating that the mode of action of these drugs depends upon the nature of tissues. This difference in action gives hope that by selectively targeting some of the pathways by novel approaches, the untoward development of cardiomyopathy and CHF among Dox treated patients can be avoided. Thus, a new generation of interest has arisen to combat the oxidative stress and to identify and target these pathways. In this chapter we summarize the research that has been carried out to determine the mechanisms of the induction of stress inducible proteins, specifically heat shock proteins (Hsps), in the heart upon treatment with Dox.

2 Differential Mechanisms of Action of Doxorubicin: The Oxidative Stress in the Heart Anthracyclines are known to kill cancer cells by DNA intercalation and topoisomerase II inhibition, while the loss of cardiomyocytes in the heart has been attributed to oxidative stress, caused by oxidants such as oxygen-derived free radicals

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Fig. 1  Differential mechanisms of Dox-induced cell death in malignant tissue and the heart. In cancer cells, the primary mechanism of cell death involves DNA intercalation, FasR mediated apoptosis, and topo II inhibition. Cardiomyocyte death in the heart is mainly by mechanisms involving oxidative stress-related apoptosis (endogenous p53/Bax pathway), mTOR inhibition, TLR 2 activation, etc. The differences in the mechanisms are mainly due to differences in the nature of the cells (differentiating versus terminally differentiated cardiomyocytes)

(O2•−, OH•) and generation of H2O2 (Fig. 1) [9]. If cell death in the heart is avoided by selectively targeting the cardiomyocyte death pathways, the intended outcome of Dox treatment could be greatly improved. Indeed, supplementation of antioxidants or specific overexpression of endogenous antioxidants has been found to be very effective in alleviating Dox-induced heart failure in animal models [10]. However, some clinical trials with antioxidants such as vitamin E showed no significant improvement [11, 12]. Thus, to realize the full potential of antioxidantbased adjuvant therapy, the complete mechanism of cardiomyocyte death needs to be understood. Even the precise mechanisms of action of anthracyclines in tumor cells remain a matter of controversy [13]. Suggested mechanisms include (1) intercalation into DNA, leading to inhibition of synthesis of macromolecules; (2) generation of reactive oxygen species (ROS), leading to DNA damage or lipid peroxidation; (3) DNA binding and alkylation; (4) DNA cross-linking; (5) interference with DNA unwinding or DNA strand separation and helicase activity; (6) direct membrane effects; (7) initiation of DNA damage via inhibition of topoisomerase II; and (8) induction of apoptosis in response to topoisomerase II inhibition [9, 13, 14]. Likewise, several mechanisms have been proposed to explain adriamycin-induced myocardial damage; the most important among them is generation of oxygen-derived ROS and subsequent “oxidative stress.” Increased oxidative stress and antioxidant deficit have been suggested to play a major role in adriamycin-induced cardiomyopathy and CHF. Previously, EPR spin-trapping has been successfully used to show the free radicals generated during Dox treatment [15]. There are two major pathways by which Dox causes free radical formation [16–19], an enzymatic mechanism catalyzed by several mono-electronic oxidoreductases and a nonenzymatic mechanism involving anthracycline–iron complexes. In the enzymatic mechanism, one electron reduction of the quinone moiety in the C ring of anthracyclines results in

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the formation of a semiquinone radical that quickly regenerates its parent quinone by reducing molecular oxygen to superoxide anion (O2•–) and subsequently to hydrogen peroxide (H2O2) via dismutation, thus increasing ROS concentrations above the physiologic levels generated during normal aerobic metabolism [20, 21]. This futile cycle is catalyzed by a number of flavin-centered oxidoreductases located primarily in mitochondria (NADH-ubiquinone oxidoreductase) [22, 23], as well as in microsomes (NADPH-cytochrome P450 or NADPH-cytochrome b5 reductases) [24, 25], nucleus (NADPH-cytochrome b5 reductases) [19] and cytosol (xanthine dehydrogenase, nitric oxide synthase) [26–28]. Anthracycline-free radicals may arise via a nonenzymatic mechanism involving reactions of anthracyclines and iron. For example, iron (III) readily interacts with Dox. This is followed by a redox reaction, wherein the iron atom accepts an electron, generating an iron (II)-doxorubicin-free radical complex to shuttle electrons to O2 forming redox cycle [29]. Anthracycline aglycones may generate ROS as well, as another pathway of nonenzymatic ROS generation. Due to their higher lipophilicity, aglycones can intercalate into mitochondrial membranes better than the parent anthracyclines and thus divert more electrons towards oxygen, thereby generating ROS in close proximity to sensitive targets [30, 31]. Additionally, Dox treatment may also increase nitric oxide (NO) production by upregulating the expression of the inducible isoform of NO synthase (iNOS) [32]. The concurrent over-production of O2•– and NO yields reactive nitrogen species (RNS) and, in particular peroxynitrite (ONOO–) [33, 34], a powerful oxidant that may directly damage every type of cellular macromolecules, resulting in lipid peroxidation, protein covalent modifications, and DNA strand breaks. Anthracycline-related oxidative and nitrosative stress have been reported to interfere with many aspects of cardiac function by inducing mitochondrial dysfunction [5, 35–37], energy imbalance [38, 39], disruption of the cardiac specific gene expression program [40, 41], and apoptosis [42–44]. In most cells, ROS and RNS formation is kept to a minimum by the presence of detoxifying enzymes, like superoxide dismutase, catalase, and glutathione peroxidase. Unfortunately, cardiomyocytes are more susceptible than other cells to oxidative and nitrosative damage for several reasons (a) high metabolic activity [45]; (b) high concentration of cardiolipin, a mitochondrial membrane-bound phospholipid critical to cell respiration for which anthracyclines have a high affinity [46, 47]; and (c) weak antioxidative defenses due to their low content of catalase, superoxide dismutase and glutathione peroxidase [48, 49]. A growing number of reports indicate that free radical damage to the mitochondria and defenses against free radicals may contribute to cumulative cardiomyopathy [4]. This concept of oxidative stress is directly supported by the detection of Dox-generated ROS using electron spin resonance spectroscopy [27, 50, 51]. In addition, indirect support is offered by studies showing that Dox increases levels of tissue malondialdehyde, which is a product of lipid peroxidation [52, 53]. There is also evidence that Dox metabolites induce myocardial damage. Doxorubicinol, the primary C-13 alcohol metabolite of Dox, has been found to accumulate in ­cardiac tissue in a time- and dose-dependent manner [54]. Anthracycline alcohol metabolites can affect myocardial energy metabolism, ionic gradients, and Ca2+

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movements. All of these effects impair cardiac contraction and relaxation. Research has proven that, at concentrations that are relatively low when compared to those of Dox, Doxorubicinol compromises both systolic and diastolic function of isolated heart preparations and block ATPase activity of the sarcoplasmic reticulum, mitochondria, and sarcolemma [55].

3 Enhanced Heat Shock Proteins Expression in Dox-Treated Hearts Cells respond to stressful conditions by activating genetic programs whose evolutionarily conserved mechanisms have common ancestral origins, from the simplest bacteria to complex organisms including humans [56]. One such genetic program that has gained increased attention is the expression of Hsps under genotoxic environments such as Dox treatment. Hsp is expressed in all living cells, in response to exerted stress. Both in vivo and in vitro studies have shown that various stressors transiently increase the production of Hsp as a protection against harmful insults. Hsp induction was first identified in the salivary glands of Drosophila upon application of heat shock [57]. Since then, studies of individual genes have shown that the cellular heat shock response is conserved across kingdoms and is characterized by the strong induction of numerous heat shock proteins. Hsps expression was found to correlate with the acquisition of thermotolerance and cytoprotection. Thus initially it was thought that Hsps expression was a unique mechanism to adapt to hypothermia [58]. Later, the interest in Hsps expanded tremendously as more and more processes in normal resting cells involving Hsp were uncovered. The multifunctional roles of Hsps in cells show that Hsps are major regulatory proteins in the cell, and vital functions of the cell, such as the maintenance of the cell cycle, are associated with Hsps [59, 60]. In mammalian cells, the stress response involves the induction of four major classes of Hsp families, namely, the small Hsp exemplified by Hsp27, Hsp60, Hsp70, and Hsp90 [61–64]. Increased expression of Hsps in Dox-treated hearts was reported as early as 1990. The Schoeppe group reported increased expression of a 30 kDa “stress protein” in cultured cardiomyocytes, upon treated with cisplatin [65] and inhibition of this protein upon being treated with Dox. Although initially the purpose of the expression of these proteins was unknown, only later was it proven that Hsp induction was an endogenous defensive mechanism of cells in response to oxidative stress. Initial experiments demonstrated that co-injection of carnitine with Doxenhanced Hsp25 expression threefold, and this provided significantly enhanced cardioprotection [65, 66]. These studies were further supported by different groups demonstrating the effect of thermal preconditioning [67] and physical exercise [68, 69] on the Dox-induced cardiotoxicity. Despite the fact that these early studies established increased expression of Hsps as cardioprotective, the mechanistic details of such enhancement, the signaling that followed such an increased expression, were lacking prior to the identification of the transcription factors that translated the stress into expression of Hsps.

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4 Transcription of Hsps in Dox-Treated Hearts: The Role of Heat Shock Factors Environmental insults provoke a variety of adaptive physiological responses to help organisms cope with specific stressors. The dramatic induction of Hsps is an important unifying component of most of these responses for survival under stressful conditions. Regulation of Hsp expression is intricate, with multiple layers of redundancy and feedback control (Fig. 2). Systemic stresses, genotoxic (i.e., Dox treatment) and proteotoxic (accumulation of denatured proteins) stresses, activate a small family of transcription factors called heat shock factors (HSFs) that are the primary regulators of stress-inducible expression in eukaryotic cells. HSFs, originally found to respond to heat shock, but they are now known to respond to any environmental stress, are activated in toxic environments such as the genotoxic agents by a complicated mechanism. The structure and function of HSFs have been conserved for more than a billion years [70]. Several HSFs are present in mammalian cells (i.e., murine and human HSF-1, -2, and -4 and a unique avian HSF-3), but HSF-1 is clearly the dominant factor controlling cellular responses to stress.

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HSF-1 was first identified in cellular extracts from Saccharomyces cerevisiae and Drosophila melanogaster. It oligomerizes on activation and binds to heat shock element (HSE) sequences to control stress-inducible expression of Hsp genes [71]. In mice, HSF-1 is ubiquitously expressed but is particularly abundant in ovary, placenta, heart, and fetal brain [72]. HSF-1 is dispensable for growth and survival under controlled laboratory conditions but essential for survival following stresses such as high temperature and endotoxin challenge [73]. Transgenic animal models have demonstrated the importance of HSFs in reproduction and survival [74]. HSF1-deficient mouse embryos suffer from defects in placental development and are recovered from cross-breeding in lower numbers than expected by Mendelian segregation. Other than being ~20% smaller than wild-type mice, however, HSF-1deficient mice display no overt organ system abnormalities and, in the absence of acute stress, live to late adulthood [73]. Although the HSF-2 transcription factor is refractory to typical stress stimuli, its ability to bind DNA is specifically enhanced in certain development situations. Studies using specific polyclonal antibodies clearly showed that only HSF-1 can respond to heat and other physiological stress [75–77]. HSF-1 was found to be activated by diverse forms of stress and the activation of HSF-1 occurs via a multistep process [78]. Under nonstress conditions, mammalian HSF-1 appears as a monomeric form that exhibits little DNA binding activity. Activation of HSF-1 is accomplished by a stress-induced conversion of monomers to trimers, translocation of HSF-1 from cytosol to the nucleus, posttranslational modifications including phosphorylation of serine residues, and transcriptional competence with enhanced heat shock gene expression [79] (Fig.  2). HSF binds to a conserved DNA sequence motif, the HSE, which is characterized by an array of inverted repeats of the motif nGAAn. Copies of the HSE are found in the promoters of genes encoding several known Hsps [80]. HSF–HSE DNA binding is not sufficient to elicit maximal transcription of the Hsp genes, and it is necessary for HSF-1 to be modified by phosphorylation and sumoylation to increase its transcriptional activity [71, 81]. It has been suggested that HSF-1 is repressed by GSK-3b (Ser303), ERK (Ser307), and JNK (Ser363) under normal conditions, whereas it is activated by hyperphosphorylation (Ser230) upon exposure to various stresses (Fig. 2) [81, 82]. As illustrated in Fig. 2, HSF-1 contains two distinct carboxyl-terminal activation domains, AD1 and AD2, which are under the control of a centrally located, heat-responsive regulatory domain (RD) [83]. Since AD1 does not appear to be heat-regulated by itself, the regulatory domain of HSF-1 has been proposed to play a key role in sensing heat stress in humans [84]. Constitutive phosphorylation of two specific serine/proline motifs, S303 and S307, is important for the function of the RD and may be critical for negative regulation of HSF-1 transcriptional activity at normal temperatures, since substitution of serine to alanine causes constitutive transcriptional competence [84–86]. Constitutive phosphorylation of S363 has also been found to negatively regulate HSF-1 under normal growth conditions [87]. The positive role of phosphorylation in regulation of HSF-1 transcriptional activity is only emerging. So far, one phosphorylated site (S230), which has a positive effect on HSF-1 transactivating capacity, has been characterized [88]. A number of studies have shown that HSF-1

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and Hsps confer protection against cardiovascular disease. Induction of HSF-1 and Hsp expression by various stimuli, such as heat shock, reduces the size of infarcts after ischemia/reperfusion [89]. Transgenic mice overexpressing a constitutively active form of HSF-1 or inducible Hsp70 in the heart show more resistance to ischemia/reperfusion injury compared with wild-type mice [90]. In contrast, the cardiac function of inducible Hsp70 knockout mice is markedly impaired by ischemia/reperfusion injury [91]. In addition to a protective effect against ischemia/ reperfusion injury and Dox-induced cardiomyopathy, it has been reported that Hsps have a beneficial role in myocardial infarction and atrial fibrillation [92, 93]. ROS activates HSF-1, which in turn increases the expression of Hsps including Hsp90, Hsp70, Hsp60 and Hsp25. In addition to their well-characterized role as molecular chaperones, previous studies have found involvement of Hsps in various cellular signaling [94]. Hsp90 has been extensively studied in cellular systems demonstrating that it activates nitric oxide synthase (NOS) [95–97]. A recent study using cardiac cell has reported that Hsp90 activates endothelial NOS to increase NO generation. The excess NO produced by such an activation can block the mitochondrial electron transport chain (ETC) by binding at the cytochrome c oxidase of complex IV [98]. Hsp27 and its murine ortholog Hsp25 have been shown to play a very important role in the protection of cardiomyocytes under oxidative stress. Ischemia–reperfusion injury and hypertrophy were found to be reduced by small Hsps in the heart and cardiomyocytes [99–106]. Studies have also shown that ROS and lipid peroxidation could be attenuated by the induction and phosphorylation of small Hsps [107, 108]. Dox has been found to show a different effect on the activation of HSF-1 in cells and animal models. HSF-1 activation by heat shock is different from that induced by anticancer drugs such as Dox. Unlike the extensive phosphorylation and transcription of a full spectrum of Hsps produced by heat shock, anticancer drugs induced oxidative stress which induces a selective expression of Hsps. For example the anticancer drug vincristine and vinblastine activate HSF-1 by selective phosphorylation via the c-JNK pathway [109]. HSF-1 has also been implicated in MDR-1 expression in Dox-resistant cells. However, with the exception of our initial report [110], the Dox-induced activation of HSF-1 in the heart or in cardiomyocytes has not been studied in detail. Such an activation of HSF-1 is expected to have a wide range of implications and needs to be studied in detail to unravel the complete mechanism of Dox-induced cardiotoxicity.

5 Mechanism of Heat Shock Protein Cardioprotection Against Dox Toxicity Small Hsps have been found to play many critical roles in cardioprotection. Various in vitro and in vivo models have shown convincingly that these proteins are vital in protecting the structural integrity of myocardium, in myogenesis, and in preventing various cardiovascular diseases. Gene deletion/mutations in both the HSF-1 as well the small Hsps such as aB-crystallin or Hsp25 (the murine ortholog of human Hsp27),

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resulted in CHF with age [111]. However, very little insight has been gained on the underlying mechanism of how small Hsps provide cardioprotection against oxidative stress including that caused by Dox. Using in vitro cardiomyocyte cultures and in  vivo small animal models, various groups have studied this problem. These ­studies have examined the issue from two angles (1) studies on the induction mechanism of the small Hsps, such as Hsp27, due to Dox-induced oxidative stress; and (2) studies on the effect of preinduction of Hsps by activation of HSFs due to heat shock and other activation procedures or by overexpression in mice. In neonatal cardiomyocytes-derived H9c2 cells, Dox was found to enhance Hsp27 expression [92]. In low concentrations, Dox-induced toxicity was higher and with increase in Dox concentration its survival reached a leveling off (Fig. 3a). Hsp27 induction increased in parallel with increasing Dox concentrations, suggesting that the Dox-induced Hsp27 offers protection from the Dox in these higher concentrations. Moreover, in preheat shocked cells (heat shock for 4  h at 42°C and incubated for the next 24 h at 37°C in order to activate HSF-1 and induce the Hsps), where the basal Hsp27 was higher (Fig. 3b), the cells showed increased resistance against Dox-induced toxicity [92]. EMSA analysis of HSF-1 binding to consensus HSE showed increased DNA binding of the transcription factor (Fig. 3c). Overall, it appears that Hsp27 induction in response to Dox treatment is likely a response aimed at protecting the cells (Fig.  3d). However this study did not establish a definitive mechanism for either the increased Hsp27 expression, or how Hsp27 offered protection from Dox. In another study, we saw that heat shock can protect the cells from mitochondrial-based apoptotic pathways, attenuating OH radical generation from mitochondrial aconitase [112]. We found that the deactivation of aconitase and subsequent generation OH radicals from the free Fe is reduced in Hsp27 overexpressing cells. Thus, it seems that the heat shock-induced activation of HSF-1 and induction of Hsps play important roles in retaining the mitochondrial integrity, even in the presence of Dox. While this could be one of the possible mechanisms, it is clear that there may be additional mechanisms involved. We recently reported that Dox-induced Hsp25 and its phosphorylation may protect myocytes by modulating p53 activity [110]. In cancer cells, Hsp25 overexpression is known to increase p53 degradation through increased ubiquitination and protect the cells by triggering cell cycle arrest/repair mechanism. Also Hsp25 is known to protect Akt and enhance its phosphorylation to activate p21, to induce cell cycle arrest/release and cell survival. As such, the question arises whether a similar Hsp25/p53 interaction is possible in the heart. However, the mechanism cannot be simply extended to cardiomyocytes because cardiomyocytes are terminally differentiated cells, and thus, unlike in cancer cells, there is no active cell cycling in cardiomyocytes. Thus Hsp25 cannot protect cardiomyocytes by altering cell cycle regulation, but it can protect cardiomyocytes by affecting p53-regulated apoptosis, by modulating p53 stability. However, studies with the use of cardiac H9c2 cells [113] were required for the initial understanding of Hsp25/p53 signaling upon treatment with Dox. The immortalized cardiac H9c2 cells that undergo active proliferation, and cardiac fibroblasts (with wild-type HSF-1 and HSF-1 knockout), which cannot be directly

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NECROSIS Fig. 3  Toxicity, HSF-1 binding to HSE and Hsp27 expression in Dox-treated cardiac H9c2 cells. (a) MTT assay. Control and heat-shocked cells, at 70–80% confluency were treated with various concentrations of Dox for 6 h followed by incubation in drug-free medium for 24 h and subjected to MTT assay. Data presented as relative percentage of cell viability, with respect to control cells with no drug treatment. In all the Dox concentrations studied (0.25–10.0 mM), significant protection was observed in heat-shocked cells (p < 0.01) [92]. (b) Representative western blots of Hsp27 expression in control and heat-shocked cells after treatment with Dox. The control and heatshocked cells were treated with various concentrations of Dox for 6 h and then exchanged with regular medium. After 24 h, the cells were lysed and used (20 mg of protein) for Western blots. The same membrane was reprobed for actin, to ensure equal loading of protein samples. The quantitative data presented (plotted as normalized with respect to control with no Dox) were obtained from a set of three experiments. Compared to the control treated with Dox, Hsp27

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compared with primary cardiomyocytes. These studies revealed that silencing the HSF-1 or Hsp27 in cardiac H9c2 cells by applying siRNA, enhanced cell death, while overexpression of Hsp27 protected the cells by modulating p21 expression. Using HSF-1 wild-type (HSF-1+/+) and HSF-1-knockout (HSF-1–/–) mouse embryonic fibroblasts and cardiac H9c2 cells, we found that p53 transcriptional activity (in terms of expression of its target proteins such as p21, Bax, etc.) depended on Hsp27 level [113]. Inhibition of p53 by Pifithrin-a (PFT-a) provided different ­levels of protection from Dox that correlated with Hsp27 levels in these cells. In HSF-1+/+ cells, PFT-α attenuated Dox-induced toxicity. However, in HSF-1–/– cells (which express a very low-level of Hsp27 compared with HSF-1+/+ cells), there was no such attenuation, indicating an important role of Hsp27 in p53-dependent cell death. Immunoprecipitation of p53 was found to coimmunoprecipitate Hsp27 and vice versa (confirmed by western blotting and matrix-assisted laser desorption/ionization time of flight) in Dox-treated cells, indicating that Hsp27 is binding to p53 in Dox-treated cells. Moreover, upregulation of p21 was observed in HSF-1+/+ and H9c2 cells, demonstrating that Hsp27 binding transactivates p53 and enhances transcription of p21 in response to Dox treatment. Further analysis with flow cytometry showed that increased expression of p21 results in G(2)/M phase cell cycle arrest in Dox-treated cells (Fig.  4) [113]. These studies clearly demonstrated p53 activation upon DOX and Hsp25 role in p53 transcriptional activity. In in vivo models also, p53 activation was reported in Dox-treated mouse hearts. Chua group found that Dox treatment increased p53 mRNA and the protein in the heart [115]. Upon using p53 inhibitors, namely PFT-a, the cardiac dysfunction was significantly reduced in Dox-treated mice. Addition of PFT-a also reduced the apoptosis by altering Bax [116]. Studying HSF-1 in vivo is much more complex compared to in  vitro studies, mainly due to the multiple mechanisms of HSF-1 activation. In our recent studies, we characterized the induction of Hsp27 in the Dox-treated hearts and found that HSF-1 is activated by Dox and increased the expression of Hsp27 mRNA, as in the case of in vitro studies. We found an increase in Hsp25 in Dox-treated hearts along with p53 (Fig.  5a–d). Hsp27/HSF-1 was shown to protect primary cardiomyocytes from mitochondrial-dependent apoptosis [110]. Furthermore, we demonstrated that Hsp27 regulates transcriptional activity of p53 in Dox-treated cells. We also found increased Bax, a proapoptotic protein that is transcriptionally activated by p53 upon treatment with Dox (Fig.  5e). Fig. 3  (continued) expression was statistically higher in heat-shocked cells (p > 0.05). (c) Nuclear extracts were prepared from control and Dox-treated cells and incubated with HSE, tagged with biotin. Binding was analyzed by blotting of biotin. Compared to control, HSF-1 binding to HSE is higher in Dox-treated mice. Consistently, addition of HSF-1 antibody (ab) shifted the band. (d) Schematic illustration of the proposed mechanism of protection from Dox toxicity. Doxinduced cell death occurs through activation of stress-induced protein kinases and subsequent signal transduction for apoptosis. On the other hand, hyperthermia also induces similar stressinduced kinases and small heat-shock proteins, which can be phosphorylated by MAP kinase. The phosphorylated small Hsps protect the cells

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Fig.  4  Regulation of p53 transcriptional activity by Hsp27 in Dox-treated H9c2 cells. Dox generates ROS which in turn activates p53. Activation of p53 results in increased transcription of the Bax and increases the p53 depended apoptosis and cell death [114]. However, increased expression of Hsp27 (by modes such as heat shock, oxidative stress or any other mode of HSF-1 activation) and phosphorylation by MAP kinase, results in p53 binding and increased transcription of p21. Such an increased transcription of p21 results in cell cycle arrest, DNA repair and cell survival [113]

Hsp25 was found to stabilize the p53 and transactivate it to increase Bax transcription. Thus, in the heart, Dox treatment increases accumulation of Hsp25 due to activation of HSF-1 and the accumulated Hsp25 transactivates p53 to increase the transcription of proapoptotic protein Bax. Although several aspects of the mechanism of Dox-induced heart failure, using various cellular and animal models, and human heart studies [9] have been determined, none of these studies has revealed the complete role of HSF-1 activation and dynamic induction of Hsp25 in Dox-treated hearts, as a response to the oxidative stress. Our study [110] was the first to demonstrate a link between HSF-1 activation and Dox-induced heart failure, and that Hsp25 could be triggering a signaling cascade causing the loss of cardiomyocytes. More importantly, HSF-1–/– showed increased survival against Dox, indicating that targeting HSF-1 or suppression of Hsp25 accumulation may be a potential therapeutic approach to prevent heart failure in Dox-treated patients. Very recently induction of proapototic Bax increase by increased activation of p53 was Fig. 5  (continued) increased more than 10 times in Dox-treated samples. Cleaved PARP-1 (89 kDa band) was observed to be higher in Dox-treated heart lysates due to increased Caspase-3 activity, consistent with higher Bax. (f) Schematic of the HSF-1 activation and cardiomyocyte loss in the heart upon Dox treatment. Dox-derived ROS activates HSF-1 to increase the transcription of Hsps, including Hsp25. Due to chronic oxidative stress, Hsp25 is phosphorylated by p38-MAP kinase (MAPKAP-2) and associated with p53. Then p53 activates the transcription of the proapoptotic protein Bax, which triggers apoptosis via the mitochondria-dependent signaling [110]

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Fig. 5  Hsp25 overexpression in Dox-treated failing hearts of mice. (a) Representative western blots of total, s-15 and s-86 phosphorylated Hsp25, Hsp70 and Hsp90. (b–d) Quantitative densitometric plots of total and s-15 and s-86 phosphorylated Hsp25, respectively, normalized with respect to GAPDH (n = 5). The error bars are the SEM. (e) Western blots of various p53 target proteins. Among the p53 target proteins probed, namely Bax, p21, MDM2, Bcl2, only the Bax

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independently reported [117]. This paradoxical observation, compared to the findings in in vitro secondary cardiomyocytes-derived cell lines, may be due to the difference in the activation mechanism of p53. Perhaps in cardiac cell lines, p53 preferably enhances the transcription of cell-cycle-arrest proteins/survival, as in the case of cancer cells, whereas in the terminally differentiated cardiomyocytes the same p53 is enhancing the transcription of proapoptotic proteins. This hypothetical duality of the function of p53 requires further experiments to confirm. On the other hand, small Hsps overexpressing mice such Hsp27 or Hsp20 overexpressing mice also showed improved cardiac function compared to their wild-type counterparts [118, 119]. In Human Hsp27 overexpressed mice, Liu et al reported an improved cardiac function compared to wild-type litter mates [120]. They found far-reduced ROS generation and protein carbonylation in these mice. In another study, overexpression of Hsp20 was found to improve cardiac function and reduced oxidative stress upon treating with Dox [118]. Although these studies reported overall protection of the heart from Dox-induced toxicity, the actual mechanism of such protection still remains elusive. The signaling pathways that are associated with such a protective mechanism have begun to emerge. Several of these studies determined the magnitude ROS generation in Hsp27 overexpressing mice or cell culture. We recently determined that the redox reaction of Dox-generated ROS could be minimized in cardiac cells [92]. The EPR spectra of DMPO spin adducts obtained from cells treated with various concentrations of Dox showed spectral features that are similar to the DMPO-OH adduct, indicating that the drug treatment causes generation of OH radicals either as a direct product or that there is iron and H2O2 for sets Fenton reaction. It is important to note that the magnitude of this spectrum decreased for heat-shocked cells as shown in Fig. 3d [92]. The quantitative plots have indicated lower values for the trapped radicals in heat-shocked cells, even in the higher Dox concentration range, indicating that the free radical burst generated by Dox is reduced by heat shock. Further studies on the nature of the free radicals generated (•OH or O2•−), their correlation to the magnitude of Hsp27, and the magnitude of the Dox-induced injury are needed. Another important observation that we have made is that p53 and s-86-phosphoHsp25 coimmunoprecipitate, indicating that phosphorylation of Hsp25 induces an interaction with p53, and in Dox-treated hearts this association is higher (Fig. 5). This observed Hsp25/p53 interaction was previously unknown, especially in the heart; however it was recently shown that HSF-1 knock out suppressed cutaneous tumerogenesis [70] and it was proposed that HSF-1 is a multifaceted factor that regulates p53 transcriptional activity. Hsps are also known to interact with many other receptor molecules. For example, recent studies have demonstrated that TLRs are activated by endogenous signals, including Hsps and oxidative stress, which may contribute to CHF [121]. TLRs are members of the interleukin-1 receptor family and are involved in the responsiveness to pathogen-associated molecular patterns. TLRs were also found to be taking part in the Dox-induced heart failure. Cardiac dysfunction, induced by a single injection of Dox (20  mg/kg IP) into wild-type (WT) mice and TLR-2-knockout (KO) mice, showed better cardiac function TLR-2 KO mice than WT mice [121]. Nuclear factor-kappaB activation and production of

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proinflammatory cytokines after Dox were also shown to be suppressed in TLR KO mice compared with WT counterparts. Survival rate was significantly higher in TLR-2 null mice than in WT mice 10 days after Dox injection. These findings suggest that TLR-2 may play a role in the regulation of inflammatory and apoptotic mediators in the heart after Dox administration. These results agree, in general with the hypothesis that Dox-induced apoptosis is primarily responsible for the loss of cardiomyocytes in the heart, that eventually lead to heart failure. However, it was recently reported that activation of the mammalian target of rapamycin, mTOR, protects the heart from Dox-induced heart failure. These authors have proposed that apoptosis could only be a secondary effect of the activated mTOR signaling in the overall pathogenesis [122]. However, there is no reported link between Hsp25 to mTOR function and hence further studies are required to delineate the relationship between the Hsp25 and mTOR in Dox-treated hearts.

6 Doxorubicin-Activated MAP Kinase and Small Hsps Phosphorylation Dox-induced ROS not only activate HSF-1 as discussed above, but it also activates protein kinases. ROS, generated by Dox-redox reactions, are aggravated by many factors in cardiac cells. For example, free redox cycling iron species have been found to play important roles in the development of Dox-induced cardiomyopathy [123]. Evidence of iron participation was obtained from the addition of membranepermeable iron chelators and their ability to attenuate the Dox-induced apoptosis. Mitogen-activated protein kinase (MAP kinase) signaling pathways are the primary intermediator of induction of apoptosis by oxidative stress. There are three major MAP kinase families, including extracellular signal-regulated kinases (ERKs), p38, and c-Jun NH2-terminal kinases (JNKs). In the cardiovascular system, ERK1/2 are potently and rapidly activated by growth factors and hypertrophic agents, thereby mediating cell survival as well as offering cytoprotection [124, 125]. Conversely, JNKs and p38-MAP kinases are activated by cellular stresses, including oxidative stress, and are thought to correlate with cardiomyocyte apoptosis and cardiac pathologies [126–128]. It has also been reported that inhibition of p38-MAP kinase by selective cardiac-specific overexpression of cysteine-rich metallothioneins reduced the Dox-induced apoptosis in the heart [129]. Interestingly, Hsps, which are induced as a stress response, have been found to be phosphorylated by MAP kinase activated protein-2 (MAPKAP-2) (downstream of p38-MAP kinase), and phosphorylated Hsps prevent apoptosis [130–133]. Elaborate studies have been carried out on the phosphorylation of small Hsps by different MAP kinases. Since MAP kinases are also activated by Dox, we have carried out studies on how these kinases especially MAPKAP-2 can phosphorylate Hsp27 in the Dox-treated cardiac H9c2 cells [92]. Similarly, proteomic analyses of Hsp25 in the Dox-treated hearts have shown that extensive oligomerization of Hsp25 occurs when the protein is overaccumulated due to activation of

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HSF-1 [110]. Phosphorylated Hsp25 is increased in Dox-treated hearts (Fig.  5); however, the ratio of phosphorylated Hsp25 to nonphosphorylated Hsp25 is not altered. At the same time, various MAP kinases are also activated due to oxidative stress, and p38-MAP kinase has been shown to phosphorylate the downstream target MAPKAP-2 which in turn phosphorylates Hsp25 [134]. Indeed phosphorylation of Hsp25/27 determines aggregation/disaggregation and functionalization of Hsp25/27 phosphorylation of Hsp25 in Dox-treated mouse hearts. Although the aggregation/disaggregation behavior of Hsp25 has been found to be regulated by phosphorylation of Hsp25, the observation of six spots in the 2D western blots separated by very narrow pI (observed almost as a continuum) could not be interpreted based on phosphorylation status alone [92]. Similar to our observation of multiple spots in 2D western blots, the Dohke research group observed an increase in the number of Hsp27 spots in 2D western blots of congestive failure hearts lysates, caused by increased pacing (tachycardia) [135]. Three prominent Hsp27 spots were observed in control hearts. These three spots were assigned to nonphosphorylated, monophosphorylated and diphosphorylated Hsp27 [136, 137]. In some studies Pro-Q staining (a marker of phosphorylated proteins) have stained all three of the spots, and these studies have assigned these spots as mono-, di- and tri-phosphosphorylated Hsp27 [135]. However, our study using MS/MS and phospho-specific antibodies has confirmed that all three spots were predominantly nonphosphorylated; rather, only a fraction of each spot represents a phosphorylated species in these aggregates [92]. This indicates that separation of Hsp27 in the first dimension (i.e., pH gradient) of the 2D gel is primarily determined by Hsp25 concentration and local pH dependent aggregation (especially at pH <6 [138]) rather than phosphorylatation status. Indeed, previous studies on the aggregation pattern of this protein have reported that aggregation behavior is dependent on both the pH and phosphorylation status of the protein. Aggregation has been found to be pH-independent about pH 7, but at lower pH ranges the aggregation is dependent on its pH [138]. Moreover, the aggregates were also found with phospho- and nonphospho-HSP25. Thus the different spots observed in the 2D western blots are aggregates of Hsp25 of different sizes, with both phosphorylated and nonphosphorylated Hsp25.

7 Effect of Dox-Induced Small Heat Shock Proteins on Antioxidant Enzymes Free radicals, involved in many biochemical and physiological processes, are formed due to the continuous use of oxygen by aerobic cells, and their levels are kept low by the different endogenous antioxidants, which remove oxidizing species at a rate similar to the rate in which they are formed. Balance is important, given the consequences of the continuous exposure of biological systems to an uncontrolled oxygen radical flux, functional damage, structural damage, and, ultimately, cell death [139]. Lipid peroxidation is a common consequence of

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free-radical-mediated chain reactions and some of the end-products, such as lipid hydroperoxides and other species that contain unpaired electrons, like alkoxyl and peroxyl radicals, are associated with damage to lipids, protein, and DNA [140]. Increased myocardial lipid peroxidation and protein oxidation are both important indices of oxidative injury on cellular structures in adriamycin administered rats [141]. MDA is the major abundant and reactive aldehyde resulting from the peroxidation of biological membranes [142]. MDA, a secondary product of LPO and protein carbonyl groups was used as an indicator of oxidative tissue damage by a series of chain reactions. LPO may directly induce DNA chain break and lipid peroxyl and alkoxyl may cause base oxidation in DNA [143]. Many studies have looked into the protection of endogeneous antioxidant enzymes by Hsps. Particularly, Hsps have been found to protect against the Dox-induced damage to the antioxidant enzymes. Overexpression of Hsp72 was found to enhance the MnSOD activity in ischemia/reperfused hearts [144]. The Hayward group has established that exercise can avoid Dox-related cardiac dysfunction through a mechanism that is related to induction of Hsps. They have found that the Hsp72 can protect the heart by enhancing antioxidant enzymes [145, 146]. In our recent work, we found that some of the antioxidant enzymes can be activated by small Hsps such as Hsp27 [123]. Changing the Hsp27 levels in cardiac cells, by using siRNA, was found to correlate with antioxidant enzyme (MnSOD) activity. Furthermore, we also found that the mitochondrial aconitase activity is protected in Hsp27 overexpressing cells, whereas the cells that have lower Hsp27 levels, such as HSF-1, were found to be more susceptible for aconitase inactivation. Based on these results, we proposed a novel mechanism that Hsp27 plays an important role in protecting aconitase from Dox-generated O2•−, by increasing SOD activity (Fig. 6). According to this mechanism, the protection of aconitase integrity by Hsp27 eliminates the catalytic recycling of aconitase-released Fe (II) and its deleterious effects in cardiac cells.

8 Future Perspective Due to their efficacy as anticancer drugs, Dox will certainly remain an important component of numerous chemotherapeutic regimens. Since treating cardiac complications is very troublesome and expensive, clinicians should provide the best available level of cardioprotection to patients undergoing chemotherapy. Welldesigned pharmacological cardioprotective intervention can be an effective option; however, the ability to rationally develop such agents largely depends on our understanding of the molecular mechanisms involved in both the cardiotoxic and anticancer effects of Dox. The studies that have been published so far clearly demonstrate that Hsps play an important role in Dox-induced cardiotoxicity. Perhaps Hsp25/p53 interaction may be an important area that remains poorly understood in Dox-treated hearts. This interaction may play a pivotal role in the overall pathogenesis of Dox-induced

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c

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CASPASE 3

+ H 2O 2 Fe2 +

{[4Fe-4 S

F ] 2+ -OO . [3 }

]+ 3S e

]2+ [4Fe-4S ACONITASE

APOPTOSIS Fig. 6  Mitochondrial aconitase and Hsp27 expression in Dox-treated cardiac H9c2 cells. (a) Western blot analysis of Hsp27, Hsp70, and Hsp90 in normal and siRNA-treated H9c2 cells. After the cells were maintained with siRNA for 24 h, the cells were either used for Western blot analysis or a toxicity assay. (b) Quantitative plots of Hsps, obtained from densometric analyses of blots. Error bars are SE from three independent measurements. (c) Cell viability (MTT assay) for normal (open symbols) and siRNA treated cells (closed symbols), measured at 24 h postdrug treatment. (d) Schematic illustration of the mechanism of Hsp27-offered protection of cardiac cells against the Dox-induced toxicity. Dox generates O2 in a redox reaction with NADPH. The generated O2 can potentially lead to necrosis or apoptosis pathways as depicted, depending on the quantity of O2 being generated. In cardiac cells, the aconitase becomes the target for the O2, which releases an Fe(II) from the [4Fe–4S] complex. The released iron redox cycles the NADP+, generating more superoxide. If Hsp27 is present, it increases the SOD activity, which will stop the aconitase disintegration because the O2 is dismutated at a faster rate, so that cells can survive. However, the accumulated H2O2 can inactivate the aconitase without the disintegration pathway (RSH, thiols in general)

heart failure. The overall mechanism seems to be very speculative at this time. Figure 5 shows the schematic illustration of the emerging mechanism. Dox-induced oxidative stress activates HSF-1, producing more Hsp25, which in turn binds and

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transactivates p53, resulting in the transcription of Bax. Bax targets mitochondria to trigger apoptosis and produces a slow loss of cardiomyocytes in the heart. Overall, it has been found that the increased induction of Hsp25 in Dox-treated hearts sensitizes cardiomyocyte death. Immunoprecipitation and immunoblotting have confirmed the association of Hsp25 and p53. Application of a p53 inhibitor increased the survival of Dox-treated HSF-1 mice (unpublished data), and survival also increased in the HSF-1 mice, where there was no significant accumulation of Hsp25. Hence, Dox-induced oxidative stress and HSF-1 activation play important roles in the development of heart failure in Dox-treated mice. This suggests that oxidative stress may be neither the main trigger nor the key executor of Dox cardiotoxicity, but rather increased ROS formation may be merely a secondary consequence of previous cellular and mitochondrial damage. Further studies using modern molecular methodologies, clinically relevant doses of Dox, and preferably in vivo experiments involving chronic Dox treatment, are needed to shed more light on these questions. A thorough understanding of how the small Hsp interaction with cellular function during Dox treatment will significantly advance our understanding of the mechanisms of Dox cardiotoxicity and allow us to rationally design novel cardioprotectants that are both safer and more effective. Acknowledgment  The studies that are summarized in this chapter, were supported by NIH grants R01HL078796, R21EB004658, R21HL094881 (to G.I.).

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Oxidative Stress in Cardiovascular Disease: Potential Biomarkers and Their Measurements Subhendu Mukherjee and Dipak K. Das

Abstract  The oxygen atmosphere surrounding us produces continuous oxidative stress because of the incomplete reduction of the O2 molecule. Oxidative stress mainly occurs in any system when the generation of reactive oxygen species (ROS) exceeds the system’s ability to neutralize and eliminate them. This imbalance of ROS can result from various pathways. Overproduction of ROS and their limited removal can result from mitochondrial respiratory chain, a lack of antioxidant capacity, exposure to environmental or behavioral stressors, etc. Such accumulation of ROS or oxidative stress can cause damage to all biomolecules, including lipids, proteins, and DNA. For this reason, oxidative stress has been implicated in a growing list of human diseases such as cancer, atherosclerosis, Parkinson’s disease, heart failure, myocardial infarction, Alzheimer’s disease, fragile X syndrome, etc., as well as in the aging process. Chronic heart disease is the major cause of death worldwide in the present era, and it is known that oxidative stress plays a crucial role in the morbidity and mortality due to cardiovascular disease. It is therefore very important to measure oxidative stress to check health. There are many techniques available to measure it. The major techniques include measurement of lipid peroxidation products, volatile hydrocarbons in breath, and oxidized DNA bases in urine. In this review, we will discuss potential biomarkers in cardiovascular disease and the methods of measuring oxidative stress. Keywords  Free radical • Hydroxyl ion • Malonaldehyde • Nitric oxide • Oxidative stress • Reactive oxygen species

D.K. Das (*) Cardiovascular Research Center, University of Connecticut Health Center, School of Medicine, 263 Farmington Avenue, Farmington, CT 06030-1110, USA e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_6, © Springer Science+Business Media, LLC 2011

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1 Introduction Oxidative stress is caused by excess accumulation of highly reactive molecules such as reactive oxygen species (ROS), due to an imbalance between the production and removal of ROS by detoxifying them in a biological system. In chemical terms, oxidative stress is defined by a large rise in the cellular reduction potential, or a large decrease in the reducing capacity of the cellular redox complexes. All forms of life maintain a reducing environment within their cells, which are preserved by enzymes that maintain the reduced state through a constant input of metabolic energy. Imbalance in this normal redox homeostasis can cause toxic effects through the production of peroxides and free radicals. These peroxides and free radicals damage all biomolecules, including proteins, lipids and DNA. The effects of oxidative stress depend on the amount of the production of the total stress. It can cause cell death; trigger apoptosis, even intense oxidative stress may cause necrosis [1]. Oxidative stress is formed mainly due to overproduction of both ROS and reactive nitrogen species (RNS) [2, 3]. ROS are ions or very small molecules that include free radicals such as superoxide (O2•−), hydroxyl (•OH), peroxyl (•RO2), hydroperoxyl (HRO2•−), hydrogen peroxide (H2O2) and hydrochlorous acid (HOCl) [2, 4]. RNS are active ions and/or molecules derived from nitric oxide, produced via the enzymatic activity of inducible nitric oxide synthase 2 (NOS2). RNS include nitric oxide (•NO), nitrogen dioxide (•NO2−), peroxynitrite (ONOO−), nitrous oxide (HNO2) and alkyl peroxynitrates (RONOO) [2, 4]. When RNS act together with ROS and cause damage to cells, nitrosative stress is produced. Free radicals are generated by the hemolytic bond fission of a covalent bond, and it contains unpaired electron. Because of the unpaired electron, these free radicals are highly excited and active.

2 Production of Free Radicals In mitochondrial respiratory chains, oxygen is converted to water in the final ­reaction. However, in mitochondrial respiratory chains, oxygen is frequently only “partially reduced” to form superoxide. The production of superoxide by the mitochondrial respiratory chain occurs continuously during normal aerobic metabolism. All the electrons moving through mitochondrial respiratory chains are never converted into water and some part of the electrons are converted into superoxide instead of water. The mitochondrial respiratory chain is a major source of ROS. Complex I and complex III of electron transport chains are the major sites for ROS production [5, 6]. Different pathologic conditions such as hypoxia [7], ischemia [8], reperfusion [9, 10], and aging [11, 12] induce the overproduction of ROS. Apart from mitochondria, there are other cellular sources of superoxide radicals. For example, xanthine oxidase is an important source of oxygen-free radicals. Xanthine oxidase catalyzes the reaction of hypoxanthine to xanthine and xanthine to uric acid. Molecular oxygen is reduced to superoxide anion in the first step and

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Fig. 1  Major source of reactive oxygen species

to hydrogen peroxide in the second step [13]. Cytochrome P450 has also been ­proposed as a source of ROS [14] (Fig. 1). Microsomes and peroxisomes are also endogenous sources of ROS [15]. In addition, white blood cells such as neutrophils produce oxygen radicals, which kill microorganisms or other pathogens that invade our bodies. In addition to these endogenous sources, there are other exogenous sources of superoxide production (Fig. 1). Abnormal environment such as hypoxia generate abundant and damaging ROS. Exposure to cigarette smoke and ozone gas also generates superoxide. Once formed, superoxide is converted to other ROS (Fig. 1). O2•− is produced by several different oxidases, including NAD(P)H oxidase, ­xanthine oxidase, cyclooxygenase and eNOS, via removal of one electron from oxygen. Initially formed O2•− is quickly eliminated by antioxidant defense mechanisms. O2•− is converted into H2O2 via the enzymatic activity of manganese superoxide dismutase (Mn-SOD) in the mitochondria and Copper/Zink superoxide dismutase (Cu/Zn-SOD) in cytosol [12/12]. This H2O2 is then converted into H2O and O2 by glutathione peroxidase (GSH-Px) or catalase (Fig. 1). In the presence of small amounts of iron or copper, H2O2 is converted into hydroxyl radicals (•OH) [Fenton reaction]. Hydroxyl radicals are extremely reactive and can cause severe damage to cells and tissues. NO is normally generated from l-arginine by endothelial nitric oxide synthase (eNOS) in the vasculature [2]. RNS are produced in animals starting with the

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r­ eaction of nitric oxide (•NO) with superoxide (O2•−) to form peroxynitrite (ONOO−) [16, 17]. •

NO (nitric oxide ) + O2 • − (superoxide ) →• ONOO − (peroxynitrite )

Peroxynitrite itself is a highly reactive species, which can directly react with various components of the cell. Alternatively, peroxynitrites can react with other molecules to form additional types of RNS including nitrogen dioxide (•NO2) and dinitrogen trioxide (N2O3) as well as other types of chemically reactive radicals. Important reactions involving RNS include: ONOO − + H + → ONOOH(peroxynitrous acid) → NO 2 (nitrogen dioxide) + • OH (hydroxyl radical) ONOO − + CO2 (carbon dioxide) → ONOOCO2 − (nitrosoperoxycarbonate) ONOOCO2 − → NO2 (nitrogen dioxide) + O = C(O• )O − (carbonate radical) •

NO + • NO2 = N 2 O3 (dinitrogen trioxide)

Production of one ROS or RNS may lead to the formation of other ROSs through a radical chain reaction.

3 Oxidative Stress in Health and Disease During the last three decades, scientists from different parts of the world showed their interest in the role of oxygen-free radicals in experimental and clinical medicine [11]. ROS, along with RNS, are well recognized for playing dual roles in both cell survival and cell death. While ROS are generated under the physiological conditions and involved in signaling cascade as secondary messengers, in defense mechanisms such as in phagocytosis, excess amounts of oxidative stress due to excess production of ROS/RNS cause damage to biomolecules such as DNA, lipids, and proteins. Nucleic acids are the main target of oxidative stress. DNA damage caused by oxygen-derived species is the most frequent type of injury encountered in aerobic cells. When this type of damage occurs to DNA, it is called oxidative DNA damage. The hydroxyl radical is known to react with both the purine and pyrimidine bases as well as the deoxyribose backbone, and thus damage the DNA [18]. Oxidative damage of DNA causes changes in DNA such as single- or double-stranded DNA breaks, purine, pyrimidine, or deoxyribose modifications, and DNA cross-links. Arrest or induction of transcription, induction of signal transduction pathways, replication errors and genomic instability can also occur due to oxidative DNA damage. All of these changes may occur in carcinogenesis [19, 20]. Ionizing ­radiation,

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a carcinogenic and exogenous source of ROS, can induce oxidative DNA damage [11]. Cigarette smoking also increases the DNA damage by 35–50% [5]. In addition to ROS, RNS have also been implicated in DNA damage [21]. Peroxynitrites and nitrogen oxides are mainly known to be involved in DNA damage. Peroxynitrite reacts with guanine and converts it to 8-nitroguanine. Due to its structure, this adduct has the potential to induce G:C→T:A transversions. While the stability of this lesion in DNA is low, in RNA, however, this nitrogen adduct is stable. It is known that mitochondrial oxidative DNA damage is involved in the carcinogenesis process [22]. It is known that oxygen radicals not only attack DNA in the cell, but also they attack polyunsaturated fatty acid residues of phospholipids [23, 24]. ROS can stimulate oxidation of low-density lipoprotein (LDL) and ox-LDL, which is not recognized by the LDL receptor. The lipid peroxidation process consists of three stages: initiation, propagation and termination. Initiation is the step of fatty acid radical formation. The initiators in living cells are most notably ROS such as OH•, which combines with a hydrogen atom to make water and a fatty acid radical. The fatty acid radical is not a very stable molecule, so it reacts readily with molecular oxygen, thereby creating a peroxyl radical. Once formed, peroxyl radicals (ROO•) react with PUFA leading to the formation of peroxyl-fatty acid radical. This cycle continues as the new fatty acid radical reacts in the same way, and finally as a result of peroxidation reaction malonaldehyde (MDA) is formed [24]. This compound is highly reactive and is one of the many reactive electrophilic toxic agents in cells. Malonaldehyde forms covalent protein adducts, which are referred to as advanced lipoxidation end-products (ALE), in analogy to advanced glycation end-products (AGE). MDA is mutagenic in bacterial and mammalian cells and carcinogenic in rats. Hydroxynonenal (4-hydroxy-2-nonenal, HNE) is another major aldehyde product of lipid peroxidation. This is weakly mutagenic but appears to be the major toxic product of lipid peroxidation. In addition, HNE has powerful effects on signal transduction pathways, which in turn have a major effect on the phenotypic characteristics of cells. The termination step is the step where the chain reaction comes to an end. When the concentration of radical species is high, then there is a high probability of two radicals to collide and form a nonradical species. MDA is also known to react with DNA bases and form M1G, M1A and M1C by reacting with G, A and C bases of DNA respectively [24]. Hydroxyl radicals react with proteins, which lead to the abstraction of a hydrogen atom from the protein polypeptide backbone to form carbon-centered radicals, which react with dioxygen to form peroxyl radicals under aerobic conditions [25]. The peroxyl radicals are then converted into the alkyl peroxides by reaction with the protonated form of superoxide (HO2•). Hydroxyl radicals can initiate the same reaction in the absence of ionizing radiation under in vivo conditions by the Fenton reaction. Thus in the absence of radiation, proteins are resistant to damage by H2O2 and by other simple oxidants unless transition metals are present. The amino acid side chains are most vulnerable to attack by various ROS and RNS. Nitric acid rapidly reacts with superoxide radical and produces peroxynitrite anion ONOO−, which reacts with cysteine sulphydryl groups to form tyrosine and tryptophan

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r­ esidues of protein and oxidize methionine residue to methionine sulphoxide. Protein oxidation by ROS is associated with the formation of many different kinds of inter- and intraprotein cross-linkages. Oxidation of proteins is associated with a number of age-related diseases including aging [26, 27]. Studies have shown that aging is often associated with the accumulation of oxidized forms of proteins. The accumulation of oxidized proteins in living systems may be due to (1) an increase in the steady state level of ROS/RNS and/or to a decrease in the capacity of an organism, or (2) a decrease in the ability to degrade oxidized proteins due to either a decrease in the protease concentrations and/or to an increase in the levels of protease inhibitors. ROS can activate several damaging pathways, which include accelerated formation of AGE, polyol pathway, hexos amine pathway and PKC. These metabolic pathways are known to induce micro- as well as macrovascular complications. O2•− and H2O2 stimulate stress-related signaling mechanisms through the activation of NF-kB, p38-MAPK and JAK-STAT, resulting in vascular smooth muscle cell (VSMC) migration and proliferation. In endothelial cells, H2O2 mediates apoptosis and pathological angiogenesis [28].

3.1 ROS in Vascular Disease Cardiovascular disease is the major cause of death worldwide. A large number of studies have shown that excess generation of highly reactive free radicals and accumulation of ROS play a key role in the pathophysiology of cardiovascular disease. Overproduction and/or insufficient removal of ROS results in vascular dysfunction, damage of cellular proteins, membrane lipids and nucleic acids. In this section we will discuss the role of ROS in different cardiovascular complications.

3.2 Atherosclerosis Atherosclerosis is an inflammatory disease of the artery characterized by the formation of atherosclerotic plaque – a focal accumulation of foam cells, smooth muscle cells, mononuclear leukocytes, lipids and extracellular matrix components [29], induction of inflammatory genes [30], inactivation of NO•, activation of matrix metalloproteinase [31], and increased smooth muscle cell growth [32]. Several studies suggest that oxidative stress and the production of ROS play a key role in endothelial cell dysfunction associated with atherosclerosis. Hyperlipidemia, ­diabetes, hypertension and smoking are the major risk factors for atherosclerosis. A current hypothesis suggests that modulation of the expression of certain vascular inflammatory genes via intracellular oxidative signal can regulate atherosclerosis [31, 33]. According to this hypothesis, various pro-inflammatory and pro-oxidant stimuli, such as tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b), ­oxidatively

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modified low-density lipoprotein (oxLDL), and angiotensin II may directly stimulate vascular cells to generate ROS. One of the most consistent hypotheses for atherogenesis is that this disease is triggered by the LDL oxidation caused by ROS from vascular wall cells. In rodents, atherogenesis is accompanied by increasing lipid peroxidation in  vivo. In case of LDL receptor knockout mice, the levels of the F2 isoprostane (iP), iPF2a -IV, are increased in both urine and aortic tissue [34, 35]. Vitamin E is known to limit atherogenesis [36–38]. Studies with animals carrying homozygous deletion of either CD36 (a receptor for oxidized LDL) or the 12/15-lipoxygenase (one of the enzymes that initiates lipid peroxidation) showed the role of macrophage-derived ROS in atherogenesis. When those animals were crossed with animals deficient in ApoE, the atherosclerosis lesion formation was shown to inhibit dramatically [39, 40], suggesting that macrophage-derived ROS play an important role in atherogenesis. Two recent studies showed that disruption of p47phox or gp91phox in ApoE mice had no effect on lesion development in the ascending aorta, but p47phox/ApoE double-knockout mice showed marked reduction in lesions in the descending aortas [41–43]. Several other studies have also supported the potential importance of the generation of intracellular ROS in the cells. Rabbits, fed with high cholesterol food for a period of weeks, were shown to produce higher amounts of O2• in the aorta [44], which in turn alters the endothelial-dependent relaxation. But treatment with polyethylene-glycolated SOD19 or probucol could reverse to the effect [45]. Several reports show that a high-cholesterol diet leads to elevation of O2•− and inactivation of •NO [46]. In rabbits, treatment of angiotensin II receptor antagonist resulted in an improvement in the endothelial dysfunction and lesion formation [45]. Similarly, angiotensin II-induced hypertensive rats showed higher production of O2•− and NAD(P) H oxidase subunit expression; such modification could be corrected by the treatment with modified forms of SOD [47]. It has also been shown that angiotensin II increases the lesion formation in ApoE−/− mice [48]. Angiotensin II upregulates the expression of receptors for the oxidized LDL [49], and thus contribute to oxidation of LDL. Angiotensin II is also responsible for hypertrophy of vascular smooth muscle in an ROS-dependent manner, which can participate in arterial thickening. These studies suggest that rennin–angiotensin systems play a key role in modulating oxidative stress. Matrix metalloproteinases (MMPs) are capable of degrading all kinds of extracellular matrix proteins. The MMPs play an important role in tissue remodeling associated with various physiological and pathological processes such as morphogenesis, angiogenesis, tissue repair, arthritis and metastasis. Excess MMPs degrade the structural proteins of the aortic wall. MMP-2 (gelatinase A, which degrades collagen IV) and MMP-9 (gelatinase B, which acts on collagen I fibers) are secreted by macrophages and vascular myocytes. Other studies have shown that ROS can activate MMP-2 and MMP-9 [31], which can modulate collagen degradation, leading to lesion instability. MMP activation by ROS could thus ­contribute to plaque rupture.

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3.3 Ischemia–Reperfusion Injury Absolute or relative shortage of blood supply resulting in a shortage of oxygen, glucose and other blood-borne fuels to a region of the body for a certain period is known as ischemia. Return of blood supply after a period of ischemia is known as reperfusion. Ischemia followed by reperfusion results in varying degrees of tissue damage, depending on the duration and extent of the ischemia. This type of injury caused by ischemia–reperfusion is known as ischemia–reperfusion injury. The reperfusion is accompanied by a burst of oxygen-free-radical generation, and thus myocardial damage caused by ischemia–reperfusion is due, at least in part, to the generation of ROS [50–54]. Reactive oxygen and nitrogen species generated during ischemia reperfusion can damage membrane lipids, protein, carbohydrates, and DNA. The pathophysiological consequences of such uncontrolled injury are widespread tissue damage and inflammation. There is evidence to support the role of free radicals in ischemia–reperfusion injury. Several studies show that consumption of endogenous and exogenous antioxidants, intake of free radical scavengers or agents that can induce antioxidants have a protective effect against ischemia–reperfusion injury [55–66]. The expression profile of the genes participating in the antioxidant defense has been shown to alter during ischemia–reperfusion injury [67–76]. These studies indirectly support the finding that free radicals play a key role in ischemia–reperfusion injury. Ischemia–reperfusion reduces oxygen to superoxide and hydrogen peroxide. Highly reactive hydroxyl radicals are formed from superoxide and hydrogen peroxide in the presence of redox-active transition metals by the Fenton type reaction [77]. One of the main sources of ROS in myocardial ischemia–reperfusion injury is the electron transport chain in mitochondria due to inadequate supply of oxygen. Early reperfusion results in a decrease in electron transport chain of mitochondria [78–81]. Inadequate supply of oxygen to organs leads to the accumulation of toxic metabolites and depletion of oxygen and adenosine triphosphate (ATP) and conversion of xanthin dehydrogenase to xanthin oxidase. Upon reperfusion, xanthin oxidase catalyzes the conversion of hypoxanthine to xanthine with the concomitant production of ROS. Elevated levels of ROS reduce the bioavailability of NO by converting it to ­peroxinitrites [82]. The reduced NO aggravates local oxidative stress and reduces blood flow. It is known that ROS disrupt the integrity of the endothelial cell ­junctions and increase the endothelial permeability, tissue edema and protein ­leakage [83]. Not only are ROS known to produce proinflammatory cytokines, platelet activation factor (PAF), and thromboxane A2 (TXA2), they also activate complement systems, reduce the production of prostacyclin (PGI2) and overexpress the adhesion molecules [84]. During ischemia, there is a shortage of oxygen supply at the mitochondria level, which results in intracellular acidosis and an increased concentration of inorganic phosphate by the breakdown of high-energy phosphates [85]. The anaerobic

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r­ espiration and lactate release during ischemia produce a small amount of ATP without the consumption of oxygen. During ischemia, the ionic conditions of the myocytes are altered, with an increase of the intracellular Na+ and a decrease of the K+ and Mg2+. Due to lack of ATP, sodium ion extrusion through sarcolemmal Na+/K+-ATPase is inhibited. Since the Na+/Ca2+ exchanger operates in reverse mode under Na+-overload, Na+ influx leads to Ca2+ influx in the ischemic tissue. As a result, ischemic tissue develops cytosolic Ca2+-overload. A Ca2+-overloaded ischemic cardiomyocytes may develop uncontrolled activation of the contractile machinery (contracture) upon reoxygenation or reperfusion [86, 87]. Reperfusion leads to rapid oxidative ATP production, which brings about the rapid recovery of the cytochrome oxidase system of the energy for cardiomyocytes to recover from cytosolic ion imbalance and reactivate the contractile function. This suggests that ROS formation and Ca2+ surge might be involved in the contractile dysfunction of the ischemic myocardium.

3.4 Diabetes Mellitus Diabetes is a risk factor for cardiovascular disease [88]. Micro- and macrovascular complications of diabetes include nephropathy, retinopathy, and atherosclerotic cardiovascular disease such as coronary artery disease, cerebrovascular disease and peripheral vascular disease. These cardiovascular complications are the major cause of death in the diabetic population [89]. There is evidence that oxidative stress is increased in diabetes conditions. Hyperglycemia-induced generation of free radicals contributes to the development and progression of diabetes. It has been suggested that ROS plays a key role in development of insulin resistance, b-cell dysfunction, impaired glucose tolerance and type 2 diabetes mellitus. A sedentary lifestyle and eating too much fast foods results in glucose and fatty acid accumulation within muscle, adipose tissue and pancreatic cells. High glucose and fatty acid accumulation accelerate the formation of ROS, mainly superoxide anions [90, 91]. It has been shown that fluctuating blood glucose concentrations contributes significantly to oxidative stress [92, 93], and overexpress the markers of oxidative stress and inflammation in the cells [94–96]. NO also plays a crucial role in diabetes; the reduction in NO availability is the key factor for endothelial dysfunction and diabetic angiopathy [97]. Hyperglycemia works paradoxically. It can both increase the production of NO and decrease the availability of NO [97–101]. Hyperglycemia produces superoxide anions, which can activate the nuclear factor kB (NF-kB). NF-kB is a transcription factor and it can overexpress inducible nitric oxide synthase (iNOS) [101], which in turn amplify the generation of NO. If the concentration of superoxide anions is high, then they rapidly react with the newly created NO to form the strong oxidant peroxynitrite [100]. Peroxynitrite being toxic to the endothelial cells, can initiate lipid peroxidation and nitration of amino acids [100]. Thus, hyperglycemia induces NO production but

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reduces NO availability. On the other hand, superoxide anions formed due to hyperglycemia inhibits endothelial NOS (eNOS) as a result of the reduction of NO ­production resulting in microvascular and macrovascular complications. Existing studies suggest that lipid peroxidation is, in part, responsible for the development of cardiovascular disease in diabetes [102]. ROS formed during hyperglycemia can initiate lipid peroxidation [103]. Once formed, lipid peroxides, after going through a series of complex reactions, ultimately bind to proteins and produce advanced lipoxidation end products (ALEs) [23, 104, 105]. Thus, lipoxidation is the covalent binding of products of lipid peroxidation reactions to proteins [106]. Oxidized LDL and ALE-containing LDL have proatherogenic effects, including an increase in smooth-muscle-cell proliferation, endothelin-1, matrix production, apoptosis in endothelial cells, macrophage-derived foam cell formation, protein kinase-C activation, transforming growth factor-beta (TGF-b) activation and decrease in nitric oxide bioavailability, proinflammatory effects, proclotting effects and inhibition of antioxidant enzymes [107].

4 Potential Biomarkers of Oxidative Stress As discussed in the previous section, oxidative stress causes major damage to nucleic acid bases, lipids, and proteins, which in turn induce a variety of cellular responses through the generation of secondary reactive species and ultimately lead to cell death by necrosis and/or apoptosis. Oxidative damage of these biomolecules should be checked regularly; otherwise it can contribute to the development of disease. Indeed, there is some evidence that supports the fact that oxidative damage is associated with several acute and degenerative diseases. Biomarkers are the compounds that can be measured experimentally and evaluated as indicators of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. In recent years, biomarkers are used in research to study the results of environmental prooxidant exposures and dietary antioxidant intake or to monitor surrogate measure of a disease process. Several in  vitro markers of oxidative/nitrosative stresses are available, including ROS/RNS themselves, but these biomarkers are not suitable for in vivo studies since they lack sensitivity and/or specificity. More importantly, ROS/RNS have very short half-lives. There should be criteria for choosing perfect biomarkers. To function as suitable biomarkers of oxidative modifications in relation to disease, it is necessary that such oxidation products be stable and easily detectable, and they should accumulate in detectable concentrations and reflect specific oxidation pathways. Also, they should correlate with the severity of the disease to use them as diagnostic tools. There are many markers of oxidative stress, but some are more easily detected in certain sample types such as cell, tissue, urine, blood, etc. In this section, we briefly discuss some of the more commonly used biomarkers of oxidative/nitrosative stress.

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The most common biomarkers used for the detection of DNA/RNA damages are 8-hydroxyguanosine (8-OHG), 8-hydroxydeoxyguanosine (8-OHdG), Abasic (AP) sites, and Double-strand DNA breaks. Among these markers, all can be used to check the oxidative stress in cells; 8-OHG and 8-OHdG can be used as biomarkers in all types of samples such as cells, tissues, blood, urine etc. 4-Hydroxynonenal (4-HNE), Malonaldehyde (MDA), 8-iso-Prostaglandin F2a (8-isoprostane) are the common biomarkers for lipid peroxidation. These can be detected in all types of samples. Carbonylated protein, 3-nitrotyrosine and advanced oxidation protein products are also used as biomarkers of oxidative protein damage. To check the ROS and antioxidant status of the system, the most useful biomarkers are catalase, superoxide dismutase, glutathione and S-glutathionylated proteins. These biomarkers are successfully used to measure oxidative stress in biological systems.

5 Measurement of Oxidative Stress From the above discussion, it is clear that oxidative stress is one of the main modulators/regulators of various kinds of diseases including cancer, cardiovascular disease and diabetes, which threaten all of our lives. For these reasons, oxidative stress measurement appears to be of utmost importance. There are several benefits to oxidative stress measurement. For example, measurement of LDL can predict susceptibility to developing atherosclerosis [108, 109]. Similarly, the measurement of pentane in breath and TBARS in blood can show the vitamin status, which can influence the disease [110]. Oxidative stress can be measured by several methods, among them the measurement of lipid oxidation products such as malonaldehyde or TBARS in blood or urine [111–114]; modified DNA bases and/on DNA adducts in peripheral blood cells [115, 116] in urine [117–119]; volatile compounds such as ethane and pentane in expired breath [46, 120, 121]; glutathione/glutathione disulfide in blood fractions [122–124]; and the “TRAP” assay that measures the total peroxyl radical-trapping antioxidant power of blood serum [125]. Some of these methods are discussed in the following sections.

5.1 Detection of Malonaldehyde for Oxidative Stress Measurement Lipid peroxidation is a well-established mechanism of cellular injury and is used as an indicator of oxidative stress in cells and tissues. Detection of oxidative stress is most frequently performed by measuring lipid peroxidation. Lipid peroxides are very unstable and form a complex series of compounds. These include reactive carbonyl compounds, of which the most abundant is malondialdehyde (MDA). An increased level of lipid peroxidation is associated with various

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Fig. 2  Chemical structure of MDA–TBA adduct

chronic diseases [126–128], and measurement of MDA is widely used as an indicator of disease [24]. MDA, formed after lipid peroxidation, reacts readily with amino groups on proteins and other biomolecules to form a variety of adducts [24]. The most popular method of MDA estimation consists of the measurement of thiobarbituric acid reactive substance (TBARS), formed from the reaction of MDA and TBA. MDA forms a 1:2 adduct with thiobarbituric acid (Fig.  2). TBARS levels are determined from a malondialdehyde equivalence standard. Previously, MDA–TBA adduct was measured by using spectrophotometric assay method [129]. This method is relatively simple but the reaction is nonspecific, because TBA not only forms a colored complex with MDA, but also reacts with many other compounds including ribose, biliverdin, amino pyrimidice and sialic acid [111]. So TBA reaction is likely to lead to incorrect interpretation of the results [129]. MDA estimation by the TBA method significantly overestimates MDA content [129]. A modified method is also available that uses a high-performance liquid chromatographic (HPLC) technique to quantitate the MDA–TBA adducts [129]. We showed that MDA can be estimated after being derivatized with 2,4-dinitrophenylhydrazine and extracted with pentane followed by estimation using HPLC [129]. This method allows rapid and accurate estimation of MDA to monitor the oxidative stress. However, the HPLC method has not gained popularity because of the length and complexity of the method. Nowadays the MDA–TBA adduct formed from the reaction of MDA in samples with TBA is also measured colorimetrically or fluorometrically. The procedure of estimating MDA is available in the literature and is very easy to perform. In brief, after collecting 1 ml of sample homogenate or body fluid in a test tube containing 0.05% butylated hydroxytoluene (BHT), 0.2 ml of 15% trichloroacetic acid is added. Then 1 ml of 0.75% TBA in 0.5% sodium acetate is added to the mixture. The final mixture is boiled for 15 min and centrifuged at 3,000 × g. Red color TBA–MDA complex comes in the aqueous layer that can be measure spectrophotometrically at 535 nm wavelength. To check DNPH-derivatized MDA, the sample homogenate is mixed with DNPH reagent and water. After vortexing this mixture, DNPH-derivatized MDA is extracted with pentane. Pentane extract is then dried and re-extracted with acetonitrile, which can be directly injected onto the Beckman Ultrasphere ODS C18 HPLC column. The DNPH derivative is detected at 307 nm.

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5.2 Measurement of Oxidative Stress by Measuring Exhaled Volatile Alkanes Lipid peroxidation generates volatile hydrocarbons, so the measurement of volatile hydrocarbons such as ethane and pentane in exhaled breath has been proposed as a method of measuring lipid peroxidation in vivo [130]. Ethane is formed from n − 3 polyunsaturated fatty acids such as linolenic acid, and pentane is formed from n − 6 fatty acids such as linolenic and arachidonic acids. These two hydrocarbons are noninvasive markers of lipid peroxidation. Alkyl ­radicals are formed from lipoalkoxyl radical by b-scission, these alkyl radicals then converted to alkanes by abstracting hydrogen. Actually, a very small amount of the lipid peroxides produces ethane or pentane and their concentration depends on the breakdown rate of peroxide [131], but the production of these alkanes reflects the extent of lipid peroxidation and thus measures the oxidative stress in vivo. These exhaled hydrocarbons can be measured by chromatographic methods. Exhaled breath is collected into a collapsible reservoir. During exhalation, the air coming from the dead space, contaminated with nasal and ambient ethane, is discarded in the atmosphere through a three-way valve. Once the exhaled air is ­collected, the reservoir is immediately sealed and the sample should be kept at room temperature for not more than 48  h. Before adding the sample in a gas ­chromatographic column, the column is conditioned overnight at 200°C, the oven temperature is set at 60°C, injector temperature at 140°C, and detector temperature at 160°C. The carrier gas used in this method is nitrogen. A sample of exhaled air is injected into the gas chromatograph using a gas-tight Hamilton syringe.

5.3 Measurement of Oxidative Stress by Measuring Isoprostanes Several markers are available to check the oxidative stress in biological systems but most of them are working in vitro and they have limited value in vivo because of their lack of sensitivity and/or specificity or require invasive methods [132]. Isoprostanes are the example of anylates formed in vivo and used as in vivo markers of oxidative stress. Isoprostanes are prostaglandin (PG)-like substances that are produced in vivo primarily by free-radical-induced peroxidation of arachidonic acid [133]. These compounds are a series of isomers containing a prostane ring of prostaglandins; they are termed F2-isoprostanes (isomeric in structure to cyclooxygenase-derived PGF2a). Other prostaglandin H2-like intermediates of isoprostane pathway are also formed in vivo and become reduced to F2-isoprostane but also undergo rearrangement to form E2-, D2-, A2-, J2-isoprostanes isothromboxanes, and highly reactive g-ketoaldehydes, termed isoketals. It has now been established that measurement of F2-isoprostanes is the best available biomarker of oxidative stress status and lipid peroxidation in vivo [132]. F2-isoprostanes are initially formed in situ esterified in phospholipids, then by the

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action of released phospholipase it becomes free and is released [134]. F2-isoprostanes are detectable in both esterified form and free form. Esterified forms are detectable in all normal biological tissues and in free form in all normal biological fluids such as plasma and urine. The amount of isoprostane indicates the “physiological” levels of oxidative stress [132, 135]. GC/MS is used to measure isoprostanes in biological tissues and fluids, but this method is expensive and time-consuming. Lately, immunoassay is being used for isoprostanes measurement. Samples of plasma or urine are generally enriched with a deuterated (iPF2a-III-D4) standard. To liberate bound isoprostanes, the plasma or urine sample is treated with KOH, and then the alkali is neutralized with KH2PO4 and loaded onto an ­isoprostane affinity column. The bound isoprostane is eluted with 95% ethanol. The concentrated sample is then injected into a C-18 HPLC column and the elution of importance is done by acetonitrile gradient in water and analyzed by electrospray negative ionization.

5.4 Detection of Oxidative DNA Damage by Measuring 8-Hydroxyguanosine Formation DNA is a well-known target for free-radical attack. ROS such as OH•, O2•−, and H2O2 produced during oxidative stress cause damage to DNA, which in turn leads to degenerative diseases such as cancer, cardiovascular disease and other agerelated diseases. Hydroxyl radical formed during oxidative stress attacks the guanosine residue of DNA and damages DNA [136]. Damaged DNA is then enzymatically repaired, and 8-hydroxydeoxyguanosine (8-OHDG) is produced and excreted in urine without further metabolism [136]. Hydroxyl radical (OH•)DNA adducts (8-OHDG) can be measured by using GC-MS [137]. But the method of measurement using GC-MS often requires derivatization and, as a result, may produce erroneous results [138]. The most common method of 8-OHDG measurement is to use a simple HPLC with electrochemical detection (LCEC) [136, 139, 140]. This method has better limits of detection than optical methods. In addition, this EC detection has greater selectivity with complex samples. Using capillary electrophoresis is a better choice than using LC because it provides higher separation efficiency. Capillary electrophoresis with electrochemical detection (CEEC) provides high efficiency separation and is good to use in case of complex biological samples. To check 8-OHDG in DNA, at first the DNA is isolated from the tissue or cell and then hydrolyzed with nuclease P1. Then the hydrolyzed DNA is injected onto an HPLC column. 8-OHDG is detected by using a wavelength of 297 nm. Further, purity of 8-OHDG is accomplished by the comparison with a standard by UV scanning. To measure 8-OHDG in urine samples, the sample is first frozen at −20°C. During analysis, the sample is brought to room temperature by putting it in a water bath followed by vortexing. Then the mixture is filtered and loaded to

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a solid-phase extraction column. 8-OHDG is eluted from the column with 20 or 30% MeOH. The measurement is done by comprising the value with standard, which is extracted in same condition.

5.5 Measurement of ROS Generation by Dihydroethidium Dihydroethidium (DHE) is a reduced and nonfluorescent precursor of ethidium bromide; it can easily enter the cell. In the presence of oxidative stress, hydroethidium is transformed intracellularly into the fluorescent compound ethidium bromide, which binds to DNA and can be detected by red fluorescence [141]. Hydroethidium is especially sensitive to superoxide anion, and to a lesser degree to H2O2 [142]. By measuring the fluorescent intensity, the amount of ROS generation can be measured. But it has recently been found that this is not precisely correct. 2-Hydroxyethidium is formed when DHE reacts with O2•−. 2-Hydroxyethidium has a molecular weight 16 U greater than DHE [143, 144]. The HPLC technique can be used to separate 2-hydroxyethidium from its parent DHE and ethidium, and the 2-hydroxyethidium peak can then be used to estimate the intracellular production of O2•− [145]. To measure the intracellular O2•− by determining DHE, the sample is at first incubated with the inhibitors of xanthine oxidase, NADPH oxidase and endothelial NO synthase. The sample is then homogenized in methanol. After filtering, the sample is loaded onto the HPLC column (C-18 reverse-phase column). Ethidium and 2-hydroxyethidium are detected with a fluorescence detector using an emission wavelength of 580 nm and an excitation of 480 nm. UV absorption is used for the detection of DHE. The 2-hydroxyethidium peak reflects the amount of O2•− formed in the tissue.

5.6 Measuring of Oxidative Stress by Using Dichlorofluorescein (DCFH) Diacetate 2¢,7¢-Dichlorodihydrofluorescein diacetate (DCFHDA) is one of the most widely used techniques for directly measuring the redox state of a cell. It has many advantages over other techniques. It is very easy to handle and quite economical. DCFHDA is a stable and relatively nonfluorescent molecule. It can easily diffuse into cells. Cellular esterases cleave DCFH-DA into 2¢,7¢-dichlorodihydrofluorescein (DCFH2), which can be oxidized to 2¢,7¢-dichlorofluorescein (DCFH) by inorganic and organic hydroperoxidases such as peroxidases, cytochrome c, Fe2+, etc. [146]. DCFH is a fluorescent molecule, so accumulation of DCFH may be measured by an increment in fluorescence. Fluorescence is assumed to be proportional to the concentration of hydrogen peroxide in the cells [147, 148]. Although DCFH-DA is a widely used method of measuring hydrogen peroxide concentration in cells, there

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are some drawbacks to this method. Superoxide and NO generation are also capable of oxidizing DCFH2 [149]. All types of SOD, such as cytosolic SOD (Cu, Zn-SOD), mitochondrial SOD (Mn-SOD) and extracellular space SOD (Ec-SOD), convert superoxide into hydrogen peroxide, which in turn oxidize DCFH2 into DCFH. To eliminate this drawback, inhibitors of SOD can be used. DCFH2 may also be oxidized independently of hydrogen peroxide. Peroxynitrite, a product of reaction between nitric oxide and superoxide, can oxidize DCFH2 to DCFH. Therefore, inhibitors of NOS should be used to prevent NO mediated DCFH2 oxidation [150]. For measurement of H2O2, the sample is incubated with DCFH-DA for 30 min at room temperature. H2O2 detection can be confirmed by treatment with polyethylene glycolcatalase, which can be used as negative control. From the fluorescence of DCFH formed via the reaction of H2O2 and DCFH-DA can be measured by checking the fluorescence. All of the images are acquired at identical settings. For cultured cells, DCFH-DA fluorescence can also be measured using fluorescenceactivated cell sorter analysis.

5.7 Measuring Cytochrome c Reduction Diradical oxygen contains two unpaired electrons. In the presence of various stimuli, oxygen gains an additional electron and becomes O2•−, which is highly reactive. Superoxides are very good oxidants because in most cases they extract an electron from another molecule and forms of H2O2. But in the case of ferricytochrome c, the mechanism is a bit different. As the electron potential of O2•− is higher, it donates its extra electron to ferricytochrome c and reduces it to ferrocytochrome c. When ferricytochrome c is reduced, its spectrophotometric absorbance is altered in a very specific fashion. Absorbance at 540 and 560 nm remain unaltered, but absorbance at 550 nm is increased. From this increase in absorbance, we can measure the amount of O2•−. But there are some drawbacks to this method; some enzymes or other molecules can reduce ferricytochrome c which can increase in absorbance at 550 nm but this change in absorbance is not specific for O2•−. To avoid this problem, the assay should be performed both in the presence and absence of SOD. The amount of O2•− produced can be measured from the difference between the increase in optical density both with SOD treatment and without it. In studies of the cardiovascular system, cytochrome c reduction has been used to measure O2•− produced by mouse aortic segments [151], human vessels [152], mesenteric arterioles [153], segments of the cardiac ventricles, and pieces of the left atrium [154]. To measure O2•− from a segment, there should be at least two sets of experiments – one in the presence of SOD and the other in its absence. One set of samples is placed in a buffer [mixture of NaCl, KCl, NaH2PO4, CaCl2, MgSO4, glucose, deferoxamine mesylate, and acetylated ferricytochrome c] and the other is placed in the same buffer with manganese superoxide dismutase. The samples are incubated in a 96-well plate. Catalase, which removes H2O2, is added to the reaction mixture to stop the reoxidation of reduced ferricytochrome c. After incubating for

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30  min at room temperature, the absorbances of the buffer are measured at 540, 550, and 560 nm, using a 96-well plate reader. The amount of O2•− is determined by the differences of optical density between the SOD containing buffer and the buffer without SOD at 550  nm. The amount of O2•− formed is calculated from the formula: O2 • − = (∆ OD without SOD − ∆ OD with SOD) / 21.1 mmol / L−1cm −1 where DODs are the shift of absorbance at 550 nm above the average of OD at 540 and 560  nm, 21.1  mmol/L−1  cm−1 is the extinction coefficient for reduced cytochrome c.

5.8 Measurement of Oxidative Stress by Reduced Glutathione Method To maintain the redox homeostasis against the oxidative challenge inside the ­biological system, cells have developed a complicated antioxidant system. Reduced glutathione (GSH) is the major nonenzymatic antioxidant in the biological system. It is a tripeptide containing a sulphydryl group and plays a central role in the defense against oxidative damage and toxins. GSH acts as a cofactor for glutathione peroxidase and glutathione S-transferase and can react directly with hydrogen peroxide, superoxide anion, and hydroxyl and alkoxyl radicals by its free sulphydryl groups. The extracellular GSH can react with cytotoxic aldehydes produced during lipid peroxidation [155]. The oxidized glutathione (GSSG) is formed by enzymatic oxidation catalyzed by glutathione peroxidase. Therefore the measurement of GSH level and/or the ratio of GSH to GSSG are valuable indicators of the oxidative stress status in the biological system. Numerous methods have been developed to quantify GSH and GSSG. Due to the chemical structure of GSH and GSSG, it is very difficult to find appropriate chromophores and fluorophores for them. To carry out this method successfully, it needs to be derivatized to increase sensitivity and thus increase the sample processing time, which may lead to autooxidation of GSH. Recently, a simple HPLC method with electrochemical detection has been used to quantify GSH and GSSG simultaneously. This method takes a very short time for sample preparation, so this method has less of a chance of GSH autooxidation; also, the sensitivity of the reaction is much higher than other methods. In this assay, samples are deproteinized with perchloric acid and then directly injected into HPLC system. C18 reverse column is used for the separation of GSH and GSSG. The detection of GSH and GSSG is performed on a coulometric electrochemical detector. To measure GSH and GSSG molecules in tissue, the tissue sample is homogenized with the buffer containing EDTA, NaClO4 and H3PO4. Then the homogenate is mixed with metaphosphoric acid to precipitate proteins. The mixture is then vortexed and centrifuged at 8,000 × g for 10 min at 4°C. Supernatant is collected and loaded onto the HPLC column. The analysis of glutathione molecule is done in 5–7 min.

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5.9 Measurement of Oxidative Stress by Using Electron Spin Resonance Electron spin resonance (ESR) or electron paramagnetic resonance (EPR) spectroscopy is the technique used for studying chemical species containing one or more unpaired electrons, such as organic and inorganic free radicals or inorganic complexes possessing a transition metal ion. ESR is based on the absorption of microwave radiation stimulated by an electromagnetic field in molecules with unpaired electrons. The typical ESR spectrometer consists of a microwave generator, a resonator cavity flanked by a pair of electrical magnets. When a paramagnetic molecule is kept in a magnetic field, the unpaired electrons start to act like a tinny magnet and orient either in a parallel or antiparallel direction to the magnetic field. Due to these orientations of unpaired electrons, two distinct energy levels are formed when microwave energy is supplied, such that microwave energy absorption causes transition from a lower energy level to an excited energy level. This transition occurs when the difference between the two states of electron energy due to magnetic field and microwave energy become similar. In summary, the absorption of microwave energy, which occurs due to the transition of unpaired electrons in an applied magnetic field, is measured by ESR. The amplitude of the ESR signal is proportional to the number of the unpaired electrons present in the sample. The free radicals in biological systems are not stable; they generally have a very short half-life, so it is not possible to detect those free radicals by using ESR. To avoid this difficulty, compounds known as “spin traps,” which can form stable adducts with free radicals, are used. Examples of such spin traps are N-t-butyl-a-phenylnitrone, a-(4-pyridyl 1-oxide)-N-tert-butylnitrone, 5,5-dimethyl-1-pyrroline-N-oxide, 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide, and 2- ethoxycarbonyl-2methyl-3,4-dihydro-2H-pyrrole-1-oxide [156]. Spin traps-free radical adducts yield specific spectra when subjected to ESR spectroscopy. Spin traps can be used to both identify and quantify the free radicals formed. Theoretically, spin trapping with nitrones should be an ideal way to detect free ­radicals in a biological system. However, there are problems in using nitrones because nitrone-free radical adducts are unstable and can easily split within seconds to minutes, and nitrones have low reactivity with O2•− compared to other O2•− probes. More importantly, nitrone radical adducts are very susceptible to bioreduction when exposed to cells or tissues, and flavins, thiols, or ascorbate can convert them to ESR silent species by reduction of their 1-electron. To solve this problem of nitrone spin traps, investigators have turned to other compounds, such as cyclic hydroxylamines, which are not spin traps but can oxidize free radicals to form stable radicals with a longer half-life period of several hours, which can be easily detected by ESR. Examples of cyclic hydroxylamines are 1-hydroxy-3-carboxy-2,2,5,-tetramethyl-pyrrolidine hydrochloride (CPH), 1-hydroxy-3-methoxycarbonyl-2,2,5,5- tetramethylpyrrolidine (CMH), 1-hydroxy2,2,6,6-tetramethylpiperidin-4-yltrimethylammonium.

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5.10 Measurement of Oxidative Stress by Measuring Hydroxyl Free Radical Formation Hydroxyl radicals (•OH) are generally formed by hemolytic cleavage of water caused by exposure to high energy radiation (gamma rays, X-rays, UV light). Hydroxyl radicals are highly studied among oxygen-derived free radicals because of their extreme reactivity and toxicity. Hydroxyl radicals are very reactive and their halflives are very short. Therefore, it is very difficult to measure •OH directly, and it must be treated with an •OH scavenger that can trap •OH and form a relatively stable compound that can be measured. It has been demonstrated that salicylate can be used to trap •OH. 2,3-Dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA) are formed after salicylate reacts with •OH. At least one of its products, 2,3-DHBA, does not occur endogenously and once formed cannot be further meta­ bolized. With the high sensitivity of coulometric electrochemical detection, salicylate trapping has been successfully used in the measurement of •OH. In this method, samples are incubated with salicylate for several hours. Samples are then collected and treated with perchloric acid to deproteinize. The deproteinized samples are injected directly onto HPLC for analysis. The separation of salicylate adducts is performed on a C18 reversed column and detection is performed on a coulometric electrochemical detector.

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In vivo Imaging of Antioxidant Effects on NF-k B Activity in Reporter Mice Ingvild Paur, Harald Carlsen, and Rune Blomhoff

Abstract  In this chapter, we describe how we have utilized transgenic reporter mice and cell lines to investigate how phytochemicals/antioxidants and dietary plants can modulate NF-kB transcription factor activity. Several dietary plants are found to be potent modulators of NF-kB activity in vivo. Even though these dietary plants are rich in antioxidants/phytochemicals, the contribution of antioxidant properties per se, to these effects on NF-kB remain uncertain. Also, the relationship and interactions between antioxidants/ROS and the NF-kB signalling pathway is complex and still not fully elicidated. In line with this discussion, we find different dietary plants to be able to both inhibit and induce NF-kB activity, and we speculate that both might contribute to the preventive effects of diets rich in plant-based foods towards chronic diseases. Keywords  In vivo imaging • NF-kB • Nutrition • Phytochemicals • ROS

1 Introduction Inflammation is a complex series of responses to stimuli or agents that are perceived as harmful to the organism. The acute inflammatory response is initiated mainly by resident macrophages that release inflammatory mediators upon activation. Vasodilation and increased permeability of the blood vessels lead to leakage of plasma proteins and fluid as well as migration of leukocytes into the affected tissue. If the acute responses lead to elimination of the cause of inflammation, resolution and, if necessary, tissue repair follows. If prolonged, inflammation may progress into a chronic inflammation, which in turn may contribute to the pathogenesis of

R. Blomhoff (*) Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo, Blindern, 0316 Oslo, Norway e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_7, © Springer Science+Business Media, LLC 2011

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chronic diseases such as CVD, diabetes, neurodegenerative diseases and several cancers [1, 2]. Dampening of inflammation may thus delay development of such diseases.

1.1 Inflammation in Cancer and Cardiovascular Diseases Inflammation and leukocyte infiltration is a common feature of atherosclerosis and cancers. In atherosclerosis, cholesterol is a common target for prevention of CVD. Still, even the most effective cholesterol-lowering drugs will only reduce coronary events by one-third [3]. In addition to elevated Low-Density Lipoprotein (LDL) levels, low levels of High-Density Lipoproteins (HDL), hyperglycemia in diabetes and access adipose tissue that produces pro-inflammatory cytokines can all contribute to the inflammation in atherosclerosis. Inflammatory cells infiltrate the intima of the arteries and can contribute to both initiation and progression of the plaque development [3]. As early as in the nineteenth century, Virchow described the link between cancer and inflammation on the basis of observations that tumors arose at sites subsequent to chronic inflammation, and that inflammatory cells were present in tumors [2]. Still, evidence for molecular links between cancer and inflammation has only emerged within the last few years [4, 5]. Epidemiological studies have found strong associations between chronic inflammatory diseases, and cancers of the corresponding site – for example, between inflammatory bowel diseases and colon cancer [6], and between Helicobacter pylori infection and gastric cancer [7], whereas the administration of non-steroid anti-inflammatory drugs reduces the risk of, and mortality caused by, colon cancers and possibly several other cancer that are linked to chronic inflammation [8]. The complex microenvironment has great impact on the progression and aggressiveness of a tumor [reviewed in [2, 9]]. Solid tumors do not only consist of malignant cells from the affected tissue, but also various non-malignant cells such as endothelial cells and inflammatory cells. Of the myeloid cells, tumorassociated macrophages (TAMs) have been shown to be important in onset, growth, and metastasis of tumors [10, 11]. Also, over-expression of inflammatory cytokines can promote tumor development (reviewed in [12, 13]). When inflammatory cells, cytokines or transcription factors mediating inflammation is targeted in experimental models, both incidence and metastasis of cancers is reduced [2, 4, 12, 14, 15]. Whether inflammation is sufficient for initiation of cancers, or if carcinogens have to be present, remains to be fully elucidated. However, cancer may arise without known exposure to exogenous carcinogens. If carcinogens are necessary for initiation of cancers, the presence of endogenous carcinogens is a likely explanation for the absence of exogenous carcinogens in many cancer cases. Reactive oxygen species is a candidate for such endogenous carcinogens.

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2 Nuclear Factor Kappa B 2.1 The NF-k B Family of Transcription Factors The family of transcription factors known as Rel/NF-kB is crucial for normal physiology and plays a critical role in cellular stress-, immune- and inflammatory responses [16]. Rel/NF-kB was first described by Sen and Baltimore in 1986 [17], and has since become one of the most intensely studied signaling pathways. Although NF-kB is essential in normal physiology, a number of human disorders involve inappropriate regulation of NF-kB. Several chronic diseases [18–22] and various human cancers [23] have been associated with an aberrant up-regulation of NF-kB activity. Also, recent studies have identified NF-kB as a direct link between inflammation and cancer [4, 5, 24]. In vertebrates, the NF-kB family of transcription factors is comprised of five structurally related proteins p65 (Rel A), Rel B, c-Rel, p50 (and the precursor p105), p52 (and the precursor p100) which form hetero- and homodimers [16]. These five proteins all contain a conserved N-terminal Rel homology domain (RHD) of 300 amino acids. The RHD is responsible for DNA binding, dimerization and interaction with the inhibitors (IkBs). p65, Rel B and c-Rel also contain a C-terminal transactivation domain (TAD), which interacts with general transcription factors, such as TFIID and TFIIB, and co-activators such as p300 and CBP to promote transcription. Both p50 and p52 are synthesized as precursors which are cleaved to produce p50 and p52. Homodimers of p50 and p52 can repress NF-kB-mediated transcription, since these lack the transactivation domain [16, 25]. The best characterized dimer is p65/p50, and the name NF-kB is often used synonymously with p65/p50. Normally, the NF-kB dimers are mainly sequestered in the cytosol bound to the inhibitor factors, the IkBs (Fig. 1). Numerous stimuli can activate NF-kB through membrane bound receptors and phosphorylation of IkB by the IkB-kinase complex (IKK) with the two catalytic subunits, - IKKa and IKKb and regulatory subunit NF-kB Essential Modulator (NEMO). Upon stimulation, IkBa is phosphorylated, ubiquitinated and degraded in S26 proteaosomes. IKKb phosphorylates IkBa at the serine residues (S) 32 and S36. The release of IkBa unmasks a nuclear import signal on the p65/p50 dimer, which, in turn, will delocalize to the nucleus. In the nucleus, the NF-kB dimer binds to specific kB-sites with the consensus DNA sequence 5¢-GGGRNNYYCC-3¢ (G = Guanine, C = Cytosine, R = Purine, Y = Pyrimidine, N = any base) [26]. For maximal effect of NF-kB on transcription, further modifications of the NF-kB dimer and the chromatin fiber are needed. These modifications include sitespecific phosphorylation and acetylation of p65, p50 and histone tails. For example on p65, phosphorylation of S276 or S311 facilitates the recruitment of co-activators p300 and CBP; phosphorylation of S529 and S536 facilitates the assembly of p65 with other basic transcription proteins; acetylation at lysine (K) K221 and K218

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Fig. 1  The classical NF-kB signaling pathway. The NF-kB complex is inactivated by binding to IkB in the cytoplasm. Upon stimulation and phosphorylation by IKK, IkB is degraded. The p65/p50 dimer translocates to the nucleus, where it binds to kB-sites and promotes transcription. IkBa can shuffle nucleic p65/p50 dimers to the cytoplasm to terminate transcription

hinders reassembly with newly synthesized IkBa, and acetylation at K310 is necessary for maximum transcriptional activity of p65 [25]. Yang et al. [27] found IKKb to be essential in the phosphorylation of S536 on p65 in monocytes/macrophages stimulated by lipopolysaccharide (LPS). The NF-kB signaling pathway is activated through a range of membranebound receptors (e.g., toll-like receptors) and their corresponding adaptor proteins and kinases upstream of the NEMO komplex/IKKs. Furthermore, the acetylation and phosphorylation of histone tails by the co-activators help remodel or open the chromatin fiber in order to facilitate the assembly of the basal transcription complex and transcription of the genes. All these events together lead to NF-kB specific transcription of genes with a kB-site in its promoter region. NF-kB target genes, more than 150, codes for proteins that are central players in inflammation, activation of the innate immune system and anti-apoptotic signaling [28]. IkBa does not only bind and inhibit NF-kB in the cytoplasm, this inhibitor can also bind NF-kB dimers in the nucleus and shuffle these back to the cytoplasm (Fig.  4), thereby terminating NF-kB transcription. The IkBa is a p65/p50 target gene, induced within 15–30 min. after activation of NF-kB, and thus lead to negative feedback of NF-kB signal [29].

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Two main pathways lead to delocalization of NF-kB dimers to the nucleus and consequent transcriptional regulation of target genes [30]. The classical NF-kB signaling pathway (described above) is activated through IKKb by pro-inflammatory stimuli, bacterial- or viral infection, and various forms of stress, such as UV-radiation and environmental toxins [28], and is essential for the innate immune system and anti-apoptotic signaling. The alternative NF-kB pathway plays a critical role in B-cell mediated responses and development of lymphoid organs and is activated by members of the TNF family other than TNF-a [30]. This pathway is dependent on IKKa, which mainly activates the Rel B/p52 heterodimer. Lawrence et al. [31] have also shown that the alternative pathway contributes to the resolution of inflammation. IKKa is essential for B-cell maturation and microstructure of the lymphoid organs. On the other hand, B-cell responses to inflammatory signals, such as LPS, is independent of IKKa, since this response is mediated through the classical NF-kB pathway [32]. Ghosh and Karin [33] suggest that a similar separation of function between IKKa and IKKb might also be present in macrophages, proliferation and activation depending on IKKb and differentiation depending on IKKa. NF-kB is identified as a promising therapeutic target in inflammation, neurodegenerative diseases and cancer. Currently known NF-kB inhibitors include glucocorticoids, ibuprofen, ubiquitin ligase inhibitors, and several structurally unrelated antioxidants such as ascorbic acid, glutathione and N-acetyl-cysteine [34]. Foods or food substances with the ability to modulate NF-kB activity can be of both nutritional and pharmacological interest.

2.2 NF-kB in Disease Targeted knock-out mice for genes encoding the NF-kB transcription factors, the IkB inhibitors and the IKKs have been developed. Most of these knock-out mice are viable, but display impaired immune responses, and some have defective lymphoid organ and skin development [35], showing that the different components of the signaling pathway to a certain extent can compensate for each other. Knockouts for p65 [36], IKKb [37] and NEMO [38] however, display embryonic lethality due to massive liver apoptosis. Although NF-kB is essential in normal physiology and organ development, several human disorders involve inappropriate regulation of NF-kB. Aberrant regulation of factors in the NF-kB pathway has been noted in several human cancers [23]. Also, atherosclerosis [18] and several cardiac pathologies [39], neurodegenerative diseases [19], rheumatoid arthritis [20], asthma [21] and inflammatory bowel disease [22] have been associated with a persistent increase in NF-kB activity. NF-kB is also activated after myocardial infarctions [40]; however, the NF-kB activation in atherosclerosis is possibly even more significant for public health with the potential of reducing the risk of CVD by dietary means. C-Reactive Protein

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(CRP) and IL-6 are both indicators of systemic inflammation, and also considered predictors of vascular events [41, 42]. IL-6 is produced by smooth muscle cells that surround the lipid pool of the atherosclerotic plaque [43], and furthermore IL-6 induces the production of CRP and other proinflammatory cytokines by the liver. The expression of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular-cell adhesion molecule-1 (VCAM-1) on the endothelium promote leukocyte infiltration into plaques [3, 44]. Furthermore, the strength of the fibrous cap is crucial for the stability of a plaque, which is highly dependent on the collagen content. Inflammatory mediators (e.g., interferon-g (IFN-g)) can both inhibit the synthesis of collagen and induce the levels of metalloproteinases (MMPs) which break down collagen [45]. Remarkably, CRP [35, 46], IL-6 [47, 48], ICAM-1 [49], VCAM-1 [50], IFN-g [51, 52], and several MMPs [53–55] have been identified as NF-kB target genes, and although this is far from a complete review of the mechanisms leading to the formation of atherosclerosis, this association is quite striking. NF-kB target genes code for proteins that are involved in both promotion and progression of cancers [56]. Mutations in genes coding for NF-kB proteins have been found to lead to dysregulation of gene expression, cell transformation and, ultimately, cancer. Mechanisms behind this include persistent NF-kB activation due to constitutive activation of IKK activity, or defective IkBa activity [57, 58]. NF-kB activation has key roles in both the tumor cells and the associated inflammatory cells such as the TAMs. NF-kB is essential both for the survival of monocytes and macrophages and for their cytokine production [59, 60]. Activation of NF-kB in TAM is also pivotal for the initiation of inflammation-associated cancers [14, 61]. In murine models of colon and liver cancers, knocking out IKK-b in inflammatory cells only, reduce both colitis-related cancer and hepatocellular carcinoma [14, 15]. These studies do not only provide evidence for a molecular link between inflammation and cancer, but also emphasize the essential role of inflammatory cells in carcinogenesis. The same studies also underline the very complex role of the NF-kB signaling pathway on carcinogenesis. Knocking out IKK-b in intestinal epithelial cells also reduces colitis-related cancer by 75% [15], whereas mice lacking functional IKK-b in hepatocytes have increased loads of hepatocellular carcinoma [14]. In the intestinal epithelial cells IKK-b induced NF-kB activity apparently drive carcinogenesis, whereas in the liver the NF-kB activation had the opposite effect. In the hepatocytes, NF-kB activation is necessary in order to protect against cytotoxins and the consequent compensatory proliferation that predisposes for carcinogenesis. Hagemann and coworkers recently showed that by inhibiting NF-kB specifically in TAMs, these cells can be “re-educated” to become cytotoxic towards malignant cells and consequently promote tumor regression [62]. Still, the inactivation of the NF-kB signaling pathway may lead to either tumor promotion or regression, depending on the cells in which NF-kB is inhibited and the stage of tumor development [63]. The discrepancy between the role of NF-kB in the tumor cells during carcinogenesis emphasize the critical and complex role of the NF-kB signaling

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pathway in both normal physiology and pathophysiology, and highlights the risk of uncritically inhibiting NF-kB. With the central role of NF-kB activation in inflammation, cancer and anti-apoptosis, substances with the ability to modulate NF-kB activation may be valuable both in chemoprevention [64] and in combination with chemotherapy [65].

3 Effects of Antioxidants on NF-kB Activation In the search for mechanisms behind the protective effects of plant-based diets against a wide range of non-communicable diseases, reductionistic approaches studying isolated antioxidants/phytochemicals have dominated. Such an approach might fit well into pharmacological research, but in doing so, we might overlook additive, synergistic or antagonistic effects that arise from combinations of compounds found in plant foods and diets, and therefore foods or dietary patterns may be better suited as the basic unit for research on these preventive effects [66]. Using foods as the basic unit, we lose information on the individual components and their potential effects; however, we are more likely to mimic the effect of the particular food item and in so doing the relevance to nutrition is increased. The relevance to pharmacology and the possibility of dissecting the data into mechanistic effects of individual components of the extracts is to some extent sacrificed. With nutrition as the focus, the classical reductionistic approaches are not well suited, since we expect that health effects may arise from combinations of compounds and/or from concurrent effects on multiple targets. Thus, in the following studies, both purified phytochemicals/antioxidants and dietary plant extracts were used. In this chapter, we will summarize several projects where we have studied the ability of antioxidatnts, phytochemicals and plant extracts to modulate NF-kB activation.

3.1 Extracts of Dietary Plants Are Efficient Modulators of Nuclear Factor Kappa B In Vitro [67] Since NF-kB has been identified as a promising therapeutic target both in cancer and chronic inflammation, and we screened dietary plants and phytochemicals for their ability to modulate NF-kB activity. We used a monocytic cell line stably transfected with an NF-kB-luciferase reporter construct in the screening of 34 dietary plant extracts and additionally tested 12 phytochemicals for their potency as NF-kB modulators. Our aim was to identify dietary components that could induce basal NF-kB activity to produce a preconditioning effect, or inhibit induction of disease-related NF-kB activity. We observed that 23 dietary plant extracts induced basal NF-kB activity, and 15 extracts attenuated LPS-induced NF-kB activation.

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The most potent inducers of basal NF-kB activity were sulforaphane and curcumin, for the phytochemicals and raspberry and chili among the extracts, while carnosol and apigenin (phytocheimcals) and oregano, thyme and coffee were the most potent inhibitors of LPS-induced NF-kB activity. Since various structurally unrelated antioxidants have been shown to inhibit NF-kB, we proceeded to investigate whether there was a correlation between the total antioxidant capacity of the extracts and their ability to modulate NF-kB activity. To measure the total antioxidant capacity of extracts, we used the FRAP (ferric reducing ability of plasma) method to determine the contents of redox-active compounds with reducing potential above −0.200 mV. We found no correlation between the FRAP value of the extracts, and their ability to modulate either basal- or LPSinduced NF-kB activity, indicating that properties other than redox activity are important in the modulation of NF-kB activity. These results indicate that dietary plants can efficiently modulate NF-kB activity in  vitro and provided the basis for the continuation of our work in transgenic reporter mice.

3.2 The Transgenic NF-kB Reporter Mice Detection of bioluminescence emitted from mammals was first performed by Contag and co-workers in 1995 [68]. They infected mice with salmonella bacteria marked with a constitutively activated luciferase, and were able to trace the bioluminescence by in vivo imaging. At the same time, we created several transgenic luciferase-based reporter mice [69], and these mice included the first generation of NF-kB-luciferase mice with the luciferase gene incorporated into the genome. The NF-kB-LUC mice used in the work presented here are all second-generation NF-kB reporter mice. This second generation of NF-kB reporter mice has the same construct of 3 kB-sites coupled to the luciferase gene, but the construct is flanked by an insulator sequence [70]. This insulator sequence serves to shield the promoter from up-stream or down-steam activator/repressor elements. Furthermore, the insulator also serves to hinder chromosomal position effects, such as epigenetic patterns that could otherwise render the promoter inactivated [71, 72]. These NF-kB-LUC mice report NF-kB activity in both a spacial and temporal manner, when combined with in  vivo imaging technology: The mice have been proven to report NF-kB activation in a wide range of conditions such as systemic inflammation [69], heart infarction [40], autoimmune disease [73], UV-induced inflammation [74], vitamin A deficiency [75], diet-induced obesity [76], hypoxia/ reoxygenation [77], and infections [78]. We have used LPS to induce a systemic inflammatory response and mimic infection. The following section summarizes our findings on modulation of LPS-induced NF-kB activation by dietary plant components.

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3.3 Extract of Oregano, Coffee, Thyme, Clove and Walnuts Inhibits Nuclear Factor Kappa B in Transgenic Reporter Mice [79] In vitro, we found a combined extract of clove, oregano, thyme, walnuts and coffee to synergistically inhibit LPS-induced NF-kB activation in a monocytic cell line, as compared to the sum of effects from the single extracts. Therefore, we continued by investigating the potency of the same dietary plant extracts in inhibition of NF-kB activation in vivo. Transgenic NF-kB-LUC reporter mice were administered a single dose of the combined extract, and subsequently challenged with LPS. When measured as redox-active compounds by the FRAP-assay, the extract administered to the mice corresponds to ~10 times the average daily intake of redoxactive compounds in the Norwegian diet. NF-kB activation was monitored by in  vivo imaging for 6  h (Fig.  2). Based on the area under the curve, the extract decreased whole-body LPS-induced NF-kB activity the during the 6-h period by 35%, as compared to control mice. Organ-specific NF-kB activation was inhibited in intestine and liver (to 53% and 67% of controls, respectively), and as well as in testis and epididymis (to 66% and 48%, respectively) of the mice receiving the combination extract. Furthermore, the mRNA levels of a range of NF-kB-related genes were significantly different in the livers of mice receiving the combination extract. Also, the activity of the transcription factor Nrf2 was increased in mice after administration of the extract, indicating an upregulation of cytoprotective proteins.

3.4 Apple, Cherry and Blackcurrant Increases Nuclear Factor Kappa B Activation in Liver of Transgenic Mice [80] The goal of this study was to investigate how a longer-term supplementation of fruits in the diet could affect NF-kB activation. In transgenic NF-kB-luciferase mice, we investigated the effects of diets supplemented with apple, blackcurrant or cherries (or a control diet) on LPS-induced NF-kB activation. The mice had ad libitum access to the respective experimental diets for 7 days. Whole-body and organ-specific NF-kB activities were determined. On day 7, all mice were given an LPS-injection, and NF-kB activation was monitored by in vivo imaging for the following 6 h. After imaging, blood samples were taken, the mice were euthanized, and ex vivo imaging of organs was performed. Compared to the control group, the apple and cherry groups had slightly higher whole-body NF-kB activation at 4 h, and all three experimental groups had higher NF-kB activation at 6 h. This increase was probably attributed to an increased LPS-induced NF-kB activation in liver with all three experimental diets (Fig. 3). On the contrary, the NF-kB activity of the head and neck area was lower in the mice on the fruit-supplemented diet as

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Fig. 2  Extract of thyme, oregano, clove, walnuts and coffee inhibits NF-kB in vivo. Transgenic NF-kB-luciferase reporter mice were given a single dose of extract or vehicle by oral gavage 3 h before s.c. LPS injection (at 0 h). (a) The luciferase activity was measured by in vivo imaging at 0, 2, 4, and 6 h (one representative mouse from each group is shown). (b) The average fold change (± SEM) of emittance of photons/s/cm2/steradian (normalized to 0 h) at 0, 2, 4, and 6 h from the whole mice minus the head, limbs, and tail (Square LPS + extract; Bullet LPS only; n = 16). The average photons emitted were compared using the Student’s t test. *P < 0.05. Reprinted from Ref. [79]

compared to the controls. Our findings indicate that high intakes of lyophilized fruits modulate in  vivo NF-kB signaling in the liver following LPS-induced stress; however, consequences of this NF-kB modulation in hepatic tissue need further investigation.

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Fig. 3  Ex vivo imaging of organs from NF-kB-LUC mice fed fruit-supplemented diets. Transgenic NF-kB-luciferase mice were given apple-, cherry- or blackcurrant- supplemented, or control diet for 7 days. On day 7, all mice were treated with LPS s.c. at 0 h. Luciferase activity in organs was measured by ex vivo imaging immediately following in  vivo imaging at 6  h. Mean ± SEM. (Control, Apple and Cherry: n = 8, Blackcurrant: n = 6). The groups were compared statistically using the Mann–Whitney U Test. *P < 0.05. Reprinted from Ref. [80], with permission from Taylor & Francis

3.5 Degree of Roasting Is the Main Determinant of the Effects of Coffee on NF-k B and Nrf2/EpRE [81] Coffee, one of the most popular beverages worldwide, is a major contributor of redox active compounds/phytochemicals in the diet and contributes more than 50% of dietary antioxidants in many countries. A moderate intake of coffee has been linked to reduced risk of chronic diseases. Furthermore, experimental studies demonstrate bioactivity of coffee or coffee compounds towards inflammation and oxidative stress. In a study focusing on coffee, we show that the degree of roasting correlates with the efficiency to dampen LPS-induced NF-kB activity and induce Nrf2 activity. The effects on NF-kB and Nrf2 were investgated in parallel, since there have been reports on a crosstalk between the NF-kB and Nrf2/EpRE signaling pathways, and since both are important in inflammatory responses. Extracts of dark roasted coffee inhibit NF-kB activity by more than 80% and induce Nrf2 activity more than 25-fold. In transgenic NF-kB-LUC mice, a single dose of dark roasted coffee extract per os inhibits NF-kB activation by 63% in whole mice (Fig. 4), with the liver being the main target with a 68% reduction in activity. In transgenic Nrf2/EpRE-LUC mice, the extract of coffee increased overall EpRE activity by 30%, again with the liver as the main contributor with a 2.7-fold increase. These results demonstrate that dark roasted coffee modulates both NF-kB and Nrf2 activity in vivo.

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Fig. 4  Extract of coffee inhibits NF-kB activation in vivo in transgenic mice. Transgenic NF-kB reporter mice were given a single dose of coffee extract by oral gavage 3 h prior to s.c. LPS injection. The luciferase activity was measured by in vivo imaging at 0 h, 3 h and 6 h. The AUC for each mouse was calculated and compared between the two groups. Mean ± SEM shown in text box. n = 6. *P = 0.016. The AUC were compared between the two groups using the Mann–Whitney U test. Reprinted from Ref. [81], with permission from Elsevier

4 Discussion of Results In the studies described above, we have shown that dietary plant and phyto­ chemicals can be potent modulators of the transcription factor NF-kB, both in vitro and in vivo in transgenic reporter mice.

4.1 The Luciferase Reporter Firefly luciferase is used as a reporter gene in our studies. The luciferase enzyme catalyzes the luciferin → oxyluciferin reaction, in which the yellow light normally seen from fireflies is produced. This bioluminescence has virtually no background, and correspondingly high sensitivity. Also, the turnover of luciferase is rapid (~2 h half-life) and can therefore reflect dynamic changes in transcription factor activity over a relatively short time frame. The luciferin → oxyluciferin reaction involves oxidation of luciferin, and thus there is a possibility that high concentrations of antioxidants could interfere with this reaction. The relevance of this for in vitro and in vivo experiments is unknown; however, results in  vitro showed no correlation between the FRAP-values and the ability to inhibit NF-kB. A selection of extracts and compounds have also been tested for their ability to inhibit the luciferin → oxyluciferin reaction by the use of recombinant luciferase in a cell-free assay. The only compound found to extensively inhibit this reaction was

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resveratrol (data not shown). Also, there have been reports on direct effects on the luciferase enzyme for b-naphtoflavone [82] and resveratrol [83]. It is therefore unlikely that direct inhibitory effects on luciferase have had a major impact on our results. Since the promoters consist of isolated kB-sites in front of the minimal promoter, the activation of our NF-kB reporters will specifically reflect the transcription factor activity. Numerous distinct binding elements are commonly found in the promoter of a gene, so that the transcriptional activation is more complex and is often dependent on several transcription factors, their activators or repressors, and possibly the interplay between transcription factors. Therefore, by using the insulator, we gain exclusivity in terms of measuring the activity of only the transcription factor that can bind to this specific DNA element. Since the activity of our reporters is “isolated”, we might, however, lose in terms of generalization specific target genes that may contain several different binding elements. For NF-kB, the specificity of the target gene regulation is most likely to depend on the nature of the stimuli, the abundance of dimers, regulated recruitment of dimers to the chromatin and simultaneous activation of other pathways and transcription factors [26].

4.2 Dietary Plants as Modulators of NF-kB Activity We have shown that dietary plant and phytochemicals can be potent modulators of NF-kB, transcription factors that are crucial in inflammation and orchestrating the immune system. There is limited data on the effects of plant extracts on NF-kB activation, especially in vivo. Some berry extracts have been reported to have inhibitory effects on activation of NF-kB in vitro. Huang et al. [84] showed that different fractions of black raspberry extracts could inhibit NF-kB activation, at least partly, by inhibiting IkBa phosphorylation. Extracts of lingonberries [85], and strawberries [86], have been shown to inhibit NF-kB activation in different in vitro systems. In our study, extracts of crowberries, wild strawberries, and blackberries inhibited LPS-induced NF-kB activation; strawberries, however, had no effect. Extract of pomegranate has been found to inhibit NF-kB activation both in experimental lung cancer models [87] and in a prostate cancer xenograft model [88]. Wild blueberries [89] and white currant [90] have also been shown to inhibit NF-kB in vivo. A combination extract of clove, oregano, thyme, coffee and walnut inhibited LPS-induced NF-kB activity in vivo, and this is, to the best of our knowledge, the first report to show inhibitory effects on NF-kB in vivo by extracts of any of these five dietary plants. Extracts of oregano, thyme, clove and turmeric were potent NF-kB inhibitors in our cell system. A few phytochemicals from spices have been fairly well studied with regards to anti-inflammatory and chemopreventive properties; however, similar studies with whole spice extracts are very limited in number. Thyme- and oregano-essential oils in combination decreased the levels of IL-1b and IL-6, as well as inflammation-related tissue damage in a model of colitis [91], both of which may be a result of NF-kB inhibition in the colon. Also, thyme induced the level of

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endogenous cytoprotective proteins in mouse liver [92]. Furthermore, rosmarinic acid [93] in thyme and oregano [94], gallic acid [95] in clove, and eugenol [96] found in clove [94] have been reported to inhibit NF-kB; however, thymol found in thyme did not modulate NF-kB activity. Even though the literature on the effects of whole foods or whole food extracts on health is limited, the number of studies investigating the possible health effects of single phytochemicals is much higher. Resveratrol, curcumin, genistein, capsaicin, epigallocatechin gallate (EGCG), quercetin, b-carotene, and lycopene are among the most widely studied phytochemicals. Phytochemicals can alter the activity of several cell signaling pathways, which can lead to modulation of inflammatory processes, regulation of cytoprotective mechanisms, and regulation of cell growth (extensively reviewed in [64, 97]). In terms of anti-inflammatory effects, we and others [98] have found several phytochemicals to be equally or even more efficient NF-kB inhibitors than classical NF-kB inhibitors and anti-inflammatory drugs such as dexamethasone. One of the most widely studied phytochemicals found in spices is curcumin from turmeric [99]. The effect of turmeric extract may be attributed to the content of curcumin, since this phytochemical is a well-characterized NF-kB inhibitor [98, 100], and also inhibited LPS-induced NF-kB activation in U937 3xkB-LUC cells. Curcumin is reported to have both anti-inflammatory and chemopreventive properties; however, these effects are mainly found in the gastrointestinal tract and the skin (reviewed in [99]). Despite low bioavailability [101], orally administered curcumin has, however, also been shown to inhibit toxin-induced liver injury in rodents by mechanisms such as inhibition of NF-kB and cytokine production [102] and Nrf2 dependent induction of HO-1 [103]. Coffee consumption has been associated with reduced risk of Parkinson’s disease [104], gallstones [105], cardiovascular disease [106], chronic liver disease [107, 108], liver cancers [109, 110] and type II diabetes mellitus [111], as well as reduced risk of death due to inflammatory diseases [112]. We found that extracts of coffee had the ability to inhibit LPS-induced NF-kB activity and induce EpRE activity both in vitro and in vivo. The potency of these abilities increased with an increasing degree of roasting. Maillard reaction products, such as those produced during roasting, have previously been reported to be bioavailable and induce the expression of EpRE target genes such as GST [113]. Also, the coffee diterpenes have been found to increase plasma levels of GST in humans [114], as well as GST and UDP-glucuronosyltransferase [115] in rat liver. Caffeic acid, a phenolic acid provided by coffee, has been shown to inhibit tumor growth through the inhibition of MMP-9 and NF-kB activity [116]. Seemingly in contrast to this, Muscat et al. [117] found activation of NF-kB in macrophages by an extract of roasted coffee beans, but not an extract of raw coffee beans. This activation was, however, from the basal NF-kB level, and might be comparable to the slight activation we also found for a number of dietary plants at the basal level. Cancer chemoprevention by phytochemicals [64] or plant foods is mainly found in organs that directly exposed to the substance without absorption, e.g., gastrointestinal tract for oral administration, and skin for topical administration, plus in the

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liver, which is a major metabolic site for phytochemicals and in the reproductive organs [118, 119]. Whether these organs are the main target organs for chemoprevention by phytochemicals, or this distribution is simply a result of a higher number of studies focusing on these organs, remains to be elucidated. With technologies such as genetically modified animals, genomics, proteomics and metabolomics in combination, effects of a treatment on multiple organs, signaling pathways and biomarkers can be detected from the same study. Our transgenic animals allow simultaneous investigation of the transcription factor activation in all organs, in addition to the overall, whole-body activity. Such investigation is important as dietary plant foods or phytochemicals may have profound effects in several organs, and modulation of transcription factor activity in one organ, e.g., the liver, may lead to subsequent systemic effects that are not a direct result of dietary intake. Interestingly, there is a marked overlap between organs in which chemoprevention is mainly found and the organ-specific effects on NF-kB in our work.

4.3 Organ-Specific Effects There is limited knowledge about tissue distribution of phytochemicals. Nevertheless, quercetin, resveratrol and EGCG have previously been detected in a wide range of tissues, but the highest levels are normally found in the intestine, liver, blood and kidneys [120–124]. In the work presented here, the distribution of the phytochemicals was not measured; rather, in the animal studies, the effect in specific organs was determined. A summary of the organ-specific regulation of transcription factor activity by dietary treatments of transgenic mice is shown in Table 1. Most notably, all treatments had significant effects on the liver. Coffee inhibited NF-kB activity, both alone and in combination with walnuts, clove, oregano and thyme. After a longer-term supplementation with apple, cherries or black currant, however, LPS-induced NF-kB activity was further attenuated compared to mice on control feed. Even though the dietary treatments differ, these results are somewhat in dissonance. The explanation could merely be the individual combinations of compounds provided by the different dietary plants. Additionally, the differences might be explained by distinctions between acute and chronic stress. We find acute Table 1  Organ-specific regulation of transcription factor activity by dietary treatments Transcription Dietary treatment factor Organ Coffee, walnuts, clove, thyme Liver, intestine, testis, NF-kB and oregano epididymis Spleen, (heart) Apple, cherry, or black currant NF-kB Liver NF-kB Liver, kidney Coffee Liver Nrf2 Thymus

Effect ↓ ↑ ↑ ↓ ↑ ↓

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Fig. 5  Ex vivo imaging of organs. A representative image of ex vivo imaging of the organs from an NF-kB-luciferase reporter mouse 6 h after an s.c. LPS-injection. The image shows the high NF-kB activity in liver, brain and intestine, and also enables comparison of the size of the organs

effects on NF-kB in the liver, while the mice were fed fruit-supplemented diets for 1 week prior to LPS-challenge. Under acute stress, it may be advantageous to dampen (not block) the inflammatory response, since an excessive inflammation may in itself be harmful. Over a longer period of time, both NF-kB (and Nrf2) activation is necessary for the handling of both xenobiotics and phytochemicals [14, 125, 126], and this may be reflected by the induced NF-kB activity in liver seen in the longer-term experiment. Also, this emphasizes the caution against taking supplements with high doses of one or a few phytochemicals [119]. Since phytochemicals are mainly metabolized in the intestine and liver, other organs will mainly be exposed to metabolites or conjugates of the ingested compounds. This could result in organ-specific NF-kB (and Nrf2) responses. NF-kB (and Nrf2) activity in the spleen (and thymus), was affected in the opposite way as in the liver. This is noteworthy as both organs are lymphoid organs, and at least for NF-kB, an alternative signaling pathway is important in both development and maintenance of these organs [30]. We speculate that it would be an advantage to maintain a high NF-kB activity in the spleen during an infection to ensure the activity of adaptive immune responses. It should be kept in mind, however, that the absolute NF-kB activity in the liver is much higher than in the lymphoid organs (illustrated in Fig. 5). Also, the liver is large in size, which implies that the liver is contributing considerably more than any other organ to the overall NF-kB activity in the mice.

4.4 Redox Regulation of NF-k B Activation Although NF-kB was initially described to be directly redox regulated, activated by oxidants and inhibited by antioxidants, there has been quite some controversy about this redox regulation [127–129]. As reviewed by Bubici et al. [129], NF-kB and

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ROS are unquestionably closely linked. It seems, however that ROS can both induce and inhibit NF-kB activation, and that this is tissue- and stimuli-dependent. To further complicate the picture, NF-kB can inhibit ROS production (discussed below). Karin et al. [127] conclude that even though selected oxidants such as H2O2 seem to activate NF-kB in some cell lines, ROS in general are not activators of NF-kB. Also, alternative mechanisms are identified for the NF-kB inhibitory effects of NAC and PDTC, two antioxidants commonly used to explain antioxidant inhibition of NF-kB. NAC blocked the binding of TNF to its receptor, and PDTC decreased the NF-kB activity by inhibiting IkB-ubiquitin ligase activity [130]. In the cytoplasm, NF-kB is present in an oxidized state; however, in the nucleus, a reduced state of the dimer is necessary for DNA-binding and full transcriptional activity. Thiol-containing molecules such as glutathione and thioredoxin regulate intracellular redox state, and these molecules have also been implicated in the redox regulation of NF-kB [131]. In the nucleus, reduction of the cysteine residue Cys-62 on p50 is essential for DNA-binding activity; however, this cysteine is apparently reduced by the nuclear protein redox factor-1 (Ref-1), not by thiols [132]. Recently, a new aspect has emerged in the relationship between NF-kB and ROS. NF-kB has actually been found to inhibit ROS production (reviewed in [129]). Signaling through TNF receptors can lead to programmed cell death (PCD); however, NF-kB may prevent the consequent PCD through control of ROS production. Upon TNF-a stimulation, wild-type cells are able to maintain ROS homeostasis, while in NF-kB-deficient cells, an elevation of ROS will occur after such stimuli. Also, the treatment with antioxidants drastically reduced TNF-a-induced PCD in the NF-kB-deficient cells [133–135]. Lines of evidence also suggest that NF-kB protect against PCD through stimuli other than TNF-a, and thus the control of ROS through NF-kB might be a general protective mechanism towards PCD [136–138]. Since the NF-kB dimer needs to be reduced in the nucleus to bind to DNA, antioxidants may promote this step of the NF-kB-signaling pathway. Also, phyto­ chemicals and extracts of selected dietary plants have been shown to inhibit the NF-kB signaling pathway by inhibiting IKK activity, IKKb phosphorylation, phosphorylation of IkBa, IkBa degradation, nuclear translocation, DNA binding, and transactivation (reviewed in [34] and [35]). Thus, it seems that dietary plants or compounds from these might inhibit the activity of the NF-kB signaling pathway, both through antioxidant properties and, perhaps even more importantly, through targeting specific steps in the pathway. Almost any kind of stress, from infection through environmental hazards to physical stress, can induce NF-kB activation. Thus, it is not surprising that compounds from dietary plants may also be perceived as stressful to the body and induce NF-kB activity. Still, the pharmacology industry has searched through plants for NF-kB inhibitors only. Compounds such as resveratrol and quercetin have been reported to both inhibit and induce NF-kB activity (reviewed in [35]). These seemingly contradictory results may be due to biphasic activity at different doses or different responses, depending on the cell type or physiological setting.

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We found extracts of several dietary plants to have the dual ability to induce basal NF-kB activity and inhibit LPS-induced NF-kB activity in a monocytic cell line. Also, we found increased NF-kB activity in liver of mice fed fruit-supplemented diets. These observations might fit with the notion that high doses of compounds found in dietary plant food can be perceived as stressors or xenobiotics. We hypothesize that these properties of plant foods may produce preconditioning and/or hormetic effects.

4.5 Preconditioning, Hormesis and Antioxidants The term preconditioning refers to a situation where one or several small insults can protect against later stronger insults. A subject, organ or cell is exposed to stressors or stimuli that will render the subject, tissue or cell better prepared to handle similar, subsequent stimuli. In 1986, Murry et  al. [139] first demonstrated that brief episodes of ischemia delayed cell death in a subsequent more severe ischemia to the heart. Later preconditioning effects have been found in other organs and by other stressors such as exercise, hypoxia and activators of the innate immune system (reviewed in [140–143]), indicating that this might be a more general mechanism for protection against stressors. NF-kB has been described to be involved in protective preconditioning effects in coronary heart disease [144, 145], sepsis [146], cerebral diseases such as ischemia or epilepsy [147], hepatic ischemia injury [148] and apoptosis [149]. Furthermore, induction of cytoprotective proteins through Nrf2 can be seen as preconditioning effects that result in subsequent protection against chemically induced cancer [126] and hepatotoxicity [150, 151]. Since we found that several dietary plant extracts were potent inducers of basal NF-kB activity, we proceeded to test preconditioning in  vitro. This work is currently under way, and initial experiments are promising with respect to precondition effects. We found that incubation of U937 3xkB-LUC cells with both a low or a high dose of LPS gave a lower NF-kB response to LPS the following day, as compared to cells that had not been pre-treated with LPS (Fig. 6). This shows that such preconditioning effects may also occur in the U937 3xkB-LUC cells. Interestingly, the low and high doses of LPS were seemingly equally efficient for preconditioning against later LPS insults. We further hypothesize that dietary extracts may also exert such preconditioning effects. Small, diet-induced episodes of NF-kB activation may protect tissues and cells against later, stronger NF-kB activating insults. The ability of several dietary plant extracts to induce basal NF-kB activity may thus play a part in the preventive effects of plant-rich diets on the development of chronic non-communicable diseases. Preconditioning effects are specific cases of a more general phenomenon called hormesis, and preconditioning has also been referred to as “conditional hormesis” [152] or “stress-response hormesis” [153]. The term hormesis was first used by Southam and Erlich in 1943 [152, 154], and is a phenomenon found in biological sciences and characterized by biphasic or non-linear dose–response curves [155, 156].

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Fig. 6  Preconditioning of NF-kB activity by LPS. U937 3xkB-LUC cells were preincubated with no LPS or LPS at 0.01 mg/ml or 1 mg/ml for 12 h. Cell culture medium was changed, each overnight batch divided into three, and cells were incubated an additional 6 h with no LPS or LPS at 0.01 mg/ml or 1 mg/ml (a total of nine different treatments). LPS-induced NF-kB activity was measured in a Synergy 2 plate-reader after the addition of 0.2 mg/ml luciferin and 5 min. incubation at 37°C. Bars represent mean ± SEM of three experiments each performed in triplicates. By courtesy of Marit Kolberg and Siril G. Johansen

Examples of such biphasic dose–response curves are the U-shaped curve (Fig. 7a) and the J-shaped curve (Fig.  7b). The concept of hormesis has been somewhat discredited, supposedly due to an association with homeopathy [157]; however, over the last few years the concept has been more frequently used in such fields as toxicology [158], the biology of aging [153] and exercise [159], and nutrition [160]. Hormesis-like dose–response curves have, however, been found for a wide selection of compounds including drugs, cytokines, toxins and phytochemicals [161]. Nutritional hormesis describes how small quantities of a food or food compound often have opposite or different health effects from those of large doses (reviewed in [162, 163]). In the U-shaped curve, zero or extremely limited as well as extremely high intake will be detrimental to health, whereas a moderate intake is optimal for reducing the risk of disease. The U-shaped curve seems to be particularly central in nutrition, illustrated by the fact that even the most basic nutritional needs, such as water and energy, adopt a U-shaped dose–response curve. We hypothesize that for the non-vitamin phytochemicals, adaptation to mild cellular stress and the modulation of signaling pathways (e.g., NF-kB) causes preconditioning effects that may at least partly explain the bottom of a proposed U- or J-shaped curve for the relationship between the risk of disease and intake of phytochemicals. Thus, the concepts of preconditioning and hormesis should be kept in mind when discussing the effects of diets rich in plant-based foods on the risk of diseases.

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Fig. 7  Dose–response relationship curves between intake and risk of disease

Of the thousands of phytochemicals found in foods, only a few have been extensively tested in clinical trials. A recent Cochrane review, including 67 studies with a total of more than 230,000 participants, found increased mortality by intake of vitamin E or b-carotene supplements [164]. Also, the World Cancer Research Fund advises against supplements for the prevention of cancers [119], and some clinical trials have even found increased risk of cancers [165–168]. These clinical trials that have led to statements of precaution against such supplements from academia, nevertheless in the general public and commercial health food industry, antioxidant supplements are still increasing in both number and popularity. A warning against taking high doses of antioxidants should be a result of these trials; however, these trials are not designed to the reject the “antioxidant hypothesis”: “Oxidative stress causes disease” + “Phytochemicals are antioxidants” ergo “Phytochemicals reduce the risk of oxidative stress related diseases through their antioxidant function in vivo”.

Especially with the U- and J-shaped hormesis curves in mind, we cannot rule out an antioxidant effect in vivo based on negative or no findings in these interventions with high doses of selected phytochemicals; neither can we confirm such effects. Most phytochemicals are potent antioxidants in vitro, as well as in the plants themselves. However, whether the various phytochemicals work as antioxidants in vivo after absorption (and metabolism) still remains to be elucidated. Clinical trials to evaluate antioxidant effects of phytochemicals in vivo are, however, extremely difficult to design as numerous phytochemicals are present in plant foods. In addition, the phytochemicals are variably absorbed, they are heavily metabolized and excreted, they are of course very reactive, and they appear in low concentrations as compared to endogenous antioxidants such as glutathione.

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Just like the clinical trials described above, our studies are neither suited to confirm nor reject the “antioxidant hypothesis”. Nonetheless, these results contribute to the discussion of how a diet rich in plant-based foods can reduce the risk of chronic oxidative stress-related diseases by showing that dietary plant compounds are bioavailable and can modulate signaling pathways involved in oxidative stressrelated processes, and induce the endogenous antioxidant defense in vivo.

5 Conclusion We have utilized transgenic reporter mice and cell lines to investigate how phytochemicals/antioxidants and dietary plants can modulate NF-kB transcription factor activity. Several dietary plants are found to be potent modulators of NF-kB activity in  vivo. Even though these dietary plants are rich in antioxidants/ phytochemicals, the contribution of antioxidant properties per se to these effects on NF-kB remains uncertain. Also, the relationship and interactions between antioxidants/ROS and the NF-kB signaling pathway, is complex and still not fully elicidated. In line with this discussion, we find various dietary plants to be able to both inhibit and induce NF-kB activity, and we speculate that both might contribute to the preventive effects of diets rich in plant-based foods towards chronic diseases. The mechanisms behind preconditioning effects and the potential of dietaryinduced preconditioning in prevention of disease should be further investigated.

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Part III

Neurology

MPTP and Oxidative Stress: It’s Complicated! V. Jackson-Lewis, M.A. Tocilescu, R. DeVries, D.M. Alessi, and S. Przedborski

Abstract  1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is the tool of choice for modeling Parkinson’s Disease (PD) in animals. Originally synthesized as a “designer drug” by drug users, MPTP, while not giving us the definitive answer as to the etiology of Parkinson’s disease, has enlightened many of us researchers about the molecules and mechanisms involved in dopamine neuron death. Herein, we provide information about some of the events that are involved here and try to make it clear just how complicated the death of the dopamine neuron in the MPTP model and possibly in PD seems to be. Keywords  Cytokines • Dopamine • Mitochondria • Monoamine oxidase • MPTP • Nitric oxide • Superoxide

1 Introduction Most neurodegenerative diseases involve specific subsets of neurons. In the case of Parkinson’s disease (PD), a common neurodegenerative disorder characterized behaviorally by resting tremor, rigidity, akinesia/bradykinesia and postural instability [1], these are mainly, though not exclusively, the dopaminergic neurons in the substantia nigra pars compacta (SNpc) [1]. PD affects mainly those individuals over 50 years of age and the risk of developing PD increases as we age [1]. Furthermore, the overall course of disease development is slow (decades) and there is no laboratory test for diagnosing PD. We do not know the etiology of PD; however, what we do know, aside from the death of the dopaminergic neurons in the SNpc, is: (1) that there is a greater loss of dopaminergic terminals in the striatum than the loss of dopaminergic neurons in the SNpc [1]; (2) that there is an upregulation V. Jackson-Lewis (*) Center for Motor Neuron Biology and Disease, Departments of Neurology, Columbia University, Room 4-401, 630 West 168th Street, New York, NY 10032, USA e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_8, © Springer Science+Business Media, LLC 2011

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of certain cytokines in the SNpc [2–4]; (3) that there is an inflammatory ­component to PD that we and others suspect to be the cause of PD’s progressive nature [5, 6]; (4) that the superoxide radical and nitric oxide have been implicated in PD [7, 8]; and (5) that dysregulation of mitochondria is somehow in play here (reviewed in Ref. [9–11]). The central hypothesis that encompasses what we know is that following the initiation of the disease, a cascade of deleterious events leading to oxidative injury, macromolecule damage and evoked inflammation all conspire to produce mitochondrial dysfunction and energy failure that ends in the death of the dopaminergic neuron. Taken together, these events are indicative of an oxidative stress situation that is taking place in a relatively small area of the brain that has a big impact on the body. Although PD affects other brain areas, its principle effects are in the brain’s nigrostriatal dopaminergic system. This brain area is located in the mesencephalon (midbrain) and projects its fibers to the striatum. Due to high levels of melanin within dopaminergic neurons, the SNpc appears black in color. The SNpc acts mainly as an input to the basal ganglia as it supplies dopamine (DA) to the striatum. Of the huge number of DA neurons in the SNpc (~450,000/SNpc at birth), these cells decline normally over time, in a linear fashion, to reach a level of ~275,000/ SNpc by the age of 60 [12]. However, in PD patients, cell counts fall below 140,000/SNpc [12]. The most prominent function of the SNpc is motor control as evidenced by the lack thereof in PD due to the loss of the SNpc dopaminergic neurons. Regardless of the cause of neuronal death, Parkinsonian symptoms do not appear until about 50–80% of SNpc dopaminergic neurons have died [13]. Most of the problems in PD, as cited above, seem to occur at the neurochemical level in the nigrostriatal pathway as DA transport systems are slowed, allowing DA to linger for longer periods, which sets the stage for big trouble in this little brain area via oxidative stress. DA neurons exhibit a unique susceptibility to oxidative stress in that although they represent only a small percentage of all of the neurons in the SNpc, this small portion is enough to upset its microenvironment, both inside of and outside of the neuron. In order to investigate the mechanisms that are involved in the death of the dopamine neuron in the SNpc, we and others have used MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), a neurotoxin that replicates, thus far, in humans [14], in non-human primates [15] and in other mammals such as mice [16], most of the hallmarks of PD. Thus, this chapter is an attempt to analyze the oxidative stress situation going on within and outside of the nigrostriatal system in the PD brain through the use of MPTP.

2 PD and Oxidative Stress Oxidative stress has consistently been demonstrated to be a hallmark of PD [17]. In the normal course of the brain’s cellular function, oxidative events are necessary for reactions that take place within the neuron. Of the oxygen that is taken into the body, the brain alone uses ~25%. Reactive oxygen species (ROS), like the

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s­ uperoxide radical and H2O2, are the results of aerobic metabolism in the brain as this organ, more than any other, produces a significant amount of ROS. Antioxidant systems, like superoxide dismutase and glutathione (GSH) and small molecules such as vitamins E and C, under normal conditions, can metabolize ROS, such as H2O2 and the superoxide radicals that are produced by these reactions. However, as we age and in neurodegenerative disorders like PD, the antioxidant defense systems dwindle and the oxidative load increases [18]. Post-mortem tissues from PD brains have provided evidence that the ROS load is excessive in the SNpc and that oxidative stress has occurred. One of the earliest changes to be noted outside of the loss of DA levels were the decreases in GSH levels and the increases in its oxidized form, GSSG, which most likely originated from the oxidation of DA [18]. DA within the neuron is metabolized by MAO-A to dihydroxyphenylacetic acid (DOPAC), H2O2 is kicked out in the process, and oxygen and water are consumed [19]. DA also undergoes auto-oxidation to DA-quinone and generates H2O2 in a reaction that involves a nucleophilic addition with protein sulfhydryl groups [20] and the reduction of GSH, the major cellular buffer that metabolizes H2O2 and prevents oxidative stress in the neuron [21]. If there is decreased GSH, then there is no buffer to the H2O2 that is present in nigral tissues. In fact, H2O2 injected directly into the SNpc causes extensive damage to this brain structure (Jackson-Lewis, unpublished data) which demonstrates the toxicity of H2O2. The latter can be converted to the hydroxyl radical via the Fenton reaction with iron, which was shown to be elevated in the PD brain [22, 23]. Another marker of the presence of oxidative stress in PD is protein carbonyl modification which results from ROS attack on amino acid residues [24]. Lipid peroxidation is yet another part of the PD DA neurodegenerative process, as elevated levels of both the aldehyde 4-hydroxy-2,3-nonenal (HNE) [25], which has also been shown to modify proteins [26] and block mitochondrial respiration [27], and malondialdehyde [23] have been recorded as evidence that lipid peroxidation has occurred in the ­nigrostriatal DA system. Furthermore, Zhang et al. demonstrated the presence of elevated levels of 8-hydroxyguanosine, a nucleoside oxidation product, within the cytoplasm of DA neurons and within the cell’s mitochondrial DNA and its RNA [26].

3 Other Toxin PD Models Several toxins have been used as models of PD, yet, none seems to cover the entire range of hallmarks noted at the neurochemical level in PD. For instance, 6-hydroxydopamine (6-OHDA) has been used in animals as a model of PD for more than 40 years and is still being used. This compound is the result of hydroxyl radical attack on DA, which makes it an oxidation product of DA [28]. 6-OHDA does not cross the blood–brain barrier (BBB), so it has to be injected into the SNpc. It is believed that the compound that causes PD, if there is such, must be able to cross the BBB to do its damage. 6-OHDA shares some structural similarities with both DA and NE and kills both noradrenergic (NE) and DA neurons; therefore, to avoid damage to

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the NE neurons, desipramine must be given prior to 6-OHDA injection. Also, a monoamine oxidase (MAO) inhibitor must also be given to prevent 6-OHDA metabolism by MAO. It is interesting to note that although 6-OHDA cannot cross the BBB, it can be taken up by both the DA transporter (DAT) and the NE transporter (NET). Injected into the SNpc, the striatum or the median forebrain bundle stereotaxically, the lesion produced by 6-OHDA is essentially focal. 6-OHDA can also be injected systemically; however, at times, this lesion is both variable and bilateral and animals often die because they cannot care for themselves [29]. Thus, the lesion of choice for 6-OHDA is a unilateral injection into the SNpc. This type of lesion produces DA neuron death within 24 h of injection, whereas a unilateral injection of this compound into the striatum kills DA terminals and produces a dying back process [30] in which the SNpc DA neurons die at a later time and the morphology of the dying neurons is that of apoptosis [31]. Thus, the type of cell death produced by 6-OHDA depends on how and where it is injected. 6-OHDAinduced nigrostriatal damage to DA neurons and DA terminals is ROS-mediated [32], and involves inhibition of mitochondrial complexes I and IV [33] and caspase activation [32]. There is some information on an inflammatory response in the SNpc to 6-OHDA [34], but the timing of this response has been shown to occur at least 6 days after 6-OHDA application. The insecticide rotenone has also been used to model PD. This compound is highly lipophilic, readily crosses the BBB [35] and accumulates within the ­mitochondria and other subcellular organelles. In mitochondria, rotenone can affect oxidative phosphorylation [36]. Routes of administration are important when using rotenone as some routes of administration have no to variable effects on DA ­neurons [37–41]. Interestingly, the chronic infusion of rotenone can produce ­proteinaceous inclusions [42] within DA neurons. These bodies resemble the Lewy bodies (LB) that are the definitive diagnosis for PD. If rotenone produces proteinaceous inclusions and the production of the other hallmarks are iffy at best, then rotenone is definitely not the tool of choice for investigations into the mechanisms producing PD. The use of rotenone as an animal model for PD leaves a number of questions to be pondered, the most important being how does it not necessarily affect the DA system, yet produce Lewy-like inclusions particularly when it has been shown that DA plays a role in synuclein aggregation [43, 44]. Paraquat is yet another herbicide that has been reported to cause PD-like lesions in the brains of mice and rats [45] through oxidative and nitrative stress mechanisms [46]. Paraquat’s heavy use in agriculture has raised some concerns about its being an environmental toxin, thus increasing the risk of developing PD; however, there is no evidence that direct exposure of humans to paraquat causes brain lesions. Paraquat belongs to a class of compounds called bipyridyl derivatives that include diquat and cyperquat (better known as MPP+). Because of its highly polar nature, there is some controversy [47, 48] about paraquat’s ability to cross the BBB; however, studies have shown that this ability is age- and speciesdependent [45, 49]. There are also differences of opinion as to the cellular targets of paraquat [50–52] due mainly to the mechanism through which paraquat operates, which is redox-cycling. Basic biochemistry tells us that as long as there is an

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a­ dequate electron donor, redox-cyclers can operate in whatever system that they just happen to occupy at the moment. So the cell type is an issue here. We know that paraquat toxicity to lung tissue, kidney tissues and hepatocytes is similar, but the question of paraquat and DA toxicity is still unclear as degeneration in DA neurons has been observed in rats only with high doses of this compound [53]. Interestingly, paraquat has been shown to produce apoptosis [54] through activation of the JNK-signaling pathway.

4 Why MPTP? The etiology of PD is still a mystery. Decades of research, prior to the discovery of MPTP, had essentially left us in the dark in terms of not only the etiology of PD, but also the mechanisms involved in its progression. Some successes in the ­treatment of this debilitating disorder have been achieved with the discovery of levodopa (l-DOPA) to treat PD symptoms and DA agonists that can mimic the actions of DA. However, these compounds have their own set of baggage and there is some question as to whether they really help or hinder here. For instance, l-DOPA is the drug of choice for the treatment of PD symptoms, yet it does not stop the progression of PD, which leads to the question of its role in the progression of PD since l-DOPA blocks mitochondria [30], the cornerstones of the oxidative stress hypothesis of PD, at the complex I site. For a true animal model of any disease, it is a must that whatever is used needs to cause the hallmarks of that disease. In the case of PD, the above-listed hallmarks must be replicated. Thus far, the tool of choice for modeling PD is the “designer heroin” MPTP.

5 MAO-B and MPTP MPTP is the tool of choice for investigations into the mechanisms of PD [55]. Its most prominent features include a greater loss of DA content in the striatum and a lesser loss of the DA-containing neurons in the SNpc of mice and other mammals [16]. As in PD, also found is an inflammatory response situation [56], mitochondrial dysfunction [57], oxidative damage to proteins, lipids, amino acids and DNA damage [58]. The story of how MPTP was discovered is interesting in itself, as this “designer heroin” was first used by humans before it became the gold standard for PD investigations using animals. MPTP itself is not toxic. However, once injected or ingested or via absorption through the skin, because of its lipophilic nature MPTP is easily absorbed into the bloodstream, crosses the blood brain barrier and is taken up into astrocytes [55] where it is metabolized to MPDP+ (1-methyl-4phenyl-2,3-dihydropyridium) by MAO-B. MPDP+ is quite unstable and immediately deprotonates to MPP+. MAO-B may be just the start of the oxidative stress situation within the nigrostriatal system. It has been shown that this enzyme is elevated with aging and in

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neurodegenerative diseases such as PD [59]. MAO-B is expressed primarily within astrocytes [59] and the SNpc contains many more astrocytes than other brain areas [60]. This significant number of astrocytes may, in part, be a clue to the vulnerability of the DA neuron. Astrocytes were originally thought to be the keepers of the DA neurons since they supply the nutrients necessary for the good health of these neurons [61]. However, it is now known that following MPTP administration to mice, MAO-B activity increases and, in the metabolism of MPTP by MAO-B, excess levels of H2O2 are produced. This excess H2O2, while not toxic to astrocytes because of their high levels of glutathione and glutathione peroxidase [59], is toxic to DA neurons as DA neurons contain much lower levels of these antioxidants. In fact, H2O2 injected directly into the SNpc causes significant tyrosine hydroxylase (TH)-positive neuron loss (Jackson-Lewis, unpublished data). Aside from metabolizing MPTP to MPP+, MAO-B also metabolizes DA to DOPAC and homovanyllic acid (HVA) and, in the process, also produces significant amounts of H2O2 [62]. It has been demonstrated that high levels of H2O2 can interfere with the normal function of the neuron such as damaging cellular proteins and inhibiting mitochondrial ­complex I activity, which can throw the DA neuron into a dysfunctional state that can lead to its death. Furthermore, because it is not a charged particle, H2O2 can freely traverse membranes, thereby affecting both the internal and external environment of the DA neuron. In fact, mice, engineered to overexpress MAO-B within ­astrocytes, have been shown to exhibit a selective degeneration of DA neurons [59]. However, DA can act as a two-edged sword in that it can decrease significantly thiobarbituric acid reagent substance formation in mitochondrial preparations [63] in the presence of iron and, at the same time, increase carbonyl content in ­mitochondrial proteins along with a significant reduction of thiol content in ­mitochondrial proteins. MAO-mediated metabolism of DA can increase MAO-B activity, but iron inhibits both MAO-A and MAO-B activities. Thus, DA can have antioxidant properties in the presence of iron and lipid peroxidation but can show pro-oxidant activity by causing the generation •OH and in the oxidation of ­mitochondrial proteins. In the SNpc and in the striatum, excess DA can be metabolized by MAO to several toxic metabolites. For instance, one quite interesting metabolite is DOPAL (3,4-dihydroxyphenylacetaldehyde), a compound that is predicted to be highly reactive and toxic to cells whose in vitro toxicity was demonstrated by Lamensdorf et al. [64]. using dose-response curves in PC-12 cells and later confirmed by Kristal and colleagues [64, 65]. The in  vivo toxicity of DOPAL was shown in injection experiments into the SNpc of rats, comparing DOPAL toxicity to DA toxicity. Here it was demonstrated that DOPAL, in doses as low as 50 ngs, was far more toxic to DA neurons than DA (500 ngs) itself, in that DOPAL killed TH-positive neurons and produced a glial reaction whereas DA did not [66]. Furthermore, while DA is metabolized by MAO-A to DOPAL within the neuron, DA is metabolized to the same compound by MAO-B extracellularly [67]. Thus, it is possible that when excess DA is in the cytosol, as a result of being pushed out of the storage vesicles by MPP+ or out of the terminals in the striatum, DA is metabolized to DOPAL by MAO-B with the occurrence of H2O2 in the process. The produced H2O2 can

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g­ enerate the hydroxyl radical (•OH) which will attack DA [68], thus producing the known neurotoxin, 6-OHDA. And, although 6-OHDA has never been recovered from the brain, recent evidence [69] notes that 6-OHDA is measurable in MPTPtreated mice that have been primed with l-DOPA, which increases DA levels in the brain. Thus, it is reasonable to think that the high levels of H2O2 here are the result of the metabolism of DA to its toxic metabolites and the metabolism of MPTP to MPP+ within the nigrostriatal system. This may also explain why the DA terminals are not only damaged first but the damage is greater here than in the neurons and may well be relevant to PD and to the oxidative stress within the nigrostriatal ­system. Furthermore, if the work of Borah and Mohanakumar does pan out, then l-DOPA, although it does alleviate PD symptoms for a time, may actually be a promoter of PD and oxidative stress.

6 DA Neurons, DA Terminals and MPTP Due to its positive charge, an ever-pressing question for a long time was how did MPP+ escape astrocytes. Since it is a charged compound, MPP+ cannot freely cross membranes. Recently, thanks to some excellent research by Tieu and colleagues [70], we now know that MPP+ is carried out of the astrocytes by the organic cation transporter, OCT-3. These researchers demonstrated first that the OCT-3 transporter is present on non-DA cells within the nigrostriatal DA system and that the release of MPP+ into the extracellular space is dependent on the OCT-3 transporter. They also noted that both the deletion (knockout mice) and the inhibition (dycenium-22) of this transporter protect against the damaging effects of MPTP to the nigrostriatal DA system. As with the DAT, there is a question as to whether the OCT-3 are ­damaged in the MPP+ release process. The answer to this is no, simply because, as mentioned earlier, astrocytes have high levels of antioxidants unlike neurons, which do not [59]. What is so interesting about this study is that the OCT-3 transporter was shown to be bidirectional, which means that this transporter has the capability of being a two-way street. Thus, this OCT-3 transporter, depending on its behavior, can affect the environment of the DA neuron either positively or negatively. Once in the extracellular space, MPP+ is taken up into the DA neuron via the DA transporter (DAT) [71]. Although this transporter is damaged by MPP+ in the uptake process [72], enough MPP+ is taken up by it to be further taken up by VMAT-2 (vescicular monoamine transporter-2) into the neuron’s vescicles, the same vescicles that store DA. Because these vescicles have a limited capacity, DA may be pushed out of these storage vescicles by MPP+ [73], leaving DA open to the metabolic activity of MAO-B and other metabolic activities. Furthermore, VMAT2 may also be damaged in the MPTP neurotoxic process [74]. Another toxic metabolite of DA that is produced following MPTP administration to mice is 5-cysteinyl-DA [75]. This stable modification of DA is formed by the oxidation of DA to DA quinine, which also kicks out the superoxide radical and H2O2 [20]. After formation of the DA quinone, this intermediate molecule interacts

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with the sulfhydryl group on the amino acid cysteine via a nucleophilic addition to form 5-cysteinyl-DA. Oxidation of DA can be facilitated by a number of enzymes including the cyclooxygenase-2 (COX-2) enzyme which is upregulated following MPTP treatment [75], as well as the enzymes lipoxygenase [76], tyrosinase [77] and xanthine oxidase [78]. We and others have measured 5-cysteinyl-DA in the striata of MPTP-treated mice. And, this permanent protein alteration of DA ­irreversibly alters protein function and may thus contribute to the toxicity of DA to the DA terminals as part of the oxidative stress in the nigrostriatal system. Although we have identified a few toxic metabolites of DA that probably contribute to the oxidative damage in the nigrostriatal system, there are, most likely, other DA metabolites that are toxic and that need to be examined in light of the possibility that they, too, might contribute to this oxidative stress and damage.

7 Cytokines and MPTP Aside from the severe loss of dopaminergic neurons in the SNpc and DA terminals in the striatum, there is a marked gliosis in the nigrostriatal pathway in PD. Astrocytes and microglia, the two main components of the glial response, are both activated [14]; however, the magnitude of their responses is quite different. The astrocytic response, both in number and in immunoreactivity, is, in general, mild, and, in only a few instances, has this response been dramatic [79, 80]. In contrast, the activation of microglia has been consistently strongest in the SNpc, the area of the brain most affected by the neurodegenerative process [81]. Moreover, activated microglia are found in close proximity to the remaining dopaminergic neurons [5], around which they sometimes cluster to produce what resembles neurophagia. This same situation has been noted in the three autopsied brains that were recovered from those individuals who self-administered MPTP [14]. Furthermore, for reasons that we do not know, the SNpc seems to contain a significant number of microglia in its microenvironment compared to other brain areas. Microglial activation and neurophagia are indicative of an active ongoing process of cell death that is consistent with the progressive nature of PD. Yet, data from PD brains and from the brains of the few individuals who ingested MPTP do not provide us with information about the temporal relationship between SNpc dopaminergic neuron death and microglial activation. To this end, the MPTP mouse model of PD was again used to glean information about microglial activation in the MPTP neurotoxic process and to sort out the process of gliosis as it relates to the time course of microglial and astrocytic activation in SNpc dopaminergic neuron death. While microglia are upregulated early on in the MPTP neurotoxic process and reaches maximum before the peak of dopamine neuron death in the SNpc, astrocytes, mildly activated early in the neuronal death process, are further upregulated and remain upregulated for a lengthy period following MPTP administration [82]. Thus, it is reasonable to believe that astrocytes participate in the death of the dopaminergic neurons in the SNpc in both PD and in the MPTP mouse model of PD.

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Microglial cells are the resident macrophage cells in the brain that have the a­ bility to react promptly in response to brain injury and to subtle changes in the microenvironment surrounding their charges, the neurons [83]. In the normal brain, microglial cells are in a resting state in which they are barely visible and very few, if any, ramified processes are detected. In contrast, in a pathological situation such as that of the MPTP-treated mouse, microglia quickly proliferate, become hypertrophic, increase in size to resemble spiders and produce a number of marker ­molecules that are either pro- or anti-inflammatory in nature. Thus, microglial cells are a double-edged sword type of cell producing such proinflammatory compounds as the superoxide radical, nitric oxide (NO), prostaglandin E2 (PGE2), excitatory amino acids, and proinflammatory cytokines such as interleukin-1-beta (IL-1-ß), or such anti-inflammatory compounds as interleukin-4 (IL-4) and transforming growth factor-beta-1 (TGF-ß1) [84–86]. Since activated microglia have been found in PD patients and in the MPTP mouse model of PD, and microglia can produce such damaging molecules, it may be that microglial cells contribute to the progressive nature of DA neuron death as seen in PD. Blocking microglial activation is indeed important to the treatment and alleviation of PD, but ascertaining the mechanisms by which microglia are activated in PD will help us to better identify those therapies that can dampen microglial activation and, maybe in the process, prevent the progressive nature of PD and possibly of other neurodegenerative disorders. An ever-pressing question in neuroinflammation is what triggers the inflammatory response in PD. Although microglia and astrocytes are an integral part of this response, it is believed that their activation here is not primary. Signaling studies suggest that the pro-inflammatory cytokines might actually initiate the oxidative stress within the nigrostriatal system that leads to the death of the DA neurons. These are small molecules that recruit immunocompetent cells to the site of injury and that influence the behavior of these immunocompetent cells [87]. A number of cytokines and chemokines are noted to be upregulated in PD. TNF-a is elevated following MPTP administration to mice [88, 89]. However, very few studies exist that deal with the time course of expression of the cytokines in relation to microglial and astrocytic activation. Herbert et al., Ciesielska et al., and Pattarini et al. did make several observations about cytokines and neuroinflammation in time-related experiments using the MPTP mouse model of PD [90–92]. These authors found that upregulation of certain cytokines in the striatum occurred as early as 6–24 h after MPTP administration. Among these were TNF-a, Interleukin-1b (IL-1b), IL-1a, IL-6 and matrix metalloproteinase-9 (MMP-9). The most significant response came from TNF-a. Documented was a sixfold increase in the expression of TNF-a mRNA in the striatum 1 day after MPTP injection [90], whereas there was a corresponding decrease in this same protein in the ventral midbrain of the treated mice. In fact, TNF-a in the ventral midbrain was not ­elevated until 3 days after MPTP treatment. Within the brain, TNF-a is expressed primarily in glial cells and particularly in microglia [93] and although several investigators have demonstrated that a deficiency in TNF-a is protective to DA neurons in the SNpc [93, 94], it is still unclear what the relationship is between TNF-a upregulation and microglial activation. Based on the work of Hebert et al. and others, what happens in the striatum acts

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like a delayed reaction in the SNpc. We do know that microglial ­activation in the SNpc peaks at 2 days after MPTP administration [95] which is 1 day after TNF-a activation in the striatum after MPTP treatment. This leads one to think that TNF-a might be instrumental in the activation of glial cells. However, if microglial activation peaks at the 2-day time point in the SNpc after MPTP administration and TNF-a peaks at the 3-day time point here after MPTP treatment, then the relationship between TNF-a and microglial activation is not necessarily the suggested initiation of the microglial response. Or, it may be possible that only a small increase in TNF-a is needed to start the microglial activation process. IL-1b, IL-1a, IL-6 and MMP-9 were also found to be elevated in the striatum of mice 1 day after MPTP exposure [90]. The greatest elevation of these cytokines did not occur in the SNpc until day 3 after MPTP, which may be related to the dying back process in that injury to the terminals could stimulate cytokine production in the SNpc. In trying to elucidate the mechanism of microglial activation by going backwards, Yasuda et al. suggest the involvement of S100B release from the ­astrocytes as a player in the microglial response [96]. S100B released from ­astrocytes normally seems to help in the maintenance of homeostasis in the ­extracellular environment of the DA neurons, but according to these authors, it can be beneficial with low doses of MPTP and detrimental with high doses of this compound. Since they did observe a morphological response from both microglia and astrocytes early on in the MPTP neurotoxic process that was consistent with an inflammatory response and a reduction in S100B in the cerebrospinal fluid as early as 1–3  h after MPTP treatment in B6 mice, they suggest that S100B may be regulatory to the cytokines and may be what occurs in the early stages of PD. Furthermore, it has already been reported that astrocytes play a role in the ­regulation of cytokines in that they secrete TNF-a in the central nervous system [97]. However, since we noted that the microglial response peaks at 2 days after MPTP administration and the astrocytic response is even later [98], the “how” of microglial activation by cytokines remains unclear. Another cytokine that may figure in the activation of glial cells in the MPTP neurotoxic process is NF-kappaB. This cytokine has been found to be elevated in both astrocytes and microglia in PD brains [99] and, although this NF-kappaB is known to have some protective qualities [100], Aoki et al. [101]. found evidence that NF-kappaB may also regulate glial activation. In fact, in some circles [99] it is thought to be a sensor of oxidative stress. NF-kappaB is found mainly in astrocytes, but it is also found in neurons of the SNpc and in the striatum [102]. MPTP ­investigations into the role of NF-kappaB in glial activation and oxidative stress in the nigrostriatal system found that NF-kappaB begins to increase in astrocytes in the SNpc as early as 5 h after MPTP administration and this elevation lasted up to 3 days post MPTP. There was also some presence of NF-kappaB in DA neurons of the SNpc. Of interest, microglia did not become activated until after astrocytes were already activated and DA neuron death was not apparent before microglial ­activation. This suggests that if NF-kappaB is indeed a sensor for oxidative stress, then it makes sense that NF-kappaB would be elevated prior to the onslaught of an oxidative event. As to the relationship of NF-kappaB to TNF-a activation, the ­time-dependency of this interaction remains to be investigated.

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8 Superoxide and MPTP ROS have been implicated in a number of neurodegenerative disorders including PD [103]. The oxidative stress hypothesis proposes that, within the DA neuron, ROS, like the superoxide radical and the hydroxyl radical, are instrumental in causing damage to essential cellular components such as mitochondria, lipids, DNA, RNA and proteins [58], which disrupt normal cellular function and eventually leads to the death of the neuron. In the course of metabolism within the DA neuron, the superoxide radical can be produced in several different ways. First of all, DA itself is metabolized by MAO, an outer mitochondrial membrane enzyme, which results in a two-electron reduction of oxygen and the production of both the superoxide and the hydroxyl radical [58]. In PD, as a result of the loss of the DA neurons, the remaining DA neurons become hyperactive and tend to exhibit increased DA turnover which results in increased ROS production [104]. Furthermore, presumably, since l-DOPA therapy increases brain levels of DA [58], DA can auto-oxidize to increase the ROS load in the brain that includes the superoxide radical [58]. Also, NADPH oxidase and cyclo-oxygenase-2 (COX-2), both inducible enzymes in pathological situations such as PD [95, 105], generate the superoxide radical. Moreover, superoxide anion can also be generated due to electron leakage during any of the electron transfer reactions of the electron transport chain. The superoxide radical generated in the mitochondrial matrix is converted to H2O2 by manganese SOD. Part of the superoxide radical can go to the inner membrane space (IMS) and part can escape by leakage into the cytosol of the neuron [104]. These scenarios represent at least three oxidative stress situations (mitochondrial, cytosolic and inflammatory) during which the superoxide radical is produced. In mimicking the situation proposed to occur in PD, our previous studies have demonstrated that the superoxide radical is involved in the oxidative stress produced by the MPTP neurotoxic process [106] and that the source of this radical appears to be the microglia [56, 95]. Antioxidant defense systems exist to protect the brain in oxidative stress situations. For the three different oxidative stress situations noted above involving the superoxide radical in the brain, different SOD [107] have evolved to handle these situations. There are three different isoforms of the SOD enzyme: copper/zinc SOD (SOD1), manganese SOD (SOD2) and extra-cellular SOD (SOD3). Each isoform is encoded by different genes and is localized to specific cellular compartments. SOD1 is found mainly in the cytosol, although a substantial amount, estimated at between 1 and 2% of total SOD1, is localized in various mitochondrial compartments [108–113] and in the nucleus of mammalian cells [114]. SOD2 is localized to the mitochondrial membrane and is considered the primary defense in the mitochondria against the superoxide radical [115]. SOD3 is a copper-containing enzyme that is secreted into the extracellular space. Although it was originally thought that their sole purpose was to dismutate the superoxide radical, it is now known that SODs contribute to the copper and manganese cycles that are vital to cell survival in an oxygenated environment [116].

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To unequivocally prove that the superoxide radical is involved in the MPTP n­ eurotoxic process, we first used mice with increased expression SOD1 [117]. We found that transgenic mice expressing increased levels of SOD1 were resistant to MPTP-induced toxicity compared to their non-transgenic littermates. This effect was not attributed to a lower delivery of MPTP to the brain nor to a reduced ­conversion of MPTP to MPP+ nor to a lower level of MAO-B activity. Our results ­suggested that protection was most likely due to the increased levels of SOD1 and that some of the noxious effects of MPTP were probably due to the increased ­presence of the superoxide radical. Other studies involving SOD2-deficient mice [115] ­demonstrated increased MPTP toxicity in the SNpc and striatum of these mice. To demonstrate that the superoxide radical is indeed present in the SNpc ­following MPTP administration, we injected MPTP-treated mice with hydroethidium at 1 and 2 days after treatment [95] and, 15 min later, observed hydroethidium fluorescence in the SNpc that seemed to be localized to the microglia. Hydroethidium is oxidized by the superoxide radical to ethidium, which is also a dismutation reaction for superoxide [118]. MPTP-induced SNpc DA neuron degeneration and microglial activation seem to go hand in hand. In our studies, we observed a significant amount of SNpc DA neuron death as early as 24 h after MPTP administration [16]. When assessed with fluorojade, an anionic fluorescein dye [119], SNpc DA neuron death is noted to occur earlier than 24 h after MPTP dosing [120]. We also saw an upregulation of macrophage antigen complex-1 (MAC-1), indicative of microglial activation, as early as 1 day after MPTP intoxication that peaked at the 2-day time point [56, 95, 98]. Thus, the injury process had already begun before microglial activation, and it is the injury that elicits the microglial response and not the other way around. In order to propagate further injury, microglia must exhibit a sustained activation. Epidemiological studies suggest that a chronic inflammation may increase the risk for developing a neurodegenerative condition such as PD [121] and animal studies using MPTP have shown that a chronic inflammatory state can augment MPTP-induced DA neuronal damage [103]. Since both PD brains and experimental models of PD demonstrate significant microglial activation, this ­suggests that inflammatory factors may mediate SNpc DA neuronal death. Among the inflammatory mediators capable of promoting DA neurodegeneration are ­microglial-derived ROS that probably contribute to the oxidative stress that is reportedly part of the pathogenic hypothesis of PD [121] and certainly part of the MPTP ­neurotoxic process. A significant source of ROS, particularly the superoxide radical during neuroinflammation, is the enzyme NADPH oxidase. This multimeric enzyme is composed of four subunits (gp91phox, p22phox, p47phox and p40phox) and is ­inactive in resting microglia because p47phox, p67phox and p40phox are all present in the cytosol as a complex and are separated from the transmembrane proteins, gp91phox and p22phox Ref122. When microglia become activated, p47phox is phosphorylated and the entire complex translocates to the plasma membrane where it assembles with gp91phox and p22phox to form the NADPH oxidase complex, which is now capable of reducing oxygen to the superoxide radical [122]. NADPH oxidase is known to be instrumental in the killing of invading microorganisms in infections through the

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sustained production of superoxide [122]. Once activated, microglia in the SNpc produce significant amounts of the superoxide radical [95] as noted above. Although the superoxide radical in and of itself is not extremely toxic, it can give rise to secondary reactive oxygen species [122] that can be far more damaging. Our studies using gp91-deficient mice confirmed the role of NADPH oxidase in ­activated microglia following MPTP treatment in that TH neurons seemed to be protected in these mice and there was a dampening of the microglial response. Furthermore, when we infused SOD1 into the striatum prior to MPTP administration, both TH-positive fibers in the striatum and the TH-positive neurons in the SNpc were protected against the damaging effects of MPTP. These studies ­confirmed several of the findings in human PD related to the upregulation of NADPH oxidase, which is only one of the many contributors to oxidative stress in the brain. Besides the above-listed sources, COX-2 [75, 95] also generates the superoxide radical and is instrumental in the oxidation of DA in the striatum. Two SOD mimetics, M40401, a non-peptidyl SOD mimetic and Tempol, a nitroxide antioxidant, both significantly blocked the production of the superoxide radical in the SNpc of MPTP-treated mice (manuscripts in preparation) as assessed by dihydroethidium.

9 Nitric Oxide and MPTP One of the things that we noted in our MPTP studies was that although we were able to block superoxide radical production, which afforded some protection to the DA neurons in the SNpc, blockade of microglial activation was not 100%, nor was protection of the SNpc DA neurons [117]. Thus, we surmised that other compounds had to be involved here. A likely candidate is nitric oxide (NO) as this compound is abundant in the CNS [56, 123], is generated by the nitric oxide ­synthase (NOS) enzymes, can cross membranes freely without the aid of carriers and has many diverse functions such as that of a physiological messenger and a mediator of toxic insults to the nervous system [124]. Of the three isoforms of the NOS enzyme [125], eNOS, found mainly in blood vessels, does not seem to have any role in the MPTP neurotoxic process in the SNpc or in the striatum (Wu et al., unpublished data). However, the other two isoforms, neuronal NOS [120, 126] and inducible NOS [56, 98], have been implicated in MPTP-induced oxidative stress in the SNpc. Of these two NOS isoforms, immunostaining documented that iNOS is upregulated in activated microglia. To demonstrate how microglia might be ­activated in the MPTP neurotoxic process, studies [90] show that astrocyte-derived cytokines such as TNF-a, IL-1b and IFN-g activate the microglial cell surface ­antigen CD23. In turn, CD23 induces iNOS up-regulation and NO production in the microglia [127]. NO can turn around and activate more cytokines and the process of microglial activation appears to become cyclic as the toxins produced by ­activated microglia can affect the external environment of the neurons in the SNpc in an adverse manner. In the striatum, the nNOS isoform of the NOS enzyme is present in high

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amounts within medium-sized neurons whereas in the midbrain, nNOS is found in both cholinergic and serotonin fibers [126]. Although thus far, nNOS has not been found within DA neurons of the nigrostriatal pathway in the in  vivo setting, by virtue of the fact that it is ever-present around the neurons in this pathway, the belief is that nNOS plays some role in the MPTP-induced damage to DA neurons. This reasoning comes from the fact that iNOS-deficient mice exhibit only a partial protection of the DA neurons when treated with MPTP [56]. Furthermore, minocycline, a second-generation tetracycline antibiotic, was shown to inhibit iNOS up-regulation following MPTP [93, 98, 128]; however, it did not block TNF-a upregulation [93], which may be the reason that SNpc protection in the iNOSdeficient mice was incomplete.

10 Peroxynitrite and MPTP Countless studies have suggested that reactive species capable of inflicting damage such as nitration of proteins, aromatic amino acids, and DNA is a part of the oxidative stress situation that has been characterized as being a part of the PD pathological picture. This involves the post-translational modification of cellular components necessary for the normal function of the cell. Post-translational modification here is the interaction of these components with peroxynitrite, one of the most damaging substances known to man [129]. Peroxynitrite is a shortlived oxygen species that is the result of the reaction between the superoxide radical and NO at diffusion-controlled rates [129]. Its site of formation must be near that of superoxide because, while NO can diffuse across membranes, superoxide, by virtue of its being a charged particle, cannot [129]. Thus, since both compounds appear to emanate from microglia, the site of formation of peroxynitrite is most likely the microglia. Peroxynitrite does not seem to damage microglia as these cells contain high levels of antioxidants such as glutathione and glutathione peroxidase [130] which act as a barrier against peroxynitrite-induced damage. In relation to the oxidative stress induced by MPTP, we have demonstrated that NADPH oxidase [98] and iNOS [56] are induced to produce high levels of the superoxide radical and NO, respectively. Using immunoprecipitation assays, we showed that several tyrosine residues (125, 133 and 136) of alpha-synuclein are nitrated following the application of peroxynitrite in an in  vitro situation and following MPTP administration in an in  vivo situation [124] In this same study, the footprint of tyrosine nitration, 3-nitrotyrosine, was also demonstrated both in the striatum and in the ventral midbrain of the treated mice. Furthermore, we have previously reported that the rate-limiting enzyme in dopamine synthesis, TH, is a selective target for nitration following exposure of PC12 cells to either peroxynitrite or MPP+131. We also showed that the nitration of TH occurs in mouse striatum after MPTP administration [131]. Nitration of tyrosine residues in TH results in loss of its enzymatic activity. In the mouse striatum, tyrosine nitration-mediated loss in TH activity paralleled the decrease

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in dopamine levels, whereas the levels of TH protein remain unchanged for the first 6 h after MPTP injection. Not only can peroxynitrite interact with TH and alpha-synuclein [124, 131], it can also react with the SOD dismutase enzymes [132], catecholamines [133], DNA and a host of other cellular components [134]. The rates of peroxynitrite formation have been estimated to be as high as 50–100 micromoles per minute; however, during oxidative stress, its steady-state contents in specific cellular compartments can be sustained in the nanomolar range for several hours [135]. And, at the aforementioned steady-state level, peroxynitrite can influence several signaling pathways, many transcription factors and even the apoptopsis-necrosis continuum of cell death that have been produced by the MPTP neurotoxic process.

11 Mitochondria, MPTP and Oxidative Stress Mitochondria are the power plants of the cell. Their main function is the production of ATP. For this, electrons are passed down the electron transport chain (ETC) through a series of membrane proteins (complexes I–IV) to oxygen [136]. In this process, protons are pumped across the inner mitochondrial membrane (IMM), giving rise to an electrochemical gradient that drives ATP synthase and thus ATP ­production [137–139], the cell’s energy source. However, the ETC is also the main source of ROS [140] due to the leakage of single electrons to oxygen, forming superoxide [141]. Most ROS are produced at the catalytic sites of complex III [142–145] and complex I [146–149]. As these studies were demonstrated, considerably in higher rates of superoxide radical production occur in conditions of reverse electron transport in complex I and when complex I is blocked downstream of the FMN site by inhibitors such as MPP+ and rotenone. In order to evaluate the effects of MPTP on the ETC, Desai et  al. [150]. have examined the effects of 200  mM MPP+ on mitochondrial complexes I–IV by incubating mitochondria from mice of three different ages. They then monitored enzyme activities at 5, 10, 15, 30, 60, and 120 min. Significant inhibition of complex I, III, and IV was noted within 15 min in all of the age groups studied, followed by some recovery of enzyme activity upon further incubation for complexes I and IV. Complex II was not affected by MPP+ in any of the studied age groups. Inhibition of complex I, III, and IV by MPP+ blocked the flow of electrons down the ETC, which led to a decrease in ATP production. It was noted that inhibition was most severe in mitochondria from the older mice, which suggests that age is a factor in ETC inhibition and, therefore, oxidative stress may be more severe in the aged brain. The fact that MPP+ inhibition at the complex I site is weak [151] has led to some ­controversy as to the legimacy of the claim that MPP+ blocks at the complex I site in the ETC. To prove that MPP+ blockade at the mitochondrial complex I was indeed the legitimate mechanism of MPTP toxicity and damage, Richardson et al. [152]. Complemented SK-N-MC neuroblastoma cells with NDI1, a reduced nicotinamide adenine nucleotide dehydrogenase that is rotenone-insensitive [152]. The

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cells were then transfected with human DAT so that MPP+ could be taken up by these cells. This NDI1 subunit exhibited a number of effects that proved unequivocally that complex I was indeed blocked significantly by MPP+ since bypassing complex I attenuated MPP+ toxicity in the neuroblastoma cells. Delivery of NDI1 via a viral vector to the mouse brain prevented MPTP-induced damage to the nigrostriatal system in these animals as well as astrocytic and microglial activation. Behavioral deficits were also ­prevented by NDI1. Also noted was protection of VMAT and DAT as well as the TH-expressing neurons in the SNpc. Mitochondrial involvement in the MPTP neurotoxic process also extends to peroxynitrite as mitochondria are also a site for peroxynitrite formation. MPP+ blocks at the complex I site in the ETC, thus production of the superoxide radical is excessive [55]. And since NO can either come to the mitochondria or be formed within the mitochondria, by virtue of the fact that superoxide is a charged substance and NO is not, peroxynitrite is most likely formed within the mitochondria. Remember that NO can react faster with superoxide than mitochondrial SOD2. As one of its many effects during oxidative stress, peroxynitrite can nitrate SOD2 [153] at its tyrosine residues, thus inactivating SOD2 and leaving more superoxide available for peroxynitrite production. Since peroxynitrite is highly reactive, it can react with other components of the METC. Most of these reactions depend on the actual concentration of the compound being reacted with and on the rate constants of the reactions [129]. And, these reactions result primarily in the inactivation of such compounds as complex I, complex II, ATPase, aconitase, cytochrome c oxidase, and creatine kinase.

12 Conclusion MPTP is indeed the tool of choice for investigations into the etiology of PD. It has pointed us in the right direction of DA neuron death in PD by replicating all of the known hallmarks of PD. With this compound, we have been able to sort out a ­number of the individual events such as MAO-B upregulation, DA metabolism, cytokine formation, superoxide formation, nitric oxide upregulation and blockade of mitochondrial complex I and how these events all conspire to contribute to the death of the DA neuron. Furthermore, what MPTP has also demonstrated is that the interplay between these sited factors in the death of the DA neuron is indeed a complicated matter.

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Oxidative Stress in Alzheimer’s Disease: A Critical Appraisal of the Causes and the Consequences Jaewon Chang, Sandra Siedlak, Paula Moreira, Akihiko Nunomura, Rudy J. Castellani, Mark A. Smith, Xiongwei Zhu, George Perry, and Gemma Casadesus

Abstract  Recent advances have shown oxidative damage as one of the hallmark characteristics in neurons in Alzheimer’s Disease (AD). Importantly, such damage is present at the very earliest stages of disease, including mild cognitive impairment, and persists throughout the course of the disease. Therefore, oxidative imbalance is likely important not only as an initiator of disease but may also contribute in propagating the disease process. One aspect of critical importance is developing treatments that target the source rather than the “collateral damage,” but of course this requires knowledge of the source. This review highlights the role of oxidative stress in AD with the aim of critically evaluating the role of oxidative stress as a cause or effect in the development of this disease. In doing so, we consider the sources of reactive oxidative species and their role in AD as well as how oxidative responses intertwine with the pathological hallmarks of the disease. Keywords  Alzheimer’s disease • Amyloid • Antioxidant • Neurofibrillary tangles • Oxidative stress • Redox balance • Senile plaques • Tau phosphorylation

1 Introduction Advances in medicine have allowed humans to live longer and reach far greater average ages than in the past. With this growing and aging population, age-related neurodegenerative diseases can only become more prevalent. Studies have shown that both forms of Alzheimer’s Disease (AD), sporadic and familial, do not stem from a single source. Rather it is now accepted that AD is a syndrome, one that is impacted by events ranging from living habits and environment factors to hereditary genes [1].

G. Casadesus (*) Department of Neuroscience, Case Western Reserve University, Cleveland, OH, USA e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_9, © Springer Science+Business Media, LLC 2011

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There have been overwhelming findings of a disturbance in the cellular metabolic equilibrium during the course of AD [2–4]. This metabolic imbalance results in the cellular production of oxidative stress [5]. The process of oxidative stress production by cells is a fine equilibrium between the production of reactive oxygen species (ROS) and the removal of such species by endogenous antioxidant defenses [6]. This imbalance has been at the core of many studies, emphasizing the effects of the oxidative stress in AD [7, 8]. On the other hand, excessive free radical production can be disputed whether it is a cause or an effect of the appearance of AD pathology. Senile plaques and intraneuronal neurofibrillary [9] tangles are two pathological markers of AD that have been intimately associated with oxidative stress production. For example, amyloid-b (Ab) plaques can form quickly after a head injury in both adults and children [10], suggesting that the deposition of Ab is a consequence of the acute phase response of nerve cells to any stress in susceptible individuals, including oxidative stress [9]. Increased oxidative stress levels are also observed in the AbPP transgenic mouse prior to the deposition of Ab [11] and recent studies demonstrate that Ab may serve as an antioxidant [12–14]. Importantly, mitochondrial SOD2 deficiency exacerbates amyloid burden, significantly reduces metal levels in the brain, and increases levels of Ser-396 phosphorylated tau, providing a mechanistic link between mitochondrial oxidative stress and the pathological features of AD [15]. Nevertheless, the current amyloid hypothesis postulates oxidative stress as a downstream insult driven by Ab oligomer and fibril deposition, primarily based on studies demonstrating that brains of familial AD patients have higher levels of oxidative stress [16] and that the addition of oligomers in culture leads to increased free-radical production [17]. The objective of this review is to provide a critical review of the cause and effects of the oxidative stress imbalance found in AD and how these may be linked to AD pathology.

2 AD Neuropathology Pathologically, AD is delineated by two distinct lesions, extracellular senile plaques and intraneuronal neurofibrillary tangles (NFT). Neuropil threads and a selective loss of neurons are also distinct features accompanying NFT formation. NFT consist mainly of the microtubule-associated protein tau present as paired helical filaments (PHF). In regard to Ab containing senile plaques, recent advances have focused primarily on the multiplicity of mutations in the amyloid-b protein precursor (AbPP) encoding genes as well as the altered AbPP proteolytic processing. These mutations end with increasing Ab1–42 [18]. The fact is that the mutations only account for the familial cases of AD. Transgenic mice that overexpress AbPP, or any of the other human mutant AD-related proteins, exhibit many neuropathologic as well as behavioral features of the human disease: development of senile plaques, neurotoxicity, and NFT develop in mice that expressed mutant tau protein [19]. The animal models that are used to develop and test methods of treatment have shown reduced levels of Ab protein, neuritic plaque load and glial activation. Various ­treatments have even

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restored learning and memory function [20]. Recent data ­indicates some pathogenic mutations that affect Ab processing lead to decreased, rather than the expected increased, levels of Ab [21–23]. The persistent central dogma has been the aggregation of Ab into toxic fibrillar deposits within the extracellular space of the brain initiates the onset of AD, highlighting the evidence that suggests that the mature senile plaque is nontoxic [24]. Other efforts that examine the soluble oligomers of Ab that seem to be mere intermediates in the aggregation process [25–28] have not clarified the relevance of in vitro neurotoxic effects to in vivo disease. Of the two major theories of neuronal loss in AD, it is continuously debated as to whether senile plaques or NFT are the cause, as well as which plays a more vital role in the neuronal loss and subsequent cognitive decline. The majority of those in the field favor the amyloid cascade hypothesis, the theory of Ab aggregation into toxic fibrillar deposits within the extracellular space of the brain. These toxic fibrillar deposits disrupt neuronal and synaptic function, making way to neuronal degeneration and eventually dementia [29]. Yet Ab, formed upon proteolytic processing of AbPP by b- and g-secretases, has a widespread distribution through the brain and body in healthy individuals throughout life. One interesting point is that tau pathology and NFT correlate with cognitive symptoms in patients, contrary to a lack of association with senile plaques [30]. Tau regulates microtubule assembly and transport, and in AD becomes hyperphosphorylated (up to 22 unique sites) and begins to lose its stability and binding functions of the microtubules, aggregating into PHF [31]. This hyperphosphorylation of tau shows an abnormal action of kinases as well as lowered phosphatase activity [32]. The hyperphosphorylated tau is self-destructive as well, as it cuts off normal tau, removing any possible chance of recovering microtubule maintenance [33, 34]. Hyperphosphorylated tau also binds to proteasomes, which is inhibitory to the proteasome activity in AD. This dysfunction of the proteasome could cause degeneration and death of neurons in AD [35]. Whether NFT deposition is reversible remains uncertain; it is clear, however, that NFT aggregation leads to a permanent insolubility that is catalyzed by processes under influence of oxidative stress [36, 37]. Even with all the studies, it is possible that senile plaques and NFT may be mere effects rather than sources of AD cognitive changes [38]. One theory is that senile plaques and NFT deposition is a mere attempt of the brain to nullify the oxidative stress placed upon itself.

3 Oxidative Stress in AD Oxidative stress refers to the imbalance between the biochemical processes of producing ROS and the removal of ROS. ROS cause damage to virtually all biomacromolecules [39–41], a process that is intimately linked to failure of biological function and ultimately the demise of the cell [42] in aging and neurodegeneration [43]. Oxidative stress on biomacromolecules is associated with the neuronal ­degeneration in the central nervous system of AD brains [44] (1) DNA and RNA

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oxidation is marked by increased levels of 8-hydroxy-2-deoxyguanosine (8OHdG) and 8-hydroxyguanosine (8OHG) [45, 46] and increased DNA oxidation and decreased repair in cerebrospinal fluid [47, 48]; (2) Protein oxidation is marked by elevated levels of protein carbonyls and nitrotyrosine [49–51]; (3) Lipid peroxidation is marked by higher levels of malondialdehyde and isoprostanes, as well as protein modification by HNE [51–57] and by acrolein [58]; (4) Sugar oxidation is marked by increased protein glycation and glycoxidation [59–63]. Oxidative stressinduced by-products accumulate even in normal neurons in AD as well as in preNFT stages [55, 64–66] and perhaps the modifications induced by the by-products play a more causative role in the pathology of AD. Studies on the sources of oxidative stress, i.e., the primary cause of increased ROS in AD focus primarily on the mitochondria because of their susceptibility to ROS production [67], and their degradation of heme proteins that release iron [68]. Heme oxygenase-1 is increased in AD [69–71] and this protein catalyzes converting heme to biliverdin, an antioxidant, with a by-product of iron. An occurrence of downregulation of the mitochondrial respiratory chain has been observed in AD [72], as well as increased mitochondrial DNA and cytochrome oxidase in neuronal cytoplasm, indicating perhaps an increased turnover of mitochondria. Along with these mitochondriarelated changes, observations of structural abnormalities within the mitochondria themselves and mtDNA deletions are also disease related. Some believe these to occur before amyloid deposition as well as neuronal degeneration [73]. Upregulation of superoxide dismutase (SOD) and glucose-6-phosphate dehydrogenase provide immunocytochemical evidence that constant oxidative stress may cause some of the damage found in AD [74, 75]. This increased oxidative damage exhibits the inability of the neurons to compensate through endogenous antioxidant activity [76]. It is possible, however, that these oxidative stress imbalances are mere markers for the initial stages of AD, and could be treated through rebalancing with mechanisms that diminish the oxidative damage [77, 78]. Deposition of tau and Ab in NFT and senile plaques, respectively, might actually be responses to an oxidatively stressed condition. The deposition of these proteins could represent an attempt at repair, with the function to trap oxidative stress agents [79]. NFT have actually been shown to persist in neurons for years and even decades. With the ability to bind and stabilize iron in vitro [80], it seems that tau accumulation could be used by the cell as a simple shield against ROS. Ab may also have a similar function against ROS as increased Ab aggregation is found to be associated with decreased neuronal levels of 8OHG [45, 81]. 8OHG is an oxidation product of RNA, and with RNA having a high turnover rate, it seemed to have a distribution similar to nitrotyrosine. Nitrotyrosine is usually produced from tyrosine after attack by peroxynitrite and 8OHG by •OH attack of guanosine. In this case, when tissue is treated with nitrite and H2O2, nitrotyrosine is produced. Looking at nitrotyrosine, it was found to be in the cytoplasm of neurons lacking NFT [49], in contrast to NFT-containing neurons that showed reduced levels. Amyloid plaques may be able to trap ROS due to the ability of Ab to bind to transition metals, a strong chelating property of Ab. Under some conditions, Ab does have some antioxidant effects [82], but the question of how much the effect in the big picture is still questionable. While more studies are required to understand

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the overall picture of the role of NFT and senile plaque aggregation, that they may be initially protecting responses to oxidative stress marks them as possible therapeutic mechanisms evolved physiologically.

4 Sources of Oxidative Stress Defined 4.1 Mitochondrial Dysfunction in AD Mitochondria are the “powerhouses” of cells, providing the high-energy requirement of neuronal function. This high-energy requirement requires aerobic oxidative phosphorylation, a major source of ROS. Superoxide anion radicals produced from the side reaction of O2− receiving single electrons from the electron transport chain, is primarily from where ROS arise. Superoxide can also be produced from mutant or damaged metalloenzymes that are involved in oxidative metabolism, or generated by the respiratory burst of neutrophils. It can be then transformed into other classical ROS species such as H2O2 and •OH. These mitochondrial-generated reactive species have several cellular targets including mitochondrial components. Mitochondrial DNA lacks histones, and having no ability to repair DNA, mitochondria are easily damaged by oxidative stress, leading to dysfunction. Changes occur in multiple mitochondrial proteins, which are also linked to cognitive impairment [83]. All of these features have been implicated in the pathogenesis of AD, as well as other neurodegenerative disorders [84].

4.2 The Role of Metals in Oxidative Stress Production and AD In most biological systems, redox-active transition metals such as iron and copper are necessary, in trace amounts. The accumulation of these metals in tissues, as well as metalloproteins, can produce higher concentrations of free metal ions. These ions, which are present to usually mediate oxidative stress reactions [81, 85] are only needed in trace amounts to catalyze “redox cycling.” Redox cycling is the process when cellular reductants such as thiols or vitamin C reduce the oxidized transition metal ions. This superoxide radical is chemically unreactive, but serves as the reductant of oxidized metal ions, which are available to produce hydroxyl radical from H2O2 through the Haber–Weiss reaction. The hydroxyl radical reacts with all biomacromolecules at diffusion-controlled rates, i.e., distances from its site of generation. It is most commonly generated by the Fenton reaction between reduced transition metals and H2O2. Under regular conditions, ROS damage is prevented by the antioxidant cascade. This protection is provided by two different methods (1) converting superoxide to O2− and H2O2 through cytosolic copper–zinc ­superoxide dismutase (CuZnSOD) and mitochondrial manganese superoxide ­dismutase (MnSOD) and (2) removal of by-products of oxygen reduction by ­oxidase

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(i.e., monoamine oxidase) by enzymes (catalases and peroxidases). In AD, a link between metals and oxidative stress modifications is well established [86–88].

5 Conclusion Looking at oxidative stress and its role in AD, several questions arise. First, is oxidative stress a cause or an effect? This is hardly a question that a simple study will answer; rather, it may be a starting point in looking for a solution. Secondly, what are the causes of oxidative stress? Oxidative stress comes from a variety of sources, but primarily the redox cycling as well as the by-products of aerobic oxidative phosphorylation. Third, is the pathology of AD primarily an effect or a last-resort attempt at protecting against oxidative stress? These questions may seem simple, but the answers are complex and ever changing as studies progress. How much influence does oxidative stress have upon the development of AD? For example, the aggregation of Ab into senile plaques is still being debated – whether it is a cause of AD or a result – and does it play a prooxidant or antioxidant role in AD? Then, what role do the other factors, inherited genes as well as outside factors of environment, enrichment, and lifestyle play in association with Ab and AD [89]? Under controlled conditions, oxidative stress can readily induce cell death and antioxidants can protect and prolong a cell’s lifespan under pressure. On the other hand, in complex disease states, it is difficult to show the relationship of oxidative stress to AD. The studies are unclear, complex, and open-ended, with narrow conclusions being promoted. In order to advance the field, the traditional view of only examining end-point lesions in AD will not provide the solutions that are sought. If oxidative stress is the cause, therapeutic studies would focus on prevention through use of antioxidants, transition metal chelators, and other ways of enhancing the ROS removal processes [89–91], keeping in mind that antioxidants also have metal-reducing capabilities often similar to the very agents that are being removed. On the other hand, the role of Ab and tau accumulation must be defined, since any efforts to remove Ab and tau may be a risk as they may provide a protective biochemical process that has yet to be confirmed. Nevertheless, achieving a preventative method for AD without any drawbacks persists as an arduous and complex goal.

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Retinal Disturbances in Patients and Animal Models with Huntington’s, Parkinson’s and Alzheimer’s Disease C. Santano, M. Pérez de Lara, and J. Pintor

Abstract  Huntington’s disease (HD), Parkinson’s disease (PD) and Alzheimer’s disease (AD) are neurodegenerative disorders associated with aging. The main hallmarks of these pathologies are deposits of mutant proteins, mitochondrial dysfunctions, reactive oxygen species (ROS) and neuronal death. These diseases affect from heterogeneous form to different brain regions. In fact, psychophysical, electrophysiological and morphological evidence shows that retinal and higher visual centers disturbances occur in HD, PD and AD. Thus, patients with these disorders suffer contrast sensitivity deficits, altered color vision and damaged conscious and unconscious visual perception. These visual deficiencies may contribute to symptoms of these pathologies related to behavior, memory and difficulty in performing daily tasks such as driving, reading or keeping their balance. This chapter tries to integrate the visual changes in pathogenesis and symptomatology of patients with HD, PD and AD and reviews the recent findings in animal models of these diseases to provide an insight into how retinal changes might contribute to symptoms of HD, PD and AD. Keywords  Alzheimer’s disease • Huntington’s disease • Melatonin • Neurodegenerative disorders • Parkinson’s disease • Retina • Reactive oxygen species Abbreviations 6-OHDA AD APP APPswe

6-Hydroxydopamine Alzheimer’s disease Amyloid precursor protein Amyloid precursor protein with the double Swedish mutation

C. Santano (*) Departamento de Bioquímica y Biología Molecular, Escuela de Optica UCM, Madrid, Spain e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_10, © Springer Science+Business Media, LLC 2011

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Amyloid b peptides Contrast sensitivity Electroretinogram Huntington’s disease Huntingtin l-3,4-Dihydroxyphenylalanine 1-Methyl-4-phenylpyridinium 1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine Neuron-restrictive silencer elements Neuron-restrictive silencer factor Parkinson’s disease, PERG, pattern electroretinogram Phosphatase-tensin homologue induced putative kinase 1 Presenilin 1 Repressor-element 1 silencing transcription factor Reactive oxygen species Visual evoked potentials

1 Introduction The vertebrate retina is developed from the neural ectoderm of the embryo [1]. It is a thin sheet of nervous tissue of approximately 0.5  mm thick situated inside the back of the eyeball and its function is to convert light into electrical signals that will be sent through the optic nerve to the lateral geniculate nucleus that projects to the visual cortex. The retinal structure is well known and contains a high diversity of neuronal types. For instance, in mammalian retinas, up to 50 neuronal types can be distinguished based on their morphological, biochemical and functional characteristics [2].

1.1 Retinal Organization The retina is in contact with the retinal pigmented epithelium, which develops from the same sheet of neuroepithelium and its function is tightly coupled with retina [3]. In fact, the retinal pigmented epithelium is essential for proper development of the retina, promoting photoreceptor survival and providing them with vital nutrients and oxygen. Among others, one of the roles of the epithelial cells is to phagocytose shed photoreceptor outer segments, which is an essential function for the maintenance of the photoreceptor excitability [4]. Understanding the retina architecture leads to an understanding of its function. The retina is a multi-layered structure that is isolated from other ocular structures by the inner and outer limiting membranes. The outer limiting membrane is not a real membrane but is a site rich in occluding junctions between Müller cells

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Fig. 1  Schematic drawing of main neurotransmission systems implicated in vertical and lateral pathways of the mammalian retina. Receptor localization in plexiform layers is summarized at the bottom table. AC amacrine cell, C cone, cBC cone bipolar cell, dA displaced amacrine cell, GABAR g-aminobutyric acid receptor, GC ganglion cell, GCL ganglion cell layer, GlyR glycine receptor, HC horizontal cell, INL inner nuclear layer, IPC interplexiform cell, IPL interplexiform layer, mGluR metabotropic glutamate receptor, ONL outer nuclear layer, OPL outer plexiform layer, R rod, rBC cone bipolar cell

and photoreceptor inner segments that lead to an optical effect visible with the light microscope. The inner limiting membrane is a basement membrane secreted by endfeet of Müller cells. Somata of neurons and glial cells present in the retina are distributed into three nuclear layers and their processes form two plexiform layers (Fig. 1): the outer plexiform layer and the inner plexiform layer, which are where synaptic contacts are established. The outer nuclear layer contains the cell bodies of the photoreceptors: rods and cones. In the inner nuclear layer somata of bipolar, horizontal, amacrines, interplexiforms, Müller cells and displaced ganglion cells are located. The ganglion cell layer is formed of ganglion cells, displaced amacrine and glial cells. Rods are responsible for low-light vision, called scotopic vision, and cones are responsible for daylight, called bright color vision or photopic vision. The predominance of these sensory cells depends on the different habitats or life styles of animals [1]. For example, rodents are nocturnal animals and have rod-dominant retinas. In humans, rod and cone distribution it is not uniform along the retina. The macula is localized at the center of the retina, on the optical axis where

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incoming light is principally focused, and is characterized by a greater photoreceptor concentration than peripheral retina. Moreover, the center of the macula, called the fovea, contains only one type of photoreceptor, the cone family, which is involved in visual acuity. However, cats and dogs do not have a macula but have a specialized area, the area centralis where cones predominate. As in the central nervous system, there are many glial cells in the retina. The subtypes of glial cells identified in the mammalian retina are Müller cells (radial glia), astrocytes and microglia. Oligodendrocytes have been found in the myelinated streak of rabbit retina [5–9]. These glial cells have not been identified in the adult human retina but myelinated axons have been documented in the retinal nerve fiber layer [8–12]. Moreover, oligondredocyte progenitors cannot migrate to the retina and myelinate the axons of ganglion cells because of a well-developed lamina cribosa in the optic nerve head [6]. However, oligodendrocyte characteristic molecules such as myelin-associated glycoprotein [13] and myelin oligodendrocyte specific protein [14] are expressed by Müller cells, and thus these cells may be responsible for retinal myelinization. Human intra-retinal myelinization occurs as a developmental variant in 1% of human eyes, although retinal myelinate patches have been found in patients with ocular pathologies, such as myopia, amblyopia and strabismus [11, 12, 15, 16].

1.2 Transmission of Visual Information The rods and cones respond to luminous information, transforming it into electrical impulses by means of a biochemical process termed phototransduction. In dark conditions, photoreceptors are depolarized and constantly release glutamate. Its effects are mediated by ionotropic glutamate AMPA receptors [17] present in bipolar cells. The activation of these receptors hyperpolarizes bipolar cells, which inhibit the generation of action potentials by ganglion cells and their propagation through the optic nerve. Under photopic conditions, electrical changes are generated in photoreceptors during phototransduction processes that are transmitted from bipolar cells and then send messages to ganglion cells by synaptic connections in the vertical visual pathway that it is modulated by the lateral visual pathway (Fig. 1). For neuronal communication in the retina, the presence of excitatory and inhibitory neurotransmitters (Fig. 1) is necessary, including a diversity of transmitter substances. Recent works show that adenosine and ATP have important roles as co-neurotransmitters in retina [18–23]. Depending on electrophysiological response, cone bipolar cells can be classified as ON bipolar cone cells when they respond with a depolarization by metabotropic glutamate receptors and OFF cone bipolar cells that respond with a hyperpolarization by AMPA receptors. However, rod bipolar cells are always depolarizated after receiving inputs from rods. Thus, visual information is transmitted through the vertical pathways that can be divided in ON and OFF sub-circuits (Fig. 2). Thereby, rod bipolar cells receive inputs from rods although the visual information is not sent

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Fig.  2  ON and OFF pathways of visual information transmission in the mammalian retina. The schematic retinal diagram shows the vertical pathways in accordance with which bipolar cells are depolarized (ON) or hyperpolarized (OFF). In the rod pathway, information is not transmitted directly from rod bipolar cells (rBCs) to cone bipolar cells (cBCs), but type AII amacrine cells (AII) are implicated in feeding the messages to ON cBCs through electric synapses by conexin 36 (C×36) and through glycinergic synapses to OFF cBCs. Cones send visual information to ON cBCs or OFF cBCs and then transmitted to GCs. GCL ganglion cell layer, INL inner nuclear layer, ONL outer nuclear layer

directly to ganglion cells but through type AII amacrine cells. These cells integrate inputs and transmit them to cone bipolar circuitry exciting to ON cone bipolar cells through electrical synapses or inhibiting to OFF bipolar cells through glycinergic synapses [24]. Finally, ON cone bipolar cells feed the message to ON ganglion cells and OFF bipolar cells send the inputs to OFF ganglion cells (see Fig.  2). Cone bipolar cells respond to inputs from cones and transmit information to ganglion cells by direct vertical pathway (see Fig. 2) [24]. Modulation of the visual information transmission is carried out through parallel lateral pathways where horizontal, amacrine and interplexiform cells are involved in modulating inputs from photoreceptors to bipolar cells in the outer plexiform layer. Amacrine and interplexiform cells also modulate the transmission between bipolar cells and ganglion cells in the inner plexiform layer mediated by GABA, glycine and electrical synapses. Retinal feedback mediated by horizontal and amacrine cells provides a lateral inhibition preventing the spread of excitatory signals over a wide area through dendritic arborizations of bipolar and ganglion cells, causing the so-called “receptive fields” of these cells. Receptive fields are concentric regions

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consisting of two parts, a central region and an antagonistic peripheral region. Receptive field of ON bipolar and ganglion cells correspond to a depolarizated central region and hyperpolarizated peripheral region while the effects of center and surround are reversed in receptive field of OFF bipolar and ganglion cells [25]. Discrimination of contrast and edge detection is based on this mechanism [1, 26]. Color vision is due to the visual pigments of the outer segments of cones. Trichromatic vision in primates is transmitted through three types of ganglion cells, named M, P and midget, according to their size and their projection to the lateral geniculate nucleus (magno, parvo and koniocellular layers, respectively). The M ganglion cells convey motion information [27] and they project to the most ventral layers, 1 and 2, of the lateral geniculate nucleus [28]. The smaller P neurons convey red–green color information [27] and send the visual information to dorsal layer 4 [28]. The midget cells are involved in blue–yellow color processing and connect with koniocellular cells of the lateral geniculate nucleus [29]. Once the visual signal reaches the ganglion cells, it is sent a message through the optic nerve to the lateral geniculate nucleus that feeds information to the V1 area of the primary visual cortex of primates. Then, visual information is sent through the dorsal visual pathway to posterior parietal cortex to integrate the spatial relationships between our bodies and our environment (unconscious vision) [30]. The message concerned with identifying objects (conscious vision) is sent to the temporal lobe by a ventral pathway [30]. Basal ganglia are connected with both pathways and are implicated in the integration of the conscious and unconscious vision and motor processes.

2 Huntington’s, Parkinson’s and Alzheimer’s Diseases Huntington’s disease (HD), Parkinson’s disease (PD) and Alzheimer’s disease (AD) are a heterogeneous group of age-related and chronic illnesses. AD is the most common dementia in developed countries, and PD and HD are characterized by increasing severity of motor dysfunctions, although cognitive and emotional changes, dysfunctions of the autonomic nervous system and visual impairments are also symptoms of both these neurodegenerative diseases.

2.1 Etiology of Huntington’s, Parkinson’s and Alzheimer’s Diseases HD is a monogenic disorder belonging to a group of inherited neurodegenerative diseases termed polyglutamine disorders (see Table 1). However, the etiologies of AD and PD are not clear but they include environmental, genetic and aging risk factors. Nine genes with pathogenic mutations associated with familial PD (10% of cases) have been identified in 13 chromosomal loci (Table 2) [31–35]. Exposure to neurotoxins such as 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP), rotenone and paraquat [36, 37] or production of endogenous toxins by abnormal

Retinal Disturbances in Patients and Animal Models Table 1  Genes identified for HD Gene Locus Mode of inheritance 4p16.3 AD htt

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Probable function Intracellular trafficking and signalling Regulation of transcription Endocytosis Neuronal survival Embryonic development

AD autosomal dominant, htt huntingtin

Table 2  Genes identified for PD Gene Locus 4q21 SNCA Parkin 6q25.2–27

Mode of inheritance AD AR

Probable function Presynaptic protein Protection against apoptosis Mitochondrial biogenesis Spr 2p13 AD BH4 Biosynthesis UCHL1 4p14 AD Degradation mitochondrial proteins PINK1 1p35–36 AR Protection against apoptosis DJ-1 1p36 AR Antioxidant LRRK2 12p11.2–q13.1 AD Protection against apoptosis ATP13A2 1p36 AR Lysosomal degradation GIGYF2 2q36–37 AD IGF and/or insulin signalling pathways 2q12 Unknown Degradation mitochondrial HTRA2 proteins, Apoptosis AD autosomal dominant, AR autosomal recessive, ATP13A2 Type 5 P-type ATPase, BH4 tetrahydrobiopterin, GIGYF2 IGF and insulin signaling pathway, HTRA2 HTRA serine peptidase 2, LRRK2 leucin-rich repeat kinase 2, PINK1 phosphatase-tensin homologue induced putative kinase 1, SNCA a-synuclein, Spr sepiapterin reductase, UCHL1 ubiquitin carboxyl-terminal hydrolase L1

Table 3  Genes identified for AD Gene Locus Mode of inheritance Probable function 21q21.3 AD APP APP PS1 14q24.3 AD Regulate proteolysis indirectly (Notch and APP) PS2 1q31-42 AD 19q13.32 Complex (risk increase) Transport and mebolism of lipids APOE AD autosomal dominant, APOE apolipoprotein E (e4-allele), APP amyloid precursor protein, PS1 presenilin 1, PS2 presenilin 2

dopaminergic metabolism [38, 39] appear to increase the risk of suffering idiopathic or sporadic PD (90% of cases). With respect to AD, highly penetrant mutations in three genes (Table 3) cause familial early-onset (£65 years) autosomal dominant forms. Common polymorphisms with low penetrance, termed non-familial sporadic late-onset (³65 years) forms, are transmitted without familial segregation and appear to increase significantly the risk for AD (Table 3) [40, 41].

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Phenotypic variations between patients could be associated with a multifactorial cascade of damaging pathogenic mechanisms [42, 43], aging and genetic variations, for instance: a-synuclein and parkin polymorphisms implicated in PD [44, 45] and size of CAG sequence differences of the huntingtin (htt) gene in HD [46].

2.2 Pathologic Mechanisms HD, PD and AD have different symptoms and effects resulting in altered biochemical and cellular mechanisms. These neurodegenerative diseases have in common differential vulnerability among the affected brain regions and distinct neuronal subtypes that determine the characteristic symptoms of these pathologies. Some of the signs of these neurodegenerative processes, such as aggregation of proteins [47], imbalance of neurotransmitters and oxidative damage [48], have been strongly implicated in the pathogenesis of HD, PD and AD. Neuroinflammatory mechanisms and abnormally high levels of redox-active metals, mainly iron, aluminium and copper, can contribute to a raise in ROS generation [49, 50]. Mitochondrial dysfunctions play a crucial role in the pathogenesis of HD, PD and AD. Mainly, the impairment of the multienzymatic complex of the mitochondrial electron transport chain, deficiencies in ATP production, abnormal Ca2+ buffering and generation of ROS [51–54] bring about neuronal death [54–57] by apoptosis and autophagy, both processes occurring simultaneously. Furthermore, a high increase in intracellular Ca2+ can lead to glutamatergic excitotoxicity, and dysregulation of voltage-dependent Ca2+ channels. Alteration of the translocation nuclear transcription factors related to genes that regulate neuronal survival has been demonstrated. Moreover, disruption in the balance of ROS and reactive nitrogen species, and the appearance of neuroinflammatory processes contribute to the cascade of events that induce apoptosis in HD, PD and AD [49, 53]. Autophagy is a removal system for large proteins and organelles in normal physiological conditions. The degradation products are recycled to synthesize new macromolecules. However, reduced or defective autophagic activity occurs in HD, PD and AD, probably due to the accumulation of aberrant proteins and damaged organelles in abnormal autophagic vacuoles, all leading to subsequent cellular death [57, 58]. The ubiquitin proteasomal system is involved in substrate-specific proteolysis and allows the removal of mutant, misfolded, damaged, and accumulated proteins that are non-useful for the cells [59]. Abnormal aggregates of htt, oxidatively modified a-synuclein and hyperphosphorylated tau protein, result in ROS production by mitochondrial dysfunction in HD, PD and AD, respectively. These abnormal proteins can interact with proteins of the ubiquitin proteasomal system forming accumulations of indigestible mutant proteins, termed aggresomes. This produces an impairment of the neuronal ubiquitin proteasomal system and autophagic degradation and, ultimately, proteolytic and autophagic stress and neuronal death [58, 59].

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3 Retinal Disturbances in Patients with Huntington’s, Parkinson’s and Alzheimer’s Diseases There is a lot of evidence for visual deficiencies in PD and AD patients. These may involve difficulty in reading, driving or balance because of visuo-cognitive deficiencies caused by neurochemical and physiological alterations in the retina, lateral geniculate nucleus and visual and associative cortex [26, 60]. In relation to the involvement of visual dysfunctions in patients with HD, the results are contradictory for the time being.

3.1 Visual Disturbances and Huntington’s Disease Visual alterations associated with HD have been diagnosed by psychophysical techniques using incremental threshold measurements. These have shown disturbances in the activity of the cone-triad (cones-bipolar cells-horizontal cells) in the outer plexiform layer of retina of patients with HD [61]. However, a histological study performed by Petrasch-Parwez et al. [62], did not reveal any sign of neurodegeneration in the postmortem retina of patients with HD. These results are contradictory and more information is needed.

3.2 Visual Disturbances and Parkinson’s Disease The primary visual impairment in PD seems to be caused by a deficiency in retinal dopaminergic neurotransmission. In fact, a decrease in dopamine and homovanillic acid levels has been observed in the postmortem retinas of patients with PD [63]. The retinal dopamine content is similar in patients treated with l-3, 4-dihydroxyphenylalanine (l-dopa) before death and in untreated controls [64]. This depletion of dopamine levels was related to a decrease in tyrosine hydroxylase immunoreactivity in dopaminergic amacrine cells. Also changes such as swollen neurons and distorted morphological deterioration of the peri-foveal dopaminergic plexus have been observed [65]. However, Lewy bodies formation (inclusions containing a-synuclein) has not been investigated in the human retina. Other morphological changes include a significant reduction of retinal nerve fiber layer thickness in the inferotemporal circumpapillary region [66] and in the macula in PD patients [67, 68]. Electrophysiological changes that are associated with visual impairments in PD have been recorded. The b-wave amplitude of the scotopic electroretinogram (ERG) in PD patients was significantly more reduced than in controls [69–75]. Also, the latency of the b-wave (ON bipolar cells current) was increased in patients with PD [60, 76, 77]. Modulation of electrical synapses between horizontal

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cells is mediated by dopamine in the mammalian retina and leads a decrease in cell-to-cell coupling among horizontal cells, which has an indirect effect upon receptive field of bipolar cells ([78, 166, 167]). Thus, deficiencies in dopamine could lead to altered modulation of receptive field properties of bipolar cells due to a defective coupling among horizontal cells in the retinas of patients with PD. This abnormal functioning of the outer retina reflects changes recorded in the ERG. Pattern electroretinogram (PERG) alterations have been found in the early stages of PD. The amplitude of PERG is reduced particularly at medium spatial frequency stimuli [75, 79, 80] while patients treated with l-dopa exhibit a normal tuning of PERG shape [81]. This suggests that the receptive field properties of ganglion cells can be disrupted by dopamine deficiency that would produce an imbalance of dopaminergic, GABAergic, glycinergic neurotransmission mediated by amacrine and interplexiform cells that are implicated in modulation of these receptive fields. Several studies performed using visual-evoked potentials (VEP) have revealed an increase in latency [75, 78, 79, 82, 83] due to retinal dopaminergic alteration and the thinning retinal nerve fiber layer and, probably, reduction of dopaminergic activity in the lateral geniculate nucleus and visual cortex. In addition, these changes in VEP are coincident with a decrease in metabolism in the visual cortex of non-demented PD patients [84–86]; thus, deficits of nutrients and oxygen can also contribute to delay in VEP. Many studies have revealed CS deficits [87–92] and impaired color discrimination in PD [80, 92]. Patients with PD exhibited a loss in CS that was more evident around a temporal frequency of 8 Hz and more marked at 4.8 cycles per degree, the normal peak of the spatial CS curve in control subjects [87]. A deficiency in dopamine could be implicated in CS disturbances through altered modulation of receptive field properties of ganglion and amacrine cells in the peripheral and foveal retina. This could explain some of the CS loss in PD, but this loss is also related to the orientation of the stimulus, mainly with the middle spatial frequencies [93]. Therefore, cortical components could be involved in the abnormal CS in PD. Patients with PD exhibit impairments in three pathways implicated in color vision; in fact, psychophysical tests have revealed that the deficiencies in color vision are more marked along the protan/deutan axis (red–green) than the tritan axis (blue–yellow) [92]. However, recordings in chromatic PERGs show significantly longer latencies and more reduced amplitudes for blue–yellow stimuli than red–green stimuli [80]. These results are contradictory but this may be because of significant important methodological differences between the two works that probably depend on the stimulus configuration [80, 92]. Deficits in CS and color vision, and electrophysiological changes in PD cannot be understood completely in terms of abnormal lateral coupling between horizontal and amacrine cells and disturbances in receptive field properties of bipolar and ganglion cells due to an anomalous modulation owing to dopamine deficiency. This reflects only an alteration in the tuning of peripheral and foveal vision. Visual changes in PD are not entirely dependant on abnormal retinal transmission and processing of visual information, but higher visual areas and those that control

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ocular movements are also affected in this disease. Studies using electroencephalography and electrooculography have shown that the g-wave of the electroencephalogram and saccadic movements are desynchronized in non-demented PD patients [86, 94]. These are why Bodis-Wollner proposed that g-wave desynchronization in the visual cortex and saccades are associated with basal ganglia and frontal cortex dysfunctions in PD [86]. Therefore, neuronal degeneration of basal ganglia may contribute to the dysregulation of ocular movements that would affect attention deficits in PD.

3.3 Visual Disturbances and Alzheimer’s Disease Visuo-spatial cognition associated with memory and visuo-spatial tasks dissociated from memory are compromised in AD patients because of the preferential degeneration in the dorsal visual pathway (see Sect.  1.2), although the primary visual cortex of these patients is also damaged [60, 95]. Moreover, a visual variant of AD has been described that is characterized by Ab (amyloid b peptides) plaques, neurofibrillary tangles and brain atrophy, predominantly in cortical occipitoparietal areas [96, 97]. Unlike typical AD, patients with this variant of AD suffer visual disturbances before mental decline [97]. There is evidence that pathological changes in the retina and optic nerve can contribute significantly to visual deficiencies of typical AD, although pathogenesis in the visual system may be distinct and less severe than that observed in some brain regions. Morphometric analysis and histological studies have revealed that the thicknesses of ganglion cell layer and retinal nerve fiber layer are reduced in post mortem retina of patients with AD, and this being correlated with a loss of ganglion cells and their axons [98, 99]. The loss of ganglion cells is more prominent in temporal region of central retina, particularly in fovea [98], than in peripheral retina [99]. Furthermore, ganglion cells’ dysfunction and reduced retinal nerve fiber layer thickness [100–102] are consistent with abnormalities in the PERG, which has a longer latency and reduced amplitude [100, 103–105]. Tsai et al. [105] carried out a photographic analysis to study disturbances in the optic nerve head of AD patients and they showed an increase of cup-to-disc ratio and cup volume and a thinning of the disc rim, probably as a result of retinal nerve fiber layer abnormalities. In addition, the astrocyte:neuron ratio in ganglion cell layer of patients was increased twofold with respect to controls, coinciding with a significantly increased of expression of glial fibrillary acidic protein in astrocytes and, possibly, Müller cells [99]. Thus, reactive gliosis (proliferation and molecular changes in astrocytes) is produced in response to loss of ganglion cells. Cerebral amyloid angiopathy occurs in AD and is characterized by Ab deposition in the walls of cerebral arterioles and arteries leading to a decrease in blood flow [106, 107] possibly due to a constrictor effect of Ab on cerebral arteries [108]. Also, disturbances of retinal hemodynamic parameters have been related to AD. Paquet et  al. [102] detected that the retinal venous blood column diameter and

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blood flow rate in AD are lower in patients than in controls. Given that Ab plaques have not been identified in retinal vessels, abnormal retinal circulation in AD maybe caused by cerebral blood flow disturbances and disruptions of the optic nerve head, where the retinal artery and vein enter and leave the retina, and lead to deficiencies in retinal nutrient and oxygen supply. 3.3.1 Role of Melatonin in the Retina of Patients with Alzheimer’s Disease Melatonin is a neurohormone secreted by the pineal gland, intraocular epithelium, retina, and other tissues. Its main functions include regulation of endogenous circadian rhythms [109]. Patients with AD suffer frequent behavioral alterations related to circadian rhythms, such as daytime agitation, nighttime restlessness and nocturnal insomnia [110–112], and these may contribute to cognitive deterioration. A decrease in melatonin level in serum [111, 113] and cerebrospinal fluid [112] is correlated with Ab plaques and neurofibrillary tangles in the pineal gland and dysfunction of the noradrenergic innervation in this gland [112]. Additionally, Sevaskan et al. [114] have found that expression of melatonin receptors (MT1 and MT2) is diminished in the retina of AD patients. Therefore, it is probable that visual information transmission to the lateral geniculate nucleus and primary visual cortex, and their feedback to the pineal gland are damaged in AD. This can be due to the role of melatonin in the modulation of retinal dopaminergic function and regulation of the retinal dark adaptation processes mediated by dopamine [168]. It is possible that the deficiencies in color vision detected in patients with AD [115] are produced by dysfunctions of melatoninergic actions in the retina that affect the dopaminergic system implicated in color processing. Melatonin is an efficient antioxidant due to activate antioxidant enzymes like glutathione peroxidase, glutathione reductase and superoxide dismutase [116–119] and it could be used as antioxidant therapy for AD [48]. The reduction of melatonin receptors would cause a loss of protection from oxidative damage in the retina of AD patients, thus therapeutic application of melatonin probably would not have the expected effect in retina. 3.3.2 Retinal Pathologies and Alzheimer’s Disease To date, pathologic brain hallmarks (Ab plaques and neurofibrillary tangles) of AD have been observed in subcortical visual centers, the lateral geniculate nucleus and superior colliculus, [120] and primary and associative visual cortex [121] but not in the retina of these patients. However, Ab deposits have been found in drusen (lipoproteinaceous deposits located between retinal pigmented epithelium and Brunch’s membrane) of age-related macular degeneration [122, 123], and it is suggested that their accumulation may promote degeneration of photoreceptor and retinal pigmented epithelium cells in this disease.

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Loss of ganglion cells and damage to the optic nerve head are typical changes in AD that occur in glaucoma, a major cause of blindness in the world. An increased incidence of primary angle-open glaucoma has been demonstrated in patients with AD [124]. In addition, Nelson et al. [125] reported two cases of secondary glaucoma with amyloidosis characterized by perivascular Ab deposits around epiescleral veins and amyloid particles in the aqueous humor and on the lens surface. Studies using rat-induced glaucoma models and DBA/2J mice with glaucoma have shown Ab deposits in ganglion cells and the optic nerve head [126, 127]. Based on these findings, it is hypothesized that glaucoma may be an ocular AD that appears in the early stages of AD [128, 130].

4 Retinal Impairments in Animal Models of Huntington’s, Parkinson’s and Alzheimer’s Diseases Human visual deficiencies related to HD, PD and AD are poorly understood and how retinal dysfunctions contribute to non-visual symptoms of sufferers is not clear. Thus, transgenic models and toxin-based animal models of HD, PD and AD are used to investigate the involvement of neurodegenerative mechanisms in visual pathogenesis of these neurodegenerative disorders.

4.1 Retinal Alterations in Animal Models with Huntington’s Disease Animal models of HD have been generated to understand the neuropathological, biochemical and molecular changes of this illness. The R6 line of mouse is the model that is the most frequently used to study HD, specifically R6/1 and R6/2 mice present 116 and 144 CAG repeats in their transgenes, respectively, encoding for mutant polyglutamine expansion in the N-terminal region (Table 4). The retinae of R6/1 and R6/2 mice have an altered morphology level with an irregular thickness and the appearance of waves disrupting the outer nuclear layer (see Fig. 3). Some nuclei of the photoreceptors become atopic, positioning themselves in the layer of the photoreceptor segments and outer plexiform layer, and the thickness of the inner and outer segments is decreased [129]. Huntingtin also accumulates, forming inclusion bodies in the nuclear layers of the retina (Fig. 3). In addition, some of the aggregates of the ganglion cell layer contain chaperones (Hdj-1 and Hdj-2 and Hsp70) and the transcriptional co-activator CREB binding protein [129]. These morphological alterations are correlated with disturbances in the ERG performed on the R6/1 mice under scotopic conditions (response to photopic stimulus under adaptation to darkness conditions) in which the amplitude of the a and b waves is reduced [129]. These results indicate a dysfunction of the rods and of the

Table 4  Transgenic mice for HD with retinal disturbances Name Transgene Promoter Pathologic hallmarks in brain R6/1

First 90 amino acids of exon 1 of human htt

Human htt

htt inclusions Atrophy of striatum and cortex 1 months old onset

R6/2

Human First 90 amino htt acids of exon 1 of human htt

Atrophy of hipoccampus 2.5 months old onset

Retinal impairments ↓ differentiation and phototransduction genes expression Morphological changes in outer layers ↓ thickness of photoreceptor segments Disturbances of ERG (a and b-wave) in photopic and scotopic conditions ↓ thickness of photoreceptor segments

ERG electroretinogram, htt huntingtin

Fig.  3  Hypothesis of mutant huntingtin (muhtt) effects in retina of R6/1 mice model. RESTNRSF complex would not interact with muhtt and it would increase the number of translocated complexes in the nucleus. This would lead to a defective polymerase II RNA binding to promoter region and consequently the down regulation of genes (Stat3, Crx and Nrl) implicated in rod and cone differentiation. Also, it would diminish expression of the main phototransduction genes: rhodopsin (Rho), transducin alpha subunit (Gnat) and arrestin (Arr). Moreover, muhtt aggregates may damage the ubiquitin proteasomal system and subsequently cause autophagic stress [59]. Therefore, mitochondrial dysfunctions resulting from muhtt interaction with complex II of mitochondrial electron chain [52] would lead to inhibition of oxidative phosphorylation (OXPHOS) and then ATP decrease, ROS generation and imbalance of intracytoplasmatic Ca2+ levels. These pathologic mechanisms and altered differentiation of photoreceptors would have a synergistic effect at the same time as R6/1 mouse is aging. The photomicrograph shows the molecular and morphological changes in retina of a 9-month-old R6/1 mouse. The outer nuclear layer (blue) adopts an irregular shape associated with disorganization and shrinkage of the outer and inner segments. The muhtt forms intranuclear inclusions in the nuclear layer (red) and glial fibrillary acidic protein expression (green) increases in astrocytes (arrow) and inner prolongation of Müller cells (arrow head). GCL ganglion cell layer, INL inner nuclear layer, ONL outer nuclear layer

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rod bipolar cells exits. As occurs with the electrophysiological responses under photopic conditions, no response is detected in the ERG [129], which indicates that the synaptic connections established between the cones and the cone bipolar cells are seriously damaged. Therefore, these mice are practically blind. The phenotype of the R6/1 and R6/2 mice may be due to the dysregulation of the expression of many genes. Specifically, a decrease in the expression of genes that encode for transcription factors implicated in the differentiation of the photoreceptors has been shown [131]. Also, a decreased expression has been detected in genes that encode for proteins involved in the phototransduction process of rods and cones in the R6/2 mice [131, 132]. These results suggest that the dysregulation of the transcription factors implicated in genetic differentiation program of photoreceptors is damaged, which would determine the appearance of defective cones and rods in the outer nuclear layer, causing impairment in the rest of the retinal layers. In addition, a downregulation of genes encoding neurotransmitters, receptors and other proteins implicated in the neuronal signalling has been detected in the striate neurons of the R6/2 mice [133]. Although the mechanism has not been identified, it is believed that it is related to the dysregulation of the neuron-restrictive silencer factor (NRSF), a transcriptional factor that controls genes regulating neuronal differentiation in the later stages of the development. In fact, it has been proposed that NRSF protein may bind repressor-element-1 transcription factor (REST), forming the REST–NRSF complex in the cytoplasm of normal mice [134]. Then this complex would interact with htt, thus reducing its availability to bind neuron-restrictive silencer elements (NRSE) sites (Fig.  3) located in the promoters of those genes related to differentiation processes. The interaction of the REST–NRSF complex and NRSE brings about the recruitment of activators and binding to promoters of genes involved in differentiation and transcription enhancement [134]. However, it seems that mutant htt cannot interact with the REST–NRSF complex, so intranuclear levels of this complex would be increased, leading to a transcriptional downregulation of regulated genes by REST–NRSF and the neuronal precursors not differentiated correctly (Fig. 3) [133]. The Müller cells and the various neuronal types of the retina are generated from neuroepithelial multi-precursors following a temporal and spatial pattern very well preserved in the retina of all vertebrates. Ganglion and horizontal cells, cones and some types of amacrine cells are the first differentiated cells, and rods, bipolar and Müller cells finish their differentiation at the late stages of development or early in postnatal stages [135, 136]. Differentiation of photoreceptors is controlled by transcriptional factors such as Stat3, implicated in retinal progenitor differentiation, and Crx and Nrl, controlling retinal progenitor proliferation, differentiation and maintenance of cones and rods. These transcriptional factors act to regulate the expression of proteins implicated in differentiation and phototransduction process, such as rhodopsin, transducin alpha subunit or arrestin [131, 132]. Mutant htt may lead to transcriptional dysregulation of genes related with differentiation of photoreceptors, expression of phototransduction-specific proteins, regulation of distal growth of their neurites and establishment of contact with horizontal cells. All of this will contribute to an abnormal synaptogenesis, mainly in retinal outer layers. Indeed, these changes would determine the appearance of the morphological

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alterations in outer nuclear layers and dysfunction of photoreceptors. Thus, the organization of the plexiform layers and several systems of neurotransmitters, mainly glutamate, would also be altered in the R6/2 and R6/1 mice, although for the time being it has not been studied. Based on this, it is very probable that the ERG changes may be due to morphological and biochemical disturbances in outer segments. In fact, the translation of mRNAs coding for rhodopsin, transducin alpha subunit or arrestin, and other proteins of the outer segments membrane is altered in both models [131, 132]. In other words, photoreceptors would not follow the normal differentiation pattern nor synaptogenesis process, and finally, visual information that is transmitted to higher visual centers would deteriorate progressively. The expression of htt is ubiquitous in all cells of the organism and, in some way its function is related to regulation of transcription, vesicular and axonal transport and endocytosic processes. However, it has been found that the expression of some genes is altered in the striated muscle of R6/2 mice [133] and overexpression of annexin A1, TAF7, P2Y receptor and ROCK1 mRNAs [137] has been observed in blood cells of patients with HD. These facts invite the consideration of HD as a systemic disease.

4.2 Retinal Alterations in Animal Models with Parkinson’s Disease Mouse models based on the mutants of affected genes in PD, such as a-synuclein, parkin, phosphatase-tensin homologue induced putative kinase 1 (PINK1), DJ1 and leucin rich repeat kinase 2, or the PINK1 and DJ1 knockout mice, do not show the typical degeneration of PD [138–140], nor have they been used to investigate retinal deficiencies. However, retinal disturbances associated with PD are studied in toxin-based models (see Table 5). These toxins are potent inhibitors of complex I of the mitochondrial electron transport chain and may enhance production of ROS (Fig. 4), leading to the death of dopaminergic neurons [37, 141, 142]. However, the neuropathological abnormalities are different in each model [138, 140]. 4.2.1 6-Hydroxydopamine Model 6-hydroxydopamine (6-OHDA) is an analogue of dopamine and norepinephrine with a high affinity for the plasma membrane transporter of these catecholamines (Fig. 4). It is administered locally because it does not cross either the blood–brainbarrier or the blood–retinal-barrier. Intraocular injection of 6-OHDA in monkeys leads to the destruction of dopaminergic neurons and a reduction in dopamine and homovanillic acid levels, although it does not produce extra-nigral pathology or Lewy bodies [71, 143, 144]. Furthermore, reduced amplitude of PERG and CS profiles similar to patients with PD have been observed in monkeys treated with this toxin, and these symptoms improve after l-dopa administration [71, 144].

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Table 5  Toxins-induced models of PD and retinal disturbances Mode Pathologic hallmarks in Name Animal administration brain 6-OHDA Monkey Intraocular Not detected

Retinal impairments ↓ DA and HVA ↓ amplitude of PERG ↓ CS ↓ amplitude and delay of MPTP Monkey Intraperitoneal Loss dopaminergic latency of ERG and neurons in substancia VEP nigra and LB ↓ DA and TH intracytoplasmatic ↑ lipofucsine in ACs Deficiencies of GABAergic synapsis Mouse Intraocular Not detected ↓ DA and TH ↑ lipofucsine in ACs Disturbances of Mouse Intraperitoneal Loss dopaminergic mitochondrial neurons in substancia Pyknotic nuclei nigra and LB Reactive gliosis intracytoplasmatic Changes of members of Bcl-2 family Intraperitoneal Degeneration in mid-brain ↓ ACs Rotenone Rat ↓ thickness of GCL and Mouse Intravitreal Not detected RNFL ↑ oxidative damage in GCs ACs amacrine cells, CS contrast sensitivity, DA dopamine, ERG electroretinogram, GCs ganglion cell layer, GCL ganglion cell layer; HVA homovanillic acid, LB Lewy bodies, PERG pattern electroretinogram, RNFL retinal nerve fiber layer, TH tyrosine hydroxylase, VEP visual evoked potentials

6-OHDA may cause the degeneration of dopaminergic amacrine cells due to a process of auto-oxidation and enzymatic degradation by monoamino oxidase A and the subsequent generation of ROS and quinones that also contribute to potentiate the cascade of pathologic mechanisms (Fig. 4). 4.2.2 1-Methyl-4-phenyl-1, 2, 3, 6-Tetrahydropyridine Model PD is a progressive neurodegenerative disease, whereas systemic MPTP injections induce neuropathological changes in the substancia nigra and parkinsonian motor symptoms in a few days after toxin administration [140, 145]. A variety of animal species (non-human primates, rodents, cats and pigs) have been used to generate MPTP models of PD, but there are clear differences among mice strains and interspecific differences of MPTP potency [37, 140, 145]. MPP + (a toxic metabolite of MPTP) leads to inhibition of complex I of electron transport chain causing decreased ATP content and increased ROS generation (Fig. 4). MPP + content in the retina is lower than in the striatum [146] so this toxin does not induce death of dopaminergic amacrine cells but produces degeneration of

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Fig.  4  Hypothetical pathological mechanisms of 6-OHDA, MPTP and rotenone in retinal neurons of toxins-based models of PD. 6-OHDA enters inside of amacrine cells through dopamine transporter (DAT) and it is metabolized enzymatically by monoamino oxidase A (MAO-A) and auto-oxidized leading to generation of quinones and ROS [141, 142]. Quinones react with proteins, for instance, a-synuclein (SNCA), and they are accumulated in aggregate forms. ROS cause upregulation of expression of genes implicated in inflammatory processes and cell death, such as pro-apoptotic molecules (Bax and Bak), which lead to releasing of cytochrome c (Cyt c) from mitochondria to cytoplasm and activation of caspases pathway. 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) is metabolized to 1-methyl-4-phenylpyridinium (MPP+) by the enzyme monoamine oxidase B (MAO-B) of astrocytes and, probably, Müller cells. MPP+ and rotenone inhibit the complex I of mitochondrial electron chain [37, 141, 142] and oxidative phosphorylation (OXPHOS) resulting in decreasing of ATP and collapse of mitochondrial membrane potential (MMP). These can open permeability transition pores (PTP) that promote elevation of cytoplasmatic Ca2+ and release of cytochrome c towards cytoplasm and consequent apoptotic cell death. Also, the mitochondrial ROS can damage mitochondrial and nuclear DNA and lipids, sugars and proteins of cellular membranes. BRB blood-retinal-barrier, COX cyclooxigenase, iNOS inducible nitric oxide synthase, nNOS neuronal nitric oxide synthase

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dopaminergic neurons in the substancia nigra. This is probably due to differences in metabolism of MPTP, vulnerability or resistance of retinal and midbrain dopaminergic neurons to toxic effects of MPP+ [37, 146]. Decreased levels of tyrosine hydroxylase and dopamine in amacrine cells have been documented in the monkey retina treated with systemic injection of MPTP [147, 148]. Immunohistochemistry studies performed by Cuenca et al. [148] have revealed that type AII amacrine cells are mainly damaged by MPTP, and that gap junction-type electrical synapses among type AII amacrine cells and chemical synapses between these neurons and rod bipolar cells have highly deteriorated. In addition, they also observed a less intense staining in glycinergic and GABAergic amacrine cells in this model. Therefore, they have suggested that the scotopic visual pathway was seriously damaged and other pathways could disturb horizontal processing of retinal information [148]. Electrophysiological studies in monkeys treated systematically with MPTP have shown decreased amplitude and increased latency in ERG and VEP [144, 147, 149], similar to the described results in parkinsonian patients (see Sect. 3.2). These changes could be temporarily reversed by the administration of l-dopa [147]. Histopathological changes such as decrease of immunoreactivity dopamine and tyrosine hydroxylase and increased lipofuscin in dopaminergic amacrine cells were found in the retinas of mice treated with MPTP intraperitoneal injection [150, 151] and MPP+ intraocular injection [152]. Lipofuscins are intracellular autofluorescent non-degradable polymeric substances, and their formation is associated with normal aging processes. Given that lipofuscin accumulates in lysosomes causing a reduction in their degradative capacity and lysosomal fusion with autophagic structures [153], we think that lipofuscin-loaded lysosomes may lead to a defective autophagosomal clearance and, subsequently, degeneration of amacrine cells. In addition, changes typical of neurodegenerative disorders, such as disturbances of mitochondria in some retinal layers, pyknotic nuclei in the inner nuclear layer, upregulation of genes encoding glial fibrillary acidic protein and glutamine synthetase in Müller cells and astrocytes together with changes in expression of pro-apoptotic and antiapoptotic members of the Bcl-2 family in retinal glia or neurons and capillary damaged have also been documented in mice treated with MPTP [154, 155]. 4.2.3 Rotenone Model Retinal disturbances associated with PD have been studied using rodent models in which neurons are damaged by the toxic effect of rotenone that inhibits complex I of electron transport chain (Fig. 4). Rats treated with intraperitoneal injections of rotenone showed a decrease of 28% in the density of retinal dopaminergic amacrine cells, the number of other retinal cell populations being unchanged [156]. These results show that dopaminergic amacrine cells are more sensitive to rotenone than other retinal neurons, just as in the substancia nigra. On the other hand, mice treated with intravitreal injections of rotenone [157, 158] exhibit a pattern of retinal neurodegeneration different from that caused by systemic administration of this toxin. The neuropathological changes were observed

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in ganglion cell layer and retinal nerve fiber layer where the thickness of both layers was reduced by 21 and 89%, respectively [157, 158]. Cells of the ganglion cell layer show a higher degree of oxidative stress compared with other retinal layers [158]. In addition, studies in vitro have revealed toxic effects of rotenone affecting ganglion cells that show enhanced oxidative stress, excitotoxicity by glutamate and cell death [159, 160]. Deficiencies of the retinal dopaminergic system associated with PD have not been documented in this model. We should bear in mind that when intravitreal injection of rotenone is carried out, this substance reaches the inner retinal layer first, although it is not known if the effects of rotenone are dosedependent or if period of treatment is enough to diffuse into amacrine and bipolar cells or photoreceptors and damage these cells. Rotenone enters the neuronal cytoplasmatic space independently of transport mechanisms and it exerts its toxic effects on all types of neurons (Fig. 4), although it seems that dopaminergic neurons are more vulnerable than GABAergic or glutamatergic neurons [51, 158, 160, 161]. Therefore, this toxin is not only used to generate PD in animal models [37, 140, 161], but it is also used as a mitochondrial poison in cell cultures and living animals to study impairment oxidative phosphorylation causing of oxidative stress and cellular death. Several models of diseases such as progressive supranuclear palsy, ataxia, retinitis pigmentosa and Leber’s hereditary optic uses this compound to mimic the real pathologies [51, 158, 160]. Therefore, the question that arises is to know whether rotenone treatment is a good retinal model of PD since this toxin is not selective for dopaminergic amacrine cells.

4.3 Retinal Alterations in Animal Models with Alzheimer’s Disease Transgenic mice expressing mutant proteins associated with AD exhibit Alzheimer-like pathology and have been used to study retinal disturbances associated with this disease (see Table 6). Studies using heterozygous transgenic mice expressing human amyloid precursor protein with the double Swedish mutation (APPswe) have reported expression of APP in ganglion cell layer and inner nuclear layer at 14 months of age and Ab deposits around vessels of ganglion cell layer and corneal and lens epithelia. However, Ab levels are lower in the retina than the brain [162] and ganglion cell death has not been investigated in these transgenic mice. Based on possible disturbances of retinal blood flow of humans with AD, it is thought that transgenic APPswe mice have abnormal circulation limiting the supply of oxygen and nutrients to the retinal inner layer. The presence of APP and Ab deposits in APPswe mice have not been identified in human retina, and this may correspond to a specific difference as well as dissimilarities of rate progression of AD. On the other hand, bi-transgenic mice overexpressing APPswe and mutant presenilin 1 (PS1) develop AD, but it is more accelerated than in the transgenic APPswe mice because mutant PS1 causes an acceleration of amyloid deposition [163].

APPswePS1 DeltaE9

Tg(APPswe,PSEN1dE9)

Hamster PrP, PDGF

Mouse PrP

Ab plaques in around vessels, corneal and lens epithelia

Ab plaques Hyperphosphorylated tau Memory deficits 9–11 months old onset Ab plaques Hyperphosphorylatde tau Memory deficits 6–7 months old onset

APP and Ab in plaques IPL, GCL, RNFL Reactive gliosis Activated microglia ↓ amplitude of scotopic ERG APP and Ab in plaques IPL, GCL, RNFLActivated Apoptosis of GCs

Retinal impairments

Pathologic hallmarks in brain

Ab plaques Hyperphosphorylated tau Memory deficits 6 months old onset Ab amyloid b peptides, APP amyloid precursor protein, APPswe amyloid precursor protein with the double Swedish mutation, ERG electroretinogram, GCs ganglion cells, GCL ganglion cells layer, IPL inner plexiform layer, PDGF platelet derived growth factor, PrP prion protein promoter, RNFL retinal nerve fiber layer

APPswePS1 DeltaE9

Tg(APPswe,PSEN1dE9)

Table 6  Transgenic mice for AD with retinal disturbances Name Mutant gene Promoter Tg(APPswe) Hamster PrP APPswe

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Expression of APP and Ab deposits have been observed in the inner layer of bi-transgenic mice retina, although it has been detected at different ages, at 9 months [162] or between 12 and 16 months [164], possibly due to methodological differences. Reactive gliosis and activation of microglia have shown in inner plexiform layer and ganglion cell layer of these APPswe/mutant PS1 mice [164, 165] and matching with Ab plaques located close to strata of cholinergic amacrine cells [164] retinal nerve fiber layer and retinal inner vessels [165]. Furthermore, scotopic ERG recordings at low-light intensity have revealed a significant reduced amplitude of both a and b-waves in 12–16-month-olds in APPswe/mutant PS1mice when compared with controls [164]. These electrophysiological changes reflect a defective phototransduction (corresponding to a-wave) and synaptic transmission between rods and bipolar cells (b-wave). Alterations of the inner layer may indirectly affect components of the ERG and optic nerve response; in fact, ganglion cells of bi-transgenic mice die by apoptosis [165], coinciding with altered PERG. Morphological and biochemical changes in the retinal outer layer have not been documented in APPswe/mutant PS1 mice but they should be investigated in the future. Ning et  al. [165] have compared two strains of APPswe/mutant PS1 mice that differ in prion promoter of transgenes. One strain bears a hamster prion promoter and a platelet derived growth factor promoter, and the other strain, a mouse prion promoter for both transgenes. They observed similar results in the expression of APP, Ab deposits, and detected ganglion cell apoptosis and activated microglia to both strains. These pathological changes were more severe at the same time as the mouse was aging. However, they found different levels of intensity and distribution of protein between both strains, which may be promoter-specific [165].

5 Conclusion Patients with HD, PD or AD suffer visual dysfunctions that may be less relevant than other symptoms such as mental decline and motor disorders from the therapeutic and clinical point of view. However, the retina is a part of the brain that it is easy accessible and there are not invasive techniques that provide us with information about retinal changes without necessity of any type of intervention. Thus, understanding of retinal neurodegeneration may get an insight into pathologic mechanisms of these diseases. Moreover, ERG, PERG and VEP are not invasive techniques and may be useful in the assessment of therapeutic drugs for the treatment of these pathologies. Acknowledgments  We thank Dr. Charles H.V. Hoyle for reading this manuscript and providing useful criticism. This work has been supported by The Comunidad de Madrid NEUROTRANS CM S-SAL-0253-2006.

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Stress Gene Deregulation in Alzheimer Peripheral Blood Mononuclear Cells Olivier C. Maes, Howard M. Chertkow, Eugenia Wang, and Hyman M. Schipper

Abstract  Alzheimer’s disease (AD) is a primary degenerative brain disorder that also affects peripheral tissues such as peripheral blood mononuclear cells (PBMC). Several hypotheses for the pathogenesis of AD are intrinsically related to mechanisms of aging and include excitotoxicity, neuroinflammation, mitochondrial dysfunction, and enhanced oxidative stress. Delineation of biochemical alterations in AD blood components may provide insights into the pathogenesis of sporadic AD and facilitate the development of diagnostic and prognostic biomarkers. Here we describe our published experimental approaches and PBMC gene expression data in sporadic AD, with emphasis on the role of altered redox homeostasis in the development of this common affliction. Keywords  Alzheimer’s disease • Gene expression • Oxidative stress • Peripheral blood mononuclear cells

1 Alzheimer’s Disease and Peripheral Blood Mononuclear Cells Alzheimer’s disease (AD) is an age-dependent dementing disorder characterized by progressive cognitive, neuropsychological and behavioral impairments resulting from neuronal degeneration and gliosis in the basal forebrain, hippocampus and E. Wang  (*) Gheens Center on Aging, and Department of Biochemistry and Molecular Biology, School of Medicine, University of Louisville, Louisville, KY 40202, USA e-mail: [email protected] H.M. Schipper  (*) Bloomfield Centre for Research in Aging, Lady Davis Institute for Medical Research, Montréal, Québec, Canada H3T1E2 and Departments of Neurology, Neurosurgery and Medicine (Geriatrics), McGill University, Montréal, Québec, Canada H3G 1Y6 e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_11, © Springer Science+Business Media, LLC 2011

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associated cortices [1]. The familial form of AD (FAD) includes autosomal dominant mutations in amyloid precursor protein (APP) and presenilin-1 or presenilin-2 genes [2]. The sporadic form accounts for over 90% of cases and has incompletely delineated genetic and environmental risk factors [2–5]. The AD brain is characterized pathologically by the accumulation of extracellular amyloid-b (Ab; senile plaques) and intracellular inclusions of hyperphosphorylated tau (neurofibrillary tangles, NFT) within affected regions [6, 7]. Senile plaques arise mainly from the accumulation of 1-40 and 1-42 Ab peptide residues following the sequential cleavage of the APP protein by b-secretase (b-site APP cleaving enzyme 1, BACE1) and g-secretase complex (PSEN1 and PSEN2) [8]. The FAD mutations have provided valuable insights into mechanisms leading to the production of Ab. The extent to which Ab is directly neurotoxic or interacts with concomitant inflammatory and vascular changes in sporadic AD remains uncertain [9]. It is also suggested that the initial formation of NFTs may be a ­prosurvival response to sequester redox active metals and to attenuate oxidative stress [10]. However, the accumulation of NFTs may eventually compromise cytoskeletal functions and promote mitochondrial damage [11]. The accruing ­cellular injury triggers a sustained neuroinflammatory response which, in turn, engenders further oxidative stress and neurodegeneration [12]. Converging lines of evidence have implicated aberrant metal deposition, ­mitochondrial insufficiency, and oxidative substrate damage as key features of AD and other aging-linked neurodegenerations [13]. Potential sources of augmented oxidative stress in AD brain are varied and include mitochondria-derived reactive oxygen species (ROS), and ROS generated from Ab, redox-active transition metals (iron, copper) and proinflammatory cytokines [13–16]. The upregulation of heme oxygenase-1 in affected astrocytes may be an important transducer of mitochondrial iron sequestration and oxidative damage in AD brain [17]. Aging-related declines in glutathione concentrations [18] and reduced glutathione S-transferase activity in AD brain [19] may also contribute to enhanced oxidative modification of proteins, lipids and nucleic acids in this condition [20, 21]. Importantly, and for reasons that remain obscure, oxidative damage also occurs in AD peripheral tissues such as lymphocyte DNA [22] and blood proteins [23, 24]. Although the mechanisms responsible for the systemic manifestations of AD remain enigmatic, considerable effort has been expended towards the development of blood-based and other peripheral biomarkers of this common illness [25, 26]. Combinations of altered plasma protein concentrations [27] and peripheral blood mononuclear cell (PBMC) gene expression patterns [28] and discriminatory spectra on near-infrared spectrometry of blood plasma [29] have recently been implicated as potential diagnostic markers of sporadic AD. Conceivably, these systemic changes may originate, in whole or in part, from aberrant central nervous system (CNS) signaling [27, 30] and thereby provide a “window” into CNS dysfunction in AD and other neurological disorders [25]. PBMC are an excellent model to study gene expression in AD patients and have provided clues into dysfunctions associated with aberrant redox homeostasis [31–33]. We recently identified an impaired genetic expression network in sporadic AD that

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β-Amyloid production

Oxidative Stress

Cellular damage

Reactive metals

Tau hyperphosporylation Mitochondrial damage

ROS

Inflammatory response Injury response Stress response

Fig. 1  Oxidative stress in Alzheimer’s disease (AD). Putative sources of reactive oxygen species (ROS) in AD brain include b-amyloid metabolism, transition metals, proinflammatory cytokines and dysfunctional mitochondria. The accruing oxidative stress may, in turn, promote cellular dysfunction, injury and inflammatory responses

may coordinate oxidative stress defense mechanisms with DNA repair responses [31]. Here, we revisited our PBMC gene expression dataset in sporadic AD [32], using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) bioinformatics resource [34]. We specifically report genes that have been annotated into Gene Ontology (GO) functional categories associated with oxidative stress response or mitochondrial functions as illustrated in Fig.  1. The results complement our previous analysis and provide specific leads into oxidative stressassociated pathomechanisms in sporadic AD.

2 Recruitment of Participants and Experimental Designs Research involving human subjects requires approval of an ethics committee from the institutions concerned and informed written consent from all participants or their primary caregivers. The study described here was approved by the Research Ethics Committee of the Jewish General Hospital (JGH, Montreal, QC). Participants and patients were recruited on a voluntary basis at the JGH-McGill University Memory Clinic, as well as through publicity in family practice clinics at the JGH

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Experimental Designs for Profiling in AD Selection criteria: 1) 2) 3) 4) 5)

Ethnicity and AD family history Age-matching Risk factor matching (education, health status, lifestyle) Gender balance or selection High-risk alleles/polymorphisms (i.e. APOE µ4, etc)

Fig.  2  Sample selection criteria for Alzheimer’s disease expression profiling. The criteria for experimental designs in AD research has been previously described [28], with permission from Oxford University Press

and community. Early sporadic AD patients and normal elderly controls (NEC) were evaluated at the clinic [1], and details of the methodology have been published [28, 32]. Whole blood was collected between 09:00 and 11:00 A.M. in order to minimize potential circadian variation and PBMC were isolated from blood within 2  h of procurement. In our experience, the yield and integrity of the total RNA extracted from the PBMC samples did not differ between diagnostic groups [28], despite theoretical concerns for disease-related oxidative damage of RNA in the AD samples. In the design of AD expression profiling studies, genetic and epigenetic risk factors as well as confounding sources of variation should be carefully considered [28]. For pilot experiments comparing NEC and AD expression, it is recommended that stringent PBMC sample selection be applied in order to control for potential sources of variation (Fig.  2). Compiled demographic information of participants, such as first-order family history of AD, age, ethnicity, education, health status, medications, nutritional status, antioxidant exposure, and alcohol and tobacco consumption, facilitate the proper selection of samples. For example, the first order of selection might involve differentiation of familial from sporadic AD cases. Next, ethnicity should be controlled for, since genetic risk factors differ among the various ethnic backgrounds. Genetic and environmental AD risk factors, including age, education, health status, gender and at-risk alleles or polymorphisms, should be carefully selected for or equally represented between diagnostic groups. Experimental designs concerned with the impact of a risk allele or mutations, such as apolipoprotein E e4 (APOEe4) or other gene polymorphisms [35], may be readily implemented using PBMC gene-expression profiling. For example, we recently delineated the impact of a functional single nucleotide polymorphism (SNP) in glutathione S-transferase M3 (GSTM3) on gene deregulation in AD [31]. The GSTM3 polymorphism was positively associated with the risk of AD among women and APOEe4-negative carriers, as well as with a hub of transcriptional networks that may coordinate antioxidant defense with DNA repair mechanisms [31]. In this study, the presence of the risk allele was balanced between the diagnostic groups. This approach can be applied to APOE [36] and other known risk alleles [35] to ascertain discrete expression networks associated with various pathogenetic pathways.

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Table 1  Demographics of the PBMC expression profiling experiment Diagnosis Gender n value Age (years) Education (years) MMSE (score/30) NEC Women 7 78.0 ± 6 10.4 ± 3 29.3 ± 1 Men 7 78.5 ± 4 14.5 ± 3 29.0 ± 2 Women 7 79.0 ± 3 10.3 ± 5 22.6 ± 2** AD Men 7 81.1 ± 4 12.0 ± 3 24.1 ± 3** Mean values compared by the Newman–Keuls multiple comparison post hoc test ** p < 0.001 versus NEC. The demographics of this experiment have been previously described [32], with permission from Elsevier

We selected NEC and sporadic AD subjects matched for gender, ethnicity, age, level of education and health status [31, 32]. Subjects with chronic metabolic and inflammatory conditions (e.g., diabetes, rheumatoid arthritis, chronic active hepatitis) as well as those with depression, head injury, morning alcohol consumption or tobacco usage, were excluded. We also selected samples procured during spring and summer months in order to minimize potential seasonal variations associated with the Canadian temperate climate. The demographics of our pilot study [32] are summarized in Table 1. Since lower educational level and old age are risk factors for sporadic AD, NEC and AD subjects were matched for both age and years of formal education. Men and women were equally represented in each diagnostic group to control for possible gender influences. Only the mean Mini-Mental State Examination (MMSE) scores, a gross measure of cognitive ability [37], differed significantly between the NEC and sporadic AD subjects, as anticipated.

3 Functional Annotations Associated with Oxidative Stress We analyzed mRNA expression in PBMC using the NIA human MGC cDNA microarray, which contains approximately 6,424 unique full-length cDNA genes [38]. Description of the methodology of profiling and analysis are found in our previous publications [28, 32], and the dataset is available at the gene expression omnibus (GEO) repository under GEO accession series GSE4226 (http://www. ncbi.nlm.nih.gov/). Over 78% of selected significant genes altered in AD were ­validated by relative quantitative real time PCR [31, 32]. In our study, we observed approximately 1,000 genes altered in sporadic Alzheimer PBMC. Of the impacted genes, relatively few were found to be upregulated in AD, whereas over 90% were downregulated [32]. The PBMC gene expression dataset was reclassified into GO categories using DAVID and selected categories specifically involved in oxidative stress mechanisms are reported in Table 2. The statistics generated by DAVID bioinformatics refer to the probability of an enriched functional group within the dataset. Among the upregulated genes, three functional groups were specifically identified as concerned with mitochondrial oxidoreductase, metal binding and response to stress (Table  2). Interestingly, genes involved in mitochondrial oxidoreductase

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(b) Downregulated genes Oxidoreduction NAD–NADP <0.001 6.51 0.001 0 SDHD, ACOX1, AKR1C2, DHCR24, ACADM, MECR, ACAD9, AKR1C3, DECR2, DPYD, IVD, TM7SF2 Reduction of oxygen 0.75 0.97 1.00 100 COQ6, HIF1AN, HMOX2, PTGS2, EGLN2, Peroxide acceptor 0.49 1.82 1.00 100 GSTK1, PTGS2, PRDX4 Mitochondrion <0.001 1.82 <0.001 0 COQ6, MRPL12, CABC1, HTRA2, SHMT1, ATP5J2, SFXN3, FDXR, ACOX1, BDH2, CKB, UQCRC1, SLC25A39, OAS1, PANK2, PRDX4, ENDOG, SDHD, MRPS6, ACOT9, BIK, TUFM, CAV2, CTSD, COX7A2L, PPP2R1A, MPST, MUT, DHX30, LETM1, IVD, RHOT2, MRPS35, NDUFS2, ATP5E, BCL2L1, NIPSNAP ATP synthesis 0.77 1.07 1.00 100 NDUFS2, COX10, NDUFS3 Mitochondrion biogenesis 0.0001 3.65 0.046 0 MRPL12, CXADR, BCL2L1, HTRA2, CDKN2A, AKT1, TIMM8A, PIM2, UCP2, COX10, SFN, MPV17, BAX, TOMM40

Table 2  Classification of (a) upregulated genes and (b) downregulated genes with oxidative stress related functions Functional group P value Fold enrichment Benjamini (a) Upregulated genes Oxidoreductase-mitochondrion 0.01 2.49 1.00 NDUFA6, PYCR2, RPL4, PECR, DHRS7, AKR1B1, L2HGDH, HSD3B7, COX7C, MOSC1 Metal binding 0.59 1.04 1.00 NLN, RPL37A, NRD1, TCF19, MOSC1, SIRT5, NUPL2, ZCCHC17, TGFB1I1, SF1, THRA, NR1I2, ZCWPW1 Response to stress 0.68 1.08 1.00 NDUFA6, CHEK1, AKR1B1, F2RL1, STK25

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Fold enrichment

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a The genes classified in each functional group by DAVID bioinformatics, are denoted by their gene symbol. The fold enrichment score is base on a modified Fisher Exact p-value for gene enrichment analysis [34]

Response to stress 0.0006 1.51 0.145 1 HSF1, SERPING1, PRKRIR, DNAJB6, PTAFR, DHCR24, RTN3, ZAK, HTRA2, APOL2, SCAP, RBX1, PTPN6, EIF2B5, SSRP1, ATOX1, ERCC3, BRSK1, EGLN2, HERPUD1, CD9, AGT, DDAH2, CX3CL1, FOS, CHAF1B, H2AFX, PARK7, FGG, ELF3, PTGS2, IGFBP4, RAD51L3, NDRG4, CSNK1E, MRPS35 Heat shock protein 0.04 2.06 0.57 49 ATOX1, DNAJB6, TBCE, PARK7, CABC1, BAG3, PFDN4, DNAJC7, CIP29, TIMM8A, DNAJC11 Stress signaling 0.23 1.47 0.98 99 ITPKB, PPP2R1A, ZAK, MAP3K6, SH2D3A, MAP3K3, SHC1, MYD88, FPR1, AGT 1.67 41 Inflammatory response 0.03 0.71 SERPING1, BLNK, PTAFR, APOL2, AKT1, NFATC3, FPR1, CFHR1, ALOX5AP, STAT3, CX3CL1, S100A8, FOS, LTB4R, MIF, CXCL10, PTGS2, ELF3, IGFBP4, MYD88, TOLLIP DNA damage response 0.01 1.70 0.65 28 RAD18, GTF2H1, LIG3, ZAK, KIF22, CDC2, CDKN2D, MCM7, RBX1, SSRP1, HTATIP, SFN, CHAF1B, H2AFX, ERCC3, PRPF19, RAD51L3, BRSK1, ATM, BAX, CSNK1E, MRPS35, ATRX Transition metal ion transport 0.06 2.47 0.85 71 LTF, ATOX1, SLC39A7, TCN1, SLC31A1, SFXN3, SLC11A2 Metal ion homeostasis 0.91 0.74 1.00 100 EDG6, THY1, ANXA7, CD52 Heme metabolism 0.17 4.00 0.96 97 COX10, HMOX2, HMBS Xenobiotic metabolism 0.36 1.50 0.97 100 GSTK1, AKR1C2, GSTA2, ADH5, GSTM3, AKR1C3

Functional group

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were highly enriched (P value 0.01). Although the two other functional groups were not significantly enriched, DAVID bioinformatics nevertheless provided an efficient means to identify genes of interest for oxidative stress-related research. Further information on the relationship between genes and their functions are shown for two upregulated groups, Oxidoreductase-mitochondrion (Fig.  3a) and Response to Stress (Fig. 3b). As expected, several genes in the group Oxidoreductasemitochondrion have oxidoreductase activity; genes associated with the group Response to Stress included CHEK1, STK25, NDUFA6, F2RL1 and AKR1B1. As mentioned above, the great majority of altered genes in Alzheimer PBMC was found to be downregulated. Among the several downregulated functional ­categories, genes annotated for injury and stress responses represented 4% of all the altered genes observed [32]. The downregulated genes enriched in oxidative ­stress-related functions by DAVID bioinformatics are reported in Table  2.

a Cytochrome c oxidase subunit VIIc (COX7C) L-2-hydroxyglutarate dehydrogenase (L2HGDH) NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 6 (NDUFA6) Neurolysin (metallopeptidase M3 family) (NLN) WAS protein family, member 1 (WASF1) ADP-ribosylation factor-like 2 binding protein (ARL2BP) MOCO sulphurase C-terminal domain containing 1 (MOSC1) Pyrroline-5-carboxylate reductase family, member 2 (PYCR2) Steroidogenic acute regulatory protein (STAR) Peroxisomal trans 2-enoyl CoA reductase (PECR) Nucleoporin-like protein 2 (NUPL2) Hydroxy-delta-5-steroid dehydrogenase (HSD3B7) Dehydrogenase/reductase (SDR family) member 7 (DHRS7) Aldo-keto reductase family 1, member B1 (AKR1B1) Heme binding protein 1 (HEBP1) Ribosomal protein L4 (RPL4) Peroxisomal biogenesis factor 5 (PEX5) transit peptide:Mitochondrion organelle inner membrane mitochondrial inner membrane transit peptide generation of precursor metabolites and energy electron transport mitochondrial membrane organelle membrane oxidoreductase oxidoreductase activity mitochondrial part mitochondrial envelope Mitochondrion envelope organelle envelope mitochondrion

Fig. 3  Association of upregulated genes in Alzheimer PBMC with shared functional annotation. Two-dimensional representations of the functional groups (a) Oxidoreductase-mitochondrion and (b) Response to stress were generated by DAVID bioinformatics. Gene names and symbols are provided to the right of the functional groups. Green areas within the enriched group denote corresponding genes having common functions

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b CHK1 checkpoint homolog (CHEK1) Serine/threonine kinase 25 (STK25) Protein tyrosine phosphatase, non-receptor type 2 (PTPN2) OTU domain, ubiquitin aldehyde binding 1 (OTUB1) Ectodermal-neural cortex (ENC1) Sirtuin 5 (SIRT5) Cystatin a (CSTA) NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 6 (NDUFA6) Nuclear receptor subfamily 1, group i, member 2 (NR1I2) CBP/p300-interacting transactivator glu/asp-rich carboxy-terminal domain 1 (CITED1) Transcription factor 19 (TCF19) SWI/SNF related matrix associated actin dependent regulator of chromatin d3 (SMARCD3) Transforming growth factor beta 1 induced transcript 1 (TGFB1I1) Ribosomal protein l6 (RPL6) Splicing factor 1 (SF1) Activating transcription factor 3 (ATF3) Cleavage stimulation factor, 3' pre-rna, subunit 1 (CSTF1) Thyroid hormone receptor, alpha (THRA) Coagulation factor ii (thrombin) receptor-like 1 (F2RL1) Aldo-keto reductase family 1, member b1 (AKR1B1) Myelin protein zero (MPZ) phosphorylation response to stress phosphate metabolic process phosphorus metabolic process response to stimulus biopolymer modification protein modification process post-translational protein modification biopolymer metabolic process

Fig. 3  (continued)

The major functional groups having the greatest number of downregulated genes enriched were mitochondrial (p < 0.001) and response to stress (p < 0.0006). Equally significant, several downregulated genes were annotated with functions of ­oxidoreduction of nicotinamide adenine dinucleotide (NAD)/phosphate (NADP) (p < 0.001), mitochondrion biogenesis (p < 0.0001), DNA damage response (p < 0.01), inflammatory response (p < 0.03) and heat shock proteins (p < 0.04). Also noteworthy were the downregulated functional groups involved in stress signaling, metal homeostasis and transport, and heme and xenobiotic metabolism.

4 Working Model for Alzheimer’s Disease Oxidative stress has been heavily implicated in normal brain aging and in a host of associated neurodegenerative diseases. Augmented levels of oxidative stress and mitochondrial ROS generation occur early in AD-affected neural tissues [21, 39, 40]. Altered gene expression patterns in peripheral PBMC, whether or not they have intrinsic biological significance per se, may reflect various dysfunctions and

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responses within the CNS of persons with AD and thereby provide important clues concerning the etiopathogenesis of this prevalent dementing condition. Herein, we describe methodology for the use of PBMC as a “probe” of altered gene regulation in sporadic AD and the application of DAVID bioinformatics resources on an Alzheimer PBMC gene expression dataset deposited at the GEO repository [32]. By emphasizing stress genes, the current findings corroborate and extend our initial investigation on the impact of mild sporadic AD on PBMC gene expression [32]. In AD PBMC, we observed elevated mRNA levels of mitochondrial genes ­manifesting oxidoreductase activity. The latter may constitute an adaptive response to mitigate oxidative damage previously documented in AD blood constituents [22–24]. On the other hand, a number of genes subserving antioxidant protection and xenobiotic detoxification were downregulated in AD PBMC relative to NEC values, as summarized in Fig.  4. Similarly, genes implicated in the cellular heat shock response and DNA damage repair were also shown to be downregulated in these cells. Of note, and as previously highlighted [32], analogous changes in gene expression profiles have been documented in AD-affected brain [41]. Taken together, the data indicate pervasive (neural and systemic) deregulation of genes subserving key antioxidant, cytoprotective and mitochondrial functions in mild sporadic AD relative to normal aging. A model of AD pathophysiology has been conceptualized based on the PBMC gene expression signatures: In AD neural and peripheral tissues, enhanced oxidative stress stemming from genetic and environmental factors promotes oxidative modification of nucleic acids and other chemical substrates. The affected tissues undergo a cellular stress response characterized by widespread repression of the transcriptome [32], an event possibly mediated by upregulation of key microRNA transcripts [33]. Downregulation of antioxidant defense, heat shock and DNA repair genes in AD exacerbates oxidative tissue damage relative to normal aging. The muted responses to perturbed cellular redox homeostasis triggers a cascade of deteriorating cytoskeletal functions, aberrant protein and vesicular trafficking and

Aging Oxidative stress

Normal response Organismal maintenance

Alzheimer BMC Model Mitochondrial functions Stress response Oxidoreduction activity DNA damage response Heat shock response Xenobiotic metabolism

Altered response Clues to pathogenetic pathways

Fig. 4  Oxidative stress-related dysfunctions as suggested by the PBMC model of sporadic AD. See text for details

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mitochondrial insufficiency [32]. In the AD CNS, the oxidative stress, bioenergetic failure and cytoskeletal dysfunctions may promote Ab production and deposition, NFT formation, neuritic degeneration/synaptolysis and apoptosis – the neuropathological substrates of cognitive decline. Although useful as a conceptual framework and for hypothesis generation, much additional data mining and benchwork (e.g., cell-based functional assays, transgenic mice) will be required to validate this model. The main objective of this chapter was to provide justification and methodology for the use of peripheral PBMC as a probe for aberrant stress gene expression in sporadic AD (and possibly other human neurodegenerative disorders). We underscored the importance of stringent matching of patient and control samples based on salient clinical and demographic variables in order to account for risk factors and confounding sources of variation when comparing subjects of heterogeneous genetic and environmental backgrounds. Further details concerning these and other aspects of experimental design involving the use of human PBMC for genomic and proteomic investigations of neurological disorders are provided in a recent publication from our laboratories [28]. Acknowledgment  The content of this chapter is a collection of previously published material, with permission from Elsevier (Neurobiology of Aging, 28/12, Maes et al., Transcriptional profiling of Alzheimer blood mononuclear cells by microarray, pp. 1795–1809, 2007) and Oxford University Press (Journal of Gerontology, Series A, 64(6), Maes et al., Methodology for Discovery of Alzheimer’s Disease Blood-Based Biomarkers, pp. 636–645, 2009). The work was supported by an internal grant from the Sir Mortimer B. Davis-Jewish General Hospital (JGH). The authors gratefully acknowledge the contribution of the medical staff, neuropsychologists, nurses, patients, research coordinators and secretaries of the JGH-McGill Memory Clinic.

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29. Burns DH, Rosendahl S, Bandilla D, Maes OC, Chertkow HM, Schipper HM. 2009. Near-Infrared Spectroscopy of Blood Plasma for Diagnosis of Sporadic Alzheimer’s Disease. J Alzheimers Dis. 30. Gladkevich A, Kauffman HF, Korf J. 2004. Lymphocytes as a neural probe: potential for studying psychiatric disorders. Progress in Neuro-Psychopharmacology & Biological Psychiatry 28(3):559–576. 31. Maes OC, Schipper HM, Chong G, Chertkow HM, Wang E. 2008. A GSTM3 polymorphism associated with an etiopathogenetic mechanism in Alzheimer’s disease. Neurobiol Aging doi:10.1016/j.neurobiolaging.2008.03.007. 32. Maes OC, Xu S, Yu B, Chertkow HM, Wang E, Schipper HM. 2007. Transcriptional profiling of Alzheimer blood mononuclear cells by microarray. Neurobiol Aging 28(12):1795–1809. 33. Schipper HM, Maes OC, Chertkow HM, Wang E. 2007. MicroRNA expression in Alzheimer blood mononuclear cells. Gene Regulation and Systems Biology 1:263–274. 34. Huang da W, Sherman BT, Lempicki RA. 2009. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature protocols 4(1):44–57. 35. Bertram L, McQueen MB, Mullin K, Blacker D, Tanzi RE. 2007. Systematic meta-analyses of Alzheimer’s disease genetic association studies: the AlzGene database. Nat Genet 39(1):17–23. 36. Xu PT, Li YJ, Qin XJ, Scherzer CR, Xu H, Schmechel DE, Hulette CM, Ervin J, Gullans SR, Haines J, Pericak-Vance MA, Gilbert JR. 2006. Differences in apolipoprotein E3/3 and E4/4 allele-specific gene expression in hippocampus in Alzheimer’s disease. Neurobiology of disease 21(2):256–275. 37. Folstein MF, Folstein SE, McHugh PR. 1975. “Mini-mental state”. A practical method for grading the cognitive state of patients for the clinician. J Psychiatr Res 12(3):189–198. 38. Nadon NL, Mohr D, Becker KG. 2005. National Institute on Aging microarray facilityresources for gerontology research. Journals of Gerontology Series A-Biological Sciences & Medical Sciences 60(4):413–415. 39. Lovell MA, Markesbery WR. 2007a. Oxidative damage in mild cognitive impairment and early Alzheimer’s disease. J Neurosci Res 85(14):3036–3040. 40. Pratico D. 2008. Oxidative stress hypothesis in Alzheimer’s disease: a reappraisal. Trends in pharmacological sciences 29(12):609–615. 41. Blalock EM, Geddes JW, Chen KC, Porter NM, Markesbery WR, Landfield PW. 2004. Incipient Alzheimer’s disease: microarray correlation analyses reveal major transcriptional and tumor suppressor responses. Proc Natl Acad Sci U S A 101(7):2173–2178.

The Use of Gpx4 Knockout Mice and Transgenic Mice to Study the Roles of Lipid Peroxidation in Diseases and Aging Qitao Ran and Hanyu Liang

Abstract  Lipid peroxidation, a primary event of oxidative stress, is considered to be important in disease pathogenesis and aging. Glutathione peroxidase 4 (Gpx4) is an antioxidant defense enzyme that can directly reduce lipid hyproperoxides in membrane lipids. Several Gpx4 knockout mouse models and Gpx4 transgenic mouse models have been generated over the past 7 years, and data collected from those models indicate that Gpx4 plays an essential role in protecting against lipid peroxidation. Because Gpx4 knockout mice and Gpx4 transgenic mice show increased lipid peroxidation and decreased lipid peroxidation, respectively, those models were used to investigate the roles of lipid peroxidation in aging and disease. For example, lifespan studies showed that Gpx4 heterozygous knockout mice and Gpx4 transgenic mice altered lifespans, indicating that mild change in lipid peroxidation does not affect aging. However, studies using Gpx4 transgenic and knockout models showed that overexpression of Gpx4 protected against Ab toxicity and that deficiency of Gpx4 resulted in increased amyloidogenesis in brain, indicating that lipid peroxidation is important in the pathogenesis of Alzheimer’s disease. Studies using Gpx4 transgenic mice also showed that overexpression of Gpx4 reduced atherosclerosis and attenuated cardiac ischemia/reperfusion injury, suggesting that lipid peroxidation is important in cardiovascular disease. Therefore, Gpx4 knockout and transgenic mouse models are useful in ­dissecting the in vivo roles of lipid peroxidation in diseases and aging. Keywords  Aging • Glutathione peroxidase 4 • Gpx4 isoforms • Lipid peroxidation Q. Ran  (*) Department of Cellular and Structural Biology, University of Texas Health Science Centre at San Antonio, San Antonio, TX 78229, USA and Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA and South Texas Veterans Health Care System, San Antonio, TX 78229, USA e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_12, © Springer Science+Business Media, LLC 2011

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1 Introduction Reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, are constantly generated in aerobic organisms by a variety of pathways. Although ROS at physiological concentrations may be beneficial for normal cellular functions such as cell signaling, excessive amounts of ROS cause oxidative damage to various macromolecules, leading to oxidative stress. Due to the presence of allylic hydrogens, polyunsaturated fatty acids, which are found predominantly in membranes, are especially vulnerable to attack by ROS [1]. Thus, the peroxidation of membrane lipids is a major consequence of oxidative stress [2]. The peroxidation of membrane lipids is detrimental to cells, because lipid peroxidation decreases membrane fluidity, facilitates phospholipid exchange between the two monolayers, and increases the leakiness of the membrane bilayer to substances that do not normally cross membrane other than through specific channels [3]. Lipid peroxidation can also cause cross-linking and inactivation of membrane proteins [3]. In addition, lipid hydroperoxides can undergo iron and oxygen mediated chain-breaking lipid peroxidation to generate reactive aldehydes such as 4-hydroxyl nonenal (4-HNE) and malondialdehyde (MDA) [4], which can attack other cellular targets, such as ­proteins and DNA, thereby propagating the initial damage in cellular membranes to other macromolecules and to other parts of cells. Increased lipid peroxidation is associated with conditions that induce oxidative stress, such as irradiation, ischemia/reperfusion, heavy metal and pesticides exposure [5–8]. Furthermore, increased lipid peroxidation is associated with pathogenesis of diseases such as neurodegenerative diseases, cardiovascular disease, cancer [9–12] and aging [13–15]. However, despite the large body of data implicating lipid peroxidation in disease and aging, the importance of lipid peroxidation in disease pathogenesis and aging remains largely unknown.

2 Glutathione Peroxidase 4 Hydroperoxides in membrane lipids can be detoxified by antioxidant defense enzymes, thereby alleviating their damage to cells [4]. Glutathione peroxidase 4 (Gpx4) is a key enzyme in reducing hydroperoxides in membrane lipids [16, 17]. Gpx4 is a member of the selenoprotein glutathione peroxidase family, which consists of four members in mammals [18]. Among them, Gpx1 is ubiquitously distributed in the cytosol and mitochondria of all tissues [19], Gpx2 is expressed in the gastrointestinal tract, and Gpx3 is expressed in various tissues in contact with the body fluids. Gpx4, also called phospholipid hydroperoxide glutathione peroxidase (PHGPx), is present at relatively low levels in mitochondria, cytosol, and nucleus of somatic tissues, while the expression of Gpx4 is very high in testes [20, 21]. All members of the selenoprotein glutathione peroxidase family reduce hydrogen ­peroxide and alkyl hydroperoxides (e.g., cumene hydroperoxide, tert-butylhydroperoxide, and hydroperoxy fatty acids) using glutathione as reducing agent. However, Gpx4 is unique in that it also reduces hydroperoxides in complex lipids

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such as phospholipids, cholesterol and cholesterol esters [22]. And Gpx4 has the ability to reduce hydroperoxides integrated in membranes. This unique characteristic of Gpx4 is rooted in the structure of Gpx4 protein: in contrast to the other glutathione peroxidases, which are all homotetramers, Gpx4 is a monomeric enzyme with a molecular size smaller than the subunits of the other glutathione peroxidases [18]. It is believed that the small size and unique structure of Gpx4 protein allow it to react directly with lipid hydroperoxide in membranes [23]. Gpx4 efficiently reduces lipid hydroperoxides in phospholipids, cholesterol and cholesterol ester [4, 24]. However, the role of Gpx4 in reducing cardiolipin (CL) hydroperoxides received the most attention in recent years. CL is a phospholipid with unusual structure and almost exclusively localized within the mitochondrial inner membrane. CL plays a pivotal role in facilitating the activities of mitochondrial inner membrane proteins including F0F1-ATPase, ANT, cyt c, complex I, III, and IV [25]. In addition, CL exerts a major impact on apoptosis because cyt c, one of the important apoptogenic proteins in mitochondria, is tightly associated with CL [26]. Because CL is rich in polyunsaturated fatty acids (90% of fatty acid in CL is linoleic acid) [27], CL is prone to oxidation. When CL is oxidized, it results in the dissociation of cyt c from the mitochondrial inner membrane [26] and the opening of the mitochondrial PT pore, leading to the release of cyt c and other apoptogenic proteins [25]. Using the oxidative lipidomics approach, Kagan et  al. [28] identified CL oxidation as an early event in apoptosis. Gpx4 is associated with the mitochondrial inner membranes [29], and is shown to reduce CL hydroperoxides [26]. The reduction of CL hydroperoxides by Gpx4 prevents cyt c dissociation from ­mitochondrial inner membrane and its subsequent release from mitochondria [25]. Therefore, Gpx4’s ability to detoxify membrane lipid hydroperoxides in mitochondria allows it to regulate an important apoptosis pathway. Studies in cell lines and animal models indeed showed that altered levels of Gpx4 expression affect the sensitivity of apoptosis [29–32]. Gpx4 exhibits pleiotropic roles in vivo. In addition to its role in reducing lipid hydroperoxides, Gpx4 is also implicated in the regulation of activities of cyclooxygenases (COXs) and lipoxygenases (LOXs). In vitro assay and studies in cell lines showed that Gpx4 inhibited enzymatic activities of COX-1, COX-2 and LOXs [33, 34], thereby regulating eicosanoids production [35]. Also, Gpx4 appears to inhibit the activities of LOXs and COXs by reducing the peroxide tone that is necessary for activation of these enzymes [36, 37]. Recently, the role of Gpx4 in regulating activities of LOXs was reported in a mouse model of inducible Gpx4 knockout [38]. Gpx4 is a major selenoprotein in testes, and plays an essential role in the maturation and function of sperm [39, 40]. Thus, Gpx4 is also important in male fertility.

3 Isoforms of Gpx4 Three isoforms of Gpx4 proteins are generated from the Gpx4 gene in mammals: a long form (lGpx4), a short form (sGpx4), and a nuclear form (nGpx4). The N-terminal of nGpx4 protein is encoded by an alternative first exon called exon Ib,

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and nGpx4 protein is mainly expressed in sperm nuclei [41]. Because mice with targeted deletion of exon 1b of the Gpx4 gene were viable and fertile, the nGpx4 protein is dispensable for both somatic functions and fertility [41]. The lGpx4 protein has a mitochondrial targeting signal at the N-terminal [42]. The sGpx4 protein lacks the N-terminal mitochondrial targeting signal found in lGpx4 protein, and is thus believed to be the nonmitochondrial Gpx4 protein found in subcellular locations other than mitochondria, such as cytoplasm, nucleus, and microsome [42]. Recently, we generated transgenic mice using mutated GPX4 genes that encode either the sGpx4 protein or the lGpx4 protein. Using these novel models, we demonstrated that the sGpx4 protein was targeted to mitochondria and cytosol of somatic tissues, and that sGpx4 protein regulated mitochondrial functions such as apoptosis. Furthermore, the expression of sGpx4 protein rescued the lethal phenotype of Gpx4 null mutation. Thus, our results indicate that sGpx4 protein is responsible for the Gpx4’s functions in somatic tissues [43]. Our results also showed that although the Gpx4 knockout mice rescued by the sGpx4 gene, which had no lGpx4 protein, were viable, the male mice were infertile due to sperm malformation. Schneider et al. [44] generated knockout mice with targeted deletion of the N-terminal mitochondrial targeting signal found in lGpx4 protein, and reported that the knockout mice without lGpx4 were viable but the male mice exhibited severe structural abnormalities in the mid-piece of sperm and were infertile. Thus, both studies demonstrated independently the vital role of lGpx4 protein in male fertility.

4 Gpx4 Knockout Mice Two groups reported the lethal phenotype of Gpx4 knockout mice independently [45, 46]. Our group showed that the Gpx4−/− mice died at embryonic day E7.5 to E8.5, indicating that Gpx4 is an essential gene for survival of mouse [45, 46]. However, because the Gpx4−/− mice die very early in embryonic development, the cause of death was not clear from the studies using the whole-body Gpx4 knockout mice. Recently, Seiler et  al. [38] reported the generation of the first floxed Gpx4 mouse model. Using this model, they showed that null mutation of Gpx4 led to massive lipid peroxidation and cell death in mouse embryonic fibroblasts (MEFs). Furthermore, deletion of the Gpx4 gene in neurons resulted in neurodegeneration because of increased lipid peroxidation. Therefore, these data ­confirmed the essential role of Gpx4 in protection against lipid peroxidation in  vivo and demonstrated that increased lipid peroxidation is a major cause of death in Gpx4−/− mice. Compared to the lethal phenotype of the Gpx4−/− mice, the heterozygous Gpx4 knockout mice (Gpx4+/−) develop normally. Because of haploid deficiency, Gpx4+/− mice have reduced levels of Gpx4 expression in all tissues. Using MEFs derived from Gpx4+/− mice, we showed that cells deficient in Gpx4 had increased lipid ­peroxidation and had increased apoptosis after exposure to oxidizing agents [47]. We subsequently showed that Gpx4+/− mice had significantly higher levels of

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F2-isoprostanes than WT mice after exposure to an oxidizing agent (diquat), and that Gpx4+/− mice showed increased apoptosis, which appeared to be mediated by increased CL peroxidation in mitochondria [29]. We also showed that middle-aged and old Gpx4+/− mice had increased levels of lipid peroxidation than their agematched WT controls [48, 49]. Those data demonstrate that deficiency in Gpx4 results in increased lipid peroxidation. Thus, the Gpx4+/− mice are useful to investigate the in vivo role of increased lipid peroxidation.

5 Gpx4 Transgenic Mice We also generated the first transgenic mice overexpressing Gpx4, Tg(GPX4)+/0 mice, which overexpress Gpx4 by about 2- to 3-fold in all tissues [32]. When exposed to pesticide diquat, Tg(GPX4)+/0 mice had reduced levels of lipid peroxidation, and the induction of hepatotoxicity and apoptosis in those mice was dramatically reduced. In addition, cells from Tg(GPX4)+/0 mice, such as cortical neurons, were protected from oxidative stress and Ab toxicity [50], and Tg(GPX4)+/0 mice had increased mitochondrial functions compared to WT mice after oxidative stress [51]. Thus, our results indicate that overexpression of Gpx4 suppresses the induction of lipid peroxidation in vivo. As described earlier, three isoforms of Gpx4 proteins are produced from the same Gpx4 gene, with the sGpx4 protein and lGpx4 protein being the two major forms. To study the specific roles of sGpx4 protein and lGpx4 protein in vivo, we generated transgenic mice overexpressing either the sGpx4 protein or the lGpx4 protein using mutated human GPX4 genes [43]. Our results showed that transgenic mice generated with the sGPX4 gene had increased total Gpx4 protein in all tissues and were protected against diquat-induced apoptosis in liver. In ­addition, the sGPX4 gene was able to rescue the lethal phenotype of the Gpx4 null mutation. In Gpx4 null mice rescued by the sGPX4 gene, the Gpx4 protein was present in mitochondria isolated from somatic tissues, and the submitochondrial distribution pattern of the Gpx4 protein in these mice was identical to that in WT mice, indicating that the sGpx4 protein is targeted to mitochondria. In contrast, transgenic mice generated with the lGPX4 gene had increased Gpx4 protein only in the testes, and the lGPX4 gene failed to rescue the lethal phenotype of the mouse Gpx4 null mutation. Interestingly, the male Gpx4 null mice rescued by the sGPX4 gene, which have no lGpx4 protein, were infertile and exhibited sperm malformation, indicating that lGpx4 protein is important for male fertility. Thus, transgenic mice overexpressing Gpx4 isoforms played important roles in clarifying the specific roles of the Gpx4 isoforms in vivo. Transgenic mice overexpressing Gpx4 in specific tissues and cell types have also been generated. Lu et al. [52] generated transgenic mice overexpressing Gpx4 in photoreceptors. Using these mice, they showed that overexpresssion of Gpx4 protected retina against oxidative stress-induced degeneration. Dabkowski et al. [53] generated transgenic mice overexpressing Gpx4 in heart and showed that transgenic

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mice overexpressing Gpx4 in heart had significantly reduced levels of lipid peroxidation and increased mitochondrial function after undergoing ischemia/reperfusion surgery. Furthermore, transgenic mice overexpressing Gpx4 in the heart showed reduced cardiac injury, as demonstrated by parameters such as increased rates of contraction, increased developed pressures and peak-systolic pressures in those mice.

6 The Use of Gpx4 Knockout and Transgenic Mice in Studying the In Vivo Role of Lipid Peroxidation in Aging and Disease A large body of data indicates that increased lipid peroxidation is important in the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease, cancer, and cardiovascular disease as well as in aging [9–12]. Because of the essential role of Gpx4 in regulating lipid peroxidation, the Gpx4 knockout mouse models and Gpx4 transgenic mouse models are expected to be useful to illustrate the mechanistic roles of lipid peroxidation in those pathological events and to determine the ­effectiveness of interventions that reduce lipid peroxidation in prevention and therapy of diseases.

6.1 The Use of Gpx4 Knockout and Transgenic Mice to Study Lipid Peroxidation in Aging One of the most popular theories of aging is the free radical or oxidative stress theory of aging [54, 55]. Based on this theory, the steady-state accumulation of oxidative damage is an important mechanism underlying aging [56, 57]. Because polyunsaturated fatty acids in lipids are mostly prone to oxidative damage, ­membrane lipid peroxidation is believed to be especially pivotal in aging [14, 15]. The critical role of lipid peroxidation in aging is supported by studies showing increased levels of lipid peroxidation in aged animals [58, 59], as well as by studies showing that calorie restriction, a manipulation known to retard aging, attenuates the age-related increase in lipid peroxidation [58, 60]. However, whether manipulation of membrane oxidation has a direct effect on lifespan was unknown. Because Gpx4+/− mice have a 50% reduction in Gpx4 levels in all tissues due to haploid deficiency [46], to determine the effect of increased lipid peroxidation on lifespan we measured the lifespans of Gpx4+/− mice [49]. The mean, median and top 10% survival for Gpx4+/− mice were 964 days, 1,029 days and 1,126 days, respectively, whereas the mean, median and top 10% survival for WT mice were 915 days, 963 days and 1,220 days, respectively. Thus, Gpx4+/− mice did not have a shortened lifespan compared to WT mice. In fact, Gpx4+/− mice had a significantly increased median survival. The increase in the median survival of Gpx4+/− was likely a result of improved pathology, because our pathological analysis of mice in the survival

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groups revealed that Gpx4+/− mice had delayed occurrence of fatal lymphoma and reduced severity of glomerulonephritis. One explanation for the improved pathology is an increase in apoptosis that would remove damaged/abnormal cells [61]. Because Gpx4+/− mice had increased sensitivity to stress-induced apoptosis compared to WT mice, the increased apoptosis could eliminate damaged and dysfunctional cells in Gpx4+/− mice, leading to reduced pathology [49]. We had expected to see a detrimental effect of increased lipid peroxidation on aging in Gpx4+/− mice. Unexpectedly, we observed that a mild increase in lipid peroxidation was beneficial for lifespan. Clearly, Gpx4+/− mice showed no sign of accelerated aging, as would be predicated by the oxidative stress theory of aging. The lifespan data of Gpx4+/− mice were consistent with data from mouse models with deficiency in other antioxidant defense enzymes such as MnSOD [62], indicating that mild, global increase in oxidative damage does not lead to shortened lifespan and accelerated aging. Recently, we also measured the lifespans of Gpx4 transgenic mice [63]. The lifespan of the Gpx4 transgenic mice (mean, median and top 10% survival) was not different from the lifespan of WT mice, indicating that increased protection against lipid peroxidation does not directly lead to retarded aging. However, the reduction of Gpx4 in Gpx4+/− mice and the overexpression of Gpx4 in Gpx4 transgenic mice were moderate and occurred in every cell type throughout the lifespan. Because reducing mitochondrial ROS by overexpressing catalase retarded aging in mice [64], it is possible that oxidative stress in particular cell types or in particular subcellular organelles may be more important for aging, Therefore, our lifespan studies of the Gpx4+/− mice and Gpx4 transgenic mice do not necessarily rule out the effect of lipid peroxidation on aging. Future studies using tissue-specific Gpx4 knockout model and inducible Gpx4 knockout mice should provide more insight into the roles of lipid peroxidation in aging.

6.2 The Use of Gpx4 Knockout Mice and Transgenic Mice to Study Lipid Peroxidation in Alzheimer’s Disease Oxidative stress in the central nervous system predominantly manifests as lipid peroxidation because of the brain’s high metabolism and high concentration of polyunsaturated fatty acids [7, 65]. Increased lipid peroxidation has been shown in many neurodegenerative diseases such as Alzheimer’s disease (AD) [7, 66], and accumulating data suggest that increased lipid peroxidation is an early event of Alzheimer’s disease (AD). For example, Pratico et  al. [67] reported that subjects with mild cognitive impairment (MCI) had significantly elevated levels of F2-isoprostanes in their cerebrospinal fluid, plasma and urine, and increased levels of 4-hydroxynonenal (4-HNE) and acrolein were also found in brain regions of subjects with MCI and patients with early AD by Williams et al. [68] and Butterfield et al. [9]. b-amyloid (Ab) is believed to play a central role in AD pathogenesis [69]. Ab peptides have been shown to generate reactive oxygen species (ROS) such as hydrogen peroxide and hydroxyl radical [70–72],

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and Ab neurotoxicity is believed to be mediated through lipid peroxidation [7]. To determine whether reducing lipid peroxidation ameliorates Ab neurotoxicity, we compared Ab neurotoxicity in cortical neurons isolated from Gpx4 transgenic mice and WT mice [50]. Our results show that cortical neurons from Gpx4 transgenic mice had a significantly higher survival than their WT counterparts after exposure to Ab25-35 and Ab1-40 peptides. In addition, apoptosis induced by Ab in cortical neurons from Gpx4 transgenic mice was attenuated. Thus, our data indicated that lipid peroxidation is an important mechanism of Ab toxicity and that reducing lipid peroxidation could ameliorate Ab neurotoxicity. Although increased lipid peroxidation appears to be an early event of AD [9, 67, 68], whether increased lipid peroxidation could enhance Ab production and accumulation in brain (amyloidogenesis) was not clear. To investigate the effect of increased lipid peroxidation on amyloidogenesis, we studied Ab level in Gpx4+/− mice. Our results indicate that middle-aged Gpx4+/− mice had increased endogenous Ab, and that the increase in Ab in Gpx4+/− mice was directly correlated with the increased level of lipid peroxidation. Transgenic mice overexpressing amyloid precursor protein (APP) are wildly as used AD mouse models [73]. To further study the causal role of increased lipid peroxidation in amyloidogenesis, we generated APP transgenic mice with deficiency in Gpx4 by crossbreeding APP transgenic mice with Gpx4+/− mice. Our results showed that compared to APP mice that are WT for Gpx4, APP mice with deficiency in Gpx4 had increased amyloid plaque burdens and significantly elevated levels of both Ab1-40 and Ab1-42. Therefore, our results from both Gpx4+/− mice and APP transgenic mice with deficiency in Gpx4 demonstrated that increased lipid peroxidation promotes amyloidogenesis, suggesting that interventions to prevent/suppress lipid peroxidation may be effective in reducing amyloidogenesis at the early stages of AD. Our results also showed that the activity of b-secretase, one of the two enzymes responsible for Ab production, was significantly elevated by increased lipid peroxidation. Our data further indicated that the increased b-secretase activity was a result of increased BACE1 expression at protein level. Although previous studies showed that BACE1 expression was inducible by oxidative stress [74–76], using the Gpx4+/− mice in which lipid peroxidation is the primary form of oxidative stress, we showed that increased lipid peroxidation directly upregulates BACE1 expression in vivo.

6.3 The Use of Gpx4 Knockout and Transgenic Mice to Study the Role of Lipid Peroxidation in Cardiovascular Disease Cardiovascular disease (CVD) is the leading cause of death in the US and Europe. While it is a multifactorial disease, a large body of evidence indicates that lipid peroxidation plays an important role in the pathogenesis of CVD. For example, elevated levels of lipid peroxidation have been observed in a number of risk factors that promote CVD, including diabetes [77], obesity [78, 79], hypercholesterolemia

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[80], cigarette smoking [81], and decreased estrogen in postmenopausal women [82]. Atherosclerosis, a chronic inflammatory process characterized by thickening of the vessel wall, is a primary event in CVD. It is believed that the oxidative ­modification of low-density lipoprotein (LDL) or other lipoproteins plays a central role in atherosclerosis [83]. For example, oxidized lipids such as F2-IsoPs and ­oxysterols have been detected in atherosclerotic lesions [84, 85]. Because of the essential role of Gpx4 in regulating lipid peroxidation, Gpx4 transgenic mice were used to determine whether suppressing lipid peroxidation protects against atherosclerosis and CVD. In one study, Guo et al. [86] generated apoE-deficient mice with overexpression of Gpx4 by crossbreeding Gpx4 ­transgenic mice with apoE-deficient mice. They showed that apoE-deficient mice with overexpression of Gpx4 had a significantly reduced development of atherosclerosis that was correlated with a decrease in the level of F2-isoprostanes in the aorta [86]. In addition, oxidized lipid-induced adhesion of monocytes to endothelial cells as well as endothelial cell deaths was diminished in the apoE-deficient mice with overexpression of Gpx4. These results indicate that the overexpression of Gpx4 in mice suppressed atherosclerosis through inhibition of lipid peroxidation. In another study, transgenic mice overexpressing the mitochondrial form of Gpx4 were generated to determine the protective effect of Gpx4 overexpression on myocardial ischemia/referfusion (I/R) injury [53]. Heart isolated from Gpx4 transgenic mice exhibited enhanced contractile functions when compared to controls, and the mitochondria from Gpx4 transgenic mice showed reduced lipid peroxidation and increased the electron transport chain complex activities. These results indicate that the overexpression of Gpx4 critically protects the heart from I/R injury by reducing mitochondrial lipid peroxidation. Therefore, transgenic mice overexpressing Gpx4 have shown promises in illustrating the role of lipid peroxidation in CVD.

7 Conclusion Oxidative stress plays complex roles in many diseases and aging. Because of Gpx4’s essential role in detoxifying hydroperoxides in membrane lipids, mouse models with increased or reduced Gpx4 levels are valuable models to study the in vivo roles of lipid peroxidation. Studies employing the Gpx4 knockout mice and Gpx4 transgenic mice have provided us with some important insights about the roles of lipid peroxidation in aging, Alzheimer’s disease and cardiovascular ­disease. With the develop of new Gpx4 mouse models, such as the floxed Gpx4 mouse that allows deleting Gpx4 in a cell-specific fashion and/or an inducible fashion, future studies using Gpx4 knockout/transgenic mice should yield more mechanistic ­information about the importance of lipid peroxidation in diseases and aging. Acknowledgments  The studies were supported by a Merit Award (Q.R.) from the Department of Veteran Affairs.

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An Experimental Model of Myocardial and Cerebral Global Ischemia and Reperfusion Lars Wiklund and Samar Basu

Abstract  Despite many programs aimed at better immediate care of cardiac arrest victims, the subsequent mortality rate remains high, with myocardial and central nervous system injuries as the most common causes of death. Preclinical research is badly needed to produce a sound base for future clinical trials and possible improvements in clinical outcome. Our continued use of a porcine model for studies of cerebral effects of anoxia and reperfusion has shown that this model results in standardized effects, where time of cardiac arrest and reperfusion are approximately proportional to the ischemic neurological injury. Free radical damage is proved to be an important pathophysiological mechanism in the early development of this nervous injury. Hence, not unexpectedly, early experimental treatment after total ischemia during early reperfusion results in improved measures of cerebral tissue damage. Keywords  Brain • CPR • Inflammation • Ischemia–reperfusion • Isoprostanes • Neurology • Oxidative stress • Prostaglandins

1 Introduction In critical care medicine, a substantial percentage of the patients suffer from pathological conditions attributable to ischemic mechanisms as well as consequences of reperfusion injury. As many of these conditions are lethal, basic methodological research must be carried out experimentally in animals. In our case, the interest has been devoted to improvement of care for patients afflicted by a cardiac arrest. Generally, an experimental cardiac arrest is generated by electrical induction of ventricular fibrillation as this arrhythmia is a clinically common cause of circulatory arrest. This circumstance has made it necessary to use rather large animals, L. Wiklund (*) Department of Surgical Sciences/Anaesthesiology and Intensive Care Medicine, Uppsala University, 751 85 Uppsala, Sweden e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_13, © Springer Science+Business Media, LLC 2011

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such as piglets, as rodents, for example, have hearts that are too small for creating ventricular fibrillation lasting more than a few seconds. Circulatory arrest is one of the major causes of death in the Western world, and afflicts approximately 100 per 100,000 inhabitants annually. In Sweden, with a population of 9,000,0000, approximately 15,000 persons die each year due to cardiovascular disease, two-thirds of whom suffer cardiac arrest outside a hospital. About 2,000 of these out-of-hospital patients undergo cardiopulmonary resuscitation (CPR) by ambulance staff. Upon arrival at the hospital, 15–50% of these patients are still alive, but only 5% are alive 1 month after their cardiac arrest [1, 2]. Despite major efforts to improve these results, little has changed during the past 25 years. It is commonly agreed that the human brain cannot survive much more than 5 min of normothermic untreated circulatory arrest. Recent statistics from Sweden [1, 2] reveal that the median time from witnessed arrest to alarm is 3–4 min, and there is a median time of 4–10 min from arrest to ambulance arrival, and 6–10 min from alarm to defibrillation, indicating that by current standards, only a minority of out-of-hospital patients are within reach of effective treatment. Twenty-five percent of cardiac arrests strike patients between 20 and 60 years of age. Similar data are available from most Western countries, necessitating improvements of care, such as pharmacological augmentation of central nervous system (CNS) tolerance for ischemia/reperfusion and its consequences. Thus, the aim of our investigations has been to use the porcine cardiac arrest model to study circulatory and CNS effects of global ischemia, the pathophysiology leading to neurological damage, and possibilities for enhancing circulatory and neurological function after cardiac arrest and subsequent CPR.

2 Methodology of Studies of Experimental Cardiac Arrest and Resuscitation Studies [3] In the experimental investigations described, piglets weighing approximately 25 kg and 10 weeks of age were used after approval from the local Institutional Review Board. They were anesthetized by intramuscular injection of 6 mg/kg of Zoletil® and 2 mg/kg of Rompun® in combination with an intravenous injection of 7 mg/kg of thiopental supplemented by intravenous (I.V.) injection of 100  mg morphine. Anesthesia was maintained by continuous I.V. administration of 4 mg/kg/h of pentobarbital. During preparation, nitrous oxide was used as an analgesic. Where applicable, catheters were inserted into a small branch of the carotid artery, the external jugular vein, the pulmonary artery, the internal jugular vein and a superior sagital vein. Pressures were registered in the arterial, pulmonary arterial, right atrial, sinus sagital and intracranial compartments. Blood flow in the aorta, carotid and pulmonary arteries was measured by ultrasonic flowmetry. Cerebral cortical blood flow was determined by laser-Doppler flowmetry. Acid–base status and blood gases were determined in arterial, internal jugular, sagital sinus and mixed venous blood. Cerebral tissue pH and PCO2 were also determined. Ventilation was

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adjusted to maintain a normal PaCO2. After preparation and discontinuation of nitrous oxide and a resting period of 1 h, control measurements were performed. Thereafter ventricular fibrillation, i.e., circulatory arrest defined as a typical EKG with loss of systolic blood pressure, was induced by a transthoracic alternating current of 40–60  V. This circulatory arrest received no treatment for a period of 2–15 min, according to the protocol in the specific investigation. Thereafter, ventilation with 100% oxygen and thoracic compressions were administered, using a mechanical hand-driven compressor (Rentsch Cardiac Press Device) (80 compressions/min) or, later, an automatic pneumatic compressor (Lukas®) (100 com­ pressions/min) for 8–15 min, according to the specific study protocol. The pigs were ventilated with pure oxygen during CPR and during the early reperfusion phase, after which the oxygen concentration was reduced to 30%. After the scheduled circulatory arrest, the heart was defibrillated by an AC electric shock. A maximum of 5 min was allowed for regaining spontaneous circulation. Restoration of spontaneous circulation (ROSC) was defined as a pulsatile cardiac rhythm with a systolic blood pressure of at least 60 mm Hg. The follow-up time after ROSC varied from 4 to 30 h. Neurological outcome was determined by the neurological deficit score and the overall performance categorization [4] 24  h after the incident where applicable. In order to describe cerebral blood flow (CBF) and oxygen metabolism during the early reperfusion phase immediately after ROSC, we have conducted a few studies where the positron emission technique has been combined with magnetic resonance imaging (MRI) in order to achieve good anatomical resolution of the CNS [5, 6]. At the end of the experiment, after termination of the experimental protocol and after potassium chloride induced circulatory arrest, Evans blue dye was administered, the skull was opened and the brain taken out. The brains were immersed in 4% buffered formalin and stored at room temperature for 1 week. Coronal sections (3–5 mm thick) of the pig brain (one cerebral hemisphere) were made and photographed. Small tissue pieces (»3 × 5 mm) from the parietal, temporal, occipital and pyriform cortices were dissected. In addition, portions of the thalamus (median thalamus) and brain stem were also taken out. These tissue pieces were dehydrated in a graded series of alcohol, rinsed in xylene and embedded in low-temperature paraffin (56–58°C) according to standard protocol [7]. About 3–5 mm-thick multiple sections (6–8) were cut from each tissue block and collected on glass slides. After deparaffinization, duplicate sections were stained with either nissl (cresyl violet) or haematoxylin and eosin stain using a commercial protocol [7].

2.1 Analytical Methods Plasma concentrations of a major F2-isoprostane, 8-iso-PGF2a, and 15-ketodihydro-PGF2a, the latter a major metabolite of prostaglandin2a were measured according to methods previously described [8–11]. Analysis of 8-iso-PGF2a, is of

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relevance in studies of oxidative injury as an index of in vivo lipid peroxidation by free radical catalysis of arachidonic acid [10]. Prostaglandin F2a (PGF2a) is one of the major prostaglandins formed at the site of inflammation and is quantified by measurement of 15-keto-dihydro-PGF2a [11]. Serum S-100b levels, an indicator of neurological injury, were measured by an immunoluminometric assay (LIA-mat, Sangtec® 100). CK-MB and troponin I were measured with monoclonal/polyclonal mass immunoassays (ADVIA Centaur, Bayer), with normal values below 0.12 mg/L for troponin I and below 6 mg/L for CK-MB.

3 Review of Results As it has been demonstrated that human brain function is dependent upon its ­nutrient blood flow, delivery of oxygen and elimination of carbon dioxide, much of the effort to improve neurological outcomes after cardiac arrest has focused on the possibilities of increasing systemic and CBF during CPR [12, 13]. Thus, the ­magnitude and mechanism for generation of the blood flow have been of considerable interest. Through comparison of open-chest and closed-chest CPR, it was ­demonstrated [14] that the systemic perfusion pressure during open-chest CPR was considerably greater than that during closed-chest CPR, and in both cases this ­perfusion pressure was increased by administration of epinephrine (Fig.  1). In ­contrast, the pulmonary arterial blood flow, i.e., the cardiac output, during CPR, as well as the pulmonary perfusion pressure, were decreased by epinephrine administration (Figs. 2 and 3). This seems to be an adequate explanation for why ­open-chest cardiac resuscitation, e.g., during cardiac surgery, yields better survival results than closed-chest CPR. Arterial carbon dioxide (CO2) elimination was shown to ­correlate well with cardiac output [15]. CBF fluctuates considerably during the first few hours after ROSC from cardiac arrest in young pigs [5, 6]. In all cases, there was an initial transient period of hypertension and hyperemia during the first 10–20 min after ROSC (Fig. 4). This was followed by a 1-h interval of hypoperfusion with a subsequent slow recovery towards baseline values. The initial hyperperfusion [6] in the CBF maps was most pronounced in the diencephalon, mesencephalon, and in the cerebellum. A similar spatial pattern of hyperperfusion in humans with resuscitation from cardiac arrest was also noted by Schaafsma et al. [16]. The regions with pronounced hyperperfusion are mainly irrigated from the basilar artery, which is known to have an ­autoregulatory capacity over a wide range, and to be sensitive to endothelial, but not neuronal, nitric oxide (NO) [17]. The vascular paralysis due to ischemia [18] in conjunction with local production of nitric oxide is the plausible cause of the ­brainstem and cerebellar hyperperfusion. However, cerebellar hyperperfusion was notably absent in an earlier study [6]. This difference suggests the presence of a time threshold for untreated cardiac arrest prior to successful ROSC in the anesthetized pig, with the development of cerebellar hyperperfusion, occurring only as a consequence of the more severe conditions occurring after prolonged cardiac

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Fig. 1  Systemic perfusion pressure (SPP) (top) and pulmonary perfusion pressure (PPP) (bottom) before, during, and after cardiopulmonary resuscitation (CPR) expressed as mean values ± SEM in the open-chest CPR groups (open squares and solid circles) and in the closed-chest CPR groups (solid squares and open circles). SPP was significantly higher during open-chest CPR. There was no statistical difference in PPP between the groups. VF ventricular fibrillation; DC direct current. By courtesy of Crit Care Med [14]

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Fig.  2  Mean pulmonary arterial blood flow (cardiac output) (MPAF) before, during, and after cardiopulmonary resuscitation (CPR) expressed as mean values ± SEM in the open-chest CPR groups (open squares and solid circles) and in the closed-chest CPR groups (solid squares and open circles). MPAF was significantly higher during open-chest CPR. VF ventricular fibrillation; DC direct current. By courtesy of Crit Care Med [14]

arrests, i.e., ROSC after ten minutes and more of cardiac arrest. In general, ROSC does not always establish reperfusion: The “no-reflow” phenomenon can occur as a result of impaired vascular patency. The extent of this failure to establish reflow is dependent on the duration of the global ischemia [19–22] and is determined in part by the coagulation system [23–25]. A dysregulation of coagulation, anticoagulation, inflammation and fibrinolysis can result in the formation of microthrombi within the cerebral vessels during the interval of circulatory arrest [24], thus impairing reperfusion. Our results suggest that the cardiac arrest produced a more pronounced no-reflow phenomenon in the cerebral cortex, but to a lesser extent in central regions, in which the CBF did not decrease as low as it did in the cortical regions. This finding implies that vasculature in the cerebral cortex may be especially vulnerable to impaired vascular patency. Indeed, the cerebral cortex has been shown to be exceptionally vulnerable to global ischemia [26, 27]. The need for effective reperfusion after ROSC has received even more emphasis [12], and we [28] were therefore surprised to find that large or repeated dosages of epinephrine actually decreased the cerebral cortical blood flow, both in the period

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Fig. 3  Systemic perfusion pressure (SPP) (open squares) (top) and pulmonary perfusion pressure (PPP) (solid squares) (bottom) before and during cardiopulmonary resuscitation (CPR). Values are expressed as mean ± SEM. Two minutes after the administration of epinephrine, SPP significantly increased while PPP decreased. VF ventricular fibrillation; DC direct current. By courtesy of Crit Care Med [14]

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Fig. 4  Mean parametric maps of cerebral blood flow (CBF), cerebral metabolic rate of oxygen (CMRO2) and oxygen extraction factor (OEF) at baseline and at intervals following resuscitation. Each image is the mean of four separate PET scans, presented in sagittal and horizontal planes and projected upon magnetic resonance images in common with stereotaxic coordinates. By courtesy of Resuscitation [5]

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before as well as after ROSC [28]. Thus, alternatives to epinephrine could be less harmful. Occlusion of the abdominal aorta by means of an aortic occlusion catheter, thereby increasing myocardial and carotid blood flow, proved to be such a method [29, 30]. This method, in combination with aortic administration of vasopressin, enhanced not only the cerebral perfusion pressure but also the cerebral cortical blood flow and the cerebral elimination of CO2 (Figs. 5 and 6), especially during the reperfusion phase subsequent to cardiac arrest [30]. Vasopressin administered intravenously had previously been proven to exert a hypertensive effect similar to that of epinephrine [32, 33], but with a longer duration [34] in experimental models; however, in a subsequent multicenter clinical trial, it resulted in only marginally better survival [35]. However, we consider these results to be due to shortcomings in this particular trial, which also included unwitnessed arrests, and may be also regarding the way in which CPR was delivered [36, 37]. Maximization of CBF as well as its adjusted distribution [38] have been considered desirable goals, and expansion of the plasma volume has therefore been tried with some success, especially with respect to CBF [31, 39]. Further enhancement of CBF can be achieved by the use of automatic mechanical compression of the thoracic cage [40–42]. Global cerebral metabolic rate of oxygen (CMRO2) remained at approximately 70% of baseline values for the 5 h after resuscitation; the maintenance of oxygen metabolism after ROSC was apparently sustained by increased oxygen extraction

Fig.  5  Cerebral perfusion pressure (mm Hg) at baseline, during closed-chest CPR, and after restoration of spontaneous circulation (ROSC). All values expressed as mean values ± SEM. Solid diamond group given intravenous vasopressin, open square group given intra-aortic vasopressin above abdominal aortic occlusion. $Significant difference between the groups. By courtesy of Acta Anaesthesiol Scand [31]

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Fig.  6  Cerebral cortical blood flow as a fraction of the steady-state baseline level. All values expressed as mean values ± SEM. Solid diamond group given intravenous vasopressin, open square group given intra-aortic vasopressin above abdominal aortic occlusion. $Significant difference between the groups. By courtesy of Acta Anaesthesiol Scand [31]

fraction (OEF) during periods of low blood flow [5]. However, at 60  min after resuscitation, we found low CBF in some brain regions and particularly high OEF, a circumstance indeed indicative of ischemia, or at least suggesting the occurrence of decreased hemodynamic reserve, as has been reported clinically in comatose survivors with resuscitation after cardiac arrest. In all cortical regions, there was distinct hypoperfusion compared to lesser declines in subcortical CBF for at least 5 h after ROSC. In addition, the regional CMRO2 declined post-ROSC in the pooled cortical regions by a mean of 27%, versus a 19% reduction of subcortical structures. Indeed, results of previous studies showed the cerebral cortex to be especially vulnerable to global ischemia. Thus, we speculate that the declining CMRO2 reflects the onset of secondary brain damage in the hours after ROSC. Pigs subjected to a 5-min survival after cardiac arrest showed a mild to moderate degree of nerve cell damage in the cortical regions that also exhibited extravasation of Evans blue dye (Fig.  7a) [3]. Moderate to severe nerve cell damage was also present in the subcortical regions, e.g., the mid-thalamic nuclei and the brain stem reticular formation. The magnitude of nerve cell damage was most pronounced in the brain stem region. In the cortex, sporadic nerve cell damage was observed in cortical cell layers III to IV. The other cellular layers in the same cortical region contained normal nerve cells (Fig. 7b). The pathological studies of ischemic damage can encompass possibilities of determining how this injury arises. In most reviews of this field, the effects of the reactive oxygen and nitrogen species are considered of importance. During the reperfusion phase in particular, the action of these compounds is regarded as crucial. However, a difficulty related to assessing the pathophysiology of reperfusion injury

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Fig. 7  Coronal section of the pig brain passing through parietal cerebral cortex shows extravasation of Evans blue in some parts of the cortex (arrows). The animal was allowed to survive 5 min after the cardiac arrest. Bar = 3 mm. By courtesy of Ann NY Acad Sci [3]. Nissl-stained, paraffin section (3 mm thick) from pig brain obtained from the parietal cerebral cortex region in the vicinity of Evans blue extravasation. Many dark and distorted nerve cells (arrows) are seen in deeper layers of the cortex. Some normal nerve cells (arrowheads) are also seen in the periphery. Sponginess and edema (asterisk) are clearly evident. Bar = 50 mm. By courtesy of Ann NY Acad Sci [3]

has been the limitations in the current practice of in  vivo measurements of free ­radical concentrations or their generation of stable end-products after free radical oxidation of CNS lipids. In order to circumvent this problem, we have developed methods for determination of nonenzymatic free radical, and enzymatic (cyclooxygenase (COX) catalyzed) products of lipid peroxidation (8-iso-PGF2a and 15-ketodihydro-PGF2a, respectively) [8]. They represent reliable biomarkers of oxidative stress and cyclooxygenase-mediated inflammation, respectively [43, 44]. We were able to demonstrate that both these processes were more active in the CNS than in the rest of the body (Figs. 8 and 9) [8]. Figure 8 (upper panel) describes the levels of 8-iso-PGF2a in the mixed venous plasma at baseline and after ROSC. Figure 7 (lower panel) shows the levels of 8-iso-PGF2a in the jugular bulb plasma collected

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Fig. 8  Mixed venous plasma levels of 8-iso-PGF2a at baseline (before induction of cardiac arrest) and after ROSC (upper panel). Jugular bulb plasma levels of 8-iso-PGF2a at baseline and after ROSC (lower panel). Solid diamond group subjected to 5  minutes cardiac arrest; solid square group subjected to 2 min cardiac arrest; and solid triangle control group not subjected to cardiac arrest. *Significant difference versus baseline; and §significant difference between group subjected to 5  min cardiac arrest versus other groups. Values are expressed as mean values ± SEM. By courtesy of FEBS letters [8]

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Fig.  9  Mixed venous plasma levels of 15-keto-dihydro-PGF2a at baseline (before induction of cardiac arrest) and after ROSC (upper panel). Jugular bulb plasma levels of 15-keto-dihydro-PGF2a at baseline and after ROSC (lower panel). Solid diamond group subjected to 5 min cardiac arrest; solid square group subjected to 2 min cardiac arrest; and solid triangle control group not subjected to cardiac arrest. *Significant difference versus baseline; and §significant difference between group subjected to 5 min cardiac arrest versus other groups. Values are expressed as mean values ± SEM. By courtesy of FEBS letters [8]

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from the brain at baseline and after induction of ROSC. These illustrate that oxidative stress is clearly evident in ischemia and reperfusion in this model. Similarly, the levels of COX-mediated product 15-keto-dihydro-PGF2a in mixed venous plasma at the baseline and after ROSC is shown in Fig. 9 (upper panel). The lower panel (Fig. 9) shows the levels of 15-keto-dihydro-PGF2a in jugular bulb plasma at the baseline and after induction of ROSC. These show that inflammation is also present during ischemia and reperfusion. Thus, the described pig model of cardiac arrest and CPR is an excellent model to study the early phase of cardiac arrest leading to oxidative stress and inflammation. This model could also be used for evaluating specific drugs or radical scavengers in addition to other therapeutic interventions. The increase of F2-isoprostanes in the brain following experimental cardiac arrest and CPR has been counteracted at different degrees by the administration of various free radical scavengers such as PBN [45], S-PBN [46] and methylene blue [9] that advocate for the fact that oxidative stress is involved in ischemia–reperfusion related brain injury, and counteraction by various agents could be studied through this pig model. In addition, this model has also been used to evaluate neurological damage, oxidative stress and inflammatory responses by extending the duration of cardiac arrest time with success [47]. As already stated, it has been claimed that the efficacy of reperfusion is of paramount importance regarding the possibility of mitigating neurological damage after a circulatory arrest [12, 45, 48]. We therefore undertook a series of experiments where we compared piglets in which reperfusion had been maximized but where, in addition, one group had received vasopressin instead of epinephrine and PBN (a-phenyl-N-tert-butyl nitrone), a scavenger of the so-called spin-trap type [45, 46]. The animals were studied for 24 h after the incident. At that point, the neurological deficit score that relates to brain function/damage was significantly less in those that had received PBN and vasopressin, despite equal perfusion pressure in the two groups (Figs.  10 and 11). In fact, the neurological deficit was somewhat greater in the piglets where reperfusion had been maximized without use of vasopressin and PBN, and worse than that registered in a control group where reperfusion had not been maximized [45]. The mode of action of PBN was elucidated in a subsequent study where the water-soluble and sulphonated derivative of PBN was administered. It was demonstrated that autoregulation during the reperfusion phase was better preserved when S-PBN was administered than in a group given a weak vasodilator [46], where autoregulation of cerebral cortical blood flow was not functioning during early reperfusion after ROSC. Thus, our results imply that the jugular bulb plasma concentration of 8-iso-PGF2a (indicating free radical-mediated lipid peroxidation) is proportional to both the duration of ischemia [47] (Figs. 12 and 13) and the resulting neurological deficit after experimental cardiac arrest and CPR [45] (Fig. 14). Particular interest has been devoted to the possible neuroprotective effects of methylene blue (MB) as being a compound known to have direct inhibitory effects on nitric oxide synthases, both constitutive and inducible [49, 50]. An additional time-limited inhibitory effect on soluble gyanylyl cyclase, which normally leads to formation of cyclic guanosine monophosphate (c-GMP) as the second messenger

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of NO, adds to the inhibition of the NO/c-GMP pathway [51, 52]. Thus, MB i­ nhibits guanylyl cyclase by binding to the heme group of the enzyme and blocks the catalytic functions of NOS by oxidation of enzyme-bound ferrous iron [53, 54]. It has been suggested that MB is a more specific and potent inhibitor of NOS than gyanylyl cyclase, as direct NO-donating compounds in the presence of MB can still partially activate c-GMP signaling pathways [49, 50]. MB has also been claimed to possess inhibitory effects on platelet activation, adhesion and aggregation [55–60] synergistically with an inhibition of platelet thromboxane A2 and endothelial prostacyclin I2 (PGI-2) production [61, 62]. The latter effects are mediated by a G-protein coupled adenylyl cyclase and cyclic adenosine monophosphate (c-AMP) in both platelets and endothelial cells [63, 64], while the endothelial antiplatelet adhesion properties are regulated both by the NO/cGMP and cyclooxygenase (COX) pathways on arachidonic acid by PGI-2 [65–67]. MB has also been claimed to block the effect of xanthin oxidase [68, 69].

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Fig. 11  Neurological deficit score in each of the surviving animals. CT group = group treated with CPR including aortic balloon occlusion, vasopressin and PBN; B group = group treated with CPR including aortic balloon occlusion; C group = group receiving standard CPR only. By courtesy of Acta Anaesthesiol Scand [45]

Fig. 12  Mean jugular bulb plasma levels of 8-iso-PGF2a at baseline and after restoration of spontaneous circulation with various durations of circulatory arrest (VF) and in control animals as defined in the upper right corner. VF2 ventricular fibrillation of 2 min; VF5 ventricular fibrillation of 5 min; VF8 ventricular fibrillation of 8 min; VF10 ventricular fibrillation of 10  min; VF12 ventricular fibrillation of 12 min. Values in animals not subjected to cardiac arrest are described as controls. By courtesy of Free Radical Research [47]

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Fig. 13  Levels of maximum increase (in fold) of plasma eicosanoids compared to baseline (8-iso-PGF2a in left panel, 15-keto-dihydro-PGF2a in right panel) and the various durations of ventricular fibrillation (VF) and cardiopulmonary resuscitation (CPR). Values in animals not subjected to cardiac arrest (control animals = 0) are set to onefold. Actual values of fold increase are also mentioned adjacent to and together with separate experiments. Y-axis represents the maximum increase of eicosanoids in fold compared to the baseline in various duration groups of VF and CPR. By courtesy of Free Radical Research [47]

Fig.  14  Linear correlation between neurologic outcome and 8-iso-PGF2a (AUC) 0–2  h after return of spontaneous circulation (ROSC). Solid circle group treated with CPR including aortic balloon occlusion, vasopressin and PBN; open circle groups treated with CPR including aortic balloon occlusion or receiving standard CPR only. By courtesy of Acta Anaesthesiol Scand [45]

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4 Conclusion ROSC can frequently be achieved after up to 15  min or more of normothermic circulatory arrest. Ischemic injury in the porcine brain is observed after only 5 min of normothermic circulatory arrest, and 8 min of circulatory arrest results in very pronounced neurological dysfunction and deficit. Maximization of systemic arterial blood pressure and CBF during CPR and after ROSC does not ameliorate the ­neurological deficit as observed 24 h after ROSC. In contrast, treatment with free radical scavengers seems to reduce the neurological damage considerably, even if administered after the incident. These findings as well as the fact that blood-borne indicators of lipid peroxidation and inflammation are increased during the early stage of reperfusion indicate the importance of this pathophysiological mechanism for the development of neurological injury. This experimental model has also proved to be a sensitive tool for detection and evaluation of therapeutic effects of neuroprotective pharmaceutical agents in general. Acknowledgment  Continued financial support over the years from The Laerdal Foundation for Acute Medicine is gratefully acknowledged.

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12. Hossmann KA. Resuscitation potentials after prolonged global cerebral ischemia in cats. Crit Care Med. 1988; 16(10):964–971. 13. Hossmann KA. The hypoxic brain. Insights from ischemia research. Advances in Experimental Medicine and Biology. 1999; 474:155–69. 14. Rubertsson S, Grenvik A, Wiklund L. Blood flow and perfusion pressure during open-chest versus closed-chest cardiopulmonary resuscitation in pigs. Crit Care Med. 1995; 23(4):715–725. 15. Wiklund L, Soderberg D, Henneberg S, Rubertsson S, Stjernstrom H, Groth T. Kinetics of carbon dioxide during cardiopulmonary resuscitation. Crit Care Med. 1986; 14(12):1015–1022. 16. Schaafsma A, de Jong BM, Bams JL, Haaxma-Reiche H, Pruim J, Zijlstra JG. Cerebral perfusion and metabolism in resuscitated patients with severe post-hypoxic encephalopathy. J Neurol Sci. 2003 Jun 15; 210(1-2):23–30. 17. Toyoda K, Fujii K, Ibayashi S, Nagao T, Kitazono T, Fujishima M. Role of nitric oxide in regulation of brain stem circulation during hypotension. J Cereb Blood Flow Metab. 1997 Oct; 17(10):1089–1096. 18. Tsuchidate R, He QP, Smith ML, Siesjo BK. Regional cerebral blood flow during and after 2 hours of middle cerebral artery occlusion in the rat. J Cereb Blood Flow Metab. 1997 Oct; 17(10):1066–1073. 19. Ames A, 3rd, Wright RL, Kowada M, Thurston JM, Majno G. Cerebral ischemia. II. The noreflow phenomenon. Am J Pathol. 1968 Feb; 52(2):437–53. 20. Fischer EG, Ames A, 3rd, Lorenzo AV. Cerebral blood flow immediately following brief circulatory stasis. Stroke. 1979 Jul-Aug; 10(4):423–427. 21. Fischer M, Hossmann KA. No-reflow after cardiac arrest. Intensive Care Med. 1995;21(2):132–141. 22. Kagstrom E, Smith ML, Siesjo BK. Local cerebral blood flow in the recovery period following complete cerebral ischemia in the rat. J Cereb Blood Flow Metab. 1983 Jun; 3(2):170–182. 23. Gaszynski W. Research work on blood clotting system during cardiorespiratory resuscitation. Anaesth Resusc Intensive Ther. 1974 Oct-Dec; 2(4):303–316. 24. Hekmatpanah J. Cerebral blood flow dynamics in hypotension and cardiac arrest. Neurology. 1973 Feb; 23(2):174–180. 25. Hossmann V, Hossmann KA, Takagi S. Effect of intravascular platelet aggregation on blood recirculation following prolonged ischemia of the cat brain. J Neurol. 1980 Jan; 222(3):159–170. 26. Geraghty MC, Torbey MT. Neuroimaging and serologic markers of neurologic injury after cardiac arrest. Neurol Clin. 2006 Feb; 24(1):107–121, vii. 27. Martin LJ, Brambrink A, Koehler RC, Traystman RJ. Primary sensory and forebrain motor systems in the newborn brain are preferentially damaged by hypoxia-ischemia. J Comp Neurol. 1997 Jan 13; 377(2):262–285. 28. Gedeborg R, Silander HC, Ronne-Engstrom E, Rubertsson S, Wiklund L. Adverse effects of high-dose epinephrine on cerebral blood flow during experimental cardiopulmonary resuscitation. Crit Care Med. 2000; 28(5):1423–1430. 29. Gedeborg R, Rubertsson S, Wiklund L. Improved haemodynamics and restoration of spontaneous circulation with constant aortic occlusion during experimental cardiopulmonary resuscitation. Resuscitation. 1999; 40(3):171–180. 30. Gedeborg R, Silander CsH, Rubertsson S, Wiklund L. Cerebral ischemia in experimental cardiopulmonary resuscitation - comparison of epinephrine and aortic occlusion. Resuscitation. 2001(50):319–329. 31. Nozari A, Rubertsson S, Wiklund L. Improved cerebral blood supply and oxygenation by aortic balloon occlusion combined with intra-aortic vasopressin administration during experimental cardiopulmonary resuscitation. Acta Anaesthesiol Scand. 2000; 44(10):1209–1219. 32. Lindner KH, Brinkmann A, Pfenninger EG, Lurie KG, Goertz A, Lindner IM. Effect of vasopressin on hemodynamic variables, organ blood flow, and acid-base status in a pig model of cardiopulmonary resuscitation. Anesth Analg. 1993; 77(3):427–435. 33. Lindner KH, Dirks B, Strohmenger HU, Prengel AW, Lindner IM, Lurie KG. Randomised comparison of epinephrine and vasopressin in patients with out-of-hospital ventricular fibrillation. Lancet. 1997; 349(9051):535–537.

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34. Nozari A, Rubertsson S, Wiklund L. Differences in the pharmacodynamics of epinephrine and vasopressin during and after experimental cardiopulmonary resuscitation. Resuscitation. 2001; 49(1):59–72. 35. Wenzel V, Krismer AC, Arntz HR, Sitter H, Stadlbauer KH, Lindner KH. A comparison of vasopressin and epinephrine for out-of-hospital cardiopulmonary resuscitation. N Engl J Med. 2004 Jan 8; 350(2):105–113. 36. Wik L, Kramer-Johansen JO, Myklebust H, Sørebø H, Svensson L, Fellows B, et al. Quality of cardiopulmonary resuscitation during out-of-hospital cardiac arrest. JAMA. 2005; 293:299–304. 37. Sunde K, Wik L, Steen PA. Quality of mechanical, manual standard and active compressiondecompression CPR on the arrest site and during transport in a manikin model. Resuscitation. 1997 Jun; 34(3):235–242. 38. Hossmann KA. Reperfusion of the brain after global ischemia: hemodynamic disturbances. Shock (Augusta, Ga). 1997 Aug 8(2):95-101; discussion 2–3. 39. Fischer M, Dahmen A, Standop J, Hagendorff A, Hoeft A, Krep H. Effects of hypertonic saline on myocardial blood flow in a porcine model of prolonged cardiac arrest. Resuscitation. 2002 Sep; 54(3):269–280. 40. Ward KR MJ, Zelenak RR, Sullivan RJ, McSwain NE Jr. A comparison of chest compressions between mechanical and manual CPR by monitoring end-tidal PCO2 during human cardiac arrest. Ann Emerg Med. 1993; 22(4):669–674. 41. Rubertsson S KR. Increased cortical cerebral blood flow with LUCAS: a new device for mechanical chest compressions as compared to standard external compressions during experimental cardiopulmonary resuscitation. Resuscitation. 2005; In Press. 42. Steen S, Liao Q, Pierre L, Paskevicius A, Sjoberg T. Evaluation of LUCAS, a new device for automatic mechanical compression and active decompression resuscitation. Resuscitation. 2002 Dec; 55(3):285–299. 43. Basu S. F2-isoprostanes in human health and diseases: from molecular mechanisms to clinical implications. Antioxid Redox Signal. 2008 Aug; 10(8):1405–1434. 44. Basu S. Novel cyclooxygenase-catalyzed bioactive prostaglandin F2alpha from physiology to new principles in inflammation. Med Res Rev. 2007 Jul; 27(4):435–468. 45. Liu XL, Nozari A, Basu S, Ronquist G, Rubertsson S, Wiklund L. Neurological outcome after experimental cardiopulmonary resuscitation: A result of delayed and potentially treatable neuronal injury? Acta Anaesthesiologica Scandinavica. 2002; 46(5):537–546. 46. Liu XL, Wiklund L, Nozari A, Rubertsson S, Basu S. Differences in cerebral reperfusion and oxidative injury after cardiac arrest in pigs. Acta Anaesthesiol Scand. 2003 Sep; 47(8): 958–967. 47. Basu S, Liu X, Nozari A, Rubertsson S, Miclescu A, Wiklund L. Evidence for time-dependent maximum increase of free radical damage and eicosanoid formation in the brain as related to duration of cardiac arrest and cardio-pulmonary resuscitation. Free Radic Res. 2003 Mar; 37(3):251–256. 48. Nozari A, Rubertsson S, Gedeborg R, Nordgren A, Wiklund L. Maximisation of cerebral blood flow during experimental cardiopulmonary resuscitation does not ameliorate postresuscitation hypoperfusion. Resuscitation. 1999; 40(1):27–35. 49. Mayer B, Brunner F, Schmidt K. Inhibition of nitric oxide synthesis by methylene blue. Biochem Pharmacol. 1993; 45(2):367–374. 50. Mayer B, Brunner F, Schmidt K. Novel actions of methylene blue. Eur Heart J. 1993;14 Suppl I:22–26. 51. Gruetter C, Kadowitz P, Ignarro L. Methylene blue inhibits coronary arterial relaxation and guanylate cyclase activation by nitroglycerin, sodium nitrate, and amyl nitrite. Can J Physiol Pharmacol. 1981; 59:150–156. 52. Gruetter CA, Gruetter DY, Lyon JE, Kadowitz PJ, Ignarro LJ. Relationship between cyclic guanosine 3,5-monophosphate formation and relaxation of coronary smooth muscle by glyceryl trinitrate, nitroprusside, nitrite and nitric oxide, effects of methylene blue and methemoglobin. J Pharm Exp Therapeutics. 1981; 219:181–186.

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53. Hay DW, Martin SA, Sugata R, Lichtin NN. Disproportionation of semimethylene blue and oxidation of leucomethylene blue by methylene blue and iron(III). Kinetics, equilibriums, and medium effects. J Phys Chem. 1981; 85:1474–1479. 54. Sevcik P, Dunford HB. Kinetics of the oxidation of NADH by methylene blue in a closed system. J Phys Chem. 1991; 95:2411–2415. 55. Johnstone MT, Lam JYT, Lacoste L, al. e. Methylene blue inhibits the antithrombotic effect of nitroglycerin. J Am Coll Cardiol. 1993; 20:255–259. 56. Diodati J, Theroux P, Latour JG, al e. Effect of nitroglycerin at therapeutic doses on platelet aggregation in unstable angina pectoris and unstable angina. Am J Cardiol. 1990; 66:683–688. 57. Schmidt HH, Pollock JS, Nakane M, Forstermann U, Murad F. Ca2+/calmodulin-regulated nitric oxide synthases. Cell Calcium. 1992; 13:427–434. 58. Radomski MW, Palmer RM, Moncada S. Endogenous nitric oxide inhibits human platelet adhesion to vascular endothelium. Lancet. 1987; ii:1057–1058. 59. Azuma H, Ishikawa M, Sekizaki S. Endothelium-dependent inhibition of platelet aggregation. Br J Pharmacol. 1986; 88:411–415. 60. Shafer AI, Alexander RW, Handin RI. Inhibition of platelet function by organic nitrate vasodilators. Blood. 1980; 55:649–554. 61. Schroer K, Grodzinska L, Darius H. Stimulation of coronary vascular prostacyclin and inhibition of human platelet thromboxane A2 after low-dose nitroglycerin. Thrombosis Res. 1981; 23:59–67. 62. Salvemini D, Currie MG, Mollace V. Nitric oxide-mediated cyclooxygenase activation. J Clin Invest.  1996; 97:2562–2568. 63. Stamler JS, Loscalzo J. The antiplatelet effects of organic nitrates and related nitrous compounds in vivo and in vitro and their relevance to cardiovascular disorders. J Am Coll Cardiol. 1991; 18:1529–1536. 64. Vane JR, Anggard EE, Botting RM. Regulatory functions of the vascular endothelium. NEJM. 1990; 323:27–36. 65. Wen FQ, Watanabe K, Yoshida M. Nitric oxide enhances PGI2 production by human pulmonary artery smooth muscle cells. Prostaglandins Leucotrienes Ess Fatty Cids. 2000; 62:369–378. 66. Tateson JE, Moncada S, Vane JR. Effects of prostacyclin (PGI) on cyclic AMP concentrations in human platelets. Prostaglandins. 1977; 13:389–397. 67. Levin RI, Jaffe EA, Weksler BB. Nitroglycerin stimulates synthesis of prostacyclin by cultured human endothelial cells. J Clin Invest. 1981; 67:762–769. 68. Salaris SC, Babbs CF, Voorhees WD, 3rd. Methylene blue as an inhibitor of superoxide generation by xanthine oxidase. A potential new drug for the attenuation of ischemia/reperfusion injury. Biochem Pharmacol. 1991 Jul 15; 42(3):499–506. 69. Kelner MJ, Bagnell R, Hale B, Alexander NM. Potential of methylene blue to block oxygen radical generation in reperfusion injury. Basic Life Sci. 1988; 49:895–898.

Part IV

Ocular Diseases

Neovascular Models of the Rabbit Eye Induced By Hydroperoxide Toshihiko Ueda, Takako Nakanishi, Kazushi Tamai, Shinichi Iwai, and Donald Armstrong

Abstract  The eye is rich in oxidizable substrates which often increase in ocular disease. In vivo rabbit models described in this chapter provide valuable clinical and biomolecular data, whereas in vitro models are used to define physiological observations such as endothelial cell proliferation, migration and vascular tube ­formation. Antioxidant supplementation significantly inhibits these phenomena and is facilitated when multiple antioxidants are given simultaneously. The 13-­isomer of linoleic acid hydroperoxide (HpODE) stimulates neovascularization when injected into the corneal stroma, the vitreous, or subretinal to create appropriate animal ­models. During this process, nuclear factors, metalloproteinase and angiogenic cytokines are ­upregulated within 3–6  h to initiate the neovascular response. Fluorescein angiography­provides direct visualization of new vessel growth. A variety of oxidative stress biomarkers can be measured in plasma, ocular homogenates and tissue culture cells, or media. Histology confirms location of injected HpODE, presence or absence of an inflammatory response, hemorrhage and illustrate features that resemble human disease. New vessels grow at the rate of 0.3 mm/day and plateau at 3–4 weeks as the HpODE stimulus is metabolized. Repeat injections result in an accelerated growth rate. Vessel length in diabetic rabbits is double that observed in normal animals. In contrast to surgical or laser-induced neovascularization ­models, the HpODE model uses a natural substrate and is considered a ­non-traumatic ­procedure. All models are valuable to validate antioxidant intervention studies in clinical trials. Keywords  Angiogenic cytokines • Antioxidants • Choroid • Cornea • Fluorescein angiography • Hyperglycemia • Light and electron microscopy • Lipid hydroperoxides • Matrix metalloproteinase • Neovascularization • Nuclear factors • Retina • Tissue culture

D. Armstrong (*) Department of Ophthalmology, University of Florida College of Medicine, Gainesville, Florida, USA e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_14, © Springer Science+Business Media, LLC 2011

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1 Introduction Demonstrable micromolar levels of lipid hydroperoxides (LHP) are normally found in ocular tissues, especially the retina [1]. We use LHP as a general term for all peroxides, but each of the four classes is specifically referred to if they are 18, 20, or 22 carbons in length [2]. These lipids are derived from endogenous polyunsaturated fatty acid precursors that can be peroxidized to the various LHPs during conditions favoring oxidative stress (OS) that results in elevated levels associated with human ocular disorders, especially age-related diseases. For example, a PubMed search indicates there are over 1,250 articles published through 2009 linking OS to eye disease. The association is greatest in cataract, followed in rank order by proliferative diabetic retinopathy, macular degeneration, glaucoma, retinopathy of prematurity, retinitis pigmentosa and Eales’s disease. In the millimolar range, levels of LHP become cytotoxic through hydroxyl-, superoxide- and peroxynitrite-free radical reactions [1]. We have used the 13 – hydroperoxide isomer of linoleic acid (HpODE) in our models because it is the most common form in blood. A paradox exists where low levels at <1 mM, considered a priming dose, stimulate a molecular growth spurt and a neovascular (NV) response in animal models instead of histological tissue injury that is observed with high dose. At >19 mM, HpODE leads to pathological change. Under these conditions, exogenous LHP becomes a “trigger” for generating radical species leading to propagation reactions that are detrimental to ocular tissues. This free radical ­phenomena is initiated during hypoxia, which activates a sub-acute inflammatory response and is exacerbated during reperfusion where transcriptional nuclear activation and upregulation of several angiogenic cytokines, especially vascular endothelial growth factor (VEGF) along with other NV factors, are generated in a time-dependent manner in animal models following intra-corneal, intra-vitreal, or sub-retinal injections [2–4]. NV is also a prominent feature in the streptozotocin animal model of diabetes where intravitreal injections cause accelerated NV compared to control that do not have dibetes [5]. Since NV depends on disruption of the cellular matrix allowing invasion and migration of endothelial cells, it is not surprising that matrix metalloproteinase is also upregulated by HpODE [6]. When LHP accumulate as a result of the aging process, they and their metabolites are deposited in Bruch’s membrane [7]. There is a strong correlation p < 0.01, between NV length and LHP concentration [8]. The AOX capacity of the retina is transiently decreased following HpODE [9] and can be restored by AOX supplementation [10]. Combinations are known to have a synergistic action [11]. Furthermore, the effect of AOXs on antioxidant capacity can be determined in whole retina, as well as isolated photoreceptor outer segments and retinal pigment epithelium and correlated with increased lipid peroxidation [12]. HpODE has similar effects in  vitro. Thus, corneal, epithelial, endothelial and stromal cells have been used to study the modulating effect on vascular

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endothelial cell proliferation, migration and tube formation [13, 14]. Conjunctival epithelial cells show regional differences to HpODE stimulation primarily associated with cell migration [15]. These models in culture are cost-effective and provide rapid, reproducible results regarding vascular growth and inhibition by AOXs. The models described in this chapter summarize our research over the past 17 years and are useful not only to define OS reactions, but also in comparing the protective effect of AOX supplementation to evaluate single, or combinatorial therapy. Recently, the NV effect of HpODE in the choroid has been validated [16] and reproduced in a rat model [17]. When compared to human cadaver eyes from patients with age-related disease, the similarities between human cadaver eyes with AMD and the rodent model were strikingly similar.

2 Materials and Methods 2.1 Lipid Hydroperoxide Synthesis In all studies, the 13-HpODE isomer was synthesized from HODE, i.e., linoleic acid. The reaction is catalyzed using soybean lipoxidase as previously reported [18]. Alternatively, HpODE can be purchased from Cayman Chem Co.

2.2 Corneal Model Adult male rabbits weighing 3 kg were given intravenous Ketaset and Rompun and under an operating microscope, the injection site was positioned 5  mm from the superior limbal arcade and a single 10 ml volume containing 30 mg of HpODE was given [2]. Vessel growth was measured with a caliper at 1, 2, 3 and 4 weeks postinjection. A portion of the superior and inferior quadrant was excised, homogenized, sonicated, centrifuged and analyzed for biomarkers. Corneal samples were also used for tissue culture studies. Photographic images were obtained directly from the corneal surface during fluorescein angiography (FAG).

2.3 Retinal Model Rabbits were given general and topical anesthesia that was followed by intravitreal injection of 600 mg of LHP in a 100 mL volume [3]. The lipid was injected into the center of the vitreous cavity at the 10 o’clock position. At 1-, 2-, 3- and 4-week intervals, animals are sacrificed, the eyes enucleated, the anterior segment removed,

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the vitreous expressed, the retina pulled away from the underlying eye cup, weighed, homogenized in cold HEPES buffer, pH 7.3 which is gently pipetted against the apical surface and the displaced RPE collected for biochemical and molecular biology analyses, or diced and transferred for tissue culture studies. Computer-enhanced images of retinal NV allow visualization of early sprouting not detected with standard FAG. Photographic images during FAG of the retinal surface are taken with a fundus camera.

2.4 Choroidal Model Subretinal injections of HpODE are used to create a model for choroidal NV where with the aid of an operating microscope, a 50 mL bolus is injected at the RPE interface using a 45° angle 41 ga needle [4]. Photographic images during FAG are taken with a confocal microscope to confirm delivery of HpODE.

2.5 Biomarkers The thiobarbituric acid reducing substances OxiTech assay kit was obtained from ZeptoMetrix Corp, Buffalo, NY, used to determine lipid peroxidation levels [19] in samples at various points in time and LHP confirmed by HPLC, cytokine assays by Western blots, nuclear factors by EMSA and MMP by gelatin zymography.

2.6 Histology Tissues are fixed in Zamboni’s paraformaldehyde, sectioned, viewed by light, or electron microscopy and photographed [20].

2.7 Electrophysiology Electroretinograms (ERG) are recorded with a pediatric contact lens placed onto the corneal surface and an electrical action potential elicited following a bright flash of light from a strobe lamp. A decrease in amplitude indicates impaired function [21].

2.8 Tissue Culture Full thickness cornea was removed from enucleated eyes along the corneal-scleral junction. Endothelial, epithelial and stromal cells were obtained by sequential

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dissection of the cornea and grown to confluence in DMEM with 20% FBS and antibiotics. Cells in passage 2 or 3 are used for study [13–15].

3 Clinical Results 3.1 Corneal Model Slight corneal edema and superficial erosion is noted at the time of HpODE ­injection, but this clears within 2 days. No clinically evident inflammatory response was observed. Vasodilation is seen as early as 3  h post-injection and vascular sprouts are apparent by 24 h in the superior quadrant above the injection site. Vessel growth increases at a linear rate of approximately 0.3 mm/day in 100% of animals over the next 3-week period and then plateaued. Vessels do not grow if the LHP is <7.5 mg. Anterior segment angiography at 3 weeks post-injection showed fluorescein dye in vessels during the early phase documenting the NV response (Fig. 1a). In the late

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phase, fluorescein can be seen leaking into tissue at the distal tips of the new vessels (Fig. 1b). This is due to the immature nature of these vessels as seen on microscopic examination. Vessels are both superficial and deep . The NV response is linear over this 3-week period (Fig.  1c). In contrast, cholesterol hydroperoxide induced NV, but was not observed until day 3, at which time the length of these vessels was ­considerably muted and the curve remained flat. Rabbits made hyperglycemic with alloxan to induce diabetes and then given 40 mM HpODE. These vessels grow roughly double the length of normoglycemic animals injected with HpODE (Fig. 2). Interestingly, VEGF levels doubled at 24 h and were four times higher at 72 h than in control animals, remaining elevated up to the 14-day time point.

3.2 Retinal Model The NV response was successful in 100% of the animals. Time curves for vitreous and retinal LHP transport into the retina are shown in Fig. 3. LHP levels in the vitreous, peak within the first hour after injection and the progressive rise in retinal LHP ­indicative of transport was maximal at 12 h post-injection. LHP can be detected very early in the vortex vein that drains the eye, and its curve parallels that of retinal transport. As compared to sham (Fig. 4a) clinically, fluorescein angiography of the retina provides clear evidence of NV following an intravitreal injection of 600  mg LHP (Fig.  4b). Other features of clinical relevance include NV sprouting, neovascular fronds, toruosity and late-phase leakage. In addition to new surface vessels, capillaries also penetrate into the retinal stroma [3]. The b-wave electroretinogram is reduced in amplitude from 490 to 45 mV within just 1 h following high-dose LHP. By 14 days post-injection, the ERG is isoelectric.

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Fig. 4  Three week fluorescein angiogram following 600 mg intra-vitreal injection of HpODE (b) in retinal model (a =sham injection). Prominent features are cork-screw-shaped tortuosity and NV fronds that formed from dual, horizontal medullary ray vessels in 100% of the animals. Optic disc is to left of angiogram

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3.3 Choroidal Model A subretinal bleb measuring 1.5 optic disc diameters indicates a successful injection. Blebs create a transient retinal detachment that quickly resolves and the retina is reattached within a few hours. CNV is confirmed histologically in approximately 84% of the animals. FAG shows the size of the CNV at 4 weeks post-injection (Fig. 5a).

Fig.  5   Choroidal model showing (a) fluorescein angiogram of injection site measured 200–250 mm at 4 weeks post-injection; (b) macrophage-like cell in sub-retinal space with lipid droplets in residual body; (c) early formation of drusen and disruption of Bruch’s membrane; (d) light micrograph of retina–choroid interface showing penetration of choroidal vessel; and (d1) TEM with insert showing positive immunohistochemical reaction for LHP in lipid droplets within vacuole; (b) and (c) appeared at 1 week, (d) formed by 4 weeks

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If retinal hemorrhage is detected, the eye is not used for further study since hemoglobin is a potent source of free radicals. Exogenous and endogenously produced LHP from injection of HpODE are most likely phagocytized by macrophages of RPE origin (Fig. 5b), or by blood-borne cells, and we observe basal laminar and linear deposits that appear to represent drusen-like material [4] accumulating under the RPE basal membrane within 3 h of injection (Fig. 5c). As the LHP reach the RPE-choroidal interface, micro-ruptures occur in Bruch’s membrane, so choroidal vascular endothelial cells can migrate into the retina (Fig. 5d). LHP is also stored in vacuoles (see D1 insert) that serve as a continuous source of de novo LHP to prolong the NV response without need to re-inject, which always carries a risk for trauma. Similar accumulation has been reported in the hypercholerolemic pig retina [22]. As described for LHP, the cytotoxic nature of 7-ketocholesterol peroxide [23] has been reported in primate retina and its biochemical profile resembles oxidized plasma LDL, which also damages Bruch’s membrane [24]. OS is reported to contribute to several types of ocular NV [25].

3.4 Tissue Culture Models HpODE stimulates cell migration in corneal cells in vitro through upregulation of VEGF, which is maximal at 10–5  M, but remains effective down to 10–7  M ­concentrations. This model is important in determining the effect of antioxidants on reducing the NV response. Sub-toxic effects have also been described [26].

4 Biochemical and Molecular Biology Results Angiogenic cytokine patterns are shown in Fig.  6, where upregulation of TNF-a was the major event occuring at 12  h post-injection. A Western blot of vascular endothelial growth factor (VEGF) is shown where peak synthesis occurs at 24 h post-injection.This response was sustained through 21 days, in contrast to TNF-a, which is induced at 12 h and declines rapidly by 24 h. In data not shown, PDGF had a bi-phasic response, while b-FGF had a slow increase over time. Upregulation was less prominent for TGF-b and IL-1. Of the nitric oxide synthase isozymes, only iNOS and bNOS are constituitivly expressed. We could also demonstrate stimulation of glutathione peroxidase, presumably due to the excess of substrate shown in Fig. 7. The curve reflected that observed for LHP and peaked at 12 h post-injection. Superoxide dismutase was mildly stimulated; however, its response indicates that free radical mechanisms are present in retinal tissue. Matrix metalloproteinase is required to digest stromal layers so new vessels can proliferate and migrate into the deeper retina. Figure  8 shows time curves for the ­vitreous and retina. These compounds peaked at 24 and 48 h respectively. MMP-2 is

Fig. 6  Relationship between LHP peak (12 h) and cytokine expression as a function of time: 12-h peak for TNF-a and 24-h peak for VEGF

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Fig. 8  Geletin zymography of matrix metalloproteinase activity in vitreous. The 92 kDa pro-and active forms of MMP-9 were not detectable by Western blot up to 12  h post-injection, then increased through 24 h and peaked at 7 days. Pro-, or active MMP-2 was constituently expressed and unchanged by exposure to LHP

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the main proteinase of the retina and normally, there is no MMP-9. Only the ­expression of MMP-9 activity was observed after exposure to LHP, but had no effect on MMP-2. A dihydrobenzofuran derivative can inhibit MMP-9 in the corneal NV model and restrict vessel length from 27 to 10 mm [27].

5 Histological Results In the corneal model, proliferation and maturation of vessels can be followed and correlated with biochemical findings, i.e., MMP and peri-vascular edema. In the retinal model, NV migration and early-late pathological events can be observed in various cell layers as a function of LHP concentration. In the retinal model, evidence is presented that the outer photoreceptor segments and RPE are the site of damage. It was recently reported that the RPE is essential in the formation of CNV [27]. In our choroidal model, one can see the movement of new vessels from choroid through Bruch’s membrane, detachment of retinal pigment epithelium and phagocytic activity, which we suspect to represent detached RPE cells in the subretinal space. These vessels are immature when they first start to grow, but become mature as they penetrate deeper into the tissue. In transgenic mice that develop diabetes, there are changes in the RPE and Bruch’s membrane coupled with retinal NV [25], which contributes to diabetic complications [28], yet there is apparently no model that fully resembles diabetic retinopathy in humans [29]. NV is expected to influence retinal physiology and it has been shown that LHP enhances leukocyte–endothelium interaction [30]. In fact, this disturbance in the microcirculation can be inhibited 95% with a 5,000 unit/kg intravenous injection of the antioxidant enzyme PEG-SOD [31].

6 Tissue Culture Results In cultures, endothelial cell proliferation, migration and vascular tube formation can be studied independently, or simultaneously in longer experiments [13]. We have found that AOXs primarily inhibit the migration phenomena [14]. In the retinal ex vivo model, various antioxidants can be tested in isolated ­photoreceptor outer segment and retinal pigment epithelial cell preparations and compared to whole retina. These preparations were exposed to iron-induced peroxidation of retinal lipids to generate LHP and then the antioxidants were added before the oxidation step and the per cent inhibition calculated. In general, inhibition ranged from 38 to 79% in the retina, from 30 to 71% in outer segments, and from 35 to 83% in RPE cells. These studies also demonstrated the synergistic effect of a multiple antioxidant formulation. For example, quercetin plus vitamin E significantly inhibited oxidation relative to either of these antioxidants given alone. In separate experiments, catechin plus coenzyme Q10 also showed synergy.

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7 Conclusion The unique feature of the LHP-induced NV models are (1) the stimulus is a naturally occurring substance; (2) NV is amplified on re-injection; (3) the injection techniques are simple to perform; (4) the eye is large for multiple biochemical, molecular biology and histological analysis; (5) the models are reproducible and have been validated; (6) initial, progressive and terminal changes can be documented; (7) injections are less traumatic than other reported animal models; (8) several types of cells can be studied; (9) blood samples from the ear vein and vortex vein provide an index of systemic and local LHP transport; and (10) the models are cost-effective, especially when adapted to rodents. As described in this chapter, the LHP-NV models have many applications and utilize many techniques for evaluating pre-clinical, laboratory and histologic changes and efficacy of AOX in tissue culture. These have enhanced our understanding of the initial response to LHP and the progressive steps leading to NV. The models emphasize the importance of LHP in ocular disease. They offer opportunities to simultaneously evaluate anti-NV, or anti-inflammatory activity against oxidative stress pathology. It is well accepted that reactive oxygen species contribute to OS -induced NV [25] that can begin even in the 50–150 mM sub-toxic range, as we have demonstrated in this chapter and our group has previously summarized. Therefore, the LHP-induced NV eye models represent a natural response to endogenous LHP and a non-traumatic approach to the study of the origin and progression of retinal-vascular disorders, such as corneal inflammation, uveitis, glaucoma, age-related macular degeneration, progressive diabetic retinopathy, retinitis pigmentosa, Eales disease and retinopathy of prematurity. As described in our numerous publications defining LHP-induced NV, these models are highly reproducible and clinically relevant because ocular examinations can be observed directly. The retinal model can also can be used to record changes in electrophysiologic activity before and after treatment as well as study the effects of LHP and AOXs on leukocyte -vascular endothelial dynamics. The rabbit eye is large enough to allow injections of materials of interest and it can provide tissue at necropsy for biochemistry, histology and culture from a single specimen. The low-dose models are indicative of early events in the NV process, i.e., ­sub-clinical inflammation, edema and retinal degeneration. These various models allow molecular interpretation of steps within the overall process, such as priming, cell proliferation, vessel maturation, time-dependent regression of vessel growth when LHP is no longer at optimal stimulation levels, tissue re-modeling and AOX protection during induced -OS. Based on our NV research, the rabbit models help define the relationship between mitochondrial bioenergenics, activation of nuclear factors, cytokine ­synthesis, AOX synergy and, in addition, they document differences in adjacent cell types, apoptotic phenomena, necrosis and gene regulation. Immunohistochemistry for OS ­biomarkers allow precise localization of OS reactions in membranes and the cytosol. NV is also a major factor in other non-ocular diseases, and information gained from these eye models can be applied to further understanding the inflammation

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hypothesis in wound healing, cancer, the menstrual cycle and virtually any disease where NV is present. In conjunction with the CNV model, tissue culture models can be used to screen efficacy of new AOX products, or formulations that modify the growth characteristics of NV. For example, the ARENDS (Age Related Eye Disease Study) project has shown that select AOXs found in the normal macula can slow the progression of AMD [32]. Using a compound score based on dietary AOX intake, it has been found that high scores are associated with lower risk of AMD [33]. The groups represented are alpha/gamma tocopherols and lycopene and carotenoids such as lutein and zeaxanthin [34]. These authors determined that a combination of AOXs best protects against late-stage AMD [35]. Clinically, abnormal retinal blood flow in humans is related to OS-induced dysfunction that is improved by the ARENDS supplement [32]. We suggest that ARENDS medication can be applied to our tissue culture and choroidal models as well as another trial medication, i.e., CARMA (Carotenoid in Age-Related Maculopathy), which studies only the late form of disease [34]. However, the reported AOX protocols have not yet confirmed whether or not they can decrease risk [36]. Such studies can easily be conducted with our models. Acknowledgements  The authors thank the following scientists who assisted in these various studies: Akira Higa, Shohei Fukuda, Eichi Nishimura, Richard Browne, Ahmad Aljada, Kathleen Dorey, Ram Sasisekharan, Tadahisa Hiramitsu, Shigehiro Iwabushi, Yuichiro Ogura, Alihisa Matsubara, Mary E. Hartnett, Kaori Watanabe, Miho Chida, Maki Kamegawa, Richard Spaide, Wang Ho-Spaide, Maria Grant and E. Ann Ellis. We also thank Professors Ryohei Koide and Hajimi Yasuhara for department and institutional support and technical assistance and Oxidative Stress Associates for administrative financial aid.

References 1. Armstrong, D. et al., 1998. Dose dependent mechanisms of lipid hydroperoxide induce retinal pathology. In, Yagi, K, ed, Pathophysiology of Lipid Peroxides and Related Radicals, Japan Sci. Soc. Press, Tokyo/S. Karger, Basel, pp 57–76. 2. Ueda, T. et al., 1997. Lipid hydroperoxide-induced tumor necrosis factor (TNF) -a,vascular endothelial growth factor and neovascularization in the rabbit cornea: effect of TNF inhibition. Angiogenesis 1: 174–184. 3. Armstrong, D, et al., 1998. Lipid hydroperoxide stimulates retinal neovascularization in rabbit retina through expression of tumor necrosis factor-a, vascular endothelial growth factor and platelet-derived growth factor. Angiogensis 2: 93–104. 4. Tamai, K et al., 2002. Lipid hydroperoxide stimulates subretinal choroidal neovascularization in the rabbit. Exp Eye Res 74: 301–308. 5. Higa, A., et al., 2002. Lipid hydroperoxide induced corneal neovascularization hyperglycemic rabbits. Curr Eye Res 25: 49–53. 6. Iwai, S., et al., 2006. Activation of AP-1 and increased synthesis of MMP-9 in the rabbit retina by lipid hydroperoxides. Curr Eye Res 31: 337–346. 7. Spaide, RF, et al., 1999. Characterization of peroxidized lipids in Bruch’s membrane. Retina 19: 141–147. 8. Watanabe, K et al., 2000. Quantitative analysis of rabbit corneal neovascularization induced by lipid hydroperoxide. Atarashi Ganka (J of Eye) 17: 710–714.

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9. Kamegawa, M et al., 2007. Effect of lipid hydroperoxide-induced oxidative stress on vitamin E, ascorbate and glutathione in the rabbit retina. Ophthalmic Res 39: 49–54. 10. Ogura, M et  al., 2007. Effect of dihydrobenzofuran derivatives on lipid hydroperoxideinduced rabbit corneal neovascularization. J Pharmacol Sci 103: 234–240. 11. Ueda, T, et al., 1996. Preventive effect of natural and synthetic antioxidants on lipid peroxidation in the mammalian eye. Ophthalmic Res 28: 184–192. 12. Chida, M, et, al, 1999. In vitro testing of antioxidants and biochemical end-points in bovine retinal tissue. Ophthalmic Res 31: 407–415. 13. Nishimura, E, et al., 2001. Upregulation of vascular endothelial growth factor in cultured rabbit corneal cells during lipid hydroperoxide oxidative stress. Showa Univ J Med Sci 13: 35–41. 14. Yamada, Y et al., 1998. Angiogenic effect of lipid hydroperoxide on bovine aortic endothelial cells. J Clin Biochem Nutr 25: 121–130. 15. Yamamoto, YM, et al., 2000. UV-B and lipid hydroperoxides promote conjunctival epithelial cell migration. Pathophysiology 6: 225–230. 16. Framme, C et al., 2008. Clinical evaluation of experimentally induced choroidal neovascularizations in pigmented rabbits by subretinal injection of lipid hydroperoxide and consecutive preliminary photdynamic treatment with Tookad. Ophthalmologica 222: 254–264. 17. Baba, T., et al., 2009. A rat model for choroidal neovascularization using subretinal injection of lipid hydroperoxide. ARVO Abstract 2055: 140. 18. Armstrong, D and Browne, R, 1998. Synthesis of lipid and cholesterol hydroperoxide standards. Meth Mol Biol 108: 139–145. 19. Armstrong, D and Browne, R 1994. The analysis of free radicals, lipid peroxides, antioxidant enzymes and compounds related to oxidative stress as applied to the clinical chemistry laboratory. In, Armstrong, (ed), Free Radicals in Diagnostic Medicine, Adv Exp Med Biol,vol 366, pp 43–58. 20. Ellis, EA, 2008. Correlative transmission microscopy: cytochemical localization and immunocytochemical localization in studies of oxidative and nitrosative stress. Meth Mol Biol, Adv Protocols 1, 477: 41–48. 21. Armstrong, D, et al., 1982. Studies on experimentally induced retinal degeneration.1. Effect of lipid peroxides on electroretinographic activity in the albino rabbit. Exp Eye Res 35: 157–171. 22. Fernandez-Robredo, P et al., 2005. Vitamins C and E reduce retinal oxidative stress and nitric oxide metabolites and prevent ultrastructural alterations in porcine hypercholesterolemia. Invest Ophthalmol Vis Sci 46: 1140–1146. 23. Moreira, EF, et al., 2009. 7-Ketocholesterol is present in lipid deposits in the primate retina: potential implications in the induction of VEGF and CNV formation. Invest Ophthalmol Vis Sci 50: 523–532. 24. Wang, L. et al., 2009. Lipoprotein particles of intraocular origin in human Bruch membrane: an unusual lipid profile. Invest Ophthalmol Vis Sci 50: 870–877. 25. Dong, A, et al., 2009. Oxidative stress promotes ocular neovascularization. J Cell Physiol 219: 544–552. 26. Xu, AI, et al., 2009. Subtoxic oxidative stress induces senescence in retinal pigment epithelial cells via TGF-beta release. Invest Ophthalmol vis Sci 50: 926–935. 27. Garcia-Layana, A et al., 2009. Development of laser-induced choroidal neovascularization in rats after retinal damage by sodium iodate injection. Ophthal Res 42: 205–212. 28. Niedowicz, DM and Daleke, DL, 2005. The role of oxidative stress in diabetic complications. Cell Biochem Biophys 43: 289–330. 29. Ogarte, M, 2009. Animal models of diabetic retinopathy: how are they helping us? Arch Soc Esp Oftalmol 84: 277–279. 30. Tamai, K, et al, 2002. Lipid hydroperoxide stimulates leukocyte-endothelium interaction in the retinal microcirculation. Exp Eye Res 75: 69–75. 31. Matsubara, A et al., 2005. Protective effect of polyethylene glycol-superoxide dismutase on leukocyte dynamics in rat retinal microcirculation under lipid hydroperoxide-induced oxidative stress. Exp Eye Res, 81: 193–199.

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32. Pemp, B, et al., 2009. Effects of antioxidants (AREDS medication) on ocular blood flow and endothelial function in an endotoxin-induced model of oxidative stress in humans. Invest Ophthalmol Vis Sci Aug 13 (Epub ahead of print). 33. Chiu, CJ, et al., 2009. Dietary compound score and risk of age-related macular degeneration in the age-related eye study. Ophthalmology 116: 939–946. 34. Rehak, MN et  al., 2008. Lutein and antioxidants in the prevention of age-related macular degeneration. Ophthalmologe 105: 37–38, 40–45. 35. Neelam, K, et al., 2008. Carotenoids and co-loclization in age-related maculopathy: design and methods. Ophthalmic Epidemiol 15: 389–40. 36. Michikawa, T, et al., 2009. Serum antioxidants and age-related macular degeneration among older Japanese. Asia Pac J Clin Nutr 18: 1–7.

Purinergic Signaling Involved in the Volume Regulation of Glial Cells in the Rat Retina: Alteration in Experimental Diabetes Andreas Bringmann

Abstract  Impairment of the glial fluid clearance from the retinal tissue is an essential step in the formation of retinal edema in diabetics. Glial fluid clearance is thought to be mainly mediated by a transcellular water and ion transport facilitated by potassium (Kir4.1) channels and aquaporins. Retinal glial cells of diabetic rats display a functional inactivation and dislocation of Kir4.1 channels which is associated with cytotoxic edema (cellular swelling) under hypoosmotic conditions. Retinal glial cells possess an intrinsic purinergic signaling cascade that prevents hypoosmotic swelling. This cascade is initiated by glutamate, and involves the consecutive activation of P2Y1 and adenosine A1 receptors, the action of the nucleoside triphosphate diphosphohydrolase-2 (NTPDase2), and a nucleoside transporter-mediated increase in extracellular adenosine. The most downstream event of the cascade, activation of A1 receptors by adenosine, results in opening of potassium and chloride channels; ion efflux equalizes the osmotic gradient across the glial membrane. Glial cells of control rat retinas express NTPDase2 and ecto-5¢-nucleotidase. Diabetic retinopathy is characterized by an upregulation of glial NTPDase1. Thus, the increase in extracellular adenosine occurs mainly by a transporter-mediated mechanism in control retinas, and by both nucleoside transporters and extracellular degradation of ATP in diabetic retinas. This may result in an increased extracellular availability of the glio- and neuroprotectant adenosine in the diabetic retina. Opening of ion channels upon A1 receptor activation inhibits cytotoxic glial edema and may restore the glial capacity of fluid clearance from the edematous tissue. Keywords  Cytotoxic edema • Diabetes • Glial cell • Purinergic receptor • Rat • Retina

A. Bringmann (*) Department of Ophthalmology and Eye Hospital, University of Leipzig, Liebigstrasse 10-14, 04103 Leipzig, Germany e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_15, © Springer Science+Business Media, LLC 2011

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Abbreviations ADP AMP ATP Kir NTPDase P2X P2Y VEGF

Adenosine 5¢-diphosphate Adenosine 5¢-monophosphate Adenosine 5¢-triphosphate Inwardly rectifying potassium Nucleoside triphosphate diphosphohydrolase Ionotropic purinergic receptor Metabotropic purinergic receptor Vascular endothelial growth factor

1 Introduction 1.1 Retinal Edema in Diabetics Diabetic retinopathy is the leading cause of reduced visual acuity and acquired blindness in working-age adults. In patients with diabetic retinopathy, the presence of retinal (macular) edema is the major cause of visual impairment [1]. By compression of retinal neurons, nerve fibers, and perifoveal blood vessels, retinal edema contributes to the ischemic–hypoxic conditions, functional impairment of photoreceptors, and degeneration of retinal neurons. Retinal edema is characterized by the accumulation of water in the macular tissue resulting in a thickening of the tissue. Water may accumulate within retinal cells (intracellular or cytotoxic edema resulting in cellular swelling) and in interstitial spaces (extracellular edema resulting in cell compression). There are systemic and retinal factors that underlie the development of macular edema. Systemic factors cause an inflow of excess water from the blood into the retinal tissue, either as the result of an increase in the hydrostatic pressure (hypertension) or a decrease in the blood osmolarity (e.g., in cases of renal or hepatic failures resulting in hyponatremia and hypoalbuminemia) [2]. Retinal factors are ischemia–hypoxia (which is associated with oxidative stress) and inflammation [1, 3–7]. Diabetic retinal edema is thought to be primarily caused by a breakdown of the blood–retinal barrier resulting in vascular leakage (vasogenic edema) [4, 6]. The major vessel-permeabilizing factor induced by retinal hypoxia is the vascular endothelial growth factor (VEGF) [8–11]. In addition to VEGF, other growth factors such as basic fibroblast growth factor, inflammatory factors such as tumor necrosis factor-a, interleukin-1b, and prostaglandins, as well as proteins of the blood coagulation cascade enhance the permeability of retinal vessels [10, 12, 13]. Generally, water accumulation within the retinal tissue results from an imbalance between the fluid inflow (from the blood into the retina) and the fluid clearance from the retinal tissue into the blood [14]. It has been shown that an impairment of the fluid absorption from the retinal tissue is an essential step in edema formation.

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Clinically significant diabetic retinal edema develops only when (in addition to vascular leakage) the active transport mechanisms of the blood–retinal barriers are dysfunctional [15]. Any anomalies in vessel permeability need to be accompanied by ineffective edema-resolving mechanisms to cause chronic edema [16]. Moreover, there are cases of retinal edema without angiographic vascular leakage, suggesting that an impairment of the fluid clearance from the tissue is the major pathogenic event in these cases.

1.2 Retinal Fluid Clearance The redistribution of excess fluid from the retinal tissue into the blood is normally carried out by the retinal pigment epithelium (which dehydrates the subretinal space around the photoreceptor segments) and retinal glial (Müller) cells, which dehydrate the inner retina [14]. The fluid clearance is mediated by a transcellular water transport that is osmotically driven by a transport of osmolytes, especially of potassium ions. A major function of retinal glial cells is the spatial buffering of the extracellular potassium concentration [17, 18]. Glial cells take up excess potassium from regions of intense neuronal activity and release a similar amount of ­potassium into extraretinal fluid-filled spaces, i.e., the blood and the vitreous humor. Excess water, which is metabolically generated especially in regions of intense neuronal activity, is transported out of the retina in association with the transglial potassium currents [14]. The ion and water transport through glial cells is facilitated by the colocalization of distinct potassium and water channels. The major potassium channel expressed by retinal glial cells is the inwardly rectifying potassium (Kir) channel, Kir4.1 [19–21]. Kir4.1 is a weakly rectifying channel that mediates inward and outward potassium currents with similar amplitudes [22, 23]. The Kir4.1 channel protein displays a prominent localization in such membrane domains across which retinal glial cells export potassium into spaces outside of the neural retina, i.e., in perivascular membranes, in membranes of the endfeet that have contact to the vitreous chamber, and in the microvilli that extend into the subretinal space (Fig.  1a) [20, 21, 26]. In addition to Kir4.1, retinal glial cells express other subtypes of Kir channels [27], e.g., Kir2.1 which is a strongly rectifying channel that mediates uptake of excess potassium from the extracellular space [21]. Aquaporin water channels facilitate the bidirectional water transport across membranes in dependence on alterations in the hydrostatic pressure and osmotic gradients [28]. In the sensory retina, various aquaporins are expressed in a cellspecific manner [29–33]. Retinal glial cells express the glial water channel ­aquaporin-4 in membrane domains through which excess water is taken up from the extracellular space (in both plexiform layers) and released into extraretinal spaces (around the vessels and at the limiting membranes of the retina; Fig. 1a) [25, 26, 30, 31]. The direction of the coupled water and ion transport out of the tissue into the blood is determined by the potassium gradients within the tissue and the rectification properties of Kir2.1 and Kir4.1 channels (Fig. 2a).

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Fig. 1  Retinal gliosis in experimental diabetes is associated with a decrease in glial potassium conductance. (a) Immunostaining of retinal slices derived from age-matched control (above) and diabetic animals (below). Retinal gliosis is indicated by the upregulation of the intermediate filament glial fibrillary acidic protein (GFAP). In the control tissue, GFAP is restricted to the astrocytes in the ganglion cell layer (GCL). In the diabetic retina, GFAP is also expressed by retinal glial (Müller) cell fibers that traverse the whole retinal tissue. In control retinas, the Kir4.1 protein displays a prominent localization around the blood vessels (arrows) and at both limiting membranes of the retina (arrowheads). In retinas of diabetic animals, the Kir4.1 protein is redistributed from these prominent expression sites, and is localized diffusely to glial (Müller) cells. In contrast, the retinal distribution of the glial water channel protein aquaporin-4 remains largely unaltered in the course of diabetes. (b) Examples of whole-cell potassium currents recorded in freshly isolated retinal glial (Müller) cells. Cells from diabetic animals display a reduction in the potassium conductance when compared to cells from control animals, with a substantial variation of the current amplitude in different cells. The currents were elicited from a holding potential of −80 mV up to +20  mV and −180  mV, respectively (increment, 20  mV). INL inner nuclear layer; IPL inner plexiform layer; ONL outer nuclear layer. Bars 20 mm. With permission from Pannicke et al. [24] and Iandiev et al. [25]

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Fig. 2  Mechanisms of osmotic glial cell swelling in the rat retina. (a) Under normal conditions, retinal glial (Müller) cells mediate the fluid clearance from the retinal tissue into the blood mainly by a transcellular cotransport of water (facilitated by aquaporin-4 water channels, AQP4) and distinct osmolytes, predominantly of potassium ions (facilitated by Kir4.1 and Kir2.1 channels). (b) Under ischemic–hypoxic and inflammatory conditions, vascular leakage occurs due to the action of inflammatory factors and VEGF. Oxygen radicals stimulate the formation of arachidonic acid (AA) and prostaglandins (PGs) by phospholipase A2 and cyclooxygenase in glial cells. These inflammatory lipids inhibit the activity of the Na, K-ATPase, resulting in sodium influx that leads to osmotically driven water influx and cellular swelling under hypotonic conditions. In the normal, healthy retina, the sodium influx can be compensated by a potassium efflux through Kir4.1 and (after autocrine activation of adenosine A1 receptors) two-pore domain potassium channels. Glial cells of the diabetic retina downregulate the expression of functional Kir4.1 channels, which impairs the compensatory release of potassium ions under hypotonic conditions. Because the cells are still capable of taking up potassium ions from interstitial spaces through Kir2.1, this results in an accumulation of potassium within the cells. An influx of sodium ions via electrogenic glutamate uptake carriers and endocytosis of extravasated serum proteins may further enhance the osmotic pressure of the cell interior. The downregulation of Kir4.1 impairs the glial release of potassium into the blood resulting in a disruption of the transglial fluid clearance from the retinal tissue

2 Osmotic Swelling of Retinal Glial Cells 2.1 Diabetes Alters the Osmotic Swelling Properties of Retinal Glial Cells It has been suggested that (in addition to ischemic changes in the retinal microvasculature) cytotoxic edema, i.e., a swelling, of retinal glial cells contributes to the development of diabetic retinal edema [14, 34]. A swelling of glial cells in the macular

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Fig. 3  Involvement of nucleoside transporters and ecto-nucleotidases in the purinergic inhibition of osmotic glial cell swelling. Diabetes was induced in male Wistar (a) or Long Evans rats (b, c) with a single intravenous injection of streptozotocin (65 mg/kg body weight). The retinas were removed 6–10 months after streptozotocin injection. The cross-sectional area of glial somata was recorded in freshly isolated retinal slices that were perfused with a hypotonic solution (containing 60% of control osmolarity) for 4  min. Glial swelling in retinal slices from age-matched control animals was induced by a barium (1  mM)-containing hypoosmolar solution. The soma area is

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tissue was suggested to precede the formation of extracellular edema [35]. In animal models of retinal ischemia–hypoxia, vascular leakage is associated with cellular edema of glial cells [36, 37]. To investigate the swelling properties of retinal glial cells under osmotic stress conditions in an experimental model, we used freshly isolated retinal slices that were superfused with a hypoosmolar solution (containing 60% of control osmolarity); this situation resembles hypoxia-induced cytotoxic edema in the brain. Retinal glial cells of diabetic rats display a characteristic alteration in the osmotic swelling properties as compared to cells from nondiabetic animals. Glial cell bodies in retinal slices from adult healthy animals held their volume constant (up to ~20 min [38]) when the osmolarity of the extracellular medium abruptly decreased (Fig. 3a). Apparently, retinal glial cells have a highly efficient cell volume regulation that compensate for changes in extracellular osmolarity. However, glial cell bodies in slices from diabetic animals swelled rapidly upon hypoosmotic challenge (Fig. 3a) [24, 39]. The data indicate that diabetes results in an alteration of the osmotic swelling characteristics of retinal glial cells. Cellular swelling reflects a dysregulation of the osmotically driven water movement across glial membranes. Diabetic retinopathy is associated with oxidative stress [40] and involves a chronic, low-grade retinal inflammation [2, 41]. Similar alterations of the glial swelling properties as in retinal tissues from diabetic rats were found in various other retinopathies that are characterized by retinal ischemia–hypoxia and/or inflammation including ischemia–reperfusion [42], branch retinal vein occlusion [43], lipopolysaccharide-induced ocular inflammation [44], retinal detachment [45], and blue light-evoked retinal degeneration [46]. This suggests that induction of osmotic glial cell swelling is a general phenomenon in response to oxidative stress and inflammatory conditions in the retina.

Fig. 3 (continued) expressed in percent of the values obtained before hypotonic challenge (100%). (a) Under hypoosmotic conditions, glial cells in retinal slices from diabetic rats display a rapid time-dependent swelling of their somata, a response not observed in cells from control animals. Insets: Soma of a glial cell from a diabetic animal before (left) and during (right) hypotonic exposure. Bar, 5 mm. (b) Administration of glutamate (1 mM) inhibits the hypoosmotic swelling of glial somata in tissues from both control and diabetic animals. The effect of glutamate is prevented in the presence of the inhibitor of nucleoside transporters, N-nitrobenzylthioinosine (NBTI; 10 mM). Glial swelling in retinal slices from control animals was induced by a barium (1 mM)-containing hypoosmolar solution. Glial swelling in retinal slices from control animals was induced by a barium (1 mM)-containing hypoosmolar solution. (c) In retinal tissues from control animals, the swellinginhibitory effect of glutamate (1  mM) is inhibited by the NTPDase inhibitor, 6-N,N-diethyl-db,g-dibromomethylene ATP (ARL-67156; 50  mM), but not by the ectonucleotidase inhibitor, adenosine-5¢-O-(a,b-methylene)-diphosphate (AOPCP; 250 mM). In contrast, both inhibitors prevent the effect of glutamate in retinal tissues from diabetic animals. Data are mean (± SEM) soma areas obtained in 5–19 cells per bar. Significant differences vs. control (100%): **P < 0.01; ***P < 0.001. Significant blocking effects: ••P < 0.01; •••P < 0.001. Significant inhibition of the glutamate effect: ••P < 0.01; •••○P < 0.001. With permission from Pannicke et al. [24] and Wurm et al. [39]

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2.2 Factors Involved in Evoking Glial Swelling 2.2.1 Decrease in Potassium Conductance Ion efflux through plasma membrane channels, resulting in equalization of the transmembrane osmotic gradient, is one mechanism to prevent cellular swelling under hypotonic conditions. The major ion channel that mediates passive ion efflux from retinal glial cells in response to a decrease in the extracellular osmolarity is the Kir4.1 potassium channel [14, 42]. Kir channels have a high open-state probability (>0.8) over a wide voltage range around the resting membrane potential [47, 48]. This high open-state probability is a precondition for the mediation of prompt potassium currents across the plasma membrane when the level of extracellular potassium is altered. A passive Kir4.1 channel-mediated release of ­potassium is suggested to prevent cellular swelling when the osmolarity of the extracellular fluid decreases [42, 49]. In retinas of diabetic rats, there is a dislocation of the Kir4.1 channel protein, as indicated by the absence of the prominent expression at both limiting membranes of the retina and around the blood vessels (Fig.  1a). The dislocation of the Kir4.1 channel protein is accompanied by a decrease in the Kir channel-mediated currents across glial membranes (Fig.  1b), suggesting a functional inactivation of the ­channels [24]. A similar dislocation and functional inactivation of Kir4.1 was observed in various other rat models of retinopathies including retinal ischemia– reperfusion and ocular inflammation [42, 44]. The functional inactivation of Kir4.1 channels results in an almost fully absence of outward potassium currents from Müller cells when the extracellular concentration of potassium decreases [42]. Inactivation of Kir4.1 should also disturb the potassium efflux under hypotonic conditions and thus will contribute to the induction of osmotic swelling. Indeed, there is a correlation between the decrease in Kir channel-mediated potassium currents and the increase in the amplitude of osmotic swelling in retinal glial cells [45]. (In addition, there is a correlation between the extent of osmotic cell swelling and other signs of gliosis, such as cellular hypertrophy [44].) The importance of Kir4.1mediated potassium efflux for the prevention of glial swelling is also indicated by the observation that retinal glial cells from healthy control animals display a swelling under hypotonic conditions in the presence (but not absence) of barium ions [42]; barium ions are known to block Kir channels in retinal glial cells [50–52]. These ex-vivo data may have importance for the understanding of retinal edema development in situ. Because retinal glial cells are still capable of taking up excess potassium from the extracellular space (through strongly rectifying Kir2.1 channels that are not altered in their expression after ischemia [53]), the impairment in the release of potassium from retinal glial cells will result in an accumulation of potassium ions within the cells and, thus, in an increase in the intracellular osmotic pressure. The increased osmotic pressure will draw water into the cells, resulting in cellular swelling (Fig. 2b). Swelling of perivascular glial endfeet may occur under pathological conditions in situ when the osmotic pressure of the neural tissue

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increases due to neuronal hyperexcitation while the osmotic pressure of the blood remains constant, causing an osmotic gradient across the glio-vascular interface. The water inflow is supported by aquaporin-4 water channels that are not altered (with the exception of glial membranes that surround the superficial retinal vessels) (Fig. 1a) or even increased in their expression [25, 37, 54]. In addition to inducing glial swelling, the absence of outward currents through Kir4.1 channels will interrupt the spatial buffering potassium currents through retinal glial cells and the export of excess potassium into the blood and vitreous. The disruption of the transglial potassium currents will also disturb the transport of water through the cells, resulting in an impairment of the fluid clearance from the edematous retinal tissue [14]. 2.2.2 Oxidative Stress and Inflammatory Lipids The action of arachidonic acid and of its metabolites, especially prostaglandin E2, was causally implicated in the development of retinal edema [5, 7]. In addition to the downregulation/inactivation of Kir4.1 channels that impairs a rapid ion efflux in response to hypoosmotic challenge, oxidative stress and the formation of inflammatory lipids are causative factors of glial swelling in the retina of diabetic rats (Fig. 2b). Acute administration of arachidonic acid, prostaglandin E2, or hydrogen peroxide to retinal slices from healthy control animals evokes glial swelling under hypotonic conditions, which is not observed under isotonic conditions or in the absence of the agents [24, 55]. The swelling-evoking effect of hydrogen peroxide was diminished in the presence of the anti-inflammatory glucocorticoide, triamcinolone acetonide (9a-fluoro-16a-hydroxyprednisolone), suggesting that oxidative stress induced activation of inflammatory mechanisms [24]. The osmotic swelling of retinal glial cells from diabetic animals is fully prevented when the activities of the phospholipase A2 (that forms arachidonic acid) or the cyclooxygenase (that forms prostaglandins) are pharmacologically blocked [24, 39]. This suggests that the formation of arachidonic acid and prostaglandins is crucially involved in evoking cellular swelling. The swelling is also inhibited in the presence of a cell-permeable reducing agent (dithiothreitol), suggesting the involvement of oxidative stress, and of a sodium-free extracellular solution, suggesting that an influx of sodium from the extracellular space into the cells is a step in evoking glial swelling [24, 39]. Apparently, osmotic challenge causes oxidative stress in the cells. It is known that the activity of the phospholipase A2 is increased in response to osmotic challenge and oxidative stress, resulting in peroxidation of membrane phospholipids and the release of arachidonic acid [56–59]. Free radicals and hydroperoxides stimulate also the activity of the cyclooxygenase [60]. Ischemic retinopathies are characterized by an increased arachidonic acid metabolism [61]. Retinal glial cells strongly increase the expression of cyclooxygenase-2 under various pathological conditions [45, 62]. It has been shown that the retinal expression of cyclooxygenase-2 increases early in diabetes [63]. Arachidonic acid and prostaglandins are potent inhibitors of the Na, K-ATPase activity; inhibition of the sodium pump leads to intracellular sodium overload and

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swelling of cultured cells [64, 65]. Apparently, the sodium influx into retinal glial cells cannot be compensated by a rapid channel-mediated efflux of potassium ions or of other osmolytes, a situation leading to water influx and cellular swelling. Arachidonic acid also blocks membrane channels such as volume-regulated anion and (in retinal glial cells) outwardly rectifying potassium channels [66, 67], which otherwise mediate a compensatory efflux of osmolytes such as amino acids, chloride and potassium ions. It is likely that under in situ conditions, an influx of sodium ions driven by sodium-dependent glutamate transporters [68, 69] and endocytosis of serum-derived proteins extravasated across the walls of leaky vessels contribute to the enhancement of the intracellular osmotic pressure and cellular swelling (Fig. 2b). Serum-derived molecules such as thrombin may also close Kir channels of retinal glial cells [70]. However, further investigations are necessary to determine the exact intracellular pathways involved in osmotic swelling of retinal glial cells.

3 Purinergic Inhibition of Glial Swelling 3.1 Purinergic Receptor Cascade Glial cells in the rodent retina possess an autocrine purinergic signaling mechanism that mediates the homeostasis of cellular volume under conditions of hypoosmotic stress [71, 72]. This autocrine signaling mechanism is active in cells from both control and diabetic animals, and involves the release of adenosine 5¢-triphosphate (ATP) from the cells; ATP is extracellularly converted to adenosine 5¢-diphosphate (ADP) that activates P2Y1 [73]. Activation of P2Y1 triggers a nucleoside ­transporter-dependent release of adenosine (Fig. 4) [71, 72]. The most downstream event of the cascade is activation of A1 receptors by adenosine. Activation of A1 receptors leads to opening of potassium and chloride channels in glial membranes; the ion efflux results in equalization of the transmembrane osmotic gradient and thus prevents glial swelling. The opening of the channels is mediated by intracellular signaling cascades including activation of the adenylyl cyclase and protein kinase A, and of the phosphatidylinositol-3 kinase [72]. The kind of the second messenger-activated potassium channels that open after A1 receptor activation is unclear. These channels are distinct from Kir channels and likely represent two pore-domain potassium channels that are expressed in retinal glial cells [74]. The purinergic cascade of swelling inhibition in rat retinal glial cells is fully independent from intracellular calcium signaling [71, 72]. Retinal glial cells release ATP upon different stimuli including electrical and mechanical stimulation, and upon stimulation with receptor agonists such as ATP, dopamine, thrombin, and glutamate [71, 72, 75, 76]. A major activator of the purinergic swelling-inhibitory signaling cascade is glutamate [71, 72]. Glutamate activates group I/II metabotropic glutamate receptors on glial cells that results in a calcium-independent release of ATP from the cells (Fig. 4). The observation of a

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Fig. 4  Scheme of the purinergic signaling cascade that inhibits the osmotic swelling of glial cells in the retina of control and diabetic rats. Glutamate activates metabotropic glutamate receptors (mGluRs) resulting in a release of ATP from retinal glial cells. ATP is extracellularly converted to ADP by the action of NTPDase2. ADP activates P2Y1 receptors that results in a transportermediated release of adenosine. Activation of A1 receptors by adenosine causes a cAMP-, protein kinase A (PKA)-, and phosphatidylinositol-3 kinase (PI3K)-dependent opening of potassium and chloride channels; the ion efflux equalizes the osmotic gradient across the plasma membrane and thus prevents water influx and cellular swelling under hypoosmotic conditions. In swollen cells, the ion efflux is associated with a water flow out of the cells, resulting in a decrease in the cell volume. All steps in the cascade are calcium-independent. In the retina of diabetic rats, upregulation of NTPDase1 enables extracellular formation of AMP as a substrate of the ecto-5¢nucleotidase, resulting in extracellular formation of adenosine

calcium-independent release of purinergic receptor agonists is in agreement with the fact that glutamate does not evoke calcium responses in retinal glial cells of the rat [77–79]. Glutamate is released from retinal glial cells in a calcium-dependent, exocytotic fashion [72]. Various receptor agonists were found to prevent osmotic swelling of retinal glial cells via stimulation of a calcium-dependent release of glutamate from the cells, for example, neuropeptide Y [71], heparin-binding epidermal growth factor-like growth factor [80], natriuretic peptides [81], and VEGF [72]. In addition to glia-derived glutamate, neuron-derived glutamate may evoke a release of purinergic receptor agonists from glial cells; this neuron-to-glia signaling might be involved in regulation of the extracellular space volume and the neurovascular coupling under conditions of intense neuronal activity (see Sect. 4.1).

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3.2 Involvement of Ecto-Nucleotidases in Swelling Inhibition In retinal tissues from control rats, the purinergic cascade of swelling inhibition involves the extracellular degradation of ATP to ADP (which both are agonists of P2Y1 [73]) and a nucleoside transporter-dependent increase in extracellular adenosine (Fig. 4) [39, 71, 72]. Pharmacological inhibition of nucleoside transporters prevents the swelling-inhibitory effect of glutamate (Fig. 3b), suggesting that adenosine is liberated from glial cells via transporters. In addition to a transporter-mediated release, extracellular adenosine can be formed by phosphohydrolysis of adenosine 5¢-monophosphate (AMP) through the action of the ecto-5¢-nucleotidase (CD73). In addition to neuronal cell bodies in the ganglion cell and inner nuclear layers, retinal glial cells display immunoreactivity and histochemical activity of ecto-5¢nucleotidase [82–84]. However, extracellular adenosine formation is not involved in the purinergic regulation of glial cell volume in retinal slices from control rats, as an inhibitor of the ecto-5¢-nucleotidase does not prevent the swelling-inhibitory effect of glutamate (Fig. 3c). On the other hand, an inhibitor of ecto-ATPases, ARL67156, prevented the effect of glutamate (Fig. 3c), suggesting the involvement of extracellular degradation of ATP. There are various extracellular ATP-degrading ecto-enzymes, for example, nucleoside triphosphate diphosphohydrolases (NTPDases). NTPDase1 (CD39, ecto-apyrase) and NTPDase2 (CD39L1, ecto-ATPase), as well as NTPDases 3 and 8, are capable of hydrolyzing nucleoside 5¢-tri- and diphosphates such as ATP and ADP, albeit with considerably different substrate preference. Whereas NTPDase1 hydrolyzes ATP and ADP about equally well (resulting in the immediate formation of the ecto-5¢-nucleotidase substrate AMP), NTPDase2 preferentially hydrolyzes ATP to ADP, with a delayed and very slow hydrolysis of ADP to AMP (the ATP to ADP hydrolysis ratio is approximately 10:1 [85]). NTPDases3 and -8 reveal intermediate substrate preferences [86, 87]. In the neural retina of the rat (Fig.  5) and man, predominantly two ATPdegrading enzymes were found in different compartments: Whereas NTPDase1 immunoreactivity and activity is exclusively localized to the vasculature [84, 88, 89], NTPDase2 is localized to the retinal parenchyma, e.g., to retinal glial cells [84]. Ecto-ATPase activity was also found in synaptic membranes [90], suggesting that both retinal neurons and glial cells express NTPDase2. The different distribution of NTPDase1 and -2 proteins in the rat retina corresponds well with enzyme histochemical data obtained in slices of the mouse retina that showed that ADP is degraded solely in the vasculature but not in the retinal parenchyma, whereas ATP is hydrolyzed in the whole retinal tissue [84]. The data suggest that retinal glial cells express enzymes for the rapid extracellular degradation of ATP and AMP (NTPDase2 and ecto-5¢-nucleotidase), but not of ADP (due to the absence of NTPDase1 and the very low ADPase activity of NTPDase2). This explains the observation that an inhibitor of the ecto-5¢-nucleotidase did not prevent the swellinginhibitory effect of glutamate (Fig. 3c), because the substrate of adenosine formation (AMP) is generated only at very low amounts at the surface of retinal glial cells.

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Fig. 5  Distribution of NTPDase1 and NTPDase2 immunolabeling in the neural retina of the rat. Retinal slices (left) and freshly isolated retinal glial cells (right) were derived from diabetic and age-matched control animals. Isolated cells were counterstained against the glial cell marker vimentin. In retinal slices from control animals, immunoreactivity for NTPDase1 is predominantly localized to large blood vessels within the nerve fiber/ganglion cell layers (filled arrowheads). In addition, faint staining is observed in the microvessels of the inner nuclear layer (INL). In retinal slices from diabetic animals, immunoreactivity for NTPDase1 is localized also to retinal structures outside of the vasculature, e.g., in glial cell fibers traversing the inner plexiform layer (IPL; filled arrow). NTPDase2 is localized throughout the whole retinal tissue, for example, in glial cell fibers that traverse the IPL. In addition to glial cell fibers, NTPDase2 is expressed in both plexiform (synaptic) layers. Unfilled arrows, glial cell somata. Unfilled arrowheads, glial cell endfeet. GCL ganglion cell layer; ONL outer nuclear layer; PRS photoreceptor segments. Bars, 20 mm. With permission from Wurm et al. [39]

3.3 Diabetes Alters Glial Expression of Ecto-Nucleotidases In the course of diabetes, there is an alteration in the source of extracellular adenosine, which is involved in regulation of glial cell volume [39]. In retinal glial cells from control rats, the swelling-inhibitory effect of glutamate is prevented by an inhibitor of nucleoside transporters (Fig.  3b) but not by an inhibitor of the ecto-5¢nucleotidase (Fig.  3c). In contrast, in glial cells from diabetic animals, both the inhibitor of nucleoside transporters (Fig.  3b) and the inhibitor of the ecto-5¢nucleotidase (Fig. 3c) prevented the effect of glutamate. The data suggest that, in

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control retinas, glutamate evokes a release of adenosine from glial cells via facilitated transport, whereas in retinas from diabetic animals, glutamate evokes both a transportermediated liberation of adenosine and an extracellular generation of adenosine by the action of the ecto-5¢-nucleotidase. These data imply that the substrate of extracellular adenosine formation, AMP, should be present in sufficient amounts in the extracellular space. Indeed, we found an increase in the mRNA expression of NTPDase1 (but not NTPDase2) in the retina of diabetic rats compared to control, as well as an immunolabeling of retinal glial cells against NTPDase1 [39]. Glial labeling against NTPDase1 was observed in both retinal slices and isolated retinal glial cells from diabetic but not control animals (Fig. 5). As in the case of NTPDase2 (Fig. 5), the distribution of the ecto-5¢-nucleotidase protein was not different in retinal slices and isolated glial cells from control and diabetic animals (not shown) [39].

4 Functional and Clinical Implications 4.1 Functional Implications The data may suggest that the glial availability of extracellular adenosine is increased in the retina of diabetic animals as compared to the retina of control animals. In control retinas, adenosine involved in glial volume regulation is liberated from the cells by facilitated transport, whereas in retinas from diabetic animals, adenosine is released from glial cells via transporters and is extracellularly formed from ATP by the actions of NTPDase1 and ecto-5¢-nucleotidase (Fig. 4). The functional significance of the alteration in the extracellular nucleotide metabolism remains to be determined. The upregulation of NTPDase1 may reflect a requirement of a higher extracellular level of adenosine in the diabetic retina as compared to control. By the prevention of cytotoxic edema, adenosine acts as glioprotectant. Adenosine has also anti-inflammatory and neuroprotective effects in the retina [91, 92]. Generally, an increase in the extracellular level of adenosine is an important component of the retinal response to ischemic–hypoxic stress, including hypoxic states of photoreceptors in the dark [93, 94]. Activation of A1/A2 receptors by exogenous adenosine, or ischemic preconditioning mediated by endogenous adenosine and A1 receptor activation, protects the retina from ischemic injury [91, 92, 95]. Adenosine induces retinal hyperemia after ischemia [96] and protects retinal neurons from glutamate toxicity by suppression of excitatory neurotransmission [97]. Adenosine also protects retinal ganglion cells from P2X7-induced apoptosis [98]. The inhibitory effect on glial cell swelling may contribute to the neuroprotective effect of A1 receptor activation, via prevention of detrimental reductions of the extracellular space volume which otherwise would result in neuronal hyperexcitability [99, 100]. Inhibition of glial cell swelling, or even shrinkage of glial cells, is especially important in the retina where overstimulation of neuronal ionotropic glutamate receptors results in a swelling of neuronal cell bodies and synapses, and a decrease in the extracellular space volume [78].

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In addition to an enhanced extracellular level of adenosine, expression of NTPDase1 may result in a decreased extracellular amount of the purinergic receptor agonists ATP and ADP in the retina of diabetic animals [84]. Under pathological conditions, mechanical and osmotic pertubations in the tissue, as well as cell degeneration, may result in an increased liberation of ATP from retinal cells. It has been shown in animal models of different retinopathies, including ischemia–reperfusion, that retinal glial cells display a strong enhancement of ATP-evoked calcium responses under pathological conditions [101–103]. This has been ascribed (at least in part) to an increase in the expression of P2Y proteins [103] and a resensitization of P2Y by the action of growth factors [104]. The predominant P2Y receptor subtype that evokes calcium responses in rat retinal glial cells is P2Y1 [105]; this receptor subtype is also involved in the calcium-independent glial cell volume regulation (Fig. 4). It could be, but remains to be proven, that facilitated degradation of extracellular ATP/ADP may protect glial cells from overstimulation of P2Y1, to avoid cytotoxic calcium overload. The action of NTPDase1 may avoid overstimulation of glial P2Y1 without an impairment of the regulation of the glial cell volume. In addition, the action of NTPDase1 may reduce the availability of the P2X7 ligand ATP and thus may be protective for retinal ganglion cells. We found that hypotonic conditions are crucial for glial swelling. This has importance for both normal and pathological conditions. Under normal conditions, retinal glial cells are surrounded by a hypoosmotic environment during periods of intense neuronal activity. Light-evoked changes in the ionic composition of the extracellular space fluid, with a decrease in sodium that exceeds the increase in potassium by a factor of two, cause a decrease in osmolarity of the extracellular space fluid [106]. An activity-dependent decrease in the osmolarity of the extracellular space fluid was also found in the brain [107]. In addition, the uptake of ­neuron-derived osmolytes such as potassium and sodium-glutamate [18] may increase the osmotic pressure of the glial cell interior relative to the extracellular space fluid. Thus, neuronal activity in the retina generates an osmotic gradient that favors water flux from extracellular to intracellular spaces. These osmotic conditions are exacerbated under pathological conditions that are characterized by glutamate-evoked hyperexcitation. Under pathological conditions, hypoosmotic conditions might be also present at the glio-vascular interface. An uptake of excess neuron-derived osmolytes by glial cells enhances the osmotic pressure of the glial cell interior in comparison to the blood. Glial accumulation of neuron-derived potassium is supported by the functional inactivation of Kir4.1, which is the sole channel that mediates passive potassium outward currents in dependence on the momentary extracellular potassium concentration (see Sect.  1.2). In addition, a decrease in blood osmolarity, e.g., in cases of renal and hepatic failures resulting in hyponatremia and hypoalbuminemia, represents a major pathogenic factor in the formation of retinal edema [2]. Both an increase in the osmolarity of the glial cell interior and a decrease in blood osmolarity will result in a water flux from the blood into the perivascular processes of glial cells, resulting in cytotoxic edema. Neuron-to-glia signaling, mediated by the neurotransmitters glutamate and ATP, may activate the volume-regulatory purinergic signaling cascade in glial cells in

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dependence on the neuronal activity. It has been shown that light stimulation of the rat retina evokes a purinergic neuron-to-glia signaling that is implicated in neurovascular coupling, i.e., the regulation of the local blood-flow rate in dependence on the neuronal activity [108]. The purinergic neuron-to-glia signaling is suggested to occur in the inner plexiform (synaptic) layer; the blood-flow regulatory signals transmit to the inner surface of the retina (where the arterioles are localized) via propagation of intracellular calcium waves in glial cells [79, 108]. The propagation of intracellular calcium waves is mediated by autocrine purinergic signaling via release of ATP from glial cells and subsequent activation of P2Y receptors [75]. It is conceivable that these calcium waves also transmit cell-volume regulatory signals simultaneously with the spread of neuronal activity to prevent a swelling of glial cells when activated retinal ganglion cells swell and the extracellular osmolarity decreases.

4.2 Clinical Implications The osmotic swelling of retinal glial cells from diabetic animals reflects a dysregulation of the transmembrane ion and water transport that may underlie the impaired fluid clearance from the edematous retinal tissue in patients with diabetes [14]. The anti-inflammatory glucocorticoid, triamcinolone acetonide, is used clinically for the rapid resolution of diabetic retinal edema [109]. Steroids are also effective in the resolution of retinal edema in cases not associated with angiographic vascular leakage, suggesting that they (in addition to a preventive effect on vasogenic edema) also stimulate the fluid clearance from the retinal tissue. It has been shown in patients with diabetic retinopathy that triamcinolone induces a very rapid reduction in retinal edema that is evident as early as 1 h after intravitreal administration [110]. Since this rapid effect cannot be explained by an alteration of gene expression (which needs several hours), the authors suggested that triamcinolone acts also through a receptor-dependent mechanism. It has been shown that triamcinolone inhibits the osmotic swelling of retinal glial cells from diabetic animals [39], the swelling of retinal glial cells in animal models of retinal ischemia, hypoxia, and inflammation [45, 46, 55], as well as the swelling of glial cells from control animals evoked by oxidative stress and the inflammatory lipids arachidonic acid and prostaglandin E2, respectively [45, 55]. Triamcinolone activates the last steps of the swelling-inhibitory purinergic signaling cascade, i.e., it stimulates the transportermediated release of adenosine, A1 receptor activation, and protein kinase A-mediated opening of potassium and chloride channels [55]. It has been suggested that the triamcinolone-evoked opening of ion channels may restore the ion and water transport through retinal glial cells and, thus, may improve the glial fluid clearance from the edematous retinal tissue and may also protect the tissue from potassium-evoked neuronal hyperexcitation [111]. In situ, the direction of the potassium and, therefore, water flow through retinal glial cells, will be determined by the potassium gradient within the tissue (high potassium in the retinal parenchyma and within

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glial cells, normal potassium in the blood and vitreal fluid). A similar mechanism has been described in the case of the retinal pigment epithelium; here, activation of different receptor subtypes, for example P2Y2, stimulates the fluid absorption from the subretinal space in cases of retinal detachment [112, 113]. Receptor activation results in an increased rate of the ion transport across the pigment epithelium and thus of the water absorption from the subretinal space. Since triamcinolone treatment can be associated with systemic and local deleterious side effects, novel drugs targeting A1 receptors or openers of two pore-domain channels may represent promising ways for the rapid resolution of diabetic retinal edema. Acknowledgments  This work was supported by grants from the Deutsche Forschungsgemein­ schaft (FOR/748, GRK 1097/1).

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Part V

Toxicology/Environmental

Helicobacter pylori-Induced Oxidative Stress and Inflammation Hyeyoung Kim and Young-Joon Surh

Abstract  Reactive oxygen species (ROS) are considered to be the important regulator in the pathogenesis of Helicobacter pylori-induced gastric ulceration and carcinogenesis. The sources of ROS are thought to be activated NADPH oxidase and CagA in H. pylori-infected gastric epithelial cells. Redox-sensitive transcription factors, nuclear factor-kB (NF-kB) and activator protein-1 (AP-1), are the master regulators of inducible genes such as interleukin-8, monocyte chemoattractant protein-1, inducible nitric oxide synthase and cyclooxygenase-2, which are involved in gastric inflammation. Therefore, scavenging of ROS or specific inhibition of NF-kB and AP-1 may be an effective strategy for prevention or treatment of gastric inflammation associated with H. pylori infection. Proteinase-activated receptor 2 (PAR2), which is activated by trypsin, is known to regulate expression of adhesion molecule integrins. H. pylori induces the expressions of PAR2 and integrin a5 and b1 as well as cell adhesion to fibronectin. H. pylori increases mRNA expression of trypsinogen 1 and 2 as well as the level and the activity of trypsin in gastric epithelial cells. H. pylori-induced production of ROS may mediate PAR2-mediated expression of integrins in gastric epithelial cells. Therefore, PAR2 may have an important role in gastric cell adhesion and possibly carcinogenesis associated with H. pylori infection. The genetic differences of H. pylori isolates, particularly in H. pylori virulence-associated genes, cag A, vac A and ice A genes, may influence the differential expression of inflammatory genes and clinical outcome of the infection. In this chapter we will discuss oxidative stress and inflammation in gastric mucosa caused by H. pylori infection in the context of roles of ROS, transcription factors, signaling mediators, proinflammatory molecules and PAR2. Animal models used for studying H. pylori-induced gastritis and gastric cancer are also described. Keywords  Activator protein-1 • Apoptosis • Chemokine • Cyclooxygenase-2 • Extracellular matrix • Gastric epithelial cells • Helicobacter pylori • Inducible H. Kim (*) Department of Food and Nutrition, Brain Korea 21 Project, College of Human Ecology, Yonsei University, Seoul 120-749, South Korea e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_16, © Springer Science+Business Media, LLC 2011

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nitric oxide synthase • Integrin • Mitogen-activated protein kinase • Nuclear factor-kB • Proteinase-activated receptor 2 • Toll-like receptor

1 Oxygen Radicals, NADPH Oxidase and Cag A Helicobacter pylori infection is an important risk factor for chronic gastritis, peptic ulcer and gastric carcinoma [1]. There are multiple lines of compelling evidence supporting the fact that activated neutrophils play an important role in the pathogenesis of H. pylori-induced gastric diseases [2, 3]. One of the potential causative factors involved in H. pylori-induced gastric injury is reactive oxygen radicals species (ROS), which are released from the activated neutrophils infiltrated into the subepithelial gastric lamina propria [2, 3]. Therefore, neutrophil infiltration of the gastric epithelium is the initial pathological abnormality described for H. pylori gastritis and remains a hallmark of active infection. Superoxide anion (O2−) and related ROS, such as hydrogen peroxide (H2O2), produced by the activated NADPH oxidase during the respiratory burst of phagocytic cells, account not only for microbicidal and tumoricidal activities [4, 5] but also for tissue injury accompanying inflammatory and immunologic reactions [6]. In addition, H. pylori produce a soluble factor known as a neutrophil-activating protein (NAP) that activates neutrophils in H. pylori-infected gastric mucosa [7]. Activation of NADPH oxidase requires the assembly of membrane-integrated components (a heterodimer formed by Nox1 and p22phox) with cytosolic components p47phox, p67phox, and the small GTPase Rac [8]. Recently, six homologs of gp91phox have been identified and collectively named the “NADPH oxidase/dual oxidase (Duox) family” [9, 10]. During the activation of the oxidase, Rac translocates to the membrane independently of the other cytosolic factors. At the membrane, GTPbound Rac directly interacts with p67phox and p47phox [11–13], thereby participating in electron transfer from NADPH to molecular oxygen [14–16]. Even though neutrophils are the major source of ROS in H. pylori-induced gastric mucosa, H. pylori can stimulate the ROS production in gastric epithelial cells in the absence of inflammatory cells [17, 18]. Thus, H. pylori may turn directly on transcription levels of inflammatory genes in gastric epithelial cells prior to the recruitment of inflammatory cells (Fig. 1). With regard to activation of NADPH oxidase, the gastric epithelial cells are primed with H. pylori lipopolysaccharide (LPS). Stimulation of toll-like receptor 4 (TLR4) by H. pylori LPS activates a phosphoinositide 3-kinase (PI3K) that converts GDP-bound Rac to the GTP-bound Rac form. The activated GTP-bound Rac directly interacts with p67phox and p47phox to form an assembly of membraneintegrated components (a heterodimer formed by Nox1 and p22phox) with cytosolic components p47phox, p67phox, and thereby upregulates the production of superoxide [19]. It activates redox-sensitive transcription factor NF-kB which, in turn, induces prolonged expression of proinflammatory cytokines and cycooyxgenase-2 (COX-2). In this context, NADPH oxidase may be one of the key molecules representing the

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LPS MD2 Cag A

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Fig. 1  The production of ROS and inflammatory gene expression in H. pylori-infected gastric epithelial cells. H. pylori CagA infiltrated into the gastric epithelial cells induces the production of ROS in mitochondria possibly by desregulating the function of mitochondria electron transport chain. On the other hand, the gastric epithelial cells are primed with H. pylori LPS. Stimulation of Toll-like receptor 4 (TLR4) by H. pylori LPS upregulates the production of ROS by activating NADPH oxidase. Activation of NADPH oxidase requires the assembly of membrane-integrated components (a heterodimer formed by Nox1 and p22phox) with cytosolic components p47phox, p67phox, and the small GTPase Rac. During the activation of the oxidase, Rac translocates to the membrane. Binding of H. pylori LPS to TRL4 activates PI3K, which converts GDP-bound Rac to GTP-bound Rac. The activated GTP-bound Rac directly interacts with p67phox and p47phox to form the assembly of NADPH oxidase components, thereby participating in electron transfer from NADPH to molecular O2. MD2 may interact with H. pylori LPS-TLR4. ROS activate two principal redox-sensitive transcription factors NF-kB and AP-1 and thus induce prolonged expression of inflammatory chemokines and inflammaroty enzymes in H. pylori-infected gastric epithelial cells. In addition, H. pylori-induced COX-2 expression enhances the cancer cell invasion and angiogenesis via TLR2 and TLR9. NF-kB activation is induced by phosphorylation (P), ubiquitination (U), and degradation of IkBa, which is an inhibitory protein of the NF-kB complex. The activated NF-kB subunits (p50, p65) and the AP-1 components (c-Fos, c-Jun) are translocated to the nucleus and bind to DNA recognition sites in target genes. However, depending on the genetic differences of H. pylori isolates, particularly in H. pylori virulence-associated genes, cag A, vac A and ice A genes, the expression profiles of inflammatory genes and clinical outcome may be different. This figure is adapted and modified from illustrations reported in [19, 20]

initial trigger for the host innate and immune response against H. pylori. NADPH oxidase is specifically expressed in gastric adenocarcinomas, while expression of this enzyme in normal human stomach is not substantial [19]. Although it is not evident whether all the subunits of NADPH oxidase complex are expressed in human gastric epithelial cells, NADPH oxidase-derived ROS may induce the expression of inflammatory genes by activating redox-sensitive transcription factors and stimulating ROS-mediated signaling cascade.

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CagA positive H. pylori species induce an increased oxidative burst in polymorphonuclaer neutrophils (PMNs) with higher ROS production [21]. CagA itself induces the production of ROS in mitochondria of gastric mucosa possibly by desregulating the function of mitochondrial electron transport chain and produce primarily superoxide [20]. In addition, mitochondrial DNA is more vulnerable to the oxidative damage than nuclear DNA because of its close proximity to the electron transport chain and its lack of protective histones or DNA-binding proteins [22]. Consequently, the activated NADHP oxidase and/or Cag A may be the main sources of ROS in H. pylori-infected gastric epithelial cells (Fig. 2).

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Fig.  2  H. pylori-induced inflammatory signaling in gastric epithelial cells. ROS produced by H. pylori infection activates Ras, the upstream activator of MAPKs. This leads to the activation of both NF-kB and AP-1. Ras activates MAPK kinase kinase (MAPKKK) which, in turn, induces the activation of MAPKK (MAPK kinase), which in turn activates MAPK. All three MAPKs, ERK1/2, JNK and p38, may phosphorylate the transcription factor Elk-1, a member of the tertiary complex family. Elk-1 binds the serum response element motif (SRE) in the c-fos promoter, thereby inducing c-fos transcription (red line). c-Jun is regulated both transcriptionally and posttranscriptionally. c-Jun transcription is up-regulated by activated ERK. Subsequently, c-Jun may increase its own transcription by binding to the TRE motif in its promoter. c-Jun is activated via phosphorylation of serine residues 63 and 73 by JNK. Novel c-Fos synthesis leads to the formation of a c-Jun/c-Fos heterodimer, resulting in increased AP-1 activity. H. pylori activates AP-1 through ERK, JNK, and p38 signaling in gastric epithelial cells. The activation of AP-1 and NF-kB by H. pylori was abolished by treatment with the MAPK inhibitors and transfection with ras-dominant negative mutant gene (ras N-17), demonstrating that H. pylori-induced activation of both NF-kB (dotted line) and AP-1 involves Ras and MAPKs in AGS cells. For NF-kB activation, ROS activate IkB kinase(s) which phosphorylate(s) an inhibitory IkBa protein and IkBa is ubiquitinated and degraded. The NF-kB subunits (p50, p65) migrate into the nucleus and bind DNA recognition sites in the regulatory regions of the target genes in H. pylori-infected gastric epithelial cells. Adapted and modified from the illustration in [23]

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2 Nuclear Factor-k B Nuclear Factor-kB (NF-kB) is a member of the Rel family including p50 (NF-kB1), p52 (NF-kB2), Rel A (p65), c-Rel, Rel B, and Drosophila morphogen dorsal gene product [24]. In resting cells, NF-kB is localized in the cytoplasm as a hetero- or homodimer, which are noncovalently associated with cytoplasmic inhibitory proteins, including IkBa. Upon stimulation by a variety of pathogenic inducers such as viruses, mitogens, bacteria, and agents generating ROS and inflammatory cytokines, the NF-kB complex migrates into the nucleus and binds to DNA recognition sites in the regulatory regions of the target genes [25]. H. pylori increased lipid peroxidation, indicative of oxidative damage, and induced the activation of two species of NF-kB dimers (a p50/p65 heterodimer and a p50 homodimer) in gastric epithelial cells [17, 18]. Pyrrolidine dithiocarbamate (PDTC), a proven free radical scavenger and a NF-kB inhibitor, potentially inhibited NF-kB interaction with its regulatory binding site, thereby preventing NF-kB-mediated transcriptional activation in H. pylori-infected gastric epithelial cells [26]. Direct contact of H. pylori with gastric epithelial cells activates NF-kB to induce inflammatory cytokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) in gastric epithelial cells [27, 28]. As described above, NF-kB activation is tightly regulated by its endogenous inhibitor, IkB, which sequesters NF-kB in the cytoplasm. The most extensively investigated IkB protein is IkBa (36 kDa) encoded by the human MAD-3 gene or its homologues in different species [29]. The mechanisms responsible for the degradation of IkBa proteins involve changes in the phosphorylation state of IkBa [25]. Two serine residues in the N-terminal domain of IkBa, serine 32 and 36, were shown to be critical for determining IkBa stability [30]. Substitution of these two serine residues by alanine residues rendered IkBa undegradable by cellular NF-kB activators [30]. Among many proteins exhibiting IkB function, IkBa is the only inhibitor that in response to cell stimulation dissociates from NF-kB complex, with kinetics matching NF-kB translocation to the nucleus [31]. Thus, activation of NF-kB requires its dissociation from IkBa. A mutant IkBa has been shown to act as an NF-kB super-repressor [32]. The cells transfected with a mutant IkBa inhibited H. pylori-induced activation of NF-kB and induction of inflammatory cytokines IL-8 and MCP-1 in gastric epithelial cells [33].

3 Activator Protein-1 Activator Protein-1 (AP-1) is composed of homodimers or heterodimers of members of the Fos and Jun families, which binds to the TRE (TPA responsive element) motif to mediate gene transcription. AP-1 regulates the expression of a battery of genes in response to signals generated by a wide array of extracellular stimuli including growth factors, tumor promoters, and cytokines [34]. The activity of AP-1 is regulated by the transcription factors involved in the activation of c-fos and c-jun promoter elements as well as by the post-translational modification of AP-1

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members, which ensure the immediate induction of AP-1-dependent transcription. The transcriptional and post-translational regulation of AP-1 activity is regulated through different signaling cascades. This may explain the versatility of AP-1 to respond to such a wide spectrum of external stimuli [35]. AP-1 has been shown to behave as a redox-sensitive transcription factor that may be induced under both pro-oxidative and antioxidative conditions depending on the cell types [36]. In relation to H. pylori infection, transcription of IL-8 genes requires the activation of NF-kB and, to a lesser extent, AP-1 in human gastric epithelial MKN 45 [37], Kato III [38] and AGS cells [39]. H. pylori strongly induced AP-1 DNA binding and selectively activated the mitogen-activated protein kinase (MAPK) cascade in gastric epithelial cells [23]. Therefore, chemokine gene transcription may require the activation of the combination of transcription factor NF-kB and AP-1 or that of NF-kB and still another important transcription factor C/EBP (CCAAT/ enhancer binding protein), depending on the types of cells and/or stimuli since the binding sites for these transcription factors exist in the promoter regions of IL-8 and MCP-1 genes. AP-1 component activated by H. pylori was characterized as a heterodimer of c-Fos/c-Jun, which might be induced by ROS [39]. The antioxidant N-acetylcysteine inhibited the activation of this transcription factor and IL-8 expression in AGS cells. As N-acetylcysteine can increase the intracellular stores of glutathione in the cells, and it can potentiate endogenous antioxidative defense, thereby mitigating H. pylori-mediated inflammatory signaling mediated by AP-1 [40]. Even though NF-kB and AP-1 represent distinct mammalian transcription factors that target unique DNA enhancer elements, the heterodimer of NF-kB (p65/p50) shares structural homology with the c-rel proto-oncogene product. Similarly the AP-1 transcription factor complex is comprised of dimmers of c-fos and c-jun proto-oncogene products [41]. c-Fos and c-Jun are capable of physically interacting with NF-kB p65 through the Rel homology domain. This complex of NF-kB p65 and Jun or Fos exhibits enhanced DNA binding and the biological function via both the kB and AP-1 response elements [41]. Since oxidative stress overactivates AP-1 and NF-kB, these transcription factors can be important molecular targets for prevention of ROS-induced carcinogenesis [42].

4 Mitogen-Activated Protein Kinases MAPKs are a family of ubiquitous and well-characterized serine/threonine kinases thought to play a critical role in regulating cellular events required for cell growth, differentiation, and apoptosis in response to a variety of extracellular stimuli [43]. Three major subfamilies of MAPKs have been identified in mammalian cells: the extracellular signal-regulated kinase (ERK), the c-Jun NH2-terminal protein kinase (JNK) or stress-activated protein kinase (SAPK), and the p38 MAPK [42]. AP-1 activity is regulated by all three MAPK pathways [44]. Regulation occurs at both

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the transcriptional and at the post-transcriptional levels. Expression of c-Fos, which is nearly absent in quiescent cells, is controlled at the level of transcription. All three MAPK, ERK1/2, SAPK/JNK and p38, may phosphorylate the transcription factor Elk-1, a member of the tertiary complex family [45]. Elk-1 binds the serum response element motif in the c-Fos promoter, thereby inducing c-Fos transcription [29]. In contrast, c-Jun is regulated both transcriptionally and post-transcriptionally. A few c-Jun homodimers preexist in the resting cells. In addition, c-Jun transcription is upregulated by activated ERK [46]. Subsequently, c-Jun may increase its own transcription by binding to the TRE motif in its promoter [47]. c-Jun is activated via phosphorylation of serine residues 63 and 73 of c-Jun by JNK. Novel c-Fos synthesis leads to the formation of Jun/Fos heterodimers, resulting in increased AP-1 activity [48]. H. pylori activated AP-1 through ERK signaling [23] and JNK signaling [49] in gastric epithelial cells. A variety of extracellular stimuli induce ras activation, resulting in activation of the ras/raf/ERK kinase/MAPK cascade [50]. The Ras–Raf pathway increases the transcriptional activity of c-Jun, an important component of AP-1 [51]. Ras dominant negative gene expression almost blocked p38 phosphorylation in smooth muscle cells [52]. Therefore, Ras is the upstream activator of MAPK, and thus may be related to AP-1 activation. H. pylori-induced IL-8 expression is mediated by AP-1 via ERK signaling in gastric cancer cells [53]. H. pylori cagA induces ras activation and Elkphosphorylation in gastric epithelial cells [54]. The H. pylori-induced activation of AP-1 and NF-kB by was abrogated by U0126 (an ERK inhibitor) and SB203580 (a p38 inhibitor), and transfection with ras-dominant negative mutant gene (ras N-17) and c-Jun dominant negative mutant gene (TAM-67) constructs. Therefore, H. pyloriinduced activation of NF-kB and AP-1 involves Ras, MAPK (p38 and ERK), and c-Jun in AGS cells [33].

5 Chemokines Chemoattractant cytokines (chemokines) form a superfamily of closely related secreted proteins [55]. These inducible proinflammatory peptides potently stimulate leukocyte migration along a chemotactic gradient. They also modulate leukocyte adhesion, activate signal transduction cascades leading to expression of a distinct set of genes, and mediate other leukocyte functions necessary for leukocytes to leave the circulation and infiltrate tissues. Therefore, increased chemokine production and release are the important mechanisms responsible for leukocyte recruitment in response to injury or infection. Chemokines are divided into groups that are defined by characteristic cysteine motifs. Four families of chemokines, such as CXC, CC, C, and CX3C (C denotes a conserved cysteine residue and X is any other amino acid), have been described [56]. Among these chemokines, IL-8, a prototype CXC chemokine, seems to play an essential role in recruiting and activating neutrophils [57]

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in the gastric mucosa. Several reports have revealed that gastric epithelial cells represent an important source of IL-8 production [57]. In addition to chemotactic potential, IL-8 is capable of stimulating polymorphonuclear degranulation, the respiratory burst, and the 5-lipoxygenase pathway [55]. Thus, IL-8 may be a key component of the inflammatory cascade. Indeed, gastric mucosal levels of IL-8 correlate with histological severity in patients with H. pylori-induced gastritis [55]. Prolonged IL-8 production by gastric epithelial cells following H. pylori infection could result in the recruitment of leukocytes to infected tissues and therefore may be important in the regulation of proinflammatory and immune processes in response to H. pylori infection [58]. MCP-1 is a CC chemokine that stimulates mononuclear leukocytes. Like IL-8 induction, elevated expression of MCP-1 is observed in the gastric mucosa and gastric epithelial cells following H. pylori infection [59]. H. pylori-induced expression of MCP-1 as well as IL-8 was shown in gastric epithelial cells [60] and gastric mucosa tissues of the patients [61]. Expression of inflammatory genes such as IL-8 and MCP-1 is primarily controlled at the transcriptional level. NF-kB and AP-1 are inducible transcription factors and their binding sites are found in the promoter region of both IL-8 and MCP-1 genes [62]. Since these transcription factors are thought to be activated by ROS [63], oxidative stress induced by H. pylori may trigger their activation in gastric epithelial cells.

6 Proinflammatory Enzyme-Inducible Nitric Oxide Synthase and Cyclooxygenase-2 Cyclooxygenase (COX), the enzyme that catalyzes the oxidation of arachidonic acid, is present in two isoforms, COX-1 and COX-2. COX-1 is constitutively expressed in most tissues and maintains cellular homeostasis [64]. In contrast, COX-2 is barely detectable in normal tissues, but its expression is readily induced in gastrointestinal epithelial cells in response to inflammatory cytokines, LPS, mitogens and oxygen radicals [65]. H. pylori-associated acute and chronic antral inflammation was associated with expression of COX-2 in gastric epithelial cells [66], which was strongly correlated with the extent of chronic inflammatory cell infiltration [67]. Chronic expression of COX-2 may account for H. pylori-associated gastric carcinogenesis in addition to propagation of gastric inflammation since prostaglandins (PGs) produced via COX-2 are believed to be the major contributors to the inflammatory process [68] and can be mutagenic [69] and tumorigenic [70]. In the studies on human antral levels of prostaglandin E2 (PGE2) [71] and COX-2 expression in gastric mucosa of patients with H. pylori gastritis [72], higher expression of COX-2 was associated with a concomitant increase in the functional activity in H. pylori infection. Immunohistochemical analysis revealed that the increased expression of COX-2 appeared in macrophages and monocytes infiltrated into gastric mucosa of the patients with H. pylori gastritis [73]. However, an in vitro study showed that H. pylori regulates transcription of COX-2 in gastric

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epithelial cells in the absence of inflammatory cells such as macrophages and monocytes [74]. PGE2 is a principal product of the arachidonic acid cascade in which COX-2 acts as a rate-limiting enzyme. Relatively large amounts of PGE2 produced by COX-2 contribute to inflammatory events. Thus, in H. pylori-infected gastric cancer cells (MKN45), PGE2 induced the production of proinflammatory chemokine IL-8 that was inhibited by pretreatment with antagonists of EP2 and EP4 that are major PGE2 receptors [75]. H. pylori-induced COX-2 expression enhances the cancer cell invasion and angiogenesis via TLR2 and TLR9, which can be attenuated by the specific COX-2 inhibitor NS398 or celecoxib [76]. Such intracellular networks that drive the COX-2-dependent PGE2 release can contribute to not only inflammation, but also to cell invasion, metastasis and angiogenesis. Nitric oxide (NO) generated by nitric oxide synthase is known to be an important modulator of the mucosal inflammatory response [77]. Infection with cagA+ H. pylori has greater degree of gastric inflammation and epithelial cell damage as a consequence of prolonged production of NO by inducible nitric oxide synthase (iNOS) [78]. iNOS is a part of the host innate defense system against bacterial infection. During chronic inflammation, as provoked by H. pylori infection, constant NO production may lead to tissue and DNA damage, thereby increasing the patient’s risk for developing cancer [79]. The activity of iNOS was enhanced in the gastric mucosa of patients with H. pylori-positive duodenal ulcers [80]. Furthermore, iNOS expression and activity were augmented by H. pylori-infection in a macrophage cell line [81]. Mannick et al. [82] reported that dietary supplement with antioxidant b-carotene in H. pyloriinfected patients reduced nitrotyrosine staining, a marker for peroxynitrite, in gastric mucosa. NO may cause cellular damage by complex formation with ferrous-sulfatecontaining proteins [83]. It may also be plausible that NO derivates, such as nitrosamine or nitroso-compound, are generated in H. pylori-infected gastric mucosa. There is a close relation between carcinogenesis and nitrative damage to nucleic acids, which occurs during inflammation through the generation of reactive nitrogen species, such as peroxynitrite, nitroxyl and nitrogen dioxide. Enhanced formation of 8-nitroguanine, a representative nitrative damage to nucleobases, has been detected in various inflammatory conditions [84]. CagA+ H. pylori strains are associated with the expression and activity of iNOS, and therefore might contribute to the development of intestinal metaplasia leading to gastric cancer of the intestinal type [85]. Expression of proinflammatory genes including iNOS has been reported to be regulated by NF-kB [86]. Transcription of COX-2 gene also requires the activation of NF-kB and AP-1 in H. pylori-infected human gastric epithelial AGS cells [40]. Increased expression of COX-2 and iNOS was shown in H. pylori gastritis [72] and gastric cancer [87]. Since H. pylori activate TLR4 and produce ROS by activating NADPH oxidase, and H. pylori-induced COX-2 expression enhances the cancer cell invasion and angiogenesis via TLR2 and TLR9, various kinds of TLRs may represent potential preventive/therapeutic targets for the management of inflammation and carcinogenesis associated with H. pylori infection.

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7 NF-kB, iNOS, and Apoptosis Apoptosis, or programmed cell death, has been characterized morphologically by cell shrinkage and chromatin condensation, and biochemically by internucleosomal DNA fragmentation [88]. NO-induced apoptotic cell death in several cell systems has been described [89, 90]. H. pylori infection has been reported to induce apoptosis of gastric epithelial cells [91, 92]. Therefore, NO produced by iNOS is likely to contribute to apoptosis in H. pylori-stimulated gastric epithelial cells. H. pylori affects the normal balance between gastric epithelial proliferation and epithelial cell death. Several investigators reported that H. pylori activated NF-kB and directly induced apoptosis in human gastric mucosa and gastric epithelial cells both in  vivo and in  vitro [53, 91–93]. The promoter of iNOS gene harbors an NF-kB consensus site in its promoter region [94], and NO production by iNOS is known to be associated with apoptosis of cells [89, 90]. Pharmacologic inhibition of iNOS attenuated H. pylori-mediated cell growth inhibition and cytotoxicity while an NO donor SIN-1 augmented H. pylori-induced cytotoxicity in AGS cells [26]. The exact mechanism by which NO induces DNA damage in cells undergoing apoptosis has not been clarified. One possible explanation is that the reaction of NO with oxygen leads to the formation of extremely reactive N2O3 (peroxynitrite). Peroxynitrite attacks aromatic amines such as pyrimidine and purine, leading to DNA strand breaks [95]. The effect of NO on nucleic acids has been determined for naked DNA and in normal cells [96]. Inhibition of NF-kB activation using antioxidants and antisense oligonucleotide (AS ODN) to NF-kB subunit p50 suppressed H. pylori-mediated growth inhibition and cytotoxicity in AGS cells. Furthermore, the treatment of antioxidants and AS ODN to p50 mitigated the H. pylori-mediated apoptosis in AGS cells. In line with the notion that activation of NF-kB is associated with cell apoptosis [97], NF-kB stimulates the tumor suppressor gene p53, and the p65 subunit of NF-kB interacts with the cell cycle inhibitor p21WAF1 [98, 99]. Grill et al. [100] reported neuroprotection achieved by blocking NF-kB. Additionally, NF-kB is concomitantly activated during TNF-a-induced apoptosis [101], and inhibition of NF-kB by a certain antioxidant prevented apoptosis [102]. Apoptosis in gastric mucosa was reduced by supplementation with either ascorbic acid or b-carotene in H. pylori-positive patients [82]. These findings suggest that NF-kB functions to promote apoptosis in gastric epithelial cells infected with H. pylori. While these data suggest that NF-kB activation is detrimental, several other studies have shown that NF-kB activation blocks apoptosis in response to a variety of insults [103, 104].

8 Protease-Activated Receptors Protease-activated receptors (PARs) are G protein-coupled receptors that are activated by the cleavage of their N-terminal domains by proteases such as trypsin [105]. PARs regulate a broad range of cellular functions and are involved in the

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pathogenesis of inflammatory disorders. The family of PARs currently includes four members: PAR1, PAR2, PAR3, and PAR4. The coagulant protease thrombin is the physiological activator of PAR1, PAR3, and PAR4. PAR2 is activated by multiple trypsin-like serine proteases including trypsin, tryptase, and coagulation proteases that are the upstream of thrombin, factors VIIa and Xa, but not by thrombin [106]. PAR2 activation induces intracellular calcium mobilization, and the release of granulocyte macrophage-colony stimulating factor, IL-6 and -8, and PGs [107]. PAR2-mediated signaling is triggered by the activation of the receptor, a process that requires the cleavage of the amino-terminal exodomain of PAR2, and generation of a new amino-terminal sequence that binds to the core receptor and serves as a tethered ligand [106]. PAR2 is activated by multiple enzymes, including trypsin and mast cell tryptase. Activated PAR2 stimulates generation of inositol triphosphate and mobilization of intracellular Ca2+ [108, 109], activities of ERK1/2 [110], and cell growth [111] in leukocytes and vascular endothelial cells. PAR2 peptide 2F-LIGRLO strongly induced the nuclear translocation of the p65 subunit of NF-kB [112]. PAR2 expression is correlated with severity of wall invasion, lymphatic invasion, venous invasion, and liver metastasis [113]. Therefore, PAR2 may play an important role in carcinogenesis by regulating mediators including Ca2+, MAPK, and NF-kB in gastric epithelial cells. Kajikawa et al. demonstrated that H. pylori-derived protease may activate gastric epithelial cells to produce inflammatory cytokine IL-8 through PAR2. H. pyloriinduced IL-8 expression was inhibited by a serine protease inhibitor, nafamostat mesilate [114]. PAR2 is implicated in normal cell growth, and possibly in the gastrointestinal carcinogenesis when there is abnormally high and prolonged activation [115]. Invasive breast and pancreatic cancer cells express high levels of PAR2 compared with normal cells [116]. PAR2 was activated by trypsin in human pancreatic cancer cells in which PAR2 is highly expressed. Trypsin is a ubiquitous enzyme and expressed in various epithelial tissues including the gastrointestinal tract, kidney, liver, airway, skin and endothelial cells, leukocytes, and neuronal cells [117]. Tumor-derived trypsin contributes to growth, invasion, and metastasis of human cancer cells, not only by proteolysis of surrounding extracellular matrix (ECM) proteins, but also by activation of PAR2. Normal epithelial cells surrounding gastric cancer cells are likely a source of active trypsin [117]. Blood vessels surrounding tumors also express trypsin [118]. Of the many factors secreted by tumor cells, trypsin has been correlated to the stage and the type of carcinoma and is associated with cell invasion and ECM degradation [119, 120]. The activity and the level of trypsin as well as the expression of trypsinogen increased by H. pylori infection in gastric epithelial cells [121]. The zymogen of trypsin, trypsinogen, is activated into the mature form through autoactivation [122] or by endogenous activators, such as enterokinase, in colorectal cancer tissues [123] and the tissues of the gastrointestinal tract [124]. Therefore, it is postulated that trypsinogen may be activated to trypsin by enterokinase and trypsin may activate PAR2 in H. pylori-infected gastric epithelial cells. Trypsin is produced in excess in gastric cancer cells. Trypsinogen, secreted by tumor cells, could be activated to trypsin and stimulates the growth and adhesiveness of the producer tumor

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cells in an autocrine manner [125]. Therefore, a high level of trypsin is reported to be related to carcinogenesis in the context of enhanced cell proliferation and adhesion. Further studies should be performed to elucidate the mechanism underlying activation of trypsin by H. pylori and the presence of enterokinase in gastric epithelial cells. The activation of both ERK1/2 and p38 MAPK is critical for mitochondrial generation or oxygen radicals that are key mediators of the signaling pathways triggered by PAR1 and PAR2 in endothelial cells [126]. PAR2 agonist peptide augmented H. pylori-induced COX-2 expression in AGS cells [127]. It is likely that oxygen radicals induce NF-kB-mediated PAR2 upregulation is related to COX-2 expression in H. pylori-infected gastric epithelial cells. Since leukocyte-endothelial cell interaction and the production of ROS are hallmarks of the development of inflammation, Lim et al. [128] investigated the effects of PAR2 activation by trypsin on lymphocyte adhesion and the generation of ROS using PAR2 wild type and knockout (PAR2−/−) mice. They found that trypsin increased the production of ROS in isolated mouse lymphocytes in a dose-dependent manner. The increase in lymphocyte adhesion and the production of ROS in response to trypsin were abolished in PAR2−/− mice. Therefore, activation of PAR2 may be a critical factor in modulating lymphocyte adhesion and the production of ROS. Since inflammatory cells mainly produce large amounts of ROS in H. pylori-infected gastric mucosa, activation of PAR2 may contribute to the production of ROS and increased cell adhesion to ECM proteins.

9 Integrins Integrins are heterodimeric transmembrane receptors consisting of one a and one b subunits. The two subunits collaborate to bind ligands, which are ECM proteins, including fibronectin, collagen, laminin, and vitronectin [129] or the counter-receptor of the Ig subfamily. The most abundant, constitutive integrins in the epidermis are a2b1 (collagen receptor), a3b1 (predominantly a laminin 5 receptor), a6b4 (laminin) [130]. a5b1 (fibronectin receptor) and avb6 (receptor for fibronectin and tenasin) are induced in culture and on wounding [131]. The adhesion of cancer cells to ECM proteins plays a pivotal role in invasion and metastasis [132]. Many a subunits associate with only one b subunit. The b1 subfamily includes the fibronectin receptor, laminin receptor and collagen receptor of epithelial cells, fibroblasts and lymphocytes. H. pylori induces the expression of integrins a5 and b1 in gastric epithelial AGS cells [133]. PAR2 mediates integrin a5b1-dependent adhesion of the cells to fibronectin and proliferation of human gastric carcinoma cells [123]. These studies suggest that the expression of integrins and cell adhesion to ECM protein may be mediated by PAR2 in H. pylori-infected gastric epithelial cells, which may account for gastric carcinogenesis associated with H. pylori infection. Integrin expression in cancer tissues demonstrates its possible contribution to tumor progression, invasion and metastasis [134]. Increased adhesion of tumor cells

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by H. pylori has been suggested to be important for H. pylori-induced metastasis of gastric epithelial cells. It has been reported that the expression of integrin subunits such as integrin a2, a3, a5 and a6 was correlated with cell adhesion, invasion and metastasis of gastric carcinoma cells [125, 134, 135]. Increased adhesion of tumor cells is considered to be important for their metastasis. H. pylori induced upregulation of integrin aM and aX in Kato 3 cells [136] and integrin a7 [137], integrins a5 and b1 in AGS cells [133]. Integrin activation is supposed to depend on the conformational change of the integrin molecule induced by the interaction of its cytoplasmic domain with some G protein-coupled receptor (GPCR)-signaling molecules [138]. The adhesion of H. pylori-infected AGS cells to ECM protein fibronectin was suppressed by treatment with a trypsin inhibitor or anti-integrin a5 and b1 antibodies as well as inhibition of PAR2 expression by use of AS ODN for PAR2 [121]. The study suggests that trypsin activates PAR2, which stimulates integrin a5b1-mediated cell adhesion to fibronectin in H. pylori-infected AGS cells. Therefore, inhibition of H. pyloriinduced PAR2 expression may be beneficial for prevention and treatment of gastric cell adhesion and possibly carcinogenesis associated with H. pylori infection. Recent studies show that NADPH oxidase controls the persistence of directed cell migration by a Rho-dependent switch of a2/a3 integrins in colon epithelial cells [139], and that oxidative stress that accompanies senescence may increase fibronectin production by human omentum-derived peritoneal mesothelial cells and thus facilitate the binding of ovarian cancer cells [140]. The role of ROS in H. pylori-induced integrin expression in the infected gastric mucosa merits further investigation.

10 Genetic Variability of H. pylori Strain H. pylori isolates exhibit a high degree of genetic variability. It has been shown that approximately 50–60% of H. pylori strains have cytotoxin-associated gene (cagA) pathogenicity island (PAI) [141]. Some of the proteins encoded by cagA PAI genes are responsible for activation of NF-kB and MAPKs in gastric epithelial cells [142]. Infection by cagA strain is more likely to result in peptic ulceration, atrophic gastritis, and gastric carcinoma [143]. Therefore, the expression of cagA may be important in signal transduction mediating H. pylori-induced gene expression, regulating inflammation, proliferation and carcinogenesis. The genetic differences in H. pylori may play a role in the clinical outcome of the infection, particularly that manifested by vacA, cagA, and iceA genes [144]. VacA, which encodes the vacuolating toxin, is present in all strains and comprises two regions of allelic variation [145]. The s-region, located at the 5¢ end of the gene, and the m-region, located in the middle of vacA, has three allelic forms [146]. The production of the cytotoxin is related to the mosaic combination of s- and m-region alleles in vacA. The vacA s1/m1 strains are more virulent than s1/m2 strains [147], and the vacA m1-type strain can cause greater gastric epithelial damage than the m2 strain [145].

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Another putative virulence gene is iceA [148]. The iceA locus comprises two main variants, iceA1 and iceA2. The function of these variants is not yet clear. The expression of iceA1 is upregulated by contact of the bacteria with human gastric epithelial cells and in some populations is associated with peptic ulcer disease [146, 148]. After examining 94 gastric biopsy specimens from patients in the Netherlands, van Doorn et  al. [146] reported that the iceA allelic type was independent of the cagA and vacA status, and there was a significant association between the presence of the iceA1 allele and peptic ulcer disease. Figueiredo and colleagues [149] demonstrated that vacA s1 and cagA+ H. pylori strains are responsible for duodenal ulcer, gastric ulcer or gastric carcinoma in Portugal. However, vacA m1 infection is associated with gastric ulcer or carcinoma but not with duodenal ulcer. Since the predominant genotype of H. pylori in Korea has been reported to be the cagA positive (cag A+) and vacA positive (vac A+) genotype [150], the high incidence of gastric cancer in Korea may be related to the cag A+ H. pylori strain. It has been shown that the dominant H. pylori strain (HP99) from Korean isolates is the cagA+, vacA s1b m2, iceA1 H. pylori strain. This H. pylori strain induces the expression of chemokines (IL-8, MCP-1) [33] and the two representative proinflammatory enzymes iNOS and COX-2 [74], which is mediated by MAPKs (ERK, JNK, and p38), NF-kB and AP-1 in gastric epithelial AGS cells. Further studies on the signaling pathways and mediators induced by H. pylori infection are necessary to provide rational approaches for the control of H. pylori-induced gastritis and gastric carcinogenesis.

11 Experimental Models of H. pylori Infection H. pylori infection triggers a cascade of mucosal changes from acute/chronic gastritis into the atrophic gastritis with intestinal metaplasia and finally to dysplasia and gastric cancer. H. pylori infection is considered to be the major initiator of the inflammatory and atrophic changes in gastric mucosa accompanied by an overexpression of certain growth factors such as gastrin as well as of COX-2 and antiapoptotic proteins including survivin and Bcl2, leading to proliferation of mutated atrophic cells, excessive angiogenesis, inhibition of apoptosis and formation of gastric tumor [151]. Considering that H. pylori is a major risk factor for human gastric adenocarcinomas, there is a need for reliable animal models to better understand the carcinogenic process and to develop preventive or therapeutic strategies. However, there is no animal model that exactly mimics the histopathology associated with H. pylori infection in humans. Nonetheless, the animal models are starting to provide valuable information with respect to factors involved in the colonization of the gastric mucosa and the importance of host factors in the development of gastric atrophy, as well as making possible the screening of potential therapeutic agents and vaccine candidates [152].

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11.1 Mongolian Gerbil Model H. pylori infection in Mongolian gerbils is an established experimental model of gastric carcinogenesis that resembles H. pylori-positive patients developing peptic ulcer and gastric cancer. Indeed, the gastric histopathologic changes in H. pyloriinfected gerbils are closely similar to those observed in patients with H. pylori infection [153]. The eradication therapy was proven in several prospective clinical trials to greatly attenuate the development of gastric cancer associated with H. pylori infection, and this was confirmed in the gerbil model of H. pylori-induced gastric carcinogenesis [151]. Moreover, the H. pylori-induced inflammatory response in gerbils is considerably greater than in murine models [154]. Thus, Mongolian gerbils are considered to be a good animal model for understanding the development of H. pylori-associated diseases. However, limitations regarding the genetic information available for this animal species hamper the elucidation of underlying mechanisms. The nucleotide sequences of Mongolian gerbil cDNAs encoding inflammatory proteins, such as IL-1b, TNF-a, COX-2 and iNOS have been identified and their expression in the glandular stomach after H. pylori infection was determined [155]. The NF-kB–IkB complex is a major transcription regulator of inflammatory and immune responses. H. pylori infection causes chronic inflammation in gastric mucosa by inducing liberation of NF-kB from the complex. Comparative immunohistochemical staining revealed a differential distribution of IkB proteins in the Mongolian gerbils [156]. Macrophages play an important role in producing proinflammatory cytokines during inflammation. Activated macrophages were found to be significantly increased in gastric mucosa of H. pylori-infected gerbils. Macrophage depletion by the administration of clodronate resulted in milder inflammation in gerbils infected with H. pylori. In macrophages, the inhibition of IkB kinase beta (IKKb), a critical upstream kinase for NF-kB activation, resulted in lower proinflammatory cytokine expression caused by heat-killed H. pylori cells [157]. Furthermore, treatment with an IKKb inhibitor resulted in milder inflammation in gerbils with H. pylori gastritis. These findings suggest that H. pylori-mediated gastric inflammation critically depends on the efficient recruitment and activation of macrophages, with sufficient NF-kB activation [157]. Though the majority of morphological and biochemical changes associated with the H. pylori-induced transformation of cultured mucosal cells into the cancer cells have been traced in Mongolian gerbils, recent advances in the mouse models allow us to dissect bacterial host interactions in a novel manner due to the availability of a wide range of immunological reagents and numerous mutant or transgenic strains [152].

11.2 Mouse Model Mouse models are preferred by most researchers because of cost effectiveness, rapid reproduction, choice of laboratory reagents, and availability of genetically ­engineered

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mutants to study specific gene functions in vivo. Murine models have validated the once-provocative hypothesis that H. pylori infection is a major risk factor for gastric carcinoma, dispelling early skepticism over the pathogenic nature of this organism in human stomach [158]. Thus the first evidence that H. pylori infection induces a chronic inflammation that progresses to gastric atrophy, the precursor lesion to gastric adenocarcinoma in humans, has first come from the mouse model. The severity of inflammation is dependent on the type of mouse strain used, highlighting the importance of host factors in the development of gastritis [159]. The pathogenesis of H. pylori infection in vivo was studied by adapting fresh clinical isolates of bacteria to colonize the stomachs of mice. A gastric pathology resembling human disease was observed in infections with cytotoxin-producing strains but not with noncytotoxic strains. Oral immunization with purified H. pylori antigens protected mice from bacterial infection. Besides Mongolian gerbils, a mouse model can also allow the development of therapeutic agents and vaccines against H. pylori infection in humans [160]. After screening a range of fresh clinical isolates and long-term adaptation in mice, a CagA and VacA positive Sydney strain of H. pylori has been isolated with a very good colonizing ability in C57BL/6 mice. The phenotype was stable with colonizing ability remaining after 20 subcultures in vitro. The bacterium attached firmly to gastric epithelium. During 8 months, a chronic active gastritis slowly developed, progressing to severe atrophy in both C57BL/6 and BALB/c mice [161]. When CD1 mice were infected with a type I (CagA+/VacA+) H. pylori strain, the majority of gastric tissue biopsies were positive for the bacteria in culture for up to 4 weeks. Immunohistochemical analysis showed that inflammatory changes started to occur after 3 weeks. In 50% of the infected mice also an increased number of mast cells was seen [162]. Another murine model for H. pylori infection was established by intragastrically challenging BALB/c mice with bacterial suspension for two consecutive days. Although the animals developed mild duodenitis and gastritis, the BALB/c mouse is not susceptible to developing peptic ulcers during H. pylori infection [163]. To analyze the influence of Helicobacter on inflammatory responses and cell proliferation, an animal model of H. pylori-induced gastritis in p53-knockout mice was employed. The bacteria were introduced by gastric intubation into p53-knockout C57BL/6 mice. The animals were then followed-up for 1 year and compared with uninfected controls of the same genotype. Histologically, p53-knockout mice exhibited increased scores of chronic and active inflammation compared with uninfected controls. In the p53-knockout mice, H. pylori infection caused severe inflammatory reactions [164]. Despite increasing knowledge on the biology of H. pylori, little is known about the expression pattern of its genome during infection. By employing a quantitative reverse transcriptase PCR (RT-PCR) that allowed the detection of minute amounts of mRNA within the gastric mucosa, the expression of four genes, 16S rRNA, ureA (encoding urease A subunit), katA (catalase), and alpA (an adhesin), was monitored during the course of a 6-month infection of mice and in biopsy samples from of 15 infected humans. These selected genes were all expressed in both mouse and human infected mucosae [165].

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Different Helicobacter isolates were tested for their ability to induce NF-kBdependent responses in murine gastric epithelial cells and in transgenic mice harboring an NF-kB-responsive lacZ reporter gene. Helicobacter infection of transgenic mice resulted in increased numbers of gastric epithelial cells with nuclear betagalactosidase activity, which is indicative of specific NF-kB activation [166]. The expression of COX-2 is induced in Helicobacter-associated chronic gastritis, which results in the induction of proinflammatory PGE2 expression. The COX-2-PGE2 pathway plays a key role in gastric tumorigenesis. The activation of Wnt/betacatenin signaling is found in 30-50% of gastric cancers, thus suggesting that Wnt signaling plays a causal role in gastric cancer development. Accordingly, it is possible that Wnt activation can cause gastric tumorigenesis through enhancement of the undifferentiated status of epithelial cells. Recent mouse model studies have indicated that induction of the PGE2 pathway is required for the development of gastric adenocarcinoma in the Wnt-activated gastric mucosa [167]. In contrast to these findings, pharmacologic inhibition of the COX-2 activity accelerated early preneoplasia, while systemic administration of synthetic PGE2 prevented development of premalignant pathology and completely reversed preexisting lesions by suppressing interferon-gamma production in the infected stomachs. The protective effect of PGE2 was accompanied by increased Helicobacter colonization in all models [168]. K19-C2mE transgenic (Tg) mice, simultaneously expressing COX-2 and microsomal prostaglandin E synthase-1 (mPGES-1) in the gastric mucosa were treated with N-methyl-N-nitrosourea (MNU) and inoculated with H. pylori to investigate gastric carcinogenesis. Wild-type (WT) and Tg mice after MNU treatment developed tumors in the pyloric region; multiplicity in Tg was higher than that in WT with H. pylori infection. No gastric tumors were observed without MNU treatment. Transcripts of TNF-a, iNOS, IL-1b, and CXCL14 were up-regulated with H. pylori infection in both genotypes and were also increased more in Tg than in WT within H. pylori-inoculated animals. Immunohistochemical analysis demonstrated significantly greater b-catenin accumulation in pyloric tumors, suggesting the of Wnt-b-catenin activation in H. pylori-induced gastric carcinogenesis as well as inflammation. Induction of various inflammatory cytokines in addition to overexpression of COX-2/mPGES-1 could be risk factors of gastric carcinogenesis and may serve as a better gastric carcinogenesis model [169].

12 H. pylori as a Putative Promoter of Gastric Carcinogenesis The role for H. pylori infection in the development of gastric cancer in humans is well established, but evidence for its carcinogenicity in animals remains inadequate. Mongolian gerbils and mice are commonly used to investigate the carcinogenicity of H. pylori, yet it is unclear whether H. pylori infection per se causes gastric cancer or duodenal ulcers in these animal models. The development of invasive adenocarcinoma in inbred mice is rare regardless of the mouse or bacterial strain, and many long-term studies have failed to induce gastric cancer in these animals [170]. Despite the World

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Health Organization’s definition of H. pylori as a class I carcinogen, debate that H. pylori might play a causative role in gastric carcinogenesis still exists. After 80 weeks of infection, almost all the H. pylori-infected mice developed hyperplastic gastritis, but did not show any evidence of gastric adenoma, dysplasia or carcinoma. Hence, H. pylori infection alone does not appear to be sufficient to induce gastric cancer in experimental animals, and additional carcinogenic insult is required [171]. In order to define the exact role of H. pylori infection in gastric carcinogenesis, female C57BL/6 mice were treated with MNU alone or MNU administration followed by H. pylori infection. The incidence of gastric adenocarcinoma at the 50th week was much higher in mice with both MNU treatment plus H. pylori infection than that in mice treated with MNU alone. Although H. pylori caused marked expansion of the proliferative zone at the surface epithelium, the bacterial infection alone caused only chronic atrophic gastritis without any evidence of carcinomas until 80 weeks. The combination of MNU and H. pylori infection also resulted in a significantly higher incidence of gastric adenoma and adenocarcinoma. Significantly higher expression of proliferating cell nuclear antigen was also noted in the gastric mucosa of mice infected with H. pylori compared to that of controls [172]. Likewise, BALB/c mice were given MNU and administered broth culture of H. pylori. The incidence of pepsinogen-altered pyloric glands, considered as precancerous lesions, was increased in the H. pylori inoculated group pre-treated with MNU [173]. H. pylori is responsible for most human stomach cancers. Gastric cancer also is overrepresented in populations consuming high-salt diets. A high-salt diet in humans and experimental animals is known to cause gastritis, has been associated with a high risk of atrophic gastritis, and is considered a gastric tumor promoter. In laboratory rodents, salt is known to cause gastritis, and when coadministered, it promotes the carcinogenic effects of known gastric carcinogens. Excessive NaCl intake enhances H. pylori colonization in mice and in humans and that chronic salt intake may exacerbate gastritis by increasing H. pylori colonization. Furthermore, elevated salt intake may potentiate H. pylori-associated carcinogenesis by inducing proliferation, pit cell hyperplasia, and glandular atrophy [174]. In an attempt to test the hypothesis that high salt promotes H. pylori carcinogenesis, C57BL/6 × 129S6/SvEv (B6129) mice were gavaged with H. pylori Sydney strain-1 or vehicle only and divided into four groups based on infection status and maintenance on a basal (0.25%) or high (7.5%) salt diet. In uninfected mice, the high-salt diet enhanced proliferation and marginally increased parietal cell mucous metaplasia with oxyntic atrophy. Infected mice on the high-salt diet exhibited significantly higher colonization and a qualitative increase in infiltrating eosinophils [175]. The role of H. pylori infections in the induction of mutagenic events in gastric epithelial cells was investigated. Big Blue transgenic male mice (C57Bl/6) were inoculated with H. pylori (SS1). After 6 months, the gastric mutant frequency was 4-fold higher in mice infected with H. pylori, than in uninfected mice. It was associated with a high frequency of transversions (AT → CG and GC → TA) known to result from oxidative damages. This genotoxicity can be attributable to oxidative DNA

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damage involving the inflammatory host response. The H. pylori-infected mice exhibited severe gastritis and a high level of iNOS messenger RNA expression. Hyperplasia developed 12 months after inoculation. No synergistic effects of a high-salt diet and H. pylori infection were observed regarding the frequency of gastric mutation [176].

13 Concluding Remarks The discovery of H. pylori in the human stomach in 1983 by two Australian scientists, B.J. Marshall and R.J. Warren, is considered as a major breakthrough in pathophysiology of gastritis and peptic ulcer, which can be definitively cured by simple eradication of germ infecting stomach [177]. The long-term infection with H. pylori causes gastritis, which predisposes to gastric cancer through cellular changes resulting from oxidative and inflammatory damages. Numerous intracellular signaling molecules have been identified that mediate the pro-oxidant and pro-inflammatory processes as a consequence of H. pylori infection. Since the recognition of the pathogenicity of H. pylori, several animal models, mainly using Monolian gerbils or mice, have been developed to link H. pylori and gastric carcinogenesis. Although several features of animal models differ from those seen in human beings, experimental models are useful in development of preventive and therapeutic strategies and studying the host-bacteria interactions. Recent advances in genetic engineering of laboratory animals, in vivo imaging, and system-wide genomics and proteomics will provide more insight into the role of H. pylori in gastric carcinogenesis [178]. Acknowledgment  This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-002916) and a grant (Joint Research Project under the Korea–Japan Basic Scientific Cooperation Program) from NRF (F01-2009-000-10101-0). H. Kim is grateful to the Brain Korea 21 Project, college of Human Ecology, Yonsei University.

References 1. Marshall, B.J. (1994) Helicobacter pylori Am J Gastroenterol 89, S116–28. 2. Yoshida, N., Granger, D. N., Evans, D. J. Jr, Evans D. J., Graham D. Y., Anderson D. C., Wolf R. E., and Kvietys, P. R. (1993) Mechanisms involved in Helicobacter pylori-induced inflammation Gastroenterology 105, 1431–40. 3. Blaser, M. J. (1992) Hypothesis on the pathogenesis and natural history of Helicobacter pylori-induced inflammation Gastroenterology 102, 720 –7. 4. Nathan, C. F., Silverstein, S. C., Brukner, L. H., and Cohn, Z. A. (1979) Extracellular cytolysis by activated macrophages and granulocytes. II. Hydrogen peroxide as a mediator of cytotoxicity J Exp Med 149, 100–13. 5. Babior, B. M. (1984) Oxidants from phagocytes: agents of defense and destruction Blood 64, 959–66.

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Experimental Models for Ionizing Radiation Research Kristin Fabre, William DeGraff, John A. Cook, Murali C. Krishna, and James B. Mitchell

Abstract  Ionizing radiation is a valuable tool used for cancer treatment as well as for basic molecular research. More recent interest stems from a need to provide countermeasures against accidental or intentional exposure to radiation through nuclear devices. This chapter provides an overview of the biological effects of radiation and highlights models used to study radiation-induced damage and repair. In vitro and in  vivo endpoints including DNA damage, cell survival, apoptosis, cytogenetic aberrations, oxidative stress, tumor response, and genomic instability are discussed. Appropriate use of these models will facilitate the advancement of radiation research as novel molecular mechanisms are elucidated. Keywords  Cell killing • DNA damage • DNA repair • IR protectors • Radiation • Radiosensitizers • ROS Abbreviations ATM AZD Bcl-2 BRCA1/2 BSE BUdR Chk1/2 CHO CMXRos DCF

Ataxia telangectasia mutant Astrazeneca Chk1 inhibitor B-cell lymphoma 2 Breast cancer associated 1/2 Bystander effect Bromodeoxyuridine Checkpoint kinase 1/2 Chinese hamster ovary cell line Chloromethyl-X-rosamine 2¢,7¢-Dichlorofluorescein

M.C. Krishna (*) Radiation Biology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_17, © Springer Science+Business Media, LLC 2011

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DHE 5-Ethyl-5,6-dihydro-6-phenyl-3,8-diaminophenanthridine, hydroethidine DiOC6 3,3¢-Dihexyloxacarbocyanine iodide DMF Dose modifying factor DNA-PKcs DNA-dependent protein kinase catalytic subunit DSB Double-strand break EGFR Epidermal growth factor receptor ELISA Enzyme-linked immunosorbent assay FPG Fluorescence plus Giemsa FX Fractionated IR GI Genomic instability Gy Gray H2AX Histone H2A H2DCF-DA 2¢,7¢-Dihydrodichlorofluorescein HDAC Histone deactetylase HGPRT Hypoxanthine–guanine phosphoribosyltransferase HPLC High performance liquid chromatography HR Homologous recombination Hsp-90 Heat-shock protein 90 IR Ionizing radiation IUdR Iododeoxyurdine LC3 Microtubule-associated protein 1 light chain 3 LD50/30 Lethal dose for 50% at 30 days LET Linear energy transfer MN Micronuclei MRN Mre11–Rad50–Nbs1 mTor Mammalian target of rapamycin MTS 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium MTT 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide NFkB Nuclear factor kappa-light-chain-enhancer of activated B cells NHEJ Nonhomologous end-joining PARP-1 Poly (ADP-ribose) polymerase-1 PF Protector factor PFGE Pulse field gel electrophoresis PI3K Phosphoinositide 3-kinases PT Permeability transition pores ROS Reactive oxygen species RPA Replication protein A SCE Sister chromatid exchange SNP Single nucleotide polymorphism SSB Single strand break ssDNA Single strand DNA Tumor control dose for 50% TCD50 TMRE Tetramethylrhodamine

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TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling VEGF Vascular endothelial growth factor XIAP X-linked Inhibitor of apoptosis protein XRCC4 X-ray repair complementing defective repair in CHO 4 XTT 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5carboxanilide gH2AX Gamma (phosphorylated) H2AX Dy m Membrane potential difference

1 Introduction Ionizing radiation (IR) is increasingly being used in biomedical research for several reasons. A major use for IR is as a valuable tool for cancer treatment. While radiation therapy is effective and useful in the control of some tumors, there is a definite need to improve local tumor control without increasing injury to normal tissues within the treatment field. Research in this area involves a host of in  vitro and in vivo experimental techniques used to evaluate agents that might enhance tumor response to IR or protect normal tissue. Unfortunately, another area of radiation research is currently directed to identify countermeasures to radiation lethality/ injury that might result following a terrorist attack using nuclear devices. Advances in this line of research may not only provide protective or mitigative applications to populations of people either accidentally or intentionally exposed to radiation, but may also benefit cancer patients being treated with radiation. Lastly, radiation is utilized in basic science research because it is a “relatively clean” method of imposing oxidative stress on cells or experimental animals. Oxidative stress, free radicals, and reactive oxygen species (ROS) have emerged as major contributors to a number of disease states as well as being involved in a variety of molecular signaling pathways. Unlike free radical-generating drugs or chemicals, radiation can be delivered quickly and precisely to cells or tissues foregoing the complications of physiological and metabolic barriers that often accompany drug delivery, particularly in  vivo. This chapter will provide the reader with a basic understanding of radiation and radiation biology, with emphasis on techniques, assays, and experimental models used to quantify radiation effects. More comprehensive, in depth information can be found in several excellent textbooks [1–3].

2 Radiation Chemistry Basics Radiation sources commonly used in biomedical research include X-ray or g-ray units (cesium-137 or cobalt-60) and various radioisotopes that decay through emission of electrons, g-rays, or alpha particles. X-rays and g-rays are two forms radiation that are a part of the electromagnetic spectrum. The electromagnetic spectrum

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H2O

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Fig. 1  Exposure of water to IR results in a number of chemical reactions yielding free radical species (radiolysis of water)

includes radio waves, microwaves, infrared, visible light, ultraviolet radiation, X-rays and g-rays (ionizing radiation; IR) that, starting with radio waves, exhibit decreasing wavelength, increasing frequency, and increased photon energy, respectively. The increased frequency and photon energy of X- and g-rays makes them unique to other members of the electromagnetic spectrum in that upon interaction with biological matter they are capable of breaking chemical bonds either directly or through free radical reactions, which can lead to biological effects [3]. In addition to X- and g-rays, other types of IR include electrons, protons, a-particles, neutrons, and heavy charged ions. Radiation can impart damage to biological molecules through direct and indirect action. Direct action involves either a secondary electron or particle damaging a biological molecule directly through chemical bond breakage. In the case of X- and g-rays, direct action account for approximately 20% of a given radiation dose, whereas the remaining 80% of damage occurs through indirect action via the generation of free radical species. Radicals produced by IR predominantly come from water, since it is by far the most abundant molecule (80%) in biological material. Exposure of water to radiation can result in generation of a number of free radicals (Fig. 1) over a subsecond time period. The free radical species thought to impart the most biological damage is the hydroxyl free radical; however, superoxide, hydrogen peroxide, aquated electrons, and hydrogen radical can contribute to damage and can most likely initiate signaling pathways. Distinction can be made among the various types of IR by the energy transferred to biological material as a function of the unit of track length, termed linear energy transfer (LET, keV/mm). For example, low LET radiation (X- and g-rays) imparts less damage to cells/tissues than high LET radiation (a-particles, neutrons, and heavy charged ions) for a given radiation dose and hence exerts less damage/ cytotoxicity [1].

3 Radiation-Induced Damage: Molecular Target There is considerable evidence suggesting that the primary target for radiationinduced cell killing and mutations is the DNA molecule [4–6]. For a 2  Gy dose (typically used in daily radiation treatments to cancer patients), approximately

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Dose (Gy) Fig. 2  Radiation survival curves for different cells and microorganisms. Note the extremely wide range of radiosensitivities, with D. radiodurans being extremely IR resistant compared to mammalian cells [1, 8, 9] (Adapted with permission from [10])

2,000 single-strand and 80 double-strand DNA breaks are registered per cell along with ~6,000 base damages [5, 7]. The vast majority of this damage is repaired by cellular enzymatic systems over a 2–8  h time period; however, the proportion of DNA double strand breaks not repaired ultimately leads to chromosome aberrations that can be lethal to cells. For example, there is a linear relationship between cell survival and the induction of radiation-induced lethal dicentrics/rings [6]. While there are many lines of evidence pointing to the DNA as the target for radiationinduced cell death, the survival of cells and microorganisms exposed to radiation depends on the volume of total DNA contained within the nucleus [1]. Mammalian cells are more sensitive to radiation and contain significantly more DNA per cell compared to other organisms, such as yeast or different strains of bacteria as shown in Fig. 2. Thus, mammalian cells present a “larger target volume” for free radical damage interaction than microorganisms [11]. Micrococcus radiodurans are extremely resistant to radiation due to their small DNA volume, high number of gene copies, and extremely fast DNA repair capabilities [8–10, 12].

4 Radiation-Induced Damage: Cell Killing 4.1 Clonogenic Assay Cells may experience several forms of cell death following exposure to IR, including loss of reproductive integrity, apoptosis, necrosis, senescence, and autophagy [13]. With respect to oncology research, the most relevant assessment of cell survival is the clonogenic assay, which assesses the ability of a single cell to undergo multiple

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Surviving Fraction

n

α

β

Dose (Gy) Fig.  3  Mammalian IR survival curve showing the surviving fraction (log) as a function of IR dose. This IR survival curve is typical for most mammalian cells including tumor cell lines. Note that for low IR doses there is a “shoulder” on the curve indicating that cells are capable of accumulating and repairing sublethal IR damage [1, 14, 15]. The shoulder region is followed by an exponential slope. Various models are used to fit IR survival data. The single-hit, multitarget model uses the extrapolation number n, which a qualitative measure of the repair capacity of a cell to IR and the terminal slope is defined as the D0 (IR dose required to reduce survival by 37%). The linear quadratic model uses the linear term (a) to describe the initial slope (nonrepairable damage) and the quadratic term (b) to define the repairable damage [16]. The parameters from both models are useful in comparing the IR sensitivity of different cell types or modification of IR sensitivity by various sensitizers or protectors [17, 18] (Curve adapted with permission from [10])

cell divisions to form a macroscopic colony [14, 15]. The assay is particularly relevant to oncology in that inhibition of uncontrolled cell proliferation, which is characteristic of cancer cells, is the major goal of oncology research. Likewise, the clonogenic assay measures cell survival inclusive of the other types of cell death listed above. A typical survival curve for mammalian cells exposed to X-rays is shown in Fig. 3. The “shouldered” segment of the survival curve is representative of most mammalian cells exposed to X- or g-rays, indicating that cells can accumulate and repair sublethal radiation damage for low doses of radiation. As the radiation dose increases, the survival response follows an exponential terminal slope. Radiation survival curve data can be fit to various mathematical models, the most utilized of which are the single hit/multiple target model or the linear quadratic model [16]. Both models provide survival curve parameters that are useful in comparing and contrasting the radiosensitivity of different types of mammalian cells with or without treatment with agents that might modify the radiation response.

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4.2 Apoptosis Apoptosis, or programmed cell death, is a systematic series of biochemical events that lead to caspase-mediated loss of mitochondrial membrane potential, the release of cytochrome c, DNA fragmentation, loss of cellular membrane asymmetry and finally, cell death [20]. Cells undergoing apoptosis shrink in size, with condensed nuclei and often exhibit membrane blebbing, features that can be readily observed microscopically. Given the number of biochemical events that occur during apoptosis, a number of assays that quantify apoptosis have emerged. In normal cells, mitochondria have negatively-charged inner membranes that create a potential difference (Dy m) due to asymmetrical distribution of H+ ions. This membrane potential can be detected using cationic lipophilic dyes such as CMXRos (chloromethyl-X-rosamine), TMRE (tetramethylrhodamine), 3,3¢dihexyloxacarbocyanine iodide (DiOC6) and rhodamine 123 that can be readily detected flow cytometrically. During apoptosis, permeability transition pores (PT) or mitochondrial mega-channels form and create loss of mitochondrial potential resulting in less dye taken by the cell, which can be detected by flow cytometry [21]. Nuclear DNA fragmentation occurs when caspase-activated endonucleases cleave DNA, which can be detected using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay [22]. In the TUNEL assay, labeled dUTP is added onto free DNA ends and readily detected using flow cytometry. DNA fragmentation can also be examined by labeling specific proteins that are entwined with genomic DNA, which can be measured by an enzyme-linked immunosorbent (ELISA) assay. When cells undergo apoptosis, phospholipids in the cell membrane become flipped, changing the hydrophobicity on the outer surface of the cell. This phenomenon can be detected using immunofluorescence and flow cytometry along with labeled annexin V, a protein that binds to phospholipids on cell surfaces [22]. Cells derived from the hematopoietic system, either normal or malignant, readily undergo apoptosis following treatment with a wide variety of stresses including IR. In contrast, fibroblasts or cells from solid tumors are more resistant to apoptosis. Hence using apoptosis assays to assess cell killing of solid tumor cells following IR may underestimate the true level of cell killing determined by the clonogenic assay [13, 23–25].

4.3 Necrosis and Autophagy Necrosis occurs when exogenous factors (i.e., inflammation, poisons, extreme microenvironmental conditions, IR) damage cells and cause premature or accidental death. Unlike apoptosis, necrosis does not involve regulated molecular pathways but rather a rapid depletion of ATP and mechanical breakdown of cellular components including the cell membrane, nuclear chromatin, and organelles. Finally the cell ruptures and leaves behind necrotic debris, which can lead to local inflammation. A hallmark of necrosis is cellular and mitochondrial swelling which can be

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detected using electron microscopy. Proteolytic enzymes released from the breakdown of lysosomes begin to demolish surrounding cellular components and perforate the cell membrane, allowing for an influx of extracellular fluid. The degraded membrane allows dyes (such as trypan blue or fluorescent dyes such as propidium iodide) that are normally excluded from healthy cells to penetrate, which can be detected using light microscopy or flow cytometry, respectively [26, 27]. Autophagy, or self-digestion, is a tightly regulated process that becomes activated upon inhibition of the PI3-K/mTor (mammalian target of rapamycin) pathway and driven by the atg gene family [28–31]. This mechanism is utilized to degrade aged/damaged proteins and organelles or can be activated by outside stresses like nutrient starvation, oxidative stress and oxygen depletion. During autophagy, doublemembrane structures, called autophagosomes, engulf cellular components and then fuse to lysosomes, which degrade the contents using acidic hydrolases. One of the standard methods used to detect autophagy is through cellular morphological changes (granular cytoplasmic ubiquitin inclusions) identified using electron microscopy; however, the use of electron microscopy is not adequate when attempting to monitor autophagy [32]. Therefore, additional markers that are associated with autophagy have been identified. A central marker used for detection is microtubuleassociated protein 1 light chain 3 (LC3) [32]. During autophagy, LC3 is activated by Atg4-induced cleavage and then conjugated to the Atg7, Atg3 and Atg12–Atg5– Atg16L multimer to become LC3II. The function of LC3II is to transport cargo to the autophagosome for degradation. The induction of LC3II can be visualized using immnuoblotting or immunofluorescent techniques. Another marker used to study autophagy is to measure p62 expression levels [32]. p62 interacts with ubiquitinated proteins and the ubiquitin-binding domain of LC3 and targets them for degradation. This marker can help identify defects in the autophagy machinery by observing an accumulation of p62. Detection of p62 can also be achieved using immnuoblotting or immunofluorescent protocols [33]. There has been recent clinical interest in developing agents that inhibit autophagy as a method to enhance cancer treatment. The majority of tumors have regions that contain quiescent, nondividing cells that may be resistant to chemotherapy and radiation therapy. Evidence suggests that these cells use autophagy to survive due to lack of nutrients and oxygen [34]. Therefore, targeting molecular components of autophagy could enhance cell killing in this subpopulation of tumor cells.

4.4 Colorimetric Assays for Cytotoxicity Assessment Colorimetric assays utilize compounds that are reduced by cells to become products that visibly change color, which can be detected spectrophotometrically [35]. Tetrazolium salts [MTT (3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide), XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium5-carboxanilide) and MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium)], are good candidates for this because

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they are reduced by cellular enzymes such as dehydrogenases and reductases to become detectable formazan dyes [36]. As such, colorimetric assays are used for analyzing cell growth by measuring the difference in formazan amount generated by control untreated cells to that of cells receiving various treatments (drugs, IR, etc.) [37, 38]. Colorimetric assays have advantages over the clonogenic survival assay because parts of the assay can be automated, making it easier and less time-­ consuming than the clonogenic assay. However, results from colorimetric assays can often be misleading. Cell killing can be wrongly interpreted from colorimetric assays for agents (drugs, IR, or combinations of both) that delay cell growth. A treatment might slow or delay growth, but it does not necessarily mean or slows cell growth does not necessarily mean that the treatment kills the cell. Likewise, it is not uncommon for cells exposed to IR to undergo several cell divisions before death occurs, making the length of time the colorimetric assay is conducted crucial to obtaining valid data [39]. Lastly, some tumor cell lines do not readily reduce the tetrazolium salt, creating ambiguities in obtaining statistically significant spectral data. Overall, colorimetric assays should be limited to use in largescale screening of drugs with or without IR treatment to identify candidate agents/ combinations for further testing by more reliable cell survival assays such as the clonogenic assay.

5 Radiation-Induced Damage: DNA Damage and Repair 5.1 DNA Repair As mentioned above, radiation produces a multitude of DNA lesions including base and nucleotide damage, cross-links with protein or DNA, single-strand breaks (SSBs) and double-strand breaks (DSBs). DNA damage can also trigger arrests in the cell cycle, presumably to allow time for repair of damage [40, 41] and initiate apoptosis or some other forms of cell death depending on p53 status [13]. Misrepaired DNA can occur when the cell attempts to resolve the insult but does so in an error prone manner. This can lead to point mutations, small deletions, inversions, and amplifications, which are not necessarily lethal to the cell, allowing for further propagation. Given enough time, and enough population doublings, these misrepaired cells have the potential to mutate and transform, eventually gaining carcinogenic potential. Because of the potentially devastating effects from IR as a result of the induction of DSBs, it is imperative to have mechanisms capable of repairing such lesions to maintain the integrity of the genome. While there are several pathways that are responsible for DNA damage repair, the two major pathways responsible for repairing DSBs are nonhomologous end-joining (NHEJ) and homologous recombination (HR) [42].

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5.2 Nonhomologous End-Joining The nonhomologous end-joining (NHEJ) pathway repairs DSB damage throughout the cell cycle and is the primary means for IR-induced DSB repair in mammalian cells (Fig. 4) [45]. DSBs produced by IR exposure are first recognized by the Ku70/ Ku80 complex, which then recruits DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to damaged site, one on each strand (Fig. 4). Once DNA-PKcs binds to Ku and the DNA, it forms the holoenzyme DNA-PK and becomes activated. In this state, DNA-PKcs is able to autophosphorylate, which is imperative for enzymatic function [46]. DNA-PK is responsible for juxtaposition of the DSB ends and recruitment of other NHEJ proteins such as the LigaseIV/XRCC4 complex that ligates the broken DSBs together. Defects in NHEJ DNA repair genes such as DNA-PKcs and ATM have been linked to cancer predisposition [47, 48]. Polymorphisms in the LigaseIV, Ku70 and a

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XRCC4 have been shown to correspond to breast cancer risk [49–52]. A more recent study has found that specific single nucleotide polymorphisms (SNPs) in Prkdc are associated with increased risk of breast cancer in Radiation Technologists [53].

5.3 Homologous Recombination HR takes place in S/G2-phase of the cell cycle and uses the undamaged sister chromatid as a template to repair the damaged chromosome arm in a relatively errorfree manner [54, 55]. Initially, the DSBs are recognized by the Mre11/Rad50/Nbs1 (MRN) complex and BRCA1 to process the ends into a 3¢ overhang. The singlestranded DNA (ssDNA) is recognized by replication protein A (RPA), which protects the strand and provides access for other HR proteins to the site. RPA is removed from the single-strand portion of the break and replaced Rad51. BRCA2 aides in the RAD51 nucleoprotein filament strand invasion at a homologous region of the sister chromatid to form a D-loop. The second DSB end invades a portion of the D-loop to form a Holliday junction. DNA polymerase is then recruited to the Holliday junction to fill in the missing sequence followed by the ligase/resolvase complex that resolves the complex and completes the repair. HR proteins are essential for cell survival, and mutations in genes coding for these proteins can greatly increase the risk of carcinogenesis. The most documented case linking HR and disease was the discovery of mutations in BRCA1/2, which has been fundamental in better understanding the link between genetic mutations and elevated breast cancer risk [56, 57].

5.4 Measurement of DNA Damage Repair While an understanding of the various mechanisms of DNA damage repair is important, having dependable assays to assess DNA damage and repair kinetics is vital to the development of radiation sensitivity modifiers (see below). Early techniques used to study DNA damage and repair kinetics involved exposing cells to high doses of IR and then evaluating DNA fragments produced by sucrose gradient centrifugation techniques [58], alkaline and neutral elution [59], and gel electrophoresis (PFGE) [60, 61]. With PFGE, smaller DNA fragments generated following IR exposure have a faster motility and will travel further down an agarose gel compared to larger fragments (see Fig.  5). As the DNA is repaired, less DNA elutes from the well, therefore by quantifying the DNA in the well vs. the DNA that elutes from the well, an accurate assessment of DNA damage repair over time can be determined. Although this method is good for measuring repair kinetics, the assay is laborious and requires high doses of radiation. Contemporary methods that are common for analyzing DSB repair include singlecell electrophoresis (the comet assay) [64] and phosphorylation of H2AX (gH2AX) [65]. The principle of the comet assay is similar to PFGE in that smaller DNA

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Fig. 5  (a) Pulsed-field gel electrophoresis of HT-29 cells exposed to 20 Gy and evaluated at 0, 3, 6, and 24 h postradiation treatment. Field inversion gel electrophoresis was carried out according to the methods of Stamato and Denko [62]. Briefly, irradiated cells were imbedded in agarose, digested at 55°C overnight in EDTA, Sarkosyl and Proteinase K, and then electrophoreses for 24 h at 56 V with a 3:1 forward:reverse pulse time ratio. After IR exposure, DNA repair occurs, which decreases the amount of DNA that can travel into the gel (as seen at the 3-h and 24-h time points). (b) Comet assay (or single-cell electrophoresis) showing an unirradiated cell versus a cell exposed to 8 Gy of g-rays at 0 h. The nucleus represents the head of the comet while the fragmented DNA appears scattered like the tail moment of the comet [63]. As DNA repair occurs over time, the amount of fragmented DNA decreases and the size of the tail moment is reduced

fragments travel faster than larger fragments through electrophoresis. This assay is beneficial because it requires less IR exposure, individual cells can be examined, and either SSB or DSBs can be assessed [63]. Irradiated cells are suspended in agarose, positioned on a slide, lysed, and then exposed to electrophoresis. DNA is then stained and examined under a microscope as seen in Fig. 5. Smaller fragments of DNA will migrate out of the nucleus under electrophoresis, generating an image that resembles a comet (the head being the nucleus and the tail moment is the fragmented DNA that traveled out of the nucleus). With time after IR, the comet tail decreases in size, representing repair of DNA damage. This assay is more sensitive than PFGE and easier to perform; however, scoring of the cells can be timeconsuming and tedious. Quantifying gamma histone H2A (gH2AX) is another technique utilized to measure DNA damage and repair, particularly for DSBs [65, 66]. In fact, gH2AX is the most common method to date that is applied to examine DSB repair kinetics. In the nucleus, chromosomal DNA is wrapped around protein scaffolds called histones: H2A, H2B, H3 and H4. It has been shown that histone H2A becomes phosphorylated and activated by damage response proteins like ATM at sites where DSB occurs; this activated form of H2A is called gH2AX. Fluorescent-tagged antibodies

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recognizing the phosphorylated gH2AX enable individual foci generated at each DSB site to be evaluated by microscopy (see Fig. 6a), by flow cytometry [68, 69] or and by western immunoblotting (see Fig. 6b). gH2AX is very useful in identifying agents that may inhibit IR repair (as shown in Fig. 6b) and also has applications as a biological IR dosimeter [67, 70].

6 Radiation-Induced Damage: Genomic Instability/Bystander Effects The acute effects of IR exposure at the cellular level are mediated through DNA damage to the nucleus of the cell as discussed above. Late effects of IR exposure, such as mutation and carcinogenesis, have traditionally been thought to be a result of damage to the nucleus of target cells and misrepair of the DNA. The consequence of such damage has been termed genomic instability (GI) and arises in the progeny of irradiated cells that can become apparent multiple generations after IR exposure [71–73]. In fact, the progeny that ultimately displays the phenotype of mutation or carcinogenesis were never directly exposed to IR. Such nontargeted genetic effects have received considerable attention over the past few years. Yet another recent development has emerged, termed the “bystander effect (BSE).” BSE is another nontargeted effect of IR where nuclear damage (DNA)

Fig. 6  Use of gH2AX to access IR-induced damage and repair [65, 66]. (a) Immunofluorescence using an anti-gH2AX primary antibody to detect DSB foci in unirradiated cells and cells exposed to 1  Gy. The number of foci (green dots over the cell nucleus) is elevated 15  min post-IR and decreases as a function of time after exposure, representing repair of DSB. (b) Western analysis of phosphorylated gH2AX from HT29 cells as a function of time following treatment with AZD7762 and radiation (8 Gy). Normalized density levels of protein bands from westerns, using H2A as the loading control for HT29 cells [67]. Note that gH2AX is elevated 0.5 h after IR and with time gH2AX levels return to near control levels by 24 h; however, with AZD7762 treatment, the levels of gH2AX do not return to control levels by 24 h, indicating that AZD7762 inhibits the repair of IR-induced DNA DSB (Adapted with permission from [67])

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can be observed by selectively irradiating the cytoplasm or a specific group of neighboring cells and subsequently observing DNA damage in the unirradiated cells [74–76]. BSE can also be studied using transfer protocols such as media transfer or cell transfer (coculturing irradiated cells with unirradiated cells) [77, 78]. DNA damage from the BSE is thought to be mediated in part by ROS [79]. Both GI and BSE exhibit similar types of DNA damage, including various cytogenetic alterations, mutations, and cell populations that exhibit oxidative stress and ROS production [72]. Thus, it is reasonable to hypothesize that GI and BSEinduced GI play an important role in radiation-induced carcinogenesis and mutation.

6.1 Measurements of Nontargeted Effects Genetic alterations resulting from IR-mediated GI or BSE include chromosomal aberrations and rearrangements, micronuclei, deletions and mutations. Additionally, another phenotype observed in various post-IR nontargeted cell populations is

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oxidative stress. An example of chromosomal aberrations and rearrangements that is measured in GI and BSE is shown in Fig. 7. Figure 7a shows chromatid aberrations (i.e., gaps and breaks) that arise from the previous cell cycle. Because of the nature of the aberration, it is an ideal assay to measure instability multiple generations after radiation exposure. If chromatid abnormalities persist to the next cell cycle, they form chromosomal aberrations such as terminal deletions, translocations, inversions and abnormal fusions [80]. Figure 7b shows an example of sister chromatid exchanges that is often used to measure the secondary effects of IR-induced BSE. When cells replicate DNA, genetically identical chromatids called sister chromatids are produced; these can be differentiated from each other detected using fluorescence plus Giemsa (FPG) [81]. A halogenated thymidine analog (5-bromodeoxyuridine, BUdR) is added to proliferating cells that will incorporate into newly synthesized DNA strands. Equal, or harlequin, ­chromatid staining is accomplished by incubating cells with BUdR for two ­population doublings so that the newly synthesized chromatid has the thymidine analog incorporated in both strands. Addition of Hoechst 33258 (a fluorescent stain) and exposure to UV light causes degradation of the newly synthesized stand, which stains lighter than the original strand when stained with Giemsa. Nontargeted bystander

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Fig. 7  Endpoints used to measure nontargeted effects of IR. (a) Chromatid aberrations such as gaps and breaks are elevated in irradiated cells due to IR-induced DNA damage. Chromatid aberrations are also observed in nontargeted cells that are progeny from irradiated cells. This observation is indicative of cytogenetic instability because chromatid aberrations only arise from the previous cell division [80]. (b) Sister chromatid exchanges (SCEs) (arrows) are induced in nontargeted cells and can be identified using FPG [76, 81]. Quantification of SCE frequency following treatment with IR, cytotoxic agents or carcinogens is widely used to assess GI and BSE

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effects of IR produce a significant increase in SCE ­frequencies in cells that have not been irradiated. In fact, this observation was how the BSE phenomenon was discovered [76]. Another method that is applied to analyzing GI and BSE of IR is to measure small DNA fragments that fail to incorporate into the nucleus after cell division, aptly named micronuclei (Fig.  8a). A micronucleus is relatively simple to detect and does not require a block in mitosis. Irradiated cells display a significant increase in MN due to the clastogenic effects of IR as shown in Fig. 8b, left panel [83]. There is also evidence demonstrating that the nontargeted effects of IR can also induce MN in unirradiated cells (Fig.  8b, right panel), potentially through signaling mechanisms or ROS. It is also worth noting that MN frequencies produced by the secondary effects do not appear to be dose dependent, unlike the direct effects of IR. MN are associated with cell division, and in order to properly evaluate this phenomenon, cells that have gone through only one round of replication (binucleated cells) are selected. This can be achieved by blocking cytokinesis using toxins such as cytochalasin B (Fig. 8a) [82]. Measuring mutation frequency is another endpoint used to study the effects of IR in unirradiated cells. Large chromosomal deletions are hallmark events that are a consequence of direct exposure to IR [84]. Secondary/nontargeted effects can produce more subtle changes such as small deletions, inversions, or point ­mutations. Examples of models used to measure elevated mutations frequencies resulting from GI and BSE are the hypoxanthine–guanine phosphoribosyltransferase (HGPRT) system­and the CHO AL system [85, 86]. HGPRT is a gene located on the X-chromosome and is involved in purine synthesis. Cells with normal HGRPT are able to grow in HAT media that contains

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Fig. 8  (a) Micronuclei are unrepaired DNA fragments that fail to incorporate into the nucleus during cell division. Bi-nucleated cells, obtained using cytochlasin B, are normally scored to ensure accurate measurement of IR-induced damage [82]. (b) Similar to chromatid aberrations, directly irradiated cells exhibit an increase of MN with increasing IR dose (left panel) [83]. Nontargeted effects of IR also show an increase of MN in unirradiated cells, independent of IR dose, indicative of the BSE (Adapted with permission from [83])

animopterin (blocks de novo purine synthesis), hypoxanthine, and thymidine, but die in TG (6-thioguanine: a toxic thymidine analog) media. However, HAT media is cytotoxic to cells that harbor HGRPT mutations but grow in TG media. There are several studies that demonstrate an increase in HGPRT mutations in nontargeted cells, which suggests that nontargeted effects produced by IR effect can have ­carcinogenic potential [87–89].

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The Chinese hamster ovary (CHO) CHO AL/human hybrid cell line was formed by fusion of human amniotic fluid fibroblasts with the gly-A mutant CHO-K1 cell line in the presence of the hybrid-inducing Sendai virus and media that is selective for CHO/human hybrid cells [90–92]. This cell line contains human chromosome 11 that expresses several human genes including AL, which encodes the cell surface protein CD59 [86, 90, 93]. After exposure to mutagens, mutants lacking CD59 antigen can be scored by using complement plus anti-CD59 antibody. While mutants will survive from the treatment and form colonies, wild-type cells are efficiently lysed. A more recent approach to mutation detection in this cell line is the use of fluorescent-labeled CD59 antibodies and flow cytometry [86, 93]. Similar to the HGPRT assay, the CHO AL mutation assay demonstrates an induction in mutations, providing further evidence that the nontargeted effects of IR have mutagenic potential [94, 95]. Several reports demonstrate that oxidative stress increases in nontargeted cells affected by IR. Although there is no definitive explanation as to why this is the case, signaling molecules, the induction of chromosomal instability and mutation frequency could all contribute to the phenomenon. Methods designed to measure oxidative stress including dihydroethidium (5-ethyl-5,6-dihydro-6-phenyl-3, 8-diaminophenanthridine, hydroethidine, DHE) and 2¢,7¢-dihydrodichlorofluorescein (H2DCF-DA). DHE and H2DCF-DA are lipophilic compounds that are able to cross cell membranes and upon oxidation become fluorescent, which can then be detected using flow cytometry. The acetate groups of H2DCF-DA are cleaved in the presence of oxidation, mainly hydrogen peroxide, which then becomes the highly fluorescent 2¢,7¢-dichlorofluorescein (DCF). DHE is mainly used to study superoxide, which converts DHE to ethidium and 2-hydroxyethidium that can intercalate into DNA and cause fluorescence. However the specificity of DHE for superoxide has recently been questioned and the recommendation of limiting this assay to HPLC instrumentation has been suggested [96, 97].

7 Radiation Responses Using In Vivo Models In vitro models enable the elucidation of mechanisms of action of IR effects under controlled laboratory conditions. In the case of translational research, principles learned from cell studies are ultimately applied to in vivo models. Animal models take into account the complexities of metabolism, physiology, and, in the case of tumors, the influence of tumor microenvironment. A host of quantitative IR models has been developed over the past 40 years to assess the radiation response of normal tissues and tumors predominantly using mouse models. It is beyond the scope of this chapter to cover all of these protocols. Many of the normal tissue assays are described elsewhere [1, 98, 99]. We will focus on several methods used to evaluate the response of tumors to IR, as these models are heavily used in the development of novel radiation modifiers for clinical applications.

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A wide range of tumor cell types and their responses to IR can be evaluated using laboratory animals and transplanted tumor systems. Both murine tumors and human tumors grown as xenografts in immune-compromised mice (athymic nude or SCID) are used. Tumor growth delay and tumor cure dose (TCD50) assays are the most common models used. For growth delay experiments, tumor cells are injected into donor animals and tumors are allowed to grow to a specific size. Animals are then divided randomly into various treatment groups (i.e., with/without IR; single/ fractionated doses; with/without a drug of interest). Generally, in response to treatment, the tumor will decrease in size due to cell death but will gradually regrow over a period of time that is dependent on the IR dose utilized. This delay between the original tumor size and time required for the tumor to regrow to 2–3 times the original tumor volume represents the regrowth delay. An example of an IR tumor regrowth model with and without an experimental targeted radiation sensitizer is shown in Fig.  9a. As can be seen, the combination of the Chk1 inhibitor (AZD) with fractionated radiation (FX) gave an enhanced tumor growth delay over the drug alone or IR alone [67]. The TCD50 assay is similar to growth delay studies but utilizes a different endpoint. In TCD50 experiments, the objective is to determine the IR dose (with or without an IR modifier) that results in tumor cure in 50% of the animals [100] (see Fig. 9b). This model, along with the tumor regrowth assay, is very useful in evaluating novel radiation modifiers for clinical applications. While these models might be criticized because they represent the IR response of mouse tumors or human tumor b

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growing in a murine host, they nonetheless represent an in  vivo model to either confirm or reject initial in vitro assessments and provide some assurance that the tested agents will exhibit activity in humans.

8 Modifiers of the Radiation Response Perhaps the largest application of IR models (in  vitro and in  vivo) is directed to means of modifying the radiation response, either for enhancement (IR sensitizers) or protection (IR protectors). For oncology research identifying nontoxic agents that selectively modify the IR response of tumor and/or normal tissues would allow for a higher IR dose to be delivered to the tumor, which could translate into a higher local tumor control probability.

8.1 Radiosensitizers An example of a radiation sensitizer is shown in Fig. 10. Halogenated pyrimidines (such as bromo- and iododeoxyurdine, BUdR or IUdR) have chemical structures similar to the DNA base thymidine [19]. Replacement of thymidine in the cell culture medium with either BUdR or IUdR results in cells incorporating these molecules into the DNA molecule in the place of thymidine. As a result of this replacement, the cells are made more sensitive to radiation as can be seen in Fig. 10. The extent of enhancement/sensitization can be quantified in terms of a dose-modifying factor (DMF) calculated at an iso-survival level, typically at the 10% survival. In this example, cells were made 1.8 times more sensitive to radiation by the agents. The DMF is a useful parameter in comparing and contrasting different radiation sensitizers for consideration of further testing in vivo. In general, agents yielding DMF values ³1.5 are thought to be worthy of additional in vivo studies. In conducting in vitro radiation sensitizer studies, there are several guidelines to consider, including the mechanism of action of the agent, and whether the agent exerts cytotoxicity alone or induces cell cycle blocks. Most of the radiation sensitizers in radiotherapy today are cytotoxic chemotherapy agents, such as cisplatin, 5-fluorouracil, and gemcitabine [103–105]. In addition to being radiation sensitizers, these agents exert cytotoxicity on their own. Further, since none of these agents provides selective radiation sensitization to tumor cells, the enhancement of normal tissue within the radiation treatment field can be problematic. A major emphasis at present is to target various molecular pathways that tumor cells exploit to perhaps obtain nontoxic tumor selective radiation sensitizers. Molecular agents that target survival, apoptosis, angiogenesis, DNA repair, and cell cycle pathways are currently under development [106]. Several drugs now exist that inhibit the function of DNA repair proteins, such as ATM, DNA-PKcs and poly (ADP-ribose) polymerase-1 (PARP-1) [107–109].

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Fig.  10  Enhancement of the IR sensitivity of Chinese hamster V79 cells by two halogenated pyrimidines [19]. Pretreatment of cells with halogenated pyrimidines (IUdR, BUdR) made them more sensitive to radiation. The extent of enhancement is determined by calculating a dose modification factor (DMF) from the survival curves. An iso-survival level is chosen typically £10% and the IR dose for control cells is divided by the IR dose for drug treated cells. In this example, the DMF was 1.8, meaning that the drug treatment rendered the cells 1.8-fold more sensitive to IR. Survival curve analysis can be used for any agent thought to modify radiation sensitivity. The DMF value is useful in comparing the enhancement or protection among diverse agents (Adapted with permission from [10])

Promising compounds, such as checkpoint kinase inhibitors (i.e., Chk1/2) [110], target the cell cycle and allow IR-induced damaged cells to slip past checkpoints and progress through the mitosis that increases the likelihood for apoptosis. Targeting proteins involved with survival, such as epidermal growth factor receptor (EGFR) [111], phosphoinositide 3-kinases (PI3K) [112], AKT [113], and nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) [114], have all been exploited and show promise as radiosensitizers. Inhibiting apoptosis proteins such as survivan [115], p53 [116], Bcl-2 [117] and X-linked inhibitor of apoptosis protein (XIAP) [118] have also been shown to improve cell-killing after IR exposure. Vascular endothelial growth factor (VEGF) [119] is of particular interest because it plays a role in the induction of tumor angiogenesis, and inhibition of this protein has also shown increased IR-treated tumor control. Most recently, inhibition of epigenetic proteins that modulate chromatin structure, such as heat shock protein-90 (Hsp-90) or histone deacetylase (HDAC), has also demonstrated enhanced radiosensitivity [120, 121].

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8.2 IR Protectors The purpose of radioprotectors is to provide selective protection of normal tissue in cancer patients receiving radiotherapy as well as protection of people against accidental or unintentional exposure to IR. Classical chemical radiation protectors scavenge IR-induced free radicals and decrease cellular damage [1, 3]. Aminothiol chemical radiation protectors were discovered in the late 1940s [122]. Subsequent studies funded by the U.S. Army identified a thiophosphate compound named S-2-(3-aminopropylamino), or amifostine, that demonstrated impressive radioprotection in vitro and in vivo [123]. Amifostine is the only FDA approved radiation protector. Nitroxides, another class of chemical radioprotectors, efficiently scavenge IR-induced radicals and provide significant protection against IR both in vitro and in vivo. In addition to the in vitro studies outlined above, in vivo IR protection studies often evaluate the efficacy of an agent to protect animals against IR-induced lethality. LD50/30 values (lethal IR dose in 50% of the animals, 30 days post-IR) are obtained for IR alone and IR plus the potential protector. Typically a protector factor (PF, LD50/30 value of IR + protector divided by LD50/30 value of IR alone) is calculated with PFs >1.0 demonstrating IR protection. Other types of radiation protectors/mitigators are currently under development or evaluation [124–127]. The in vitro and in vivo models outlined in this chapter can be used in the assessment of potential IR protectors.

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Experimental Models to Study Cigarette Smoke-Induced Oxidative Stress In Vitro and In Vivo in Preclinical Models, and in Smokers and Patients with Airways Disease Hongwei Yao and Irfan Rahman

Abstract  Oxidative stress is one of the important hallmarks of various chronic inflammatory lung diseases including chronic obstructive pulmonary disease (COPD). Cigarette smoke contains high concentrations of oxidants/free radicals and different chemical compounds including reactive aldehydes, quinones, and semiquinones. It has been shown that cigarette smoke is the major risk factor for the development of COPD by inducing oxidative stress and lung inflammation. Therefore, studying cigarette smoke-induced oxidative stress in  vitro and in  vivo models is useful for understanding the pathogenesis of chronic inflammatory lung diseases, and developing the potential antioxidants and anti-inflammatory therapies to intervene in the progression of COPD/emphysema. In this chapter, we discuss these in vitro and in vivo experimental models induced by cigarette smoke exposures. Furthermore, human samples, such as exhaled breath condensate, induced sputum, and bronchoalveolar lavage from smokers and patients with COPD, are also useful for investigating the association of oxidative stress with the progression of COPD. However, all these models cannot mimic the exact phenotypic alterations seen in human smokers and COPD patients. Oxidative stress biomarkers are also used to monitor the progression and prognosis of cigarette smoke-induced diseases, such as COPD. Keywords  Biomarkers • Cigarette smoke • COPD • Experimental models • Oxidative stress

I. Rahman (*) Department of Environmental Medicine, Lung Biology and Disease Program, University of Rochester Medical Center, Box 850, 601 Elmwood Avenue, Rochester, 14642, NY, USA e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_18, © Springer Science+Business Media, LLC 2011

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1 Introduction Reactive oxygen species (ROS) are shown to cause oxidation of proteins, DNA, and lipids leading to cell dysfunction. The lung is susceptible to injury induced by endogenous and exogenous oxidants and pollutants (e.g., cigarette smoke, mineral dust, and ozone) owing to its large surface area and extensive blood supply. Indeed, increased oxidative stress is one of the important events in various chronic inflammatory lung diseases including asthma, pulmonary fibrosis, and chronic obstructive pulmonary disease (COPD). Increased concentrations of ROS in the lungs of smokers and in patients with COPD are reflected by elevated levels of oxidative stress biomarkers in exhaled breath, airways, lung tissue and blood. A variety of approaches are currently being used to target oxidative stress with antioxidants or boost the endogenous levels of antioxidants to intervene in the progression of these diseases. Cigarette smoke is a major risk factor for the development of COPD, and it ­contains an estimated 1015 to 1017 oxidants/free radicals and ~4,700 different chemical compounds including reactive aldehydes, quinones, and semiquinones per puff. We and others have shown that oxidative stress mediated by cigarette smoke is shown to induce inflammation, apoptosis, senescence, and autophagy in lung cells in  vitro and in  vivo, which are associated with the pathogenesis of COPD [1–9]. Therefore, these cigarette smoke-induced in vitro and in vivo models are useful for studying the pathogenesis and developing the potential antioxidants and anti-inflammatory agents to treat inflammatory lung diseases including COPD/emphysema. In this chapter, we have discussed these in vitro and in vivo experimental models for studying the effects of cigarette smoke-induced oxidative stress in lung inflammatory response. Human samples, such as exhaled breath condensate (EBC), induced sputum, bronchoalveolar lavage fluid (BALF), and lung tissues from smokers and patients with COPD, are useful for investigating the association between oxidative stress in smokers and the progression of COPD. Furthermore, we have also provided information on a variety of oxidative stress biomarkers for monitoring the progression and prognosis of oxidative stress/cigarette smoke-induced diseases.

2 In Vitro Cigarette Smoke Treatment Model A variety of lung cells, including epithelial/endothelial cells, fibroblasts, and immune-inflammatory cell macrophages and lymphocytes are used to study oxidative stress in response to cigarette smoke extract (CSE) or cigarette smoke condensate (CSC) treatments. In addition, air–liquid interface exposure system is also utilized to expose cells (e.g., epithelial cells) to mainstream smoke. This is to allow both the particulate matter and the vapor phase of cigarette smoke to make contact with the cells, as opposed to both CSE and CSC, which contain only the particulates [10]. The purpose of an in vitro model is to simplify the experiment and to limit variability. Thus, specific and focused scientific questions, such as cell type-dependent effect and signaling pathway mediated by cigarette smoke can be determined using these in  vitro models (Table  1). However, the extrapolation of

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Table 1  Pros and cons of in vitro cigarette smoke exposure CSE and CSC treatment Air–liquid exposure Usage of cells Cell lines or primary cells Primary cells Components of Particulate matter Particulate matter and vapor phase cigarette smoke Pros 1. Limitation of variability in vivo 2. Study the signaling pathway/mechanism by cigarette smoke using manipulating system Cons Loss of volatile and rapidly reactive components in cigarette

these data to humans in  vivo should be applied with care, since CSE or CSC is aqueous of cigarette smoke.

2.1 Preparation of Aqueous CSE Research grade cigarettes (e.g., 1R3F and 2R4F) are routinely obtained from the Kentucky Tobacco Research and Development Center at the University of Kentucky, Lexington, KY, USA. CSE (10%) is prepared by bubbling smoke from one cigarette in 10 ml of culture media supplemented with 1% FBS at a rate of one cigarette per minute [4, 5]. A representative apparatus for CSE preparation is shown in Fig. 1. The pH of the CSE is adjusted to 7.4 and is sterile filtered through a 0.45 mm filter (25 mm Acrodisc; Pall Corporation, Ann Arbor, MI). CSE preparation is standardized by measuring the optical density (OD, 0.735 ± 0.05) at 320 nm. The pattern of absorbance (spectrogram) at l320 usually shows very little variation between different preparations of CSE. CSE is freshly prepared for each experiment and diluted with culture media supplemented with 1% FBS as required immediately before use. Control medium is prepared by bubbling air through 10 ml of culture media supplemented with 1% FBS, adjusting pH to 7.4, and sterile filtering as described for 10% CSE. Treatment with CSE, which is prepared according to the above protocol, significantly caused oxidative stress in a variety of alveolar epithelial cell lines as well as in primary human small airway epithelial cells (SAEC) [5]. Interestingly, the NF-kB activation and proinflammatory cytokine release occurred in SAEC, but not in any of the transformed epithelial cell lines studied, suggesting that primary, but not transformed, lung epithelial cells are an appropriate model for studying the inflammatory mechanisms in response to cigarette smoke in an in vitro system [5].

2.2 Preparation of CSC CSC is generated by passing cigarette smoke through Ca2+/Mg2+-free PBS using a 25 ml round shaped tonometer [6, 11]. Briefly, cigarette smoke is extracted using a syringe on an automatic pump system; this is then passed through rubber tubing to the tonometer that contained the Ca2+/Mg2+-free PBS. The tonometer rotates, causing

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Vacuum 1

2

4

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Fig. 1  A representative apparatus to prepare CSE. For CSE preparation, a cigarette is lit (1) which is attached to a cigarette holder and is connected with a Pasteur pipette (2). When the vacuum is switched to ‘ON’ cigarette smoke is drawn into 50-ml falcon tube (3) through Pasteur pipette (2), where the smoke bubbles through 10 ml of culture medium supplemented with 1% FBS (4). Label (4) is the 10% of CSE used for in vitro experiments after sterile-filtered through a 0.45 mm filter and standardized by measuring the optical density at a wavelength of 320 nm. One cigarette is burned in 1 min by adjusting the vacuum in order to obtain similar optical density between different preparations of CSE

the Ca2+/Mg2+-free PBS to form a large meniscus around the bulb of the tonometer, thus representing the fluid in the alveoli air sac. The cigarette smoke is pumped out of the tonometer through a barrel that is used for fresh cigarette smoke being pumped in. A ratio of one cigarette per 1 ml of Ca2+/Mg2+-free PBS is used, with a minimum of 2 ml and maximum of 4 ml made up at any one time. This ratio is taken as 100% of CSC. CSC is freshly made just before the use for every experiment. CSC can also be prepared by using a McChesney-Jaeger CSM-SSM Single Cigarette Machine (CH-Technologies Inc.). Briefly, CSC from a 1R3F researchgrade cigarette is generated according to international smoking standards or Federal Trade Commission protocol, and a 35 ml puff is taken over a 2-s time period every minute and trapped on a preweighed Cambridge filter paper. The collected CSC on filter paper is weighed and subsequently dissolved in dimethyl sulfoxide (DMSO) and expressed as mg of CSE/ml. The optical density is 0.75 ± 0.03 at a wavelength of 320  nm, which was found to be similar to our CSE preparation as described above. Similar to CSE, treatment with CSC caused oxidative stress, but not induced NF-kB activation and proinflammatory cytokines release in a transformed epithelial cell line (A549 cell) [6, 11]. However, the pro-inflammatory effect of CSE was seen in primary small airway epithelial cells [5].

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2.3 Biophysical Comparison and Standardization of Different Research Grade Cigarettes Based on Their Total Particular Matter Concentration In order to standardize CSE preparation based on total TPM (total particular ­matter), 1R3F (TPM concentration 18.1 mg/cigarette) and 2R4F (TPM concentration 11.7 mg/cigarette), cigarettes are bubbled into 10 ml of culture media with 1% FBS as mentioned above to achieve 10% CSE. The optical density measured for 1R3F is 0.74 ± 0.05, whereas that of 2R4F is 0.58 ± 0.03 at a wavelength of 320 nm. In order to optimize the CSE concentration based on TPM concentration, one cigarette of 2R4F is bubbled in 6.5 ml of culture media supplemented with 1% FBS and the optical density obtained at 320  nm is 0.73 ± 0.04, which was similar to that of 1R3F research cigarette bubbled in 10 ml of media. Hence, the optical density of CSE from different brand of cigarettes has to be determined based on TPM/tar concentration to achieve the comparable results before cell treatment. This is critical, since we found that treatment of human monocyte– macrophage cell line (MonoMac6 cells) and SAEC with CSE (1%) from 1R3F and 2R4F significantly depleted the intracellular glutathione (GSH) and increased ROS release. However, treatment with CSE (1%) from smokeless cigarettes did not cause any alteration of intracellular GSH levels and ROS release in these cells (Fig. 2). These differential effects were due to the different TPM in these cigarettes (Table 2).

2.4 Air–Liquid Interface System Cigarette smoke is a mixture of several thousand chemicals distributed in vapor phase (~95%) and particulate phase (5%). However, both CSE and CSC only contain the particulate fraction of smoke, and volatile and reactive components are lost during their aqueous solution preparation. Thus, air–liquid interface exposure system is introduced to mimic in vivo smoke exposure to lung airway epithelial cells [10]. Primary lung epithelial cells are grown on a porous membrane and placed inside a special smoke chamber. Through peristaltic motion, warm media is moved over the bottom of the cells, while cigarette smoke flows over the top of the cells at a concentration of 150 mg/m3 TPM, which is shown to cause a significant inflammatory response with acceptable levels of cell toxicity. In control, cells are exposed to filtered air alone. At different time points after smoke exposure, cells are transferred into 24-well plate with 300 ml of basal serum-free medium, and returned to cell culture incubator at 37°C for a selected time period. Our preliminary studies demonstrated that exposure to cigarette smoke for 5 min using the air–liquid interface system, then incubated at 37°C for 24 h, led to a significant increase in IL-8 release in lung epithelial H292 cells.

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Fig.  2  Effect of CSE from different type of cigarettes on GSH level and ROS release in macrophages and small airway epithelial cells. Human monocyte–macrophage cell line (MonoMac6 cells) and human small airway epithelial cells (SAEC) are seeded in a 6-well plate at a density of 1.0–1.5 × 106/well. After starvation overnight in 2 ml of culture media supplemented with 1% FBS, the cells are treated with CSE (1.0%) from smokeless, nicotine-free, 2R4F, and 1R3F cigarettes for 24  h. After harvesting, the cells are lysed or spun on cytospin slides for determination of GSH levels and ROS release (using CH2-DCFDA staining) respectively, as described in Section 5.2. MonoMac6 cells and SAEC treated with CSE (1%) from nicotine-free, 1R3F, and 2R4F cigarettes significantly depleted the intracellular glutathione (GSH) and increased ROS release. However, treatment with 1% of CSE from smokeless cigarettes did not cause any alteration of intracellular GSH levels and ROS release in these cells. ***P < 0.001, compared with respective control Table 2  Comparison of quit-smoking products and research grade cigarettes

Cigarettes Smokeless

TPM (mg/cig) 0

TAR (mg/cig) 0 7.0

Nicotine (mg/cig) 0

OD at 320 nm of 10% CSE −0.008 ± 0.002

0

0.53 ± 0.002

Nicotinefree

Not known

2R4F

11.7

11.0

0.80

0.56 ± 0.002

1R3F

17.1

15

1.16

0.74 ± 0.002

Equivalent commercial Manufacturer cigarette Grant Light-free works, FL cigarette Smoke free NonInc., MI nicotine cigarette University of Marlboro Light Kentucky, KY Marlboro Regular

3 In Vivo Models to Investigate Cigarette Smoke-Induced Oxidative Stress Cigarette smoke exposure to in vitro models is useful for studying the acute effect and signaling pathway mediated by oxidative stress. However, both CSE and CSC differ from gaseous smoke, making it difficult to determine what dose and exposure

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time best extrapolate and discern the effect of smoking in the lung. Thus, cigarette smoke exposure in in vivo animal models have been developed to investigate the inflammatory and injurious lung responses to cigarette smoke [12–15]. Here we have discussed the acute, subchronic and chronic cigarette smoke exposures in mice and rats using different smoke generating machines.

3.1 Cigarette Smoke Exposure to Mice Eight- to ten-week-old littermate mice (22–25  g) are used for cigarette smoke exposure at two different concentrations of TPM for 3 days [12]. In brief, mice are housed in an individual wire cage compartment, which is placed inside an aerated plastic box connected to the smoke source. Research grade cigarettes (e.g., 2R4F and 3R4F) are used to generate smoke, and mice are exposed according to the Federal Trade Commission protocol (1 puff/min of 2-s duration and 35-ml volume) using a Baumgartner-Jaeger CSM2072i automatic cigarette smoke-generating and exposure machine (CH Technologies, Westwood, NJ). Mains-tream cigarette smoke is diluted with filtered air and directed into the exposure chamber. The smoke exposure (TPM in per cubic meter of air) is monitored in real time with a MicroDust Pro-aerosol monitor (Casella CEL, Bedford, UK) and verified daily by gravimetric sampling [7, 16–19]. Meanwhile, the chamber atmosphere is also monitored for TPM by adjusting the flow rate of diluted medical air [7, 17–19]. The mice receive two 1-h cigarette smoke exposures, 1 h apart so as to avoid CO poisoning since higher concentrations of TPM lead to intolerable carboxyhaemoglobin (CoHb) levels, for 3 consecutive days with a concentration of 300 mg/m3 TPM, and are sacrificed at 2 h and 24 h post-last exposure. For 80 mg/m3 TPM cigarette smoke exposure, the mice are exposed for 5–6 h per day for 3 consecutive days, and sacrificed at 24 h post-last exposure. The carbon monoxide (CO) level is 279 and 79.4 ppm in chamber with the 300 and 80 mg/m3 TPM exposure protocols respectively. No significant change in body weight in mice is observed in response to 3 days of cigarette smoke exposure. Control mice are exposed to filtered air in an identical chamber, according to the same protocol described for cigarette smoke exposure. The neutrophils influx into BALF in response to CS exposure is dose-dependent, using a Baumgartner-Jaeger CSM2072i automatic cigarette smoking machine according to above exposure protocol when mice are sacrificed at 24 h of post-last exposure [12]. Furthermore, the number of neutrophils is found to be much higher in BALF of mice killed at 24 h than that at 2 h after the last CS exposure, i.e., five times higher in C57BL/6J mice. This suggests that the acute effects of CS exposure (within hours after the end of CS exposures) are related to suppression of the number of neutrophils recovered in the lavage, possibly due to capillary trapping or increased adhesion in the interstitium or alveolar compartment. For subchronic and chronic cigarette smoke exposure, 3R4F or 2R4F cigarettes (University of Kentucky) are used to generate a mixture of sidestream smoke (89%) and mainstream smoke (11%) by a Teague smoking machine (Model TE-10,

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Teague Enterprises, Woodland, CA) at a concentration of ~100 mg/m3 TPM in the chamber [20]. Each smoldering cigarette is puffed for 2 s once every minute for a total of 8 puffs, at a flow rate of 1.05 l/min, to provide a standard puff of 35 cm3. The CO level in chamber during cigarette smoke exposure is 375  ppm. Mice receive 5-h exposures per day, 5 days/week for 2, 4, or 6 months, and are sacrificed at 24 h post-last exposure. It is interesting to note that no neutrophils are infiltrated into the BALF using a Teague machine to expose mice to cigarette smoke according to the above protocols. However, the number of total cells and macrophages in BALF increases in a time-dependent manner between 2 and 6 months of cigarette smoke exposure in mice [20].

3.2 Cigarette Smoke Exposure in Rats Sprague-Dawley rats (300-350 g) (Charles River) are exposed to air or cigarette smoke for 3 days, 8 weeks, 6 months, or 8 months [7, 21, 22] or using the Baumgartner-Jaeger CSM2072i automatic cigarette smoking machine according to the above exposure protocol [12]. The smoke concentration is set at a nominal value of 600 mg/m3 TPM by adjusting the flow rate of the dilution air. Rats receive two 1-h exposures, 1 h apart, according to the Federal Trade Commission protocol (1 puff/min of 2 s duration and 35 ml volume) for 3 days. Rats are sacrified at 1, 4 and 24 h after the last exposure by an intraperitoneal injection of 50 mg/kg (body weight) sodium pentobarbital. Rats are also exposed to cigarette smoke for 3 days, 8 weeks, 6 months, or 8 months using the whole body cigarette smoke exposure generated from 2R4F research cigarettes (University of Kentucky, Louisville, KY, USA) in 7 liters smoking chambers at 4 cigarettes per day, 5 days per week [7, 21, 22]. The TPM consists of 27.1 ± 0.8 mg/cigarette. To ensure a consistent exposure across the exposed animals, cotinine levels are measured. Plasma cotinine levels are 2.66 ± 0.12 mM and 0.51 ± 0.07  mM at 1  h and 24  h post-last exposure, respectively. Cotinine is not detectable in the air-exposed animals. There was no change in cotinine levels during 1 week of exposures. Carboxyhemaglobin (COHb) levels are measured immediately after the animals are removed from the chambers. A peak level of COHb (42 ± 4.0 mM) is reached after the fourth cigarette, which quickly decreased after exposure was stopped. Air-exposed animals are exposed to medical grade air in 7  liters smoking chambers, 5 days per week. Rats are sacrificed at 2  h post-last exposure by intraperitoneal injection of an over-dose of sodium pentobarbital. It is shown that 6 months of cigarette smoke exposure did not cause airspace enlargement, whereas 8 months of cigarette smoke exposure caused emphysema in rats [22]. Therefore, a long-term exposure to cigarette smoke is carried out when using rats to induce airspace enlargement/emphysema, although emphysematous lung in rats does recapitulate many of the phenotypic changes observed in patients with COPD.

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3.3 Characteristics of Cigarette Smoke-Induced Lung Inflammationand Emphysema It has been shown that acute or short-term cigarette smoke exposure is a useful model to study the molecular mechanism of airways diseases, and pharmacological intervention of certain agents in cigarette smoke-induced oxidative stress and inflammation [12, 18, 23]. Since cigarette smoke is the predominant cause of COPD, animal models using cigarette smoke exposure would be the logical choice for investigation. The mechanism to induce emphysema by cigarette smoke is associated with apoptosis, premature aging, inflammation, extracellular matrix and vascular remodeling. Mice are generally used to establish cigarette smoke-induced COPD/emphysema in terms of low cost, availability of gene/protein sequence and commercial antibodies, and the feasibility of genetic modifications in mice. Six months of cigarette smoke exposure is shown to cause emphysema in C57BL/6J mice. However, induction of lung inflammation and emphysema varies between different strains of mice [12, 15, 24–26] (Table 3). For example, the AKR strain is highly susceptible, whereas the A/J and C57BL/6J strains are moderately sensitive, and NZW and ICR mice are resistant to cigarette smoke-induced emphysema. This may be due to the differential thiol/antioxidant status in the lung of these mouse strains [27] as well as variability in genetic make-up. Lack of respiratory bronchioles in mice renders them unable to develop centrilobular emphysema, which is often seen in COPD, in response to chronic cigarette smoke exposure. Furthermore, the severity of cigarette smoke-induced emphysema never reaches the same degree as seen in human smokers, even after prolonged exposure. This may be the reason that structure emphysema does not correlate with lung compliance in mice after chronic cigarette smoke exposure, since pulmonary function tests are less sensitive than morphometry and may detect only severe degrees of parenchymal destruction [28]. The mechanism underlying these variations is not clear and remains to be elucidated. Subchronic (2 and 4 months) cigarette smoke exposure can be used to study the gradual progression of COPD/emphysema. Moreover, Table 3  Characteristics of cigarette smoke-induced lung inflammation and emphysema Strain Species Smoke machine

Emphysema location

Emphysema severity

Strain-dependent lung inflammation and emphysema by cigarette smoke Cigarette smoke-induced emphysema occurs in mice whereas no emphysema is seen in rats after 6 months’ exposure Cigarette smoke exposure using a Teague machine does not cause neutrophils influx in bronchoalveolar lavage fluid of mice whereas the Baumgartner-Jaeger CSM2072i automatic cigarette smoking machine does Cigarette smoke-induced emphysema is patchy rather than centrilobular in mice since mice do not have the respiratory bronchioles The severity of cigarette smoke-induced emphysema in mice never reaches to the degree, which is seen in human smokers

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these subchronic models are also important to determine/identify the susceptible genes/factors in pathogenesis of COPD.

4 Collection of Samples from Smokers and COPD Patients to Determine Oxidative Stress Markers It is known that oxidative stress plays an important role in the pathogenesis of COPD/emphysema. Oxidative stress is shown to be an important biomarker to monitor the progression and prognosis of this disease. A variety of samples/­ biofluids, such as EBC, induced sputum, and urine or blood and BALF can be collected from smokers and COPD patients though noninvasive and invasive approaches to determine the association of oxidative stress with the disease.

4.1 Exhaled Breath Condensate The collection of EBC has been described in detail [29–31]. In brief, the EcoScreen (Jaeger, Wurzburg, Germany) condenser is used to collect EBC after rinsing out the mouth with saline. Subjects usually breathe at normal frequency and tidal volume for 15 min through the mouthpiece (with a two-way, non-rebreathing value) wearing nose clips during the entire procedure. The collected EBC (approximately 2.0 ml) is stored immediately (lipid peroxidation inhibitor butylated hydroxytoluene and 0.1 mM of EDTA are added prior to freezing) in aliquots at –70°C, and is analyzed within 3 months. EBC is condensed from water, but also contains droplets that carry solute from the lower respiratory tract. Collecting EBC by cooling exhaled air is simple to conduct, relatively inexpensive, noninvasive and easily repeatable [32]. It does not cause any discomfort or risk to the subject, nor does it cause airway inflammation and alteration in lung function [33–35]. Moreover, EBC collection is also suitable for mechanically ventilated patients. There is accumulating evidence suggesting that the abnormalities in biomarkers present in EBC may reflect the severity of COPD better than those assessed by either spirometry or symptoms [33, 36]. EBC mirrors the composition of the extracellular lining fluid but the major drawbacks are saliva contamination, sample dilution, and high individual variability. Significant salivary contamination can be excluded by measuring the amylase concentrations in samples, but uncertainty still exists with respect to nasal contamination [33]. Dilution of EBC can be corrected by measuring the levels of urea and/or electrolytes derived from extracellular lining fluid [37]. Other confounding factors include consumption of alcohol, diets with variable amounts of endogenous or dietary intake of antioxidants, smoking and subclinical lung diseases. Overall, the collection of EBC is easy and simple, and it is, therefore, promising for the evaluation of oxidant markers. Major limitations in the EBC include a plethora of potential markers that cannot be reliably or easily analyzed in these highly diluted samples.

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4.2 Induced Sputum Sputum induction is performed according to a standardized procedure [38]. Subjects/patients are asked to inhale nebulized hypertonic saline using a DeVilbiss UltraNeb 2000 ultrasonic nebulizer (Devilbiss Healthcare, Heston, UK) during the normal tidal breathing for 5 min with 3% saline initially, and if unsuccessful, 4 and 5% saline are given. The nebulizer had an output of approximately 2 ml per minute, with a mean particle size of 0.5–5 mm in diameter. After each period of inhalation, forced expiratory volume in 1  s (FEV1) is measured and nebulization continues only if the FEV1 does not fall by more than 20%. All sputum samples were processed within 2 h of production by the method of Popov et al. [39]. In brief, sputum plugs are separated from contaminating saliva by macroscopic examination, and the weight is recorded. Sputum is dispersed using 0.1% dithiothreitol (Sputolysin 10%; Calbiochem, La Jolla, CA, USA), a solution prepared in PBS, which is four times the weight of the selected sputum that is added, vortexed for 30 s and mixed on ice for 15 min to homogenize the mucus. A volume of PBS equivalent to the dithiothreitol volume is then added, and the sample filtered through 48  mm nylon gauze to remove mucus and debris, but not the cells. The cells are centrifuged at 1,200 rpm for 10 min at 4°C. The supernatant is decanted, centrifuged at 2,500 rpm for 8 min at 4°C, and stored in aliquots at –70°C. The cells are washed with PBS, and centrifuged at 1,000 rpm for 8  min at 4°C. Supernatant was aspirated and stored at –70°C, and cytospin slides are prepared from the resuspended cell pellet, and a total cell count of nonsquamous cells and viability are performed. The total cell count, viability (exclusion of trypan blue), and percentage of squamous cell contamination are assessed using a Neubauer haemocytometer. Cytospins slides are prepared in a Shandon Cytospin 3 by centrifugation at 300 rpm for 3 min. Induced sputum represents a nearly noninvasive technique; the collection of induced sputum by hypertonic saline is shown to be safe, effective, and relatively reproducible method for investigating airway diseases in adults and children over 6 years of age [40–45]. Induced sputum samples provide information about both the inflammatory mediators and the inflammatory cells present in the airways, and they are believed to accurately mirror the conditions at the site of oxidative damage [43, 46]. Several markers are relatively similar in the induced sputum and BALF, and the technique of collecting induced sputum is much less invasive than fiberoptic bronchoscopy. However, it has been shown that induced sputum is sampled predominantly from the large airways and may not reflect the peripheral airways [47]. Sputum induction is often difficult to accomplish in certain individuals, and it causes discomfort to the patient and might induce coughing and bronchoconstriction in patients with COPD particularly during exacerbations; hence, bronchodilators such as albuterol (2 puffs or 200 mg) may be given prior to sputum induction [48–51]. Sampling cannot be repeated within a short period of time, and the ­technique itself may induce an inflammatory response, which may persist for 24 h [48, 52]. Contamination of sputum samples by saliva may lead to reduced mediator concentrations, although differential cell counts remain relatively unchanged [53].

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Nevertheless, induced sputum is a standardized method and recommendations for sputum induction have been formulated by the European Respiratory Society/ American Thoracic Society Task Force.

4.3 Bronchoalveolar Lavage Flexible bronchoscopy is performed under local anesthesia following premedication (inhaled salbutamol 400 mg, 0.5 mg atropine I.M., and 10 mg diazepam I.M.) as previously described [54]. Following macroscopic evaluation of the bronchial tree, BAL is performed by wedging the bronchoscope in the bronchi of the middle lobe or the lingula and by administering 4 × 50 ml sterile NaCl warmed to 37°C. BALF is collected into sterile containers and placed on an ice bath. During the procedure, the patients/subjects receive oxygen intratracheally and the blood oxygen saturation is monitored with a pulse oximeter. Bronchoalveolar lavage has the advantage of sampling the distal airways and alveoli in the lung periphery, and the technique has been standardized [55]. However, the invasiveness of bronchoaveolar lavage limits its clinical application in assessing oxidative stress and airway inflammation in COPD. Generally, bronchoaveolar lavage has been performed safely on patients with stable COPD [56]. Bronchoaveolar lavage is, however, risky for patients with moderate/severe lung disease and acute exacerbations of airway disease; it is also unsuitable for children because of sedation, impairment of gas exchange and risk of infections. Furthermore, bronchoaveolar lavage fluid for COPD patients is often inadequate and not representative of the situation in the bronchioles due to airway collapse and reduced fluid recovery [57]. Saline instillation into the lungs can lead to artefacts, i.e., the dilution of the lavage may contribute to the variability of measurements. Bronchoaveolar lavage cannot be recommended for the diagnosis or follow-up of patients with COPD.

4.4 Collection of Human Lung Tissue To investigate the relevance of changes seen in animals’ lungs exposed to cigarette smoke, human lung tissue from COPD patients, smokers and nonsmokers is studied [58–61]. Resected lung tissue from an area distal to the tumor is obtained from COPD patients undergoing lung resection for peripheral bronchial carcinomas. Lung tissue is also obtained from current smokers (suspected carcinoma) of similar age, and sex-matched with no clinical or spirometric evidence of COPD. The lung samples are subject to pathological examination and then snap frozen in liquid nitrogen, and stored at −80°C until further analysis. Oxidative stress parameters are increased in the lungs of smokers, which is further augmented in smokers with

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COPD [58–61]. Furthermore, the degree of oxidative stress is negatively correlated with the FEV1 in these subjects [59].

5 Measurement of Oxidative Stress Biomarkers In addition to oxidants/free radicals contained in cigarette smoke, it also induces the endogenous production of ROS, by activating NADPH oxidase, xanthine/ xanthine oxidase, and myeloperoxidase. ROS, such as superoxide anion and hydroxyl radical, can oxidize membrane lipids leading to generation and accumulation of lipid peroxidation products, such as 4-hydroxy-2-nonenal, malondialdehyde, and F2-isoprostanes or aldehyde-adducts e.g., 4-HNE and acrolein protein adducts. Oxygen radicals are also shown to oxidize and modify DNA, resulting in gene mutation or 8-hydroxy-2¢-deoxyguanosine (8-OHdG) formation. Importantly, a variety of proteins are directly oxidatively modified (e.g., protein carbonylation) by ROS leading to degradation and loss of function. Thus, determination of oxidative stress by direct (e.g., superoxide anion levels) and indirect (e.g., oxidation of lipid, DNA and proteins) biomarkers reflect the degree of oxidative damage, and monitor the effect of antioxidants/anti-inflammatory agents in intervention of cigarette smoke-related diseases.

5.1 Measurement of Superoxide Anion Release in Bronchoaveolar Lavage Cells Superoxide anion is biologically deleterious, and contributes to the pathogenesis of many diseases, including COPD/emphysema. ROS release in bronchoaveolar lavage and cultured cells can be quantified using a spectrophotometric method based on the superoxide dismutase-inhibitable reduction of ferricytochrome c [62]. Each tube contains 105 cells, and 80  mmol of ferricytochrome c from the equine heart (Sigma) in PBS. Superoxide anion release is measured under two conditions, spontaneously and after phorbol myristate acetate stimulation for 80 min at 37°C. Phorbol myristate acetate is freshly prepared by diluting a stock solution in PBS to a final concentration of 1.6 mM. Paired tubes are established with and without superoxide dismutase from bovine erythrocytes (240  IU  ml−1 final concentration; Sigma) to assess the specificity of cytochrome-c reduction as control. Optical density is then measured with absorbance at 550 nm. The resulting change in absorbance between the experimental group and its control (same sample supplemented with superoxide dismutase) is used to calculate the nanomoles of superoxide anion produced by application of the molar extinction coefficient (∑550) of cytochrome-c (21 × 103 M−1 cm−1) using the following formula, and is expressed as nmol/105 cells/80 min: ∆absorbance / ∑ 550 × light path = concentration.

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5.2 Measurement of ROS by Flow Cytometry and Fluorescent Microscopy in Cells and Bronchoaveolar Lavage Cells 5-(and 6)-carboxy-2¢,7¢-dichlorodihydrofluorescein diacetate (CH2-DCFDA) is an oxidant-sensitive fluorescent probe [12, 63]. Inside the cell, the probe is deacetylated by esterases and formed H2DCF that is oxidized by ROS to DCF, a highly fluorescent compound, which can be assessed by flow cytometry or under fluorescent microscopy. Briefly, cells are seeded at a density of 5 × 105 cells in 6-well plates in a total final volume of 2 ml and treated with CSE (1.0–5.0%) for 24  h. Cells are washed with PBS, and incubated with CH2-DCFDA (10  mM, Molecular Probes) at 37°C for 30  min. Cells are then trypsinized, washed and resuspended in PBS. Flow cytometric analysis is performed using ELITE flow cytometer (Coulter, Hiaelea, FL, USA) [64]. A minimum of 10,000 events is acquired for each sample. Debris is gated out and analysis is performed only on the live cells population. Furthermore, ROS release can be determined by CH2DCFDA under a fluorescent microscope. Briefly, the cultured and bronchoaveolar lavage cells are prepared on cytospin slides followed by incubation with 10 mM DCF-DA for 15 min at room temperature. After being rinsed with PBS, the slides are observed under the fluorescent microscope, and assessed with a score of between 1 and 3, based on the following criteria (1) weak staining; (2) moderate staining; (3) intense staining.

5.3 Myeloperoxidase Assay Activated myeloperoxidase uses hydrogen peroxide, which can be derived from superoxide anion, to produce the strong oxidant hypochlorite resulting in further oxidative modification of lipids, proteins and DNA. A method to determine the activity of myeloperoxidase in lungs in response to cigarette smoke exposure is described [62]. In brief, the lung tissues are homogenized in 10 volumes of 100 mM phosphate buffer (pH 7.4) containing protease inhibitors. To determine the myeloperoxidase activity in an enzymatic extract, a spectrophotometric method is used with tetramethylbenzidine (TMB, Sigma-Aldrich). The assay mixture consisted of 400 ml of phosphate buffer (100 mM, pH 5.4) with 0.5% hexadecyltrimethylammonium bromide (Sigma-Aldrich), 40  ml TMB (20  mM in dimethyl sulfoxide, prepared fresh and protected from light), and H2O (23 ml). To this assay mixture, 20 ml of enzyme extract is added and the mixture is incubated at 37°C for 3 min, 17 ml of H2O2 (0.03%) is added. The mixture is further incubated for 3 min. The reaction is stopped by adding 2 ml of acetate buffer (0.2 M, pH 3.2). The tubes are kept on ice and the absorbance is read at 655 nm. The appropriate reagent blank is prepared by using a buffer instead of the tissue extract. Activity is expressed in the samples as units/mg protein.

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5.4 Determination of Intracellular 4-Hydroxy-2-nonenal Levels and Its Adducts Aldehydes generated endogenously during lipid peroxidation or contained in cigarette smoke are involved in many pathophysiological events associated with oxidative stress in cells and tissues. 4-Hydroxy-2-nonenal (4-HNE) is a highly reactive and diffusible end-product of oxidative stress-induced lipid peroxidation that can attack targets far from the original site of free radical generation. Therefore, the level of 4-HNE reflects the degree of oxidative stress. Briefly, 4-HNE levels are measured in cell lysates by using a lipid peroxidation assay kit (Calbiochem, San Diego, CA, USA) [5, 12]. After treatment of cells with CSE, CSC, or exposure to air–liquid interface smoke, cells are rinsed twice with ice-cold PBS. The pellets are resuspended in 200  ml of 20  mM Tris–HCl, pH 7.4, containing 5  mM butylated hydroxytoluene (BHT, Sigma-Aldrich, MO, USA) and kept frozen at –70°C until assayed. To each sample, 650 ml of N-methyl-2-phenylindole and 150 ml of 15.4 M methanesulfonic acid are added. The reaction mixture is mixed by vortexing and is incubated at 45°C for 60  min. After centrifugation at 15,000 × g for 10  min, the absorbance of the supernatant is determined at 586  nm. A standards curve using 4-HNE diethylacetal in methanesulfonic acid is also prepared. The values are expressed as mmol 4-HNE/mg of protein. As for the determination of lipid peroxidation adducts in lung tissues, a lung lobe is homogenized with ice-cold 20 mM Tris–HCl (pH 7.4) and diluted to approximately 10% (w/v). The homogenates are centrifuged at 3,000 rpm for 10 min at 4°C and the 200 ml of supernatants are used to determine the level of 4-HNE as described above. 4-HNE protein adducts can also be determined in lung sections by immunohistochemistry using 4-HNE antibodies [59]. The assessment of immunostaining intensity is performed semiquantitatively and in a blinded manner: 0 = no staining; 1 = weak staining; 2 = moderate staining; and 3 = intense staining.

5.5 Immunohistochemistry for 8-Hydroxy-2¢-deoxyguanosine 8-hydroxy-2¢-deoxyguanosine (8-OHdG), an indicator of oxidative DNA damage, can be assessed in lung tissue by imunohistochemistry or ELISA in response to cigarette smoke exposure [65]. Mouse lungs (which had not been lavaged) are inflated by 1% low-melting agarose at a pressure of 25 cm H2O, and then fixed with 4% neutral buffered formalin. The formalin-fixed, paraffin-embedded lung sections are deparaffinized and rehydrated by passing through a series of xylene and graded alcohol. Endogenous peroxidase activity is quenched by exposure to 3% H2O2 in methanol for 30 min. 8-OHdG was detected by immunohistochemistry in paraffin-embedded sections pretreated with 100 mg ml−1 ribonuclease A in PBS in order to inhibit nonspecific binding to RNA using a mouse monoclonal antibody (Oxis International Inc., Foster City, CA, USA). A biotinylated goat

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anti-mouse/rabbit Ig (DAKO Corp., Santa Barbara, CA, USA) is used at a titer of 1:100. Tissues are incubated with primary antibodies ­overnight at 4°C. After being washed, tissues are incubated with secondary antibodies for 30  min. 3,3¢-Diaminobenzidine (DAKO) is used as a peroxidase substrate. In each instance, sections from different time points are processed together, with equal time for color development. Tissues are counterstained with hematoxylin. The number of 8-OHdG positive cells in lung sections (three random microscopic fields per lung section in three different sections) is counted manually in a blinded manner.

5.6 Carbonylated Nuclear Erythroid-Related Factor 2: Protein Carbonyl Assay Nuclear erythroid-related factor 2 (Nrf2), a redox-sensitive transcription factor, is involved in transcriptional regulation of many phase II antioxidant genes, including glutamate–cysteine ligase. In response to oxidative stress or electrophilic compounds (aldehydes), Nrf2 can be carbonylated leading to inhibition of Nrf2 translocation into the nucleus. Furthermore, this modified Nrf2 undergoes rapid proteasomal degradation, which could be the reason for the CSE-induced decrease of nuclear Nrf2 levels at 24 h in epithelial cells [64]. Thus, Nrf2 protein carbonylation assay will reflect the degree of carbonylated proteins in response to oxidative stress/cigarette smoke exposure. Protein carbonyls are analyzed according to the method of Levine et al. [66] with slight modifications [64]. Cells are treated with CSE for 24 h. Following treatment, cytosolic protein extracts are immunoprecipitated with an Nrf2 antibody. Approximately 100  mg protein is precipitated with ice-cold trichloroacetic acid (final concentration 10% v/v) and is centrifuged at 11,000 × g for 3  min. The protein pellet obtained is resuspended in 0.5  ml of 10  mM 2,4-dinitrophenylhydrazine (DNPH) in 2  M HCl. Samples are vortexed continuously at room temperature for 1 h. The samples are precipitated with 0.5 ml of 20% trichloroacetic acid, centrifuged at 11,000 × g for 3 min. The resulting pellet is washed with 1 ml of ethanol–ethyl acetate (1:1 v/v) to remove free DNPH reagent, and allowed to stand for 10 min. The sample is centrifuged for 5 min at 11,000 × g and the supernatant is discarded. The resulting protein pellet is resuspended in 0.9 ml of 6 M guanidine (prepared in phosphate buffered saline, pH 6.5). The samples are incubated at 37°C for 15  min followed by centrifugation at 10,000 × g for 3 min to remove any insoluble materials remaining in suspension. The carbonyl content is measured at 360  nm spectrophotometrically against a blank containing 1 ml of 6 M guanidine solution (prepared in phosphate buffered saline). The molar absorption coefficient of 22,000 M−1 cm−1 is used to quantify the levels of protein carbonyls. Protein carbonyl content is expressed as nmol/mg protein.

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5.7 Measurement of Intracellular Glutathione Levels Glutathione (GSH) is a major intra- and extracellular antioxidant in the lung, and intracellular GSH is depleted in lungs in response to cigarette smoke exposure [5, 12]. Intracellular glutathione (GSH) levels in the cell extracts are measured by the 5,5¢-dithiobis-2-nitrobenzoic acid DTNB-GSSG reductase recycling method described by Tietze [67] with slight modifications [12, 68–70]. In brief, cells are rinsed twice with ice-cold PBS, scraped off from the 6-well plate using Coster cell scrapers, into 500  ml of ice-cold extraction buffer (0.1% Triton X-100 and 0.6% sulfosalicyclic acid in 0.1 M phosphate buffer with 5 mM EDTA, pH 7.5). Cells are vortexed for 20  s followed by sonication (30  s), and centrifuged (2,500  rpm for 5  min at 4°C). In order to determine GSH levels in the lung, a piece of lung is homogenized with the above extract buffer and diluted to approximately 10% (w/v). The homogenates are centrifuged at 3,000 rpm for 10 min at 4°C and supernatants are collected. Twenty microliters of the supernatant is added to 120  ml of 0.1  M phosphate buffer, 5 mM EDTA, pH 7.5, containing 100 ml of 5 mM DTNB and 0.5 units of glutathione reductase. Sixty microliters of 2.4 mM NADPH is added and the rate of change in absorbance is measured for 1 min at 410 nm using microplate reader. A standard curve using GSH in the range of 0.33–1.35  nmol is prepared prior to making measurements in the samples. The amount of GSH in the sample is expressed in nmoles GSH/mg protein. Similarly, the levels of glutathione disulfide (GSSG) are determined by derivatizing the samples/extracts with 2-vinylpyridine following by neutralization with triethanolamine as described earlier [68–70].

5.8 Electron Paramagnetic Resonance Measurement Electron paramagnetic resonance (EPR) (Magnettech X-band miniscope MS-100, Berlin, Germany) is used to establish the quenching effect of antioxidants on superoxide anion and hydroxyl radicals generated from xanthine/xanthine ­oxidase, FeCl3 + H2O2 and menadione respectively. Xanthine (100 mM)/xanthine oxidase (100 mU/ ml), FeCl3 (50  mM) + H2O2 (10  mM) and menadione (500  mM) are incubated in phosphate-buffered saline (pH 7.4, 37°C) containing the spin trap Tempone-H (1 mM) [71], oxidation of which generates 4-oxo-tempo with a characteristic threeline EPR signal centered at 3,365 G. Development of this signal is monitored for 24 h after the addition of oxidizing species and compared to parallel incubations containing antioxidants. The amplitude of first line of the spectrum is measured; data are expressed in arbitrary units. In order to establish whether oxidation of antioxidants generated a stable radical species or whether the product of this reaction is spin silent, separate experiments are also conducted with xanthine/xanthine oxidase, FeCl3 + H2O2 or menadione in the presence of antioxidants without the spin trap. The EPR parameters for these experiments are as follows: Microwave

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frequency – 9.4  GHz; microwave power – 20  mW; modulation frequency – 100 kHz; modulation amplitude – 1,500 mG; center field – 3,365 G; sweep width – 50 G; sweep time – 20 s; Number of passes – 1; receiver gain – 3 × 101.

6 Conclusions It is now well recognized that increased oxidative stress occurs in many diseases, including airway disease (COPD), which is due to cigarette smoke exposure. Cigarette smoke contains a variety of oxidants/free radicals and chemical compounds including reactive aldehydes, quinones, and semiquinones. Thus, cigarette smoke-induced oxidative stress measurements models both in vitro and in vivo are useful for studying how the oxidative stress is produced and its effects on cellular signaling and function, and to identify pharmacological targets for the design of antioxidants/anti-inflammatory agents. It should be noted that rodents are obligate nose breathers leading to particle infiltration/deposition in the nostrils and upper respiratory tract, whereas human inhale smoke via mouth. Furthermore, all of these models are complementary and not mutually exclusive, and specific models should be chosen and combined (e.g., combination in vitro and in vivo models) in terms of the purpose/feasibility of studies. For example, samples from smokers and patients with COPD will give more direct information on the changes of oxidative stress in the lung and the disease status. The mechanism on how the biomarkers of oxidative stress are changed in human samples cannot be clarified, and, therefore, in vitro and in vivo animal models are exploited for investigation. Cigarette smoke exposureinduced COPD/emphysema in animals will not progress after cessation of exposure, whereas COPD frequently progresses even after the subject stops smoking. In addition, a variety of factors, such as TPM, exposure time, type of smoke machines used, and animal species will influence the results. Therefore, more appropriate animal models remain to be developed to study the oxidative stress-mediated lung damage by cigarette smoke. Identification and validation of new biomarkers of oxidative stress will facilitate the understanding of pathogenesis, and define targets, as well as develop the pharmacological agents to intervene in the progression of cigarette smoke-induced diseases including COPD. Acknowledgments  This study was supported by the NIH R01-HL085613 and NIEHS Environmental Health Science Center grant ES-01247.

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Exhaled Breath Condensate Biomarkers of Airway Inflammation and Oxidative Stress in COPD Paolo Montuschi

Abstract  Exhaled breath condensate (EBC) is a noninvasive method for collecting airway secretions and for studying the composition of the airway lining fluid. Several biomolecules, including leukotrienes, prostaglandins, isoprostanes, hydrogen peroxide, nitric oxide-derived products, hydrogen ions, and adenosine triphosphate, have been measured in healthy subjects. Some of these inflammatory mediators are elevated in patients with chronic obstructive pulmonary disease (COPD). Analysis of biomolecules in EBC is potentially useful for monitoring of lung inflammation and oxidative stress, which is an important component of inflammation, in patients with COPD. As it is completely noninvasive, EBC might also be suitable for longitudinal studies, and for monitoring the effects of pharmacological therapy in patients with COPD. Different profiles of biomarkers in EBC might reflect different aspects of lung inflammation or oxidative stress. Identification of selective profiles of biomarkers in EBC in lung diseases might have a value for differential diagnosis in respiratory medicine. However, several methodological aspects have to be formally addressed and standardization of EBC methodology is required before this technique can be considered for application in the clinical setting. Keywords  Chronic obstructive pulmonary disease • Exhaled breath condensate • Lung inflammation • Noninvasive markers

1 Introduction Exhaled breath consists of a gaseous phase that contains volatile biomolecules (e.g., nitric oxide, carbon monoxide and volatile organic compounds) and a liquid phase, known as exhaled breath condensate (EBC), which is mainly formed by

P. Montuschi (*) Department of Pharmacology, Faculty of Medicine, Catholic University of the Sacred Heart, Largo F. Vito, 1, 00168 Rome, Italy e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_19, © Springer Science+Business Media, LLC 2011

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water vapor, but also contains aerosol particles in which nonvolatile biomolecules including leukotrienes, 8-isoprostane (15-F2t-isoprostane), a marker of lipid peroxidation, prostaglandins, hydrogen peroxide, reactive nitrogen species, and adenosine have been detected [1–3]. Measurement of exhaled nitric oxide (NO) is a standardized and well-accepted technique for assessing and monitoring airway inflammation in patients with asthma [4], but its utility in patients with chronic obstructive pulmonary disease (COPD) has to be established. EBC is a noninvasive method for collecting airway secretions and for studying the composition of the airway lining fluid [5, 6]. Respiratory fluid represents between 0.01% and 2% of EBC volumes as reported by one study in which electrolyte concentrations in EBC were measured as a dilution marker [7]. Analysis of biomolecules in EBC is potentially useful for monitoring lung inflammation and oxidative stress, an important component of inflammation, in patients with COPD [8]. As it is completely noninvasive, EBC might also be suitable for longitudinal studies, and for monitoring the effects of pharmacological therapy in patients with COPD. Different profiles of biomarkers in EBC might reflect different aspects of lung inflammation or oxidative stress. Identification of selective profiles of biomarkers in EBC in lung diseases might have a value for differential diagnosis in respiratory medicine. However, several methodological aspects have to be formally addressed before EBC analysis can be considered for applications in the clinical setting. This article describes the methodology of EBC analysis, summarizes the results of the studies aimed at measuring biomarkers of inflammation and oxidative stress in EBC in patients with COPD, presents advantages and limitations of EBC analysis, and proposes directions for future research.

2 Methodology of EBC Analysis 2.1 Experimental Setup Home-made or commercially manufactured (Figs. 1 and 2) equipment for collecting EBC consists of a mouthpiece with a one-way valve connected to a collecting system that is refrigerated (Fig. 1a) [9]. Subjects are generally asked to breathe tidally through a mouthpiece connected to the condenser. Exhaled air enters and leaves the chamber through one-way valves at the inlet and outlet while the chamber is kept closed. Volume of EBC collected can be highly variable [10, 11] depending on differences in respiratory parameters, type and efficiency of condenser, temperature and turbulent airflow. Commercially manufactured condensers include the EcoScreen® and the EcoScreen® II (Jaeger Toennies, Hoechberg, Germany) (Fig.  2a, b); the RTube (Respiratory Research, Inc., Charlottsville, VA, USA) [12] (Fig. 1b). The EcoScreen can be connected to a pneumotachograph and a computer for online recording of respiratory parameters [13]. The EcoScreen ® condenser has been used in several studies, but its production has recently been discontinued due to a ­manufacturer’s  

EBC Biomarkers of Airway Inflammation and Oxidative Stress in COPD outer glass device

a

outer glass device

423

b

saliva filter

mouthpiece

ice

one-way valve

breath condensate

Fig. 1  (a) An example of “home-made” EBC collecting system. (b) The RTube® (Respiratory Research, Inc., Charlottsville, Virginia, USA). Schematic representation of a commercially available condenser (EcoScreen®, Jaeger Toennies, Hoechberg, Germany). EBC is collected in the collecting vial, as indicated by the arrow. [Panel (a): Modified with permission of [1], copyright 2002 Macmillan Magazines Ltd; panel (b): Reprinted with permission from [2], copyright 2004]

a

b

mouthpiece one-way valve valve block cooling cuff lamellar condenser sample collection vial

Fig. 2  (a) The EcoScreen® (Jaeger Toennies, Hoechberg, Germany). EBC is collected in the collecting vial, as indicated by the arrow. (b) The EcoScreen II® (Jaeger Toennies, Hoechberg, Germany). [Panel (a): Modified with permission from [1], copyright 2002 Macmillan Magazines Ltd]

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decision. With the EcoScreen® II (Jaeger Tonnies, Hoechberg, Germany) (Fig. 2b), respiratory parameters can be measured during EBC collection. EBC derived from the airways or the alveoli can be collected at the same time into two separate collecting systems that consist of plastic bags inserted into a refrigerating system (Fig. 2b). The EcoScreen® II might be useful for studying the origin of biomarkers in the respiratory system (airways versus alveoli), but no published studies are available with this condenser. The RTube (Fig.  1b) consists of a disposable polypropylene tube that is connected to an exhalation valve that also serves as a syringe-style plunger to pull fluid off the condenser walls [12]. Breath is cooled down by placing an aluminum cooling sleeve over the polypropylene tube. As it is portable, the RTube is suitable for onsite collection of EBC samples. Generally, a 1-min test can provide a sufficient EBC for pH measurement [12]. Samples collected on site have to be transported to the laboratory for analysis. Hydrogen peroxide can be measured immediately after EBC sample collection with a selective sensor [14]. The development of selective sensors for detecting specific biomolecules in the breath is currently under investigation.

2.2 Analytical Techniques Various analytical techniques have been used to measure biomolecules in EBC in patients with COPD (Table  1). In most studies, biomolecules in EBC have been measured by immunoassays [6, 37]. The presence of leukotriene (LT) B4 [38–40], 8-isoprostane [41, 42], prostaglandin (PG) E2 [41], aldehydes [32] and free 3-nitrotyrosine [43, 44] in EBC in healthy subjects and in patients with respiratory diseases has been confirmed by mass spectrometry. LTB4 [38–40] and aldehydes [32] in EBC have been measured by liquid chromatography/mass spectrometry (LC/MS). LTB4, LTD4 and LTE4 concentrations in EBC in healthy subjects and patients with asthma have been measured with GC/MS [45]. However, the stable isotope dilution method was not used in this study [45]. Arachidonic acid was used as the internal standard because it is structurally similar to the LTs and is not detectable in EBC [45]. PGE2 in EBC has been measured by gas chromatography/mass spectrometry (GC/MS) [41]. 8-Isoprostane and free 3-nitrotyrosine have been measured by both GC/MS [41, 44] and LC/MS [42, 43]. Adenosine [46] and reduced glutathione [47] in EBC have been detected by high performance liquid chromatography (HPLC). Generally, immunoassays for biomolecules in EBC require validation with reference analytical techniques such as MS or HPLC. Two radioimmunoassays (RIAs) for 8-isoprostane and PGE2 developed in our laboratory [48], and a commercially available enzyme immunoassay (EIA) for LTB 4 [49], were qualitatively validated by HPLC. Malondialdehyde (MDA) has been quantified in EBC with fluorescence detection after HPLC separation [50].

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Table 1  Inflammatory biomarkers in exhaled breath condensate in patients with COPD Biomarker Method Values References Leukotrienes c EIA 100.6 (73.5–145.0) pg/ml [15] LTB4 a EIA 8.5 ± 0.8 pg/ml [16] b EIA 86.7 ± 19 pg/ml [17] c LTE4 EIA 23.3 (9.1–31.3) pg/ml [15] Prostanoids PGE2 PGE2 PGF2a PGD2-MOX

EIA RIA EIA EIA

c

EIA RIA EIA EIA EIA EIA EIA

a

Spectrophotometry Spectrophotometry Spectrophotometry Fluorimetry Spectrophotometry Spectrophotometry

a

NO-derived products S-Nitrosothiols Peroxynitriteg Peroxynitriteg Nitrite Nitrite/nitrates Nitrite/nitrates Nitrotyrosine Hydrogen ions

Spectrophotometry Fluorimetry Fluorimetry Spectrophotometry Fluorimetry Fluorimetry EIA pH meter

a

Aldehydes MDA hexanal heptanal

GC/MS/MS GC/MS/MS GC/MS/MS

a

ATP

Bioluminescence

a

Multiplex fluorescent bead immunoassay Multiplex fluorescent bead immunoassay EIA Multiplex fluorescent bead immunoassay

Isoprostanes 8-Isoprostane

Hydrogen peroxide

98.0 (57.0–128.4) pg/ml 93.5 (84.0–105.5) pg/ml c 15.0 (10.9–19.0) pg/ml c 11.2 (8.7–15.0) pg/ml

[15] [18] [15] [15]

45 ± 3.6 pg/ml 47.9 (40.5–51.9) pg/ml d 47 (41–53) pg/ml a 43.7 ± 2.8 pg/ml a 6.0 ± 0.7 pg/ml a 18.1 ± 2.0 pg/ml e 14.2 (9.5–26.4) pg/ml

[19] [18] [20] [21] [16] [22] [23]

0.205 ± 0.054 mM 2.6 (1.9–3.5) mM a 0.46 ± 0.03 mM b 0.32 ± 0.03 mM a 0.50 ± 0.11 mM d 0.66 (0.54–0.68) mM

[24] [25] [26] [27] [10] [20]

0.24 ± 0.04 mM 7.9 ± 3.0 nM a 295.1 ± 52.7 nM a 2.62 ± 0.52 mM a 37.8 ± 3.4 mM f 10.2 (2.0–93.8) mM a 24.6 ± 2.2 (ng/ml) d 7.16 (7.09–7.23)

[28] [29] [21] [28] [21] [30] [21] [31]

57.2 ± 2.4 nmol/l 63.5 ± 4.4 nmol/l a 26.6 ± 3.9 nmol/l

[32] [32] [32]

141 ± 44 pM

[33]

h

Not available

[34]

h

Not available

[34]

8.0 ± 0.1 pg/ml Not available

[35] [34]

e

e

c

b

a

Cytokines IL-1b IL-6

IL-8

a

h

(continued)

426 Table 1  (continued) Biomarker

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Method

Values

References

IL-10

h Multiplex fluorescent bead Not available [34] immunoassay h Not available [34] IL-12p70 Multiplex fluorescent bead immunoassay [36] Multiplex bead immunoassay c3.75 (3.15–5.02) pg/ml sICAM-1 Multiplex bead immunoassay c22.8 (16.6–35.6) pg/ml [36] MIF Multiplex bead immunoassay a37.2 ± 8.9 (pg/ml) [36] RANTES Multiplex bead immunoassay a22.5 ± 1.7 (pg/ml) [36] h Not available [34] Multiplex fluorescent bead TNF a immunoassay Abbreviations: ATP Adenosine triphospahte; sICAM-1 Soluble intracellular adhesion molecule-1; IL Interleukin; LT Leukotriene; MDA Malondialdehyde; MIF Macrophage migration inhibitory factor; MOX Methoxime, a stable PGD2 metabolite; NO Nitric oxide; PG Prostaglandin; RANTES Regulated on activation normal T cell expressed and secreted; TNF a Tumor necrosis factor a   Values are expressed as amean ± SEM, bmean ± SD, cmedian with the minimum to maximum range, dmean with 95% confidence intervals for the differences, emedian with interquartile range, or fgeometric means ± SEM. gPeroxynitrite was measured using oxidation of 2¢,7¢-dichlorofluorescin in EBC collected at high pH. hData is presented as figures; numerical data is not available For some biomarkers, there is a high variability in the concentrations reported in different studies. This can be partially explained by the lack of standardization of the exhaled breath condensate method and different analytical techniques

2.3 Methodological Aspects of EBC Analysis Standardization of EBC collection, identification and quantitative assessment of biomolecules in EBC with reference analytical techniques, and validation of the immunoassays for measuring EBC biomarkers of inflammation and oxidative stress, are the priorities in this research area. Methodological aspects of EBC analysis have been discussed in detail [6, 37]. These include the origin(s) of biomolecules in EBC; flow- and time-dependence of biomolecules in EBC; organ specificity of EBC; characterization of the physico-chemical properties of different biomolecules in EBC; the use of reference indicators to estimate the concentrations of inflammatory mediators in the airway-lining fluid and to normalize for variations in nonvolatile biomolecules in EBC that can result from variations in aerosol particle dilution by water vapor [7]; the effects of temperature, humidity, and collecting system materials on measurements; possible nasal, saliva and sputum contamination; assessment of between-day, within-day, and technical variability of measures; comparison of different condensers; storage issues; pretreatment of samples; and influence of different factors on biomolecules in EBC. 2.3.1 Origin(s) of Biomolecules in EBC The cellular source(s) of biomolecules in EBC, the site of production of biomolecules detected in EBC (airways as opposed to alveoli), and the extent to which

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EBC reflects the composition of airway lining fluid have to be clarified. EBC is not suitable for studying inflammatory cells in the respiratory system. Indirect information on the cellular origin(s) of a given biomolecule in EBC can be obtained by correlating its concentrations in EBC with counts, for example in sputum, of a given cell type. However, such correlation does not necessarily mean that the biomolecule is released by that cell type as elevated biomolecule concentrations in EBC could also result from cell activation without increased cell counts. Establishing the cellular origin(s) of biomolecules in EBC requires invasive techniques including bronchoscopy. Flow dependence might indicate whether biomolecules in EBC are principally produced in the airways as opposed to the alveolar region [51]. Flow rate affects the time available for the accumulation of a biomolecule in the respiratory tract, and, consequently, its concentrations in the EBC [51]. Hydrogen peroxide concentrations in EBC depend on expiratory flow rate indicating that this biomarker of oxidative stress is principally produced in the airways [51]. Concentrations of 8-isoprostane [13, 41] and PGE2 [13, 41] in EBC do not depend on minute ventilation; exhaled glutathione [47] and aldehydes [32] are not flow-dependent. Taken together, these data indicate that these biomolecules are mostly produced in the alveolar region where the effect of flow rate in determining EBC concentrations is less important [51]. Data on flow-dependence of biomolecules in EBC were mainly obtained from healthy subjects. Studies aimed at establishing the effects of different flow rates on biomolecule concentrations in EBC in patients with COPD and other lung diseases are warranted. In these patients, the greater turbulence of the airflow can facilitate the formation of aerosol particles that contain nonvolatile biomolecules [12]. Moreover, the activation and/ or increased numbers of selective cell types in different compartments of the respiratory system might have a different impact on the concentrations of a given biomarker in the EBC as compared with healthy subjects. As it was designed for separate sampling of EBC from the airways and the alveolar region, the EcoScreen® II could be suitable for studying the origin of EBC biomolecules in the respiratory system. 2.3.2 Flow-Dependence of Biomolecules in EBC and Effect of Respiratory Patterns The volume of EBC collected depends on ventilation patterns [52, 53]. In healthy subjects who maintained the same tidal volume for 15  min, the mean volume of EBC collected was almost twofold higher when the respiratory rate was increased from 14 to 28 breaths/min [52]. EBC volume is correlated with expiratory minute ventilation, but higher values of expiratory minute ventilation are associated with decreased condenser efficiency [53]. In healthy nonsmokers and patients with mild asthma, hydrogen peroxide concentrations in EBC depend on the expiratory flow rate [51]. In contrast, similar exhaled MDA concentrations in healthy adults [32] and exhaled glutathione and MDA concentrations in healthy children [47] were observed when EBC was collected at different flow rates indicating that these biomolecules are not flowdependent. 8-Isoprostane [13, 41], PGE2 [13], LTB4 [52], LTE4 [52], nitrite [53],

428

P. Montuschi

total protein [53] and ethanol [54] concentrations in EBC do not seem to depend on respiratory patterns. We studied the impact of ventilation patterns on 8-isoprostane and PGE2 concentrations in EBC [13]. Fifteen healthy adults were asked to breathe at different expiratory minute ventilations (10, 20, and 30  l/min) for 10, 10, and 5  min, respectively [13]. 8-Isoprostane and PGE2 concentrations in EBC were similar, indicating that the concentrations of these eicosanoids in EBC are not affected by expiratory minute ventilation [13]. Consistent with our findings, there was no correlation between 8-isoprostane concentrations in EBC and minute ventilation in mechanically ventilated patients [41]. LTB4 and LTE4 do not seem to be affected by expiratory minute ventilation as there was no difference in their concentrations in EBC when healthy subjects were asked to breathe at 14 and 28 breaths/min, maintaining the same tidal volume [52]. As airflow rate and respiratory patterns can have a different impact on the concentrations of different biomolecules in EBC, each biomolecule should be studied individually. Further studies aimed at clarifying the effects of ventilation patterns and airflow rate on the concentrations of EBC biomarkers are required as (1) data on the flow-dependence of many biomolecules are not available; (2) studies on the influence of ventilation patterns on EBC biomolecules were mainly performed on healthy subjects and the results of these studies cannot be directly extrapolated to patients with COPD and other lung diseases; and (3) comparison of data is often difficult as a different methodology was generally used in the published studies. Establishing the effect of respiratory parameters on the concentrations of exhaled biomolecules is relevant to the standardization of the EBC technique. Depending on flow dependence of a given biomolecule, EBC will be collected either at a constant flow rate for a fixed amount of time, or at a constant exhaled volume for a variable amount of time. 2.3.3 Time-Dependence of Biomolecules in EBC The mean volume of EBC collected after 20  min of tidal breathing is almost twofold higher than that collected after 10 min of tidal breathing [32]. 8-Isoprostane concentrations in EBC [41] and EBC pH [55] do not depend on the duration of EBC collection, whereas exhaled hydrogen peroxide concentrations decrease with prolonged sampling time [56]. Conflicting results have been reported for MDA. In one study, concentrations of MDA in EBC samples collected at 10  min and 20 min were similar [32], whereas in another study, MDA concentrations in EBC decreased over time [55]. The duration of EBC collection could be relevant for unstable biomolecules including cysteinyl-LTs and hydrogen peroxide. Further studies are required to establish the effect of the duration of EBC sampling on EBC concentrations of different biomolecules and identify the ideal sampling time for each of them. 2.3.4 Organ Specificity of EBC EBC analysis is likely to provide organ-specific quantitative assessment of pulmonary inflammation. However, it is possible that biomolecules produced at sites

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remote from the lungs could circulate in plasma and enter the pulmonary epithelial lining fluids, particularly when the inflammatory process increases capillary permeability. The answer to this question can be forthcoming by comparing biomolecule concentrations in EBC and in biological fluids that reflect systemic production of inflammatory mediators such as plasma and urine in patients with airway inflammatory diseases. Studies aiming at measuring biomolecules in EBC in patients with pathophysiological conditions in which systemic inflammation has a role might also be useful. However, the interpretation of results from these studies could be difficult due to possible systemic inflammation in patients with COPD. 2.3.5 Nasal and Saliva Contamination It is important to exclude nasal and salivary contamination of EBC samples. In healthy subjects and patients with atopic rhinitis, concentrations of adenosine, ammonia and thromboxane (Tx) B2 in EBC are not affected by the mode of inhalation (nasal versus oral) [46]. Salivary contamination of EBC is excluded by measuring amylase concentrations that are generally undetectable [3], even when very sensitive assays are used [7]. When amylase is detected, its concentrations in EBC are less than 0.01% than those in EBC indicating very little, if any, salivary contamination [7]. Consistent with this data, similar adenosine and TxB2 concentrations were observed in EBC samples obtained through tracheostomy in mechanically ventilated patients or through the mouth in healthy subjects [46]. Studies with nuclear magnetic resonance spectroscopy [57] and GC/MS [58] confirm that EBC is not generally contaminated by saliva. 2.3.6 Dilution Reference Indicators EBC is mainly formed by water vapor. The fact that nonvolatile biomolecules are detected in EBC indicates that droplets are also collected. The use of dilution reference indicators, including measurement of conductivity, urea and electrolytes, has been proposed as a dilution reference indicator in EBC to normalize for interindividual variations in droplet formation and to estimate the concentrations of nonvolatile biomolecules in the airway lining fluid [7, 59]. Interindividual differences in concentrations of nonvolatile biomolecules in EBC could be partially explained by differences in the dilution of respiratory droplets. However, the selective increase of biomolecules in EBC and the lack of correlation between concentrations of structurally similar biomolecules in EBC are not consistent with this hypothesis [52]. The role of dilution reference indicators in EBC analysis has not been established. Alter­ natively, EBC results can be expressed as a change in ratio in one biomolecule to another. 2.3.7 Within-Day and Between-Day Variability of Biomolecules in EBC Each biomolecule has to be studied separately to establish within-day and betweenday variability. Using RIAs, we assessed between-day reproducibility of 8-isoprostane

430

P. Montuschi

and PGE2 measurement in EBC by collecting samples on days 1, 3, and 7 from healthy subjects [13]. These RIAs were developed in our laboratory and were previously qualitatively validated by reverse phase-high performance liquid chromatography (RP-HPLC), providing evidence for their specificity [48]. RIAs for 8-isoprostane and PGE2 in EBC were highly reproducible as shown by their intraclass correlation coefficients that were 0.95 and 0.90, respectively [13]. Between-day reproducibility of RIA measurement of 8-isoprostane, PGE2, and TxB2 in EBC was also assessed in children with asthma [60, 61] and expressed as limits of agreement according to analyses described by Bland and Altman [62]. Limits of agreement for RIA measurement of these eicosanoids in EBC were as follows: 8-isoprostane, −5 and 5 pg/ml; PGE2, −5.3 and 5.1 pg/ml; TxB2, −3.7 and 4.7  pg/ml [60]. TxB2 was detectable only in some children with asthma. Measurements of leukotrienes in EBC in children with asthma using commercially available EIAs (Cayman Chemicals, Ann Arbor, MI) were less reproducible than RIAs for prostanoids, as indicated by their limits of agreement [63].

3 Analysis of EBC in Patients with COPD and Healthy Smokers Several biomolecules, including leukotrienes, prostaglandins, isoprostanes, hydrogen peroxide, nitric oxide-derived products, hydrogen ions, and adenosine triphosphate, have been measured in healthy subjects. Some inflammatory mediators are elevated in patients with COPD [8].

3.1 Eicosanoids In most studies, leukotriene and prostanoid concentrations in EBC have been measured by commercially available immunoassays that are generally not validated. The limitations of this approach and the need for a robust analytical methodology for a specific and quantitative assessment of eicosanoid concentrations in EBC have been discussed [6, 37]. The presence of LTB4 [38–40], 8-isoprostane [41, 42], and PGE2 [41] in EBC has been definitively demonstrated by mass spectrometry. RIAs for 8-isoprostane and PGE2 have been qualitatively validated by using RP-HPLC [48]. 3.1.1 Leukotrienes LTB4 concentrations in EBC are elevated in steroid-naïve and steroid-treated patients with stable COPD [15, 17] compared with healthy smokers. In patients with COPD [15], the profile of leukotrienes in EBC is different from that in

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patients with asthma [52]. LTE4 is elevated in patients with asthma [52], but not in patients with COPD [15]. Exhaled LTB4 is elevated in both patients with asthma and COPD, but this increase is more pronounced in patients with COPD [15]. In patients with exacerbations of COPD, LTB4 concentrations in EBC are decreased 2 weeks after treatment with antibiotics, and this effect persists after 2 months [16].

3.1.2 Prostanoids Compared with healthy nonsmokers, PGE2 concentrations in EBC are increased in steroid-naïve and steroid-treated patients with stable COPD [15]. There is a correlation between exhaled PGE2 and LTB4 concentrations in both COPD groups of patients [15]. This data might indicate higher production of PGE2, an endogenous biomolecule that may have anti-inflammatory effects in the airways, in conditions of increased lung inflammation. Nonselective cyclo-oxygenase (COX) inhibition by oral ibuprofen (400 mg q.i.d. for 2 days) reduces PGE2 concentrations in EBC in patients with COPD [18]. This effect might be relevant to the modulation of airway inflammation. Selective COX-2 inhibition by oral rofecoxib (25 mg once a day for 5 days) has no effect on PGE2 and LTB4 concentrations in EBC in patients with stable COPD [18]. Taken together, these findings indicate that PGE2 in EBC in patients with COPD is mainly derived from COX-1 activity [18]. The pathophysiological implications of selective and nonselective inhibition of COX for lung inflammation in patients with COPD have to be clarified.

3.1.3 Isoprostanes Isoprostanes are prostaglandin-like compounds that are produced in vivo independently of COX enzymes, principally by peroxidation of arachidonic acid due to free radicals [64]. Isoprostanes are considered among the best markers of oxidative stress [65, 66]. 8-Isoprostane, a compound that belongs to the F2 class of isoprostanes, is measurable in EBC in healthy nonsmokers [9, 19, 41], and its concentrations in EBC are elevated in healthy smokers and, to a greater extent, in current and ex-smokers with stable COPD [19]. Similar EBC 8-isoprostane concentrations in current and ex-smokers with COPD might indicate that oxidative stress due to chronic smoking reaches a point at which smoking cessation has little, if any, effect [19]. Increased 8-isoprostane concentrations in EBC in healthy smokers and patients with stable COPD has been confirmed by other authors [20, 22, 23, 31, 67]. 8-Isoprostane concentrations in EBC may reflect the degree of emphysema assessed by high-resolution computed tomography [22]. In patients with exacerbations of COPD, 8-isoprostane concentrations in EBC are higher than in healthy subjects [16, 67] and decreased after antibiotic treatment [16]. One study reported that 8-isoprostane concentrations in EBC are at least tenfold lower than those in sputum supernatants [67]. Inhalation of aminoguanidine, a relatively selective inhibitor of

432

P. Montuschi

inducible nitric oxide-synthase (iNOS), has no effect on 8-isoprostane concentrations in EBC in patients with COPD, healthy smokers and healthy nonsmokers [21].

3.2 Hydrogen Peroxide Hydrogen peroxide in EBC has been measured by spectrofluorimetry [68–70], spectrophotometry [10, 24, 71], fluorimetric assays [11, 51, 72–74], and chemiluminescence [75, 76]. A wide variability in the mean hydrogen peroxide concentrations in EBC in healthy, nonsmoking adults, has been reported (0.01 nM–0.45 mM) [68, 69]. Even under strictly controlled conditions, there is a high degree of day-to-day variability that highlights the fact that standardization of the experimental technique is required [51]. Compared with healthy nonsmokers, exhaled hydrogen peroxide concentrations are increased in healthy smokers [68] and this increase is more pronounced in patients with stable COPD [10, 24, 70]. In these patients, hydrogen peroxide concentrations in EBC do not seem to be dependent on smoking [70]. Exhaled hydrogen peroxide in EBC is further elevated in patients with COPD exacerbations [24, 26]. EBC hydrogen peroxide concentrations increase immediately after maximal exercise in patients with stable COPD [77]. The effect of inhaled glucocorticoids on exhaled hydrogen peroxide concentrations in patients with COPD is not established [25, 27]. One study showed that beclomethasone at a dose of 500 mg b.i.d. for 2 weeks had no effect on hydrogen peroxide concentrations in EBC in nonsmoking patients with stable COPD [25]. By contrast, another study reported that HFA-beclomethasone dipropionate at a dose of 400 mg b.i.d. for 4 weeks and fluticasone dipropionate at a dose of 375 mg b.i.d. for 4 weeks reduced hydrogen peroxide concentrations in EBC in patients with stable to moderate COPD [27]. In patients with COPD who required hospital admission because of exacerbations due to lower respiratory tract infections, exhaled hydrogen peroxide concentrations were not reduced during treatment with intravenous dexamethasone, inhaled salbutamol/ipratropium and, if required, antibiotics [78]. By contrast, other studies reported reduced hydrogen peroxide concentrations in EBC after intravenous glucocorticoid treatment in patients with exacerbation of COPD [26, 79]. In patients with stable COPD, treatment with oral N-acetyl-cysteine, which can have antioxidant effects, at a daily dose of 600 mg for 12 months reduced hydrogen peroxide concentrations in EBC starting from 9 months [80], whereas a similar study showed a much earlier effect starting from 15 days [81]. Despite some evidence of reduced oxidant stress, a multicenter trial has shown that N-acetyl-cysteine is ineffective at preventing deterioration in lung function and prevention of exacerbations in patients with COPD [82]. Hydrogen peroxide concentrations in EBC in patients with COPD are transiently increased 30 min after inhalation of N-acetyl-cysteine given by nebulization [83]. The clinical relevance of these findings is not currently known. More controlled studies to establish the effects of pharmacological therapy on hydrogen peroxide concentrations in

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EBC in patients with COPD are required. As with other biomolecules, a reliable methodology for EBC analysis is essential for planning and interpreting the results of interventional studies.

3.3 NO-Derived Products Patients with COPD have higher S-nitrosothiols concentrations in EBC than healthy nonsmokers [28]. Whether elevated S-nitrosothiols merely reflects NO production or whether they are functionally involved in the pathophysiology of COPD has to be clarified. Concentrations of peroxynitrite, measured using oxidation of 2¢,7¢-dichlorofluorescin in EBC collected at high pH, are increased in patients with moderately to severe COPD compared with those in healthy smokers and nonsmokers [21, 29] and are negatively correlated with FEV1 [29]. Nitrite and nitrates are end-products of NO metabolism. Nitrite has been measured in the EBC from healthy nonsmokers and was found increased in patients with COPD [28]. Another study reported similar nitrite/nitrates concentrations in EBC in patients with COPD and healthy subjects [30]. In patients with COPD, nitrite concentrations in EBC correlate with hyperinflation [84]. Smoking induces an acute increase in nitrate, but not nitrite, concentrations in EBC in healthy smokers [85]. In a double-blind, placebo-controlled study, inhibition of inducible nitric oxide synthase (iNOS) with inhalation of aminoguanidine given by nebulization resulted in a significant, but not complete, inhibition of peroxynitrite production as reflected by its concentrations in EBC in patients with COPD and healthy smokers 60  min after inhalation [21]. Inhalation of aminoguanidine has no effect on peroxynitrite concentrations in EBC in healthy nonsmokers [21]. Aminoguanidine reduces nitrite/nitrate concentrations in EBC in patients with COPD at 60 min and 120 min after inhalation, whereas in healthy smokers and healthy nonsmokers, reduction in nitrite/nitrate concentrations in EBC following aminoguanidine inhalation is observed only at 60  min [21]. iNOS inhibition with aminoguanidine has no effect on nitrotyrosine concentrations in EBC in patients with COPD, healthy smokers and healthy nonsmokers [21]. Taken together, these data indicate that both constitutive and inducible NOS isoforms might contribute to the elevated pulmonary peroxynitrite and nitrite/nitrate production in patients with COPD [21].

3.4 pH EBC pH is a robust and reproducible assay of airway acidity [12, 86]. Mean EBC pH values are lower in patients with COPD compared with patients with asthma and healthy subjects [31]. In patients with COPD, pH values in EBC are correlated with sputum neutrophilia and hydrogen peroxide concentrations in EBC [31].

434

P. Montuschi

In this study, pH values in EBC in patients with COPD were similar, irrespective of treatment with inhaled glucocorticosteroids [31]. However, controlled studies to establish the effects of glucocorticoids on EBC pH values in patients with COPD are warranted. pH values in EBC are negatively correlated with gastro-esophageal reflux disease symptoms in patients with COPD and healthy controls [87].

3.5 Aldehydes Measurement of MDA, a product of lipid peroxidation in biological fluids, is a widely used test for oxidative stress, but its reliability for assessing oxidative stress in  vivo is questionable [88]. MDA and other aldehydes (hexanal, heptanal, and nonanal) have been measured in EBC using liquid chromatography-tandem mass spectrometry [32]. MDA, hexanal, and heptanal concentrations in EBC are increased in patients with COPD as compared with nonsmoking subjects, whereas only MDA is elevated in patients with COPD compared with healthy smokers [32].

3.6 Adenosine Triphosphate Adenosine triphosphate (ATP) was detected in EBC in healthy nonsmokers, healthy smokers and patients with exacerbation of COPD [33]. There was no difference in ATP concentrations in the three study groups [33]. After treatment, oxygenation of COPD patients improved, but EBC ATP concentrations were similar to those of admission [33].

3.7 Other Markers A wide interindividual variability (from undetectable to 1.4 mg) in the amount of total proteins in EBC [89] has been reported even in healthy subjects. Although amylase concentrations can be detected by very sensitive analytical techniques, amylase concentrations in EBC are generally undetectable or very low, indicating little, if any, salivary contamination of EBC [7, 58]. Compared with those in healthy nonsmokers, healthy smokers and patients with stable COPD, interleukin (IL)-1b, IL-6, IL-8, IL-10, IL-12p70, and tumor necrosis factor (TNF) a concentrations in EBC measured by a multiplex bead array test combined with flow cytometry have been found increased in patients with exacerbation of COPD [34]. Compared with those in healthy nonsmokers, IL-1b and IL-12 concentrations in EBC were increased in patients with stable COPD [34]. Healthy smokers had increased concentrations of all cytokines in EBC compared to healthy nonsmokers and, with the exception of

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IL-1b, to patients with stable COPD [34]. In another study, IL-6 ­concentrations in EBC in patients with stable COPD were found increased compared with those in healthy nonsmokers [35]. One study reported elevated macrophage migration inhibitory factor, IL-12, regulated on activation normal T-cell-expressed and secreted (RANTES), and soluble intracellular adhesion molecule-1 in EBC in patients with moderate to severe COPD with no effect after treatment with infliximab, an antiTNF-a antibody [36]. In all published studies, cytokines in EBC were measured by immunoassays that are very sensitive and work well in buffer, but their behavior in EBC has not been established. Concentrations of cytokines measured in EBC are very low and close to the lower limit of quantification of the immunoassays used. At these concentrations, the analytical variability is very high. This makes it difficult to interpret the results. The presence of cytokines in EBC has not been definitively demonstrated and an accurate quantification of their concentrations in EBC has not yet been provided. This relevant issue needs to be formally addressed by a specific and robust analytical methodology. Metabolomics, the study of molecules generated by metabolic pathways, has been applied to the analysis of EBC [57]. Metabolomic analysis of EBC by nuclear magnetic resonance spectroscopy discriminates between patients with COPD, laringectomized patients and healthy subjects [57]. When samples were collected using a commercially available condenser, saliva spectra were highly different from corresponding EBC samples [57]. This data excludes significant salivary contamination.

4 Advantages and Limitations Measurements of biomolecules in EBC may provide insights into the pathophysiology of COPD. Measurement of surrogate markers of pulmonary inflammation in EBC might be useful for identifying subgroups of healthy chronic smokers who are more susceptible to COPD, phenotyping of COPD, assessing the effect of antiinflammatory therapy in patients with COPD, identifying those patients who are most likely to benefit from pharmacological therapy, and testing new drugs for COPD. The identification of selective profiles of inflammatory markers in patients with COPD might be useful for a more accurate selection of patients with COPD in clinical trials. At present, the lack of standardization of EBC analysis is the principal limitation of this technique. This can account for most of the variability of the results reported in various studies and makes inter-laboratory comparison of data difficult. Other current limitations of EBC analysis are the size of the published studies, which is generally relatively small; the fact that this technique is unable to provide any information on the inflammatory cells involved in the pathophysiology of COPD; the fact that the demonstration that EBC reflects lung rather than systemic inflammation has yet to be provided; and the origin (airways versus alveolar region) and cellular sources of biomolecules in EBC that have to be identified.

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5 Future Research Standardization of procedures for sample collection and storage and validation of the analytical techniques for measuring different biomolecules in EBC are currently the priorities in this research area. The methodological issues discussed above have to be addressed before EBC analysis can be considered in the clinical setting. A robust analytical methodology is essential for the development of EBC analysis. Future research in this field should aim at developing reference analytical techniques (e.g., mass spectrometry) to provide definitive evidence for the presence of biomolecules in EBC and an accurate quantitative assessment of their concentrations; development of sensitive, specific, and reproducible immunoassays or validation of those already available to be used routinely. Controlled studies are required to establish the utility of EBC analysis as a tool for assessing the response to pharmacological therapy in patients with COPD. Future research in this area should also include identification of reference values for different inflammatory biomarkers in healthy subjects and quantitative assessment of their concentrations in patients with stable COPD of different severity and in patients with COPD exacerbations; studies on the reproducibility of measurements to address between-day, diurnal, and analytical variability; large longitudinal studies in patients with COPD; and studies aiming at clarifying the relationships of EBC markers with symptoms, lung function, and other indices and/or methods for quantifying lung inflammation in patients with COPD. As the behavior of biomolecules in EBC may be different, standardized procedures need to be implemented for each individual biomolecule. Whether, and when, analysis of EBC will have clinical applications is difficult to predict. In any case, this technique should be part of an integrated approach that consists of clinical assessment, lung function testing, and possibly sputum induction. Due to the relative lack of noninvasive methods for assessing and monitoring airway inflammation and therapeutic intervention in patients with COPD, further research in this area is warranted. Acknowledgments  Supported by the Catholic University of the Sacred Heart, Fondi di Ateneo 2007–2010.

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Induction of Oxidative Stress by Iron/Ascorbate in Isolated Mitochondria and by UV Irradiation in Human Skin Ingrid Wiswedel, Wolfgang Augustin, Sven Quist, Harald Gollnick, and Andreas Gardemann

Abstract  Two different model systems that have been successfully used in our lab to induce oxidative stress are described. The first is the so-called “iron/ascorbate” system, under appropriate conditions suitable to investigate oxidative stress mediated functional impairment in isolated intact mitochondria in  vitro and the second is UV irradiation, which can be used under experimental and clinical conditions in  vitro and in  vivo to induce oxidative stress and inflammation in human skin. In the presence of iron/ascorbate, the induction of lipid and protein oxidation was investigated in isolated mitochondria. Very early events after treatment of freshly prepared, functionally intact mitochondria consist in the inhibition of active respiration mainly due to an attack of the complex III activity of the respiratory chain. The membrane potential was sustained nearly unchanged for a relatively long time and broke down abruptly at the end of the initiation phase of lipid peroxidation, when sufficient energization and antioxidant protection of mitochondria could not be maintained. UVA irradiation in combination with photosensitizing agents as 8-methoxypsoralen is successfully used as extracorporeal photoimmunotherapy (ECPI) in the treatment of several skin diseases, such as cutaneous T-cell lymphoma and systemic scleroderma. We used this system to determine markers of oxidative stress in plasma and cells from the buffy coat under experimental ECPI-relevant conditions and in patients during ECPI treatment. Only moderate oxidative stress could be demonstrated by a decrease of the antioxidant defense and a dose-dependent augmentation of hydroxyeicosatetraenoic acids and F2-isoprostanes as highly specific and sensitive biomarkers of lipid peroxidation and potential mediators of inflammatory and/or photoimmunomodulatory effects of ECPI. UVB is known as a major skin carcinogen that mainly affects keratinocytes and induces a complex cascade of inflammation, including reactive oxygen species. I. Wiswedel (*) Department of Pathological Biochemistry, Otto-von-Guericke University, Magdeburg, Germany e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_20, © Springer Science+Business Media, LLC 2011

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Effects of moderate UVB doses on the generation of biomarkers of oxidative stress and inflammation in HaCaT keratinocytes in vitro and in microdialysates of human skin in vivo were studied. F2-isoprostanes and prostaglandins were increased UVB dose-dependently, whereas functional integrity of HaCaTs was diminished. As an adaptive response, protein levels of MnSOD were enhanced, but higher UVB doses lead to irreversible cell damage. The microdialysis technique allowed for sensitively quantifying the markers of inflammation and oxidative stress and their kinetic profile in vivo following UVB exposure. Keywords  HaCaT keratinocytes • Human skin • Inflammation • Iron/ascorbate • Microdialysis • Mitochondria • Oxidative stress • UV irradiation

1 Introduction 1.1 Initiation of Oxidative Stress in Isolated Mitochondria In order to gather more detailed information concerning the mechanism, the kinetics and the causal chain of events following radical-mediated processes, several model systems were used to initiate in  vitro generation of reactive oxygen species and lipid peroxidation in different cellular systems, including mitochondria. The bestknown prooxidant tools for inducing oxidative stress in  vitro are ferrous sulfate/ ascorbate [1, 2], ferrous sulfate/hydrogen peroxide [3], NAD(P)H/ADP/ferrous sulfate [4, 5], cumene hydroperoxide [6] tert-butylhydroperoxide [7], 2,2¢-azobis (2-amidino)propane (AAPH) [8], xanthine/xanthine oxidase [9] and comparable moderate model systems applying hypoxia/reoxygenation [10]. A lot of studies are concerned to lipid peroxidation as a complex system of reactions, involving free radical intermediates, which cause oxidative degradation of unsaturated fatty acids and accumulation of a variety of peroxidized products in the membrane. The effects of such attacks on energy-linked functions of mitochondria [2, 10, 11] and membrane-associated enzymes [12–14] under different experimental conditions were reported. Mitochondria have been shown to be efficiently protected against oxidative stress by powerful defense systems including the activities of superoxide dismutase and glutathione peroxidase, whereas lipid peroxidation has been found to be strongly inhibited by various nonenzymatic antioxidants [15–18]. Development of free radical-induced lipid degradation is associated with the breakdown of the preventive mechanisms during the exponential phase of extensive peroxidation, finally resulting in irreversible damage to mitochondria [18]. Examples, mainly from the own studies, will be given for the use of iron/ascorbate as an in vitro model system to investigate free radical-induced degradation of polyunsaturated fatty acids and generation of products of lipid peroxidation, free radical-mediated functional alterations in mitochondria, changes in endogenous antioxidants and the effects of exogenously added antioxidants.

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1.2 Initiation of Oxidative Stress by UV Irradiation The second part is concerned with the effects of UV irradiation (UVA and UVB) as a model system for investigating UV dose-dependent alterations of oxidative stress and inflammation markers in vitro in HaCaT keratinocytes, in buffy coat or plasma samples and in  vivo in the interstitial space of the dermis using microdialysis technique. The ultraviolet component of sunlight on the earth’s surface is usually divided into two wavelength regions that are termed UVA (320–400 nm; near UV) and UVB (290–320  nm; mid-UV) [19]. Both of these contribute to biological changes as oxidative stress, inflammation, gene mutation and immunosuppression in the skin [20]. The reaction pattern and the time course of acute UV-induced skin inflammation are wavelength-dependent. This may be due to differences in UV penetration into skin layers (UVA > UVB) and specific reaction cascades initiated by a specific wavelength band. Depending on the radiation dose, UVA and UVB can reach the dermis and the dermal vasculature [21]. Short-wavelength UVB irradiation is believed to play a greater role for the most damaging effects of sunlight, including skin cancer, than long-wavelength UVA irradiation [22]. UVB wavelengths are absorbed by DNA, can induce mutagenic lesions such as pyrimidine dimers [23] and modulate to a higher extent than UVA-specific and nonspecific immune responses [24], which play at least a partial role in causing skin cancer. The effects of UVA are mainly mediated by the activation of endogenous and exogenous photosensitizer radicals as psoralens or reactive oxygen species [23, 25]. The effects of UVA irradiation on human plasma and buffy coat in the course of photopheresis (ECPI), as well as under ECPI-relevant experimental conditions and of UVB irradiation on HaCaT keratinocytes in vitro and human skin in vivo, are demonstrated from our own studies and discussed in light of the findings published by other authors.

2 Materials and Methods 2.1 Isolation of Functionally Intact Mitochondria from Rat Liver, Brain and Heart Liver mitochondria were prepared from fasting adult male Wistar rats in ice-cold medium containing 250 mM sucrose, 20 mM TRIS, pH 7.4, 2 mM EGTA and 1% (w/v) bovine serum albumin (BSA) using a standard procedure [26]. The functional integrity was determined by measuring the rate of oxygen uptake by a closed Clarktype electrode with succinate or glutamate plus malate as hydrogen-supplying substrates, with and without ADP (state 3 and state 4 respiration) and/or FCCP (uncoupled respiration). The mitochondria were well coupled as indicated by a respiratory control index (RCI) greater than 6. Brain mitochondria were prepared from the whole brain (without cerebellum) of adult male Wistar rats in ice-cold medium containing 250  mM mannitol, 20  mM

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TRIS, 1 mM EGTA, 1 mM EDTA, and 0.3% (w/v) BSA at pH 7.4 using a standard procedure [27]. After the initial isolation, Percoll was used for purification of mitochondria containing residual endoplasmic reticulum, Golgi apparatus and plasma membranes. The mitochondria were well coupled with glutamate plus malate as hydrogen-supplying substrates (RCI greater than 4). Heart mitochondria were prepared from adult male Wistar rats in ice-cold medium containing 150 mM KCl and 10 mM EDTA at pH 7.4, in detail described by Schaller et al. [28]. The RCI with glutamate plus malate as substrates was greater than 4.

2.2 Protein Content The mitochondrial protein content was measured according to the method of Lowry et al. [29] or the method of Bradford [30] by using BSA as the standard.

2.3 Incubation Conditions in the Presence of Iron/Ascorbate Iron/ascorbate induced peroxidation. Isolated mitochondria (1–5  mg/ml) were incubated in an air atmosphere in a medium containing 0.1 M KCl, 0.01 M TRIS, 20–50 mM iron II-sulfate, and 500 mM ascorbate (pH 7.4) at 25°C. Aliquots were withdrawn after distinct time intervals for the analysis of thiobarbituric acidreactive substances (TBARS) as an immediate measure of lipid peroxidation. Further aliquots were taken for the measurement of oxygen uptake and membrane potential as functional parameters and for the analysis of phospholipids, fatty acids, lipohydroperoxides, monohydroxyeicosatetraenoic acids, F2-isoprostanes, monofunctional aldehydes, carbonyls and others.

2.4 Respiration and Membrane Potential Oxygen uptake of mitochondria was measured at 30°C in a thermostat-controlled chamber equipped with a Clark-type electrode. For the calibration of the oxygen electrode, the oxygen content of the air-saturated incubation medium was taken to be 217 nmol O2/ml [31]. The transmembrane potential (Dy) was determined as described in detail in [2].

2.5 Phospholipid and Fatty Acid Analysis Mitochondrial phospholipids were extracted by the method of Folch et al. [32] and quantified by HPLC/UV detection [33] or HPLC/fluorescence detection [34].

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For the determination of mitochondrial fatty acids, lipids were extracted and saponified. After crown ether catalyzed derivatization to p-bromo­phenacylesters, individual fatty acids were quantified by HPLC as outlined in detail in [35].

2.6 Lipid Peroxidation Products 2.6.1 TBARS The content of TBARS was determined by HPLC/UV detection in principle according to Buege and Aust [36], modified by Jentzsch et al. [37].

2.6.2 4-Hydroxynonenal and Other Monofunctional Aldehydes Monofunctional aldehydes were determined by the method of Esterbauer and Cheeseman [38], which includes the derivatization of these compounds by 2,4-dinitrophenylhydrazine, subsequent separation of the hydrazones by thin-layer chromatography into classes of different polarities and the final separation by HPLC/UV detection [39].

2.6.3 Lipohydroperoxides The mitochondrial content of lipohydroperoxides was measured iodometrically as described in detail in [36].

2.6.4 Monohydroxyeicosatetraenoic Acids and F2-Isoprostanes The quantification of hydroxyeicosatetraenoic acids (HETEs) and isoprostanes in rat brain mitochondria was performed by gas chromatography–mass spectrometry/negative ion chemical ionization, as described by Wiswedel et al. [40].

2.6.5 Oxidized Cardiolipin [(C-18:2)3 Monohydroxylinoleic acid-CL] The specific cardiolipin oxidation product [(C-18:2)3 monohydroxylinoleic acidCL] was quantified by electrospray ionization mass spectrometry according to Valianpoor et  al. and Pope et  al. [41, 42], modified as described in detail by Wiswedel et al. [43].

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2.7 Antioxidants 2.7.1 Reduced and Oxidized Glutathione Glutathione was determined using the catalytic assay according to Tietze [44], either as the sum of the reduced and oxidized species expressed as GSH equivalents or as GSSG alone [45], so that GSH could be calculated by difference.

2.7.2 a-Tocopherol and Ubiquinol The extraction procedure and HPLC analysis of a-tocopherol and coenzyme Q was performed according to Lang et  al. [46], described in detail by Noack et al. [47].

2.8 UVA Irradiation 2.8.1 UVA Exposure of Cells and Plasma In Vitro Buffy coat (plasma containing leukocytes) and plasma from patients were diluted according to photopheresis conditions [30 ml plasma plus 13.5 ml phosphate-buffered saline (PBS)] and subsequently exposed to increasing cumulative doses of UVA (0.5, 1.0, 2.0 and 20  J/cm2), using a UV 800 lamp (Phillips; 315–400 nm, maximum 355 nm, intensity 7 mW/cm2). The temperature was kept constant at 25°C. The 8-methoxypsoralen (8-MOP) concentrations were 0, 50, 200 and 400 ng/ml. Plasma aliquots were taken immediately after the exposure, frozen in liquid nitrogen and stored at –80°C in deep freeze. Cells were prepared from the radiated buffy coat by sedimentation at 800 g and subsequent washing with PBS. The patients involved in the studies had the following diagnoses: cutaneous T-cell lymphoma (11 patients), and progressive systemic scleroderma (7 patients). Patients were treated with the UVAR system (Therakos, West Chester, PA). Blood was drawn from the patients, and erythrocytes, leukocytes and plasma were separated by low-speed centrifugation. The erythrocytes were directly reinfused into the patient. The buffy coat fraction that contained the mixed leukocytes and blood platelets was diluted with PBS (30 ml plasma plus 13.5  ml PBS), mixed with 8-MOP in a final concentration of 150  ng/ml and exposed to UVA irradiation (cumulative dose of 2  J/cm2, radiation intensity 7 mW/cm2, irradiation time 4:46 min). Then the cells were reinfused. Two ECPI sessions on consecutive days were investigated. Samples were taken from the peripheral blood immediately before and after each treatment and 24 h after the second session.

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2.8.2 Western Blotting for Detection of Oxidatively Modified Proteins This method was adopted from Levine et  al. [48] and performed as described in detail by Reinheckel et al. [49]. 2.8.3 Total Antioxidant Status Total antioxidant capacity was determined using the assay of Randox Laboratories Ltd., Crumlin, UK. The assay is based on the incubation of ABTSR (2,2-azino-di[3-ethylbenzthiazoline sulfonate]) with a peroxidase (metmyoglobin) and hydrogen peroxide to produce the radical cation ABTS+, which is measured at 600  nm. Antioxidants in the sample will suppress the ABTS+ formation proportional to their concentration. 2.8.4 Quantification of HETE Isomers and Enantiomer Separation of 5-HETE HETEs were quantified by a modified method of Thomas et  al. [50] using gas chromatography mass spectrometry described in detail in [51]. The enantiomer separation of 5-HETE, the main HETE isomer in human plasma, was carried out on a Chiralcel OB column as described by Wiesner and Kühn [52].

2.9 UVB Irradiation and HaCaT Keratinocytes In Vitro 2.9.1 UVB Irradiation and Cell Culture HaCaT keratinocytes (human adult low calcium high temperature) were obtained from the German Cancer Research Center, Heidelberg, Germany. They were routinely grown in Dulbecco’s Medium, supplemented with 10% fetal calf serum, 2% l-glutamine, and 1% antibiotic–antimycotic solution (all chemicals by GIBCO/Invitrogen, Karlsruhe, Germany) under a humidified atmosphere of 5% CO2 at 37°C [53]. For the purpose of UVB irradiation, 1 day before the start of the experiment, subconfluent cultures were harvested (0.25% trypsin-EDTA), and 1 × 105 cells/cm2 were subcultured in flat bottom tissue culture plates (nunc, Wiesbaden, Germany). HaCaT keratinocytes were irradiated with 10, 30, 50, and 100 mJ/cm2 UVB (solar UV simulator, Solar Light, Philadelphia, PA, USA). Controls underwent identical manipulations without irradiation. After an additional incubation of 24 h, supernatants were collected, centrifuged (2,000 g, 5 min) and kept cell-free frozen at −80°C until measurements of metabolites. HaCaT keratinocytes were harvested in phosphatebuffered saline (PBS). A cell aliquot was counted by means of a Neubauer cell counting chamber using trypan blue staining. Immediately, the cells were directed

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to further analysis or frozen in liquid nitrogen and stored in deep freeze (−80°C) until further analysis (e.g., for quantification of F2-isoprostanes and cardiolipin). Protein content of HaCaT keratinocytes was determined according to Lowry [29]. 2.9.2 Determination of Cell Viability: Vital Staining and TUNEL-Staining The irradiation-dependent viability of HaCaT keratinocytes was assessed by doublelabeling with propidium iodide and fluorescein diacetate. The technique is based on the phenomenon that living cells are able to hydrolyze fluorescein diacetate (10 mg/ ml PBS) by intracellular esterases resulting in a green–yellow fluorescence. Dead cells were labeled by propidium iodide (4 mg/ml PBS), which interacts with DNA to yield a red fluorescence of cell nuclei. Evaluation was carried out on a fluorescence microscope (Axiophot, Zeiss, Jena, Germany) equipped with phase-contrast, rhodamine and fluorescein optics. The technique of terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling (TUNEL) was used to detect apoptotic DNA fragmentation in the cultured HaCaT keratinocytes. Therefore, HaCaT cultures were washed and permeabilized with methanol:chloroform:acetic acid (66:33:1, 10  min) and incubated in a humidified box (37°C, 90  min) using 50  ml reaction solution: 0.4  ml terminal deoxynucleotidyl transferase (TdT, 0.5  U/ml), 2  ml cobalt chloride (2.5 mM), 4 ml TdT reaction buffer, 0.8 ml fluorescein-dUTP (2 nM), 12.8 ml MilliQ (Roche Diagnostics, Germany). Incubation was stopped by washing cultures with saline sodium citrate (3 × 5 min) and PBS (2 × 5 min). Again, cells were examined on an Axiophot fluorescence microscope. 2.9.3 Determination of Biomarkers of Oxidative Stress and Inflammation F2-isoprostanes were quantified by gas chromatography mass spectrometry/ negative ion chemical ionization (selected ion monitoring mode) described in detail elsewhere [40, 54]. 2.9.4 Mitochondria and Submitochondrial Membranes Mitochondria and submitochondrial membranes were isolated from the HaCaT cells according to Navarro et al. [55]. 2.9.5 Mitochondrial Electron Transfer Activities and Mitochondrial Nitric oxide Synthase (mtNOS) Activity The electron transfer activities of the complexes I–III, II–III, and IV were determined spectrophotometrically at 30°C in 100 mM phosphate buffer (pH 7.4) according to Navarro et al. [56]. Mitochondrial nitric oxide (NO) production was determined by the oxyhemoglobin (HbO2) oxidation assay as described by Boveris et al. [57].

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2.9.6 Quantification of Cardiolipin Species Cardiolipin species were analyzed by HPLC fluorescence detection as described in detail by Schlame et al. [34]. 2.9.7 Western Blot Analysis of MnSOD and CuZnSOD The procedure according to Lorenz et  al. [58] was used, described in detail by Wiswedel et al. [59]. The following antibodies against MnSOD (polyclonal, Biomol, Hamburg, Germany, 1:5,000) or CuZnSOD (polyclonal, Biomol, 1:800) were used. Immunoreactivity was visualized using the ECL detection system (Amersham Pharmacia Biotech Ltd., Buckinghamshire, UK) and quantification occurred by densitometric analysis using a Biometra BioDocAnalyzer. b-Actin (monoclonal, Sigma, Germany, 1:2,500) detection was used to show equal sample loading.

2.10 UVB Irradiation and Microdialysis of Human Skin In Vivo 2.10.1 Control Persons Ten healthy female volunteers, 18–30 years old, were included in the study. Inclusion criteria were skin type II, body mass index 19–25, nonsmoking, no drug intake and agreement to UV-exposure according to the protocol. Grading of skin type was performed with respect to individual case history, hair and eye color and skin coloration, as well as testing of the minimal erythema doses (MED) for UVB. Exclusion criteria were any kind of clinically relevant diseases, especially concerning the skin, the gastrointestinal tract, and the central nervous system. Before entering the study, the subjects underwent a thorough physical examination by a dermatologist. They were neither allowed to take any kind of medication during the 2 weeks before the experiments nor during the study time. 2.10.2 Induction of Skin Erythema by UVB Irradiation One week before the first UVB exposure, the MED for UVB (wavelength 290– 320 nm) irradiation was examined using a calibrated solar multitester (Saalmann multitester SBB LT 400, Saalmann Medizintechnik, Herford, Germany). For that purpose, five circular spots at the buttocks, 15 mm in diameter and at a distance of 25 mm, were treated with increasing doses of UVB. All subjects were instructed to avoid UV exposure in order to keep individual MEDs during the study as constant as possible. Besides the macular erythema test reaction, there were no further alterations in the skin areas. Erythemas were graded by visual assessment (grade 0–6) and by chromametry (CR-300 Minolta, Osaka, Japan) using the international three-dimensional color system (L, a, b).

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For the study purpose, defined areas on the volar forearm (lateral arm) of the volunteers were exposed to UVB irradiation at dosages 2× the individual minimal erythema dose (Waldmann UV 3003K, H. Waldmann GmbH & Co. KG Villingen-Schwenningen, Germany). 1% diclofenac gel or 5% diclofenac cream (both about pea-size) was topically applied under plastic film occlusion to suppress UVB erythema. 2.10.3 Microdialysis Technique Twenty-four hours after UVB-irradiation, sterilized microdialysis probes (CMA/70, CMA Microdialysis, Solna, Sweden) were intracutaneously inserted, at least 5 cm apart, beneath the irradiated (middle of a 4  cm2 field) and nonirradiated areas (depth: 0.9–1.1  mm, measured by 20  MHz ultrasound; Technos MPX, Esaote, Munich, Germany) without any prior local anesthesia. The shaft length of the probe was 25  mm, the membrane length was 10  mm, and the membrane diameter was 0.5 mm. After probe insertion, probes were connected to two microdialysis pumps (CMA/102, Microdialysis, Solna, Sweden) and perfusion was started with isotonic saline solution (E 153, Serumwerke Bernburg, Germany) at a flow rate of 1.0 ml/ min. The semipermeable membrane allows passive diffusion of molecules smaller than 20 kDa from the interstitial space into the perfusate. After probe insertion, the membrane was flushed at 5.0 ml/min for 1 h for tissue recovery and probe equilibration. Then, dialysate samples were collected in small Eppendorf tubes on ice, in 20-min fractions for up to 4–5 h. Dialysate samples were stored in liquid nitrogen during the study and at –80°C until GC-MS analysis of lipid mediators. 2.10.4 Determination of Concentrations of F2-Isoprostanes and Prostaglandins The determination of the concentrations of nonesterified F2-isoprostanes was carried out as described previously [40, 54, 60]. PGE2 was identified and quantified by the same sample work-up procedure using PGE2 (m/z 495) as authentic standard and PGE2-d4 (m/z 499) as internal standard.

3 Results and Discussion 3.1 Iron/Ascorbate Induced Peroxidation in Mitochondria In the presence of iron plus ascorbate and oxygen, isolated mitochondria generate high amounts of reactive oxygen species, as hydroxyl radicals via perferryl radicals and/or Fenton reaction [61]. This kind of oxidative stress causes degradation of mitochondrial macromolecules, as mainly lipids, proteins and nucleic acids.

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Fig.  1  Experimental system of iron/ascorbate-induced oxidative stress in mitochondria. Resp chain: repiratory chain; ox phos: oxidative phosphorylation. This figure was taken from Augustin et al. [18]

Using iron/ascorbate for the initiation of lipid peroxidation in isolated rat liver and brain mitochondria, a biphasic course of lipid oxidation can be shown, characterized by an initial lag phase with very small MDA (or TBARS – thiobarbituric acid reactive substances) generation, followed by a so-called exponential phase with high rates of TBARS formation finally reaching plateau levels of TBARS between 20 and 100 nmol/mg mitochondrial protein, depending on the iron/protein ratio, the energization of mitochondria and the antioxidative protection [2] (Fig. 1 shows a scheme of the experimental system). From the different mitochondrial species, rat brain mitochondria are more sensitive to iron/ascorbate-induced oxidative stress than rat liver mitochondria, whereas rat heart mitochondria are rather resistant to iron/ascorbate [2, 11, 40]. Rat heart mitochondria generally show a low lipid peroxidation in the presence of iron/ascorbate, but a slight enhancement of oxidative stress after exposure with ADP-iron/ NADPH [11]. The sensitivity against oxidative stress was strongly found to depend on the age of the animals. In particular, mitochondria, which were isolated from neonatal rat hearts, were very susceptible to oxidative stress [62].

3.1.1 Iron/Ascorbate Induced Generation of Lipid Peroxidation and Protein Oxidation Iron/ascorbate-induced oxidative stress has a lot of effects on isolated rat liver, rat brain and rat heart mitochondria. We studied the iron/ascorbate-mediated degradation of mitochondrially localized phospholipids, in particular cardiolipin [43],

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the decrease of polyunsaturated fatty acids, as linoleic, arachidonic and docosahexaenoic acid [39, 40], the increased generation of lipohydroperoxides [63] and more specifically the generation of monohydroxylinoleic acid-cardiolipin as oxidized (C18:2)4-cardiolipin species [43]. Furthermore, the enhanced formation of monohydroxy-eicosatetraenoic acids (HETEs), F2-isoprostanes [40], and monoand bifunctional reactive aldehydes (4-hydroxynonenal, n-hexanal, 4-hydroxydecenal, 2-octenal and malondialdehyde) as products of lipid peroxidation was shown [39]. The aldehydes have been demonstrated to have relatively long half-lives within the cells (minutes to hours), allowing for multiple interactions with cellular components [64]. Besides lipid peroxidation products, the iron/ascorbate-induced generation of protein-bound carbonyls as indicators of oxidative modification of proteins was demonstrated by Reinheckel et al. [39]. Vatassery et al. [65] reported that a significant percentage of mitochondrial cholesterol was oxidized by treatment of Fe2+ plus ascorbate. 7-keto- and 7-hydroxy­ cholesterols and small amounts of 5a,6a-epoxycholesterol were produced. A lot of biological activities of oxidized cholesterols and cholesterolesters were described in the literature [66–69]. The iron/ascorbate-induced model system may be useful to compare the different measures of oxidative stress, as lipohydroperoxides, TBARS, HETEs, isoprostanes, several monofunctional aldehydes and carbonyls. It could be shown that their time courses are highly correlated [39, 40]. But the final levels of the various oxidation products differed very much: lipohydroperoxides > MDA > n-hexanal > 4-HNE >> HETEs >> F2-isoprostanes [39, 40, 63]. For example, F2-isoprostanes, in mitochondria totally bound to phospholipids, are minor products of lipid peroxidation with about 20  pmol/mg protein as maximum level in isolated rat brain mitochondria. Under the same experimental conditions, about 5,000 times more TBARS and about 50 times more HETEs in comparison to F2-isoprostanes were generated. But interestingly, F2-isoprostanes were elevated about 100-fold after incubation with iron/ ascorbate compared to baseline levels whereas HETEs less than 10-fold [40]. The time-dependent accumulation of lipid peroxidation products coincided with the decline of phospholipids and the consumption of polyunsaturated fatty acids [40, 43]. 3.1.2 Iron/Ascorbate Mediated Impairment of Functional Intactness of Mitochondria The decline of important mitochondrial functions occurs before a significant amount of peroxidation products, e.g., TBARS, has been generated by treatment with iron/ascorbate. One of the first functional events during iron/ascorbate-induced oxidative stress in isolated intact rat liver mitochondria were found to consist in a progressive inhibition of the phosphorylating (active or state-3 respiration) and also of the uncoupled respiration (by FCCP) with succinate and glutamate/malate as hydrogen-supplying substrates, whereas the resting state respiration (state 4 respiration) during the same time period was virtually not influenced [2]. An attack at the level of the respiratory chain was established as a reason for the early inhibition of oxygen uptake during iron/ascorbate-induced lipid peroxidation [13]. By following

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the activities of the respiratory chain complexes I to IV, a close correlation between the decline of mitochondrial respiration and the activity of complex III strongly suggests that an impairment of the respiratory chain in the b–c1 region represents one of the first functional events in the causal sequence of reactions preceding the phase of massive TBARS formation [13]. The mitochondrial membrane potential, registered simultaneously with the oxygen uptake in functionally intact rat liver mitochondria, could be built up for a relatively long time following exposure to iron/ascorbate, with only minor decreases, but broke down rather promptly, when the active respiration was highly diminished and the drastic increase of TBARS formation began. Inhibition of respiration and, with some delay, the drop of membrane potential are early stages in the causal chain of events that precede the onset of intensive lipid peroxidation in rat liver mitochondria [2]. 3.1.3 Role of Endogenous and Exogenous Antioxidants During Iron/Ascorbate-Induced Oxidative Stress The mitochondrial antioxidative defense systems can be effectively regenerated during or after iron/ascorbate- induced oxidative stress and the mitochondria are not damaged, as long as they can keep a high energized state [70]. Energization of mitochondria mainly depends on the availability of suitable respiratory substrates that can provide hydrogen for the reduction of either the glutathione (GSH) or the a-tocopherol system since GSH is regenerated by glutathione reductase with the substrate NADPH and the a-tocopheroxyl radical likely by reduced coenzyme Q. After the exhaustion of antioxidative defense systems, damage of mitochondrial functions are expressed, followed by membrane injuries along with the oxidation and degradation of mitochondrial proteins and lipids finally leading to total damage of the mitochondria. In this connection, we studied the role of the mitochondrial glutathione system in the defense against iron/ascorbate-induced oxidative stress [70]. A successive diminution of the mitochondrial glutathione pool during the initiation phase of peroxidation coinciding with the decrease of active respiration was observed, essentially due to losses of GSH while the redox state of the glutathione system was not significantly influenced before the onset of massive TBARS formation. Oxidizable substrates as glutamate/malate and 3-hydroxybutyrate affected the peroxidation by preventing the early inhibition of respiration and the diminution of GSH and by extending the period of the induction phase considerably. Obviously, that was due to the supply of NADPH to recover GSH via GSSH reductase as evidenced by the correlation of the decline of NADPH and GSH [70]. The decreased GSH content could not be attributed to the accumulation of GSSG. An explanation for the loss of GSH could be the formation of mixed disulfides with proteins or CoASH via GSH transferases, the formation of adducts with several peroxidation products, in particular with aldehydes, or a direct oxidation to sulfinic or sulfonic acids. In the studies performed in the presence of iron/ascorbate, no attempts have been made to characterize the products that account for the loss of soluble GSH.

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Although the data strongly supports the fact that mitochondrial glutathione plays a central role in the defense of oxidative stress, there are other potent defense systems in mitochondria. Therefore studies were undertaken to determine the consumption of lipid-soluble antioxidants, as a-tocopherol and ubiquinols, in mitochondria following exposure to iron/ascorbate [47]. During the induction phase of iron/ascorbateinduced oxidative stress, a-tocopherol declined moderately to about 80% of initial contents and the total coenzyme Q pool (coenzyme Q9ox plus coenzyme Q9red) remained nearly unchanged, but the reduced coenzyme Q9 continuously declined, reaching its completely oxidized state at the start of massive formation of TBARS. At the same time, a-tocopherol began to decline steeply, but never became completely exhausted. Evidently, the oxidation of the coenzyme Q9 pool constitutes a prerequisite for the onset of heavy lipid peroxidation in mitochondria and for the subsequent depletion of a-tocopherol. Trapping of the glutathione by the addition of dinitrochlorobenzene (DNCB; substrate of the GSH transferase), results in a moderate acceleration of lipid peroxidation, but a-tocopherol and ubiquinol levels remained unchanged in comparison with the controls in absence of DNCB. The addition of succinate to GSH-depleted mitochondria, however, effectively suppressed the TBARS formation as well as the a-tocopherol and ubiquinol depletion [47]. The data support the assumption that the protective effect of respiratory substrates against lipid peroxidation in the absence of mitochondrial glutathione is mediated by the regeneration of the lipid-soluble antioxidants coenzyme Q and a-tocopherol, whereby coenzyme Q probably recovers a-tocopherol from the a-tocopheroxyl radical. This data does not, however, answer the question about the mutual contribution of either GSH and a-tocopherol systems in the antioxidative defense of mitochondria. It appears that both systems probably exert a synergistic action in the defense against oxidative impairment in isolated mitochondria. Extramitochondrial antioxidants may assist the mitochondrial antioxidant defense system in a more or less complex way [18, 63] and the iron/ascorbate system may be useful to test specific effects of exogenously added antioxidants alone or in combination. A few examples from own studies are presented. Different exogenously added antioxidants [BHT (butylated hydroxytoluene), BHA (tert-butyl-hydroxyanisole), and a-tocopherol] prevent the accumulation of TBARS and the cation efflux under oscillating conditions in rat liver mitochondria within a similar efficiency range in the order of BHT > BHA > a-tocopherol [71]. Half maximal inhibition of TBARS, expressed in nmol antioxidant per mg protein, was attained at 0.5 for BHT, 1.0 for BHA and 43 for a-tocopherol. The addition of a-tocopherol to rat liver mitochondria immediately before the initiation of lipid peroxidation not only delayed the TBARS formation concentration dependently, but also significantly inhibited the active respiration, thus protecting functional properties of mitochondria [18, 63]. Furthermore, it could be shown that the addition of a-dihydrolipoic acid to rat liver mitochondria, immediately before the initiation of lipid peroxidation, potentiated the antioxidant effect of a-tocopherol, whereas a-dihydrolipoic acid alone showed prooxidative effects in the lower concentration range (1  mM and less) and antioxidative effects at higher concentration (2 mM and above) [18, 63].

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The ethanolic extract from Gynostemma pentaphyllum, a wild growing plant in Asian countries known for its antioxidant and anticancer activities, was shown to protect hippocampal slices and isolated rat liver mitochondria subjected to hypoxia/ reoxygenation immediately after functional injury [72]. Given to isolated mitochondria exposed to iron/ascorbate, G. pentaphyllum induced a significant delay of TBARS formation and a decrease in the plateau level in a concentration-dependent manner.

3.2 UV Irradiation and Human Skin 3.2.1 UVA-Mediated Damage to Proteins and Lipid Mediators in Extracorporeal Photoimmunotherapy UVA-irradiation in the presence of photosensitive agents, such as 8-methoxypsoralen (8-MPO), is successfully used as extracorporeal photoimmunotherapy (ECPI) in the treatment of T-cell mediated diseases, in particular cutaneous T-cell lymphoma, systemic scleroderma, and rheumatoid arthritis [73]. Despite the clinical success of ECPI, its mode of action is still not fully elucidated. It is well known, however, that UVA in combination with 8-MOP and molecular oxygen results in the formation of reactive oxygen species (ROS). Primary species of this process are singlet oxygen, the superoxide anion radical and, to a lesser extent, the highly reactive hydroxyl radical [74]. These ROS are able to impair all biological structures by reaction with macromolecules like DNA, proteins, carbohydrates and lipids. It was the aim of our studies to investigate oxidative modification of proteins, the capacity of antioxidative defense and lipid peroxidation in plasma and/or cells from the buffy coat using conditions relevant to ECPI (cumulative UVA dose at the sample level £2 J/cm2) and in patients undergoing ECPI treatment [49]. A widely used marker for oxidative protein modification is the assessment of protein-bound carbonyls by derivatization with dinitrophenylhydrazine (DNPH). With UVA doses between 0.5 and 2.0  J/cm2 and therapeutically relevant 8-MOP concentrations between 50 and 400  ng/ml, no changes in protein patterns and no increases in protein oxidation were detectable in plasma and cells isolated from buffy coats after UV irradiation [49]. Plasma exposed to about ten times higher UVA doses of 20 J/cm2, however, caused considerable increase in oxidatively modified proteins, formation of high molecular protein aggregations, crosslinking and fragmentation. Whereas TBARS levels as markers of lipid peroxidation were also not influenced by low UVA doses and different 8-MOP concentrations (see above), the total antioxidative capacity decreased to about two-thirds of the initial value, suggesting that the antioxidative defense of plasma is able to cope with oxidative stress under ECPI conditions. The in  vitro experiments were qualitatively confirmed by studies on ten patients with scleroderma or cutaneous T-cell lymphoma during ECPI treatment. The analysis of plasma and buffy coats of the patients by electrophoresis and western blotting for oxidatively modified proteins did not reveal a changed pattern.

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Furthermore, no increase of TBARS was seen during the therapy. As shown in the in  vitro experiments, the antioxidative defense also seems to protect plasma proteins and lipids from ROS attacks in patients. Reduced glutathione decreased following ECPI therapy and recovered thereafter, pointing to a functioning of the antioxidative system [49]. Whereas the results suggest that ROS are formed during ECPI, oxidative damage may not occur to a considerable extent, findings which are in line with the clinical observation of ECPI. In order to find out if more specific products of lipid peroxidation play a role in ECPI therapy or if their specific effects may trigger the photoimmunomodulatory effects of ECPI, the generation of monohydroxyeicosateraenoic acids (HETEs) and F2-isoprostanes was analyzed in plasma samples [51, 75]. In in vitro studies we observed an UVA intensity-dependent increase of HETEs in the buffy coat with a maximal HETE generation at UVA doses of 1  J/cm2 (Fig.  2a). The enhanced HETE formation is dependent on the presence of the photosensitizer, whereby ECPI-relevant concentrations of 200–400  ng/ml 8-MOP were used (Fig. 2b). Irradiation in the absence of the photosensitizer did not lead to significant alterations in the content of HETEs. Analysis of HETEs in plasma samples obtained from patients before and after ECPI revealed a characteristic increase in total HETE levels following UVA treatment, in particular of 5-HETE which contributed with about 80% to the sum of all HETE isomers. Chiral-phase HPLC indicated almost equal amounts of 5S- and 5R HETE, suggesting that the majority of lipid peroxidation products are formed by nonenzymatic oxidation reactions [51]. This was also supported by 2.8-fold increase of F2-isoprostane plasma levels after the second ECPI session [75]. During the same time period, the a-tocopherol content in the plasma samples was diminished by about 20% [75]. 3.2.2 Effects of UVB Irradiation on HaCaT Keratinocytes In Vitro and Microdialysates of Human Skin In Vivo  VB Irradiation-Induced Damage of HaCaT Keratinocytes U and Adaptive Responses to Oxidative Stress UVB irradiation affects in particular the outer layer of the skin and induces various physiological and pathophysiological processes, including oxidative stress and acute inflammation characterized by the development of erythema, edema and skin cancer [76]. Keratinocytes, as the major cell population in the skin, are the major targets of UVB effects. They have to protect the skin from UV light and are provided with a potent antioxidant system consisting of low-molecular-weight antioxidants and cellular antioxidant enzymes [77]. We studied the effect of moderate UVB doses (10–100  mJ/cm2) on the cellular and mitochondrial integrity of HaCaT keratinocytes, on eicosanoid lipid mediators (F2-isoprostanes and PGE2), biomarkers of oxidative stress and inflammation, and on antioxidant protection by superoxide dismutases. Cell viability of HaCaTs was UVB dose-dependently

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Fig. 2  (a) Monohydroxyeicosatetraenoic acid levels after exposure of the “plasma fraction” to increasing UVA doses in the presence of 8-methoxypsoralen (8-MOP; 400 ng/ml). Human plasma containing leucocytes (so-called buffy coat) was diluted to meet ECPI conditions (30 ml plasma + 13.5  ml phosphate-buffered saline) and exposed to UVA irradiation. Zero values are without 8-MOP. Values are means ± SD of three separate determinations in the plasma fraction of one patient. (b) Monohydroxyeicosatetraenoic acid levels after exposure of the “plasma fraction” to a UVA dose of 2  J/cm in the presence of increasing concentrations of 8-MOP. Control without 8-MOP and UVA irradiation. Values are means ± SD of three separate determinations in the plasma fraction of one patient. This figure was taken from Wiswedel et al. [51]

reduced and apoptosis was enhanced [59]. The activities of mitochondrially localized enzymes as respiratory chain complexes and mitochondrial nitric oxide synthase (mt-NOS) were lowered at UVB doses of 50  mJ/cm2 and above. Cardiolipin as functionally important mitochondrial phospholipid was considerably diminished at 100 mJ/cm2 [59]. From the literature it is known that UVB exposure induces the release of inflammatory mediators such as cytokines, nitric oxide and oxidized arachidonic acid derivatives [78, 79]. The latter were generated nonenzymatically

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by free-radical mechanism or enzymatically by cyclooxygenases isoenzymes. In our investigations, we found UVB-induced increases of F2-isoprostanes at relatively low UVB doses at 30  mJ/cm2, further increases at 50  mJ/cm2 and decreases or plateau levels at higher doses. PGE2 as proinflammatory prostaglandin was definitely more intensively enhanced than F2-isoprostanes. The finding that diclofenac, a nonsteroidal anti-inflammatory drug and cyclooxygenase inhibitor did not only lower the enzymatically generated prostanoids, but also the concentrations of F2isoprostanes, showing that F2-isoprostanes are not only biomarkers of lipid peroxidation and potential indicators of oxidant stress, but also mediators of inflammation (Fig. 3) [80–82]. As an adaptation to oxidative stress, superoxide dismutases (in particular MnSOD, but also CuZnSOD) as antioxidant enzymes were increasingly expressed in dependence on the intensity of UVB exposure to HaCaT keratinocytes [59]. UVB-induced increases in SOD protein levels were maximal at 50 mJ/cm2, whereas higher UVB doses diminished the SOD protein content, leading to irreversible cell damage. The effects were more distinctly expressed for MnSOD than for CuZnSOD, emphasizing the outstanding role of MnSOD as a primary antioxidant enzyme in mitochondria. The UVB dose-dependent impairment of SOD and also of catalase in human keratinocytes was demonstrated by other authors, too [83]. Takahashi et  al. [84] reported that UVB irradiation transiently decreased CuZnSOD and MnSOD activities and protein levels. Naderi-Hachtroudi et al. [85] and Lontz et al. [86] investigated the role of M nSOD in the control of aging and tumor generation and interrelations with cytokines as IL-1a, IL-1b, TNFa and GM-CSF. Irradiation of keratinocytes with UVB induced a release of cytokines that amplified MnSOD

Fig. 3  Levels of F2-isoprostanes and prostaglandins in HaCaT keratinocytes – influence of UVB (30 mJ/cm2) irradiation and diclofenac (DCLF). Means ± SEM, n ³ 6, *p < 0.05 vs. UVB; + p < 0.05 vs. control; **p < 0.01 vs. control (Student’s t test)

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activity, MnSOD protein and RNA levels. Cutaneous overexpression of MnSOD may be regarded as a protective cellular response evoked by cytokines released from inflammatory cells invading the irradiated skin. From the data, it can be concluded that keratinocytes are sufficiently protected at low UVB doses, whereas higher doses >50 mJ/cm2 lead to irreversible mitochondrial and cell damage.

 VB Irradiation-Induced Damage of Human Skin In Vivo U Using Microdialysis Technique Microdialysis is a minimal-invasive technique [87] that allows the continuous nearin  vivo measurement of F2-isoprostanes, prostaglandins [60, 80] and cytokines [88, 89] in the interstitial space of the skin. In principle, a semipermeable membrane is inserted into the upper dermis, and due to the concentration gradient between the interstitial space of the dermis and the perfusate of the catheter, substances diffuse through the pores of the membrane and can be analyzed in the dialysate. The recovery of prostanoids determined by retroanalysis was higher than 80% using a flow rate of 1.0  ml/min [90–93]. Concentrations of F2-isoprostanes and PGE2 in microdialysate samples were enhanced about 3–4 times in the 24 h following UVB treatment compared with healthy untreated skin (Fig. 4). Diclofenac (DCLF), topically applied as cream or gel, prevented the generation of erythema and reduced the levels of prostanoids remarkably (Fig. 4) [80]. As observed in HaCaT keratinocytes [59],

Fig. 4  Levels of F2-isoprostanes and prostaglandins in microdialysis samples of human skin – influence of UVB irradiation and diclofenac (DCLF). Means ± SEM of n = 6 healthy female volunteers; UVB irradiation (200–400 mJ/cm2) caused the formation of erythema (stages 3–5), which were completely suppressed by DCLF. *p < 0.05 vs. nonirradiated control and **p < 0.05 vs. UVB without DCLF

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DCLF did not only influence the prostaglandin, but also the isoprostane pathway, which may be, at least partially, due to radical scavenging or antioxidant side effects of DCLF or cyclooxygenase-mediated F2-isoprostane generation. The additional observation that the release of F2-isoprostanes correlates with inflammatory cytokines, measured simultaneously with prostanoids in microdialysate samples [90], suggests a role of F2-isoprostanes not only as biomarkers of oxidative stress, but as mediators of inflammation as well. In a further study using microdialysis technique, the influence of green and black tea, epigallocatechin-3-gallate and theaflavin on the generation of F2isoprostanes and prostaglandins in UVB-irradiated human skin was investigated [94] Whereas epigallokatechin-3-gallate and theaflavin showed less pronounced antioxidant and anti-inflammatory effects on the release of prostanoids, green and black tea extracts inhibited the generation of enzymatically and nonenzymatically formed eicosanoids in a dose-dependent fashion. It can be summarized from these results that (1) microdialysis allows the “nearin vivo” measurement of prostanoid mediators released in the interstitial space of the dermis under inflammatory conditions; (2) both in vitro and in vivo studies have indicated associations of F2-isoprostanes and inflammatory conditions. Antiinflammatory drugs, as the cyclooxygenase inhibitor diclofenac, did not only suppress the prostaglandin, but also the F2-isoprostanes pathway, in  vivo and in vitro; (3) exogenously added antioxidants inhibit the UVB-induced prostanoid generation; and (4) cutaneous microdialysis allows the elaboration of a kinetic profile of prostanoids and cytokines as inflammation markers after an immune challenge such as UVB irradiation in human skin in vivo. Acknowledgments  The work was supported by COST B35. We would like to thank Daniela Peter for her excellent work in mass spectrometric analyses and Heidemarie Faber, Marita Lotzing, Ines Doering and Elke Wölfel for their skillful assistance with the experiments.

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Carbon Tetrachloride-Induced Hepatotoxicity: A Classic Model of Lipid Peroxidation and Oxidative Stress Samar Basu

Abstract  Carbon tetrachloride (CCl4)-induced lipid peroxidation and liver injury is a classic experimental model for comprehending the cellular mechanisms behind oxidative injury, and further estimating the therapeutic potential of drugs and antioxidants. Several methods have often been used to study free radical-induced lipid peroxidation following CCl4 induction suffer methodological discrepancies when considering the measurements in vivo, and thus the results could not be evaluated appropriately. Isoprostanes, free radical-derived prostaglandin F2-like compounds, extended a new era of determination of oxidant stress in vivo. This chapter mainly focuses on the formation of F2-isoprostanes as a marker of oxidative stress and its relation to inflammatory responses by evaluating prostaglandin F2a (PGF2a) formation following CCl4 treatment in experimental animals, and their further regulation by antioxidants. In this context, a study protocol on the induction of oxidative stress in rats is described to evaluate these eicosanoids. Both eicosanoids (F2-isoprostanes and PGF2a) are increased dramatically in liver tissue, peripheral plasma and urine, but with varied kinetics of formation, release and excretion patterns. Consequently, free radical- and cyclooxygenase-mediated oxidation of arachidonic acid products are closely associated with experimental hepatotoxicity, and thus could be used as consistent model of oxidative stress using a reliable in  vivo marker of oxidative stress. In addition, its relation to inflammation has been further verified by applying this experimental model. Antioxidants have been shown to influence both the formation of F2-isoprostanes and prostaglandin formation, but the therapeutic values and precise mechanisms of action still remain uncertain.

S. Basu (*) Laboratorie de Biochimie, Biologie Moléculaire et Nutrition, Faculté de Pharmacie, Université d’Auvergne, 28 Place Henri-Dunant, BP 38, 63001 Clermont-Ferrand, France and Oxidative stress and Inflammation, Department of Public Health and Caring Sciences, Faculty of Medicine, Uppsala University, SE-751 85 Uppsala, Sweden e-mail: [email protected]; [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_21, © Springer Science+Business Media, LLC 2011

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Keywords  Antioxidants • Carbon tetrachloride • Cyclooxygenases • Free ­radicals • Inflammation • Isoprostanes • Lipid peroxidation • Oxidative injury • Prostaglandins

1 Introduction It is well documented that acute and chronic liver injuries that cause elevated ­morbidity and mortality originate from dissimilar pathophysiological conditions. Carbon tetrachloride (CCl4), a known hepatotoxin, has been widely used to explain the mechanisms behind hepatotoxicity, and to study oxidative stress experimentally. This model has also been applied successfully to study the protective effects of antioxidants, drugs and radical scavengers, etc. [1, 2]. This experimental model is considered to be a classic model of induction of oxidative stress for decades. Carbon tetrachloride, also known as perchloromethane or tetrachloromethane, is a colorless, noninflammable volatile liquid with a discrete smell and immiscible with water, that is formed by the chlorination of methane, ethane, propane, or propene. The molecular weight of this compound is 153.82 Da. It has been acknowledged for a while that breathing the vapor of this chemical can depress the central nervous system activity and cause degeneration of liver and kidneys by exerting a deleterious and toxic effect to the cells and organs [1]. The function of this compound in human health and experimental studies has been shown to be colossal in the last 80 years since the discovery in 1926 that exposure to carbon tetrachloride ultimately leads to cirrhosis of the liver [3]. This is due to the liver damage caused by this solvent that may have originated during the metabolism of CCl4 in the liver and the subsequent initiation of lipid peroxidation by free radicals [1, 4–6]. In the end, this causes oxidative stress in vivo and seems to play a key role in the pathogenesis of both acute and chronic liver damage. Application of this compound has been further shown to be an exceptional tool for the study of experimental oxidative injury due to its rapid metabolism of CCl4 in the liver to a free radical species, and following detrimental effects of the radicals in the liver. Recently it has been shown that the administration of CCl4 to rats leads to characteristic formation of both nonenzymatically and enzymatically derived eicosanoids by activating both free radicals and cyclooxygenases [7–12]. The main aim of this chapter is to focus on the effect of this compound in lipid peroxidation – specifically on eicosanoid biosynthesis and the observed effects by some antioxidants – and to further relate it to the experimental model in rats. A number of analytical approaches have been described to assess the free radicalmediated reaction intermediates, primary products, and also their metabolites, to elucidate the episode of in vivo or in vitro lipid peroxidation. Among them, measurement of lipid hydroperoxides, conjugated dienes, ethane, pentane, malondialdehyde (MDA), thiobarbituric acid reactive substances (TBARS), and hydroxynonenol have frequently been used to measure lipid peroxidation. Measurement of ethane and pentane have been applied as a measure of decomposition products of various PUFAs [13–15]. Loss of unsaturated fatty acid has also been applied as a measure of lipid peroxidation.

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Measurement of lipid hydroper-oxides [16–18], conjugated dienes [19–21], TBA test for TBARS and MDA [22–25] have been shown to be the most common methods for detection of lipid peroxidation both in vivo and in vitro. Nevertheless, many of these above-mentioned methods are presently known to suffer from specific detection of free radical-mediated lipid peroxidation products, and the inadequacy of these methods in determining lipid peroxidation is widespread [26], especially when allowing for measurement of these compounds in biological materials. Even though measurement of MDA or TBARS has been an extensive method of detection of lipid peroxidation as indicators of oxidative stress for several years, these methods seem not to be consistent with the detection of nonenzymatic free radical reactions in vivo. Malondialdehyde is primarily a by-product of platelet activation, and it biosynthesizes during the process of thromboxane production and release through cyclooxygenase (COX) catalysis of arachidonic acid. Thus, the identification of this compound is not exclusively representative of nonenzymatic lipid peroxidation in  vivo but is somewhat a part of the enzymatically catalyzed lipid peroxidation. Additionally, the detection of this compound might make chromophores during reaction with TBA that can nonspecifically depreciate the proposed analytical conclusion. An additional problem in the measurement of MDA in the biological fluid is the possibility of formation of this compound ex vivo [27].

2 Isoprostane Formation and Release Following CCl4 Challenge The relevance of nonenzymatic formation of these prostaglandin derivatives in vivo was not disclosed until 1990. Isoprostanes, a family of prostaglandin-like compounds generated in vivo by free radical catalyzed peroxidation of arachidonic acid, opened new promises on the detection of free radical-mediated lipid peroxidation products [8, 28–32]. Isoprostanes are stable lipid peroxidation products of experimental oxidative injury [7, 28, 29]. Unlike COX-derived primary prostaglandins, isoprostanes are shown to be formed in situ in esterified form to tissue phospholipids, and later released in free acid form after hydrolyzation of the ester moiety [28]. Many studies have shown that 8-iso-PGF2a, a major F2-isoprostane, or its related derivatives, are increased dramatically in numerous syndromes that are associated with oxidant injury, and the detection of F2-isoprostanes in various tissues and body fluids is widely regarded as a reliable biomarker for in  vivo measurement of lipid peroxidation through free radical pathways (Fig. 1) [8, 29, 32–35]. 8-Iso-PGF2a generally has about 10 times higher basal levels than the enzymatically formed PGF2a and the free form of this compound is easily detectable in numerous body fluids by sensitive and specific analytical approaches. Measurement of the esterified and free isoprostanes is appropriate in the tissues after administration of CCl4 or other related compounds in rats that induces oxidative stress, and thus the determination of isoprostanes could be applied as a reliable approach of lipid peroxidation and oxidative stress measurement for tissue injury of interest [10, 36–38].

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Arachidonic acid Oxidative stress

ROS

8-iso-PGF2α

(F2-isoprostane)

COX

Inflammation

PGF2α 15-keto-dihydro- PGF2α

Fig. 1  Biosynthesis of F2-isoprostanes and 15-keto-dihydro-PGF2a, a major metabolite of PGF2a from arachidonic acid by free radicals (ROS) and cyclooxygenases (COX), respectively

3 Prostaglandin F2a Formation and Release Following CCl4 Challenge Prostaglandins are enzymatic lipid oxidation products derived from arachidonic acid through cyclooxygenases (COX) [39]. Prostaglandin F2a is one of the key primary prostaglandins that possesses vasoconstrictive properties and is implicated in a range of inflammatory processes [9, 40, 41]. Having a longer half-life and found at a much higher concentration in  vivo than the parent compound PGF2a, and not being formed artefactually, measurement of the initial and major PGF2a metabolite, 15-keto-dihydro-PGF2a or degraded metabolites of PGF2a has been successfully applied for many years as parameters of PGF2a release (Fig. 1) [9]. Thus, measurement of 15-keto-dihydro-PGF2a has been shown to be relevant for the evaluation of enzymatic lipid peroxidation in  vivo and has been applied on the induction of oxidative stress and inflammation experimentally by CCl4 [7, 12, 42]. Several of the prostaglandins are consequently released during the release of F2-isoprostanes.

4 Metabolism of Carbon Tetrachloride Carbon tetrachloride is a characteristically identified compound that causes hepatotoxicity by an acute exposer [43, 44], and it induces cirrhosis after chronic ­exposure to this compound [43–45]. It promptly reacts to the organs, mostly in the liver, through its chemical reactivity properties to the adjacent molecules while it introduces to the body internally. This occurs essentially through covalent binding of the compound to the microsomal lipids and membrane proteins [46, 47] during lipid peroxidation [43, 48, 49]. Carbon tetrachloride quickly metabolizes monooxygenases of the endoplasmic reticulum in the liver cell through a reductive dehalogenation (by one electron reduction) catalyzed by the cytochrome P450 ­system to the trichloromethyl radical (•CCl3) [50–53]. Then, the trichloromethyl radical can rapidly react with oxygen to become a trichloromethyl peroxyl radical (•CCl3 O2•) [54].

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These radicals are perhaps either alone or formed together following steps of CCl4 metabolism in  vivo by utilizing nicotinamide adenine dinucleotide phosphates (NADPH). These radicals, and perhaps other radical intermediates that are formed through the influence of low or high levels of oxygen during CCl4 metabolism, are not only involved in the lipid peroxidation processes but also may influence various membrane proteins of the body. The lipid oxidation process seems to be an instantaneous episode when considering nonenzymatic free radical-catalyzed or enzymatic peroxidation of arachidonic acid, given that the products of these reaction processes i.e., esterified and free isoprostanes or primary PGF2a, respectively, emerge in the circulation within 2 h of oral administration of CCl4 [1, 7, 10, 12]. However, the appearance of esterified isoprostanes is much sooner in tissue than in circulation [12].

5 Induction of Free Radical-Mediated Lipid Peroxidation and Oxidative Injury by Carbon Tetrachloride The function of free radicals in CCl4-induced toxicity has been shown to be a major pathway of induction of nonenzymatic lipid peroxidation that then affects various enzyme activities of the body, and thereby to enzymatically induced lipid peroxidation. In the initial phase of CCl4 toxicity following its administration, a huge amount of CCl4 bioconverts to trichloromethyl radicals or other radicals that in turn accelerate reactions of several biochemical pathways. These radicals appear to affect the adjacent lipids in the tissues to induce lipid peroxidation. Esterified arachidonic acid is an indispensable part of lipids in vivo that are seen in all tissues, which can be metabolized in the liver and other tissues by the free radicals formed through CCl4 to form esterified isoprostanes. The mechanisms of formation of esterified isoprostanes from arachidonic acid have previously been discussed [8, 29, 32, 33, 55]. Early steps of the formation of esterified isoprostanes are followed by a rapid hydrolyzation of these compounds and subsequent release of de-esterified free isoprostanes into the peripheral circulation. It is well known that various hydrolytic enzymes that are ubiquitous in the body are primarily responsible for the formation of free isoprostanes from the esterified isoprostanes in the tissues. This de-esterification phase might possibly be one of the rate limiting step for the release of free isoprostanes in the tissue, and further release into the peripheral circulation. 8-Iso-PGF2a and other related derivatives, the major F2-isoprostanes are found to be reliable parameters of lipid peroxidation and indicators of oxidative stress [32, 34]. Whether these compounds have any direct significant pathophysiological effect upon formation during CCl4 intoxication or other pathophysiological condition is yet not completely evidenced. Since F2-isoprostanes show profound biological effects including vasoconstrictive and other properties, it is assumed that excess biosynthesis and the release of these primary compounds may lead to an even more pronounced pathological state. Nonetheless, following formation, these compounds undergo a rapid metabolism to various degraded products through the same metabolic pathway as

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enzymatically formed prostaglandins, and are excreted efficiently in the urine [56]. A large amount of the free parent compound has also been found in the urine because during the extensive synthesis of isoprostanes, a substantial portion of these compounds are excreted without being metabolized. Studies have shown that isoprostanes are produced soon after the intragastric administration of large doses of CCl4 to rats [7, 10]. Since these compounds are unique products of arachidonic acid oxidation through nonenzymatic pathway measurement of isoprostanes in various body fluids have been undertaken to estimate oxidative injury in vivo upon CCl4 administration in animal models [7, 10, 12].

6 Induction of Cyclooxygenase-Mediated Lipid Peroxidation by Carbon Tetrachloride Enzyme systems of the body are also impaired by CCl4 toxicity [57]. A loss of aminopyrine desmethylase, cytochrome P-450 and glucose-6-phosphatase activity was observed after CCl4 induction [58]. Proinflammatory and cytotoxic cytokines are revealed to be involved in liver injury, and the role of inflammation in hepatotoxicity is constantly increasing [59–61]. It has also been shown lately that CCl4 can induce prostaglandin formation through the COX pathway [7, 12]. Prostaglandin, specifically PGF2a, is recognized for its proinflammatory and vasoconstrictive properties. This compound is extremely potent in vivo in exerting various pathological processes in the body [9]. As large amounts of PGF2a are formed mainly in the lungs following intraperitoneal administration of CCl4, it is thought to be that this prostaglandin may have formed mainly through the induction of cyclooxygenase-2 (COX-2), since COX-2 is shown to be involved in the inflammatory processes in the body and is inducible by a range of proinflammatory stimuli.

7 Methodological Protocol of Induction of Oxidative Stress by Carbon Tetrachloride Male rats (Sprague-Dawley) weighing about 200–300 g (~6 weeks old) were used in the study of CCl4-induced hepatotoxicity and oxidative stress after acclimatization of the animals to the laboratory conditions [7, 12]. Rats always had open access to tap water and food, and were subjected to a 12-h light/12-h dark schedule. Powdered food was prepared for all rats from commercial food pellets containing 4% total lipids, 18.5% protein, 55.6% carbohydrates, and 3.5% fiber. Rats were fed with CCl4 orally (2–2.5 ml/kg body weight) and blood and urine samples were collected at 0, 1, 2, 3, and 4  h after administration of CCl4 to induce hepatic lipid peroxidation. Samples were also collected from the control rats and rats supplemented with vitamin E (63  mg/kg, all rac-a-tocopherol succinate blended into

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powdered food at a concentration of 2 g/kg diet) prior to CCl4 treatment (when the efficacy of a vitamin or radical scavenger is needed). Urine samples were collected in a Petri dish. The rats were weighed and anesthetized with ether. During laparotomy, the livers were excised, and blood samples were drawn from the abdominal aorta. The rats were sacrificed afterwards by heart puncture. Blood samples were collected in heparinized glass vials, and plasma was prepared by centrifugation at 1,930 × g for 8 min. All samples were immediately stored at −70°C until analysis. The animal experimental procedure was approved by the Animal Ethics Committee of the Medical Faculty of Uppsala University. A large amount of esterified 8-iso-PGF2a, the major F2-isoprostane was detected at 2  h following the oral administration of (2.5  ml/kg) CCl4 in the liver tissues (Fig. 2), while the free 8-iso-PGF2a levels of this compound in the liver tissue were rather low [38]. In another study, it was revealed that free 8-iso-PGF2a levels increased 17-fold in plasma and 53-fold in urine from the basal levels at 4 h after CCl4 (2 ml/kg) administration to the rats [7]. At 6 h, when the animals were sacrificed, free 8-iso-PGF2a levels increased 7-fold in the plasma, and 87-fold in the urine (Fig. 3). The levels of F2-isoprostanes were still significantly elevated after 24 and 48 h as compared to the baseline values after the administration of CCl4 [10]. The formation of isoprostanes precedes the appearance of biochemical markers (sGPT) of hepatic necrosis, and it suggests that isoprostane formation is not a ­nonspecific consequence of cell death through necrosis, but rather a manifestation of lipid peroxidation-induced oxidative stress. It also suggested that hepatic ­dysfunction caused by CCl4 intoxication does not appear to account, to a significant extent, for the elevated levels of plasma isoprostane measured at 24–48  h after injury due to the elimination half-life of 8-iso-PGF2a that was not changed by the devascularization of the liver [10]. Consequently, CCl4-induced F2-isoprostane ­formation is evidenced to be linked to lipid peroxidation rather than to hepatic necrosis. Major parts of the esterified isoprostanes that are subsequently formed in

Fig. 2  Levels of free and total 8-iso-PGF2a in liver tissues at different times following oral administration of CCl4 (2.5 ml/kg body weight) to rats (*p <0.05 and **p <0.001 for difference of free and total 8-iso-PGF2a, compared to 0 time values) (courtesy of Södergren et al. [38])

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Fig. 3  The levels of free 8-iso-PGF2a in peripheral plasma and urine at different times following oral administration of CCl4 to the rats (*p <0.05, **p <0.001, ***p £ 0.001) (courtesy of Basu [7])

Fig. 4  The levels of 15-keto-dihydro-PGF2a in peripheral plasma at different times following oral administration of CCl4 to the rats (*p <0.05, **p <0.001, ***p £ 0.001) (courtesy of Basu [7])

the tissue are released into the peripheral circulation in free acid form since the hydrolization of the esterified compounds is very rapid. Highest levels of free 8-isoPGF2a were seen in the peripheral circulation at 4 h after the oral administration of CCl4 in rats [7, 10]. The earliest detection of PGF2a metabolite was observed in the circulation when oxidative injury was induced in rats by the oral administration of CCl4 (2  ml/kg) (Fig.  4). 15-Keto-dihydro-PGF2a, a major metabolite of PGF2a, had

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increased ­ninefold in the circulatory plasma at 4 h after the administration of CCl4 [7]. At 6 h after its administration, the increase of this metabolite was still high (a sixfold increase compared to the basal levels). Since PGF2a synthesis and release followed oxidative injury induction, it was suggested that oxidative injury might activate the COX reaction to the arachidonic acid, and is the distinct a component of the inflammatory response [7].

8 Role of Antioxidants in Carbon Tetrachloride-Induced Isoprostane and Prostaglandin Formation Although hepatotoxicity is a complex process, there is adequate evidence to show that CCl4-induced hepatotoxicity is mediated through microsomal lipid peroxidation [5, 43, 44], and anoxia and ischemia are associated with reduced cellular antioxidant levels. Based on these theories, several antioxidant nutrients had been used over the years to counteract hepatic damage and lipid peroxidation following CCl4 administration [62–67]. It is well known that endogenous vitamin E and other antioxidant nutrients have a fundamental role in the preservation of antioxidant defense in the mammalian system [68–71], although the outcomes of many of these studies are diverse and controversial. The reason for this controversy is multifactorial; however, the main reason is possibly caused by the complex reaction mechanisms involved in the free radical chemistry leading to the formation and disappearance of very shortlived radical species, and are less-well understood. Additionally, there may be different types of radical species and reactions involved in this process that are difficult to interpret due to the insufficiency of the applied methods and multiplicity in the experimental protocol. To impede an early lipid oxidation process that occurs within the cell after CCl4 administration, certain types of antioxidants, with a property of scavenging that particular type of accelerated radical species need to be selected. Consequently, it is not always possible to expect obtaining or achieving a specific inhibition of lipid peroxidation by a generally efficient scavenger of free radicals. Although some of the basic mechanisms of free radical reactions following CCl4 administration are mostly identified, a potential therapy against hepatotoxicity has not yet been achieved. Possibly the reaction mechanisms that are recognized now from the previous studies are not within the entire range of reactions that occurs in vivo following CCl4-induced liver injury, which has yet to be elucidated. A very few studies are performed to scrutinize whether antioxidant nutrients have any role upon CCl4-induced eicosanoid formation in vivo. Polyenylphos­ phatidylcholine, a mixture of phospholipids extracted from soybean, protects the development of septal fibrosis and cirrhosis in the baboon [72, 73] and attenuates hepatic fibrosis induced by CCl4 [74]. In a recent study, it was shown that carbon tetrachloride-induced lipid peroxidation (as measured by F2-isoprostanes and 4-hydroxynonenal) was attenuated, and the levels of F2-isoprostanes and 4-hydroxynonenal paralleled liver fibrotic scores and collagen accumulation [75]. Accordingly, hepatic protection of polyenylphosphatidylcholine against

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CCl4-induced lipid peroxidation appears to be somewhat due to the antioxidant effect of polyenylphosphatidylcholine through the inhibition of lipid peroxidation. Nonetheless, this study showed that the levels of a-tocopherol, the potent lipophilic membrane antioxidant, did not modify among the experimental groups (control, CCl4, CCl4 + polyenylphosphatidylcholine). Whether the high content of choline in the polyenylphosphatidylcholine had some effect on lipid peroxidation is not known. Vitamin E deprivation was shown to increased levels of F2-isoprostanes [76]. In a study, a high dose of vitamin E (20 g/kg diet of all-rac-tocopheryl succinate for 3 weeks) was administered to affect both nonenzymatic and enzymatic lipid peroxidation in rats with CCl4-induced hepatotoxicity (2.5 ml/kg) [12]. Rats supplemented with vitamin E prior to CCl4 treatment had significantly lower levels of urinary and liver free 8-iso-PGF2a and urinary 15-keto-dihydro-PGF2a than rats treated with CCl4 alone. Both nonenzymatic and enzymatic lipid peroxidation during experimental hepatic oxidative injury and inflammatory response could be reduced by daily dietary supplementation of high doses of vitamin E. Therefore, supplementation of high doses of vitamin E may influence both free radical-induced oxidative injury and inflammatory response through COX-catalyzed prostaglandin formation. This investigation is of interest since the supplementation of vitamin E (200 IU/day) to healthy subjects for 2 or 6 weeks did not affect either the basal levels nor the CLA-induced F2-isoprostane and PGF2a formation, respectively [77].

9 Conclusion Carbon tetrachloride-induced oxidative stress has been evidenced as a classical model for the experimental induction of oxidative stress and hepatotoxicity. It has also been shown in this model that inflammation is a consequence of the acute induction of oxidative stress by an elevated formation of both isoprostanes and PGF2a metabolites, the latter being recognized as a marker of inflammation. The formation of these bioactive compounds occurs in specific physiological kinetics that establishes a specific role of oxidative stress in inflammation. Hence, this model is simple and unique for studying both oxidative stress and inflammation concurrently. This pathology seems to be moderately affected by antioxidant supplementation, at least at the earlier, rather than later phase, since the later stage is merged with the activation of various endogenous biochemical cascades that usually cause irreversible deterioration of the tissues. Currently, quantitative measurement of oxidative injury and inflammatory response through the detection of F2-isoprostanes and PGF2a metabolite, respectively, are reliable methods to study the underlying mechanisms of CCl4-induced lipid peroxidation and also to further study protective mechanisms of various antioxidants, radical scavengers, drugs or other nutritional strategies. Study of induced-oxidative stress by CCl4 and measuring these eicosanoids unfolded new insight for investigating the role of various antioxidants in earlier stages of oxidative stress and inflammation. This experimental

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model further opened the prospects of studying the multifaceted interactive mechanisms behind oxidative stress and inflammation, and future therapeutic developments against oxidative stress.

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44. Recknagel RO, Glende EA, Jr., Dolak JA, Waller RL (1989) Mechanisms of carbon ­tetrachloride toxicity. Pharmacol Ther 43: 139–154 45. Rubin E, Popper H (1967) The evolution of human cirrhosis deduced from observations in experimental animals. Medicine (Baltimore) 46: 163–183 46. Castro JA, Gomez MI (1972) Studies on the irreversible binding of 14 C-CCl 4 to microsomal lipids in rats under varying experimental conditions. Toxicology and applied pharmacology 23: 541–552 47. Diaz Gomez MI, Castro JA (1973) Effect of inhibitors of drug metabolism on mitochondrial swelling and on carbon tetrachloride-induced lysosomal damage. Toxicology and applied pharmacology 24: 378–386 48. Comporti M, Saccocci C, Dianzani MU (1965) Effect of CCl-4 in vitro and in vivo on lipid peroxidation of rat liver homogenates and subcellular fractions. Enzymologia 29: 185–204 49. Ghoshal AK, Recknagel RO (1965) Positive Evidence of Acceleration of Lipoperoxidation in Rat Liver by Carbon Tetrachloride: In Vitro Experiments. Life sciences 4: 1521–1530 50. Ingail A, Lott, K. and Slater, TF (1978) Metabolic activation of carbon tetrachloride to a ­free-radical products: Studies using a spin trap. Biochem Soc Trans 6: 962–978 51. McCay PB, Lai EK, Poyer JL, DuBose CM, Janzen EG (1984) Oxygen- and carbon-centered free radical formation during carbon tetrachloride metabolism. Observation of lipid radicals in vivo and in vitro. The Journal of biological chemistry 259: 2135–2143 52. Noguchi T, Fong KL, Lai EK, et al. (1982) Specificity of a phenobarbital-induced cytochrome P-450 for metabolism of carbon tetrachloride to the trichloromethyl radical. Biochemical pharmacology 31: 615–624 53. Poyer JL, McCay PB, Lai EK, Janzen EG, Davis ER (1980) Confirmation of assignment of the trichloromethyl radical spin adduct detected by spin trapping during 13C-carbon tetrachloride metabolism in vitro and in vivo. Biochemical and biophysical research communications 94: 1154–1160 54. Packer J, Slater, TF and Willson, R.L. (1978) Reactions of carbontetrachloride-related peroxy free radical (CCl3.O2) with amino acids. Pulse radiolysis evidence. Life sciences 23: 365–362 55. Morrow JD, Tapper AR, Zackert WE, et  al. (1999) Formation of novel isoprostane-like compounds from docosahexaenoic acid. Advances in experimental medicine and biology 469: 343–347 56. Basu S (1998) Metabolism of 8-iso-prostaglandin F2alpha. FEBS Lett 428: 32–36 57. Glende EA, Jr., Recknagel RO (1969) Biochemical basis for the in vitro pro-oxidant action of carbon tetrachloride. Experimental and molecular pathology 11: 172–185 58. Gonzalez Padron A, de Toranzo EG, Castro JA (1996) Depression of liver microsomal glucose 6-phosphatase activity in carbon tetrachloride-poisoned rats. Potential synergistic effects of lipid peroxidation and of covalent binding of haloalkane-derived free radicals to cellular components in the process. Free radical biology & medicine 21: 81–87 59. Blazka ME, Wilmer JL, Holladay SD, Wilson RE, Luster MI (1995) Role of proinflammatory cytokines in acetaminophen hepatotoxicity. Toxicology and applied pharmacology 133: 43–52 60. Bruccoleri A, Gallucci R, Germolec DR, et al. (1997) Induction of early-immediate genes by tumor necrosis factor alpha contribute to liver repair following chemical-induced hepatotoxicity. Hepatology 25: 133–141 61. Luster MI, Simeonova PP, Gallucci RM, Bruccoleri A, Blazka ME, Yucesoy B (2001) Role of inflammation in chemical-induced hepatotoxicity. Toxicology letters 120: 317–321 62. Tappel AL (1980) Vitamin E and selenium protection from in vivo lipid peroxidation. Annals of the New York Academy of Sciences 355: 18–31 63. Kunert KJ, Tappel AL (1983) The effect of vitamin C on in vivo lipid peroxidation in guinea pigs as measured by pentane and ethane production. Lipids 18: 271–274 64. Knook DL, Bosma A, Seifert WF (1995) Role of vitamin A in liver fibrosis. Journal of gastroenterology and hepatology 10 Suppl 1: S47–49 65. Seifert WF, Bosma A, Hendriks HF, et al. (1995) Beta-carotene (provitamin A) decreases the severity of CCl4-induced hepatic inflammation and fibrosis in rats. Liver 15: 1–8

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66. Merino N, Gonzalez R, Gonzalez A, Remirez D (1996) Histopathological evaluation on the effect of red propolis on liver damage induced by CCl4 in rats. Arch Med Res 27: 285–289 67. Iyama T, Takasuga A, Azuma M (1996) beta-Carotene accumulation in mouse tissues and a protective role against lipid peroxidation. International journal for vitamin and nutrition research Internationale Zeitschrift fur Vitamin- und Ernahrungsforschung 66: 301–305 68. Burton GW, Ingold KU (1989) Vitamin E as an in vitro and in vivo antioxidant. Annals of the New York Academy of Sciences 570: 7–22 69. Gutteridge JM, Halliwell B (2000) Free radicals and antioxidants in the year 2000. A historical look to the future. Annals of the New York Academy of Sciences 899: 136–147 70. Halliwell B (1990) How to characterize a biological antioxidant. Free Radic Res Commun 9: 1–32 71. Halliwell B (2000) Lipid peroxidation, antioxidants and cardiovascular disease: how should we move forward? Cardiovasc Res 47: 410 – 418 72. Lieber CS, DeCarli LM, Mak KM, Kim CI, Leo MA (1990) Attenuation of alcohol-induced hepatic fibrosis by polyunsaturated lecithin. Hepatology 12: 1390–1398 73. Lieber CS, Robins SJ, Li J, et  al. (1994) Phosphatidylcholine protects against fibrosis and cirrhosis in the baboon. Gastroenterology 106: 152–159 74. Ma X, Zhao J, Lieber CS (1996) Polyenylphosphatidylcholine attenuates non-alcoholic hepatic fibrosis and accelerates its regression. J Hepatol 24: 604–613 75. Aleynik SI, Leo MA, Aleynik MK, Lieber CS (1998) Increased circulating products of lipid peroxidation in patients with alcoholic liver disease. Alcohol Clin Exp Res 22: 192–196 76. Awad JA, Morrow JD, Hill KE, Roberts LJ, 2nd, Burk RF (1994) Detection and localization of lipid peroxidation in selenium- and vitamin E-deficient rats using F2-isoprostanes. The Journal of nutrition 124: 810–816 77. Smedman A, Vessby B, Basu S (2004) Isomer-specific effects of conjugated linoleic acid on lipid peroxidation in humans: regulation by alpha-tocopherol and cyclo-oxygenase-2 inhibitor. Clin Sci (Lond) 106: 67–73

Enhanced Urinary Excretion of Lipid Metabolites Following Exposure to Structurally Diverse Toxicants: A Unique Experimental Model for the Assessment of Oxidative Stress Francis C. Lau, Manashi Bagchi, Shirley Zafra-Stone, and Debasis Bagchi

Abstract  Structurally diverse toxicants in the environment are responsible for the production of reactive oxygen species (ROS) in biological systems. The accumulation of ROS results in oxidative stress and causes damage to macromolecules such as DNA, proteins, lipids and carbohydrates. Consequently, exposure to environmental oxidants increases the occurrence of a number of diseases as well as reproductive and developmental defects. One of the hallmarks of oxidant-induced oxidative stress is the production of fat metabolites caused by lipid peroxidation. Lipid metabolites such as acetaldehyde, acetone, formaldehyde and malondialdehyde can be quantified in biological fluids including serum and urine using high-performance liquid chromatography, gas chromatography and/or mass spectrometry. In this regard, the simultaneous detection of lipid metabolites using these procedures may provide a powerful tool for the rapid, accurate, reproducible and noninvasive assessment of oxidative stress induced by environmental exposure to toxicants. This chapter reviews the application of these methodologies in the evaluation of temporal effects of toxicant exposure on lipid peroxidation in a variety of experimental systems. Keywords  Acetaldehyde • Acetone • Formaldehyde • Lipid metabolites • Lipid peroxidation • Malondialdehyde • Oxidative stress • Toxicants

1 Introduction Humans are exposed to a diverse array of environmental oxidants that facilitates the development of a number of diseases including cancer, acute organ toxicity, and chronic inflammatory conditions. Although many of these exposures are inevitable, D. Bagchi (*) InterHealth Research Center, Benicia, CA, USA and Pharmacological and Pharmaceutical Sciences, University of Houston College of Pharmacy, Houston, TX, USA e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_22, © Springer Science+Business Media, LLC 2011

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some are due to certain lifestyle factors such as carcinogenic and proinflammatory chemicals contained in tobacco smoke and air pollution, pharmaceuticals, dietary carcinogens, and occupational hazards. Exposure to these environmental oxidants results in the accumulation of reactive oxygen species (ROS) that can react with cellular macromolecules including protein, carbohydrate, lipid and DNA [1–10]. ROS collectively refers to oxygen radicals such as superoxide and hydroxyl, and nonradicals such as hydrogen peroxide (H2O2), which are readily converted to radicals [11–13]. ROS are the by-products of normal aerobic processes occurring mainly in mitochondria of mammalian cells [14–19]. In fact, approximately 90% of the total oxygen consumed by the human body is used by mitochondria [20, 21]. Biologically relevant oxygen radicals include hydroxyl radical (OH•), H2O2, perhydroxyl radical (HO2•−), and superoxide anion (O2•−) [16]. Under normal cellular conditions, superoxide is rapidly dismutated by superoxide dismutase (SOD) into O2 and H2O2 nonradicals [22]. H2O2 can be generated in the brain during enzymatic degradation of neurotransmitters such as dopamine (DA) [23–25]. Consequently, H2O2 is converted to water by catalase or glutathione peroxidase (GSHPX) [16, 26]. However, at high concentrations, H2O2 reacts with ferrous or cuprous ions to produce the highly oxidizing OH• [11, 27, 28]. OH• attacks almost all of the macromolecules in living cells [27, 29–31]. Animal studies have shown that aging induces a significant increase in cellular H2O2, the source of OH• [32]. Nitric oxide (NO) and its derivatives are referred to as reactive nitrogen ­species (RNS). NO, the molecule of the year in 1992 according to Science [33], is a ubiquitous intracellular messenger that diffuses through cells and tissues [34–38]. NO is often referred to as a double-edged sword because it can either be cytotoxic or cytoprotective, depending on a variety of factors including the level and duration of its expression [39–44]. Endogenous NO can be formed enzymatically in mammalian systems by nitric oxide synthases (NOS), or generated nonenzymatically by reaction of NO2− under acidic conditions [44]. Exogenous NO exposure occurs via air pollutants and cigarette smoking [45]. NO reacts with O2•− to form the highly toxic oxidant peroxynitrite (ONOO−) [46]. In addition, NO is the source for other RNS such as nitrogen dioxide radical (NO2•), nitrite (NO2−), nitrate (NO3−), and dinitrogen trioxide (N2O3) [44, 47, 48]. The mammalian cells have developed an efficient antioxidant system during evolution to keep oxidative stress in check. This system is comprised of antioxidant enzymes such as SOD, GSHPX and catalase as well as oxidant scavengers including glutathione, trace elements, and dietary antioxidants [14]. As a result, there is a balance between oxidative stress and antioxidant defense. However, this antioxidant defense system is not totally infallible [49]. In fact, approximately 1% of ROS escape the surveillance of this defense system every day, resulting in the accumulation of oxidative stress over time, thus tipping the balance in favor of oxidative stress [50]. The overall consequence of oxidative imbalance is the oxidation of cellular macromolecules, perturbation of cell signaling cascades and gene expression, and increased oxidative stress-induced cell death [15, 51–59]. Toxicants such as herbicides, insecticides, and pesticides, as well as metal ions such as chromium(VI) and cadmium(II), have been shown to induce oxidative stress

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leading to increased lipid peroxidation, DNA damage, and depletion endogenous antioxidants such as glutathione [60–62]. Methods for the evaluation of toxic effects induced by these compounds are essential to identify means to reduce or eradicate these toxic effects. The extent of oxidative stress induced by toxicants can be measured by the increased excretion of urinary metabolites, and compounds that reduce these metabolites may be potentially beneficial in protecting against the detrimental effects of these toxicants [63, 64]. This chapter reviews experimental models for the assessment of toxicant-induced oxidative stress.

2 Toxicant-Induced Oxidative Stress 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic compound among halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons. It was identified as a major contaminant in Agent Orange, which is a defoliant and herbicide widely used during the Vietnam War [65]. TCDD is chemically stable and poorly biodegradable. These properties make TCDD a persistent environmental toxicant [66]. TCDD also has a long half-life of 5–10 years in the human body because it is highly lipophilic and there is little or no metabolism [67]. The International Agency for Research on Cancer has classified TCDD as a known human carcinogen [68]. Today, the major source of TCDD is from incinerator emission [65]. TCDD exposure has been linked to increased oxidative stress that leads to vascular dysfunction and hypertension [65]. Endrin (1,2,3,4,10,10-hexachloro-6,7-epoxy-1,4,4a-5,6,7,8,8a-octahydroendo, endo-1,4:5,8-dimethano-naphthalene) is a persistent organic pollutant that is banned in many countries. Endrin is an organochloride that was used as an insecticide and a pesticide [69]. The toxic effects of endrin on animal models were similar to those observed in TCDD [70]. These toxic effects include hepatotoxicity, thymic hypoplasia, weight loss and death. Several lines of evidence suggest that free radicalmediated lipid peroxidation contributes to the toxicity exhibited by endrin [71–73]. In fact, protein kinase C signaling pathway might be compromised by enhanced production of ROS observed after in vitro and in vivo endrin treatment [74]. Paraquat (1,1¢-dimethyl-4,4¢-bipyridylium dichloride), a nonselective contact herbicide, is a potent human poison that induces lung injury leading to death as a result of progressive and irreversible pulmonary fibrosis [60, 64, 75, 76]. Paraquat is fast-acting, causing mortality in as few as 5 days post-ingestion [77, 78]. The chemical structure of paraquat is similar to N-methyl-4-phenylpridinium ion, a dopaminergic neurotoxin that is capable of inducing mitochondrial damage and increasing oxidative stress by stimulating ROS production [79–81]. Recent research has indicated that paraquat induces oxidative stress in the substantia nigra area of rats injected with this herbicide [82]. Rats receiving intraperitoneal injections of paraquat exhibited impaired balancing skills on a rotorod apparatus. Histological and biochemical evaluations of rat brains revealed an increased concentration of oxidative stress markers and a loss of approximately 65% of dopamine neurons in the

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substantia nigra area, indicating that exposure to paraquat induced dopaminergic neurodegeneration similar to the conditions seen in Parkinson’s disease [82]. Naphthalene is a polycyclic aromatic hydrocarbon that is widely used in commercial and industrial applications such as lavatory scent disks, soil fumigants, mothballs and surfactants [83]. Naphthalene exposure may damage or destroy red blood cells, a condition known as hemolytic anemia. Naphthalene poisoning via ingestion of mothballs may lead to prolonged hemolysis and methemoglobinemia [84]. Symptoms of naphthalene poisoning include fatigue, lack of appetite, restlessness, and pale skin. Exposure to large amounts of naphthalene may cause nausea, vomiting, diarrhea, blood in the urine, and jaundice. Although fetal human dose of naphthalene has not been established, as little as one mothball could result in toxicity upon ingestion in children [84]. Naphthalene metabolites, such as menadione and other quinines, may be involved in the redox cycling and the production of oxidative stress that may contribute to its toxicity. Depletion of endogenous antioxidants such as glutathione by reactive quinones could result in augmented lipid peroxidation [85, 86]. It has been shown that rats receiving daily doses of 1 g/kg of naphthalene displayed elevated levels of serum and liver lipid peroxides [87]. A subsequent study demonstrated that male, weanling Blue-Spruce rats treated with naphthalene showed an enhanced peroxidation in the liver as compared to that of the controls [88]. This increased peroxidation was accompanied by a reduction in the activity of the selenoenzyme GSHPX in hepatic cytosolic fractions and an associated increase in the selenium-independent GSHPX. There was no increase in peroxidation in the lung, eye or heart of naphthalene-treated rats and the activities of the selenoenzyme. The selenium-independent GSHPXs were not affected by naphthalene in these organs. Naphthalene also did not affect SOD activity in any of the organs examined. Therefore, naphthalene-induced reductions in the selenoenzyme GSHPX may contribute to peroxidation in the liver and may be a contributing factor in naphthalene toxicity in vivo [88]. Metals such as chromium (Cr) and cadmium (Cd) also induce oxidative stress in living organisms. Cr is widely used in industrial chemicals in steel, alloy, chrome, paints, metal finishes and wood treatment [61, 89]. Chromium is known to cause allergic dermatitis and cancer in animals and humans alike. The first report of carcinogenity of chromate dust was published in 1890, in which it revealed that there was an elevation of cancer risk of workers in a chromate dye company [90]. Chromium exists in a wide range of oxidation states from −2 to +6; however, the most common states in the workplace are Cr(III) and Cr(VI). Cr(III) is generally regarded as nontoxic, but Cr(III) can be reduced to Cr(II) via biological reductants. The reduced Cr(II) reacts with H2O2 to generate radical OH• that induced tissue damage [91]. Cr(VI) is more effective in inducing ROS formation and causing oxidative damage in tissues and DNA [92]. The toxic effect of Cr(VI) is exacerbated by its ability to readily cross cellular membranes via nonspecific anion carriers, while Cr(III) is poorly transported across the membrane [93]. Cd is a relatively abundant element that exists predominantly in oxidation state +2. It is widely used in electroplating, galvanizing and the production of nickel– cadmium batteries as well as in pigments, plastics and other synthetics [61, 94, 95].

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The primary route of industrial Cd poisoning is through inhalation of Cd-containing fumes that may result in metal fume fever and may progress to chemical pneumonitis, pulmonary edema, and eventual death [96, 97]. For the general population, the major source of Cd exposure is food, but smoking may account for as much as 50% of the total body burden of Cd when exposure via food is low. It has been estimated that about 10% of the Cd content of a cigarette is inhaled through smoking. The absorption of cadmium from the lungs is much more effective than that from the gut, and as much as 50% of the Cd inhaled via cigarette smoke may be absorbed [98]. On average, smokers exhibit up to five times higher blood Cd concentrations and three times higher kidney Cd concentrations than nonsmokers. Even though there is a high Cd content in cigarette smoke, there seems to be little exposure to cadmium from passive smoking. No significant effect on blood cadmium concentrations could be detected in children exposed to environmental tobacco smoke [99]. Cd and several cadmium-containing compounds are known carcinogens and can induce many types of cancer [94]. The production of ROS and oxidative stress may be attributed to Cd toxicity. It has been shown that Cd induced the most lipid peroxidation in lungs, brain and liver as compared to other tissues in rats receiving CdCl2 [100–102]. In addition, Cd has been shown to inhibit SOD activity and increase lipid peroxidation in the liver and kidney [103].

3 Urinary Lipid Metabolites Oxidative stress-induced lipid peroxidation is associated with a wide range of toxicological effects, such as perturbed mitochondrial and Golgi apparatus functions, reduced membrane fluidity, and impaired calcium homeostasis [104, 105]. Membrane dysfunction in organs and/or tissues often leads to human diseases. Uncontrolled membrane damage enhances lipid peroxidation and facilitates disease progression. Therefore, lipid peroxides and peroxidation metabolites may be used as biomarkers for oxidative stress-induced membrane damage. Indeed, lipid metabolites from fat peroxidation accumulate and leak from organs or tissues into the bloodstream, and eventually get excreted in the urine [106]. Urinary lipid metabolites, such as acetaldehyde (ACT), acetone (ACON), formaldehyde (FA) and malondialdehyde (MDA), can be quantified by chromatographic methods including high-performance liquid chromatography (HPLC) and gas chromatography-mass spectroscopy (GC/MS), which provide noninvasive procedures for the evaluation of lipid metabolism and oxidative stress [64, 107–109]. These methods may be used to measure the metabolites directly or after the metabolites are derivatized with Brady’s reagent 2,4-dinitrophenylhydrazine (DNPH) [110]. DNPH reacts with the carbonyl group on a ketone or aldehyde to form hydrazone derivatives [107, 109]. This methodology is relative simple, rapid, sensitive and reproducible, which can be used in many applications for the study of exposure to environmental pollutants and oxidative stress as well as the status of lipid metabolism in a variety of disease states [107].

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4 Experimental Models The effects of chemically induced oxidative stress on lipid metabolism was examined by measuring lipid metabolites in urinary excretion of female Sprague-Dawley rats [107]. An oxidative stress was induced by orally administering 100  mg/kg of TCDD, 75 mg/kg of paraquat, 6 mg/kg of endrin or 2.5 ml/kg carbon tetrachloride to rats. Control rats received vehicle without the toxicants. Urine samples from female Sprague-Dawley rats were collected after 24 h of treatment and derivatized with DNPH. The hydrazones of the four lipid metabolic products were simultaneously quantified by HPLC on a Waters 10-mm m-Bondapak C18 column. The identities of ACON, ACT, FA and MDA in urine were confirmed by GC/MS. Urinary excretion of the four lipid peroxidation products increased relative to control animals 24 h after treatment with all toxicants. Of the four metabolites, FA was excreted in the highest amount. The largest increase in the excretion occurred with ACON in response to paraquat, which elicited a 10.7-fold increase in ACON excretion as compared to that for the control animals. Paraquat treatment was found to induce the greatest increases in all four metabolites [107]. Although these four toxicants induce free radical-associated cell injury through different mechanisms and toxicokinetics, they all enhance the excretion of lipid metabolites. A subsequent study examined the time-dependent changes in serum and urine levels of lipid peroxidation products in response to a single oral 50 mg/kg dose of TCDD in rats [111]. The changes in ACON, ACT, FA and MDA were quantified by HPLC. The effects of TCDD were compared to ad libitum-fed control animals and pair-fed animals, since rats on TCDD-supplemented diets exhibited hypophagia. Serum and urine levels of the four metabolites were analyzed on days 0, 3, 6, 9, and 12 post-treatment. Following TCDD administration, significant increases in all four lipid metabolites were observed in serum and urine at all points in a timedependent manner. Increases in the serum and urine levels of the four metabolites were significantly greater for TCDD animals than for pair-fed control animals at most points. The thiobarbituric acid (TBA) colorimetric method was used to assess the serum levels of MA as determined by HPLC. Similar time courses were observed, although higher results were obtained for the less-specific TBA method. The results clearly demonstrate that TCDD causes markedly elevated serum and urine levels of four specific products associated with lipid metabolism [111]. The dose- and time-dependent effects of endrin on hepatic lipid peroxidation, membrane microviscosity and DNA damage were evaluated in rats. Rats were treated with control, 3.0, 4.5, or 6.0 mg/kg of endrin as a single oral dose in corn oil and were sacrificed 0, 12, 24, 48, or 72  h post-treatment. Dose-dependent increases in hepatic mitochondrial and microsomal lipid peroxidation, microviscosity, and nuclear DNA single-strand breaks were observed as early as 12 h post-treatment with maximal increases occurring 24  h post endrin administration for all doses. While the incidence in DNA damage decreased with time after 24 h, the incidence of lipid peroxidation and microviscosity of microsomal and mitochondrial membranes remained relatively constant. The findings indicated that endrin treatment

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induced hepatic lipid peroxidation, which might lead to the increased membrane microviscosity as a result of membrane damage as well as enhanced DNA damage [112]. Based on the data from this study, a dose-dependent study was conducted to evaluate the effects of endrin on urinary excretion of lipid metabolites in rats [112]. Rats were orally administered control, 1.5, 3.0, 4.5 or 6.0 mg/kg of endrin. Urine samples were collected at 12-h intervals up to 72 h post-treatment. Urinary excretion of the lipid metabolites ACON, ACT, FA and MDA was simultaneously assessed as indicators for oxidative stress and enhanced lipid peroxidation. The identities of the four lipid metabolites were confirmed by GC/MS, while the DNPH derivatives of these lipid peroxidation products were quantified by HPLC procedure. Relative to control values, maximum increases in the excretion of ACON (162%), ACT (121%), FA (93%) and MDA (160%) occurred at 24 h post-treatment at all doses, with no significant increases in excretion of these four metabolites occurring thereafter. Seventy-two hours after endrin administration, the liver weight/body weight and spleen weight/body weight ratios significantly increased, while the thymus weight/body weight ratio markedly decreased. The results demonstrate that endrin induces dose- and time-dependent oxidative stress as indicated by the alterations in lipid metabolism with the enhanced excretion of specific metabolic products in the urine [113]. Paraquat exerts a profound effect on lipid peroxidation through the formation of ROS by its cyclic oxidation–reduction reaction [114, 115]. The time-dependent effect of paraquat on urinary excretion of lipid metabolites was examined in rats fed a diet containing 75 mg/kg paraquat [60]. Urinary excretion of the lipid metabolites ACON, ACT, FA and MDA was examined at 6-h intervals over 48 h post-treatment. The urinary metabolites were identified by GC/MS and quantified by HPLC. Timedependent increases in the urinary excretion of the four metabolites were observed after paraquat administration. In comparison to the control animals, paraquat treatment increased urinary excretion of ACON in time-dependent manner. At 12, 24, 36, and 48 h post-treatment, urinary excretion of ACON increased by 317, 609, and 995% respectively, as compared to control values. Maximal increase in ACT and FA levels (331% and 155%, respectively, more than the control values) were detected at 36 h post-treatment. In contrast, maximum increase in MDA concentration (218% more than that of the control) was observed at 30 h post-treatment [60]. The results clearly demonstrate that paraquat increases the urinary excretion of four lipid metabolites, even though differences existed for each metabolite in terms of the time of maximal levels of excretion detected [60]. Quinone metabolites of naphthalene are known to generate ROS and induce lipid peroxidation [116]. Quinone metabolites of naphthalene (NAP) are known to produce lipid peroxidation. The ability of naphthalene to induce oxidative stress and the protective effect of vitamin E succinate (VES) on naphthalene-induced oxidative stress were assessed in female Sprague-Dawley rats [83]. Rats were given a single oral dose of 1.1 g/kg naphthalene in corn oil. VES-supplemented rats were given an oral dose of 100 mg VES/kg/day for 3 days prior to naphthalene treatment, and 40 mg VES/kg/day after naphthalene administration. Hepatic and brain tissues as well as urine samples were collected 0, 12, 24, 48, and 72 h after naphthalene

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treatment. After 24 h treatment, there was a 2.1-fold increase in lipid peroxidation in liver and brain mitochondria as revealed by TBA colorimetric analysis. Treatment with VES alone had no significant effect on lipid peroxidation in hepatic or brain tissue. However, pretreatment with 100 mg/kg of VES 3 days prior to naphthalene treatment, followed by 40 mg VES/kg cotreatment along with naphthalene, reduced the naphthalene-induced increases in lipid peroxidation at 12 h by 28% in hepatic mitochondria and 34% in hepatic microsomes as compared to control values. Under the same conditions, significant decreases in lipid peroxidation were found in brain tissues where a 37% reduction was observed in brain mitochondria and a 30% decrease was observed in brain microsomes as compared to the control values. Increases in hepatic and brain mitochondrial lipid peroxidation in VES plus naphthalene-treated rats were 39–46% less than naphthalene-treated rats at 24  h post-treatment. DNA-single strand breaks increased 3.0-fold in hepatic tissues in naphthalene-treated rats, while only a 1.6-fold increase was observed in VES protected rats at the 24  h. The endogenous antioxidant glutathione decreased by 83% in hepatic tissues and 49% in brain tissues in naphthalene-treated rats at the 24-h time point, while the GSH level in VES plus naphthalene-treated rats decreased 47% and 21% in hepatic and brain tissues, respectively, at the same point in time. Microsomal membrane fluidity, a measurement of membrane damage, increased 1.9-fold in hepatic and 1.7-fold in brain tissues in naphthalene-treated rats, while only 1.3-fold increase in liver and 1.2-fold increase in brain tissues were observed in VES plus naphthalene-treated rats at the 24-h time point. Urinary excretion of lipid metabolites ACON, ACT, FA and MDA was determined by GC/MS procedure from 0 to 96 h after naphthalene administration. Maximal excretion of the four urinary lipid metabolites was observed between 12 and 24 h after naphthalene administration. VES reduced the NAP-induced excretion of these urinary metabolites by 28–49% 24  h after naphthalene administration. These results support the hypothesis that naphthalene induces oxidative stress and tissue damage, and that vitamin E succinate provides significant protection. It has been shown that chromium (Cr) and its salts may cause cytotoxicity and mutagenesis, and that these adverse effects can be attenuated by vitamin E. The cytotoxic effects exerted by chromium may be mediated through the production of ROS. A study was designed to examine and compare the effects of Cr(III) in the form of chromium chloride hexahydrate and Cr(VI) in the form of sodium dichromate following single oral doses (0.50 × LD50) on the production of ROS by peritoneal macrophages, and hepatic mitochondria and microsomes in rats. The effects of Cr(III) and Cr(VI) on hepatic mitochondrial and microsomal lipid peroxidation and enhanced excretion of urinary lipid metabolites as well as the incidence of hepatic nuclear DNA damage and nitric oxide (NO) production were also examined. Forty-eight hours after the oral administration of 25 mg Cr(VI)/kg induced an increase in lipid peroxidation of 1.8- and 2.2-fold in hepatic mitochondria and microsomes, respectively. In contrast, lipid peroxidation only increased by 1.2- and 1.4-fold in hepatic mitochondria and microsomes, respectively, after administration of 895  mg Cr(III)/kg. Urinary excretion of ACON, ACT, FA and MDA were identified by GC/MS and quantified by HPCL from 0 to 96 h after Cr

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administration. Maximal excretion of the four urinary lipid metabolites was observed between 48 and 72  h post-treatment with increases of 1.5- to 5.4-fold, respectively, in Cr(VI)-treated rats. Peritoneal macrophages from Cr(VI)-treated animals resulted in 1.4- and 3.6-fold increases in chemiluminescence and iodonitrotetrazolium reduction 48 h after treatment, which indicated an enhanced production of superoxide anion. On the other hand, macrophages from Cr(III)-treated animals showed negligible increases in these same parameters. Increases in DNA single-strand breaks of 1.7-fold and 1.5-fold were observed following administration of Cr(VI) and Cr(III), respectively, at 48 h post-treatment. Enhanced production of NO by peritoneal exudate cells (primarily macrophages) was monitored following Cr(VI) administration at both 24 and 48 h post-treatment with enhanced production of NO being observed at both points in time. Urinary lipid metabolites were ­measured from 0 to 96  h post-treatment. Maximal Cr-enhanced excretion of the four metabolites was observed between 48 and 72  h post-treatment. No further increases in the excretion of these metabolites were detected beyond 72 h. Cr(VI)treated animals excreted more of each of the four lipid peroxidation metabolites than Cr(III)-treated counterparts. Although the exact mechanisms for the formation of these lipid metabolites were not completely clear, the results thus far suggested that the increased urinary excretion of lipid metabolites might be due to enhanced oxidative stress-induced lipid peroxidation. Nonetheless, the comparative data ­indicated that both Cr(VI) and Cr(III) induced an oxidative stress at equitoxic doses, while Cr(VI) induces greater oxidative stress in rats as compared to Cr(III)treated animals [92]. It is well documented that cadmium (Cd) exposure leads to lipid peroxidation and that free radical scavengers and antioxidants attenuate Cd-induced toxicity. These observations suggest that Cd-induced toxicity may be, at least in part, mediated through the production of ROS. The effects of Cd(II) chloride (CdCl2) on ROS production were evaluated in rats following a single oral dose of 44 CdCl2 mg/kg [94]. Parameters including hepatic mitochondrial and microsomal lipid peroxidation, glutathione content in the liver, the incidence of hepatic nuclear DNA damage, and excretion of urinary lipid metabolites were assessed. There were marked increases in lipid peroxidation of 4.0- and 4.2-fold that occurred in hepatic mitochondria and microsomes, respectively, 48 h after the oral administration of CdCl2, while a 65% decrease in glutathione content was observed in the liver. Increases in DNA single-strand breaks of 1.7-fold were observed 48  h after administration of CdCl2. The urinary excretion of lipid metabolites ACON, ACT, FA and MDA were identified by GC/MS and quantified by HPLC at 12-h intervals from 0 to 96 h after Cd administration. Maximal excretion of the four urinary lipid metabolites was detected in CdCl2-treated rats between 60 and 72 h post-treatment, and no further increases were observed after 72 h. Specifically, maximal levels of FA and MDA were observed at 60 h, while maximal concentrations of ACON and ACT were detected at 72 h post-treatment as compared to control values. These results support the hypothesis that cadmium induces production of ROS, which may contribute to the tissue-damaging effects of this metal ion [94].

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5 Conclusion Chronic exposure to toxicants may lead to the development of various diseases. Toxicant poisoning causes perturbations in a wide range of cellular metabolism resulting in the enhancement of oxidative stress formation and comprised cellular functions. Increased lipid peroxidation is associated with toxicant exposure, possibly due to the upsurge of ROS. ROS, or free oxygen radicals, attack the fatty acid side chains of lipids in the various membranes of the cell, especially mitochondrial membranes that are directly exposed to the superoxide anions produced during cellular respiration and enhanced ROS generated during toxicant exposure. The relevant examples presented here clearly demonstrate that urinary excretion of lipid metabolites such as acetaldehyde, acetone, formaldehyde and malondialdehyde increases significantly following toxicant exposure. This increase is accompanied by the enhancement of DNA single-strand breaks, lipid peroxidation of mitochondria and microsomes, and glutathione depletion. Taken together, these findings suggest that toxicant exposure produces ROS that leads to the accumulation of oxidative stress as a result of macromolecular damage in DNA, protein and lipids. The detection of these four lipid peroxidation products in urinary excretion after toxicant exposure provides a rapid, convenient and noninvasive way to assess oxidative stress-induced lipid peroxidation and to monitor the progression of toxicant poisoning. This method is sensitive and reproducible, which may allow it to be used in a variety of applications including the assessment of the effects of antidotes on lipid peroxidation status after toxicant exposure and the evaluation of altered lipid metabolism in disease states that induce enhanced lipid peroxidation.

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Oxidative Stress in Animal Models with Special Reference to Experimental Porcine Endotoxemia Miklós Lipcsey, Mats Eriksson, and Samar Basu

Abstract  Animal experiments offer a unique possibility to tailor study design and to standardize the experiment in a way that allows us to repeat challenges, monitoring and interventions according to scientific rationale. The animal models described in this chapter are thought to replicate human disorders where oxidative stress is a major component. These experimental settings are focused on inflammatory diseases, with particular focus on sepsis models, each animal model having its own advantages and disadvantages. Depending on the study rationale, it is possible to use specific animal models. In studies where the role of the genetic expression is thought to be evaluated, knockout mice are of utmost importance for the understanding of the synthesis of a functional gene product. When biological variables are used to determine and evaluate pathophysiological events, it is essential to know that the bioassay is applicable as well as knowledge to interpret detection limits and potential cross-reactions to other related compounds. Since inflammatory challenges differ between various types of animals, responses may not exactly reflect the reactions that occur in man. Species differences in both the innate immune systems, which offer immediate defense against infection in a nonspecific fashion, and the adaptive immune responses, which provide the vertebrate immune system with the capability to distinguish and recall identifiable pathogens are important limitations. Cascade systems and receptor expression are other examples where interpretative care must be taken. Nevertheless, animal models offer a unique ­possibility to study serious reactions and events, which are generally impossible to induce experimentally in man. Keywords  Inflammation • Isoprostanes • LPS • Oxidative stress • Prostaglandins • Sepsis

M. Eriksson (*) Department of Anesthesia and Intensive Care, Uppsala University, Uppsala, Sweden e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_23, © Springer Science+Business Media, LLC 2011

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1 Introduction to Animal Models of Inflammation Oxygen is essential for most living organisms, but oxygen is also a reactive ­molecule that can react with virtually any compound in most living organism. Free radicals are important elements of the inflammatory response, which protect the organism’s homeostasis against internal and external insults. Since the inflammatory response is highly conserved in evolution, the inflammatory processes affecting the human body can be studied in animal models. Research in animals are subject of continuous debate, but in order to improve our understanding of these patho­ physiologic mechanisms and to develop new treatment options, experimental procedures may be performed under standardized conditions, without intervention with the natural cause of the disorder in question. Thus, among other research modalities, where science and seeking knowledge and not improvement of medical practice is the primary focus, animal models are essential components in the arsenal of inflammatory research. This chapter focuses on describing animal models of sepsis specifically on aspects of oxidative injury elicited by an acute inflammatory response. The first part of this chapter discusses general principles of animal experiments that can be applied in many fields of oxidative stress. In sepsis, oxidative injury is not a single pathologic pathway, but involves multiple biochemical cascades that affect many organs under many physiologic and pathologic circumstances. Thus, the possible animal models are nearly infinite. The second part of this chapter discusses three examples of animal models of sepsis with emphasis on the endotoxemic models.

1.1 The Validity of Animal Models in Inflammatory Research Although the physiology of humans and animals is similar, research utilizing animal models has not always given results that are applicable to humans. Differences between humans and model species alter the response to pathologic processes, which might cause misleading conclusions about human pathophysiology. For instance, sensitivity to endotoxin, commonly used to elicit sepsis-like symptoms, varies considerably between different species. Primates, especially humans, are several magnitudes more sensitive to endotoxin than pigs, dogs and rats [1]. Animal models of sepsis may also be criticized since most common patients with septic shock, organ ischemia or ARDS are elderly persons with multiple systemic ­diseases, whereas most of the research animals are young and healthy. Also, many inflammation models study the animals during the early disease stages, the endpoint being mortality occurring a few hours after the onset of inflammatory ­challenge. In contrast, patient mortality is seldom so close to the first signs of inflammation [2]. Endotoxin administration to human volunteers has been used in several studies, although it must be remembered that such experiments always carry a risk for unforeseen, potentially life-threatening complications [3, 4]. The possibility of

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performing standardized animal experiments where extensive monitoring is used, in combination with the opportunity to initiate local and systemic inflammation as well as surgically induced inflammation, gives us the possibility to evaluate pathophysiologic events and reactions, and ultimately death, with a high degree of reproducibility. Thus, results obtained from animal models are still a major source of current knowledge in inflammatory research.

1.2 The Choice of Animals The choice of laboratory animal species is determined by the organ systems to be studied, number of animals needed, local legislation, project budget, housing ­possibilities, animal size, the possibility to induce the pathological process to be studied, and local traditions with existing models. In inflammatory research, ­considerable knowledge has been accumulated on models in different animal ­species. Small rodents, mice, and rats are particularly suitable for models where long-term observation or greater numbers of animals are required. These animals also offer the broadest availability of inflammatory mediator assays; moreover, these species are available with gene modifications. Rabbits offer few advantages as small rodents, still given that their blood volume is much larger than, for example, rats, and rabbits are suitable for antibody production or other rodent experiments with largevolume sampling. The size of rabbits still limits the use of advanced circulatory and respiratory equipment. Sheep can be employed in several types of inflammatory models; however their anatomy in the lower respiratory tract differs considerably from that of the human [5]. Still, ovine models for ARDS are popular among the scientist. The dog that was a sanitary problem a century ago is today a popular pet, which limits its use for ethical and emotional reasons. Pig models have many of the advantages that the dog offers. Furthermore, they are easy to obtain from local ­producers, often without long, stressful transports, and the juvenile pig is large enough for instrumentation and the use of human medical equipment during the experiment. Also, the blood volume of juvenile pigs allows extensive blood sampling, making pigs a suitable species for intensive care models. Porcine endotoxin shock models have been widely employed in experimental ­sepsis research and ARDS models. Although the circulation of the pig has been shown to be very similar to that of humans, among the non-primates [1, 6], the lung vascular smooth muscle in the pig is very sensitive to live bacteria and endotoxin, leading to a substantial increase in pulmonary vascular resistance. Increased ­pulmonary vascular resistance leads to hypodynamic circulation in porcine sepsis and ARDS models, unlike the hyperdynamic circulation seen in human septic shock. Experiments in baboons have ­contributed to unveiling important immunological mechanisms related to the development of organ dysfunction [7]. Despite the fact that these animals are biologically ideal, these models tend to be less frequently used. Apart from the animal’s species, the sex [8], age [9], strain [10] and diet [11] of the animals may all influence the experimental model and contribute to variations in the experiment.

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1.3 Ethical Considerations Before setting up an experimental model, it is essential to assure that evaluable results can be obtained from the experiments. This principle of research ethics ­corresponds with the researcher’s interest to obtain good results from the experiments performed, since data that cannot be used from an experiment indicates that time, resources and animals have been in vain. Furthermore, the suffering of the animals must be weighed against the overall benefit of the project. Since this may be difficult or impossible to determine for an individual researcher, research ethics committees have been set up worldwide. Approval is necessary for conducting animal experiments and publishing results from such experiments. A simplified but relevant approach to the key principles for conducting research in animal models are summarized in Russell and Burch’s three “R”s: reduction, replacement and refinement [12].

2 Experimental Sepsis Models As oxidative stress is a pathophysiologic process involved in many diseases, and animal models preferably mimic specific diseases, the list of possible models is virtually unlimited. Sepsis models are described in the following sections, where we especially point out the following options: endotoxemia, live bacteria and peritonitis. At the end of this chapter, we describe our model of porcine endotoxemia in the appendix. Practical considerations and suggestions are given.

2.1 Sepsis Models Although the classic models for oxidative injury include carbontetrachlorideinduced oxidative stress or some form of ischemia–reperfusion injury, oxidative injury is also a central mechanism in sepsis and sepsis models. In septic shock and its experimental counterparts, hypoxia and reperfusion are combined with the oxidative processes of the elicited immune response, e.g., oxidative burst in neutrophil granulocytes. Oxidative injury is thus one of final pathways of cell injury in sepsis and sepsis models, and may be seen shortly after endotoxin challenge [13]. Furthermore, in these sepsis models, even endotoxin doses that induce only minimal circulatory changes activate lipid peroxidation, and the extent of this oxidative process increases with increases in the endotoxin dose over a wide range [14]. The role of oxidative pathways in sepsis models is also demonstrated by the attenuation the inflammatory response by antioxidant agents [13, 15], by inhibition or gene knock-out of key enzymes of the oxidative pathways [16, 17].

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2.2 SIRS and Sepsis Models Systemic inflammatory response syndrome (SIRS) [18] is a clinical entity that can be induced by infections, burns, pancreatitis and several other causes. An advantage of the term SIRS is that, irrespective of cause, once systemic inflammatory response is established, the pathophysiologic mechanisms have several features in common. On the other hand, a problem with the concept of SIRS and sepsis definitions in experimental research is that criteria for these conditions (tachycardia, tachypnoea, hypo- or hyperthermia and leucocytosis/leucopenia and bacteremia) cannot be applied in most animal models. The heart rate, respiration rate, core temperature and white cell counts usually differ from human reference intervals. Nevertheless, the experimental setup and changes from baseline levels in these variables indicate the validity of sepsis models in animals. Even if most sepsis models induce sepsis-like systemic inflammatory syndrome, some aspects of the septic syndrome will be present or absent. This has to be taken into consideration when designing the study. Several different animal models as well as inflammatory challenges have been utilized to experimentally mimic human septic shock. For example, the most commonly used methods, endotoxemic shock and cecal ligation combined with puncture (CLP), have significant differences in the kinetics and magnitude of cytokine production [19]. Apparently, these regimens also differ regarding the arachidonic acid cascade, since COX-2 inhibition improves early survival in endotoxemic mice, but not in those with CLP [20]. The three main types of sepsis models, namely, the endotoxemic, the bacteremic and the peritonitis models are extensively described below. Continuous administration, by various routes, of bacteria, e.g., Escherichia coli, or by endotoxin in the anesthetized pig, are frequently used models for replicating human gram-negative septic shock and acute inflammation. In such experimental models, oxidative stress in addition to acute inflammatory responses can be evaluated. Utilizing the endotoxemic model, it has not only shown that oxidative stress occurs already within the first hour after endotoxemic challenge as shown by the rapid increase of F2-isoprostanes in plasma, but also elevates cyclooxygenase-catalyzed primary PGF2a formation among surviving and nonsurviving pigs [21]. However, the formation of both F2-isoprostanes and PGF2a seems to occur in diverse kinetics among the survivors and nonsurvivors (Fig. 1). Assessment of urinary excreted 8-iso-PGF2a, a major F2-isoprostane has also been performed in this model as seen in Fig. 2 [22]. Interestingly, renal clearance of 8-iso-PGF2a increased ninefold to its peak at 2 h in the survivors, compared to a 14-fold increase at 5 h in nonsurvivors. Thus, urine sampling may be performed when oxidative stress is to be evaluated in subjects where blood samples are difficult or unpractical to obtain, e.g., long-term studies in humans or animals.

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Fig. 1  The mean levels of 8-iso-PGF2a (upper) and 15-K-DH-PGF2a in plasma in surviving and/ or nonsurviving endotoxemic pigs (*P < 0.05; **P < 0.01; ***P < 0.001). Reprinted with permission from the publisher (ScienceDirect®, Elsevier B.V.)

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Fig.  2  Percentage of “0  hours”-value of 8-iso-PGF2a in urine of surviving and nonsurviving, respectively, endotoxaemic pigs (10 mg kg−1 h−1). “0 hours” denotes start of endotoxin infusion. ** P < 0.01; ***P < 0.001. Statistical evaluations were based on individual differences between 0 h and each measurement. Reprinted with permission from the publisher (Wiley InterScience)

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2.3 Endotoxins Endotoxins are extensively studied trigger factors of the inflammatory response as demonstrated in experimental sepsis model. They are potent immunostimulators of eukaryotes – from the small two-winged fruit flies used in genetic research (Drosophila melanogaster) – to the Homo sapiens [23–31]. The sensitivity to endotoxin is thus a phylogenetically conserved “alarm system” that functions as an activator of the immune system if gram-negative bacteria are present. While the term endotoxin is occasionally used to refer to any cell associated bacterial toxin, its use ought to be limited to the lipopolysaccharide complex associated with the outer membrane of gram-negative bacteria. The terms endotoxin and LPS are used synonymously, although the former is usually used in the context of biological activity while the latter is used to describe the macromolecule. LPS are a group of compounds with varying biological activity. It is important to remember that not all LPS have endotoxin activity and that not all endotoxins have LPS structure [32, 33]. For example, bacterial mutants deficient of endotoxin have been shown to elicit only a partial response in comparison with that caused by wild-type bacteria [34]. Lipopolysaccharides are complex molecules with molecular weight in the 10 kDa range. They are stable at 100°C for over 30 min due to a molecular structure with covalent bounds. Two main regions can build up the amphiphilic LPS molecule: the lipophilic lipid A region and a hydrophilic polysaccharide region [35]. Biological activity, or endotoxicity, is associated with the lipid component (Lipid A) and immunogenicity is associated with the polysaccharide components. The polysaccharide region can be further divided into the O antigen and a core region, where the latter connects the former with the lipid A region. A major antigenic determinant of the gram-negative cell wall resides in the O antigen and constitutes the chemical basis of the bacterial serological classification. The hydrophilic O antigen polysaccharide consists of up to 50 repeating oligosaccharide subunits made up of two to eight different monosaccharides. Specificity of this site is due to an extremely high structural and chemical variation of the O antigen. With minor variations, the core region is common to all members of a single bacterial genus. It is attached to one of the two N-acetylglucosamines of the lipid A region. Lipid A contains the membrane-anchoring region of LPS and consists of a phosphorylated N-acetylglucosamine (NAG) dimer with six or seven saturated fatty acids attached. Although the structure of the lipid A region is relatively conserved between bacterial species, the variations in this part of the LPS determines its endotoxicity. The LPS layer of the outer membrane of gram-negative bacteria is an effective permeability barrier against external stress factors. It is permeable only to low molecular weight, hydrophilic molecules, which makes it a barrier to e.g., lysozyme, bile acids and many antimicrobial agents. LPS is liberated in small amounts during bacterial growth [36–38]. Larger amounts of endotoxin are released with bacterial lysis irrespective of its mechanism. These may be autolysis, external lysis mediated by complement or antibiotics and lysozyme phagocytic digestion of bacterial cells. Antibiotic-induced endotoxin release varies with the type of anti­ biotics. For instance, the cephalosporin-induced endotoxin release can be reduced if tobramycin is added. To explain this phenomenon, low endotoxin liberation per

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bacteria or a nonspecific binding between aminoglycosides and endotoxin has been suggested. The finding that tobramycin does not exhibit endotoxin-neutralizing effects in the LAL assay contradicts the latter explanation [39]. Nevertheless, since endotoxin interacts with several biologic binding sites in the mammalian organism, the possibility of an antiendotoxin effect cannot be excluded in vitro.

2.4 Host–Endotoxin Interactions The mammalian organisms have several proteins and cells that, like watchmen, are constantly susceptible for activation by danger signals, such as endotoxin, to initiate an immune reaction against gram-negative “intruders.” Many of the proteins activated are serine proteases, e.g., the Hageman factor and C3, which are parts of cascades such as the coagulation and complement system, respectively [40–43]. The cells in the first line of endotoxin activation are monocytes, macrophages and neutrophil granulocytes, phagocytic cells of the innate or natural immune system [44–48]. A key immunological feature of these cells is the constitutive expression of the CD 14 antigen (CD 14) and Toll-like receptor 4 (TLR4) on their membrane [49]. Activation of the CD14 and TLR4 leads to the release of cytokines, ­biologically active lipids and free radicals.

2.5 Endotoxemic Models As LPS can be stored, easily quantified, and administered in a standardized dose, it provides a simple and reproducible tool when endotoxemic challenge is to be ­evaluated in an animal model. This is an advantage when studying effects of ­substances with specified modes of action [50]. In particular, endotoxin models are of interest when drugs or other treatments with a proposed antiendotoxin effect are being investigated [51–53]. It may be administered either as a continuous ­intravenous infusion or given as a bolus intravenously, intraperitoneally or intratracheally [54]. However, while endotoxin elicits a hyperdynamic cardiovascular response in humans, the pattern of response is characteristically biphasic in large mammals such as the pig. In the pig, the cardiac output decreases with a nadir at about 3 h after the start of continuous endotoxin administration and by 8–12  h, it reaches supranormal values. The hyperdynamic porcine models are characterized by long observation ­periods with the first cardiac output measurements after 6 h. These observation periods are cumbersome since long-term anesthesia or sedation may influence the findings, whereas preservation of intravenous access in awake animals may be problematic. Also, animal welfare regulations, based on ethical considerations, restrict the use of awake animals in experiments where considerable suffering is expected. In order to

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reach the state of multiorgan failure, relatively high endotoxin doses are used, which may be rare in clinical sepsis. This may produce a condition of acute ­intoxication rather than a gradually developing sepsis. The other consequence of these rather short observation periods, is that these models become limited to exploration of the early events during sepsis [50]. Since bacterial factors other than endotoxin may also contribute to the host response, animal models using infusion of live gram-negative bacteria [55], usually E. coli, have sometimes been proposed to be more clinically relevant than endotoxin models [56]. Nevertheless, the pathophysiological changes in live bacteria models are more or less similar to the endotoxin infusion models.

2.6 Live Bacteria Models Administration of live bacteria would seem to be the method of choice when septic shock is modeled. But since any model with live bacteria requires facilities where bacteria can be kept safely, cultured, typed and quantified, these models are more difficult to apply than most other sepsis models. Still, oxidative stress has been studied in live bacteria models [57, 58]. The decision on the species of bacteria is set by the clinical condition to be modeled. Apart from the bacteria species, the subtype of bacteria within the species may also have a substantial impact on the experimental model. For example, over 700 serotypes of E. coli have been described with varying virulence. A highly virulent E. coli serotype may be given in a low dose that corresponds to the clinical situation to be modeled. On the other hand, bacteria with low virulence may be easier to dose and quantify since they need to be administered in a higher dose. The growth cycle of bacteria should also be considered, especially in short experiments where bacteria are given intravenously. Bacteria can be administered not only intravenously, intraperitoneally or intratracheally, but also intramuscularly and subcutaneously. If a microbiology laboratory is not available, the mixed flora peritonitis models offer a clinically valid model as described in the next section.

2.7 Peritonitis Models A number of peritonitis models have been described in the literature. Three common types are cecal ligation and puncture, colon ascendens stent peritonitis, and peritoneal feces implantation models. The two former models are usually performed in small rodents. A principally important property of these models is that sepsis develops ­relatively slowly from a focal to a systemic inflammatory process, mimicking the clinical situation. Other advantages are simplicity, reproducibility, the relatively long observation time and, being rodent models, the relatively low

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cost per animal. Drawbacks of these models are also mainly due to the choice of relatively small animals making blood sampling, monitoring of pathophysiologic changes and therapeutic interventions difficult. Furthermore, the biological ­differences between rodent and human physiology may complicate the interpretation of results in the context of human sepsis. The cecal ligation and puncture model resembles sepsis with a focal abdominal infection as its source. The experimental animal is anesthetized, the cecum is ligated distal to the ileocecal valve and punctured with a small caliber needle, and the abdominal wall is closed. In about 10 h the animals develop a proinflammatory, hypermetabolic state with hyperdynamic circulation. This proceeds into a phase with hypodynamic circulation over several hours [59]. Oxidative stress has been studied in this model by several groups [60, 61]. The colon ascendens stent peritonitis model is a relatively new model of general peritonitis. This model has been used in the murine model of oxidative burst [62]. The ascending colon is perforated with two stents that are fixated and left in place. The feces leaking into the peritoneal cavity induces sepsis. The diameter of the stents greatly affects the mortality rate of the animals. Typically, the animals ­survive one to several days [63]. Implantation of feces into the peritoneal cavity can be used in small as well as in larger animals [64]. The animals in this model rapidly develop sepsis with inflammatory activation and hypodynamic circulation similar to the infusion of LPS. Some groups use fibrin clot embedded bacteria to produce slower onset of sepsis [65].

2.8 Appendix on Induction of Endotoxemia and Acute Inflammation A brief and somewhat standardized description of our experience, partly from a practical point of view, regarding the endotoxemic pig model is given below. Inclusion criteria are baseline arterial oxygen tension (PaO2) ³ 10 kPa (75 mm Hg) and a mean pulmonary arterial pressure (MPAP) < 2.7 kPa (20 mm Hg) 30 min after completion of the surgical procedure. Both these criteria are sensitive markers of respiratory or systemic infection and are thus used as a screening method in order to minimize unnecessary variation in the acquired data. Animals of both sexes are used. The pigs commonly weigh between 20 and 30 kg. Larger animals have lower frequency of infections and have gone through a significant degree of selection but pose higher demands in terms of research facilities, instrumentation and drug expenses. We give each pig an intramuscular injection in the nuchal region of 6 mg kg−1 of tilétamine–zolazépam mixed with 2.2  mg  kg−1 of xylazin in order to induce ­anesthesia, while still in its cage. The animals do not seem to react to this stimulus and they fall usually asleep within a few minutes. Although the animal’s ­airway usually remains patent and the respiratory drive is sustained, care should be taken to avoid asphyxia during this period. An intravenous cannula is easily inserted into a vein into the ear of the sleeping pig. A continuous intravenous infusion of

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sodium pentobarbital (8  mg  kg−1  h−1), pancuronium bromide (0.26  mg  kg−1  h−1) and ­morphine (0.48  mg  kg−1  h−1) is started in order to maintain the anesthesia. Twenty milligram of morphine I.V. is recommended when the animal has fallen asleep in order to deepen the anesthesia. A tracheotomy is easily performed in order to secure the airway. A tracheostomy is much easier to perform in the pig than in man. This can be ­performed through a midline incision between the ­cricoid cartilage and the area ­corresponding to the supra sternal notch. Blunt ­dissection strictly in the midline leads to the trachea that can be intubated through a semicircumferential incision between two tracheal cartilages. This whole procedure can be performed in 1 min. Oral tracheal intubation can be performed, but since the pig has a long epiglottis, intubation is somewhat more difficult to perform as compared to man. In pigs weighing more than 20 kg, a Miller blade no. 4 is preferred. Over 30 kg, even this laryngoscope blade may be too short. Thirty percent oxygen is given in N2O during the insertion of catheters, otherwise 30% oxygen is administered in N2. In case the N2O is not available, morphine or ­ketamine can be used for analgesia during surgery. A central venous line and a 7 F Swan-Ganz catheter, equipped with a thermistor, are inserted through the external jugular vein into the superior caval vein and into the pulmonary artery, respectively. It should be remembered that the distance from the insertion in the vessel to the pulmonary artery is much shorter than in man. The vein can be found a few centimeters laterally from the midline. We insert the ­vascular catheters surgically. The external jugular vein is thus divided and ligated. Below the divided vein, a cervical artery can be localized and catheterized for pressure monitoring and blood sampling. During the experimental period, 2.5% glucose with sodium chloride (70 mmol L−1) is infused at a rate of 18 ml kg−1 h−1. Since it is impossible to introduce a urinary catheter into the bladder through the urethra, we suggest that a urinary catheter be inserted through a small vesicostomy. The animals should be kept warm by a ­heating pad and ventilated mechanically. We set the ventilatory minute volume to obtain an initial PaCO2 of between 5.0 kPa and 5.5 kPa, and thereafter ventilation is kept constant during the experiments. After induction of anesthesia and surgical ­preparation, each animal is placed in the prone position to avoid atelectasis. A positive end-expiratory pressure of 5  cmH2O is applied throughout the experiment. Endotoxemia is induced by a continuous infusion of E. coli endotoxin (O111:B4; Sigma Chemical, St. Louis, MO, USA) at a dose of 0.063–16 mg kg−1 h−1, usually for 6 h. The pathophysiological responses to endotoxin vary and we recommend that a titration be performed before a large series of animals is initiated. In recent years, we usually administer endotoxin at 2–4 mg kg−1 h−1. Doubling of the initial MPAP during the first hour of the endotoxin infusion is frequently accepted as a sign of endotoxemic shock. If the mean arterial pressure equals the same level as the mean pulmonary pressure within the first hour after baseline, a single dose of adrenalin 0.1  mg is usually administered intravenously. The animals that survive the experimental period are sacrificed by an intravenous overdose of potassium chloride monitored by electrocardiogram while still under anesthesia according to the local ethical guidelines.

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46. Muzio M, Bosisio D, Polentarutti N, et al. Differential expression and regulation of toll-like receptors (TLR) in human leukocytes: selective expression of TLR3 in dendritic cells. J Immunol 2000;164:5998–6004. 47. Muzio M, Polentarutti N, Bosisio D, Prahladan MK, Mantovani A. Toll-like receptors: a growing family of immune receptors that are differentially expressed and regulated by different leukocytes. J Leukoc Biol 2000;67:450–6. 48. Zhang FX, Kirschning CJ, Mancinelli R, et al. Bacterial lipopolysaccharide activates nuclear factor-kappaB through interleukin-1 signaling mediators in cultured human dermal endothelial cells and mononuclear phagocytes. J Biol Chem 1999;274:7611–4. 49. Antal-Szalmas P, Strijp JA, Weersink AJ, Verhoef J, Van Kessel KP. Quantitation of surface CD14 on human monocytes and neutrophils. J Leukoc Biol 1997;61:721–8. 50. Parker SJ, Watkins PE. Experimental models of gram-negative sepsis. Br J Surg 2001;88:22–30. 51. Fiorini RN, Shafizadeh SF, Polito C, et al. Anti-endotoxin monoclonal antibodies are protective against hepatic ischemia/reperfusion injury in steatotic mice. American journal of transplantation 2004;4:1567–73. 52. Giacometti A, Cirioni O, Ghiselli R, et al. Antiendotoxin activity of protegrin analog IB-367 alone or in combination with piperacillin in different animal models of septic shock. Peptides 2003;24:1747–52. 53. Goscinski G, Lipcsey M, Eriksson M, Larsson A, Tano E, Sjolin J. Endotoxin neutralization and anti-inflammatory effects of tobramycin and ceftazidime in porcine endotoxin shock. Crit Care 2004;8:R35–R41. 54. Dyson A, Singer M. Animal models of sepsis: why does preclinical efficacy fail to translate to the clinical setting? Crit Care Med 2009;37:S30–7. 55. Schrauwen E, Cox E, Houvenaghel A. Escherichia coli sepsis and endotoxemia in conscious young pigs. Vet Res Commun 1988;12:295–303. 56. Crocker SH, Eddy DO, Obenauf RN, Wismar BL, Lowery BD. Bacteremia: host-specific lung clearance and pulmonary failure. J Trauma 1981;21:215–20. 57. Matejovic M, Krouzecky A, Martinkova V, et al. Effects of tempol, a free radical scavenger, on long-term hyperdynamic porcine bacteremia. Crit Care Med 2005;33:1057–63. 58. Saetre T, Hoiby EA, Aspelin T, Lermark G, Egeland T, Lyberg T. Aminoethyl-isothiourea, a nitric oxide synthase inhibitor and oxygen radical scavenger, improves survival and counteracts hemodynamic deterioration in a porcine model of streptococcal shock. Crit Care Med 2000;28:2697–706. 59. Hubbard WJ, Choudhry M, Schwacha MG, et al. Cecal ligation and puncture. Shock 2005;24 Suppl 1:52–7. 60. Zapelini PH, Rezin GT, Cardoso MR, et  al. Antioxidant treatment reverses mitochondrial dysfunction in a sepsis animal model. Mitochondrion 2008;8:211–8. 61. Devrim E, Avci A, Erguder IB, Karagenc N, Kulah B, Durak I. Activities of xanthine oxidase and superoxide dismutase enzymes in rat intestinal tissues in sepsis. J Trauma 2008;64:733–5. 62. Neumann B, Zantl N, Veihelmann A, et al. Mechanisms of acute inflammatory lung injury induced by abdominal sepsis. Int Immunol 1999;11:217–27. 63. Zantl N, Uebe A, Neumann B, et al. Essential role of gamma interferon in survival of colon ascendens stent peritonitis, a novel murine model of abdominal sepsis. Infect Immun 1998;66:2300–9. 64. Rodriguez ZZ, Guanche D, Alvarez RG, Rosales FH, Alonso Y, Schulz S. Preconditioning with ozone/oxygen mixture induces reversion of some indicators of oxidative stress and ­prevents organic damage in rats with fecal peritonitis. Inflamm Res 2009. 65. Goldfarb RD, Glock D, Kumar A, et  al. A porcine model of peritonitis and bacteremia ­simulates human septic shock. Shock 1996;6:442–51.

Models and Approaches for the Study of Reactive Oxygen Species Generation and Activities in Contracting Skeletal Muscle Malcolm J. Jackson

Abstract  Reactive oxygen and nitrogen species are generated in increased amounts in skeletal muscle during contractile activity, but most techniques to study this area have utilized end point analyses and are influenced by non-muscle cells present in the sample. This review will briefly examine the various alternative models that can be used to overcome these problems and help determine the potentially important roles that ROS appear to play in physiological and pathological processes in muscle. Keywords  Reactive oxygen species • Skeletal muscle • Microdialysis • Cell culture • Fluorescence microscopy

1 Introduction There is now a substantial body of literature demonstrating that reactive oxygen and nitrogen species are generated in increased amounts in skeletal muscle during ­contractile activity, and recent developments in the techniques available to study this area have facilitated a greater understanding of the nature of the reactive ­oxygen species (ROS) that are generated during contractile activity. Most recent data indicates that ROS are generated in a controlled and regulated manner in response to physiological and pathophysiological stimuli and play important roles in physiological processes, such as the stimulation of adaptive responses to the stresses of contractions (see [1, 2] for reviews). It now seems clear that these ­substances are not merely produced by accident in skeletal muscle cells. There is therefore a need to be able to monitor ROS in muscle. Ideally this would involve M.J. Jackson (*) School of Clinical Sciences, University of Liverpool, Liverpool L69 3GA, UK e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_24, © Springer Science+Business Media, LLC 2011

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measuring the generation of specific ROS, but by the nature of these molecules, they are generated and then rapidly react with a variety of cellular components. These components may play a role in ROS regulation (such as the superoxide ­dismutases), may become modified by the ROS in a manner described as oxidative damage (e.g., lipids, proteins and DNA) or play an “antioxidant” role in cells to scavenge or quench the reactivity of the ROS (e.g., glutathione or tocopherols). As a consequence of this reactivity, the true cellular concentrations of individual ROS is never high and cannot be readily measured in biological systems. The various approaches to monitor ROS have therefore relied upon measurements of end points of ROS reaction with biomolecules (i.e., oxidative damage markers) or use reagents with a particularly high reactivity with ROS that can compete with endogenous substrates for reaction with ROS. These latter measurements are more correctly described as measures of the activity of the ROS that are studied (see [2]).

2 Measurements of Circulating Markers Many studies have examined markers of ROS in the body fluids of exercising humans and animals and these generally describe an increase in a variety of markers resulting from the exercise. These include changes in oxidation products of lipids, DNA and proteins (e.g., see [3–6]). In addition, an increase in oxidation of antioxidant substances such as glutathione has been extensively reported [7]. These data generally appear to indicate that exercise increases the oxidation of biomolecules and they are generally assumed to reflect an increase in ROS generation, but do not provide any guide to the tissue source of the putative increase in ROS generation. In some studies, traditional physiological approaches, such as simultaneous arterio-venous sampling of blood across a bulk of muscle, have been used to demonstrate that the skeletal muscle tissue provides a source of the increased markers of ROS activity observed in the circulation [8, 9], although they do not provide information on the specific cell types involved. Many further studies have also examined the activities of key regulatory enzymes for ROS (e.g., glutathione peroxidases or catalase) or the content of antioxidant substances (e.g., vitamin E or carotenoids) that are found in the circulation and these may be modified by exercise, but these data provides little mechanistic information on the processes that occur [2].

3 Measurement of End Products of ROS Reactions in Muscle The vast majority of studies where researchers have been interested in ROS activities or generation have relied on measurements of end products of ROS reaction with lipids, proteins and DNA [10]. Although many assays used originally to study muscle were nonspecific (e.g., TBARS or diene conjugates), there has been increasing

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use of more specific assays that in some cases can provide information on the type of radical species that may have caused the oxidative damage [11]. These assays have been applied to muscles taken from experimental animals post-mortem and to biopsies of muscle obtained from healthy subjects and from a wide variety of patient groups. Muscle biopsy techniques continue to be refined. Originally, “open” biopsies taken under general anesthesia, were used for diagnostic and investigational studies and this approach was clearly able to provide substantial amounts of tissue for analyses. A variety of needle biopsy approaches under local anesthesia have mostly superseded open biopsies with the use of the “Bergstrom” needle generally the most popular [12]. This needle comes in various sizes and has also been modified in various ways, such as with the use of suction to try and increase the amount of sample obtained. Techniques for rapid freezing of samples to preserve labile metabolites have been described. The most recent development of this approach involves a semiautomated “gun” approach in which the needle is propelled rapidly into the muscle to reduce pain and trauma. This provides less sample, but multiple passes can be used because of the relative lack of discomfort caused [13]. The needle biopsy approach does allow some opportunity for repeated analyses of the same muscle in humans, and many studies with repeat biopsies have been undertaken to determine the time course of changes following exercise (e.g., [14]), but generally the number of biopsies that can be taken limit this aspect of the approach. The major problem with approaches dependent upon analyses of whole muscles from experimental animals or muscle biopsy samples from human subjects is the heterogeneous cell population that is sampled. Thus, although the major portion of the sample is likely to be muscle fibers, samples will inevitably contain vascular tissue, fibroblasts, inflammatory cells etc. in varying amounts. While this problem may be partly avoided by the use of immunohistochemical techniques and may not be a problem for muscle-specific analytes, for others (such as cytokines) it can provide a major limitation to the approach. Direct analyses of radical species by EPR have also been applied to muscle samples from animals [3] and human biopsies [15], but the amount of information that can be obtained by these approaches is limited by lack of fine detail in the EPR spectra due to the high water content of samples.

4 Techniques to Study Extracellular ROS Because of the relative ease of access to extracellular fluids, much work in this field has examined this compartment. Relatively little is known about the roles of extracellular ROS, although neutrophils and other cells are recognized to release substantial amounts of ROS into extracellular fluids during normal activation. Nevertheless, it appears likely that extracellular ROS may play roles in processes as diverse as cell–cell signaling [16, 17] and aging [18].

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Microdialysis techniques provide a potential means of monitoring the composition of the tissue extracellular fluid (ECF) that may not be available by other means. The technique involves the positioning of a small probe, containing a dialysis membrane into the tissue of interest. The membrane is perfused on the inside with a physiological medium at a very slow flow rate. The outside of the membrane is in contact with the extracellular fluid. This allows water and low molecular weight compounds in the tissue ECF to perfuse across the dialysis membrane to be collected via the outlet port. Microdialysis allows continuous sampling of compounds in the ECF in close proximity to cells, providing information on local chemical changes before such compounds are cleared, and diluted in, the circulatory system. The theory and application of the technique have been comprehensively reviewed [19]. Attempts have been made to detect ROS in ECF by microdialysis since the beginning of the 1990s with early studies examining NO release [20], spin trapping techniques to detect lipid radicals [21] and hydroxyl radical activity [22]. Our initial studies in the area examined superoxide release by skeletal muscle [23] and confirmed the extracellular release of this anion seen by Reid et al. [24]. Subsequent development of the approach allowed the measurement of hydroxyl radical activity [25], nitric oxide and hydrogen peroxide [26, 27] in microdialysates from skeletal muscle and included some validation studies to examine the robustness of the approach [28]. Microdialysis approaches have been applied to study ROS in the extracellular space of muscle in human subjects (for example, see [29]) and potentially provide a unique approach to monitoring specific ROS in human tissues. Cell culture approaches. Complementary to the use of microdialysis to study extracelluar ROS in vivo has been the use of cell culture to examine both specific ROS released from skeletal muscle cells and intracellular ROS activities. Although muscle tissue in  vivo and isolated muscles in  vitro have both been previously used to study ROS generation (for example, see [8, 24, 30]), these tissues contain endothelial cells, lymphocytes and fibroblasts that may contribute to the generation of oxygen/nitrogen free radicals. Use of skeletal muscle cells in culture may ­overcome this problem, and we showed that primary cultures of myotubes from mice (see Fig. 1) release an increased amount of superoxide during electrically stimulated contractile activity. [23] and others reported release of hydrogen peroxide and NO by primary cultures of myotubes during contractile activity [32]. We also examined the release of ROS from an immortalized muscle cell line in culture [33] and demonstrated release of NO and superoxide together with generation of hydroxyl radical in the extracellular space. Surprisingly, the release of NO and formation of hydroxyl radical was related to the frequency of stimulation of the muscle cells, but the release of superoxide was activated by contractions, but ­unrelated to the frequency of stimulation [33]. Thus these studies have indicated a relatively complex pattern of release of NO, superoxide and hydrogen peroxide from skeletal muscle cells which, only in part, reflects the metabolic activity of the contracting tissue. It appears that other factors influence the release of superoxide and potential generation of a number of secondary ROS species.

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Fig. 1  Images of cultured primary mouse muscle cells at 1 (a), 3 (b) and 5 (c) days after switching to a low-serum culture medium to induce differentiation and fusion. Fusion into multinucleate myotubes is apparent by 3 days and essentially complete by 5 days. Five-day myotubes have been used for studies of both extracellular and intracellular ROS generation [23, 31]

5 Techniques to Study Intracellular ROS A number of intracellular probes have been used to study ROS activities in different cell types, but 2¢, 7¢-dichlorodihydrofluorescin-diacetate (DCFH-DA) has been most widely used as a relatively nonspecific intracellular probe for ROS [34]. DCFH-DA is nonpolar, crosses cell membranes readily and within the cell it is hydrolyzed by cytosolic hydrolases to DCFH. This compound rapidly reacts with hydrogen peroxide in the presence of peroxidases and with some other ROS to form fluorescent dichlorofluorescin (DCF). In practice, use of DCFH-DA as an intracellular probe to directly image or measure ROS activities is limited by photo-oxidation [35] and only a few studies have directly examined the oxidation of DCFH by epifluorescence from muscle fibers [24, 35, 36]. The effect of contractile activity on DCFH oxidation had been studied by homogenization prior to fluorescence measurements in myotubes [32] and skeletal muscle [37], but the effect of the homogenization process on DCF fluorescence is unknown [10].

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Fig. 2  A single isolated fiber forms the flexor digitorum brevis muscle of the mouse photographed under white light (left) and loaded with dihydroethidium (right). Such fibers have been used for studies of intracellular ROS activities [38, 39]

We examined the use of DCFH-DA as a nonspecific probe to examine intracellular ROS generation by immortalized skeletal muscle cells prior to, during, and following a demanding period of contractile activity. We found that the myotubes at rest slowly oxidized DCFH and that this increased rapidly during a period of contractile activity [31]. The pattern of demanding contractile activity imposed on the cells would be predicted to cause a considerable increase in the muscle oxygen utilization and, in light of this, the relatively modest (~ fourfold) increase in intracellular ROS generation that was seen in these studies was surprising. One explanation for this data is that during aerobic contractile activity, skeletal muscle mitochondria are predominantly in state III, thus limiting their generation of ROS. Subsequent studies have indicated the feasibility of applying these fluorescent approaches to use other indicators, such as DAF-based probes or dihydroethidium to assess intracellular NO and superoxide [38, 39]. A recent development in this field has been the use of single isolated mature skeletal muscle fibers as a model in which to study intracellular ROS generation. Single fibers from small rodent muscles such as the flexor digitorum brevis can be isolated by collagenase digestion and cultured on a collagen-based matrix. In our hands they retain viability for ~1 week and can be stimulated to contract using external platinum electrodes in a manner similar to the approach used with myotubes [38, 39] and Fig. 2. This approach has the advantage that the fibers are mature and essentially uncontaminated with other cell types.

6 Conclusion This brief review has demonstrated that a variety of techniques are now available for the study of ROS activities in skeletal muscle. The approach to be followed will undoubtedly be dictated by how much detail is required and what the overall

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q­ uestion is. The whole topic of how to assess ROS activities in any tissue remains in flux and potential new methods are constantly being proposed. Clearly the sort of techniques that are in general use and acceptable for high-quality publications has changed over the 20+ years of study of ROS in muscle and measurements of “TBARS” and diene conjugates are no longer appropriate because of their acknowledged limitations. The “holy grail” in this area is undoubtedly the ability to ­measure changes in the activities of specific ROS in single contracting fibers in real time in association with measurements of force production. Recent studies are close to this and suggest that it is now technically feasible (e.g., by a combination of the approaches followed by Pye et al. [38] and Aydin et al. [40]), but at the time of this writing have not been reported.

References 1. Jackson MJ. (2008). Free radicals generated by contracting muscle: by-products of metabolism or key regulators of muscle function? Free Radic Biol Med. 2008, 44: 132–41. 2. Powers SK, Jackson MJ. (2008). Exercise-induced oxidative stress: cellular mechanisms and impact on muscle force production. Physiol Rev. 88: 1243–76. 3. Davies KJ, Quintanilha AT, Brooks GA, Packer L. (1982). Free radicals and tissue damage produced by exercise. Biochem. Biophys. Res. Commun. 107: 1198–205. 4. Jackson MJ, Jones DA, Edwards RH. (1983). Vitamin E and skeletal muscle. Ciba Found Symp. 101: 224–239. 5. Goldfarb AH, McIntosh MK, Boyer BT, Fatouros J. (1994). Vitamin E effects on indexes of lipid peroxidation in muscle from DHEA-treated and exercised rats. J Appl Physiol. 76: 1630–1635. 6. Kanter MM, Nolte LA, Holloszy JO. (1993). Effects of an antioxidant vitamin mixture on lipid peroxidation at rest and postexercise. J Appl Physiol. 74: 965–969. 7. Sastre J, Asensi M, Gascó E, Pallardó FV, Ferrero JA, Furukawa T, Viña J. (1992). Exhaustive physical exercise causes oxidation of glutathione status in blood: prevention by antioxidant administration. Am J Physiol. 263: R992–5. 8. O’Neill CA, Stebbins CL, Bonigut S, Halliwell B, Longhurst JC. (1996). Production of hydroxyl radicals in contracting skeletal muscle of cats. J Appl Physiol. 81: 1197–1206. 9. Bailey DM, Davies B, Young IS, Jackson MJ, Davison GW, Isaacson R, Richardson RS. (2003). Epr spectroscopic evidence of free radical outflow from an isolated muscle bed in exercising humans: functional significance of decreasing intracellular PO2 vs. increasing O2 flux. Adv Exp Med Biol.: 297–303. 10. Halliwell B, Gutteridge JMC. (1989). Free radical biology and medicine. Oxford University Press. 11. Neubauer O, Reichhold S, Nersesyan A, König D, Wagner KH. (2008). Exercise-induced DNA damage: is there a relationship with inflammatory responses? Exerc Immunol Rev. 14: 51–72. 12. Bergström J. (1979). Muscle-biopsy needles. Lancet. 1(8108): 153. 13. Morton JP, Holloway K, Woods P, Cable NT, Burniston J, Evans L, Kayani AC, McArdle A. (2009). Exercise training-induced gender-specific heat shock protein adaptations in human skeletal muscle. Muscle Nerve. 39: 230–3. 14. Khassaf M, Child RB, McArdle A, Brodie DA, Esanu C, Jackson MJ. (2001). Time course of responses of human skeletal muscle to oxidative stress induced by non-damaging exercise. J. App. Physiol. 90: 1031–1036. 15. Jackson MJ, Edwards RH, Symons MC. (1985). Electron spin resonance studies of intact mammalian skeletal muscle. Biochim Biophys Acta. 847: 185–190.

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16. Hancock JT. (1997). Superoxide, hydrogen peroxide and nitric oxide as signalling molecules: their production and role in disease. Br J Biomed Sci. 54: 38–46. 17. Jalkanen S, Salmi M. (2001). Cell surface monoamine oxidases: enzymes in search of a function. EMBO J. 20: 3893–3901. 18. de Grey AD. (2000). The reductive hotspot hypothesis: an update. Arch. Biochem. Biophys. 373: 295–301. 19. Benveniste H, Huttemeier PC. (1990). Microdialysis--theory and application. Prog Neurobiol. 35: 195–215. 20. Balcioglu A, Maher TJ. (1993). Determination of kainic acid-induced release of nitric oxide using a novel hemoglobin trapping technique with microdialysis. J Neurochem. 61: 2311–2313. 21. Zini I, Tomasi A, Grimaldi R, Vannini V, Agnati LF. (1992). Detection of free radicals during brain ischemia and reperfusion by spin trapping and microdialysis. Neurosci Lett. 138: 279–282. 22. Chiueh CC, Krishna G, Tulsi P, Obata T, Lang K, Huang SJ, Murphy DL. (1992). Intracranial microdialysis of salicylic acid to detect hydroxyl radical generation through dopamine autooxidation in the caudate nucleus: effects of MPP+. Free Radic Biol Med. 13: 581–583. 23. McArdle A, Pattwell D, Vasilaki A, Griffiths RD, Jackson MJ. (2001). Contractile activityinduced oxidative stress: Cellular origin and adaptive responses. Am. J. Physiol. (Cell Physiol.) 280: C621–C627. 24. Reid MB, Shoji T, Moody MR, Entman ML. (1992). Reactive oxygen in skeletal muscle. II. Extracellular release of free radicals. J. Appl. Physiol. 73: 1805–1809. 25. McArdle A, van der Meulen J, Close GL, Pattwell D, Van Remmen H, Huang TT, Richardson AG, Epstein CJ, Faulkner JA, Jackson MJ. (2004). Role of mitochondrial superoxide dismutase in contraction-induced generation of reactive oxygen species in skeletal muscle extracellular space. Am J Physiol Cell Physiol. 286: C1152–1158. 26. Vasilaki A, Mansouri A, Remmen H, van der Meulen JH, Larkin L, Richardson AG, McArdle A, Faulkner JA, Jackson MJ. (2006). Free radical generation by skeletal muscle of adult and old mice: effect of contractile activity. Aging Cell. 5: 109–117. 27. Vasilaki A, Csete M, Pye D, Lee S, Palomero J, McArdle F, Van Remmen H, Richardson A, McArdle A, Faulkner JA, Jackson MJ. (2006). Genetic modification of the MnSOD/GPx1 pathway influences intracellular ROS generation in quiescent, but not contracting myotubes. Free Radical Biology & Medicine 41: 1719–1725. 28. Close GC, Ashton T, McArdle A, Jackson MJ. (2005). Microdialysis studies of extracellular reactive oxygen species in skeletal muscle: Factors influencing the reduction of cytochrome c and hydroxylation of salicylate. Free Rad. Biol. Med. 39: 1460–1467. 29. Crowe AV, McArdle A, McArdle F, Pattwell DM, Bell GM, Kemp GJ, Bone JM, Griffiths RD, Jackson MJ. (2007). Markers of oxidative stress in the skeletal muscle of patients on haemodialysis. Nephrol Dial Transplant. 22: 1177–83. 30. Balon TW, Nadler JL. (1994). Nitric oxide release is present from incubated skeletal muscle preparations. J. Appl. Physiol. 77: 2519–2521. 31. McArdle F, Pattwell DM, Vasilaki A, McArdle A, Jackson MJ. (2005). Intracellular generation of reactive oxygen species by contracting skeletal muscle cells. Free Radic Biol Med. 39: 651–657. 32. Silveira LR, Pereira-Da-Silva L, Juel C, Hellsten Y. (2003). Formation of hydrogen peroxide and nitric oxide in rat skeletal muscle cells during contractions. Free Radic Biol Med. 35: 455–464. 33. Pattwell DM, McArdle A, Morgan JE, Patridge TA, Jackson MJ. (2004). Release of reactive oxygen and nitrogen species from contracting skeletal muscle cells. Free Radic Biol Med. 37: 1064–1072. 34. Zuo L, Clanton TL. (2002). Detection of reactive oxygen and nitrogen species in tissues using redox-sensitive fluorescent probes. Methods Enzymol. 352: 307–325. 35. Murrant CL, Andrade FH, Reid MB. (1999). Exogenous reactive oxygen and nitric oxide alter intracellular oxidant status of skeletal muscle fibres. Acta Physiol Scand. 166: 111–121.

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36. Arbogast S, Reid MB. (2004). Oxidant activity in skeletal muscle fibers is influenced by temperature, CO2 level, and muscle-derived nitric oxide. Am J Physiol Regul Integr Comp Physiol. 287: R698–705. 37. Bejma J, Ramires P, Ji LL. (2000). Free radical generation and oxidative stress with ageing and exercise: differential effects in the myocardium and liver. Acta Physiol Scand. 169: 343–35. 38. Pye D, Kabayo T, Palmero J, Jackson MJ. (2007). Real-time measurements of nitric oxide in mature skeletal muscle fibres during contractions. J Physiol. 581: 309–318. 39. Palomero J, Pye D, Kabayo T, Spiller DG, Jackson MJ. (2008). In situ detection and measurement of intracellular reactive oxygen species in single isolated mature skeletal muscle fibres by real-time fluorescence microscopy. Antioxidants and Redox Signalling, 10: 1463–1474. 40. Aydin J, Korhonen T, Tavi P, Allen DG, Westerblad H, Bruton JD. (2007). Activation of Ca(2+)-dependent protein kinase II during repeated contractions in single muscle fibres from mouse is dependent on the frequency of sarcoplasmic reticulum Ca(2+) release. Acta Physiol (Oxf). 191: 131–137.

Exercise as a Model to Study Interactions Between Oxidative Stress and Inflammation Christina Yfanti, Søren Nielsen, Camilla Scheele, and Bente Klarlund Pedersen

Abstract  Inflammation is involved in the pathogenesis of insulin resistance, ­atherosclerosis, neurodegeneration, and tumor growth, and low-grade chronic inflammation appears as a key player in the pathogenesis of several chronic diseases, including type 2 diabetes, cardiovascular disease, cancer and Alzheimer’s disease. Given that regular exercise offers protection against all causes of mortality, primarily by protection against atherosclerosis and insulin resistance, we suggest that exercise may exert some of its beneficial health effects by inducing anti-inflammatory actions. During exercise, skeletal muscles release IL-6 into the circulation and muscle-derived IL-6 mediates anti-inflammatory effects. Supplementation with antioxidative vitamins attenuates the systemic IL-6 response to exercise primarily via inhibition of the IL-6 protein release from contracting skeletal muscle per se. Apparently, antioxidants attenuate some of the normal physiological responses to nondamaging exercise. Therefore, the use of antioxidant supplementation may be less desirable from a long-term health perspective. This point of view is partly supported by large-scale studies showing no effect, or even a detrimental effect, of antioxidant supplementation on morbidity and mortality and by studies showing that antioxidants may blunt some of the metabolic effects of regular exercise. Keywords  Antioxidants • Exercise • IL-6 • Inflammation • Reactive oxygen species

1 Introduction Endurance exercise is associated with the generation of reactive oxygen species (ROS) by the mitochondria as by-products of oxidative metabolism. The fact that exercise-induced enhanced production of ROS can potentially lead to modifications

B.K. Pedersen (*) The Centre of Inflammation and Metabolism, the Department of Infectious Diseases, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_25, © Springer Science+Business Media, LLC 2011

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of lipids, proteins, nucleic acid and other cellular compounds [1] led to the idea that exercise-induced muscle damage potentially might be counteracted by the administration of antioxidants, which could dampen exercise-induced tissue injury and inflammation. Today, antioxidant supplementation is widely used by athletes. More than 50% of all elite endurance athletes and about 40% of the nonelite athletes in the U.S. consume vitamin supplements daily [2] and in much higher doses than the recommended daily allowance [3]. Consumption of vitamin supplements is not limited to athletes, however. Among individuals participating in a regular exercise program within the U.S., 25% of the women and 16% of the men reportedly ingest supplements on a daily basis [2]. However, there is no solid evidence that they will benefit from antioxidant supplementation. From the initial view that ROS were, in general, harmful, and that preventing their actions would be beneficial [4], it now seems possible that these substances also play fundamental roles in the regulation of gene transcription and protein synthesis [5, 6]. A number of transcription factors appear to be regulated by the intracellular redox state [7]. Two of the most well-documented redox-sensitive transcription factors are Nuclear Factor kB (NF-kB) that regulates the transcription of immune and inflammatory genes and activator protein (AP)-1 that regulates genes involved in growth and differentiation [8]. In addition, ROS have been shown to act as important ­signals for the induction of endogenous antioxidant enzymes during endurance training [9, 10].

2 Acute Exercise, Muscle Damage and Antioxidative Vitamins Let us take a closer look at exercise studies in which antioxidant supplementation has been used. The initial findings of enhanced production of ROS in response to exercise led to research showing that it was possible to inhibit ROS production during exercise [11]. Therefore, it was hypothesized that supplementation with vitamins C and E would have a positive effect on performance. The results, though, are controversial. Some early studies indicate that supplementation with ascorbic acid increases work capacity [12] while others suggest that there is no effect [13, 14]. With regard to an effect of antioxidative vitamins on muscle damage, studies have typically shown either no effect or little effect on muscle damage. We found that vitamin supplementation did not influence exercise-induced muscle damage as visualized by creatine kinase levels during down-hill running [15]. This finding was in agreement with studies by Warren et  al. [16] and Kaikkonen et  al. [17] but differed from McBride et  al. [18], Sumida et al. [19] and Rokitzki et al. [20]. The discrepancies could be a result of varied exercise protocols and different duration, quantities, and types of antioxidant supplementation used during the trials.

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3 Exercise, Cytokines and Oxidative Vitamins The rationale for antioxidant supplementation in relation to exercise has been to inhibit exercise-induced muscle damage and inflammation. However, the rationale may be wrong. The goal of inflammation is to recruit leukocytes rapidly to the site of injury/ infection. Inflammation is a double-edged sword; on one hand, inflammation is required in order to combat infections and acute injury and on the other, chronic systemic inflammation causes impaired metabolism. Along this line, one would suspect that a sufficient inflammatory reaction would allow the host to recover from acute injury. Accordingly, blocking inflammation may not be beneficial. Several exercise studies have looked at the effect of antioxidative vitamins on cytokine levels in response to exercise. However, there is no support for the idea that exercise-induced cytokine production is linked to muscle damage or reflects a classic acute inflammatory response.

3.1 The Cytokine Response to Exercise It has been consistently demonstrated that the plasma concentration of IL-6 increases during muscular exercise. This increase is followed by the appearance of an IL-1 receptor antagonist (IL-1ra) and the anti-inflammatory cytokine IL-10. In most exercise studies, tumor necrosis factor (TNF)-a does not change. In general, the cytokine response to exercise and sepsis differs with regard to TNF-a. Thus the cytokine response to exercise is not preceded by an increase in plasma TNF-a. Even though there is a moderate increase in the systemic concentration of these cytokines, the underlying fact is that the appearance of IL-6 in the circulation is by far the most marked and that its appearance precedes that of other cytokines [21]. Following exercise, the basal plasma IL-6 concentration may increase up to 100fold, although less dramatic increases are more frequent. Of note, the exerciseinduced increase of plasma IL-6 is not linear over time; repeated measurements during exercise show an accelerating increase of the IL-6 in plasma in an almost exponential manner [22]. Furthermore, the peak IL-6 level is reached at the end of the exercise or shortly thereafter followed by a rapid decrease towards pre-exercise levels. Overall, the combination of mode, intensity, and duration of the exercise determines the magnitude of the exercise-induced increase of plasma IL-6.

3.2 Exercise-Induced IL-6 Production Is Not Linked with Muscle Damage Since IL-6 is a classical inflammatory cytokine, it was first thought that the IL-6 response was related to muscle damage [23]. However, it has become evident that eccentric exercise is not associated with a larger increase in plasma IL-6 than exercise

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involving concentric “nondamaging” muscle contractions, clearly demonstrating that muscle damage is not required to increase plasma IL-6 during exercise. Rather, exercise involving eccentric contractions may result in a delayed peak and a slower decrease of plasma IL-6 during recovery [21]. Until the beginning of this millennium it was commonly thought that the exercise-induced increase in IL-6 was a consequence of an immune response due to local damage in the working muscles [24] and it was hypothesized that the immune cells were responsible for this increase [25]. It now appears that IL-6 mRNA in monocytes, the blood mononuclear cells responsible for the increase in plasma IL-6 during sepsis, do not increase as a result of exercise [26–28]. Therefore, the previously held assumption that the IL-6 response to exercise may involve immune cells does not appear to be correct. Moreover, it has become clear that the cytokine response, at least not the IL-6 response, is not related to exercise-induced muscle damage [29]. Croisier et al. reported that a single bout of eccentric exercise in untrained subjects elicited a marked increase in plasma IL-6 (~8-fold) that was associated with severe levels of delayed-onset muscle soreness and a greater than 100-fold increase in serum myoglobin, both indicators of muscle damage. However, after subjects completed 3 weeks of exercise training, the same bout of eccentric exercise elicited an identical marked increase in IL-6 despite no significant change in perceived soreness or serum myoglobin, which suggests that the ­production of IL-6 in response to exercise is not a function of damage to ­contracting myofibres. Steensberg et al. found that circulating IL-6 levels also increase dramatically during low-intensity (40% of a maximum 2-min power output), one-legged knee extensor exercise, a form of exercise that evokes little to no increase in creatine kinase levels [30]. Moreover, on the basis of femoral arteriovenous IL-6 concentration and blood flow measurements, it was found that the net release of IL-6 from the exercising leg could account for the entire increase in circulating plasma IL-6 concentration, which suggests that active muscle tissue is the source of IL-6 production during exercise. Today, it is very clear that the contracting skeletal muscle per se is the main source of the IL-6 in the circulation in response to exercise. In resting human skeletal muscle, the IL-6 mRNA content is very low, while several studies have demonstrated an induction of gene expression following exercise. In response to exercise, an increase of the IL-6 mRNA content in the contracting skeletal muscle is detectable after 30 min of exercise, and up to 100-fold increases of the IL-6 mRNA content may be present at the end of the exercise session [21]. Thus, as shown by Croisier et al., plasma-IL-6 will increase also in the absence of muscle damage [29]. Moreover, concentric exercise is associated with a much more marked increase in plasma-IL-6 compared to eccentric exercise, further supporting the fact that muscle damage and exercise-induced increases in plasma-IL-6 are not related.

3.3 Exercise-Induced IL-6 and Antioxidative Vitamins Two studies have shown that oral supplementation with antioxidants can attenuate the exercise-induced increase of IL-6 in plasma [31, 32]. We found it of interest that

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murine myotubes express IL-6 when exposed to oxidative stress [33]. Thus, we hypothesized that oral supplementation with antioxidant vitamins would inhibit the exercise-induced IL-6 response in human skeletal muscle. We were able to show that oral supplementation with vitamins C and E totally inhibits the release of IL-6 from contracting human skeletal muscle, thus causing a marked attenuation of IL-6 in the circulation, as well as blunting the increase of IL-1ra and cortisol in response to exercise [34]. Muscle-derived IL-6 mediates important autocrine, paracrine and endocrine functions. Thus, IL-6 is involved in mediating glucose uptake in muscle, fat oxidation, lipolysis and possesses strong anti-inflammatory properties [35]. Thus, we speculate that since antioxidants apparently attenuate the normal physiological response to concentric exercise, the use of antioxidant supplementation may be less desirable from a long-term health perspective. This point of view is partly supported by large-scale studies showing no or even a detrimental effect of antioxidant supplementation on morbidity and mortality [36].

4 Chronic Inflammation and Disease Type 2 diabetes, cardiovascular diseases, colon cancer, breast cancer, dementia, and depression constitute a cluster of diseases that defines “a diseasome of physical inactivity.” Both physical inactivity and abdominal adiposity, reflecting ­accumulation of visceral fat mass, are associated with the occurrence of the diseases within the diseasome. Chronic inflammation is involved in the pathogenesis of insulin ­resistance, atherosclerosis, neurodegeneration, and tumor growth. Evidence suggests that the protective effect of exercise may to some extent be ascribed to the anti-inflammatory effect of regular exercise, which can be mediated via a reduction in visceral fat mass and/or by induction of an anti-inflammatory environment with each bout of exercise. The finding that muscles produce and release so-called ­myokines, including IL-6, therefore provides a conceptual basis to understanding the ­mechanisms, whereby exercise influences metabolism and exerts anti-­ inflammatory effects [37].

5 Linking Inflammation with Disease Mounting evidence suggests that TNF-a plays a direct role in the metabolic syndrome, recently reviewed in [21]. In short, patients with diabetes demonstrate high protein expression of TNF-a in skeletal muscle and increased TNF-a levels in plasma, and it is likely that adipose tissue, which produces TNF-a, is the main source of the circulating TNF-a [38]. In vitro studies demonstrate that TNF-a has direct inhibitory effects on insulin signaling and evidence suggests that TNF-a plays a direct role in linking insulin resistance to vascular disease [39]. Importantly, also tumor initiation, promotion, and progression are stimulated by systemic elevation

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of proinflammatory cytokines. In addition, a number of neurodegenerative diseases, including Alzheimer’s disease, are linked to a local inflammatory response in the brain (neuroinflammation) [40, 41]. With regard to IL-6, its role in insulin resistance is highly controversial, as reviewed [21]. Infusion of rhIL-6 into resting healthy humans does not impair whole-body, lower-limb, or subcutaneous adipose tissue glucose uptake or endogenous glucose production (EGP), although IL-6 contributes to the contraction-induced increase in endogenous glucose production. When diabetes patients were given a recombinant human (rh) IL-6-infusion, plasma concentrations of insulin declined to levels comparable with that in age and BMI-matched healthy controls, indicating that IL-6 enhances insulin sensitivity. Furthermore, a number of studies indicate that IL-6 enhances lipolysis, as well as fat oxidation, via an activation of AMPK, reviewed in [21]. Of note, whereas it is known that both TNF-a and IL-6 induce lipolysis, only IL-6 appears to induce fat oxidation [42, 43]. Given the different biological profiles of TNF-a and IL-6 and given that TNF-a may trigger an IL-6 release, one theory holds that it is TNF-a derived from adipose tissue that is actually the major “driver” behind inflammation-induced insulin resistance and atherosclerosis. A vast number of studies during the past decade have revealed that in response to muscle contractions, both type I and type II muscle fibres express the myokine IL-6, which subsequently exerts its effects both locally within the muscle (e.g., through activation of AMPK) and – when released into the circulation – peripherally in several organs in a hormone-like fashion. Within skeletal muscle, IL-6 acts locally to signal through a gp130Rb/IL-6Ra homodimer, resulting in activation of AMPkinase and/or PI3-kinase to increase glucose uptake and fat oxidation. IL-6 may also work in an endocrine fashion to increase hepatic glucose production during exercise or lipolysis in adipose tissue, reviewed in [21]. To study whether acute exercise induces a true anti-inflammatory response, a model of “low-grade inflammation” was established in which a low dose of E. coli endotoxin was administered to healthy volunteers, who had been randomized to either rest or exercise prior to endotoxin administration. In resting subjects, endotoxin induced a two- to threefold increase in circulating levels of TNF-a. In contrast, when the subjects performed 3 h of ergometer cycling and received the endotoxin bolus at 2.5  h, the TNF-a response was totally blunted. Moreover, when the subjects were treated intravenously with recombinant human IL-6 to mimic the systemic concentrations elicited by a bout of exercise, IL-6 totally inhibited the endotoxin-induced increase in TNF-a. This study provides some evidence that the anti-inflammatory effects of acute exercise may be mediated via exercise-induced IL-6 production [44].

6 Training Studies and Antioxidative Vitamins Apparently, antioxidants attenuate some of the normal physiological responses to nondamaging exercise. In particular, it appears that antioxidant supplementation inhibits important players in the exercise-induced anti-inflammatory response.

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Therefore, the use of antioxidant supplementation may be less desirable from a long-term health perspective. Recently, the hypothesis that antioxidant supplementation during endurance training may attenuate some of the beneficial effects of exercise has received some support. Gomez-Cabrera et al. [45] found that vitamin C supplementation in rodents attenuates training-induced mitochondrial biogenesis and endurance capacity. Another recent study showed that antioxidant supplementation with vitamins C and E inhibit health beneficial effects of 4 weeks of training on insulin sensitivity and other markers of metabolic training adaptation [46]. Moreover, when a 6-week aerobic exercise training program was applied in patients with hypertension, supplementation of antioxidants (vitamins C and E, and a-lipoic acid) led to an enhancement of blood pressure and an inhibition of exercise-induced flow-mediated vasodilatation [47]. Thus, even though antioxidant treatment may improve glucose metabolism in type 2 diabetics, it may also block other positive health benefits of regular physical activity.

7 Conclusion Regular exercise offers protection against chronic diseases and this effect may in part be mediated via increased production of IL-6 during exercise. Supplementation with antioxidative vitamins attenuates the systemic IL-6 response to exercise primarily via inhibition of the IL-6 protein release from the contracting skeletal muscle per se. Apparently, antioxidants attenuate some of the normal physiological responses to nondamaging exercise. Therefore, the use of antioxidant supplementation may be less desirable from a long-term health perspective. This point of view is partly supported by large studies showing no or even a detrimental effect of antioxidant supplementation on morbidity and mortality and by studies showing that antioxidants may blunt some of the metabolic effects of regular exercise. Acknowledgment  The Centre of Inflammation and Metabolism is supported by a grant from the Danish National Research Foundation (DG 02-512-555). In addition, support was obtained from the Danish Medical Research Council, and the Commission of the European Communities (contract no. LSHM-CT-2004-005272 EXGENESIS).

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Exercise as a Model to Study Oxidative Stress Mari Carmen Gomez-Cabrera, Fabian Sanchis-Gomar, Vladimir Essau Martinez-Bello, Sandra Ibanez-Sania, Ana Lucia Nascimento, Li Li Ji, and Jose Vina

Abstract  Physical exercise generates free radicals. The major source of radicals in exercise appears to be extracellular. Our experiments show that xanthine oxidase is a key player in the generation of superoxide during exercise. Mitochondrial contribution appears to be less important: during high oxygen utilization by mitochondria in state 3, the proportion of oxygen that is converted to superoxide is on an order of magnitude lower than in resting, state 4 conditions. Exercise-induced radicals constitute a double-edged sword: high intensity ­exercise causes the generation of relatively high concentrations of radicals that cause oxidative stress and eventually damage. On the other hand, low intensity training activates the expression of antioxidant genes and other cell adaptations to exercise. This has practical implications: antioxidant supplements are useful after exhaustive exercise (which causes damage) but should not be used in training (because oxidant-dependent adaptations are prevented). Thus, exercise is an excellent physiological model to understand oxidative stress and subsequent cellular adaptations to such stress. Keywords  Antioxidants • Exercise • Muscle • Oxidative stress • RONS • ROS

1 Free Radical Generation and Oxidative Stress in Exercise Oxidative stress was first defined in 1985 as “a disturbance in the pro-oxidant– antioxidant balance in favor of the former” [1]. Oxidative stress usually leads to oxidative damage, which can be characterized by the evaluation of the damage to cellular components – mainly lipids, proteins, and DNA. Common measures of biooxidation include the measurement of protein carbonyls as an indicator of ­protein

J. Vina (*) Department of Physiology, Faculty of Medicine, University of Valencia, Fundacion Investigacion Hospital Clinico Universitario/INCLIVA, Spain e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_26, © Springer Science+Business Media, LLC 2011

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oxidation; assessment of isoprostanes, malondialdehyde, and 4-hydroxyl-2-nonenol as signs of lipid peroxidation; and evaluation of DNA oxidation by oxidized nucleotide 8-hydroxy-2¢-deoxyguanosine (8-OH-dG) [2]. It is well accepted that skeletal muscle generates RONS during contractile activity. Research in this area started in the 1950s when the first data showing that free radicals are present in muscle was published [3]. The first suggestion that exercise was associated with an increase in the formation of lipid peroxidation by-products did not appear until the late 1970s [4]. In 1980, Koren et al. showed that free radical content was elevated in frog limb muscles stimulated to contract repetitively [5]. It was in 1982 when Davies et al. [6] first showed increased free radical production in rat skeletal muscle after running to exhaustion. Since then, research in the area has mushroomed. It is now clear that intense muscular contractile activity can result in oxidative stress not only in animals but also in humans [7, 8]. There are several potential tissue sources from which RONS may be produced during exercise: heart, lungs, white blood cells and skeletal muscle have been most studied [9]. It is well known that exhaustive exercise (especially when sporadic) causes structural damage to muscle cells or inflammatory reactions within the muscles. For instance, eccentric exhaustive exercise resulted in an increase in the plasma activity of cytosolic enzymes, accompanied by sarcolemma and Z-line ­disruption [10]. Some of this damage was thought to be related to the production of free radicals by phagocytic white cells [11]. Some authors have suggested that ­common metabolic changes that occur during exercise, such as the increased release of cathecolamines may play a role in the increased ROS generation [12]. However, it is without question that skeletal and heart muscles provide the major source of free radicals during physical exercise. At the subcellular level, several well-established sites have been shown to generate RONS during muscle contraction. The role of mitochondria in the generation of O2•− and H2O2 has been studied in depth [13]. It has generally been assumed that an increase in oxygen consumption by mitochondria would cause an increase in O2•− formation in the electron transport chain [14]. This, however, is based on the misconception that the proportion of ROS formed by mitochondria is in the range of 2% of the total oxygen consumed. Early work by the group of Britton Chance [15] revealed that approximately 2% of oxygen used by mitochondria is converted to free radicals only when these mitochondria are in the resting state (state 4). However, when mitochondria are in state 3, i.e., actively producing ATP from ADP, with a high electron flow into oxygen, the proportion of oxygen converted to free radicals falls to a tenth of that found in resting state 4. In fact, recent findings suggest that mitochondria may not be the dominant source of ROS during exercise [16]. Brand and colleagues have recently reassessed the rate of production of ROS by mitochondria and have concluded that the upper estimate of the total fraction of oxygen utilized that forms O2•− is ~0.15% [17]. With all this evidence in mind, the role of mitochondria in the formation of free radicals in exercise should be reconsidered and perhaps alternative sources of ROS should be identified. NAD(P)H oxidase enzymes have been studied as relevant sources of ROS in skeletal muscle. These enzymes are associated with the sarcoplasmic reticulum [9],

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transverse tubules [18] and plasma membrane [19]. The superoxide generated by these enzymes appears to influence muscle contraction [20]. Another relevant enzyme in free radical generation in skeletal muscle is phospholipase A2 (PLA2) [21]. Nethery and colleagues have shown that PLA2 function is essential for the rise in intracellular ROS that occurs during repetitive, fatiguing contractions [22]. The role of xanthine oxidase (XO) in oxidant generation during muscle contraction has long been studied [7, 23, 24]. Xanthine oxidase and xanthine dehydrogenase (XDH) catalyze the oxidation of hypoxanthine and xanthine to urate during purine catabolism in mammals. While XDH preferentially transfers the electrons released during the oxidation process to NAD+, XO utilizes molecular oxygen, thereby generating O2•− [25]. Treatment with allopurinol or oxypurinol has been associated with a decrease in muscle damage and markers of oxidative stress after exhaustive exercise both in humans and in rats [8, 26]. Finally, NO is also generated continuously by skeletal muscle [27]. The neuronal, the endothelial and the inducible NOS are expressed in skeletal muscle and can produce NO during contractile activity [28].

2 Exercise and Antioxidants To ensure that RONS do not inevitably injure skeletal muscle cells, all aerobic organisms have developed efficient antioxidant systems to regulate free radical production and prevent their potentially deleterious effects [16]. These protective systems include antioxidant vitamins (C and E), glutathione, thiols and antioxidant enzymes [29]. The primary contributors are the enzymes superoxide dismutase (SOD), catalase and the glutathione peroxidase [30]. Each of these antioxidants plays a unique role in the cell and complements each other functionally. In general, the mitochondria-rich, slow-twitch oxidative (type I) fibers have a higher antioxidant capacity compared with fast-twitch glycolytic (type II) fibers [16, 31]. The term antioxidant has been defined as any substrate that, when present in low concentrations compared with those of an oxidizable substrate, significantly delays or prevents oxidation of that substrate [32]. Broadly speaking, the possible mechanism by which antioxidants may protect against oxygen toxicity are (a) prevention of ROS formation; (b) interception of ROS attack by scavenging the reactive metabolites and converting them to less reactive molecules and/or by enhancing the resistance of sensitive biological targets to ROS attack; (c) avoiding the transformation of less reactive ROS to more deleterious forms; (d) facilitating the repair of damage caused by ROS and triggering the expression of genes that encode antioxidant proteins; (e) providing a favorable environment for the effective functioning of other antioxidants [30]. One bout of exhaustive exercise is known to increase the activities of antioxidant enzymes, including SOD, catalase and GPX in skeletal muscle, heart and liver [31]. The threshold and magnitude of activation appear different among enzymes and tissues [33]. Consistent with the effect of acute exercise, chronic training also induces muscle antioxidant enzymes, among which the ­mitochondrial fraction of

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manganese-containing MnSOD and selenium-containing GPX show a greater induction [33]. Training effects on catalase and Cu, Zn-containing SOD in the cytosol are less prominent and there are conflicting reports. In general, training does not influence heart or liver antioxidant enzyme systems as much as in skeletal muscle, but vigorous training has been shown to increase SOD in the left ventricular tissues, and the extent of induction depends on training intensity [34]. The ­differential effect of training on various antioxidant enzymes may reflect the specific cellular locations where ROS are produced as well as the basal antioxidant capacity in various tissues. Skeletal muscle has one of the lowest antioxidant enzyme levels in the body. The large increase in ROS during exhaustive exercise is the likely trigger of antioxidant enzyme synthesis. As described in the next section, ROS produced in exercise act as signals that regulate molecular events important in muscle functional adaptations to exercise. Because these signals result in an upregulation of powerful antioxidant enzymes, moderate exercise itself can be considered as an antioxidant [14]. The practical consequence is that the recommendation of taking antioxidant supplements before moderate exercise should be revised as they may prevent useful adaptations induced by exercise [35]. In 1993, Reid and coworkers [36] showed that in unfatigued skeletal muscle, ROS have a positive effect on excitation–contraction coupling and are obligatory for optimal contractile function. Specifically, they demonstrated that addition of the antioxidant enzymes (i.e., catalase and SOD) resulted in a diminished in vitro muscle contractile force in unfatigued muscle. The contractile losses during catalase exposure were reverted by the addition of H2O2 in a dosedependent manner [36]. However, a massive increase in H2O2 concentration results in a sharp decrease in force output [33]. Further, the addition of strong synthetic antioxidants such as dithiothreitol and DMSO [37, 38] to the media of skeletal muscle contraction results in depressed force production. These data have been confirmed in other studies where the introduction of a ROS-generating system (XO and hypoxanthine) resulted in an increase in low-frequency contractility of the unfatigued diaphragm. The exact mechanism involved in this process is not completely clear. We have found that inhibition of XO with oxypurinol significantly decreased superoxide anions in skeletal muscle following a protocol of nondamaging isometric contractions. Moreover oxypurinol treatment decreased the maximum tetanic force produced by both extensor digitorum longus and soleus muscles. Thus this study indicates that XO activity contributes to the increased superoxide anion detected within the extracellular space of skeletal muscles during nondamaging contractile activity, and that XO-derived superoxide anion or derivatives of this radical have a positive effect on muscle force generation during isometric contractions of mouse skeletal muscles [39]. We recently found that supplementation with high doses of vitamin C seriously decreases improvement in running capacity associated with training. The adverse effects of vitamin C possibly resulted from its capacity to reduce the exerciseinduced expression of key transcription factors involved in mitochondrial biogenesis and in the antioxidant defense [35]. These results have been confirmed and extended to the healthy effect of exercise (and not only performance) by Michael Ristow and colleagues. They have demonstrated that exercise-induced oxidative

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stress ameliorates insulin resistance and causes an adaptive response, promoting endogenous antioxidant defense capacity. Supplementation with antioxidants ­(vitamins C and E) precludes these health promoting effects of exercise in humans [40]. Elsewhere, in humans involved in an endurance training program, acute ­supplementation of vitamins C (1 g) and E (600 I.U.) seemed to prevent exerciseinduced vasodilation [41], which, in addition to causing acute hypertension, could hypothetically also blunt the blood flow-induced stimulus for angiogenesis.

3 Redox Control of Muscle Adaptation to Exercise The idea of the deleterious effects of free radicals has been firmly entrenched in the minds of scientists for the past 30 years [14]. However, as mentioned in the previous section, there is now an appreciation that the RONS generated during muscle contraction play a physiological role in the adaptation to exercise [42]. Free ­radicals produced in skeletal muscle appear to be involved in a number of crucial physiological processes, including the regulation of vascular tone [43], excitation– contraction coupling [44], mitochondrial biogenesis [35, 45] and signal transduction [46]. RONS can interact with redox-sensitive transcription factors [42], leading to increased expression of the antioxidant enzymes such as MnSOD, cytoprotective proteins such as heat shock proteins [47], transcription factors involved in mitochondrial biogenesis [35] and other enzymes involved in muscle adaptation to exercise (i.e., iNOS and eNOS) [26]. RONS and other inducing agents can influence the activity of transcription ­factor binding in several ways (a) activating kinases so as to trigger a signaling cascade through sequential enzyme phosphorylation; (b) modulating phosphatases: some serine/threonine phosphatases and all protein tyrosine phosphatases contain a highly conserved sequence in their catalytic domain with a critical cysteine residue sensitive to redox status [48]; and (c) controlling the synthesis and degradation of transcription factors thereby determining their steady-state concentration and/or their ability to form homodimers or heterodimers [49]. The most thoroughly studied factors regulated by ROS are the nuclear factor (NF)kB, mitogen-activated protein kinase (MAPK) and, more recently, the peroxisome proliferator-activated receptor-g coactivator-1a (PGC-1a) [45, 50]. NF-kB is a dimeric transcription factor composed of members of the Rel family [51]. When activated, NF-kB migrates to the nucleus and binds to specific binding sites in the promoter sequences of many genes. NF-kB is activated by a variety of external stimulants, such as H2O2, proinflammatory cytokines and ionizing irradiation, among others. The best-known proteins and enzymes that require NF-kB binding to activate transcription are MnSOD, cyclooxygenase (COX)-2, iNOS, g-glutamylcysteine synthetase (GCS), vascular cell adhesion molecule-1 (VCAM-1), and several cytokines. These genes are involved in a wide variety of biological functions such as antioxidant, inflammation, immunity, and antiapoptosis [52]. Hollander et al. [53] first reported that an acute bout of treadmill running activated MnSOD gene expression in rat skeletal muscle, due to enhanced NF-kB binding in

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muscle nuclear extracts after exercise. In 2004, Ji et al. [42] studied the effects of an acute bout of physical exercise on the NF-kB signaling pathway in rat skeletal muscle. Highest levels of NF-kB binding were observed at two hours post-exercise. We found that the NF-kB signaling pathway can be activated in a redox-sensitive manner during muscular contraction, presumably due to increased oxidant production [42]. From this study, we concluded that ROS initiate a cascade of intracellular events that may be the overture to elevated gene expression of MnSOD, as reported earlier [54]. Activation by ROS appears to involve oxidation of key cysteine residues in the upstream activators of NF-kB and in many situations is prevented by antioxidants or reducing agents [55]. A further level of redox control is also evident within the nucleus, where binding of the NF-kB to DNA requires a reducing environment [56]. The primary function of the MAPK pathway is to modulate growth, metabolism, differentiation, transcription, translation, and remodeling. It is noteworthy that several important adaptations in skeletal muscle, such as mitochondrial biogenesis, hypertrophy, and fiber transformation are regulated primarily by MAPK signaling and they play an important role in determining the overall cellular oxidant– antioxidant homeostasis [57]. Goodyear et al. [58] first reported that key enzymes in the MAPK pathway such as JNK, ERK1/2 and p38 were activated in rat skeletal muscle after an acute bout of treadmill running. This research group soon after showed that ERK1/2 were phosphorylated after bicycle exercise in human muscle, along with activation of MEK1 and Raf-1 (MEKK), as well as downstream substrate p90 ribosomal S6 kinase [59]. Since then, numerous studies have shown that MAPK pathways can be activated by contractile activity in skeletal muscle [60]. The signals triggering the MAPK activation have been attributed to a variety of physiological stimuli associated with exercise including hormones, calcium ion, neural activity, mechanical force and RONS generation. In 2005 [26] we reported that ROS generated in exercise activate p38 and ERK1/2, which in turn activate NF-kB, which results in an increased expression of important enzymes associated with cell defense (MnSOD) and adaptation to exercise (eNOS and iNOS). However, at present, the physiological consequences of individual MAPK signaling pathways are still not totally predictable. Because of the widespread implications of RONS in almost all important biological functions, it is difficult to define all the pathways and gene targets that are affected by redox signaling during exercise. The following are some of the most relevant products that play a critical role in homeostatic regulation of muscle oxidative–antioxidant balance under various physiological and pathological conditions: MnSOD [53], selenium-dependent GPX [31], iNOS [26], g-glutamylcysteine synthetase and thioredoxin [61] and uncoupling protein 3 [62]. Mitochondrial biogenesis is critical for the normal function of cells. It is well known that mitochondria are produced and eventually, after normal functioning, they are degraded. Thus, the actual level of mitochondria in cells is dependent both on the synthesis and the degradation [63]. Mitochondrial synthesis is stimulated by the PGC1a–NRF1–Tfam pathway. PGC-1a is the first stimulator of mitochondrial biogenesis. NRF1 is an intermediate transcription factor that stimulates the synthesis of Tfam, which is a final effector activating the duplication of mitochondrial DNA

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molecules. This pathway is activated during exercise in skeletal muscle. Recent data indicates that contraction-activated PGC-1a signaling pathways in skeletal muscle are redox sensitive [35] and that nonmitochondrial ROS play an important role in stimulating mitochondrial biogenesis [45].

4 Hypoxia, Exercise and Oxidative Stress Cellular hypoxia is characterized by an increased levels of reduced equivalents due to insufficient availability of O2 to be reduced in the mitochondrial electron transport chain [64]. In both animal models’ [65] and in humans exposed to extreme altitude (3,000–8,500  m), it has been demonstrated the oxidative stress through increased lipid, protein and DNA oxidation in skeletal muscle [66]. The activity of enzymes such as cyclooxygenase, NAD(P)H oxidase and xanthine oxidase (as in exercise) are crucial for the ROS generation under hypoxic conditions [64]. In terms of exercise without exposure to altitude, the intermittent periods of anoxia–reoxygenation of muscle (ischemia–reperfusion) can be considered a frequent source of cellular hypoxia in humans. Bailey et al. demonstrated the importance of the partial pressure of O2 in the muscle on the generation of ROS, alkoxy and peroxy radicals when a group of subjects underwent a knee extension exercise with incremental intensity [67]. Taking into account this data and the previous sections of the chapter, we can consider that both hypoxic protocols and exercise are good models to study oxidative stress. Its combination (hypoxia and exercise) is a regular practice in sports. For the past few decades now, athletes have trained at high altitudes to increase their performances [68]. Among the various training regimens, that of living at high altitudes while training at sea-level, the socalled “living high, training low” (LHTL) protocol, has become an important strategy to increase performance [69]. Several studies using this strategy have been developed in humans. However, the results are confusing. Pialoux et al. observed that 13 days of LHTL did not significantly affect the prooxidant/antioxidant balance in elite swimmers [70], while a significant increase in oxidative stress was reported after 18 days of LHTL in elite runners [71]. In animal models, an increase in the level of oxidative protein damage [72] and lipid peroxidation has been shown at different altitudes [73]. Radak et  al. showed that the activity of antioxidant enzymes such as Mn-SOD, in both white and red types of skeletal muscle, increased after exercise training at a high altitude [74].

5 Role of RONS in Muscle Health/Disease Skeletal muscle is critical for human health, physical activity and function, as well as for athletic performance [75]. Improvement of muscle mass is of great interest to many populations. Athletes and body builders desire to increase muscle mass and

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strength for competitive reasons. Others, such as the elderly, astronauts, or those suffering from various muscle disorders or recovering from restricted activity or illness, need to increase muscle mass to increase functionality and quality of life. In these more stressed populations, muscle serves as a metabolic reserve to ensure glucose homoeostasis and to provide amino acid precursors to maintain protein synthesis in vital tissues and organs in the absence of amino acid absorption from the gut and by providing hepatic gluconeogenic precursors [76]. Striated muscles have a striking ability to adapt to variations in activity and working demand. Plasticity is the most appropriate wording to describe the ability of muscle to adapt [77]. While it is well known that an acute bout of exercise can result in oxidative stress as indicated by altered muscle and blood glutathione levels and an increase in both protein and DNA oxidation [78], long-term consequences of physical inactivity in a number of physiological and pathological processes in muscle is less known [79]. An imbalance of RONS generation and adequate antioxidant signaling has been found in numerous muscle diseases due to either genetic myopathy [80] or other etiological factors such as neurological and neuromuscular junction disorders [81], inflammatory myopathies [82], aging [83], or simply disuse, improper use or immobilization [84]. Several studies have demonstrated that some of these muscle pathologies could be retarded via antioxidants [85].

6 Conclusion Exercise, both acute and chronic, is an excellent model to study the physiological adaptations to oxidative stress. The general conclusion of our studies is that exhaustion generates free radicals causing oxidative stress. However, moderate exercise, and in particular exercise training, generates small amounts of free radicals that act as signals to upregulate the expression of antioxidant genes, and in this regard exercise can be considered to be an antioxidant.

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Part VI

In Vitro/Tissue Culture

Models of Mitochondrial Oxidative Stress Enrique Cadenas and Alberto Boveris

Abstract  Mitochondria are sources of H2O2 under aerobic and physiological ­conditions; the mitochondrial H2O2 generation is conceived as a consequence of the univalent and nonenzymatic reduction of oxygen to superoxide anion followed by its disproportionation to H2O2. Recognized sites for the univalent reduction of ­oxygen to superoxide are the autoxidation of ubisemiquinone in complex I and complex III and probably the autoxidation of the flavin semiquinone in complex I. The formation of H2O2 by mitochondria acquires further significance when ­considering that it reports a high mitochondrial energy charge by its diffusion to the cytosol and that it may be involved in domain-specific signaling pathways or ­signaling in localized subcellular areas. H2O2 is considered a major player in the redox regulation of cell signaling by modulating the activity of glutathione-, ­thioredoxin-, and peroxiredoxin-supported systems. Although H2O2 is highly ­diffusible across biological membranes, significant gradients are established in the cells and the involvement of mitochondrial H2O2 in the regulation of specific signaling pathways requires careful consideration of its sources, of its removal by specific enzymic systems, and of the mechanism by which H2O2 modifies the signaling pathways. Keywords  Coenzyme Q • Complex I • Hydrogen peroxide • Mitochondria • Oxidative stress • Redox signaling • ROS • Superoxide anion

E. Cadenas (*) Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90089, USA e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_27, © Springer Science+Business Media, LLC 2011

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1 Mitochondrial Production of Superoxide Anion and Hydrogen Peroxide In the early 1970s, several groups established that mitochondria are sources of H2O2 under aerobic and physiological conditions [1–4] and subsequent work showed that superoxide anion (O2•−) was the stoichiometric precursor of hydrogen peroxide (H2O2) in these organelles [5–7]. Loschen et al. [5] first detected O2•− production by mitochondrial membranes and this discovery was followed by other groups that emphasized O2•− as the main, if not sole, precursor of H2O2 production in mitochondria (see [8]), a concept strengthened by the discovery of the specific Mn-superoxide dismutase located in the mitochondrial matrix [9]. In other words, mitochondrial H2O2 production was conceived as a consequence of the univalent and nonenzymatic reduction of O2 to O2•− followed by its disproportionation to H2O2 (2O2•− +  2H+ → H2O2 + O2). The univalent reduction of O2 to O2•− occurs primarily upon autooxidation of ubisemiquinone (UQH•) in complex III (ubiquinol-cytochrome c reductase) and in complex I (NADH-ubiquinone reductase) and by auto-oxidation of the flavin semiquinone (FMNH•) of complex I [10]. An exception to the O2•−-mediated H2O2 formation in mitochondria is the monoamine oxidase (MAO) activity – attached to the outer mitochondrial membrane – that catalyzes the oxidative deamination of biogenic amines in a reaction that entails a two-electron reduction of O2 to H2O2 [11]. Recently, type A monoamine oxidase has been reported activating and regulating cell death processing with an involvement of monoamine oxidase in the apoptotic signal triggered by ­mitochondria and with implications for neurodegeneration [12].

2 Measurement of Mitochondrial Superoxide and Hydrogen Peroxide Production The primary formation of the products of the univalent and bivalent reduction of O2 (O2•−, H2O2), a concept from L. Michaelis (see [13]), during aerobic metabolism constitutes the keystone for the concepts of oxidative stress and oxidative damage. Superoxide radical is not a physiological oxidant; it should be properly recognized as a pro-oxidant, considering both its dismutation reaction to yield H2O2 and its reaction with ferritin Fe3+ to produce and release Fe2+. Both H2O2 and Fe2+ are the reactants of the Fenton reaction (H2O2 + Fe2+ → Fe3+ + HO− + HO•), which still constitutes the best explanation for the rate-limiting step of the biological phenomenon of “oxygen toxicity” [13]. The group of the three chemical species – O2•−, H2O2, and HO• (hydroxyl radical) – is frequently called “reactive oxygen species” (ROS) for an easy explanation of the biological effects. However, the three chemical species have different chemical properties, very different cellular steady-state concentrations, and different enzymic and nonenzymic reactions that utilize or remove these pro-oxidants and oxidants. Production rates and utilization rates, both considered

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numerically equal in the logic of stable biological systems, identify the two terms of the equation that establish the concept of mitochondrial and cellular steady-state concentrations. This is indeed an important concept, for which it is expected that in disease states associated with oxidative stress and damage an increased cellular steady state of oxidants should be confirmed quantitatively. Hence, consideration of the rates of generation and removal of oxidants in the calculation of cellular steady-state levels reflects subtle changes in these processes that are important for the description of mitochondrial or cellular oxidative stress. The relevant cellular sources of O2•− and H2O2, the determination of their cellular and physiological production rates, and the calculation of their cellular steady-state concentration have been considered by the steady-state approach: the premise is that in the steady state, the rate of production of an oxidant (for example, H2O2) equals the rate of its utilization (+d[H2O2]/dt = −d[H2O2]/dt) by specific enzymes or controlled reactions with cellular targets [14]. This approach allows the calculation of the steady-state levels, for instance of O2•− and H2O2, in different cellular ­compartments derived from the rates of production of O2•− and H2O2 in those ­compartments. It follows that the quantitative determinations of both, the rates of generation of the primary cellular oxidants O2•− and H2O2 and the rates of utilization of the same chemical species are critical for the establishment of the condition of mitochondrial or cellular oxidative stress. Rates of O2•− or H2O2 production by ­submitochondrial particles and isolated mitochondria are given in Table 1, where the rates have been recalculated for the physiological condition of 37°C and 22 mM O2. The physiological rates for the organs in situ can be calculated from the mitochondrial contents of 32 and 54  mg of mitochondrial protein/g of organ for liver and heart, respectively. The rates of production of O2•− and H2O2 given in Table 1 yield, after consideration of the enzymic and nonenzymic reactions that utilize them, the steady state concentrations of both O2•− and H2O2 ([O2•−]ss and [H2O2]ss) in mitochondria . Similar considerations yield the [O2•−]ss and [H2O2]ss in the cytosol (Table 2).

Table 1  Rates of production of O2•− and H2O2 in mitochondrial inner membranes Mitochondrial source Rat heart submitochondrial particles with antimycin A Rat heart mitochondria State 4 State 3 Physiological conditions (with •NO)a

DO2•−/Dt (nmol/min/mg protein) 1.40 ± 0.05

DH2O2/Dt (nmol/min/mg protein) 0.87 ± 0.04

0.29 ± 0.03 0.05 ± 0.01 0.47 ± 0.03

0.19 ± 0.02 0.03 ± 0.01 0.31 ± 0.03

Rat liver mitochondria State 4 0.18 ± 0.01 0.12 ± 0.01 State 3 0.03 ± 0.01 0.03 ± 0.01 0.42 ± 0.03 0.26 ± 0.02 Physiological conditions (with •NO)a Data taken from Boveris and Cadenas [14] and recalculated for 37°C and 22 mM [O2] a At a state 4/state 3 mitochondria ratio of 2 and in the presence of 0.30 mM •NO [15]

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E. Cadenas and A. Boveris Table 2  Steady-state concentrations of reactive oxygen species in rat organs M Source, compartment −10 •− [O2 ]ss 1.50 × 10 Heart mitochondria 0.90 × 10−10 Liver mitochondria 2.50 × 10−11 Liver cytosol Heart mitochondria [H2O2]ss 0.57 × 10−8 0.48 × 10−8 Liver mitochondria 1 × 10−8 Liver cytosol 4.0 × 10−9 Liver peroxisomes Perfused liver, hepatocytes 2.7 × 10−8 Taken from Boveris and Cadenas [14] and recalculated for 37°C and 22 mM [O2]

Considering increased steady-state concentrations of reactive oxygen species as the logical condition for cellular oxidative stress, it is easily understood that an increased rate of production of O2•− or H2O2 associated with an adaptive response correlates with an increased level of the corresponding antioxidant enzyme, superoxide dismutase or catalase, a similar steady-state concentration will be set in the considered compartment. Quantification of H2O2 is usually done by peroxidase-based assays with different hydrogen donors, preferably highly fluorescent, such as scopoletin, p-hydroxyphenylacetic acid, and Amplex red. This approach is suitable for samples with H2O2 formation rates in the range of 1 mM/min. For instance, horseradish peroxidase (HRP) oxidizes the aforementioned fluorescent hydrogen donors (AH2) in the presence of H2O2: HRP + H2O2 → HRP–H2O2; HRP–H2O2 + AH2 → HRP + 2H2O + A. The fluorescent hydrogen donors (either fluorescent in their reduced or oxidized form) are suitable for measuring H2O2 generation by mitochondrial membranes, taking into account that they are almost unaffected by the high light scattering of the mitochondrial suspensions [16]. The quantitative determination of O2•− is conventionally carried out by the ­reduction of cytochrome c (or better, acetylated cytochrome c) in a superoxide ­dismutase-sensitive spectrophotometric assay [16]. The control addition of super­ oxide dismutase confers specificity to cytochrome c reduction by O2•− but underestimation can still occur due to the reoxidation of ferrocytochrome c (especially, when measuring O2•− in submitochondrial particles due to ferrocytochrome c oxidation by cytochrome oxidase). The oxidation of epinephrine by O2•− is not very specific due to the spontaneous epinephrine autoxidation to adrenochrome in an autocatalytic process [17], but because the auto-oxidation is highly pH-dependent it can be controlled and assayed at neutral pH (for instance, 7.2–7.4) with full enzymatic activity of the respiratory enzymes. It is important to recognize that the above-mentioned methods for measuring H2O2 or O2•− are quantitative and reliable approaches to determine the rates of their production in mitochondrial preparations. Murphy [18] has recently challenged the validity of the in vitro determinations of the rates of production of O2•− and H2O2 in order to approach the values under

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physiological conditions; O2 concentration, temperature, and organelle and ­ embrane modifications after cell fractionation were considered. Concerning the m usual determination of O2•− and H2O2 production rates in isolated mitochondria and in submitochondrial particles, it is true that the assays are carried out in reaction media at 180–220  mM O2, with O2 levels that are about 10-fold higher than the physiological tissue levels. Because the rates of these auto-oxidation reactions are first order with respect to O2, the corresponding rates for physiological conditions can be calculated. However, for the consideration of the situation of mitochondrial and cellular oxidative stress, the approach is completely valid, since (a) the production rates will be similar in experimental conditions with similar organelle damage due to the isolation procedure, and (b) temperature will equally affect production and utilization rates. There are other approaches to measure O2•− and H2O2 but with a lower degree of specificity. In the case of O2•− quantification is usually difficult or not possible; EPR in connection with spin probes, such as DMPO, has been frequently used for O2•− detection and the introduction of the spin probe DEPMPO conferred a better specificity to this approach [19, 20]. Hydroethidine oxidation is important for studies involving “redox imaging,” but quantification using this probe is difficult [21]. Likewise is the use of dichlorofluorescin (2¢-7¢-dichlorofluorescenin; DCFH) fluorescence to detect H2O2 in flow cytometry and in cell fluorescence approaches. A major concern with the use of DCFH is the lack of specificity for H2O2, because peroxidases and hydroperoxides, hypochlorite, and peroxynitrite readily oxidize DCFH. A thorough analysis of the cellular mechanisms of DCFH oxidation as a measurement of H2O2 formation or oxidative stress is available [22, 23].

3 Models of Mitochondrial Oxidative Stress It should be borne in mind that isolated mitochondria, and by extension mitochondria in its intracellular location, produce H2O2 at rates that depend primarily on the mitochondrial respiratory state: the rate of H2O2 production is about five times higher in state 4 respiration, when the respiratory chain carriers are mostly reduced, than in state 3 (in the presence of ADP), when respiratory carriers are largely oxidized [3, 8]. Thus, the formation of H2O2 by mitochondria acquires further significance when considering that it reports a high mitochondrial energy charge by its diffusion to the cytosol [15] and that it may be involved in domain-specific signaling pathways or signaling in localized subcellular areas. The original observations using respiratory chain inhibitors and uncouplers of oxidative phosphorylation indicated that the main generator of H2O2 in mitochondria was an electron carrier of the electron-transport chain acting between the rotenone- and the antimycin-sensitive sites [3, 8]. A comprehensive review by Murphy points out the difficulties in the determination of the rates of O2•− and H2O2 generation in  vivo and under physiological conditions and recognizes four main determining factors: the ratios NADH/NAD and UQH2/UQ, the local mitochondrial [O2], and the Dy of the inner membrane [18].

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3.1 Formation of O2•−/H2O2 by Complex I Isolated complex I in the presence of NADH is an effective generator of O2•− [10]. The phenomenon is also recognized in isolated mitochondria with malate–glutamate as substrate and in the presence of rotenone; the rates of H2O2 production in state 4 are increased by rotenone [17, 24]. Two sites for the univalent reduction of O2 in complex I have been proposed: the reduced flavin mononucleotide semiquinone (FMNH•) and the ubisemiquinone (UQH•) at the quinone-binding site, the first indicated by studies in conditions of reversed electron transport [17]. Experimental models supporting the formation of O2•− by complex I entail (a) inhibition of electron transfer by rotenone, thereby increasing the levels of FMNH2 and FMNH· with the consequent increased O2•− formation, and (b) rotenone-sensitive reverse electrontransfer models involving entry of reducing equivalents at the coenzyme Q-binding site of complex I [17, 24, 25]. The recent scientific literature shows a long list of pathological situations (ischemia–reperfusion, aging, endotoxic shock, neurodegenerative diseases, etc.) where an inhibition of complex I activity is observed in isolated mitochondria and in submitochondrial preparations. In these reports it is frequently considered that the inhibition of electron transfer causes an over-reduction in the redox active groups of the complex with an increased production of O2•− and of H2O2 by FMNH• auto-oxidation; the phenomenon was recognized by experimental determination of O2•− and H2O2 in rat liver ischemia–reperfusion and choline deficiency [26, 27]. The concept of H2O2 index, introduced by Carreras et  al. [28] to describe adaptive changes during rat liver development, constitutes a useful approach to produce evidence indicating complex I-mediated oxidative stress in vivo. The assay would require the comparison of diseased experimental animals with normal controls. In both groups, the rates of O2•− or H2O2 production are determined in the absence and in the presence of rotenone. The ratio of the rates (no additions)/(with rotenone) is established and compared for both groups. Usually, there is no significant difference in the rates with rotenone; both groups have fully reduced FMNH2 with a maximal rate of auto-oxidation. The ratio (no additions)/(rotenone) would be increased in cases of oxidized or nitrated complex I. Noncovalent hydrophobic bonds are important in keeping together the polypeptide structure of complex I; low concentrations of detergents, natural and synthetic steroids [29], or hydrophobic pesticides [30] are effective in disrupting intracomplex I polypeptide hydrophobic bonds and in inhibiting complex I electron transfer. Mitochondrial complex I is also particularly sensitive, in terms of inhibition and inactivation by oxidants, oxygen free radicals and reactive nitrogen species. This special characteristic is frequently referred as the “complex I syndrome,” with diminished complex I activity and with reduced state 3 mitochondrial respiration with malate–glutamate as substrate, as it has been observed in Parkinson’s disease and in other neurodegenerative diseases [31–34], as well as in brain aging [35] and in ischemia–reperfusion [26]. The molecular mechanism involved in the inactivation of complex I is likely accounted for by the sum of ONOO− mediated reactions, reactions with free radical

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i­ntermediates of the lipid peroxidation process (of the type alkyl and peroxyl) and amine–aldehyde adduction reactions. It is understood that the three processes mentioned above alter the native noncovalent polypeptide interactions and synergistically promote protein damage and inactivation by shifting noncovalent bonding to covalent cross-linking in complex I [36].

3.2 Inhibition of Complex III: The Outer and Inner UQ Pools Antimycin A is the classical inhibitor of complex III and a drastic model for mitochondrial oxidative stress associated with blocked electron transfer and ATP production. Antimycin A supplementation by inhibiting electron transfer between cytochromes b and c, purports high levels of UQH2 and UQH· and high rates of UQH· autoxidation and of O2•− production [6, 14]. In experiments carried out with mitoplasts, devoid of the outer mitochondrial membrane, neither rotenone nor thenoyltrifluoroacetone (TTFA) generated an EPR signal attributable to O2•− unless antimycin A was also present, thus suggesting that under these experimental conditions neither complex I nor complex II was responsible for O2•− formation [37]. The use of either antimycin A or myxothiazol [38] helped distinguish the generation of O2•− at the outer ubiquinone pool (UQO) from that at the inner ubiquinone pool (UQI): myxothiazol binds at the UQO site of the UQ pool, thereby blocking electron transfer from ubiquinol UQOH2 to the iron–sulfur clusters and to cytochrome b1; antimycin binds to the UQI site and blocks electron transfer from cytochrome bH to UQI [39] (Fig. 1). This experimental design [37] confirms the vectorial release of O2•− towards the cytosolic side of the inner mitochondrial membrane in agreement with a functional relationship of the occurrence of Cu,Zn-superoxide dismutase in the intermembrane space. Hence, part of the H2O2 diffusing from mitochondria to cytosol originates in the intermembrane space, following to the O2•− disproportionation by Cu,Zn-superoxide dismutase in this compartment [40–42] and part in the mitochondrial matrix, following O2•− disproportionation by Mn-SOD. Hence, in the model of mitochondrial oxidative stress triggered by antimycin A, complex III releases O2•− to both sides of the inner mitochondrial membrane by engaging the inner- and outer-ubiquinone pools [37, 43]. Another model of mitochondrial oxidative stress entailing redox changes at complex III involves the inhibition of electron transfer at the cytochromes bc1 segment by nitric oxide (•NO) [44]. •NO has two targets in the mitochondrial respiratory chain: complex IV and complex III. First, it regulates mitochondrial respiration upon reversible binding to cytochrome oxidase (complex IV) with half-maximal effect at ~0.1 mM •NO [45–50]. Second, higher concentrations of •NO inhibit succinate- and NADH-cytochrome c reductase activities (half-maximal effect at ~0.30  mM •NO) [44] and under these conditions, submitochondrial particles and isolated mitochondria supplemented with succinate and treated with •NO show markedly increased O2•− and H2O2 productions, with rates that are about one-half of those observed in the presence of antimycin A. Nitric oxide addition elicits a partial

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reduction of cytochrome b whereas cytochromes c and aa3 remain oxidized – in other words, an effect similar to that of antimycin A but produced by the physiological metabolite •NO at concentrations that are in the range of those calculated for the mitochondrial matrix (0.22–0.38  mM) [15]. Interestingly, the •NO-dependent production of O2•− at complex III was able to remove – in a feedback mechanism – the •NO inhibition of cytochrome oxidase [44]. In connection with the vectorial release of O2•− into the intermembrane space (Fig.  1), mitochondria should also be viewed as cellular sources of O2•− released into cytosol via a voltage-dependent anion channel (VDAC) [51]: minute amounts of O2•−, detected by quantitative spin-trapping EPR, are measured in intact mitochondria supplemented with complex I or II substrates; inhibitors of VDAC (4¢-diisothiocyano-2,2¢-disulfonic acid stilbene; DIDS) and dextran sulfate (in a voltage-dependent manner) inhibit O2•− release from mitochondria (~50%), thus suggesting that a large portion of O2•− exit mitochondria via these channels (Fig. 1). Skulachev [52] described a high proton motive force in state 4 associated with an increased O2•− formation; subsequent work [53] determined a threshold Dy value, exceeding the state 3 Dy level, above which the increase rate in mitochondrial H2O2 production occurred. Later experiments carried out with brain mitochondria of synaptosomal and nonsynaptosomal origin oxidizing physiological NADHdependent (complex I) substrates determined that H2O2 production was regulated by Dy and by the NAD(P)H redox state over ranges that are consistent with those

intermembrane space

UQI + bh2+ antimycin UQI.– + bh3+

UQI.–

Cu,Zn-SOD O2.–

inner UQI membrane UQO

O2

UQO.–

myxothiazol matrix

O2

UQO.– + [Fe2S2]red

O2.–

UQOH+ + [Fe2S2]ox

Mn-SOD

O 2.– VDAC

outer membrane

O 2.– QO.–

ψ inner membrane

Fig.  1  Scheme showing mitochondrial O2•− production and release into the matrix and the intermembrane space in the normal function of the Q cycle and vectorial release of O2•− into cytosol via VDAC

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Fig. 2  Solid state model showing the structural interactions between complexes I, III, and IV and mtNOS and views from the matrix side and from the intermembrane space to indicate the asymmetry of the inner mitochondrial membrane in terms of constitutive proteins and redox groups

that exist at different levels of cellular energy demand [54]. A similar dependence of H2O2 and •NO production on Dy was reported for mitochondria isolated from rat heart, liver, kidney, and brain [15]. In this way, complex III, UQ-binding protein, and mtNOS form a group of membrane proteins (Fig. 2) that are regulated in their activity, in terms of O2•− and •NO formation, by the electrical potential (Dy) of the inner mitochondrial membrane. As mentioned before, the concept of “H2O2 index” [28] provides a useful approach to describe complex III-mediated oxidative stress and damage. In this case, there are two possibilities (a) a •NO mediated regulation, as the index was originally described, and (b) a ONOO− mediated structural damage. In the first case, the H2O2 index-ratio is taken between the rate of H2O2 in isolated mitochondria (with arginine)/(with antimycin); the ratio indicates a regulation by an active mtNOS and by •NO. In the second case, the ratio (no additions)/(with antimycin) indicates structural damage with nitration and nitrosylation in complex III.

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3.3 Complex II Early reports indicated that succinate dehydrogenase (complex II) was much less effective than complex I or complex III in H2O2 generation [10], that submitochondrial particles devoid of succinate dehydrogenase still produced large amounts of H2O2 [55] and that in UQ-depleted and UQ-reconstituted submitochondrial particles, complex II activity was restored to maximal activity with low levels of added UQ, whereas H2O2 production increased up to high levels of added UQ and with rates that were lineal with the level of succinate-reducible UQ [56]. A detailed analysis of O2•− formation by complex II using specific inhibitors or RNA interference to subunit assembly revealed that inhibition of the SdhB subunit of complex II was associated with increased formation of O2•− [57]. Other reports identified the subunit SdhA as a source of O2•− [58]. The experimental model entails specific inhibition of complex II components: thenoyltrifluoroacetone (TTFA) inhibits complex II at the ubiquinol-binding site in SdhD and 3-nitropriopionic acid (3-NPA) inhibits complex II at the succinate binding site on SdhA [57]. Other studies concluded that in the presence of 3-NPA, mitochondria generate O2•− from a site between the ubiquinol pool and the succinate binding site on SdhA (sensitive to 3-NPA) [59]. Forman and Kennedy [60] reported O2•− formation from a dihydroorotate-dependent activity localized at the inner mitochondrial membrane. The original question was whether O2•− originated from autoxidation of the dihydroorotate dehydrogenase flavin or from autoxidation of the reduced ubiquinone pool. The analysis of the rates of O2•− production indicated that the second possibility was more likely [8]. Other dehydrogenases in the mitochondrial matrix or in the outer side of the inner mitochondrial membrane are also sources of O2•−, such as the dihydrolipoamide dehydrogenase subunit of a-ketoglutarate dehydrogenase [61, 62] and glyceraldehyde phosphate dehydrogenase [63], respectively. Concerning the whole series of reactions of autoxidation of the reduced forms of the components of the mitochondrial respiratory chain and of the flavin dehydrogenases located at the inner and outer mitochondrial membranes, all the reactions are spontaneous from a thermodynamic point of view, but only a pair of them, auto-oxidation of complex III and of complex I, are kinetically compatible with a significant role in the O2•− production in normal conditions.

4 Cellular H2O2: Mitochondrial and Nonmitochondrial Sources Signal transduction, cell development, cell proliferation, migration, apoptosis, and necrosis, among other processes, are considered to be regulated by the redox status of the cell, understanding that “redox status” is a complex biochemical nonequilibrium situation that is mainly reflected by the [H2O2]ss and the –SH/–SS– ratio in the

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glutathione pool, and of thiol pairs of redox active cysteines. H2O2 is considered a major player in establishing these redox regulations by the activity of the glutathione-, thioredoxin-, and peroxiredoxin-supported systems [64–66]. The involvement of H2O2 in specific signaling pathways requires the consideration of its sources, of its removal by specific enzymic systems and of the mechanism by which H2O2 modifies the signaling pathways [66, 67]. Although H2O2 is highly diffusible across biological membranes, significant gradients are established in the cells. For instance, quantitative measurement of H2O2 production by titration of the catalase– H2O2 intermediate steady-state level in rat liver revealed a basal [H2O2]ss of ~10−9 M and 5.4 × 10−8 M (a ~50-times gradient) in liver slices by measuring H2O2 after diffusional equilibrium was reached [68]. Using yeast cytochrome c peroxidase, a sensitive and specific spectrophotometric indicator of H2O2, Boveris et  al. [69] provided a value of 90 nmol H2O2 per minute per gram of liver as an approximation for the overall rates of H2O2 formation by hepatocytes. Cytosolic [H2O2]ss can be estimated from the rates of H2O2 generation by subcellular sources and of its removal by catalase and glutathione peroxidase. Based on this, the pecking order of cellular sources of H2O2 was described as endoplasmic reticulum > mitochondria > peroxisomes >> cytosolic enzymes for rat liver. However, mitochondria are usually referred to as the “major cellular sources of reactive oxygen species,” with the term “reactive oxygen species” referring almost solely to H2O2, for being the mitochondrial release of O2•− into cytosol (via VDAC) much smaller [51]. Moreover, the previous consideration refers to liver. For other organs, such as heart, kidney, and whole brain (also considering cerebral cortex, striatum and hippocampus) that do not have a high mass of either endoplasmic reticulum or peroxisomes, the pecking order is clearly mitochondria >> peroxisomes > endoplasmic reticulum > cytosolic enzymes. This statement is correct for almost any organ or type of cell (excepting hepatocytes, as mentioned) insofar as cellular H2O2 production is a consequence of aerobic mitochondrial respiration. The steady-state levels of H2O2 – estimated for the mitochondrial and cytosolic compartments [14] – are based on the equal rates of production and utilization by the removal systems. The mitochondrial thioredoxin system and, more importantly, peroxiredoxin III, which is of mitochondrial compartmentalization [70, 71], were not considered in these calculations. The system function is still more complex by the translocation of sulforedoxin to mitochondria and its function in reducing the hyperoxidized peroxiredoxin III [72]. Phagocytic cells (neutrophils) generate large amounts of H2O2 upon stimulation of its plasma membrane NADPH-oxidase (Nox), in a process centered at the activation of the gp91phox catalytic subunit. In nonphagocytic cells, including spermatozoa, a gp91phox homolog also generates O2•−/H2O2, species involved not only in signal transduction but also in the modification of the extracellullar matrix [73]. This is the model that Rhee [66] uses to dissect H2O2 production, protection, and signaling. The significance of the identification of the cellular source of H2O2 and its further involvement in cell signaling is based on concepts advanced by Terada [74] that cells spatially restrict oxidant production to allow microdomain-specific signaling. For example, Nox-generated H2O2 affects a number of different pathways involved in cell migration and adhesion, immune cell function, and proliferation. Conversely,

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mitochondrial H2O2 appears to be involved in other “domain-specific signaling” regulating cellular energetics through the modulation of AMP-activated protein kinase (AMPK) [75], as also discussed in [74], and of JNK. In the former instance, the activator of AMPK seems to be an O2•− and NO-derived species, likely peroxynitrite (ONOO−). Concerning the latter, the cellular oxidative stress model involving mitochondrial H2O2 production consists of pyruvate-supplemented HeLa cells [76] with increased mitochondrial metabolic flow, O2 consumption and H2O2 formation and activation of JNK. The role of mitochondrial oxidants in this experimental model was strengthened by the use of antimycin A: the much higher H2O2 levels under these experimental conditions were associated with activation of JNK and its downstream target, probably ribosomal S6 kinase [76]. The translocation of pJNK to mitochondrion was observed under conditions of oxidative stress [77] and as a function of age [78], but the activation (bisphosphorylation) of JNK was not attributable to mitochondrial H2O2. The translocation of JNK to mitochondria triggers a phosphorylation cascade that results in inactivation (by phosphorylation) of pyruvate dehydrogenase in a pyruvate dehydrogenase kinase-dependent manner [78]. Sustained JNK activation appears to be the result of the H2O2-mediated inhibition of MAPK phosphatases and to be part of a general mechanism in redox ­signaling that involves reactive cysteines as redox targets. These reactive cysteines include those with a low pKa of the sulfhydryl group that increases its susceptibility to oxidation [79] and, depending on the type of reversible modification, the reactive cysteines signal provides different functional responses [80]. The association of glutathione S-transferase-Pi (GSTp) to the JNK pathway is disrupted in oxidative stress conditions leading to JNK activation [81]. However, these redox mechanisms involved in the regulation of signal transduction do not specifically involve mitochondrial H2O2 and the premise that “mitochondria are the major cellular source of reactive oxygen species” does not support all oxidative events in protein-specific amino acids (reactive cysteines). The concept of domain-specific signaling is useful but it needs to be supported by an experimental approach in which cellular oxidative stress is a consequence of quantifiable mitochondrial H2O2 production. Cellular quantification of H2O2 is essential. Advantage can be taken from the diffusion of H2O2 from isolated cells, tissue cubes and slices to support its function as a signaling molecule. In a pioneering work, Antunes et al. [82–84], using glucose oxidase to produce H2O2 at controlled rates, established well-determined and quantified steady-state levels of H2O2 in Jurkat cells. In such studies, three levels or phases were defined for H2O2-induced apoptosis with reference to cytosolic [H2O2]ss: values below 0.7  mM elicited no effects, values of ~0.7–3 mM induced apoptosis, and >3 mM yielded no additional apoptosis but a gradual shift towards necrosis. These observations provided solid support for the quantitative relationship between [H2O2]ss and cell survival and death pathways. The carefully regulated and determined steady state levels of H2O2 in the mentioned experiments contrasts with the high levels of added H2O2 (in the range of 10–100 mM) that are frequently used in signaling experiments with the associated risk of nonphysiological free radicals reactions. H2O2 has been also considered as an intercellular messenger; it has been reported that in HeLa cells treated with

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tumor necrosis factor (TNF), mitochondria-generated H2O2 in apoptotic cells serves as an intercellular messenger; the processing of the H2O2 signal by the recipient cells entails an increased mitochondrial production of H2O2. The authors viewed this phenomenon as an expansion of the apoptotic region around a damaged part of the tissue [85]. Hence, despite a huge number of reports on H2O2 as a signal, the source of H2O2 for intracellular signaling remains intangible. It appears that well-defined experimental models are needed to allow identification of mitochondrial H2O2 contribution to cell signaling involving the modulation of the mitochondrial respiratory chain and of major mitochondrial metabolic pathways. For example, it was recently shown that respiring mitochondria are the primary source of H2O2 for dynamic neuronal signaling in a model that required coapplication of rotenone and succinate. Interestingly, in this model, other sources of H2O2, such as monoamine oxidase and Nox did not have an effect on dopamine release [86]. Another cell model involves inactivation of cell survival pathways to the challenge of ONOO− by mitochondrial H2O2. ONOO− triggers a survival pathway in U937 cells that depends on extracellullar signal-regulated kinase 1 and 2 (ERK1/2), on mitochondrial H2O2 formation, and on the activation of a phosphotyrosine phosphatase leading to inhibition of the ERK1/2 pathway [87, 88]. The formation of O2•− (and H2O2) by mitochondria exposed to ONOO− can be understood as an oxidation of the mitochondrial ubiquinol pool by ONOO− and by the intermediate peroxynitrous acid (ONOOH; [HO•…NO2•]) (UQH− + [HO•…NO2•] → HO− + NO2• + UQ•− + H+) followed by autoxidation of UQH• (UQ•− + O2 → UQ + O2•−) [89]. It may be surmised that experimental models entailing an increased expression and activity of mtNOS, nNOS or eNOS would be associated with an enhanced H2O2 formation by mitochondria, explained on the basis of NO production in the organelles or in •NO diffusion to the organelles, which are the cellular focus of O2•− formation and thereby favorable sites for ONOO− generation (•NO + O2•− → ONOO−) [90]. Conversely, the role of mitochondrial H2O2 in MAPK trafficking through mitochondria is well defined [91] in a three-compartment model (mitochondria, cytosol, nucleus), in which responses such as cell proliferation and cell cycle arrest depended on MAPKs preferential traffic to mitochondria, where a selective ­activation of either ERK1/2 or p38-JNK1/2 by colocalized upstream kinases (MAPKKs) facilitated their further passage to the nucleus. MAPKs activation resulted from oxidation of conserved ERK1/2 or JNK2 cysteine domains to sulfinic- and sulfenic acids at a definite H2O2 concentration [91].

5 Conclusion The mitochondrial production of O2•− by autoxidation of FMNH· of complex I and of UQ•− of complex III with univalent electron transfer to O2 is considered a physiological process. Catalyzed (Mn-SOD) dismutation in the mitochondrial matrix of O2•− to H2O2 provides a molecule, frequently referred to as ROS, that is able to

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d­ iffuse to the cytosol and that supports the concept of oxidative stress, currently associated with a series of pathological situations and neurodegenerative diseases. There are acceptable assays to determine mitochondrial oxidative stress and ­damage and specific assays to determine complex I- and complex III-dependent mitochondrial oxidative stress. Although myriad reports point to ROS and H2O2 as likely intracellular signals for the regulation of cell development, proliferation, migration and apoptosis, the mechanism(s) of signaling remain(s) elusive. The use of the term ROS in signaling is not correct, ROS is not a chemical species, and measurement of the steady-state levels of O2•− and H2O2 strongly support H2O2 as a major signaling molecule. There are postulated mechanisms for H2O2 signaling through redox couples, such as –SH/–SS– in reduced and oxidized glutathione and in active cysteine–cystine pairs. The restriction of the cellular space to “specific redox domains” [74] may help to elucidate mitochondrial H2O2 signaling, considered in the scope of processes that increase or decrease the cellular mitochondrial mass as the processes of mitochondrial biogenesis, fusion and fission.

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Protection of Oxidant-Induced Neuronal Cells Injury by a Unique Cruciferous Nutraceutical Zhenquan Jia, Soumya Saha, Hong Zhu, Yunbo Li, and Hara P. Misra

Abstract  The involvement of reactive oxygen species and electrophiles in the pathogenesis of various neurodegenerative disorders has stimulated extensive studies on the use of exogenous antioxidative compounds to prevent oxidative neurodegenerative processes with few effects. In this study, we demonstrated that the cruciferous nutraceutical 3H-1,2-dithiole-3-thione (D3T) at micromolar concentrations (10–100  mM) has the potential to induce the levels of reduced glutathione (GSH) and NAD(P)H:quinone oxidoreductase 1 (NQO1), two crucial cellular defenses against oxidative and electrophilic stress in human neuroblastoma cells (SH-SY5Y), human primary neurons and astrocytes. In addition, pretreatment with D3T protected cells from oxidative and electrophilic neurocytotoxicity induced by various neurotoxicants, including acrolein, 4-hydroxy-2-nonenal (HNE), 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium (MPP+). The results of this study may have important implications for the development of novel neuroprotective strategies against neurodegenerative disorders underlying Parkinson’s disease. Keywords  Astrocytes • Neuronal cells • Neurotoxicity • ROS Abbreviations 6-OHDA BSA D3T DCIP GPx GSH

6-Hydroxydopamine Bovine serum albumin 3H-1,2-Dithiole-3-thione 2,6-Dichloroindophenol Glutathione peroxidase Reduced glutathione

H.P. Misra (*) Edward Via Virginia College of Osteopathic Medicine, 2265 Kraft Drive, Blacksburg, VA 24060, USA e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_28, © Springer Science+Business Media, LLC 2011

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GST Glutathione S-transferase HNE 4-Hydroxy-2-nonenal MPP+ 1-Methyl-4-phenylpyridinium MTT 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium NQO1 NAD(P)H:quinone oxidoreductase 1 ROS Reactive oxygen species SOD Superoxide dismutase

1 Introduction Reactive oxygen species (ROS) and electrophiles have been implicated in various neurodegenerative disorders including Parkinson’s disease and Alzheimer’s disease [1, 2]. The brain is particularly susceptible to oxidative damage due to its high oxygen consumption and relative deficiency in antioxidant defense systems, including low concentrations of reduced glutathione (GSH) [3]. The ROS-induced alteration of macromolecules including polyunsaturated fatty acids in membrane lipids, proteins, and DNA has been observed in the parkinsonian patients and animal models [4–6]. To minimize oxidative/electrophilic damage to cellular components, cells have developed a number of antioxidant defenses [7]. For example, endogenous cellular antioxidant GSH and NAD(P)H:quinone oxidoreductase 1 (NQO1), one of the extensively investigated phase-2 enzymes, have recently been demonstrated to play important roles in protecting cells against oxidative stress and electrophilic ­quinone-induced damage [8, 9]. GSH is the primary low-molecular-weight thiol antioxidant and cosubstrate for several cellular antioxidative and phase-2 enzymes. In this context, early depletion of cellular GSH and the accompanying oxidative stress are important biochemical features for the development of Parkinson’s ­disease [10–14]. Because of critical involvement of oxidative and electrophilic stress in Parkinson’s disease, extensive studies have focused on the use of exogenous ­antioxidative compounds, including antioxidant vitamins to prevent oxidative ­neurodegenerative processes [15–18]. However, the limited bioavailability, ­inefficient permeability, inability to cross the blood–brain barrier, and/or other untoward effects associated with the use of these individual exogenous antioxidative compounds have raised questions as to their effectiveness in protecting against neurodegenerative diseases [19–24]. We propose a novel strategy for protective/ therapeutic intervention in Parkinson’s disease through coordinated upregulation of endogenous antioxidative enzymes in the neuronal cells, mediated by a cruciferous dithiolethione nutraceutical. Dithiolethiones are five-membered cyclic sulfurcontaining compounds, some of which are constituents of cruciferous vegetables. 3H-1,2-Dithiole-3-thione (D3T) is the most potent member of dithiolethiones for induction of endogenous antioxidants and phase-2 enzymes in hepatic and ­cardiovascular tissues/cells [25–29]. In this study, using human SH-SY5Y ­neuroblastoma cells, human primary neurons and astrocytes as model systems, we have investigated the induction of cellular antioxidants and phase-2 enzymes by

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D3T, and the protective effects of the D3T-upregulated cellular defenses on ­cytotoxicity elicited by various neurotoxicants. Our results demonstrate that total cellular GSH and NQO1 can be potently induced by D3T in SH-SY5Y neuroblastoma cells, human neurons and astocytes, which is accompanied by increased resistance to various neurotoxicants including acrolein, 4-hydroxy-2-nonenal (HNE), 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium (MPP+).

2 Materials and Methods 2.1 Chemicals and Materials Oxidized glutathione, yeast-derived glutathione reductase, 1-chloro-2,4-dinitrobenzene; o-phthalaldehyde, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and bovine serum albumin (BSA) were from Sigma Chemical (St. Louis, MO). D3T was obtained from LKT (St. Paul, MN). Tissue culture flasks and 48-well tissue culture plates were from Corning (Corning, NY).

2.2 Cell Culture Human neuroblastoma SH-SY5Y neuroblastoma cells (ATCC, Manassas, VA) were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin in 75 or 150 cm2 tissue culture flasks at 37°C in a humidified atmosphere of 5% CO2. The cells were fed every 3–4  days and subcultured once they reached 80–90% confluence. Human neurons were purchased from ScienCell Research Labs (San Diego, CA) and cultured according to the manufacturer’s protocol in a recommended neuronal medium (ScienCell). Human primary astrocytes were purchased from Lonza Research Products (Walkersville, MD) and cultured in 75 cm2 tissue culture flasks at 37°C in a humidified atmosphere of 5% CO2. The cells were fed every other day and subcultured once they reached 80–90% confluence.

2.3 Cell Extract Preparation The cells were collected and resuspended in icecold 50 mM potassium phosphate buffer, pH 7.4, containing 2  mM EDTA and 0.1% Triton X-100. The cells were sonicated, followed by centrifugation at 13,000 × g for 10 min at 4°C to remove cell debris. The resulting supernatants were collected and the protein concentrations were measured using a Bio-Rad protein assay kit (Hercules, CA) with BSA as the standard. The supernatants were then used for measurement of the antioxidants and phase-2 enzymes.

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2.4 Assay for GSH Content The measurement of total cellular GSH was based on the o-phthalaldehyde-based fluorometric method, which is specific for determination of GSH at pH 8.0 [28, 30]. Briefly, 10 ml of the cell extract was incubated with 12.5 ml of 25% metaphosphoric acid, and 37 ml of 0.1 M sodium phosphate buffer containing 5 mM EDTA, pH 8.0 at 4°C for 10 min. The samples were centrifuged at 13,000 × g for 5 min at 4°C. The resulting supernatant (10  ml) was incubated with 0.1  ml of o-phthalaldehyde ­solution (0.1% in methanol) and 1.89 ml of the above phosphate buffer for 15 min at room temperature. Fluorescence intensity was then measured at an excitation wavelength of 350  nm and an emission wavelength of 420  nm. Cellular GSH ­content was calculated using a GSH (Sigma Chemical) standard curve and expressed as nanomoles of GSH per milligram of cellular protein.

2.5 Assay for Activities of NQO1 and Other Antioxidant Enzymes Cellular NQO1 activity was measured according to the procedures described previously [31]. Briefly, the reaction mix contained 50  mM Tris–HCl, pH 7.5, 0.08% Triton X-100, 0.25  mM NADPH, and 80 mM of 2,6-dichloroindophenol (DCIP) in the presence or absence of 60 mM dicumarol. The reaction was started by adding 5 ml of sample to 0.695 ml of reaction mixture and the reduction of DCIP was monitored at 600 nm, 25°C for 3 minutes. The inhibition of NQO1 activity by dicumarol was calculated using the extinction coefficient of 21.0  mM−1  cm−1 and expressed as nanomoles of DCIP reduced per minute per milligram of cellular protein. The activities of other antioxidant enzymes, including for superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and glutathione S-transferase (GST), were measured according to the procedures previously described by us [32, 33].

2.6 Assay for Cell Viability Cell viability was determined by a slightly modified MTT reduction assay, as described before [28]. In brief, cells were plated into 48-well tissue culture plates. After incubation of the cells with the neurotoxicants in astrocyte basal medium (Lonza Research Products), supplemented with 0.5% FBS at 37°C for 24 h, media were discarded, followed by the addition of 0.5 ml of fresh medium containing MTT (0.2 mg/ml) to each well. The plates were incubated at 37°C for another 2 h. Then media was completely removed, followed by the addition of 0.2 ml of mix of dimethyl sulfoxide, isopropanol and deionized water (1:4:5) to each well to solubilize the formazan crystals. The amount of dissolved formazan was then detected at 570 nm.

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2.7 Statistical Analysis Statistical analysis was performed with the analysis of variance (ANOVA) and the Student–Newman–Keuls test was used to establish differences between the treatment and control groups. All data are expressed as means ± SEM from four separate experiments.

3 Results 3.1 Induction of Cellular Antioxidant GSH by D3T in SH-SY5Y Neuroblastoma Cells, Primary Human Neurons and Astrocytes GSH plays a critical role in the detoxification of ROS and in protection against oxidative/electrophilic injury, including oxidative neuropathophysiology [9, 34]. We first determined whether D3T could cause elevation of GSH content in cultured human SH-SY5Y neuroblastoma cells, a widely used model for studying neurotoxicity and neuroprotection [35]. As shown in Fig. 1, incubation of SH-SY5Y neuroblastoma cells with 10, 25, 50 and 100 mM of D3T for 24 h resulted in dramatic increases in total cellular GSH content. Moreover, the increases in total cellular GSH content induced by D3T exhibited a concentration-dependent fashion. Notably, incubation of cells with D3T at a concentration as low as 10 mM led to a 35% increase of cellular GSH (Fig. 1). Because human primary neurons are considered to be more suitable for the study of the pathophysiology of neurodegenerative diseases, including Parkinson’s disease, and astrocytes are known to possess important roles in maintaining normal brain function and providing trophic support to the neurons, we further examined the effects of D3T on GSH content in these cells. As shown in Fig. 1, incubation of the neurons and astrocytes with 10–100 mM of D3T for 24 h also resulted in significant increases in cellular GSH content in a concentration-dependent fashion. Notably, a 20% induction of cellular GSH in neurons (Fig. 1) and a 32% in astrocytes were observed after treatment of the cells with D3T at a concentration as little as 10 mM. The basal cellular GSH level was found to be 28.98 ± 2.38, 20.38 ± 1.25 and 35.34 ± 2.15  mmol/mg protein in SH-SY5Y neuroblastoma cells, neurons and astrocytes, respectively.

3.2  Induction of Cellular Phase-2 Enzyme NQO1 by D3T in SH-SY5Y Neuroblastoma Cells, Neurons and Astrocytes NQO1 is a phase-2 enzyme that participates in the detoxification of quinone molecules and ROS [36–39]. As shown in Fig. 2, incubation with D3T (10–100 mM)

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Fig.  1  Concentration-dependent induction of GSH by D3T in SH-SY5Y neuroblastoma cells, human primary neurons and astrocytes. Cells were incubated with the indicated concentrations of D3T for 24 h, followed by measurement of cellular GSH content. Results are expressed as percent of the controls. Values represent mean ± SEM, n = 4. *Significantly different from the respective control groups

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Fig. 2  Concentration-dependent induction of NQO1 by D3T in SH-SY5Y neuroblastoma cells, human primary neurons and astrocytes. Cells were incubated with the indicated concentrations of D3T for 24 h, followed by measurement of cellular NQO1 activity. Results are expressed as percent of the controls. Values represent mean ± SEM, n = 4. *Significantly different from the respective control groups

caused significant increases in cellular NQO1 activity in SH-SY5Y neuroblastoma cells, neurons and astrocytes in a concentration-dependent manner. Notably, when compared to corresponding controls, D3T at a concentration as low as 10 mM significantly enhanced cellular NQO1 activity by 109%, 42%, 69% in SH-SY5Y neuroblastoma cells, neurons and astrocytes, respectively. The basal cellular NQO1 activities were found to be 7.96 ± 1.35  mmol/min/mg, 5.63 ± 1.96  mmol/min/mg and 18.01 ± 2.35 mmol/min/mg protein in SH-SY5Y neuroblastoma cells, neurons

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and astrocytes, respectively. However, D3T at concentrations of 10–100 mM did not result in any statistically significant changes in other antioxidant enzymes including SOD, catalase, GPx, and GST in all cell types (data is not shown).

3.3 D3T Pretreatment Protects SH-SY5Y Neuroblastoma Cells Against Acrolein, 4-Hydroxy-2-nonenal (HNE) or 6-OHDA-Induced Neurocytotoxicity Because both GSH and NQO1 play critical roles in protecting cells against reactive aldehyde-induced oxidative stress as well as in the detoxification of electrophilic quinone molecules [38, 40], we examined the protective effects of D3T pretreatment on cytotoxicity induced by some of the known neurotoxicants including acrolein, HNE or 6-OHDA. These neurotoxicants are widely used to investigate the pathogenesis and progression of experimental parkinsonism. To this end, SH-SY5Y neuroblastoma cells were pretreated with 100 mM D3T for 24 h and then exposed to the various concentrations of acrolein, HNE or 6-OHDA for another 24 h. As shown in Fig. 3, pretreatment of SH-SY5Y neuroblastoma cells with D3T resulted in a marked protection against the neurocytotoxicity elicited by the above neurotoxicants.

3.4 D3T Pretreatment Protects Human Primary Neurons Against HNE and 6-OHDA-Induced Neurocytotoxicity The two well-known neurotoxicants 6-OHDA and HNE were selected to further examine whether the D3T-elevated cellular defenses in the human primary neurons could also lead to neurocytoprotection against the cytotoxicity elicited by these neurotoxic agents. To this end, neurons were pretreated with 100 mM D3T and then exposed to various concentrations of 6-OHDA or HNE. As shown in Fig. 4, pretreatment of the neurons with D3T for 24 h afforded significant protection against both 6-OHDA and HNE toxicity. It is important to note that the neurocytotoxicity elicited by 75 mM of 6-OHDA or 20 mM of HNE was still significantly protected by D3T pretreatment (Fig. 4).

3.5 D3T Pretreatment Protects Human Primary Astrocytes Against 1-Methyl-4-phenylpyridinium (MPP+), HNE or 6-OHDA-Induced Neurocytotoxicity Astrocytes play important roles in maintaining normal brain function and providing trophic support to the neurons. They also suffer a range of toxic insults, being a

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6-OHDA (µM) Fig. 3  Protective effects of D3T pretreatment on (a) acrolein, (b) HNE or (c) 6-OHDA-mediated cytotoxicity in SH-SY5Y neuroblastoma cells. Cells were pretreated with 100 mM D3T in 48-well culture plates for 24 h, followed by exposure to the indicated various concentrations of acrolein, HNE or 6-OHDA for another 24 h. Cell viability was detected using MTT reduction assay. Values represent mean ± SEM, n = 4. *Significantly different from the respective control groups

chief target of prooxidants such as MPP+, 6-OHDA and HNE [41]. Accordingly, we investigated the neurocytoprotective effects of D3T pretreatment on those neurotoxicants in the astrocytes. As shown in Fig. 5, incubation of astrocytes with these neurotoxicants at micromolar concentrations resulted in decreases in cell viability

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HNE (µM) Fig. 4  Protective effects of D3T pretreatment on (a) HNE or (b) 6-OHDA-mediated cytotoxicity in human primary neurons. Human primary neurons were pretreated with 100 mM D3T in 48-well culture plates for 24 h, followed by exposure to the indicated various concentrations of HNE or 6-OHDA for another 24 h. Cell viability was detected using MTT reduction assay. Values represent mean ± SEM, n = 4. *Significantly different from the respective control groups

in a concentration-dependent manner. Pretreatment of astrocytes with 100 mM D3T afforded a remarkable protection against neurocytotoxicity elicited by MPP+, HNE or 6-OHDA (Fig. 5).

4 Discussion This study demonstrates that the cruciferous nutraceutical D3T potently induces the cellular GSH and the phase-2 enzyme NQO1 in SH-SY5Y neuroblastoma cells, neurons and astrocytes, suggesting that the effect of D3T on the antioxidant/ phase-2 enzyme induction is not cell types specific. Studies over the past decade have demonstrated that a variety of cellular antioxidants and phase 2-enzymes, especially GSH and NQO1, can protect neuronal cells against oxidative and electrophilic insults [14, 36]. Several epidemiological studies have also demonstrated that increased consumption of antioxidant-rich fruits and vegetables is associated

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6-OHDA (µM) Fig. 5  Protective effects of D3T pretreatment on (a) MPP+, (b) HNE or (c) 6-OHDA-mediated cytotoxicity in human primary astrocytes. Human primary astrocytes were pretreated with 100 mM D3T in 48-well culture plates for 24 h, followed by exposure to the indicated concentrations of MPP+, HNE or 6-OHDA for another 24 h. Cell viability was detected using MTT reduction assay. Values represent mean ± SEM, n = 4. *Significantly different from the respective control groups

with the reduced risk of ischemic stroke, dementia, and neurodegenerative diseases [42–45]. The dithiolethione nutraceutical compound D3T is rich in cruciferous vegetables. In this cortex, the coordinated induction of endogenous GSH and NQO1 by D3T (Fig. 1) may be responsible, at least partially, for the reduced risk

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of neurodegenerative disorders [43, 44, 46]. However, the exact signaling pathway by which D3T mediates elevation of GSH and NQO1 in neuronal cells remains to be defined. Recently, the nuclear E2-related factor (Nrf2) has been demonstrated to be an indispensable factor for regulating expression of antioxidative and phase-2 genes in various types of tissues and cells [47–49]. Studies are currently under way to investigate whether the above Nrf2-mediated transcriptional regulation is also involved in D3T-induced elevation of GSH and NQO1 in neuronal cells. A notable finding of this study was that the coordinated induction of various cellular defenses by D3T in SH-SY5Y neuroblastoma cells, neurons and astrocytes was accompanied by augmented resistance of these cells to neurocytotoxicity induced by various neurotoxicants. Acrolein and HNE are two major types of unsaturated aldehydes and the presence of protein-bound unsaturated aldehydes in  vivo has been detected in the dopaminergic neurons in substantia nigra of Parkinson’s disease patients [50]. It has been suggested that GSH plays an important role in the detoxification of reactive aldehydes, including acrolein and HNE [6]. In this regard, GSH is able to directly react with aldehydes to form a less reactive conjugate [6]. Accordingly, the potent induction of GSH by D3T in all cell types (Fig. 1) may account for increased resistance of D3T-pretreated cells to acrolein or HNE-induced neurocytotoxicity (Figs.  3–5). Our previous studies further demonstrated that upregulation of cellular GSH is a predominant ­mechanism for D3T-mediated cytoprotection against acrolein or HNE-induced neurotoxicity[51–53]. It is interesting to note that astrocytes expressed more basal levels of GSH (35.34 ± 2.15 mmol/mg protein) than SH-SY5Y neuroblastoma cells (28.98 ± 2.38  mmol/mg protein) and neurons (20.38 ± 1.25  mmol/mg protein). Astrocytes are known to provide GSH precursors to neighboring ­neurons [54–56]. Depletion of astrocyte GSH has been correlated with increased neuronal death in an oxidative stress/co-culture system [57]. Thus, the induction of GSH by D3T in astrocytes may contribute to the development of novel strategies for neuroprotection. Pretreatment of the astrocytes with D3T also rendered these cells more resistant to neurocytotoxicity induced by other neurotoxins including 6-OHDA (Figs. 3–5) and MPP+ (Fig.  5). The neurotoxicity of 6-OHDA has been attributed to the ­generation of ROS as well as the formation of electrophilic reactive metabolites, such as quinone molecules and semiquinone radicals [58]. NQO1, one of the extensively investigated phase-2 enzymes, has recently been demonstrated to play an important protective role in quinone-induced oxidative stress [38]. In this regard, NQO1 is found to catalyze the two-electron reduction of quinones and quinoid compounds to hydroquinones, therefore limiting the subsequent generation of ROS [38]. In addition to NQO1, GSH is also capable of reacting with quinone molecules as well as ROS to attenuate 6-OHDA-induced neurocytotoxicity [59]. Accordingly, the coordinated induction of the endogenous antioxidant GSH and phase 2 enzyme NQO1 by D3T appeared to be responsible, at least partially, for the increased resistance of D3T-pretreated cells to 6-OHDA-induced neurocytotoxicity (Figs.  3–5). MPP+ is a another well-known neurotoxicant that causes dopaminergic neuron death leading to permanent Parkinson’s disease-like syndrome [60]. MPP+ is known to induce oxidative stress, and astrocytes are a primary locus for MPP+-induced neurotoxicity

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both in vitro and in vivo [61–63]. Our labs are the first to report that ROS – including superoxide, hydrogen peroxide, and hydroxyl radical – are ­produced during the autoxidation of MPP+ [64, 65]. While a number of antioxidants are involved in the detoxification of ROS, GSH is believed to be a major cellular nucleophile that reacts with ROS, including superoxide, hydrogen peroxide, and hydroxyl radicals, thereby preventing their attack on critical cellular targets [34, 66]. In this context, the potent induction of cellular GSH by D3T (Fig. 1) may largely contribute to the increased resistance of the D3T-pretreated astrocytes to MPP+-mediated neurocytotoxicity (Fig. 5). In addition, the induction of NQO1 may also indirectly contribute to D3Tmediated neurocytoprotection against MPP+-induced oxidative stress by maintaining cellular levels of ubiquinol and vitamin E, two important biological antioxidants involved in the detoxification of ROS [36, 38]. These results suggest that cruciferous D3T might be a promising agent in protecting against oxidative neurotoxicity. The use of the cruciferous nutraceutical dithiolethione compound D3T may be a better strategy for protecting cells against degenerative processes because this compound is abundant in nature and is well tolerated by animals and humans [27]. Furthermore, the upregulation of GSH and phase-2 enzyme NQO1 by D3T, which protects cells against ROS and electrophilicinduced injury, is a more effective approach than exogenous antioxidant treatment in preventing cell injury caused by various oxidative insults. Because D3T is highly lipophilic, it is most likely to penetrate the blood–brain barrier and consequently brings about upregulation of endogenous antioxidant and phase-2 enzymes effectively in the brain. In addition, although the neuroprotective effects of D3T against Parkinson’s disease are largely unexplored, oltipraz, a synthetic analog of D3T used as a cancer chemopreventive agent in humans is currently under clinical investigation [67]. Given the profound potency and lipophilicity of indirect antioxidant D3T, it is likely that dietary consumption of D3T may afford a protective influence against neurodegenerative diseases. In conclusion, these studies have demonstrated that cellular GSH and the phase-2 enzyme NQO1 are induced by cruciferous D3T in SH-SY5Y neuroblastoma cells, human primary neurons and astrocytes. This nutraceutical-mediated coordinated upregulation of cellular defenses is accompanied by remarkably increased resistance to cells against various neurotoxicants, including acrolein, HNE, 6-OHDA and MPP+. These new findings may have implications for developing protective strategies against neurodegenerative disorders underlying Parkinson’s disease.

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Oxidatively Generated Damage to DNA and Biomarkers Jean Cadet, Thierry Douki, and Jean-Luc Ravanat

Abstract  The first part of this chapter is devoted to the description of degradation pathways for DNA bases and sugar moieties in the cell that are mediated by hydroxyl radical, one-electron oxidants and singlet oxygen. Thus, 11 single modified nucleosides and nucleobases that may be part of more complex radiationinduced DNA damage have been shown to be generated in nuclear DNA. In addition, four clustered DNA addition products and one tandem base-sugar lesion that arises from •OH-mediated hydrogen abstraction at C4 and C5 of the 2-deoxyribose moiety of DNA have also been identified in cells. Mechanisms of formation that are inferred from model studies are available for the 16 single and complex lesions thus detected in nuclear DNA. The DNA oxidation products whose radiation-induced formation in cellular was found to vary between 2 and 100 per 109 normal nucleosides per Gray may be used as biomarkers of oxidative stress. The accurate measurement of single, clustered or tandem lesions was performed, once DNA was extracted and subsequently enzymatically or chemically hydrolyzed, using accurate chromatographic methods. These involve, in most cases, the association of high performance liquid chromatography with the electrospray ionization tandem mass spectrometry (HPLC-ESI/MS–MS) detection techniques operating in the highly accurate and sensitive multiple reaction monitoring mode. Another approach that is more sensitive and less prone to artefactual oxidation consists of pre-incubating oxidized DNA with repair enzymes to reveal classes of modifications as strand breaks prior to either single-cell electrophoresis analysis or alkaline elution analysis. Keywords  Clustered damage • Enzymic assays • Hydroxyl radical • Liquid chromatographic measurements • One-electron oxidants • Oxidized nucleosides • Singlet oxygen • Tandem lesions

J. Cadet (*) Laboratoire “Lésions des Acides Nucléiques”, SCIB-UMR-E n°3 (CEA/UJF), Institut Nanosciences et Cryogénie, CEA/Grenoble, Grenoble Cedex 9 38054, France e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_29, © Springer Science+Business Media, LLC 2011

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1 Introduction Oxidatively generated damage to biomolecules, including DNA, has been shown to be involved in aging and several pathological situations implicating the free radical theory [1]. These include chronic inflammation, cancer, diabetes mellitus, atherosclerosis and neurodegenerative diseases [2–7] such as Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, multiple sclerosis and Huntington’s disease [8]. Among oxidation reactions that occur in cells, one may distinguish between endogenous and exogenous processes. Respiratory burst in mitochondria is at the origin of the rather unreactive superoxide anion radical (O2•−) that arises from one-electron reduction of molecular oxygen [9] and that is in dynamic equilibrium with the related conjugated acid and very low abundant hydroperoxide radical (HO2•) (~1%) at neutral pH. The main known reactions of O2•− include addition to oxidizing radicals as those resulting from one-electron oxidation product of guanine [10], 8-oxo-7,8-dihydroguanine, tyrosine and likely tryptophan [11, 12] together with competitive reduction of these reactive intermediates. It has been also shown that O2•− is able to reduce thymine hydroperoxyl radical, a key step in the pathways involved in hydroxyl radical (•OH), and the one-electron-mediated formation of thymine hydroperoxides [13]. Spontaneous or enzymic dismutation of O2•− gives rise to unreactive hydrogen peroxide (H2O2) that needs the presence of reduced metals to be converted into reactive oxygen species. Thus reduction of H2O2 by Fe2+ leads to the generation of highly reactive hydroxyl radical (•OH), according to the Fenton reaction [14], which reacts indifferently with molecules at the site of production. Evidence has been provided, at least in model systems, that the reaction of Cu+ and Cu2+ with H2O2 gives rise to singlet oxygen (1O2) and one-electron oxidation reaction, respectively [15–17]. One may also note that O2•− is involved in an addition reaction with NO that is generated by nitric synthase during inflammation processes. This leads to the formation of peroxynitrite (ONOO−), which exhibits both oxidative and nitrosative features with DNA. It has been proposed that ONOO−, through its reaction with bicarbonate, may act as a one-electron oxidant of guanine through the transient formation of nitrosoperoxycarbonate [18] and the subsequent release of oxidizing carbonate anion radical (CO3•−) [19]. The most common exogenous physical oxidizing agents include ionizing radiation and the UVA component of solar light. Ionizing radiation acts mostly through an indirect process that is mediated by •OH, the main reactive species produced by the radiolysis of water, whereas the direct interaction of high energy photons with DNA leads to ionization of both nucleobase and 2-deoxyribose moieties. It is well documented that the chemical reactions induced by ionizing radiation are complex due to the occurrence of several simultaneous radical and excitation events along the photon tracks. This may lead to the formation of clustered lesions involving one or several base modifications, single and double strand breaks within one or two helix turns. Further complexity is associated with the occurrence of bystander signaling effects that are mediated by gap junctions and inflammatory responses [20, 21] leading to the generation of ROS in neighboring

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cells or compartments. This is not restricted to ionizing radiation since bystander responses have been shown to be induced by other genotoxic agents including UV radiations [22] and chemotherapeutic compounds [23]. In the first section of this chapter, the main available oxidative pathways that lead to the formation of single and clustered modifications in cellular DNA and human samples such as blood lymphocytes and skin explants are reported and discussed mechanistically. Three main oxidizing agents, including hydroxyl radical (•OH), high intensity UVC nanosecond laser pulses acting as an one-electron oxidant of nucleobases and singlet oxygen (1O2), were used to generate single, clustered and tandem DNA lesions. These lesions may be considered to be bio-markers of oxidative reactions in cells. In the second part of the chapter, emphasis is placed on a critical review of available analytical methods aimed at monitoring the formation of oxidatively generated damage in cellular DNA. While high performance liquid chromatography associated with either electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) or electrochemical detection (HPLC-ECD) allow accurate and specific measurements of well-characterized DNA modifications. Enzymatic based methods implicating either agarose gel electrophoresis or alkaline elution technique for the detection of direct and enzymatically induced single-strand breaks (SSBs) are more appropriate for measuring low variations of the levels of oxidatively generated damage to DNA.

2 Radical Reactions Involving OH and One-Electron Oxidation Processes •

A large body of information is available on the reactions mediated by •OH and oneelectron oxidants of DNA components as inferred from extensive model studies that have involved detection of transient radicals, final decomposition product analysis and theoretical approaches [24–28]. Thus more than 80 modified nucleosides, including relatively unstable thymidine hydroperoxides and diastereomers of 5,6-dihydroxy-5,6-dihydropyrimidine derivatives have been isolated and characterized and detailed mechanisms of their formation, are available (for comprehensive reviews, see [29–36]). It may be added that the main degradation pathways arising from •OH-mediated hydrogen abstraction at C1, C3, C4 and C5 from the 2-deoxyribose moiety of nucleosides and DNA have been elucidated [37–39]. In contrast, there is still a large deficit of accurate information concerning the formation of oxidatively generated base and sugar damage in cellular DNA [31], the topic of this chapter. The problem is mostly due to the difficulties of accurately measuring low amounts of oxidatively generated base damage typically within the range of a few lesions per 107 normal bases, as further discussed in the second part of the chapter. A suitable way to investigate the effects of •OH on cellular DNA is to use the predominant indirect effect of ionizing radiation. By applying acute doses of ­ionizing radiation, this allows one to increase significantly the frequency of oxidized bases with respect to steady-state background level due to the occurrence

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of oxidative metabolism that mostly results from Fenton- type reactions. Formation of radical cations of the nucleobases and the 2-deoxyribose moieties that subsequently are likely to undergo hydration [40] and/or deprotonation as shown in model studies [31, 41] may be generated in cells through the direct interaction of ionizing radiation with DNA. However, one-electron oxidation of nucleobases may be achieved in a milder way, using type I photosensitizers [41], KBrO3 [42], CO3•− [18] or nanosecond UVC laser pulses [43]. The latter approach has been applied to cellular DNA [40] in order to gain mechanistic insights into the chemical reactions of the radical cations from nucleobases that have been shown to be generated in model studies with a similar efficiency. In particular, THP1 human monocytes have been used for studies aimed at characterizing 11 single base lesions and the five more complex lesions formed by exposure to •OH, one-electron oxidants or 1O2 under low temperature conditions that minimize the occurrence of DNA repair.

2.1 Single-Base Lesions Ubiquitous •OH is known to react efficiently at rates controlled by diffusion with the four main purine and pyrimidine bases, mostly by addition to unsaturated bonds. In addition, two specific hydrogen atom abstraction reactions involve the methyl group of thymine and the 2-amino group of guanine respectively. 2.1.1 Thymine Six main stable oxidation products of thymine (1) have been shown to be generated in the DNA of THP-1 human monocytes upon exposure to gamma rays and heavy ions [40, 44, 45]. These results were achieved using accurate high-performance liquid chromatography HPLC–MS/MS after quantitative enzymic digestion of extracted DNA under conditions where artifactual oxidation of overwhelming normal nucleosides was minimized [46]. The six oxidized nucleosides consist of the four cis and trans diastereomers of 5,6-dihydroxy-5,6-dihydrothymidine (8) and two methyl oxidation products, namely 5-formyl-2¢-deoxyuridine (10) and 5-(hydroxymethyl)-2¢-deoxyuridine (11).The radiation-induced formation of compounds 8, 10 and 11 was found to be linear with the dose of g-rays or 12C6+particles within the dose range of 90–450 Gy. Another key observation was a decrease in the yield of formation of each of the six oxidized nucleosides with the linear energy transfer of radiation as shown in Table 1. These results strongly suggest that •OH plays a major role in the formation of radiation-induced damage to cellular DNA. Another striking result deals with the yields of formation of thymidine oxidation products which are much lower by a factor of one to three orders of magnitude than those reported previously using the questionable GC–MS (Fig. 1). The formation of 8, 10 and 11 may be rationalized on the basis of available mechanisms that were proposed from extensive model studies in aerated aqueous solutions. Thus the reactions of •OH across the 5,6-double bond of the base moiety of 1 is

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Table  1   Yields a of degradation products of thymidine (1), 2¢-deoxyguanosine (12) and adenine in the DNA of THP-1 malignant human monocytes upon exposure to g–raysb Lesions g–rays 97 Cis and trans 5,6-dihydroxy-5,6-dihydrothymidine (8) 5-Formyl-2¢-deoxyuridine (10) 22 5-(Hydroxymethyl)-2¢-deoxyuridine (11) 29 8-Oxo-7,8-dihydro-2¢-deoxyguanosine (15) 20 2,6-Diamino-4-hydroxy-5-formamidopyrimidine (16) 39 8-Oxo-7,8-dihydro-2¢-deoxyadenosine (25) 3 4,6-Diamino-5-formamidopyrimidine (26) 5 a expressed in lesions per 109 nucleosides b from [45]

•OH

O

O CH3

HN O

O CH3

HN O

N

−H+

CH2•

HN O

N

N

2

1

3

•OH

H2 O

O

O CH3 OH

HN O

N

• 4

H

+

HN O

N

•

CH3 OH H

5

Fig.  1  Formation of 5,6-dihydroxy-5,6-dihydrothymine (8) from the 5-hydroxy-5,6-dihydrothym-6-yl radical (4)

known to generate predominantly the reducing 5-hydroxy-5,6-dihydrothym-5-yl radical (4) together with a lower amount of the oxidizing 6-hydroxy-5,6-dihydrothym-6-yl radical (5). In a subsequent step, addition of O2 to 4 and 5 leads to the formation of four cis and trans diastereomers of 6-hydroperoxy-5-hydroxy-5,6-dihydrothymidine (6) and 5-hydroperoxy-6-hydroxy-5,6-dihydrothymidine (7) [47, 48] after reduction by O2•− [13], followed by protonation of transient hydroperoxyl radicals. Reduction of the peroxide bond of 6 and 7 that has been shown in model studies

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to be highly stereospecific is the likely mechanism giving rise to the four cis and trans diastereomers of 5,6-dihydroxy-5,6-dihydrothymidine (8) [49, 50]. Formation of 5-formyl-2¢-deoxyuridine (10) and 5-(hydroxymethyl)-2¢-deoxyuridine (11) is explained by the transient formation of 5-(hydroperoxymethyl)-2¢-deoxyuridine (9) according to the sequences of reactions depicted in Fig.  2. These reactions have received support from previous mechanistic studies on the free nucleoside and isolated DNA [48, 50]. Thus, O2 addition to 5-(2¢-deoxyuridilyl) methyl radical (3) formed by •OH-mediated H atom abstraction from the methyl group of 1 gives rise to the related peroxyl radical that then undergoes reduction by O2•− and subsequent protonation. Dehydration and reduction of 9 are the two likely competitive pathways that would explain the formation of 10 and 11 (Fig. 2). It is worth noting that the relative formation of 9 and 10 with respect to that of thymidine glycol 8 is much higher by a factor of about 3 in cellular DNA than for free nucleoside 1 upon exposure to • OH. This is likely to be accounted for by a change in •OH reactivity, the accessibility of the methyl group of 1 relatively increases with respect to the 5,6-ethylenic bond when moving from the isolated nucleoside to duplex DNA. Interestingly, the one-electron oxidation of 1 in cellular DNA following UVC nanosecond laser irradiation has been found to induce a similar oxidation product pattern than that induced by •OH. Therefore the formation of 8, 10 and 11 is explained in terms of the initial formation of thymidine radical cation 2 (Fig. 3) that O

O CH3 OH

HN O

N

•

H

O2

O CH3

HN

O2−°, H+

O

OH OOH H

N

Reduction

CH3

HN O

OH OH H

N

6

5

8

Fig. 2  Formation of 5-formyluracil (10) and 5-(hydroxymethyl)uracil (11) O CHO

HN O CH2•

HN O

−H2O

O

O2 O2−°, H+

N

3

N

CH2OOH

HN O

O

10

N

O

9

CH2OH

HN

Reduction O

N

11 Fig. 3  •OH and one-electron oxidation-induced radicals in the thymine moiety (1) of DNA

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is able to undergo both hydration and deprotonation as shown in model studies [51]. Selective reaction of a water molecule with 2 gives rise exclusively to oxidizing 5 as the first step of a sequence of reactions leading to 8 through the intermediacy of 5-hydroperoxy-6-hydroxy-5,6-dihydrothymidine (7). Competitive loss of a proton from the methyl group of 2 generates neutral radical 3 that has been shown to be the precursor of the •OH-mediated formation of 10 and 11 in cellular DNA and the photosensitized one-electron oxidation of 1 in aerated aqueous solution. 2.1.2 Guanine Two main degradation products of 2¢-deoxyguanosine (12), including 8-oxo-7,8dihydro-2¢-deoxyguanosine (15) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (16) have been detected and characterized by HPLC–MS/MS in the enzymic hydrolysate of DNA of THP1 cells that were exposed to g-rays and high LET particles [40, 45]. A likely mechanism for the formation of 15 and 16 involves initial •OH addition at C8 of 12, giving rise to reducing radical 13 (Fig. 4) with a yield that has been estimated to be 17% for the free nucleoside [52]. Subsequent one-electron oxidation of 12 gives rise to 15, the most widely used DNA biomarker of oxidative stress. It may be pointed out, however, that the detection of 15 in cellular DNA cannot be used as a specific index of oxidation since the oxidized nucleoside has been shown to be generated not only by •OH but also by oneelectron oxidants, singlet oxygen, peroxynitrite [29, 30, 34, 53] and through the addition of vicinal thymine hydroperoxides [54, 55]. The opened imidazole compound 16 that is produced upon competitive one-electron reduction of 13 (Fig. 4) is generated with a two-fold higher efficiency than 15 in contrast to that observed

O

−e−

O

H2N

H2N

H N

HN

N

N

H2N

14

N

HN

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CHO N

16

+e−

H2O

NH

N

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O

12

•

OH

N

HN H2N

N

N

OH H

O

−e−

H2N

13

H N

HN

O N

N

15

Fig. 4  Formation of 8-oxo-7,8-dihydroguanine (15) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (16) by •OH and one-electron oxidation of guanine (12)

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in aerated aqueous solution of DNA where 15 predominates. This may be explained by the lower amount of O2 in nuclear DNA and also by the presence of reducing compounds such as thiols. Further support for a major involvement •OH in the radiation-induced formation of 15 and 16 is provided by an observed decrease in the yield of degradation products of 12 with an increase in the LET value of heavy particles (Table 1). 2,2-Diamino-4-[(2-deoxy-b-d-erythro-pentofuranosyl)amino]-5(2H)oxazolone (21), another •OH and one-electron oxidation product of 12 in free nucleoside [56, 58] isolated DNA has been detected in liver DNA of diabetic rats by HPLC–MS/MS [59]. The yield of formation of 21 was found to be ten-fold lower than that of 12. The mechanism of formation of 21 that is inferred from detailed model studies on nucleosides and oligonucleotides is reported in Fig. 5. The first step of a rather complicated degradation pathway involves •OH-mediated hydrogen atom abstraction from the 2-amino group of the base moiety of 12, a recently revisited mechanism as demonstrated by pulse radiolysis experiments [60]. This constitutes a relevant alternative pathway to the •OH addition reaction at C4 of 12 followed by dehydration that was proposed earlier [52]. The resulting aminyl radical 17 is likely to be in dynamic equilibrium with other tautomeric forms such as the oxyl type radical 19 and the C5 carbon centered radical 18. Addition of O2•− and not O2 to C5 of 18 as shown by flash photolysis studies on nucleosides and oligonucleotides [10] is likely to give rise after protonation to a 5-hydroperoxide that is the precursor through a complex series of reactions and rearrangements of 2-amino-5-[2-deoxy-b-d-erythro-pentofuranosyl)amino]-4Himidazol-4-one (20). These involve successively, hydration of the 7,8-double bond, a keto-hydroperoxide cleavage mechanism, decarboxylation, loss of a formamide residue and subsequent intramolecular cyclization [57, 61]. The imidazole nucleoside 20 that has been shown to be unstable in aqueous solution at neutral pH is expected to be converted into 21 (Fig. 5). Evidence was provided for the formation of 15 and 16 in cellular DNA upon exposure to high intensity nanosecond UV pulses [40]. The radical cation 14 that O• N

N

−e−

O

N

N

19

O

N

HN H2N

H2N

N

N

• N

N H2N

12

O

HO° H2 O

N

HN •HN

N

N

N

O

O2°−, H+ H 2O −CO2

H N NH

HN

18 -Formamide

N

20

O

O

NH2

HN

N

NH2

H2O

21

N

17

Fig.  5  Formation of 2,2,5-triamino(2H)-oxazolone (21) by •OH and one-electron oxidation of guanine (12)

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is generated by bi-photonic ionization of the purine base of 12 is able to undergo hydration reaction with the subsequent formation of 15 and 16 through the intermediacy of reducing 13 [62] (Fig. 4). It is interesting to note that although the radical cations of the four main purine and pyrimidine bases are generated upon UVC laser irradiation with a similar efficiency in model compounds, 15 and 16, are the predominant lesions in cellular DNA. The more specific degradation of 12 upon base ionization may be rationalized in terms of long- and short-range charge transport within DNA [63, 64] to guanine bases which exhibit the lowest ionization potential among DNA components are preferential sinks for electron holes. This also provides further support for the predominant role of •OH in the molecular effects of ionizing radiation on cellular DNA. 2.1.3 Adenine Two main degradation products of the base moiety of 2¢-deoxyadenosine (22), which include 8-oxo-7,8-dihydro-2¢-deoxyadenosine (25) and 4,6-diamino-5-formamidopyrimidine (26), have been shown to be generated in the DNA of human monocytes THP1 upon either exposure to gamma-rays or nanosecond laser UVC pulses [40]. Similar mechanisms to those proposed for either the •OH-mediated degradation or the one-electron oxidation of 12 are implicated in the radical generation of 25 and 26 (Fig. 6). Thus the formation of 8-hydroxy-7,8-dihydroadenyl radical (23), the precursor of 25 and 26 may be rationalized in terms of either •OH addition at C8 of 22 or hydration of the adenine radical cation 24. Subsequently, competitive one-electron oxidation [65] and one-electron reduction [52, 66] reactions

NH2

NH2 N

N •OH

N

N

OH H

O N

N

+e−

N N

N

H N

N

25

23

NH2 N

−e−

NH2 N

22

NH2

N

CHO N

N

N

N

H N NH

26

24

Fig.  6  Formation of 8-oxo-7,8-dihydroadenine (25) and 4,6-diamino-5-formamidopyrimidine (26) by •OH and one-electron oxidation of adenine (22)

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of 24 are likely to occur leading to the generation of 25 and 26, respectively. One may note that 25 and 26 are produced with about ten-fold lower efficiency than the corresponding guanine analogs 15 and 16. This may be explained by enhancement of •OH-mediated formation of the latter guanine degradation products through the generation of tandem base modifications [67] as discussed below. The formation of 2-hydroxy-2¢-deoxyadenosine that was reported to be one of the main radiationinduced degradation products of the adenine moiety of cellular DNA [68, 69] on the basis of questionable GC–MS measurements has been revisited. Using the accurate HPLC–MS/MS, the formation of 2¢-hydroxy-2¢-deoxyadenosine was not detectable in the DNA of THP1 cells upon exposure to g–rays at doses up to 200 Gy [70]. This questions the biological relevance of 2-hydroxy-2¢-deoxyadenosine as a biomarker of oxidative stress.

2.2 Clustered and Tandem Lesions Increasing attention is given to the formation and biological role of complex oxidatively generated DNA lesions that may be generated by either multiple simultaneous hits or single radical events. Thus evidence is accumulating from both experimental and theoretical investigations that ionizing radiation and heavy particles are able, through energy dissipation, to induce the generation of complex DNA damage that may include one- or several-strand breaks, abasic sites and base lesions on both strands within one or two helix turns. The complexity and the severity of such modifications that arise from the occurrence of simultaneous radical and excitation events were found to increase with the LET of the radiation or charged heavy ions. The identification of these modifications, each of them being unique, remains a challenging analytical issue due their extreme complexity and difficulty of isolation. Only limited information on the formation of radiation-induced clustered lesions involving base lesions, abasic sites or strand breaks is available so far on the basis of electrophoresis measurements. Double-strand breaks (DSBs) that constitute a heterogeneous class of radiation-induced clustered damage with highly deleterious features are produced in about 20-fold lower yield than single- strand breaks. Several assays, including pulsed field gel electrophoresis [71], neutral comet assay [72] and immunological detection of g-H2AX foci [73] are available for the measurement of DBSs in cells. Following the pioneering work of Box et al. [74, 75], there is growing evidence that one initial radical hit can give rise to tandem base modifications as the result of addition of either •OH- or one-electron oxidation-mediated pyrimidine peroxyl radicals to vicinal intrastrand guanine [54], adenine [67] or pyrimidine bases [76–78]. On the basis of detailed mechanistic studies [55], the addition of either 5-hydroperoxyl-6-hydroxy or 6-hydroperoxyl-5-hydroxy-5,6-dihydrothymine radicals to the C8 of guanine in dinucleoside and isolated DNA led to the formation of 15 and formylamine, a remnant of the radical oxidative degradation of thymine. It has also been shown that peroxyl pyrimidine radicals are able to promote the ­one-electron

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oxidation of distant guanine leading to double base lesions that may be separated by several normal bases [67]. However, attempts to look for the radiation-induced formation in cellular DNA of tandem base damage involving 12 and formylamine have so far been unsuccessful, probably due to a lack of sensitivity of the HPLC–MS/MS assay. Two other tandem base lesions have been shown to be generated in the DNA of HeLa cells in very low yields that are about two orders of magnitude lower than those of 5-formyl-2¢-deoxyuridine (10) upon exposure to gamma rays. This was accurately achieved using HPLC–MS/MS and further confirmed by MS3 analyses [76, 77, 79, 80]. The tandem base modification arises from intramolecular addition of 5-(uracilyl)methyl radical and 6-hydroxy-5,6-dihydrocytosyl radical to a vicinal guanine located on the 5¢end as established from experimental and theoretical studies [80–89]. Emphasis is placed in the next two subsections on the formation in cellular DNA of other tandem and clustered damage that share a common feature in their formation involving initial •OH-mediated hydrogen abstraction from the 2-deoxyribose moiety at C4 and C5, followed by addition of the reactive species or radical, thus generated to a base on the opposite strand. These represent unusual degradation pathways since hydrogen atom abstraction reactions at C3¢, C4¢ and C5¢ lead ­predominantly to single-strand breaks [37–39], whereas oxidation at C1¢ gives rise to 2-deoxyribonolactone, an oxidized abasic site [87]. 2.2.1 Cytosine Adducts Arising from •OH-Mediated-Hydrogen Abstraction at C4¢ The four possible diastereomers of 6-(2-deoxy- b-d-erythro-pentofuranosyl)-2hydroxy-3(3-hydroxy-2-oxopropyl)-2,6-dihydroimidazo[1,2-c]-pyrimidin-5(3H)one (30) were first detected in isolated DNA upon exposure to •OH in aerated aqueous solutions [91, 92]. This was achieved by HPLC–MS/MS operating in the neutral loss scan mode after suitable enzymic hydrolysis. Further insights into the structure and the mechanism of formation of 30 were gained from model studies that have involved isolated DNA, polynucleotides and nucleotides. Additional support for the implication of the C4¢ radical in the initial step of the sequence of reactions leading to 33 was provided by the formation of the latter cyclic nucleosides upon incubation of isolated and cellular DNA with bleomycin, a radiometric enedyine that is known to predominantly abstract the C4¢ hydrogen atom in doublestranded DNA [93]. The resulting carbon centered C4¢ radical that may also be generated by •OH is converted among other degradation pathways into a 4¢ oxidized abasic site 27 [39] after O2 addition and elimination of the base (Fig. 7). The abasic site, or, more likely, its acyclic form [94], is subject to b-elimination promoted by the presence of a cytosine or adenine base on the opposite strand as shown by mechanistic studies [95]. This leads to cleavage of the 3¢-phosphodiester bond and formation of conjugated keto-aldehyde 28 that is able to undergo nucleophilic addition with the 4-amino group of 2-deoxycytidine 29 on the opposite strand with the subsequent formation of 4 diastereomers of 30. Evidence for the formation of

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O

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Fig. 7  Formation of a cross-link (29) upon •OH-mediated hydrogen atom abstraction at C4¢

interstrand cross-links was provided from model studies that have involved the insertion of a ­photolabile precursor of the C4¢ radical in DNA duplexes [95, 96]. The yield of radiation-induced 33 in cellular DNA was shown to be about 1% of that of 8-oxo-7,8-dihydro-2¢-deoxyguanosine (15) and similar to that of DSBs. Similar cycloadducts to adenine are expected to be generated in isolated and cellular DNA upon either exposure to •OH or incubation with bleomycin as inferred from reactivity studies [96].

2.2.2 Formation of (5¢R)-5¢,8-cyclo-2¢-deoxyadenosine (33) through • OH-mediated hydrogen abstraction at C5¢ Earlier model studies have shown that •OH-mediated hydrogen abstraction at C5¢ of adenosine 5¢-monophosphate (AMP) and 2¢-deoxyadenosine give rise to related adenine 5¢,8-cyclonucleotide [97] and 5¢,8-cyclonucleoside [98] respectively. Evidence was provided later for the formation of two 5¢R and 5¢S diastereomers of 5¢,8-cyclo-2¢-deoxyadenosine as a result of intramolecular cyclization of the 5¢-yl radical to the C8 position of the purine ring of AMP [99], with the 5¢R diastereomer predominating. Subsequently, both 5¢R and 5¢S diastereomers of 5¢,8-cyclo-2¢-deoxyguanosine were shown to be induced by radiation in oxygenfree aqueous solution of 2¢-deoxyguanosine (12) and isolated DNA [100]. This was achieved by gas chromatography analysis of enzymatically released nucleosides. Recent ­experimental and theoretical studies have provided further mechanistic insights on the effect of oxygen in the oxidation reaction following intramolecular cyclization and the partial prevention of formation of

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5¢,8-­cyclonucleoside due to efficient competitive O2 fixation on the C5¢-yl radical [101]. In contrast to previous claims [100, 102], the presence of O2 in the aqueous solutions does not abolish the formation of purine 5¢,8-cyclonucleosides. The formation of both adenine and guanine 5¢,8-cyclonucleosides was found to decrease concomitantly with an increase in O2 concentration, the inhibitory effect being more pronounced for the diastereomers of 5¢,8-cyclo-2¢-deoxyguanosine. This is in agreement with the occurrence of an efficient competitive reaction involving fast O2 fixation on C5¢-yl radical. It is likely that the cyclization of 2¢-deoxyguanos-5¢-yl radical is faster [103] than that of 2¢-deoxyadenosyl radical whose rate constant, kc = 1.6 × 105  s−1, has been determined by flash photolysis experiments [104]. The cyclization reaction has been shown to be highly stereospecific in DNA, giving rise to a ratio of 5¢R/5¢S of ~3 and ~4 for 5¢,8-cyclo-2¢deoxyguanosine and 5¢,8-cyclo-2¢-deoxyadenosine, respectively [98]. This contrasts with previously reported data showing that the 5¢S diastereomer of either purine 5¢,8-cyclo-2¢-deoxyribonucleosides is predominantly generated in both isolated and cellular DNA. Indeed, a dose of 2 kGy was necessary to detect the bare presence of (5¢R)-cyclo-2¢-deoxyadenosine (33) with a radiation-yield of 0.2 lesions per 109 normal and per Gy. The radiation-induced formation of 33 is explained in terms of intramolecular cyclization of the 5¢-radical initially formed by •OH-mediated hydrogen atom abstraction at C5¢, followed by one-electron oxidation of the aminyl radical thereby transiently generated as depicted in Fig. 8. These data are not in agreement with previous findings suggesting that the radiation-induced formation frequency of 5¢,8-cyclonucleosides is about 200-fold higher [105] in comparison to that recently measured by HPLC–MS/MS [101]. As an indication, the yield of radiation-induced (5¢S)-5¢,8- cyclo-2¢-deoxyadenosine, (5¢R)-5¢,8-cyclo-2¢-deoxyguanoisne and (5¢S)-5¢,8-cyclo-2¢-deoxyguanosine were estimated to be 14, 30 and 100 per 109 normal nucleosides per Gy, respectively. This should be compared with the radiation yield of 8-oxo-7,8-dihydro-2¢deoxyguanosine (15), one of the main oxidation products of DNA that was shown to be 20 lesions per 109 normal nucleosides and per Gy in the DNA of gamma– irradiated monocytes cells [45]. The discrepancies between measurements achieved by either the HPLC–MS or GC–MS on the one hand, and HPLC–MS/ MS on the other hand, can be explained by the unsuitability for the two first methods operating in the single ion monitoring detection mode to deal with low amounts of DNA lesions, typically in the sub-femtomole range [106]. NH2 N •

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Fig. 8  Formation of (5¢R)-5¢,8-cyclo-2¢-deoxyadenosine (33) from •OH-mediated hydrogen atom abstraction at C5¢

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3 Singlet Oxygen Oxidation of Guanine Singlet oxygen (1O2) in the first excited 1Dg state (E = 22.4 kcal mol−1) is the only form of excited oxygen observed in cells mostly as the result of photodynamic reactions involving a type II photosensitization II mechanism. The reactivity of 1O2, a dienophile that efficiently adds to molecules rich in double bonds, is totally different from that of triplet oxygen that exhibits biradical features allowing it to react efficiently with most of the carbon-centered radicals. Evidence has been provided for the exclusive reaction of 1O2 with the guanine base of 12 and DNA, according to a Diels–Alder [4 + 2] cycloaddition reaction across the imidazole ring of the guanine moiety [31]. This gives rise to a diastereomeric pair of 4,8-endoperoxides 34 that rearrange into linear 8-hydroperoxy-2¢-deoxyguanosine 35 (Fig.  9) as inferred from mechanistic studies that have allowed characterization of the latter intermediates by low temperature NMR experiments performed on suitable model compounds [107, 108]. Subsequent reduction of 36 gives rise to the related 8-enol derivative in dynamic equilibrium with the predominant 6,8-diketo tautomeric form 15. It may be noted that the 4R* and 4S* diastereomers of spiroiminodihydantoin 2¢-deoxyribonucleosides that have been shown to be the main 1O2 oxidation products of free 2-deoxyguanosine (12) [109] have not been detected so far in DNA. Relevant information on the 1O2-mediated formation of 15 in cellular DNA was gained from experiments that have involved the use of a thermolabile [18O-]-labeled naphthalene endoperoxide derivative that is able to penetrate cells, release [18O]-1O2 and damage nuclear DNA at 37°C [110]. On the basis of HPLC–MS/MS measurement of digested 2¢-deoxyribonucleosides, it was shown that 15 thus generated in cells was subsequently labeled at C8 [111]. This is strongly indicative of the formation of 15 through 1O2 cycloaddition to guanine and not as the result of oxidative stress possibly induced by the penetration of naphthalene endoperoxide into cells. Evidence has been provided that exposure of various kinds of prokyarotic and mammalian cells gave rise to 15 (for a review, see [112]). It was shown in a comparative study involving ionizing radiation as a reference oxidizing agent that the formation of 15 in human monocytes upon exposure to UVA radiation arises from the predominant contribution of 1O2 estimated to represent 80% [113]. The remaining 20% of reactive oxygen species involved in the formation of 15 are likely •OH that is generated by Fenton-type reactions after the initial production of superoxide anion radical and its subsequent dismutation into H2O2. The nature of endogenous photosensitizers implicated through type II mechanism in the generation of 1O2 that may include quinones, porphyrins … remains to be elucidated. The sensitized formation

N

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1

O2

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H H2N

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Reduction OOH

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Fig. 9  Singlet oxygen oxidation of the guanine moiety in DNA

H N

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of 15 to UVA radiation has also been shown to occur in human explants as inferred from HPLC–MS/MS analysis [114]. A controversial issue concerning the putative cleavage activity of 1O2 on DNA strands has been clarified. It was shown by incubating human monocytes with a naphthalene endoperoxide derivative [110] that 1O2 was not able directly or indirectly to generate DNA strand breaks [115].

4 Measurement of Oxidatively Generated DNA Damage The measurement of oxidatively base and sugar damage in cellular DNA is still a great analytical challenge since initial attempts in the early 1970s to detect the formation of 5,6-dihydroxy-5,6-dihydrothymidine (8) in cells using an indirect assays [116]. This is likely due to several reasons, mostly including the very low frequency of oxidized base and 2-deoxyribose residues, ranging from a few modifications per 107 to 109 normal bases, and the high risk of artefactual oxidation reactions during DNA extraction and subsequent work-up. In that respect the use of [3H]- or [14C]-labeled thymine or [32P]-postlabeling of oxidized nucleotides has been shown to lead to overestimation of both 5,6-dihydroxy-5,6-dihydrothymine (8) and 8-oxo-7,8dihydroguanine (15) by at least three orders of magnitude as the result of self-radiolysis [117, 118]. Other sources of spurious oxidation of overwhelming normal nucleobases or nucleosides associated with the use of chromatographic methods will be discussed later. A common approach to measuring DNA damage, and, in particular, bulky DNA lesions such as bipyrimidine photoproducts, involves immunodetection of lesions carried out in either isolated cells or tissues and mostly taking advantage of sensitive fluorescence detection. However, despite a large number of immunological applications that can be found in the literature for measuring 8-oxo-7,8-dihydro-2¢deoxyguanosine (15), there are still uncertainties about the specificity of polyclonal and monoclonal antibodies raised against 15 [119, 120]. Most of the antibodies have been shown to cross-react with normal guanine bases with an efficacy of 10−4, preventing the accurate detection of 15, which is generated with a frequency at least two orders of magnitude lower. This is illustrated by the enzyme-linked immunosorbent assay measurements of 15 in human placenta DNA that were about ten-fold higher [121] than those determined by the accurate HPLC-ECD method. Evidence has been recently been obtained pointing to the lack of specificity of N45.1 monoclonal antibody raised against 15 [122]; in particular, it was found to cross-react with urea with measurements of 15 in urine [123, 124]. Other approaches including chromatographic methods and DNA repair-based assays which in most cases appear to be accurate are critically reviewed below.

4.1 HPLC-Based Methods Two chromatographic approaches, including the gas chromatography-mass spectrometry (CG–MS) and the HPLC-ECD methods, became available in the ­mid-1980s

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for oxidatively measuring base damage to cellular DNA. In pioneering work, Dizdaroglu designed a CG–MS assay including an acidic hydrolysis step followed by the derivatization of released bases prior to GC analysis [125]. The first application of GC in ­mammalian cells led to the detection of 5¢,8-cyclo-2¢-deoxyguanosine [126] and of a dozen base modifications [127] in DNA. Almost simultaneously, Floyd et al. [129] introduced the use of an electrochemical detection operating in the oxidative mode to detect 8-oxo-7,8-dihydro-2¢-deoxyguanosine (15) slightly after its discovery as a main oxidation product of 2-deoxyguanosine (12). It was later shown that other ­electroactive modified bases and nucleosides including 8-oxo-7,8dihydroadenine, 5-hydroxcytosine, 5-hydroxyuracil, 2,6-diamino-4-hydroxy-5-formamidopyrimidine (16), 4,6-diamino-5-formamidopyrimidine (26) and related nucleosides could also be detected by HPLC-ECD [130]. However, it was observed that the levels of 15 measured by GC-MS were on the average two orders of magnitude higher than those determined by HPLC-ECD [131]. The origin of these apparent discrepancies was found to be due to artifactual oxidation of guanine bases during the derivatization step of the GC–MS assay [132, 133]. Spurious oxidation was also observed for other bases including adenine and thymine [134], therefore making the GC–MS method unsuitable to accurately measure oxidatively generated base damage to DNA unless the lesions of interest are pre-purified prior to chromatographic analysis [135]. Another possibility for improving the CG–MS is to specifically release 8-oxo-7,8-dihydroguanine using formamidopyrimidine DNA glycosylase repair enzyme, thereby preventing the autoxidation of overwhelmingly normal nucleobases in the sample [136]. A second major drawback associated with the acidic hydrolysis step of GC–MS assay concerns the instability of 2,6-diamino4-hydroxy-5-formamidopyrimidine (16) and 4,6-diamino-5-formamidopyrimidine (26) that are mostly destroyed under hot acid formic treatment [137]. Another source of spurious oxidation of nucleobases that is shared by both GC and HPLC assays has been also identified. Fenton-type induced reactions were found to occur during the extraction of DNA from cells and its subsequent work-up. This may be at least partly prevented by using the chaotropic method of DNA extraction and/or adding chelating agents for trapping the released transition metals [138–140]. It may be added that under these optimized conditions, an amount of at least of 30 mg is required to further minimize the contribution of artefactual oxidation [141]. As a general remark, one may emphasize the active contribution of the European Standard Committee on Oxidative DNA Damage (ESCODD), a ­consortium of 25 European laboratories whose main aims were to compare and optimize available methods for measuring 15 in both isolated and cellular DNA [142, 143]. As one of the main recommendations, it was concluded that HPLCECD and HPLC–MS/MS were both suitable among chromatographic methods for accurately measuring 15 in cellular DNA. HPLC–MS/MS operating in the ­electrospray ionization mode [46, 144–146] is more versatile, and, on average, more sensitive than HPLC-ECD, and is presently the gold standard for monitoring the oxidative formation of single and clustered damage in cellular DNA and urine [106]. The accurate measurement of the DNA lesions requires the use of the ­multiple reaction monitoring (MRM) detection mode and isotopically labeled

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i­nternal standards. This only applies when the amount of the lesions to be measured is within the sub-femtomole range, depending on the sensitivity of detection of lesions. Therefore, the accurate monitoring of oxidatively generated damage ­present at a frequency lower than 1 lesion per 109 nucleosides may necessitate the use of MS3 detection. This has been required for the accurate measurement of ­tandem base modifications between vicinal guanine and thymine [79] and guanine and cytosine [76, 77] respectively. It is worth mentioning that the use of HPLC with a single quadrupole (HPLC–MS) for measuring the formation of oxidized nucleosides and bases in the sub-femtomole range is not appropriate. This is due to the fact that HPLC–MS is on the average 50-fold less sensitive than HPLC–MS/MS. However, by increasing the sensitivity, there is a risk of capturing fragments arising from the electrospray ionization of impurities that may interfere with the fragmentation pattern of the expected DNA lesions, even though the detection has been optimized using a single transition in MRM mode. It should be emphasized that HPLC–MS is restricted to the measurement of oxidized nucleosides when the frequency of the lesions is higher than a few modifications per 10e5 normal nucleosides, thus preventing the use of this analytical approach on cellular DNA unless hundreds of mg are ­available. This explains the report of questionable data that consists in overestimated values of the radiation-induced formation yields of 8-oxo-7,8-dihydro-2¢-deoxyadenosine (25) and 8-oxo-7,8-dihydroguanosine (15) in cellular DNA when assessed by HPLC–MS [147, 148] with respect to HPLC–MS/MS measurements [31, 40]. A similar remark applies as well to the high values of (5¢R)-5¢,8-cyclo-2¢deoxyadenosine [105] and spiroiminodihydantoin 2¢-deoxyribonucleosides [149] that were measured by HPLC–MS analysis following exposure of cellular DNA to ionizing radiation and one-electron oxidants respectively.

4.2 Enzymatic Assays HPLC–MS/MS measurements of oxidatively generated damage to DNA exhibit a fair sensitivity that is, however, hampered by the occurrence of still-uncontrolled artefactual oxidation that required acute exposure to oxidizing agents in order to observe changes. One way to overcome this difficulty is to use enzymatic assays associated with analytical methods including the alkaline gel electrophoresis (comet) assay [150, 151], the alkaline elution technique [152, 153] or the alkaline unwinding assay [154, 155] for which it was shown that adventitious oxidation to DNA one extracted from cells is negligible. In their basic version, the three assays allow the measurement of direct strand breaks and additional nicks that arise for the alkali decomposition of abasic sites and several oxidized bases, including, among others, 5,6-dihydroxy-5,6-dihydrothymine (8), 5-formyluracil (10) and 2,2,5-triaminooxazolone (21). A pre-incubation of the released DNA with DNA repair enzymes allows the conversion of classes of oxidized bases into additional DNA strand breaks. Thus bacterial formamidopyrimidine DNA N-glycosylase

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(Fpg) is able to convert mostly 8-oxo-7,8-dihydroguanine (15), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (16) and 4,6-diamino-5-formamidopyrimidine (26) into strand breaks [150]. It was shown that human oxoguanine glycosylase 1 (OGG1) was even more specific in the detection of 15 and 16 [156]. It may be added that oxidized pyrimidine bases, including 5,6-dihydroxy-5,6-dihydrothymine (8) and related cytosine or uracil glycols, may be also recognized and cleaved by bacterial endonuclease III (endo III), giving rise to abasic sites, the precursors of strand breaks under alkali conditions. The calibration of the strand breaks and enzyme-sensitive sites that are thus detected in the three biochemical assays is usually achieved using ionizing radiation as the reference-damaging agent. However, there are still some uncertainties about the quantitative measurement provided by these more global methods. In that respect, it was found in comparative studies promoted within ESCODD that the level of Fpg-sensitive sites measured by either the comet assay or the alkaline elution technique was lower than the frequency of 15 assessed by either HPLC-ECD or HPLC–MS/MS [143, 157]. A possible reason for this discrepancy would be the formation of 8-oxo-7,8-guanine containing tandem base modifications as the result of one initial •OH hit, which are refractory to enzymic cleavage by Fpg [67]. Interestingly, numerous applications of the comet assay, alkaline elution technique or alkaline elution method are available for a wide variety of studies ranging from fundamental aspects to biomonitoring environmental genotoxic effects in the human population [158–162]. It may also be noted that the alkaline unwinding method or a relaxation assay have been used to measure ­oxidatively generated damage in mitochondrial DNA [120, 163].

5 Conclusion and Future Perspectives Evidence is provided in this chapter on the formation of oxidatively generated base and sugar damage within cellular DNA that can be used as biomarkers of oxidation reactions mediated by •OH, one-electron oxidants and 1O2. The measurement of 16 single and complex lesions so far identified in cellular DNA was achieved using mostly accurate and specific HPLC-ESI/MS–MS assays that have required ­optimization of DNA extraction protocols in order to minimize the occurrence of artefactual oxidation. The HPLC method is suitable for acute conditions of ­exposure to oxidizing agents, while enzymic base assays are more appropriate, although less specific, for measuring low levels of DNA modifications as it is often the case in human biomonitoring studies. Efforts should be made for the search of still-missing lesions, including cytosine oxidation products and tandem modifications such as DNA-protein cross-links. This will require the design of dedicated assays based on the known physico-chemical features of the lesions including stability, enzymic digestibility, electrospray ionization potential... One may note the paucity of ­accurate methods for monitoring the formation of oxidatively generated damage to mitochondrial DNA at the exception of the alkaline unwinding and gene-specific repair assays [120].

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References 1. Harman D (1981) The aging process. Proc Natl Acad Sci USA 78:7124–7128. 2. Floyd RA (1999) Neuroinflammatory processes are important in neurodegenerative diseases: an hypothesis to explain the increased formation of reactive oxygen and nitrogen species as major factors involved in neurodegenerative disease development. Free Radic Biol Med 26:1346–1355. 3. Bjelland S, Seeberg E (2003) Mutagenicity, toxicity and repair of DNA base damage induced by oxidation. Mutat Res 531:37–80. 4. Halliwell B (2006) Oxidative stress and neurodegeneration: where are we now? J Neurochem 97:1634–1658. 5. Halliwell B (2007) Oxidative stress and cancer: have we moved forward? Biochemical J 401:1–11. 6. Franco R, Schonevel O, Georgakilas AG, Panayiotidis MI (2008) Oxidative stress, DNA methylation and carcinogenesis. Cancer Lett 266:6–11. 7. Møller P, Løhr M, Folkmann JK, Mikkelsen L, Loft S (2010) Aging and oxidatively damaged nuclear DNA in animal organs. Free Radic Biol Med 48:1275–1285. 8. Sayre LM, Perry G, Mark A, Smith MA (2008) Oxidative stress and neurotoxicity. Chem Res. Toxicol 21:172–188. 9. Marnett LJ (2002) Oxy radicals, lipid peroxidation and DNA damage. Toxicology 181182:219–222. 10. Misiaszek R, Crean C, Joffe A, Geacintov NE, Shafirovich V (2004) Oxidative DNA damage associated with combination of guanine and superoxide radicals and repair mechanisms via radical trapping. J Biol. Chem. 279:32106–32115. 11. Cadet J, Di Mascio P (2006) Peroxides in biological systems. In Functional Groups in Organic Chemistry – The Chemistry of Peroxides, Rappoport, Z. (ed.), Wiley and Sons, New York, Vol 2, pp. 915–1000. 12. Das AB, Nagy P, Abott HF, Winterbourn CC, Kettle AJ (2010) Reactions of superoxide with the myoglobin tyrosyl radical. Free Radic Biol Med 48:1540–1547. 13. Wagner JR, van Lier JE, Johnston LJ (1990) Quinone sensitized electron transfer photooxidation of nucleic acids: chemistry of thymine and thymidine radical cations in aqueous solution. Photochem Photobiol 52:333–343. 14. Frelon S, Douki T, Favier A, Cadet J (2002a) Comparative study of base damage induced by gamma radiation and Fenton reaction in isolated DNA. J Chem Soc Perkin Trans I 2866–2870. 15. Yamamoto K, Kawanishi S (1989) Hydroxyl free radical is not the main active species in sitespecific DNA damage induced by copper (II) ion and hydrogen peroxide. J Biol Chem 264:15435–15440. 16. Frelon S, Douki T, Favier A, Cadet J (2003) Hydroxyl radical is not the main reactive species involved in the degradation of DNA bases by copper in the presence of hydrogen peroxide. Chem Res Toxicol 16:191–197. 17. Park J-H, Troxel, AB, Harvey RG, Penning TM 2006) Polycyclic aromatic hydrocarbon (PAH) o-quinones produced by the aldo-quinones produced by the aldo-keto-reductases (AKRs) generate abasic sites, oxidized pyrimidines and 8-oxo-dGuo via reactive oxygen species. Chem Res Toxicol 19:719–728. 18. Joffe A, Geacintov NE, Shafirovich V (2003) DNA lesions derived from the site selective oxidation of guanine by carbonate radical anions. Chem Res Toxicol 16:1528–1538. 19. Medinas DB, Cerchiaro G, Trindale DF, Augusto O (2007) The carbonate radical and related oxidants derived from bicarbonate buffer. IUBMB Life 59:255–262. 20. Mothersill C, Seymour CB (2004) Radiation-induced bystander effects – implication for cancer. Nat Rev Cancer 4:158–164. 21. Prise KM, O’Sullivan JM (2009) Radiation-induced bystander signaling in cancer therapy. Nat Rev Cancer 9:351–360. 22. Bagdonas S, Dahlie J, Kaalhus O, Moan J (1999) Cooperative inactivation of cells in microcolonies treated with UVA radiation. Radiat Res 152:174–179.

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145. Glintborg B, Weimann A, Kensler TW, Poulsen HE (2006) Oltipraz chemoprevention trial in Qidong, People’s Republic of China:unaltered oxidative biomarkers. Free Radic Biol Med 41:1010–1014. 146. Singh R, Teichert F, Verschoyle RD, Kaur B, Vives M, Sharma RA, Steward WP, Gescher AJ, Farmer PB (2009) Simultaneous determination of 8-oxo-2¢-deoxyguanosine and 8-oxo2¢-deoxyadenosine in DNA using online column-switching liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom 23:151–160. 147. Tuo J, Jaruga P, Rodriguez H, Bohr VA, Dizdaroglu M (2003a) Primary and 8-hydroxyadenine resulting from oxidative stress. FASEB J 17:668–674. 148. Tuo J, Jaruga P, Rodriguez H, Dizdaroglu M, Bohr VA (2003b) The cockayne syndrome group B gene product is involved in cellular repair of 8-hydroxyadenine in DNA. J Biol Chem 277:30832–30837. 149. Hailer MK, Slad PG, Martin BD, Sugden KD (2005) Nei deficient Escherichia coli are sensitive to chromate and accumulate the oxidized guanine lesion spiroiminodihydantoin. Chem Res Toxicol 18:1378–1383. 150. Collins AR, Duthie SJ Dobson VL (1993) Direct enzymic degradation of endogenous oxidative base damage in human lymphocyte DNA. Carcinogenesis 14:1733–1735. 151. Collins AR, Oscoz AA, Brunborg G, Gaivão I, Giovannelli L, Kruszewski M, Smith CC, Stetina R (2008) The comet assay: topical issues. Mutagenesis 23:143–151. 152. Pflaum M, Will O, Epe B (1997) Determination of steady-state levels of oxidative DNA base modifications in mammalian cells by means of repair endonucleases. Carcinogenesis 18:2225–2231. 153. Pflaum M, Will O, Malher HC, Epe B (1998) DNA oxidation products determined with repair endonucleases in mammalian cells: types, basal levels and influence of cell proliferation. Free Radic Res 29:585–598. 154. Hartwig A, Dally H, Schlepegrell R (1996) Sensitive analysis of DNA damage in mammalian cells: use of the bacterial Fpg protein in combination with alkaline unwinding. Toxicol Lett 88:85–90. 155. Moreno-Villanueva M, Ffeiffer R, Sindinger T, Leake A, Müller M, Kirkwood TB, Bürkle A (2009) A modified and automated version of the ‘Fluorimetric Detection of Alkaline DNA Unwinding’ method to quantify formation and repair of DNA strand breaks. BMC Biotechnol 9:39 (9 pages). 156. Smith CC, O’Donovan MR, Martin EA (2006) hOGG1 recognizes oxidative damage using the comet assay with greater specificity than FPG or ENDOIII. Mutagenesis 21:185–190. 157. ESCODD (2003) Measurement of DNA oxidation in human cells by chromatographic and enzymic methods. Free Radic Biol Med 34:1089–1099. 158. Loft S, Högh D, Mikkelsen L, Risom L, Forochhammer L, Möller P (2008) Biomarkers of oxidative damage to DNA and repair. Biochem Soc Trans 36:1071–1076. 159. Azqueta A, Shaposhnikov S, Collins AR (2009) DNA oxidation: Investigating its key role in environmental mutagenesis with the comet assay. Mutat Res 674:101–108. 160. Lorenzo Y, Azqueta A, Luna L, Bonilla F, Domínguez G, Collins AR (2009) The carotonoid beta-cryptoxanthin stimulates the repair of DNA oxidation damage in addition to acting as an antioxidant in human cells. Carcinogenesis 30:308–314. 161. Hackenberg S, Friehs G, Froelich K, Ginzkey C, Koehler C, Scherzed A, Burghartz M, Hagen R, Kleinsasser N (2010) Intracellular distribution, geno- and cytotoxic effects of nanosized titanium dioxide particles in the anatase crystal phase on human nasal mucosa cells. Toxicol Letter 19:9–14. 162. Szaflik JP, Rusin P, Zaleska-Zmijewska A, Kowalski M, Majsterek I, Szaflik J (2010) Reactive oxygen species promote localized DNA damage in glaucoma-iris tissues of elderly patients vulnerable to diabetic injury. Mutat Res 697:19–23. 163. Trapp C, McCullough AK, Epe B (2007) The basal levels of 8-oxoG and other oxidative modifications in intact mitochondrial DNA are low even in repair-deficient (Ogg1-/-/Csb-/-) mice Mutat Res 625:155–163.

Measuring Oxidative Stress in Cell Cultures, Animals and Humans: Analysis and Validation of Oxidatively Damaged DNA Hanna L. Karlsson and Lennart Möller

Abstract  An association between oxidative stress and different diseases has been described in the literature, but there are obvious shortcomings with methods available to assess oxidative stress. This chapter focuses on the analysis of markers for oxidative stress with special attention on biomarkers for analysis of oxidatively damaged DNA. Such markers have often been used, but the levels in healthy and diseased subjects show high variation in the literature. Therefore, various efforts that have been undertaken, or are ongoing, which aim to validate different methods for analysis of oxidatively damaged DNA, will be discussed. It is concluded that measuring oxidatively damaged DNA is a useful biomarker for oxidative stress and that further validation of this biomarker is important. Oxidatively damaged DNA can be used as a marker to monitor oxidative stress in different diseases or following different exposures. However, it may also have the potential to act as a biomarker of disease development risk and may be used clinically to assess efficacy of therapy. Keywords  Comet assay • DNA damage • Oxidative stress • Proteins

1 Introduction Oxygen is essential to life but can, on the other hand, participate in the destruction of tissues and cells due to the formation of reactive oxygen species (ROS). ROS are products of normal cellular metabolism but the levels can be increased as a consequence of various environmental exposures and diseases. However, there are many defense systems available that protect from the ROS. These include enzymes, such

L. Möller (*) Unit for Analytical Toxicology, Department of Biosciences and Nutrition, Novum, Karolinska Institutet, SE-141 57 Huddinge, Stockholm, Sweden e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_30, © Springer Science+Business Media, LLC 2011

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as superoxide dismutase (SOD), which catalyses the dismutation of superoxide anion (⋅O2−) to hydrogen peroxide (H2O2) and O2, and catalase, which decomposes H2O2 to H2O. Other important enzymes are the glutathione peroxidases (GPx) that reduce hydrogen peroxides by oxidizing glutathione (GSH to GSSG). Other defense systems include dietary antioxidants such as ascorbic acid (vitamin C) and a-tocopherol (vitamin E), which can block the generation of ⋅O2− from redox cycling compounds. Metals are potentially harmful, and binding of, for example, iron by transferrin and ferritin is part of the protection against increased levels of ROS. Haem proteins (containing iron) are potentially pro-oxidants and need to be taken care of when released from damaged cells. This is done by haem oxygenase (HO) that consists of two isoforms, of which one is stress-induced (HO-1). When haem degradation takes place, bilirubin is an end product, and this compound, which is bound to albumin in the body, is considered to be an important antioxidant [1]. If these protecting systems are overwhelmed, oxidative stress can be the result. Oxidative stress can be defined as a disturbance in the pro-oxidant-antioxidant balance in favor of the former, leading to potential damage (see Fig. 1). A consequence of oxidative stress is damage to DNA, lipids and proteins. Oxidative damage to any of these molecules can contribute to disease development and there is a link between oxidative stress and many diseases, including asthma, atherosclerosis,

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Lesions of nuclear DNA

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autoimmune diseases, cardiovascular disease and cancer [1, 2]. Oxidative stress is also considered to be a phenomenon involved in the normal aging process [3]. Furthermore, on the cellular level, ROS have been shown to affect signal transduction, cell proliferation, cell death and intercellular communication [4]. It has been reported that at least 127 genes and signal transducing proteins are sensitive to reductive and oxidative states in the cell [5]. The mitogen-activated protein kinase (MAPK) and the nuclear factor-kB (NFkB) signal transduction pathways are the most examined. Definite evidence for an association between oxidative stress and diseases has often been absent due to shortcomings with methods available to assess oxidative stress. Therefore, this chapter focuses on measures of markers for oxidative stress. There will be a particular focus on biomarkers for analysis of oxidatively damaged DNA. Such markers have often been used, but the levels in healthy and diseased subjects show high variation in the literature. Therefore, various efforts that have been undertaken, or are ongoing, which aim to validate different methods for analysis of oxidatively damaged DNA, will be discussed. In the following section some commonly used markers for oxidative stress in vitro and in vivo will be discussed. Many of these methods have been used to show effects of environmental pollution, such as airborne particles. The ability of particles to cause oxidative stress has been investigated in humans, animals and cells, since oxidative stress is considered to be a key mechanism behind the adverse effects seen following such exposure [6–8]. Studies on various ambient particles as well as manufactured nanoparticles will be presented as examples of how these methods can be used to assess oxidative stress in different systems.

2 Markers for Oxidative Stress 2.1 Non-Cellular Markers Free radicals can be measured directly by using the spectroscopic technique electron spin resonance (ESR), which detects unpaired electrons. The use of specific “traps” such as DMPO (5,5-dimethyl-pyrroline N-oxide) enables the detection of short-lived free radicals via the formation of a spin adduct that yields a distinctive spectrum that is characteristic of the free radical that is trapped [9, 10]. A chemical assay that can be used to analyze redox-active compounds is the DTT-assay, which is based on the ability of redox-active compounds to transfer electrons from dithiothretiol (DTT) to oxygen. The rate of DTT consumption can be monitored by measuring non-reacted DTT with the use of the thiol reagent 5,5¢-dithio-bis(2-nitrobenzoic acid), DTNB. This assay has, for example, been used for comparing the oxidative potential of particulate matter emissions from gasoline, diesel, and biodiesel cars [11].

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Fig. 2  Karlsson and Möller

We have used another approach for measuring the oxidative potential of various particles [12, 13] as well as of other substances [14]. The principle of the method is to incubate particles/the substance with a solution of the DNA base 2¢deoxyguanosine (dG) and measure the formation of 8-oxodG by using a HPLC–UV/EC system. In one study, we showed that particles from the subway had a higher oxidative potential compared to particles from the street [12] (Fig.  2). This difference was also observed when cells were exposed and 8-oxodG was analyzed using the same method. Another approach that has been used when testing the oxidative capacity of different particles is an in vitro screening system that involves incubation of particles with a synthetic respiratory tract lining fluid (RTLF) [15]. The RTLF represents the first interface that inhaled particles will meet and has been shown to contain high concentrations of certain antioxidants. By measuring the depletion of these antioxidants, a measure of the oxidative capacity will be gained. Furthermore, these reactions may reflect reactions that are likely to occur in vivo at the air–liquid interface. A more simplified system that can be used is an “ascorbate-only” model. In this model, the rate of ascorbate depletion following incubations with various particles is analyzed by following the decrease in absorbance at 265 nm, which can be done in 96-well plates after a certain time of incubation [16].

2.2 Markers for Intracellular ROS Another approach possible to use in vitro is fluorescent probes to detect intracellular ROS. The main principle is to use leuco dyes, i.e., dyes whose molecules can acquire two forms, one of which is colorless or does not show any fluorescence. The fluorescent compounds flourescein and rhodamine can be chemically reduced to a “dihydro-form”, which rather easily can be oxidized back to the parent form

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and thus can be a measure of the oxidative capacity in a cell or a tissue. One of the most popular of these probes is 2¢,7¢-dichlorofluorescin diacetate (DCFH-DA). DCFH-DA is a non-fluorescent compound that is freely taken up by cells and hydrolyzed by esterase that removes the DA group. DCFH is then oxidized to fluorescent dichlorofluorescin (DCF) by cellular oxidants, thereby indicating the level of intracellular ROS. It is often used to detect “cellular peroxides” but it reacts only slowly with H2O2 and lipid peroxides while being more sensitive to hydroxyl radicals and peroxynitrite [9]. The method is not specific but it can be used as an assay to measure oxidative stress in general rather than any specific ROS. Another probe is dihydrorhodamine 123 (DHR) that passively diffuses across most cell membranes and can be oxidized to cationic rhodamine 123, which localizes in the mitochondria. Similar to what happens when using DCFH-DA, DHR does not directly detect superoxide but is sensible to, for example, peroxynitrite and H2O2. In a study comparing the ability of DCFH-DA and DHR, respectively, to detect cellular H2O2 in lung cancer cells, it was shown that the fluorescence increased for both substances when H2O2 was added to the cells. However, DHR seemed to be more specific for H2O2 [17]. For measuring superoxide radicals, MitoSOX Red mitochondrial superoxide indicator is an alternative. This dye is rapidly targeted to the mitochondria where it is oxidized by superoxide and shows bright red fluorescence when binding to nucleic acids. The dye seems to be oxidized by superoxide, but not by other ROS or RNS. The different dyes described here have been used by our group, as well as by others, for investigating the toxicity of various particles [18, 19].

2.3 Changes in Cellular Antioxidant Status Other markers of oxidative stress that have been used are alterations in cellular compensation mechanisms such as different antioxidants. Increases in, for example, SOD and GSH can be interpreted as oxidative stress since the cell may compensate for increased stress by upregulating the production of these antioxidants. On the other hand, decreases may also be interpreted as oxidative stress because the cellular stores may become depleted as a consequence of oxidative stress. When studying effects of particles, an induction of the enzyme HO-1 [7, 20], which responds to oxidative stress, as well as a decline in the ratio of oxidized and reduced glutathione (GSH/ GSSG), have been shown [7]. GSH is oxidized during oxidative stress to form a GSH-GSH disulfide between two GSH molecules giving rise to GSSG. The ratio of GSH and GSSG may thus be investigated by using HPLC. However, the method is time-consuming and prone to oxidation giving rise to overestimations of the amount of GSSG [21]. An alternative approach is to use a spectrophotometric/microplate reader assay method for glutathione (GSH) that involves oxidation of GSH by the sulfhydryl reagent 5,5¢-dithio-bis(2-nitrobenzoic acid) (DTNB) to form the yellow derivative (5¢-thio-2-nitrobenzoic acid, TNB), that can be measured at 412 nm [21]. DTNB is used in the presence of NADPH and glutathione reductase to convert all GSSG to GSH, and the method thus measures the sum of GSH and GSSG.

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2.4 Oxidation of Lipids and Proteins Instead of measuring the radicals directly, or indirectly with fluorescent probes, it is more common to analyze biological markers of oxidative stress. Lipids can be oxidized and products of lipid peroxidation can be measured. Malondialdehyde (MDA) has been used as a marker of oxidative stress in exhaled breath condensate, bronchoalveolar lavage and plasma [22]. An old and commonly used test for analysis of MDA is the thiobarbituric acid (TBA) test. The test material is heated with TBA under acidic conditions, and the formation of a pink color is measured. Since most TBA-reactive material in human body fluids is not related to lipid peroxidation, this test should not be used in vivo [9]. However, MDA can be analyzed reliably using other techniques such as gas chromatography with mass spectrometry detection (GC–MS) [9]. An association between personal exposure to PM2.5 and MDA in plasma for women (but not men) was reported in one study [23]. Another, and probably the best available, biomarker of lipid peroxidation is analysis of isoprostanes. These are specific end-products of the peroxidation of polyunsaturated fatty acids and most work has been done on the F2-isoprostanes arising from arachidonic acid [24]. Isoprostanes can also be analyzed in plasma and urine, using MS-based techniques and increased levels of isoprostanes have been observed in many animal and human conditions associated with oxidative stress [24, 25]. Proteins are also oxidized by ROS, and the most common marker for protein oxidation is carbonyl compounds. Carbonyls can be measured spectrophotometrically and by ELISA techniques, and the levels in tissue and plasma have shown to be elevated in many human diseases with a link to oxidative stress [25]. An association between personal exposure to carbon black and a marker for protein oxidation (plasma proteins, PLAAS) has been reported [23].

2.5 Oxidatively Damaged DNA Guanine is most easily oxidized among the DNA bases, since the oxidation potential of guanine is lower than of the other bases. The oxidative lesion 8-oxo-7,8dihydro-2¢-deoxyguanosine (8-oxodG) that is formed after oxidation of 2¢-deoxyguanosine (Fig. 3) was discovered in 1984 [26]. 8-oxodG may be formed either by oxidation of guanine in DNA, or by incorporation of an oxidized nucleotide (8-oxodGTP) during replication or repair. 8-oxodG can lead to GC → TA transversion mutations since it can form hydrogen bounds with adenine [27]. The oxidatively damaged DNA can be repaired, which is done mainly by the base excision repair (BER) enzymes that use specific glycosylases to remove the damaged DNA base. Oxyguanine glycosylase (OGG1) is considered the primary enzyme for the repair of 8-oxodG. It removes 8-oxo-guanine from opposite cytosine in the DNA strand. Another glycosylase called MYH repairs mismatches, such as adenine

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opposite oxidized guanine, that are formed due to erroneous incorporation of ­nucleotides during DNA replication. Furthermore, a specialized enzyme called MTH1 takes care of cleaning the nucleotide pool by degrading 8-oxodGTP and thereby prevents its incorporation during DNA synthesis [28].

2.5.1 Analysis of Cellular Levels of 8-oxodG The most commonly measured oxidative DNA lesion is 8-oxodG and the technique often used to measure it is high-performance liquid chromatography (HPLC) with on-line electrochemical (EC) detection. During EC-detection, a potential is applied causing oxidation (or reduction) of the compound of interest, and the subsequent current being measured is proportional to the concentration of the compound. The non-modified nucleoside dG is often analyzed using UV on-line detection and the result is presented, for example, as 8-oxodG/106 dG (Fig. 4). It is possible to analyze 8-oxodG with other methods such as GC–MS and LC–MS. When compared to HPLC–EC, fewer research groups have used GC–MS. One likely reason is that the equipment is more expensive and technically demanding. Another reason is that high baseline levels have been reported, which likely is due to artifacts formed mainly during DNA hydrolysis and derivatization that is done prior to GC–MS analysis [2]. Many advantages, such as positive identification and accurate quantification are, however, offered without any sample derivatization when using LC–MS or LC with tandem mass spectrometry (LC–MS/MS) [2]. It is, however, known that the work-up procedure in these methods can give artificial oxidation of DNA [14, 29]. When DNA is extracted from cells, the in vivo protecting enzymes and antioxidants are no longer present to handle the ROS. The chromatide structure is also destroyed, making DNA more vulnerable to damage. Previous results from our laboratory suggest that there are several ways to limit artificial oxidation of DNA during DNA extraction and preparation [14, 30, 31]. These measures include: (a) Removal of transition metals and reducing agents. This can be done by treating buffers with chelex and by adding the iron chelator desferal during sample preparation since biological samples contain iron.  

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(b) Oxidation of transition metals and reducing agents. The electron acceptor named TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl) may be used to oxidize transition metals and reducing agents to prevent them from reducing peroxides causing oxidation of dG. (c) Removal of peroxides from solutions, which can be done by using catalase that removes H2O2. (d) Reduction of temperature. Low temperatures slow down reactions and a cold work-up procedure has been developed showing low levels of 8-oxodG in lymphocytes/monocytes from healthy men.  

 

 

We have used the HPLC–EC/UV method for investigating 8-oxodG in human lymphocytes. In one study (30,) we found the mean levels in human lymphocytes/ monocytes in 60 healthy men to be around 0.7 lesions 8-oxodG per10 6 dG. In another study, we showed similar levels, 0.9 lesions 8-oxodG per106 dG in 94 healthy individuals (46 females and 48 males) [32]. We have also used this method for investigating oxidative stress in rat lungs following exposure to diesel exhaust. The levels of 8-oxodG increased in the lungs of the rats from 1 month of exposure, which likely contributed to the later development of lung cancer [33]. This method has also been used in cell cultures; we showed, for example, increased levels of 8-oxodG in cultured lung cells after exposure to subway particles, but not street particles [12].

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2.5.2 The Comet Assay A completely different method is the single-cell gel electrophoresis (SCGE) or the “Comet assay” that is used to measure DNA damage in single cells. It was first developed in 1984 to analyze strand breaks [34] but has later been modified to enable analysis of single-strand breaks (SSB) and alkali labile sites (ALS) under alkaline (pH > 13) conditions [35]. In short, cells are embedded in agarose gel on a microscope slide, after which cells are lysed and exposed to alkali to obtain singlestranded DNA. Electrophoresis is then performed under alkaline conditions and the slides are neutralized and fixed in methanol. After that, DNA is stained with, for example, ethidium bromide and the comets are examined by computerized image analysis. The electric current pulls DNA from the nucleus towards the anode, which results in an image that looks like a comet with a head and a tail (Fig. 5). The more strand breaks that are present in DNA, the more DNA will be present in the tail. By modifying the Comet assay, it is also possible to measure oxidative DNA damage. DNA is then treated with endonuclease III that nicks the DNA at sites of oxidized pyrimidines, or with formamidopyrimidine glycosylase (FPG) that recognizes, for example, 8-oxodG and FAPydG (ring-opened 8-oxodG), thereby increasing the number of DNA breaks. The difference in tail intensity before and after treatment with the enzymes gives a measure of the oxidized damage on DNA. The FPG enzyme has been used in several studies for detecting oxidative stress in humans following exposure to air pollution [36]. The enzyme is not very specific however, and in our study we could not see a correlation between the level of 8-oxodG measured with HPLC–EC and FPG-sensitive sites in human lymphocytes (n = 96) [32]. It has been reported that FPG also detects alkylated DNA sites [37], and a possible way to obtain a more specific measure of oxidatively damaged DNA is to use the hOGG1 enzyme. In an ongoing study we noted, however, that the

Fig. 5  Karlsson and Möller

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hOGG1 enzyme was less sensitive in detecting oxidatively damaged DNA when compared to FPG (unpublished results). Even though the FPG enzyme recognizes other lesions – except for 8-oxodG – the levels, when calculated as DNA lesions per 106 dG, are lower when compared to 8-oxodG measured with HPLC–EC [32, 38]. Thus, it is possible that the values obtained by using HPLC still are high due to artificial oxidation, or that the Comet assay underestimates the damage. The Comet assay has many advantages for use in biomonitoring studies, such as possibility to use material from different tissues, e.g., nasal and buccal cells, or the very limited amount of blood needed. Furthermore, it is rather economical and easy to perform. We have used the Comet assay for measuring oxidatively damaged DNA in humans and found, for example, a correlation between FPG sites and body mass index in females [32]. In animals (rats) we used the Comet assay for investigating oxidative stress caused by various carcinogenic urban air pollutants [39]. Furthermore, in lung cell lines we showed that particles from the subway caused oxidative DNA lesions [40], as well as various manufactured micrometer-sized particles and nanoparticles [18, 41]. Furthermore, the assay has also been used in various cell lines for detecting protective effects of antioxidants [42, 43]. A novel version of the Comet assay can be used to detect the activity of repair enzymes for oxidatively damaged DNA. In this assay, substrate cells that have been treated with the Ro19-8022 photosensitizer and light, which generates 8-oxodG but very few strand breaks, are used. An extract is prepared from the cells of interest (e.g., human lymphocytes) and is incubated together with the substrate cells for a certain time. The glycosylase enzymes (mainly OGG1) in the cell extract incise at the sites of oxidized bases and the repair activity is measured as the number of incisions that are generated during the incubation [44]. Using this approach, it has been shown that dietary antioxidants can alter DNA repair activity [45]. 2.5.3 Oxidatively Modified DNA Lesions in Urine As previously discussed, 8-oxodG is often analyzed in white blood cells such as lymphocytes, but it is also possible to analyze in urine. In principle, it is assumed that urinary secretion of 8-oxodG or 8-oxoguanine reflects the formation rate of oxidative damage, whereas analysis in white blood cells reflects a balance between damage and repair. The repair product from OGG1 is, however, the base 8-oxoguanine, but 8-oxodG, which often is analyzed, may result from other repair pathways, such as from MTH1. The methods that have been used for analysis of oxidatively damaged DNA in urine are either chromatographic methods or immunoassay (ELISA). While there is a consensus between various chromatographic methods, ELISA approaches seem to overestimate the levels of 8-oxodG [46]. The benefits of ELISA, on the other hand, include ease of use, rather simple equipment and high throughput. Questions have been raised whether, for example,. the diet can affect the levels of these oxidation products although this does not seem to be the case. Increase in urinary excretion has been seen in relation to air pollution [36] as well as to various diseases [2].

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3 Validation of Oxidatively Damaged DNA The various efforts that have been undertaken and are ongoing regarding validation of various methods for analysis of oxidatively damaged DNA will be described in the following section.

3.1 Results in ESCODD A meeting with participants involved in measuring oxidative damage to DNA was held at the Rowett Research Institute in Aberdeen, Scotland in 1997, and as a result ESCODD (The European Standards Committee on Oxidative Damage on DNA) was established. The aims were to critically examine various approaches to measure DNA oxidation, to identify sources of error during analysis of 8-oxodG, to produce standard and validated protocols, and to reach consensus on the likely level of oxidative damage in normal cellular DNA. In total, 28 laboratories in Europe have been involved in different interlaboratory studies, including analysis of oligonucleotides with defined amounts of 8-oxodG, calf thymus DNA and HeLa cells with experimentally induced 8-oxodG and analysis of oxidative DNA damage in pig liver [47–49]. The last round aimed at validating methods for use in human population studies. The conclusions from the ESCODD project include [29, 38]: (a) The HPLC-EC method is capable of analyzing 8-oxodG introduced experimentally in calf thymus DNA or HeLa cells. Using this method, a dose-response curve was detected and the slopes were similar among different laboratories. There was, however, no consensus on the background level. (b) In contrast, the GC–MS failed to detect a dose-response of induced 8-oxodG and should not be regarded as a reliable method for analysis of low levels of damage. (c) The methods using the FPG-enzyme (mainly Comet assay) seem less prone to artificial oxidation, and the background levels detected are much lower than those gained using chromatographic methods. These methods can be used quantitatively, but rely on careful calibration using irradiation. (d) The background level of DNA oxidation in normal human cells is likely around 0.3–4.2 lesions 8-oxodG per million dG.  

 

 

 

3.2 Work in ECVAG When using the Comet assay for assessment of oxidatively damaged DNA with the FPG enzyme, laboratories use various endpoints (e.g., per cent of DNA in

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the tail, visual classification, tail moment, tail length), which lead to different results and make it difficult to compare results between different laboratories. Furthermore, differences in protocols used by various research groups, such as differences in slide preparation, duration of enzyme treatment, duration of alkaline treatment and electrophoresis conditions lead to different estimations and there is no agreement about the normal background level of DNA lesions measured by the Comet assay. Because of this, a validation project was started within ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility) a Network of Excellence operating within the European Union Sixth Framework Program. This project, called ECNIS Comet Assay Validation Group (ECVAG), aims to minimize both intra- and inter-experiment variability in the measures of Comet assay endpoints. The aim of the first validation study in ECVAG was to investigate the interlaboratory variation in the assessment of oxidatively damaged DNA by the Comet assay. To enable the comparisons between different laboratories, primary Comet assay endpoints were converted to number of lesions/106 base pairs by calibration with ionizing radiation. Coded samples with DNA oxidation damage induced by treatment with different concentrations of photosensitizer (Ro 19-8022) plus light, and calibration samples irradiated with ionizing radiation, were distributed to the ten participating laboratories, to measure DNA damage using their own Comet assay protocols. The study showed that nine of the ten laboratories reported the same ranking of the level of damage in the coded samples. Furthermore, the variation in assessment of oxidatively damaged DNA was largely due to differences in protocols and the protocol step that influenced the results most was the alkaline treatment. After conversion of the data to lesions/106 base pairs using laboratoryspecific calibration curves, the variation between the laboratories was reduced [50]. In general, the participants in ECVAG were successful in finding a dose-response of oxidatively damaged DNA by using the Comet assay.

3.3 Work in ESCULA Another validation project within ECNIS is the European Standards Committee Urinary (DNA) Lesion Analysis (ESCULA). The primary goal of this project is to achieve consensus between different methods used to measure oxidatively modified DNA lesions in urine, and, furthermore, to establish reference ranges in healthy and diseased subjects. In a recent study, two chromatographic assays (HPLC–GC/MS and HPLC–EC) were compared with an enzyme-linked immunosorbent assay (ELISA) for analysis of 8-oxodG in urine. The chromatographic assays showed positive agreement (r = 0.89, p < 0.0001), but there was markedly worse, although still significant, agreement with the ELISA method (r = 0.43 and 0.56 for the HPLC–GC/MS and HPLC–EC methods, respectively). It was concluded that the shortcomings with the ELISA method can question its continued use in its present form [51].

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4 Oxidative DNA Damage and Relation to Disease Elevated levels of oxidatively damaged DNA have indeed been shown in many studies [2]. The question is whether the increased level seen is a cause or a consequence. Elevated levels in tumors, for example, may have occurred as a result of increased metabolism or cell turnover. Another question concerns to what degree is there enough oxidative damage to DNA in our bodies to cause cancer. Reports have suggested that there is, but as previously discussed, the reported background values of, for example,. 8-oxodG in the literature have shown high variations. Oxidative DNA damage seems to relate to an increased risk of cancer development later in life, but there is no direct “proof ” today that oxidative modification of DNA damage is a valid marker for subsequent cancer development in humans. The reason is simply that it would require a cohort study measuring, for example, 8-oxodG in lymphocytes in many thousands of healthy humans and a followup decades later to see which of them develops cancer (such a study has not yet been performed). A more practical approach would be a nested design, i.e., collection of blood samples and analysis of, for example, 8-oxodG first after the follow-up time, of samples from cases and matched controls [52]. This would require many fewer analyses, but instead storage of the samples with a risk for artificial oxidation. Urinary biomarkers of nucleotide damage are stable during storage, and may be more suitable to use [52]. Recently, Loft and colleagues showed an association between 8-oxodG urinary secretion and lung cancer risk among persons who had never been smokers [53]. Some evidence could also be gained from animal studies. Mice lacking OGG1 repair enzyme are, however, viable, and show a moderately increased level of spontaneous mutation, show no increase in tumor incidence but have increased levels of 8-oxodG in tissues such as liver [28]. Mice lacking MTH1 (that takes care of cleaning the nucleotide pool by degrading 8-oxodGTP) showed an increased mutation rate in certain genes, and increased numbers of tumors in lungs, stomach and liver about 18 months after birth compared to wild-type mice [28].

5 Conclusion This chapter has focused on the ability to analyze oxidative stress in general and oxidatively damaged DNA in particular. It concludes that measuring oxidatively damaged DNA is a useful biomarker for oxidative stress and that the validation of this biomarker is important. Such efforts have been undertaken in the interlaboratory trials within ESCODD and are also at present ongoing in ECVAG and ESCULA. Oxidatively damaged DNA can be used as a marker to monitor oxidative stress in various diseases or following different exposures. However, it may also have the potential to act as a biomarker of disease development risk and may be used ­clinically to assess efficacy of therapy.

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Acknowledgements  The authors of this paper are also partners in ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility), a network of excellence operating within the European Union 6th framework program, priority 5: “Food Quality and Safety” (Contract No. 513943), as well as ESCODD and ECVAG.

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The Roles of cAMP and G Protein Signaling in Oxidative Stress-Induced Cardiovascular Dysfunction Soumya Saha, Zhenquan Jia, Dongmin Liu, and Hara P. Misra

Abstract  Cyclic adenosine monophosphate (cAMP) is a second messenger that plays a vital role in numerous biological processes. It serves as an intracellular signal transducer in many different organisms, exerting its effect through gene expression. In addition to its effects on intermediary metabolism, it has profound effects on cellular events, including growth stimulation, cell differentiation, and development. It mediates the action of many hormones acting through the cAMP response element (CRE) at gene levels. The levels of intracellular cAMP are regulated by Adenylyl cyclase (AC) and phosphodiesterase that catalyze the formation and breakdown of cAMP, respectively. Abnormalities in cAMP levels have been implicated in the pathogenesis of vascular diseases including hypertension. Increased levels of cAMP have been shown to have anti-inflammatory and tissue-protective effects. Reactive oxygen species (ROS) that cause oxidative stress have been shown to play a major role in the initial stage of cardiovascular diseases, including hypertension, where the role of cAMP has been implicated. Although the role of cAMP-signaling during hypertension is well known, its effects on oxidative stress protection are not fully understood. Two different signaling pathways have been suggested where cAMP could play a major role in causing hypertension: (1) lowering the cAMP levels, by angiotensin II, induces ROS production that leads to over expression of Gia protein, which consequently exerts hypertensive effects, and (2) increased levels of cAMP is known to attenuate the NADPH oxidase activity in cells, resulting in decreased oxidative stress, which, in turn, would decrease hypertension. However, in hyperglycemic state, it was proposed that an altered Gi protein expression causes accumulation of cAMP that leads to oxidative stress, thus resulting in hypertension. Regardless of the actual mechanism of action, it is apparent that cAMP and G protein signaling are associated with oxidative stress and play an important role in the pathogenesis of hypertension and hyperglycemia. Keywords  cAMP • CVD • G protein • Oxidative stress

H.P. Misra (*) Edward Via Virginia College of Osteopathic Medicine, 2265 Kraft Drive, Blacksburg, VA 24060, USA e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_31, © Springer Science+Business Media, LLC 2011

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1 Cyclic AMP and G Protein Signaling Cyclic adenosine monophosphate (cAMP) is a second messenger that plays an important role in many biological processes, including intracellular signal transduction pathways. Intracellular concentration of cAMP is regulated by the activities of both adenylyl cyclase (AC) and the phosphodiesterase (PDE) enzymes. The enzyme AC, which is located at the cell membranes, converts ATP to cAMP. The decomposition of cAMP into 5¢AMP is catalyzed by an intracellular enzyme PDE. A high concentration of cAMP has been shown to posses anti-inflammatory and tissue protective effects. Erdogan et  al. have demonstrated that rats infected by Brucella melitensis had increased levels of plasma IL-12, which was suppressed by the cAMP elevating agents [1]. The polypeptide hormones (“first messengers”) cannot cross cell membranes but able to initiate their effects on their target cells via specific cell surface receptors (see Fig. 1). These first messengers encompass peptide hormones, growth factors and cytokines and mediate their intracellular effects by low molecular weight signaling molecules, such as cAMP or calcium, which are called “second messengers”. The cAMP serves as second messenger in regulating the effects of epinephrine and glucagon via the activation of protein kinases. The beta-adrenergic receptors are coupled to these first messengers to generate the second messenger, cAMP. cAMP transduces its effects on glycogen–glucose interconversion by regulating a key signaling enzyme, protein kinase A (PKA). It also regulates the passage of Ca2+ through ion channels [2]. In this regard, an elevated level of cAMP is associated with diminished vascular resistance in intact blood vessels. It also inhibits cellular migration and proliferation of vascular smooth muscle cells. An abnormal production of cAMP may play a role in the pathogenesis of vascular diseases, such as hypertension. AC, which regulates intracellular cAMP, is a lyase Catecholamines ACTH Glucagon

Angiotensin II Adenosin C-ANP4-23

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β γ αi

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Fig. 1  Adenylate cyclase pathway

5´AMP

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enzyme that catalyzes the conversion of ATP to cyclic 3¢,5¢-cyclic AMP and ­pyrophosphate. There are different isoforms of AC, which are regulated by different subunits of guanine nucleotide protein (G protein), protein kianse C and calcium–calmodulin. The AC system plays an important role in signal transduction mechanisms that regulate a variety of physiological functions, including cardiac contractility and vascular smooth muscle tone [3, 4]. This system comprises two components: receptor, and two subunits of the enzyme – the catalytic subunit and regulatory subunit. The regulatory proteins include stimulatory (Gs) and inhibitory (Gi) guanine nucleotide proteins (G proteins) [5]. The receptor is coupled to AC activation by the action of Gs. As the hormone can stimulate many Gs a-subunits, the hormone signaling is amplified. In order to avoid prolonged AC activation, the Gs a-subunit, which also possesses GTPase activity, is able to hydrolyze the GTP to switch off the AC activation. Furthermore, the prolonged action of hormone desensitizes the receptor by phosphorylation via the activation of receptor kinase. The G-protein coupled receptor, which inhibits cAMP production via inhibition of AC activity, is called the inhibitory G-protein or Gi. Thus the levels of cAMP and AC activity are regulated by Gs and Gi proteins. Alterations of Gi proteins combined with cAMP levels lead to various pathological states, including cardiovascular injury [5]. Natriuretic peptides, produced in mammalian hearts, exert their physiological effects through cell membrane receptors. There are two major classes of natriuretic peptide receptors (NPR). Those that activate guanylyl cyclase are referred to as NPR-A and NPR-B, and those that couple to AC inhibition through Gi or to activation of phospholipase C are referred to NPR-C [6]. The C-type natriuretic peptide as an endothelium-derived hyperpolarizing factor in the vasculature has recently been identified [7] and a novel signaling pathway involving activation of NPR-C has been described. It has been proposed that CNP/NPR-C signaling is a novel regulatory pathway governing coronary blood flow and protecting against ischemiareperfusion injury and provides a novel target for therapeutic intervention in ischemic cardiovascular disorders.

2 Oxidative Stress and Reactive Oxygen Species Oxidative stress is as an imbalance between reactive oxygen species production and the antioxidant capacity of the cell to prevent oxidative injury. Oxidative stress has been implicated in a large number of human diseases including atherosclerosis, pulmonary fibrosis, cancer, neurodegenerative diseases, and aging [8]. Reactive oxygen species (ROS) encompass various molecules which have wide-ranging and divergent effects on cellular functions [9]. These species are comprised of O2−, •OH, HOO•, H2O2. Although these is no consensus as to which oxidants are responsible for causing cardiovascular injury, a large number of reactive species are known to be

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formed at the sites of inflammation, including O2−, H2O2, HCl/OCl−, NOO−/ONOH. Each of these species may contribute directly or indirectly to cardiovascular injury. Chronically elevated ROS levels have been shown to play a major role in the pathophysiology of cardiovascular diseases, including hypertension. Although all ROS are derived from the reduction of molecular oxygen, the different chemical properties of individual ROS have important implications for their roles in cellular signaling. Among all species, O2− and •OH have relatively short biological halflives. The •OH radical is thus known to be the most reactive and damaging species; its half-life, measured in nanoseconds, is diffusion-limited. Superoxide (O2−) is intermediate in stability and may actually serve as either an oxidizing or reducing agent. The charge on superoxide anion (O2−) makes it unable to cross cellular membranes except possible through ion channels [9]. Accumulating evidence suggests that ROS are not only injurious by-products of cellular metabolism, but also essential participants in cell signaling and regulation [10, 11]. Although there is strong evidence of a physiological role of ROS in several non-mammalian systems, the physiological functions of ROS in vertebrates is a relatively novel concept. In bacteria, OxyR protein functions as a transcriptional regulator of H2O2− inducible genes activated by oxidants. Studies has shown that H2O2 oxidizes two conserved cysteines in OxyR to form intramolecular disulphide linkages that trigger the activation of this transcription factor, presumably by changing its confirmation [12]. Thus, in Escherichia coli, the SoxR transcription factor is activated specifically in response to O2−, generating redox cycling agents such as paraquat and menadione [13]. Activated SoxR mediates Sox gene transcription, resulting in an increase in SoxR protein that then activates the transcription of several other genes, including superoxide dismutase. In plant cells, generation of H2O2 in response to various pathogens elicits localized cell death to limit spread of the pathogen and a more systemic response involving the induction of defense genes regulating plant immunity [14]. There are several enzymatic sources of ROS in mammalian cells including the mitochondrial electron transport system, xanthene oxidase, cytochrome p450 system, the NAD(P)H oxidase(s) and nitric oxide (NO) synthase (NOS). Although all these systems may be important in certain pathophysiological processes, recent studies suggest that the main source of ROS in cardiovascular cells is a phagocyte-type NAD(P)H oxidase [15]. NADPH oxidase seems to be principal source of O2− production in several animal models of vascular diseases, including diabetes [16, 17]. NADPH is a multicomponent enzyme that comprises membrane bound subunits, p22phox and gp91phox (Nox2 or its homologues Nox 1, 3, 4 and 5), and cytosolic subunits, p47phox, p67phox and the small G protein Rac 1, that play a role in cell signaling in rat vascular smooth muscle cells and in activating NADPH oxidase. Two events are required for oxidase activation: exchange of GTP for GDP on the small G protein Rac 1 and phosphorylation of the p47phox subunit by protein kinase C (PKC). The translocation of p47phox and Rac 1 favors the assembly of active NADPH oxidase [18]. The levels of ROS have been shown to be augmented in VSMCs of spontaneously hypertensive rats, and experimental hypertension as well as in patients with various hypertensive disorders [9, 19–22]. In addition, an enhanced expression of different subunits of NADPH oxidase, such as p47phox,

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Nox4 and p22phox that has been shown in several tissues from these hypertensive rats, appears to be responsible for the enhanced activity of NADPH oxidase and ROS production. ROS, when in excess, inflict damage on macromolecules. Evidence has generally suggested that ROS act as second messengers that are required for the downstream signaling effects. ROS are involved in a variety of physiological and pathological processes. Low levels of ROS regulate cellular signal transduction and play an important role in cell proliferation [23]. High levels of ROS lead to apoptosis and necrosis, which are implicated in many disease processes including cancer, aging and neurodegenerative disorders [24]. Other than experimental animals, patients with hypertension also demonstrate increased oxidative stress and concomitant decreased activity of endogenous antioxidant enzymes in blood and mononuclear cells [25]. These patients also elicit increased oxidative DNA damage as compared to normotensive individuals [25]. In addition, patients with renovascular hypertension demonstrate increased oxidative stress together with impaired endothelium-dependent vasodilatation. Vascular ROS production is also elevated in a range of different experimental models of hypertension including AngII-induced, minerelocorticoid and renovascular hypertension [21]. ROS are also thought to play an important role in reperfusion injury. The roles of ROS as essential biomolecules in the regulation of cellular functions and toxic by-product metabolism may, at least in part, be related to differences in the concentrations of their production. ROS, particularly H2O2, are important signaling molecules involved in regulation metabolism. Significant changes in concentrations of H2O2 occur in response to cytokines, growth factors and ­biomechanical stimulation. Insulin signaling through reversible inactivation of some protein tyrosine phosphatases appears to involve H2O2 as part of the mechanism. The reactive nitrogen species such as nitric oxide (NO•), peroxynitrite (ONOO−) and NO2 play an important role in oxidative stress. NO• radicals are produced by different isoforms of nitric oxide synthase (NOS) from l-Arginine, where they serve as neurotransmitters and regulate immune response and vascular tone. NO• also has both regulatory functions and cytotoxic effects, depending on the enzymatic source and relative amount of NO• generated [26]. NO• functions as signaling molecule-mediating vasodilatation when produced in low concentrations by constitutive isoforms of nitric oxide synthase in vascular endothelial cells. NO• produced by inducible NOS in macrophages are used for microbicidal killing [27]. All three NOS isoforms have been shown to contribute to ROS production. These enzymes are susceptible to uncoupling that leads to the formation of O2− rather than NO• under certain conditions [28].

3 Messenger Action of ROS There are two not mutually exclusive theories concerning the mechanism of the messenger action of ROS that have been proposed: (1) modification of target protein molecules, and (2) changes of intracellular redox state.

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Oxidative modifications of target protein molecules.  ROS can alter protein structure and functions by modifying critical amino acid residues, inducing protein dimerization and interacting with Fe–S moieties or other metal complexes. Although protein modification could occur with oxidation of several critical amino acids, the oxidative modification of cysteine within the functional domain of proteins causing protein modification is well described in the literature. The sulfhydryl group (−SH) of a single cysteine residue may be oxidized to form sulfenic (−SOH), sulfinic (−SO2H), sulfonic (−SO3H), or S-glutathionylated (−SSG) derivatives (Fig.  2). Such alterations may alter the activity of an enzyme if the critical cysteine is located within its catalytic domain [14, 29] or the ability of a transcription factor to bind DNA, if it is located with in its DNA binding motif [30]. Shimid et  al. recently described a novel mechanism for redox priming based on the ability of phosphocreatine in combination with H2O2 to serve as an alternate phosphate donor for autpho-sphorylation [31]. Alterations in intracellular redox state.  In comparison with the extracellular environment, the cytosol is normally maintained under strong reducing conditions. This is accomplished by the redox-buffering capacity of intracellular thiols, primarily GSH and thioredoxin (TRX). The high ratios of reduced to oxidized GSH and TRX are maintained by the activity of GSH reductase and TRX reductase, respectively. Both of these thiol redox systems counteract intracellular oxidative stress by reducing both H2O2 and lipid peroxides, reactions that are catalyzed by peroxidases. GSH has been reported to regulate redox signaling by altering the levels of intracellular glutathione (GSH) and its ratio to glutathione disulphide (GSSG) [8, 32]. Cellular GSH depletion has been found to be associated with decreased cell proliferation in vascular endothelial cells [33] and increased proliferation of fibroblasts [34]. The thioredoxin (TRX) system (TRX, TRX reductase, and NADPH) is a ubiquitous thiol oxidoreductase system that regulates cellular reduction/oxidation (redox) status. TRX is a small multifunctional protein with two redox-active

Protein (active)

SH

O2− (H2O2)

Protein (inactive)

SOH Sulfenic acid derrivative

−SO2H (sulfinic acid derivative) −SO3H (sulfonic acid derivative)

GSH

Thioltransferase

Protein (reversible)

SSG (Sglutathionylation)

Fig. 2  Modification of proteins by oxidation of cysteine residues

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cysteins within a conserved active site. Recent evidence suggests that TRX can regulate the activity of some proteins by directly binding to them [14]. A mammalian two-hybrid assay revealed direct association between TRX and the glucocorticoid receptor, and this interaction was favored under oxidative conditions within nucleus [14]. There is also evidence that TRX can translocate from cytosol to the nucleus in response to oxidant stress to regulate gene expression through Ref-1. Ref-1 is redox factor 1, which is identical to a DNA repair enzyme. The activity of Ref-1 is in turn modulated by various redox active compounds, including TRX. Binding and activation of Ref-1 by TRX facilitates DNA binding of the JunFos complex to the AP-1 site to mediate transcription [35]. The essential role for TRX to limit oxidative stress directly via antioxidant effects and indirectly by protein–protein interaction with key signaling molecules, such as apoptosis signalregulating kinase1, was reviewed by Yamawaki et al. [36]. It has been proposed that TRX and its endogenous regulators may be useful in clinical therapies in oxidative stress-induced cardiovascular disorders. The production of ROS in the cells can be stimulated by different agents such as angiotensin II (AngII), endothelin I and different types of growth factors [15]. The peptide hormone AngII is a potent stimulator for NAD(P)H oxidase in vascular smooth muscle cells (VSMCs), cardiac myocytes, and isolated hearts subjected to ischemia/reperfusion [15].

3.1 G Protein Signaling and Oxidative Stress in Hypertension 3.1.1 Role of superoxide anions A role of oxidative stress in AngII-mediated cell signaling during hypertension has been established [37]. Anand-Srivastava and colleagues have shown that there is a direct role of oxidative stress in AngII-mediated increased expression of Gia protein and associated AC [38]. Angiotensin II treatment in vascular smooth muscle cells from embryonic thoracic aorta of rat enhanced the production of superoxide anions that contributes to the enhanced expression of Gia protein in these cells. It also increased the expression of Nox4 and p47phox, two major subunits of NADPH oxidase. The antioxidants diphenyleneiodonium (DPI), N-acetyll-cysteine (NAC), alpha-tocopherol and apocynin decreased the enhanced expression of Gia2 and Gia3 proteins. In addition, antioxidant treatment also restored the AngII-induced increased receptor-dependent and -independent AC activity, thus decreasing cAMP levels. The stimulatory hormones, such as forskolin and oxotremorine, blocked the Gsa-mediated stimulation of AC in AngII-treated cells that was restored to control levels by DPI, suggesting that AngII-induced enhanced levels of Gia proteins and associated functions resulting in decreased levels of cAMP in vascular smooth muscle cells. This may be attributed to the AngII-induced enhanced oxidative stress, which exerts its effects through mitogenactivated protein kinase signaling [38].

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Mitogen-activated protein kinases (MAP kinases) are serine/theronine-specific protein kinases that respond to extracellular stimuli and regulate various cellular activities, such as gene expression, mitosis, differentiation, proliferation, and cell survival/apoposis [39]. It has been shown by Anand-Srivastava et  al. that MAP kinases are coupled with G protein signaling [40]. It has also been reported that the enhanced expression of MAP kinases in vascular smooth muscle cells from spontaneously hypertensive rats was attenuated to control levels by antioxidants, suggesting a role of oxidative stress in enhancement of MAP kinase phosphorylation. An antioxidant such as DPI was shown to inhibit the expression of MAP kinases induced by Angiotensin II in vascular smooth muscle cells from embryonic thoracic aorta of rat, thus suggesting the role of oxidative stress in increased expression and function of Gia protein [38]. The vascular smooth muscle cells from spontaneously hypertensive rats (SHR) exhibit enhanced levels of Gia proteins as compared to control rats. The enhanced Gia protein levels were restored to control levels by antioxidant treatments, supporting the notion that oxidative stress plays an important role in enhancement of Gia proteins in SHR [41]. Thus, it has become apparent that G protein signaling pathway and cAMP play important roles as modulators of ROS signal transduction in VSMC. The downstream signaling molecules of Angiotensin II receptor 1 (AT1) activation including oxidative stress in SHR has received more attention recently. The expression of Gia protein that couples to AT1 receptor of the adenylate cyclaclase system was enhanced in SHR and this effect was attenuated by the antagonist of AT1 receptor and decreased oxidative stress in these animals. Furthermore, treatment of VSMC from SHR with pertussis toxin, which inactivates and decreases the levels of Gia protein, resulted in the attenuation of the enhanced levels of O2− and NADPH oxidase activity. In addition, natriuretic peptide receptor activation (that decreased Gia protein) in VSMC from SHR was also shown to restore the enhanced levels of O2− and the increased NADPH oxidase activity to control levels. This data provides further evidence that the enhanced levels of Gia proteins and decreased cyclic AMP levels may contribute to the enhanced oxidative stress in SHR. The enhanced oxidative stress in SHR has been attributed to the decreased levels of cAMP, which is a downstream signaling molecule of Gia protein [42]. There is evidence that VSMC from SHR exhibit decreased levels of cAMP, which were attributed to the decreased basal levels of AC. Taken together, it is evident that the decreased levels of cAMP, the downstream signaling molecule of Gia protein, may be one of the important factors responsible for the enhanced oxidative stress in SHR. This hypothesis was further supported by studies showing that increasing the intracellular levels of cAMP by a cAMP donor 8Br-cAMP, isoproterenol, and forskolin (FSK) resulted in the attenuation of enhanced levels of O2− in SHR almost to control WKY levels [42]. In addition, these agonists also restored the enhanced expression of p47phox and Nox 4 protein and augmented the enhanced activity of NADPH oxidase in VSMC from SHR to control WKY levels. The implication of decreased levels of cAMP in enhancing oxidative stress was further supported by our studies showing that AngII induced enhanced production of O2−, enhanced

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NADPH oxidase activity, and enhanced expression of p47phox, and Nox4 in VSMC form thoracic aorta of rat, were all attenuated to control levels by 8Br-cAMP [42]. Although there is no clear consensus on the cause or effects, a large number of agents that induce cAMP production also attenuate oxidative stress. Thus, Cholera toxin, which increases intracellular cAMP levels, and dibutyryl cAMP (dbcAMP) have been shown to decrease the production of O2− stimulated by parathyroid hormone in bone-resorbing osteoclasts [43]. Similarly, cyclic AMPelevating agents, such as forskolin (FSK), IBMX, and dbcAMP, were shown to decrease 2-deoxy-ribose-induced oxidative damage in pancreatic beta cells. The dbcAMP was also shown to attenuate the production of ROS in granulocytes from healthy subjects. In addition, theophylline (an inhibitor of cAMP phosphodiestarase) was shown to inhibit cadmium-induced lipid peroxidation and oxidative stress in rat submandibular saliva as well as diabetes-induced oxidative stress in rats [44]. Marcil et al. have reported that the enhanced levels of Gia protein and associated decreased levels of cAMP precede the development of high blood pressure in SHR [45]. Because these two signaling molecules contribute to the enhanced production of ROS, and enhanced oxidative stress also occurs before the onset of hypertension, it is possible that oxidative stress is one of the contributing factors in the pathogenesis of hypertension. In support of this contention, the increased expression of p47phox protein, which is the major cytosolic component of NADPH oxidase and translocated to cell membrane when the enzyme is activated, has been reported in kidney from prehypertensive SHR. Decreased levels of cAMP, either in SHR or rats treated with AngII, contribute to the enhanced oxidative stress in VSMC. Taken together, it is apparent that the elevation of the intracellular levels of cAMP by cAMP-elevating agents attenuates the oxidative stress by decreasing the activity as well as the expression of Nox4 and p47phox subunits of NADPH oxidase. These results also suggest that elevated levels of cAMP in SHR may be a protective response against oxidative stress and vascular complications prevalent in these hypertensive rats [42].

4 Role of Peroxynitrite in G-Protein Signaling Nitric oxide (NO) is a diffusible messenger that plays a role in a variety of physiological functions including vasorelaxation, inhibition of platelet aggregation, inflammation, neurotransmission, hormone release, cell differentiation, migration, and apoptosis [46]. S-nitroso-N-acetylpenicillamine, a nitric oxide donor, decreases the levels and functions of Gia proteins by formation of peroxynitrite (ONOO(−)) in vascular smooth muscle cells (VSMC). Peroxynitrite decreases the expression of Gi proteins and associated functions in VSMC through a cyclic Guanosine monophosphate (cGMP)-independent mechanism and may involve the MAP kinase signaling pathway [47]. In addition, it has been demonstrated that cells treated with NO donor S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitropruside (SNP) for 24 h, decreased the Gia2 and Gia3 proteins expression without affecting Gsa

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proteins in vascular smooth muscle cells from embryonic rat aorta and primary cultured cells. The decreased expression of Gia proteins was reflected in decreasing Gi functions (receptor dependent and independent), adenylyl cyclase activity and lowered cAMP levels in the cells. The decreased Gi functions were restored to control level by scavengers of peroxynitrite, such as uric acid and MnTBAP. The decreased Gi function and Gia expression by NO donors was also restored by superoxide dismutase mimetic agents that inhibit the production of peroxynitrite. These data suggest that NO, through formation of peroxynitrite, alters the levels of Gia protein and its function, thus altering the level of cAMP and influencing the G-protein signaling [46].

5 NPR-C and Oxidative Stress Natriuretic peptide receptor C (NPR-C), a 67 kDa protein, which inhibits the regulatory Gi protein and activates AC, is also known to be involved in activation of phospholipase C. It is known to have no guanylyl cyclase activity. It has been shown by Saha et al. that NPR-C activation decreases the enhanced oxidative stress in VSMC of SHR. This activation was shown to decrease the augmented levels of Gia protein by decreasing levels of NADPH oxidase [48]. This receptor activation decreased the levels of O2− and NADPH oxidase activity in aortic VSMC of SHR through the inhibition of Gia protein levels as well as the expression of p47 phox and Nox4 subunits of NADPH oxidase. Both the Gia protein levels and NADPH oxidase activity are normally higher in these species of animals. We have shown that pertussis toxin, which inactivates and decreases the levels of Gia protein by ADP-ribosylation in SHR, also resulted in the attenuation of NADPH oxidase activity and O2− production, suggesting that Gia protein may, in part, be involved in the activation of NADPH oxidase resulting in enhanced production of O2− in these animals. Marty et al. [49] have recently shown that Gia activates the NADPH oxidase promoting the interaction of p67phox and promotes the association of p67phox and p47phox subunits of this enzyme in both transfected HEK 293T cells as well as in peripheral blood polymorphonuclear leukocytes. Taken together, it appears likely that natriuretic peptide receptor C activation and pertussis toxininduced enhanced levels of Gia protein in SHR may impair the interaction between different subunits of NADPH oxidase leading to the attenuation of the enhanced oxidative stress normally found in these rats [48]. Because NPR-C is known to cause activation of cAMP, the role of cAMP in oxidative stress needs further clarification. The cAMP was shown to be decreased in SHR [50, 51]. The increased oxidative stress caused by the enhanced production of ROS in SHR and other animal models for hypertension is also well documented in the literature. The role of Ang II and endothelin I in enhanced production of O2−, via activation and enhanced expression of NADPH oxidase subunits in hypertensive animal models has been implicated. In addition, it has been shown that SHR

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VSMC, which exhibits enhanced production of O2−, was brought to control levels of O2−production by losartan, an AT1 receptor antagonist. Losartan was also able to downregulate the enhanced expression of p47phox in SHR [41]. These findings further support the role of AngII in enhanced oxidative stress in SHR. Furthermore, Gia protein was shown to enhance the AC system in SHR via AT1 receptor and may play an important role for the enhanced oxidative stress in SHR [41]. Thus, it is evident that NPR-C activation could increase the levels of cAMP in cells leading to oxidative stress.

6 G Protein Signaling and Oxidative Stress in Hyperglycemia The breakdown of glycogen to glucose in the liver is predominantly stimulated by the polypeptide hormone glucagon. This hormone is secreted by the pancreas when blood sugar is low. Binding of hormone to receptor activates AC generating cAMP, which plays a key role in intracellular transduction, leading to the conversion of glycogen to glucose. Hyperglycemia-induced alterations in G protein and adenylyl cyclase activity and its responsiveness to various hormones have been demonstrated in several tissues of both laboratory animals and humans. Increased oxidative stress has also been reported in hypertension and other cardiovascular diseases including diabetes [37]. It has recently been shown that the high-glucose-induced enhanced production of O2− in VSMCs was abolished by Tempol, O2− scavenger or Apocynin, a specific inhibitor of NADPH oxidase and unaffected by Rotenone, an inhibitor of mitochondrial respiratory chain complex 1, N-[omega]-nitro-l-arginine methyl ester (l-NAME), an inhibitor of NO synthase (NOS), or Oxypurinol, an inhibitor of Xanthene oxidase, suggesting that NADPH oxidase is primarily responsible for high glucose-induced O2− production in VSMCs [52]. The vasoactive peptides, such as Ang II and endothelin, under hyperglycemic conditions were shown to increase oxidative stress via activation of NADPH oxidase in VSM cells of diabetic animals and humans. A decreased level of Gia proteins was observed in aortic tissue of streptozotocin-induced diabetic rats and vascular smooth muscle cells from rat embryonic aorta was exposed to high glucose. They were restored to control levels by antioxidants. In addition, scavengers of peroxynitrite, such as Mn-tetralis (benzoic acid porphyrin) and uric acid, an inhibitor of nitric oxide synthase such as l-NAME but not catalase, also restored the high glucose-induced decreased expression of Gia proteins to the control levels in vascular smooth muscle from rat aorta. Furthermore, the enhanced production of superoxide anion (O2−) and increased activity of NADPH oxidase in these cells were also restored to control levels by diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase. The alteration of AC system induced by hyperglycemia was restored to control levels by antioxidants. This was the case when adenylyl cyclase activity was inhibited by inhibitory hormones or stimulated by forskolin and GTPgammaS as well as other

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agonists. These results suggest that the decreased levels of Gia proteins induced by hyperglycemia may be attributed to the enhanced oxidative stress [37]. It has been shown that antioxidants, such as alpha tocopherol, N-acetyl cysteine (NAC), scavengers of O2−, and an inhibitor of NADPH oxidase, such as DPI, restored the hyperglycemia-induced enhanced O2− production. These agents were able to restore the hyperglycemia-induced decreased expression of Gia2 and Gia3 to control levels, thereby decreasing the cyclic AMP level. Thus, it is apparent that NADPH oxidase plays an important role in the hyperglycemia-evoked decreased expression of Gia protein [37].

7 Conclusion In conclusion, it has become apparent that cAMP and G-protein signaling is involved in oxidative stress during hypertension and hyperglycemia. Decreased levels of cAMP and Gi protein functions contribute, at least in part, to the increased oxidative stress observed in these pathological states or vice versa (Fig.  3). Treatment with antioxidants decreases the superoxide and peroxynitrite production, thereby restoring the Gi protein functions by increasing the levels of cAMP. In addition, cAMP donors can restore the oxidative stress caused by augmented NADPH oxidase activity to control levels suggesting the role of cAMP and G-protein signaling in hypertension and hyperglycemia.

cAMP

Giα expression

NPR-C

Oxidative stress

NADPH oxidase

Vascular resistance Hypertension

Fig. 3  Possible mechanism involving cAMP and Gi protein signaling in oxidative stress

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Cellular and Chemical Assays for Discovery of Novel Antioxidants in Marine Organisms Tim Hofer, Tonje Engevik Eriksen, Espen Hansen, Ingrid Varmedal, Ida-Johanne Jensen, Jeanette Hammer-Andersen, and Ragnar Ludvig Olsen

Abstract  Low-molecular weight antioxidants receive increased interest both as protectors from disease and therapeutics, but are also used in other applications such as sun screens and food additives. Antioxidants are present in all organisms and are well characterized in terrestrial organisms whereas little knowledge exists for marine organisms. Only in Norwegian Arctic and sub-Arctic waters, thousands of species (invertebrates, algae, bacteria) are yet to be chemically characterized. Here we describe cell-based antioxidant assays (CAA and CLPAA in microplate format, and the Comet assay) measuring both hydrophilic and lipophilic antioxidant activities of single compounds and extract mixtures. Contrary to our chemical methods (ORAC and FRAP), cell-based assays assess cell membrane permeability and operate under more physiologically relevant conditions. A brief overview of our group’s work on finding novel antioxidants in marine organisms including testing activities of semipurified natural product extracts, identification of active compounds, purification of compounds in greater amounts, as well as how antioxidants can be tested in animals and humans is also described. Keywords  Antioxidants • Chemical assays • Comet assay • Marine organisms Abbreviations ABAP 2,2¢-azobis(2-amidinopropane) C11-BODIPY 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-sindacene-3-undecanoic acid CAA Cellular antioxidant activity CLPAA Cellular lipid peroxidation antioxidant activity cumOOH Cumene hydroperoxide

T. Hofer (*) MabCent-SFI, University of Tromsø, Tromsø 9037, Norway e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_32, © Springer Science+Business Media, LLC 2011

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2¢,7¢-Dichlorofluorescein 2¢,7¢-Dichlorofluorescin 2¢,7¢-Dichlorofluorescin diacetate Dimethyl sulfoxide Epigallocatechin gallate Ferric reducing antioxidant power Human hepatocellular liver carcinoma cell line Half maximal inhibitory concentration Oxygen radical absorbance capacity

1 Introduction The antioxidant field receives increased interest and currently about 10,000 articles per year concern antioxidants of various kinds (ISI Web of Knowledge). Dietary antioxidants, thought to protect from cancer, degenerative diseases, chronic inflammation conditions, etc. [1–4] are continuously used up and need to be re-supplied on a daily basis. Human diet phytochemicals of antioxidant relevance are classified as polyphenols (flavonoids, phenolic acids and non-flavonoids), terpenes (monoterpenes and carotenoids), organosulfur compounds and saponins [5]. Mechanisms of low-molecular weight antioxidants are commonly related for: • Free radical scavenging • Metal chelating • Signal transduction and gene expression. Where flavonoids can both scavenge radicals and chelate catalytic metal ions [6], and where effects on cellular signaling pathways can be very complex (e.g. as for resveratrol [7], a polyphenolic non-flavonoid stilbene) and remain to be clarified for many antioxidants. Phytochemicals may possibly also act as xenobiotics, ­activating protective cellular systems (cytochrome P450 enzymes, DNA repair, antioxidant enzymes) [8–11], thereby pre-conditioning against chemical stressors. So far, scientific focus related to dietary low molecular weight antioxidants has almost exclusively been on antioxidants present in terrestrial plants. Evolution has led to a nutritional dependence where higher organisms (humans, animals) are dependent on low-molecular weight essential biomolecules synthesized by lower organisms (plants, yeast), but also non-essential biomolecules appear ­beneficial to humans. For example, several carotenoids [12] and polyphenolic antioxidants [13] are permeable over the blood–brain barrier and may protect from degenerative diseases including Alzheimer’s, a disease for which a common theme concerns how to remove toxic iron that mediates oxidative damages [14]. The carotenoids lutein and zeaxanthin are present in the retina of the eye, protecting from light induced age-related macular degeneration [15]. Long-chain polyunsaturated fatty acids (Lc-PUFA), common in fatty seafood (e.g. salmon, mackerel and herring) and other marine products (e.g. fish oil), originate from microalgae ­(phytoplankton). These fatty acids are particular prone to oxidation and it is ­therefore likely that

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marine phytoplankton must have developed compounds and systems which inhibits such oxidation. Lc-PUFA may function as antioxidants both directly and indirectly by affecting the inflammatory pathways [16]. Intake of diets with higher ratios of long-chain omega-3 fatty acids (e.g. docosahexaenoic acid, DHA, and eicosapentaenoic acid, EPA) to omega-6 polyunsaturated fatty acids can reduce the production of omega-6 dependent inflammatory eicosanoids as well as increase omega-3 derived anti-inflammatory resolvins [17]. Omega-3 fatty acids are administrated therapeutically against several conditions including chronic inflammation. For small marine antioxidants novel discoveries are increasingly reported [18–21]. Interestingly, antioxidants found in terrestrial plants (catechin, gallic acid, catechol, morin, rutin) were reported present also in seaweeds (macroalgae) [22]. A potent interesting class of marine compounds are phlorotannins (e.g. eckol and derivatives thereof) isolated from seaweed that potently both inhibit reactive oxygen species (ROS) generation through anti-inflammatory effects [23] as well as scavenge free radicals [23, 24]. Another well-characterized class of marine (also terrestrial) origin are carotenoids (fucoxanthin, astaxanthin, etc.) [25], where particularly astaxanthin has been intensively studied and reported to have potentially positive effects in humans [26]. The sometimes extreme conditions of marine life suggest that quite different classes of compounds can be discovered and several phyla are unique to the oceans [27]. Sea ice algae living under extreme oxygen concentrations (89% oxygen) must present well-developed antioxidant systems [28], and several marine animals (e.g. clams, sea urchins, tubeworms, sea-turtles, whales, rock-fish) have 100–400 year life-spans [29] or longer (corals and sponges can live for thousands of years), which could partly be due to dietary components. Identification of new potent antioxidants requires adequate methodologies capable of handling and testing large numbers of samples of variable chemical nature, sensitive bioassays, and analytical techniques for structural identification. To date, antioxidant activities have mainly been assessed using chemical assays such as the Ferric Reducing Antioxidant Power (FRAP) assay [30, 31], but recent development of cellular assays should provide more relevant results and also limit the numbers of false positive hits in extracts. Below we go over three cellular assays: the Cellular Antioxidant Activity (CAA), the Cellular Lipid Peroxidation Antioxidant Activity (CLPAA), and the Comet assay, that in turn mainly measure intracellular, membrane lipid peroxidation, and DNA protecting antioxidant activities, ­respectively. We also briefly cover how marine extracts are prepared for assay ­testing, technologies to identify compounds, purification of larger quantities, and how identified antioxidants can be tested in animals and humans. Two quite ­different chemical assays: FRAP and the Oxygen Radical Absorbance Capacity (ORAC) assays were compared to the CAA (and CLPAA) assay with tests of extract mixtures and known compounds.

1.1 CAA Whereas chemical assays do not account for compound uptake through the cell membrane, presence of metabolizing enzymes or influence from other molecules,

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the Cellular Antioxidant Activity assay (CAA) does [32]. CAA is based on ­simultaneous cellular uptake of antioxidants and the ROS-sensitive non-fluorescent probe 2¢,7¢-dichlorofluorescin diacetate (DCFH-DA) which is hydrolyzed into cytosolically trapped DCFH [33] that upon oxidation by peroxyl radicals derived from thermal decomposition of 2,2¢-azobis(2-amidinopropane (ABAP or AAPH), forms 2¢,7¢-dichlorofluorescein (DCF; fluorescent green), which is monitored [32, 34]. The formation of peroxyl radicals from 2,2¢-azobis(2-amidinopropane) is ­illustrated by the equations below:

− R − N = N − R − ( + heat) → 2 − R • + N 2 − R • + O 2 → − ROO•





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Thus, reactions are intracellular as outlined in Fig. 1. Short term (1–2 h) incubations with up to 2% dimethyl sulfoxide (DMSO) or ethanol solvents did not significantly alter reaction rates or induce cell necrosis in any of the assays. This allows tests of lipophilic antioxidants (e.g. carotenoids) that can be serially diluted in DMSO, avoiding common precipitation and adherence to plastic surface (tubes, pipettes) issues, followed by direct mixing with cell medium

Fig. 1  Principle of and procedure for the CAA assay. The oxidant-sensitive probe DCFH-DA is hydrolyzed by cellular esterases and trapped within cells (we use human hepatocellular liver ­carcinoma cells, HepG2) as DCFH. Addition of ABAP generates a constant rate of peroxyl radicals that oxidize DCFH into fluorescent DCF. Only antioxidants (AOx) that have penetrated the cell membrane will prevent formation of DCF. HepG2 cells are seeded in black 96-well plates with transparent bottoms for fluorescence monitoring, and tests are performed the following day. DCF formation is recorded over 60 min and results can be expressed as area under curve (AUC), or as endpoint (t=60 min) minus starting value (t=0)

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as recommended [35]. For extremely fatty compounds (e.g. lycopene which ­dissolves poorly in DMSO), Teen emulsifier (instead of tetrahydrofuran) delivery to cells can be considered [36]. Of note is that many antioxidants strongly absorb light, and quenching effects should be tested for and subtracted when appropriate. Failure to do so can result in misleading conclusions, as observed effects can have been due to antioxidant light absorption per se (particularly at higher concentrations), not decreased probe oxidation. To rule out that the effects were not due to effects on esterases, DCFH-DA incubations can alternatively be performed prior to antioxidant uptake. We seed 80,000 cells per well (giving ~50% confluency for HepG2) both for CAA and CLPAA. A greater cell density can increase the signal-noise-ratio and reduce scatter of data for fluorescence measurements, but may result in somewhat higher IC50-values as higher antioxidant concentrations may be required to obtain the same protective effect. For optimal results, the plate reader needs to provide equal heating on all positions on the microplate. This is particularly of importance in assays employing heat-dependent ABAP decomposition, and can be tested for ­having positive controls in all 96 wells. Alternatively, microplate heating inside a cell culture chamber, and/or placed on an aluminum plate can be considered.

1.2 CLPAA To measure effects of antioxidants on cellular lipid membrane peroxidation, we have set up the Cellular Lipid Peroxidation Antioxidant Activity (CLPAA – our acronym) assay [37], where after cellular uptake of the oxidation sensitive probe C11-BODIPY (makes cell membranes fluorescent red) and subsequent antioxidant uptake, lipid peroxidation is induced by adding cumene hydroperoxide (cumOOH) [38, 39]. Free radical attack on C11-BODIPY generates two similar fluorescent green products (both having lost a phenyl group), correlative to the degree of lipid peroxidation that has taken place [39, 40]: C11 - BODIPY (red) (+ oxidation) → C11 - BODIPY oxidation products (green)

(3)

Carotenoids (highly lipophilic), several of marine origin, localize to membranes and will be interesting to study for their roles in lipid peroxidation processes. Several studies have shown positive effects for the marine carotenoid astaxanthin in vivo [26, 41, 42], but it is unclear if this is due to scavenging of free radicals or other effects (e.g. activation of genes or effects from metabolites) [43]. Carotenoids contain multiple carbon-carbon double bonds that absorb UV-light [15] and can react with singlet oxygen (1O2) produced from UV-light induced photosensitization reactions [44]. However, 1O2 is of relatively low physiological importance. Carotenoids may also react with peroxyl radicals [45] but may be incapable in halting lipid peroxidation, becoming degraded themselves [46].

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1.3 The Comet Assay Mainly intended as a follow-up assay for pure compounds we have set up the Comet assay (also called the single cell gel-electrophoresis assay) which measures cellular DNA damage [47]. In principle, DNA strand breaks relax supercoiled DNA loops which can then extend under electrophoresis to form comet-like tails outside damaged cells. This assay can test a compound’s protective effect from oxidants (peroxides, metals, irradiation) on DNA damage, effects on DNA repair rates [48], as well as test if the compound itself is genotoxic. Both freshly isolated and ­cultured cells can be studied, as well as tissues – given that the cells can be separated into intact single cells. Optionally, a DNA-lesion recognizing enzymatic (e.g. Fpg, hOGG1, endonuclease III) step can be included, where additional strand breaks are created at sites of modified DNA-bases, but this step is not necessary when using H2O2 which generates sufficient DNA strand breaks. However, the procedure is presently quite laborious and time-consuming involving many steps as schematically summarized in Fig. 2.

1.4 ORAC The Oxygen Radical Absorbance Capacity (ORAC) assay is a chemical assay commonly used for hydrophilic compounds [49], but can be adjusted for lipophilic

Fig. 2  Principle of the Comet assay. Cultured cells are incubated with antioxidants, trypsinized and dissociated into single cells, mixed with low melting point agarose cast on microscope slides on ice. After exposure to hydrogen peroxide (H2O2) in ice-cold buffer and treatment with lysis solution (detergent and high salt removes membranes and histones from the DNA, creating ­nucleoids of supercoiled DNA attached to the nuclear matrix), treatment with high alkali (unwinds and denatures DNA), electrophoresis is performed under alkaline conditions (broken DNA migrates out of the nucleoid forming the shape of a comet). Then, gels are pH neutralized, the DNA stained (Hoechst 33258) and visualized by fluorescence microscopy using a black and white video camera connected to a computer. Live scoring of 50–100 comets per gel is performed using a software (such as Comet Assay IV from Perceptive Instruments, Haverhill, U.K.) where the relative intensity of tail/head reflects DNA break frequency (“percentage of DNA in the tail” is the common parameter chosen). Antioxidants can prevent from DNA damage and decrease the tail intensity. The assay is sensitive towards over-trypsinization which can be stopped by adding serum-containing medium. Small mitochondrial DNA (mtDNA) is not studied with this ­technique

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components by including a solubility enhancer [50]. The pH and temperature used in the assay are simulating physiological conditions, and the assay measures inhibition of reactive oxygen species (ROS). Thermal decomposition of ABAP generates peroxyl radicals which oxidize the fluorescent probe fluorescein, and antioxidants have a protective effect delaying loss of fluorescence. As no living cells are included in the assay, the ability of compounds to penetrate the cell membrane is not tested.

1.5 FRAP The Ferric Reducing Antioxidant Power (FRAP) assay measures the ability of antioxidant to reduce ferric tripyridyltriazine complex (Fe3+-TPTZ) to a ferrous complex (Fe2+-TPTZ) at non-physiological low pH [51] and notably does not involve production of ROS. The chemical assays FRAP and ORAC are considerably easier to use than cell-based assays and demand no cell culture maintenance and have shorter assay procedures.

2 Materials and Methods 2.1 Cell Culture, CAA and CLPAA Experimental Procedures The used experimental procedures are based on publications for CAA [32], CLPAA [37, 39], the Comet assay [10], ORAC [49], and FRAP [51] respectively with minor modifications. Briefly, for cell-based assays, human hepatocellular liver carcinoma cells (HepG2) are cultured in minimum essential medium Earle’s (MEM Earle’s) supplemented with gentamycin, l-glutamine, non-essential amino acids, sodium pyruvate and 10% fetal bovine serum (FBS) inside 175 cm2 flasks in humidified 5% CO2 atmosphere at 37°C. After washing cells with phosphate buffered saline (PBS, Mg2+- and Ca2+-free), cells are detached using trypsinization and well resuspended in cell culture medium using a pipette. The cell concentration is determined by Bürker chamber counting, and 80,000 cells are seeded into black 96-well microplate wells having transparent bottoms (Corning or Nunc brand). For microplates, final volumes are 100 ml. For the Comet assay, cells are seeded into 6 or 12 well plates. Cells are allowed to settle at room temperature to minimize plate edge effects [52] before incubation at 37°C. After 24 h, experiments are conducted. For CAA, cells are washed with PBS and incubated with compounds (or fractionated extracts) and DCFH-DA (25 mM) simultaneously in “treatment medium” (FBS-free MEM Earle’s medium as above) for 60 min at 37°C. After a PBS wash, clear Hank’s buffer supplemented with glucose containing ABAP (600  mM) is added and the green fluorescence (485/14 nm excitation and 520/10 nm emission filters; CW-lamp: 15,000 stabilized energy) monitored for 60 min inside a Wallac VICTOR3 1420 multilabel counter (Perkin Elmer, Waltham, MA) at 37°C set at bottom reading. Control wells having no oxidant, or a known antioxidant

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(when screening fractionated extracts), are included in all plates. To correct for the ­antioxidants’ own light absorption, as well as fluorescent molecules present in the probe when purchased, CAA data records for each well are baseline corrected ­setting the lowest reading point for each well to zero, prior to performing summation integrals generating area under curve (AUC) values. This ensures that only experimentally generated DCF formation during the 60  min exposure (+ABAP) time is quantified, and implies that a “theoretically perfect” antioxidant should have zero AUC units. For CLPAA, cells take up C11-BODIPY (5 mM) for 30 min (37°C) prior to incubations with compounds (60 min, 37°C) and cumOOH exposure (60 min, 37°C) in Hank’s buffer and fluorescence monitoring over 60 min, with frequent alternating measurements of the red (590/7 nm and 632/45 nm filters; CW-lamp: 10,000) and green (485/14 nm and 520/10 nm filters; CW-lamp: 20,000) fluorescence [37]. Incubation times and wash procedures can be adjusted to test a compound’s rate of action and duration in cells. Stocks of 0.1 M cumOOH (for CLPAA) or H2O2 (for the Comet assay) are prepared immediately prior to use as diluted peroxides are unstable. Lipophilic antioxidants can be serially diluted in pure DMSO prior direct addition into wells to give 1% DMSO (1 ml is added to 99 ml medium) in all wells including controls. Antioxidants are sensitive to oxidative modifications particularly at dilute concentrations and repeated tests of different preparations (weighouts and dilutions) on different days are recommended to confirm results. Aliquots of DCFH-DA (20  mM in ethanol), ABAP (200  mM in water) and C11-BODIPY (6 mM in DMSO) are kept at −80°C and thawed immediately prior use.

2.2 Extracting, Screening and Identifying Marine Natural Compounds The process of identifying bioactive molecules from marine organisms starts with the collection and taxonomic determination of the marine animals, plants or microorganisms. A correct taxonomic determination is crucial if a bioactive compound is found, as it may be required to recollect larger amounts of the specimen for further chemical and biological characterization. When extracting chemicals from biological specimens, the objective is to release the widest possible array of compounds with respect to chemical properties. In order to ensure that the solvents are accessible to all parts of the sample and to avoid the formation of hydrophobic pockets, the marine organisms are freezedried and ground prior to extraction. It is desirable to start the sample preparation from at least 1 kg (wet weight) of the marine specimen. We commonly use a twostep extraction protocol. First, the water soluble compounds are extracted with distilled water (10×) over night at 4°C. Then, the aqueous extract is centrifuged and the supernatant freeze-dried. Next, the pellet is freeze-dried and its lipophilic ­compounds extracted with dichloromethane:methanol (1:1 vol:vol; 10×). After ­filtration, the organic extract is dried under reduced pressure in a rotary evaporator.

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The extractions take place in darkness in order to minimize the risk of degradation of photosensitive compounds and the concentrated dry organic and aqueous extracts are stored at −20°C. The crude organic and aqueous extracts of the marine samples are very complex, typically containing hundreds of different compounds at vastly different concentrations, and are therefore not suitable for immediate bioactivity screening. Thus, prior to the screening, aliquots (~200 mg) of the extracts are separated into 40 fractions using reversed phase (RP) semi-preparative HPLC (step F1 in Fig. 3) which can be done in an automated manner. When bioactivity is detected in one or more of the fractions during assay screening (S1), the fractions are re-tested after dilution (1:1, 1:2 and 1:4) for confirmation of bioactivity (S2). If confirmed, the active fraction is subjected to a second purification step (refractionation) using RP semi-preparative HPLC with focused gradients, and 40 fractions (each collected over 1 min; F2) are tested for bioactivity (S3).

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Fig. 3  Scheme over the steps involved in natural compound discovery. After homogenization of the specimen and extraction of chemicals, the extract is fractionated (F1) using semi-preparative HPLC and collected fractions screened (S1) in assays for bioactivity. Highly active fractions are re-tested for activity after dilution (S2), and if still of interest further purified by HPLC and ­collected fractions (F2) screened for activity (S3). Active fractions containing semipurified ­compounds are analyzed using mass spectrometry (MS) and results matched against compound libraries, after which a decision on up-scaled purification is taken

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If the bioactivity is recovered, the active fraction is analyzed by high-resolution mass spectrometry (HR-MS). By acquiring accurate mass data at high resolution it is possible to assign an elemental composition to the bioactive compound. The elemental composition and the biological origin are used to search databases, making it possible to eliminate known antioxidants from the pipeline at a relatively early stage. This process is known as dereplication. If the active compound appears potentially valuable, an up-scaled purification of the remaining part of the crude extract (~5 g) is required to provide sufficient material for a full structure elucidation and studies of the biological mode of action. This is labor intensive and time-consuming work typically including liquid–liquid extractions, low-pressure (flash) chromatography and semi-preparative HPLC with different stationary phases (adsorbents). The structure of the novel compound is determined using a wide array of analytical techniques such as high-resolution tandem MS, nuclear magnetic resonance (NMR), infrared (IR) and ultraviolet–visible (UV–VIS) spectroscopy, optical rotatory dispersion-circular dichroism (ORD-CD) and X-ray crystallography. Even though several of the analytical techniques have become increasingly sensitive over the last decade, milligram amounts of the pure bioactive compound are still needed for a full three dimensional structural characterization. When the structure of the bioactive compound has been determined, it is verified by database searches whether it is a truly novel compound or not.

3 Results and Discussion 3.1 CAA Effects of two oxidants, ABAP and H2O2, were compared kinetically in the CAA assay, Fig. 4. The effect of H2O2 saturated at higher concentrations (Fig. 4b), and at 5 mM quickly exerted maximum effect (Fig. 4a). ABAP displayed a 15 min lagphase likely related to cell membrane passage and heating of the microplate, but then caused constant oxidation of DCFH into DCF (Fig. 4a) likely attributable to constant peroxyl radical formation, despite at a lower (600 mM) concentration than H2O2 (5  mM). Thus, ABAP was more efficient than H2O2, which may relate to ­cellular removal of H2O2, or H2O2 being dependent on cellular ferrous iron (Fe2+) for generation of oxidizing short-lived hydroxyl radicals (HO•), whereas ABAP decomposition generates longer-lived peroxyl radicals (ROO•). Fluorescence monitoring for 60 min using ABAP as oxidant was found suitable for testing antioxidant effects of compounds, as was suggested [32]. Antioxidants can be compared in parallel at identical concentrations, or by comparing half maximal inhibitory concentrations (IC50, a measure of the effectiveness of a compound). However, as antioxidants differ greatly in efficacy, comparison of antioxidants at a fixed concentration may give little information, and sigmoidal ­dose-response curve-fits using the four parameter logistic equation can provide IC50-values and curve slopes (called the Hill slope) for comparisons, see examples for epigallocatechin gallate (EGCG) and hydroxytyrosol using CAA in Fig. 5.

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Usually, a strong concentration dependence is seen for potent antioxidants in the mM–mM range (−6 to −3 on a log-scale), and even if full protection from DCF formation cannot be achieved, approximate IC50-values can be obtained by extrapolating the curve to the bottom plateau. Absolute IC50-values are dependent on the cell density and other experimental conditions, and therefore, maintaining constant conditions and careful cell counting is necessary for reproducible results.

3.2 CLPAA In previous studies, both the decrease in red, and increase in green, C11-BODIPY fluorescence have been used to estimate lipid peroxidation, but from our experience [37] measuring the rate of increase in green fluorescence works better than measuring

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log[compound (M)] IC50 (hydroxytyrosol)=10−3.44=366 µM ; Hill slope: −0.32 IC50 (EGCG)=10−5.05=8.86 µM ; Hill slope: −0.67

Fig. 5  CAA assay dose-response tests. (a) Chemical structures for EGCG and hydroxytyrosol, antioxidants present in green tea and olive oil, respectively. Hydroxytyrosol derives from hydrolysis of oleuropein. (b) With increasing concentrations of antioxidants, DCF (fluorescent) formation from ABAP derived peroxyl radical mediated oxidation of DCFH is prevented. The “controls” (+ABAP and −ABAP) are selectively placed at concentrations distant (here at 100 pM and 100 M, respectively) from the tested range of antioxidants (0.2 mM–1.6 mM). This experiment was performed in a 96-well plate and each concentration tested in triplicate (N = 6 for controls, averages shown). Log-transformation of data and curve fits generating IC50-values and Hill slopes was performed using Prism 5.0 (GraphPad Software, San Diego, CA). High concentrations of many antioxidants give less DCF fluorescence than the “−ABAP” control, the latter being a measure of endogenous oxidation reactions inside cells. Thus, for IC50-value calculations a “theoretical zero level” of DCF formation is used as bottom plateau (y-value constrained to 0) for extrapolation of dose-response curves. The top plateau is constrained to the “+ABAP” average, a measure of ABAP as well as endogenously derived oxidants

the decrease in red fluorescence which, in relation to the increase in green, can be relatively low, Fig. 6. In addition, we found the background fluorescence from the used 96-well plastic plate to be very strong in the red light range. We also tested using endpoint green/red fluorescence ratios, but found that certain compounds affect (quench) the green and red light signals differently, thus exerting an unwanted nonlinear effect particularly evident at higher compound concentrations. Setting the

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Fig.  6  CLPAA assay kinetics and lipid peroxidation prevention by hydroxytyrosol. Cumene hydroperoxide (cumOOH; 50 mM) initiated HepG2 lipid peroxidation, indirectly monitored by (a) formation of fluorescent green C11-BODIPY oxidation product, and (b) disappearance of C11BODIPY (fluorescent red). The antioxidant hydroxytyrosol prevented C11-BODIPY oxidation, and (c) plotting the rate of fluorescently green C11-BODIPY oxidation product increase (obtained from linear regressions of slopes over 0–40 min) against the hydroxytyrosol concentration gave an IC50 of 114 mM. Each point represents averages from triplicate wells (N = 6 for controls). Top (“+cumOOH” average) and bottom plateaus (y = 0) are constrained. Abbreviation: a.u. = arbitrary units

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excitation wavelength >580 nm for red fluorescence avoids light absorption by C11BODIPY oxidation products that increase during the course of the experiment [39]. How much cumOOH and C11-BODIPY to use depends on the cell concentration, incubation time, instrument sensitivity etc., but for concentrations below 1 mM C11BODIPY (80,000 HepG2 cells per well), discriminating the red and green signals from background (cells, plastic) can become difficult from our experience.

3.3 The Comet Assay Flavonoids have been reported to both scavenge free radicals and remove iron from cells [6], and when tested, the flavonoid quercetin displayed a small protective effect (at 50 mM) from H2O2-induced DNA damage, whereas no significant increase in DNA strand breaks was seen for quercetin alone, Fig. 7.

3.4 Comparison of Chemical and Cellular Assays: FRAP, ORAC, CAA and CLPAA When the same fractionated aqueous extracts of an unusually antioxidant rich marine organism were tested for antioxidant activities with the FRAP, ORAC, CAA and CLPAA assays, activities of most fractions correlated between the assays, Fig.  8. Notably, fractions 18–31 gave strong responses in all assays. However, whereas fraction 3 (very hydrophilic) was highly active in FRAP and ORAC, it showed no activity in CAA or CLPAA which may be due to inability of compounds to be taken up into (and pass) the cell membrane. When comparing identical aliquots of commercial antioxidants using FRAP, ORAC and CAA, only few compounds correlated between the different assays, Fig. 9, which suggests that the assays measure different parameters, as suggested by others [53]. The most potent antioxidants were (in order) EGCG, quercetin and myricetin with FRAP, catechin, resveratrol, and quercetin with ORAC, and kaempferol, luteolin, quercetin with CAA, Fig.  9. The marine carotenoid astaxanthin exerted a limited effect using the CAA assay, and displayed no effect with FRAP or ORAC, although it remains possible that the conditions were not optimal in the latter two assays, as astaxanthin is highly lipophilic. The FRAP and ORAC assays commonly require several-fold dilutions of compounds and fractions can be tested in the nM range, being more sensitive than CAA where effects are commonly observed first in the mM range. When comparing antioxidative capacities during simulated gastrointestinal digestion of fish (saithe) and shrimp muscle versus blueberry using FRAP and ORAC [54], blueberry (rich in anthocyanins, a type of flavonoid) was about tenfold more effective with FRAP, whereas muscle exhibited somewhat higher antioxidant capacity using ORAC. For muscle, the antioxidant capacity correlated with

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p­ roteolytic release of amino acids. These results may suggest that amino acids in general have poor reducing power but can protect from peroxyl radicals.

3.5 Testing Antioxidants in Animals and Humans After having isolated or synthesized enough quantities of the novel compounds of interest, animal and human studies should be considered.

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Fig.  9  Comparison of identical aliquots of compounds using FRAP, ORAC and CAA assays. Structures for the most potent antioxidants for each assay are shown. For (a) FRAP and (b) ORAC, antioxidants were compared at 1.5  mM and for (c) CAA (HepG2 cells) at 10  mM. FRAP and ORAC results are expressed as Trolox (vitamin E analog) equivalents. For CAA, low values indicate high antioxidative effects, and protective effects (%) vs. controls (±ABAP) are indicated as well as significant differences (vs. +ABAP) obtained with one-way ANOVA and Dunnett’s multiple comparison test (*p < 0.05, **p < 0.01). Data are shown as average ±SEM ((a) N = 2, (b) N = 3, (c) N = 5–6). EGCG epigallocatechin gallate, AUC area under the curve

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In animals, short-term consumption or injections of antioxidants can be challenged with induced oxidative stress by administration of a free radical ­generator (e.g. carbon tetrachloride or paraquat), an inflammatory activator (lipopolysaccaride, LPS), or exposure to whole-body gamma (g) irradiation. Post-mortem analyses of tissue oxidative damages and effects on inflammation etc., can determine protective effects from antioxidants. Longer periods of administration can test for toxic systemic effects, and life-long administration for effects on life-span (free radicals cause aging). Notably, lifelong dietary administration of aspirin (acetylsalicylic acid) and nordihydroguaiaretic acid (NDGA), compounds exerting both anti-inflammatory and antioxidant properties, resulted in significant lifespan extension in male mice [55]. Moreover, administration of the radical spin trap a-phenyl-N-tert-butyl nitrone (PBN) extended lifespan in both mice [56] and rats [57]. For tests on humans, non-invasive techniques such as blood (plasma or cells) and urine analyses, or collection of tiny tissue biopsies are often more realistic. Here, analysis of urinary (or plasma) oxidative damage excretion products (e.g. 8-hydroxyguanine lesions from DNA and RNA, or F2-isoprostanes derived from peroxidation of arachidonic acid) can estimate how much oxidative damage have taken place in the body [58, 59]. The sensitive Comet assay can be employed on just drops of blood, where subsequently isolated lymphocytes are exposed to oxidants in vitro after consumption of antioxidants [60, 61]. A possible confounding factor to ­consider if measuring plasma antioxidant status is that food intake is followed by increased plasma uric acid levels, a potent antioxidant [11]. Of the few human studies performed so far where pure “antioxidants” were administrated as dietary ­supplements, one study found that vitamin E and b-carotene supplements were harmful [62], and metaanalyses suggest that supplementary intakes of vitamin E and A, and b-carotene may increase overall mortality [63]. It remains to be seen if antioxidant supplements in humans, who already consume antioxidant mixtures through foods, will increase our lifespan, but with the right compounds this might be possible. Systemic excretion and removal rates for phytochemicals are commonly rapid and reported half-lives are often hours to days [64, 65] and antioxidants can eventually be metabolized all the way into carbon dioxide (CO2) [66]. As metabolic ­patterns are considerably different for animals and humans, results obtained for animals should not be automatically converted to humans.

4 Conclusions Cellular and chemical assays were set up for discoveries of novel marine antioxidant compounds where cell-based assays appear more relevant. Comparison of assays with identical substrates gave mixed outcomes and the assays can complement one another, assessing different molecular properties. Acknowledgements  We thank Rudi Caeyers for help with graphical illustrations. We are grateful to the Norwegian Research Council and our industrial partner ABC Bioscience for financial support.

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Arsenic-Induced Oxidative Stress: Evidence on In Vitro Models of Cardiovascular, Diabetes Mellitus Type 2 and Neurodegenerative Disorders Rubén Ruíz-Ramos, Patricia Ostrosky-Wegman, and Mariano E. Cebrián

Abstract  This chapter provides an overview of the evidence of oxidative stress and compensatory responses in response to arsenic exposure in diverse in  vitro models of cardiovascular diseases, type 2 diabetes mellitus and neurodegenerative disorders. The studies described here are recent approaches related to (1) the presence of oxidative and nitrosative damage; (2) the activation of novel and sensitive oxidative stress targets providing an environment conducive to the onset and progression of these diseases. We emphasize the need for further studies exploring the usefulness of the novel oxidative and nitrosative stress targets as biological markers of As toxicity. Keywords  Arsenic • Atherosclerosis • CNS • Endothelium dysfunction • Hypertension • ROS Abbreviations AD AMPK AP-1 aP2 As C/EBPa COX-2 CVDs DMAIII DMAsIIIO DMAV

Alzheimer’s disease AMP-activated kinase Activator protein-1 Adipocyte-selective fatty acid-binding protein Arsenic CCAAT-enhancer binding protein a Cyclooxygenase-2 Cardiovascular diseases Dimethylarsinous acid Iododimethylarsine Dimethylarsinic acid

M.E. Cebrián (*) Departamento de Toxicología, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), Ave. Instituto Politécnico Nacional, 2508 México, DF, Mexico e-mail: [email protected] S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-60761-956-7_33, © Springer Science+Business Media, LLC 2011

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Extracellular signal regulated kinase Glucose transporter 4 Glutathione Human umbilical vein endothelial cells Interleukin-6 c-Jun N-terminal kinase Mitogen-activated protein kinases Microtubule-associated protein-tau Methylarsine oxide Monocyte chemoattractant protein-1 Monomethylarsonous acid Monomethylarsonic acid Neurofilament-medium Neurofilament-heavy Neurofilament-light Neurofilament high molecular weight subunit Neurofibrillary tangles Nuclear factor-kB Nitric oxide Nitric oxide synthases NF-E2-related factor 2 3-Phosphoinositide-dependent kinase-1 Proteoglycans Phosphatidylinositol 3-kinase Phosphatidylinositol-3,4,5-triphosphate Protein kinase B Peroxisome proliferative-activated receptor g Phosphatase and tensin homolog deleted on chromosome ten Reactive nitrogen species Reactive oxygen species Type 1 sphingosine-1-phosphate Stress-activated protein kinases Diabetes mellitus type 2 Tyrosine aminotransferase Tumor necrosis factor-a Vascular endothelial cadherin Vascular smooth muscle cells

1 Introduction Arsenic (As) is an ubiquitous element in the environment whose main route of human exposure is through consumption of contaminated drinking water. Arsenic is a well-known reactive oxygen species (ROS) inducer, which is also able to alter the generation of reactive nitrogen species (RNS) [1]. Free radicals have been defined

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as molecules or molecular fragments containing one or more unpaired electrons whose presence usually confers a considerable degree of reactivity [2]. ROS can be produced from both endogenous and exogenous substances. Potential endogenous sources include mitochondria, cytochrome P450 metabolism, ­peroxisomes, and inflammatory cell activation [3]. The main actors of oxidative stress and oxidative signaling are superoxide anion (O2•−), hydroxyl radical (•OH), hydrogen peroxide (H2O2), singlet oxygen (1O2) and peroxyl radical (LOO•). Superoxide anion is ­considered the primary species in As-induced oxidative stress and its formation leads to a cascade of secondary ROS such as hydrogen peroxide and hydroxyl radicals [1]. The generation of reactive oxidants during arsenic metabolism has been shown to play an important role in As-induced injury [4, 5]. Nitric oxide (NO) is an abundant reactive radical generated in biological tissues by specific nitric oxide synthases (NOS), which metabolize arginine to citrulline, and acts as an important oxidative biological signaling molecule in a large variety of physiological processes, including neurotransmission, blood pressure regulation, defense mechanisms, smooth muscle relaxation and immune regulation [6, 7]. NO is formed by nitric oxide synthase (NOS), which has neuronal, endothelial, and inducible forms, requiring Ca2+, calmodulin, flavin adenine nucleotide, flavin mononucleotide and tetrahydrobiopterin as cofactors for its activation; NO also possess toxic effects such as prooxidant activity, genotoxicity and mutagenicity [8, 9] . ROS and RNS are directly involved in the oxidative damage to lipids, proteins and DNA in As exposed cells. Oxidative and nitrosative stress are among the more documented mechanisms of As toxicity and carcinogenicity [10] and these terms refer to a serious imbalance between production of reactive species and antioxidant defense, encompassing a broad spectrum of conditions that alter cellular redox status [11]. Under pathological conditions, increased intracellular ROS content contributes to cellular impairment and metabolic remodeling through oxidative damage that may lead to physiological dysfunction and degenerative chronic diseases. Arsenic exposure has been associated with different types of cancer (skin, bladder, liver, kidney and lung) [12], diabetes mellitus type 2 (T2DM) [13], arteriosclerosis and cardiovascular diseases [14], hypertension [15] and neurological ­diseases (Alzheimer and Parkinson) [16, 17]. This chapter provides an overview of the evidence of oxidative stress and compensatory responses in response to arsenic exposure in in vitro models of cardiovascular diseases, T2DM and neurodegenerative disorders. The studies described here are recent approaches related to (1) the presence of oxidative and nitrosative damage; (2) the activation of novel and sensitive oxidative stress targets.

2 Arsenic-Induced Oxidative Stress in the Cardiovascular System Epidemiological studies have associated As exposure with an increased prevalence of cardiovascular disorders, such as atherosclerosis [18], ischemic heart disease [19], cerebral infarction [20], and hypertension [21]. These effects are consistent

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with a large body of literature implicating the cells in blood vessel walls as primary targets of arsenic toxicity. Dysregulation of the vasomotor function and/or ­structural remodeling of blood vessels results in enhanced stiffness and decreased ­compliance of the vessel walls and other impaired vascular functions, which subsequently lead to further pathological changes. Major factors for both disrupted signaling and remodeling are the changes in ROS or redox signaling in both vascular endothelial and smooth muscle cells [22, 23]. The following sections review the basic concepts of endothelial dysfunction, atherosclerosis and hypertension, and the mechanisms underlying the effects of As.

2.1 Endothelium Dysfunction The vessel wall is normally composed of an endothelial cell lining on a medial layer of vascular smooth muscle cells and wrapped by an adventitial coating of connective tissue. The vascular endothelium is a metabolically active layer that covers the luminal surface of entire blood vessels and has anticoagulant and ­antithrombotic properties. The endothelial cells provide the transduction of signals between blood and the vessel wall, and coordinate the homeostatic balance of the vessels by producing mediators regulating vascular tone, coagulation status, cell death, and inflammatory cell trafficking [24]. The endothelium regulates the release of several mediators such as nitric oxide NO, prostanoids, endothelin-1, ­angiotensin-II, tissue plasminogen activator, von Willebrand factor, adhesion molecules and cytokines [25]. Endothelial cell dysfunction has been associated with alterations of the cellular redox state and it mainly occurs due to reductions in NO ­synthesis and release, inactivation and/or uncoupling of eNOS, excessive generation of ROS, and/or decreases in the antioxidant defense mechanisms [8]. NO is an important mediator which has vasodilatory and anti-inflammatory properties, inhibits platelet adhesion and aggregation, and smooth muscle cell proliferation and migration [26]. The integrity of the endothelial barrier is maintained by proteins of the adherens junction, such as vascular endothelial cadherin (VE-cadherin) and b-catenin, and their association with the actin cytoskeleton. The partial loss of the balance between endothelium-mediated vasodilation and vasoconstriction, known as vascular endothelial dysfunction, is often associated with the pathogenesis of various chronic disorders, such as atherosclerosis [27], hypertension and diabetes [28]. Alterations in endothelial cells lead to a series of events including vasoconstriction and increased adhesiveness resulting in inflammatory cell infiltration and platelet-thrombus formation [29, 30]. Although endothelial cells are considered arsenic primary targets, the underlying molecular mechanisms of toxicity remain the subject of further study. Arsenic is well known for its oxidative stress inducing properties which may alter gene expression, inflammatory responses, and endothelial nitric oxide homeostasis, all of which play an important role in maintaining vascular functions [31, 32]. Bunderson et  al. [33] reported that sodium arsenite (0.5  mM) increased the

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p­ roduction of peroxynitrite, a strong oxidant formed from the coupling of nitric oxide and superoxide anion in bovine aortic endothelial cells. Arsenite also increased the expression of the inflammatory mediator cyclooxygenase-2 (COX-2) and the generation of prostaglandin E2 alongside with COX-2 nitration. These findings indicate that arsenite increases the formation of reactive species, induce important inflammatory mediators and may exacerbate inflammation, thus providing an environment conducive to the onset and progression of atherosclerosis. More recently, Tsou, et al. [34] showed that arsenite pretreatment (0–10 mM) potentiated tumor necrosis factor-a (TNF-a)-induced expression of vascular cell adhesion molecule-1 along with up-regulation of both activator protein-1 (AP-1) and nuclear factor-kB (NF-kB) in human umbilical vein endothelial cells (HUVEC). The study also showed that glutathione plays an important role in the enhanced response to arsenite and that modulation of proinflammatory cytokines is a potential mechanism for the induction of vascular inflammation and vascular diseases. These findings were consistent with studies on rats showing that sodium arsenite markedly diminished vascular endothelium integrity by inactivating eNOS, reducing NO generation and bioavailability, increasing oxidative stress and subsequently decreasing endothelium-dependent vasorelaxation [35]. Nuntharatanapong et al. [36] reported that sodium arsenite (0–20 mM) transactivated EGF (ErbB1) and ErbB2 receptors, leading to induction of p53 and p21Cip1/Waf1, regulatory proteins of cell cycle and apoptosis. The data indicated that arsenite induced HUVEC cell death via a mechanism involving p21Cip1/Waf1 induction, suggesting another mechanism by which As could alter endothelial function and promote vascular disease. Balakumar et al. [9] recently reviewed the potential target sites modulating vascular endothelial dysfunction and Balakumar and Kaur [37] presented an overview of the relationships between As exposure and cardiovascular disorders. In summary, in addition to increasing ROS formation, arsenite increased the production of peroxynitrite, a strong oxidant formed from the coupling of NO and superoxide anion causing nitrosative stress. Arsenite also induced important proinflammatory cytokines, thus providing an environment conducive to the onset and progression of vascular inflammation and vascular diseases. Arsenite is able to induce endothelial cell death via a mechanism involving p21Cip1/Waf1 induction. Arsenite activated PKCa potentially altering the association between VE-cadherin and b-catenin and causing a loss of endothelial monolayer integrity.

2.2 Atherosclerosis Atherogenesis is a multifactorial and complex disease of the arterial vasculature, characterized by progression from inflammation and smooth muscle cell proliferation to a late stage of thrombotic and fibrotic obliteration of vessels. Upon endothelial activation, mononuclear cells attach to the endothelium and then transmigrate into the sub-endothelial space where they subsequently transform to macrophages and foam cells. These cholesterol-rich macrophages secrete pro-inflammatory ­cytokines

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and enhance lesion inflammation and atherogenesis. At later stages, oxidized lipids stimulate migration of medial smooth muscle cells into the intima, where smooth muscle cells proliferate and engulf oxidized lipids, synthesize extracellular matrix proteins and form a fibrous cap. In addition, oxidative stress contributes to regulate the expression of genes involved in atherogenesis, being the oxidative changes of low-density lipoprotein particles among the dominant hypothesis of atherogenesis [38]. Epidemiological studies in adults [39, 40] and experimental studies in adult mice [41, 42] have shown strong correlations between arsenic exposure and atherosclerosis. However, little is known about the mechanisms by which As initiates and/ or exacerbates atherosclerosis. Arsenic rapidly stimulates both endothelial and smooth muscle NOX enzymes, which play a central role in pathogenic redox signaling in vascular disease and hypertension. Earlier studies showed that endothelial NOX-generated superoxide and hydrogen peroxide levels increased very rapidly after exposure to low arsenite concentrations [43]. In addition, Lynn et  al. [44] provided evidence that arsenite (1–10 mmol/L) increased intracellular superoxide levels and caused oxidative DNA damage in aortic human vascular smooth muscle cells (VSMCs). The authors ­suggested that the effect resulted from NADH oxidase activation along with increased p22phox mRNA levels, and that these effects played an important role in As-induced atherosclerosis. These results were consistent with those of Smith et al. [45] who reported that arsenite (0–20 mM) increased the activity of the membranebound NADPH oxidase enzyme complex associated with p67phox and Rac1 in porcine aortic endothelial cells. Rac1 is a key regulator of endothelial cell migration in angiogenesis, and its translocation may represent a sensitive mechanism for As-induced NADPH oxidase activity. Later, the same research group [46] showed in human microvascular endothelial and mouse liver sinusoidal endothelial cells that sodium arsenite (0–5 mmol/L) stimulated the type 1 sphingosine-1-phosphate (S1P1) receptor. Arsenic acted in a manner similar to S1P, the cognate ligand for S1P1, activating endothelial cell Rac1 and NADPH ox enzyme activities, stimulating NOX enzyme-mediated ROS generation. Stimulation of S1P1 receptor appeared to be a common mechanism for arsenic-initiated signaling for alterations in the angiogenic responses of both cell types, and in the pathogenic increase of barrier function in liver vasculature. The authors concluded that the inappropriate activation of S1P1 promotes selective molecular effects of As in vascular diseases and tumorigenesis and suggested that the identification of the GPCR signaling pathway as a target contributes towards the understanding of As-related vascular pathogenesis. Several studies have indicated that inflammation also plays an important role in atherosclerotic plaque formation. Mechanistic studies have shown that endothelial and vascular smooth muscle cells, and macrophages/monocytes generate chemokines and proinflammatory cytokines, such as monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), and TNF-a. Genetic deletion of these chemokines and cytokines in mice reduces formation of atherosclerotic lesions [47]. In addition, the enhanced VSMC proliferation induced by ROS is mediated via the activation of MAP kinases and Akt and their downstream transcription factors, such as NF-kB or AP-1, which are well-documented to regulate the expression of atherogenic related

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genes [48, 49]. Regarding the effects of As, it has been reported that ­individuals highly exposed to As have an increased expression of cytokine-related genes in atherosclerotic lesions [50]. In vitro mechanistic studies described that arsenite (10 mM) induced 5-lipoxygenase and subsequent increases in leukotriene E4 synthesis in bovine aortic endothelial cells, suggesting a link between formation of RNS and inflammation, both effects related to As-induced atherosclerosis [42]. In addition, Lee et al. [51] provided evidence in human and rat VSMCs that arsenite (5 mM) increased ROS/NO generation and enhanced the expression of heme ­oxygenase-1, MCP-1, and IL-6, genes involved in the regulation of VSMC proliferation. Overexpression of MCP-1 and IL-6 may also increase the attachment, penetration, and migration of monocytes, thus suggesting a possible mechanism for atherogenesis initiation in As-exposed humans. Pereira et  al. [52] exposed human aortic endothelial cells to sodium arsenite (0–10 mM) and showed that arsenite activated PKCa, leading to increased phosphorylation of b-catenin, plausibly ­altering the association between VE-cadherin and b-catenin, which along with formation of actin stress fibers resulted in intercellular gap formation and increased endothelial permeability. The authors concluded that arsenite caused a loss of endothelial monolayer integrity, potentially contributing to the development of atherosclerosis. Fujiwara et al. [53] studied the effects of As on the composition of proteoglycans (PG), important components of VSMCs intima extracellular matrix. Their results showed that arsenite but not sodium arsenate (0–10 mM) inhibited the synthesis of all PG types. The authors concluded that arsenite alters PG composition, a key event in the development and progression of atherosclerosis, proposing it as a different mechanism for As vascular toxicity. Interestingly, Klei et al. [54] showed in primary human microvascular endothelial cells, a model for the initial steps of angiogenesis, that sodium arsenite (1–5 mM) and ethanol (0.1%) interacted positively at proximal steps in signaling cascades to induce a number of angiogenic genes, including vascular endothelial cell growth factor and insulin-like growth factor-1. These findings indicate that non-cytotoxic arsenic exposures interact with other vascular toxicants to enhance signaling for pathogenic angiogenesis and ­inappropriate vascular remodeling, which could contribute to vascular disease and tumorigenesis, thus suggesting the need for further studies in this area. Bae et al. [55] reported that sodium arsenite (0–50 mM) significantly enhanced thrombin-induced procoagulant activity in freshly isolated human platelets, whereas without platelet activation As did not induce procoagulant activity. Arsenite inhibited flippase, an enzyme that restores phosphatidylserine to the inner leaflet, potentiating phosphatidylserine outer membrane exposure and microparticle formation, the major mediators of procoagulant activity. Arsenite also potentiated the increase of intracellular calcium and subsequent calpain activation. These effects were reproduced in rats and resulted in accelerated thrombus formation. Taken together, these findings suggest that platelets are an important target for As cardiovascular toxicity. Recently, the same research group reported that monomethylarsonous acid (MMAIII) (0–5  mM) and dimethylarsinous acid (DMAIII) (0–25 mM) induced procoagulant activity and apoptosis in non-activated platelets without affecting platelet aggregation. MMAIII induced phosphatidylserine

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e­ xposure and apoptosis. MMAIII and DMAIII directly induced procoagulant ­activity independently from calcium, in contrast to inorganic As that induced ­procoagulant activity by calcium mediated pathways only in activated platelets. In addition to provide evidence to suggest another mechanism by which arsenic metabolites could play a role in arsenic-associated cardiovascular diseases (CVDs), these findings highlight the toxicological significance of xenobiotic-induced ­platelet apoptosis in thrombus formation and CVD development [56]. In summary, the mechanisms by which As could induce atherosclerosis and promote vascular disease are varied. Arsenite induced 5-lipoxygenase and subsequent increases in leukotriene E4 in aortic endothelial cells suggested a link between RNS formation and inflammation. The increased ROS/NO generation induced by arsenite enhanced the expression of genes regulating VSMC ­proliferation. In particular, the overexpression of MCP-1 and IL-6 increased the attachment, penetration, and migration of monocytes. Arsenite also altered the composition of proteoglycans, important components of the VSMC intima extracellular matrix, potentially playing a key role in the development and progression of atherosclerosis. The identification of the GPCR signaling pathway as an As target, particularly S1P1 receptor stimulation, plays an important role in the understanding of As-initiated signaling for endothelial cell alterations in the angiogenic responses and the pathogenic increase of barrier function in liver vasculature. The positive interaction to induce a number of angiogenic genes in primary human microvascular endothelial cells suggest the need for further studies on the interactions among toxicants signaling for pathogenic angiogenesis and inappropriate vascular remodeling, potentially contributing to vascular disease and tumorigenesis. Arsenite enhanced thrombin-induced procoagulant activity in freshly isolated human platelets, potentiating phosphatidylserine outer membrane exposure and microparticle formation, hallmarks of procoagulant activity. MMAIII and DMAIII induced procoagulant ­activity and apoptosis in non-activated platelets independently from calcium mediated ­pathways. These findings suggested a novel mechanism by which arsenicals may act in As-associated CVDs, and highlight the toxicological significance of ­xenobiotic-induced platelet apoptosis in thrombus formation and CVD development.

2.3 Hypertension The increased contractility of blood vessels disrupts vasomotor tone regulation allowing vasoconstriction to predominate, potentially inducing hypertension. Smooth muscle contraction is regulated by neural and humoral factors, mechanical forces, and vasoactive substances produced in endothelial cells. Vascular smooth muscle contraction is triggered primarily by a rise in intracellular free Ca2+ concentration [57]. Although earlier studies reported elevated peripheral resistance in populations exposed to As via drinking water [21], its mechanisms of toxicity are still under investigation. Lee et al. [58] reported that arsenite (5–25 mM), increased

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the contraction of rat aortic rings induced by phenylephrine or serotonin. However, arsenate, monomethylarsonic acid (MMAV), and dimethylarsinic acid (DMAV) were less potent. Similar effects were observed in aortic rings devoid of endothelium, suggesting that VSMC play a key role in the enhanced vasoconstriction induced by arsenite. Vasoconstriction correlated with the extent of myosin light chain phosphorylation stimulated by phenylephrine. Direct Ca2+ measurements revealed that arsenite enhanced the vasoconstriction induced by high K+, without concomitant increases in intracellular Ca2+ elevation, suggesting that Ca2+ sensitization rather than a direct Ca2+ elevation is a major contributor to arsenite-induced vasoconstriction. The in vivo study showed that arsenite treatment potentiated the hypertensive effect of phenylephrine in rats [58]. Taken together, both studies suggest that increased vasoconstriction may be a contributing factor in the development of cardiovascular diseases. These results were consistent with ex vivo experiments reporting that intravenous administration of sodium arsenite (6  mg/kg) increased the vasoconstrictor responses of rat isolated aorta and renal arteries to phenylephrine and inhibited endothelium-dependent NO-mediated vasodilation, without altering acetylcholine-induced vasodilation mediated by NO in the aorta. Arsenite induced the expression of Hsp72, Hsp32, and Hsp90, without significantly effecting eNOS. The authors concluded that arsenic increased vascular reactivity by ­compromising basal endothelial NO function [59]. Bae et al. [60] investigated in several experimental models the effects of MMAIII (0–10 mM) on the vasomotor tone of blood vessels. In contrast to inorganic arsenite, MMAIII irreversibly suppressed normal vasoconstriction induced by phenylephrine, serotonin and endothelin-1 in the isolated rat thoracic aorta and small mesenteric arteries. The effect was also observed in aortic rings devoid of endothelium, suggesting that MMAIII directly impaired the contractily of vascular smooth muscle. Voltage-clamp assays in Xenopus oocytes showed that MMAIII inhibited ­phenylephrine-induced Ca2+ increase, resultant from the concomitant inhibition of its release from intracellular stores and blockage of the extracellular Ca2+ influx via the L-type Ca2+ channel. However, MMAIII did not significantly affect downstream processing of Ca2+, as shown in permeabilized arterial strips experiments. In rat in vivo studies, MMAIII attenuated phenylephrine-induced blood pressure increase. The authors concluded that MMAIII-induced smooth muscle dysfunction by ­disturbing Ca2+ regulation, which results in impaired vasoconstriction and aberrant blood pressure changes. Pysher et al. [61] reported that sodium arsenite (0.67 mM) induced a sustained increase in FAK–src association and activation in primary VSMCs, particularly inducing focal adhesion protein co-association with other proteins, including Akt. These effects resulted in stimulation of PAK1 and downstream extracellular signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and mitogen-activated protein kinases (MAPK) pathways, known mediators of VSMC growth, proliferation, and migration, whose chronic stimulation has been associated with vascular disease. Interestingly, arsenite activated many of the same proteins as angiotensin II, but differing on the precise pathways followed. Angiotensin II is a well-known promoter of VSMC hyperplasia and hypertrophy, which mediates signaling through alterations in ROS, intracellular Ca2+ and other

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signaling pathways (Gregg et  al., 2003). Taken together these data suggest a ­potential role for As-induced oxidative stress and angiotensin II as mediators of VSMC alterations. In summary, arsenite was more potent than MMAV and DMAV in increasing the contraction of rat aortic rings induced by phenylephrine or serotonin. In ­contrast, MMAIII irreversibly suppressed normal vasoconstriction induced by phenylephrine, serotonin and endothelin-1 in the isolated rat thoracic aorta and small mesenteric arteries, inducing smooth muscle dysfunction by altering Ca2+ regulation. These findings suggest that impaired vasoconstriction and potential aberrant blood ­pressure changes may be a contributing factor in the development of cardiovascular diseases. Intravenous arsenite increased the ex vivo vasoconstrictor responses of rat isolated aorta and renal arteries to phenylephrine by inhibiting endothelium-dependent NO-mediated vasodilation without altering eNOS expression. Arsenite induced in primary VSMCs a sustained increase in FAK–src association and ­activation, accompanied by stimulation of PAK1 and several downstream pathways, whose stimulation has been associated with vascular ­disease. Arsenite ­activated many of the same proteins as angiotensin II, suggesting a potential role for As-induced oxidative stress and angiotensin II as mediators in the loss of blood pressure control.

3 Arsenic-Induced Oxidative Stress in Diabetes Mellitus Type 2 T2DM is a metabolic disorder characterized by hyperglycemia, insulin resistance in peripheral tissues, and altered insulin secretory capacity of pancreatic b-cells that has become a major public health problem worldwide [62]. A link between ­environment and diabetes has been suggested and exposure to environmental ­contaminants, including As, has been proposed to play a role in diabetes ­development [63]. Chronic exposure to high levels of As has been associated with T2DM, as shown by epidemiologic studies thoroughly reviewed by Navas-Acien et al. [64]. In addition, recent studies carried out in Bangladesh [65], Taiwan [66], Mexico [67] and USA [68, 69] provided additional support for the importance of As exposure in the development of T2DM. Although the interference of As with pyruvic acid metabolism by inhibiting pyruvate and a-ketoglutarate dehydrogenases, essential enzymes for gluconeogenesis and glucolysis, was described by Krebs in the early 1930s, the precise mechanisms of the diabetogenic effects of arsenic are still largely undefined [70]. Oxidative stress has been clearly implicated in the etiology of T2DM and is the basis for alterations in multiple cellular pathways potentially leading to cellular injury. A key driver in the pathogenesis of T2DM is the impairment of glucosestimulated insulin secretion, the hallmark of pancreatic b-cell function [71]. Oxidative stress and free radical production have been implicated in insulin resistance involving the impairment of phosphoinositide 3 kinase (PI 3-K) activity and

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protein kinase B (PKB/Akt) functioning. Insulin controls glucose transport through insulin receptor substrate-1 and partially through Akt and protein kinase C. In ­addition, Akt signaling is tied to insulin sensitivity, since it accelerates glucose uptake in adipocytes in response to insulin by stimulating glucose transporter 4 (GLUT-4) translocation and the synthesis of GLUT-1. Conversely, the loss of Akt kinase activity is accompanied by alterations in insulin-stimulated glucose transport in muscle and adipocytes, leading to impaired glucose tolerance [72]. The control of glucose-stimulated insulin secretion in b-cells depends largely on glucose metabolism, where glycolytic and oxidative phosphorylation triggers a sequence of signaling events. In addition to ATP levels and the ATP/ADP ratio, ROS derived from glucose metabolism serve as one of the metabolic signals for glucose-stimulated insulin secretion [73]. The endogenous antioxidant enzymes that are strongly induced in response to oxidative stress have the potential to blunt the glucose triggered ROS signal, thus inhibiting glucose-stimulated insulin secretion. The NF-E2related factor 2 (Nrf2) driven induction of endogenous antioxidant enzymes, aimed to maintain intracellular redox homeostasis and defend cells against oxidative damage, may also have the potential side effect of reducing ROS that function as intracellular signals. Therefore, glucose-dependent insulin secretion, ROS production, and ­oxidative stress in pancreatic b-cells may be tightly linked processes [74]. Houstis et  al. [75] studied in 3T3-L1 adipocytes the mechanisms of insulin resistance ­associated to TNFa or dexamethasone, which act through different cellular response pathways. Genome-wide expression analyses showed that 34 genes were common to both treatments and most were responsive to ROS. They also tested the role of ROS in insulin resistance by assessing the effects of antioxidant molecules and transgenes encoding ROS-scavenging enzymes. The authors concluded that the latter treatments decreased dexamethasone- and TNF-induced ROS levels and ­insulin resistance, therefore suggesting that increased ROS levels are an important ­trigger for insulin resistance. Furthermore, the content of antioxidative enzymes in diverse tissues may be a major determinant of organ susceptibility to ROS-induced cytotoxic damage. For example, the expression and activity of antioxidant enzymes (superoxide dismutase, the hydrogen peroxide-inactivating enzyme catalase, and glutathione peroxidase) in rat pancreatic islets and insulinoma RINm5F cells are significantly lower than in liver and kidney, suggesting that pancreas is more ­vulnerable to prolonged oxidative stress [76, 77]. Regarding the effects of As, earlier studies reported that exposure of transfected mouse pancreatic b-cells (MIN6) to high sodium arsenite concentrations (0.5 mM) stimulated the homeodomain transcription factor PDX1 (pancreatic/duodenal homeobox-1), which binds to the human insulin gene promoter, increased insulin mRNA levels in response to glucose, and also stimulated PDX1 translocation from the cytoplasm to the nucleus. This response was similar to that obtained by treating cells with high glucose concentrations [78]. Wauson et al. [79] showed that incubation of C3H 101T1/2 cells with sodium arsenite (6  mM) for 2 months prevented adipocyte differentiation and reduced the mRNA levels of peroxisome proliferativeactivated receptor g (PPARg), CCAAT-enhancer binding protein a (C/EBPa), and adipocyte-selective fatty acid-binding protein (aP2). Arsenic also blocked upregulation

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of p21Cip1/Waf1, a factor whose expression is tightly regulated during adipogenesis. The differentiating effect of pioglitazone, a PPARg agonist inducer of adipogenesis and used to reduce insulin resistance, was also inhibited by arsenic, suggesting that As interferes with adipogenic signaling at or below PPARg. Bazuine et  al. [80] showed that arsenite (0–1  mM) displayed insulin-mimicking effects in 3T3-L1 adipocytes. Arsenite stimulated glucose uptake and induced translocation of the insulin-responsive GLUT4 glucose transporter from the low-density microsomal fraction towards the plasma membrane. However, the classical IR–IR substrate– PI-3¢ kinase pathway, essential for insulin-induced GLUT4 translocation, was not activated by arsenite but the induction of a tyrosine kinase was essential to promote glucose uptake. Arsenite did not interfere with early events in insulin-induced signalling. Pi et al. [81] showed in HaCaT cells, an immortalized human keratinocyte cell line, that sodium arsenite (0–10 mM) increased hydrogen peroxide production and enhanced the expression of Nrf2, a transcription factor that regulates both constitutive and inducible expression of many antioxidative response enzymes. Arsenite also caused nuclear accumulation of Nrf2 in association with downstream activation of Nrf2-mediated oxidative response genes. Arsenic simultaneously increased the expression of Keap1, a regulator of Nrf2 activity, suggesting that its importance for As-induced Nrf2 activation. Further experiments using hydrogen peroxide scavengers or compounds mimicking superoxide dismutase indicated that hydrogen peroxide was the main mediator of this effect. Exploring other mechanisms of As toxicity, Bodwell et al.[82] showed in rat EDR3 hepatoma cells that sodium arsenite (0.05–1 mM) stimulated the glucocorticoid receptor (GR)-mediated gene activation of both the endogenous tyrosine aminotransferase (TAT) gene and the reporter genes containing TAT glucocorticoid response elements; however, the effects were inhibitory at slightly higher concentrations (1–3 mM). These effects were consistent with previous studies showing that rat H4IIE hepatoma cells treated with arsenite (0.3–3.3 mM) suppressed the glucocorticoid inducible expression of the endogenous rat PEPCK gene, the rate-limiting step in gluconeogenesis [83]. More recently, Walton et al. [84] showed in 3T3-L1 adipocytes that treatment with trivalent arsenicals, arsenite (iAsIII; 20 mM), methylarsine oxide (MAsIIIO; 1 mM), and iododimethylarsine (DMAsIIIO; 2  mM) decreased insulin-stimulated glucose uptake by 35–45%, without significantly affecting basal glucose uptake. In contrast, pentavalent arsenicals (up to 1 mM) had no significant effects on either basal or insulin-stimulated glucose uptake. Trivalent arsenicals suppressed expression and phosphorylation of PKB/Akt, suggesting that trivalent arsenicals inhibited insulin-stimulated glucose uptake by interfering with the PKB/Akt-dependent mobilization of GLUT4 transporter. Later, the same research group showed that iAsIII (50  mM) or MMAIII (2  mM) had no significant effects on the activity of phosphatidylinositol 3-kinase (PI-3K), which synthesizes phosphatidylinositol3,4,5-triphosphate (PIP3), or on PTEN (phosphatase and tensin homolog deleted on chromosome ten) phosphorylation. Trivalent arsenicals did not interfere with the phosphorylation of 3-phosphoinositide-dependent kinase-1 (PDK-1), located downstream from PI-3K, although PDK-1 activity was inhibited. Consistent with these findings, PDK-1-catalyzed phosphorylation of PKB/Akt (Thr308) and PKB/

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Akt activity were suppressed. In addition, PKB/Akt (Ser473) phosphorylation, catalyzed by a putative PDK-2, was also suppressed. The expression of constitutively active PKB/Akt restored the normal insulin-stimulated glucose uptake pattern. These results suggested that inhibition of the PDK-1/PKB/Akt-mediated transduction step is the key mechanism for the inhibition of insulin-stimulated glucose uptake in adipocytes exposed to trivalent As, and ­possibly for impaired glucose tolerance associated with human exposure [85]. Further studies of this [86] and other research groups [87] described experiments in which the diabetogenic effects of As and their association with oxidative stress were reproduced in animal models. Diaz-Villaseñor et al. [88] reported that isolated rat pancreatic b-cells treated with sodium arsenite (0–10 mM) secreted less insulin, an effect partially explained by decreased insulin mRNA expression. In addition, arsenite-treated cells lost their ability to distinguish between two different extracellular glucose concentrations, secreting similar amounts of insulin when exposed to 5.6 mM (basal) or 15.6 mM glucose. The authors concluded that these effects resembled T2DM and suggested that these mechanisms play an important role in the increased incidence of T2DM observed in populations chronically exposed to As. Fu et al. [89] showed in INS1(832/13), a clonal insulin-producing pancreatic b-cell line, that sodium arsenite (0–0.5 mM) decreased glucose-stimulated insulin secretion. Arsenite also enhanced Nrf2 activity and increased intracellular GSH and intracellular H2O2 scavenging activity. The basal cellular peroxide level was increased whereas the net percentage increase in glucose-stimulated intracellular peroxide production was markedly inhibited. In contrast, insulin synthesis and the consensus glucose-stimulated insulin secretion pathway, including glucose ­transport and metabolism, were not significantly affected. The authors proposed that the Nrf2-mediated antioxidant response plays a paradoxical role in insulin secretion, on one side protecting b-cells from oxidative damage and on the other reducing ROS signaling triggered by glucose, an important component of the process of insulin secretion, resulting in reduced glucosestimulated insulin secretion. In summary, earlier studies reported that As mimicked the effects of insulin, stimulating the PDX1 transcription factor and increased insulin mRNA levels in response to glucose, a response similar to that obtained with high glucose ­concentrations. High concentrations also stimulated glucose uptake and induced translocation of the insulin-responsive GLUT4 glucose transporter. The induction of a tyrosine kinase by arsenite was essential for glucose uptake, but it did not ­significantly interfere with early events in insulin-induced signaling. Long term incubation with low As concentrations reduced PPARg, C/EBPa, and aP2 mRNA levels, and blocked p21Cip1/Waf1 upregulation, preventing adipocyte differentiation and suggesting an interference with adipogenic signaling at or below PPARg level. Arsenite also increased hydrogen peroxide production, and enhanced Keap1 and Nrf2 expression. Other studies showed that arsenite stimulated the GR-mediated activation of the TAT gene and genes containing TAT glucocorticoid response ­elements, but at slightly higher concentrations, inhibitory effects were observed, illustrating the dose-dependency of the effects. Trivalent arsenicals inhibited PDK-1 activity and PKB/Akt phosphorylation, whose activity was suppressed,

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interfering with GLUT4 transporter mobilization, thus suggesting that PDK-1/ PKB/Akt-mediated transduction step is the key mechanism for the inhibition of insulin-stimulated glucose uptake. Pancreatic b-cells treated with arsenite showed a decreased insulin mRNA expression and insulin secretion, in addition to lose their ability to distinguish between two different extracellular glucose concentrations. Arsenite also decreased glucose-stimulated insulin secretion, enhanced Nrf2 activity and increased intracellular GSH and H2O2-scavenging activity. The basal ­peroxide level was increased whereas the increase in glucose-stimulated peroxide production was markedly inhibited, suggesting that the Nrf2-mediated antioxidant response induced by As plays a paradoxical role in insulin secretion, reducing ­oxidative damage but also blunting the glucose-triggered ROS signaling needed for insulin secretion.

4 Arsenic-Induced Oxidative Stress in CNS Degeneration In addition to other pathologies, arsenicals cause neurodevelomental disorders and brain disease [90]. Central neuropathy due to As has been reported to impair ­neurological functions such as learning, short-term memory and concentration. Neuropsychological tests showed mildly impaired psychomotor speed and attentive processes, whereas verbal learning and memory were severely impaired [91]. However, much less information is available on As-induced neurodegeneration and their possible mechanisms. Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by neuronal loss and brain lesions in the form of senile plaques, neurofibrillary tangles (NFTs) and neuropil threads. The deposition of b-amyloid peptides characterizes senile plaques, whereas paired helical filaments, the predominant structures in NFTs and neuropil threads, are comprised of the hyperphosphorylated microtubule-associated protein tau [92]. Numerous kinases phosphorylate tau in  vitro, including stress-activated protein kinases (SAPKs), which are members of the MAPKs family [93]. The exposure of cells to either proliferative or stressful stimuli elicits a complex response involving one or more distinct phosphorylation cascades culminating in the activation of multiple ­members of the MAPK family, including ERK, JNK/SAPK, and p38/RK/CSBP protein kinase. Multiple lines of evidence have shown that oxidative stress is an early event that plays a significant role in AD pathogenesis. Oxidative stress not only ­temporally precedes pathology but also activates the cell signaling pathways that contribute to lesion formation and simultaneously trigger cellular responses, such as compensatory up regulation of antioxidant enzymes [94]. Regarding the effects of As, Liu et al. [95] showed that high concentrations of sodium arsenite (400  mM) activated both JNK/SAPK and p38, but moderately ­activated ERK in Rat-l embryonic fibroblasts. The activation was prevented by N-acetyl-L-cysteine, suggesting that an oxidative signal initiated these responses. These findings were in good agreement with reports from ex vivo studies showing that arsenite decreased constitutive nNOS activity accompanied with increased lipid

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peroxidation and ROS production in the striatum [96]. Other studies also reported that short oral exposure to As induced excessive ROS generation and ­significantly reduced the activities of antioxidant enzymes, causing significant ­oxidative stress and neuronal damage in rat brain tissues [97]. Namgung and Xia [98] described that sodium arsenite (0–15  mM) induced apoptosis, activated p38 and JNK3 but not JNK1 or JNK2 in primary cortical neurons prepared from ­newborn Sprague Dawley rats. The same research group reported that sodium arsenite (0–15 mM) and DMA (0–5  mM) reduced cerebellar neuron viability and induced nuclear fragmentation and condensation, as well as DNA degradation. Arsenite was more potent than DMA and selectively activated p38 and JNK3 in cerebellar neurons. The authors concluded that arsenite-induced apoptosis in neurons might explain some of the degenerative CNS effects attributed to environmental exposure to As [99]. These results are in good agreement with studies in  vivo showing that arsenite caused faulty migration, delayed maturation and alterations in rat cerebellar Purkinje cells [100]. Giasson et al. [101] showed that high concentrations of sodium arsenite (50 mM) activated SAPKg in PC12 cells, and hyperphosphorylated perikaryal neurofilament high molecular weight subunit (NFH) in cultured dorsal root ganglion neurons. The authors proposed that SAPKs are involved in the abnormal phosphorylation of NFH, a common feature of several neurological diseases. This effect provided a new lead to the mechanisms of several neurological diseases associated to As. The same research group, later investigated in CHO T40 cells the effects of arsenite (500 mM) on tau phosphorylation, since As is an inducer of heat-shock responses and a potent activator of SAPKs, both effects implicated in modulating tau phosphorylation. Their results showed that arsenite increased the phosphorylation of several amino acid residues in tau, indicating that As might be involved in the cascade leading to deregulation of tau function associated with neurodegeneration, and suggesting the need for further studies on the role of arsenic exposure in AD pathogenesis [93]. More recently, Vahidnia et al. [102] studied the effects of As species on the neuronal cytoskeleton by examining their effects on the expression of genes contributing to regulate axonal integrity in cell lines originating from peripheral (Schwannoma cells ST-8814) or central (neuroblastoma cells SK-N-SH) nervous system. Their results showed that AsIII increased microtubule-associated protein-tau (MAPT) expression in ST-8814 cells, whereas iAsV did not induce significant effects. On the other hand, MMAV increased ­neurofilament-heavy (NEFH) and neurofilament-medium (NEF3) expression in Schwannoma ST-8814 cells whereas MAPT, NEFH, NEF3 and NEFL expression was induced in neuroblastoma SK-N-SH cells. DMAV induced NEFH, NEF3 and neurofilament-light (NEFL) expression in ST-8814 cells whereas it only induced NEFH expression in SK-N-SH cells. In a subsequent publication, Vahidnia et al. [17] reviewed some mechanisms of As neurotoxicity leading to formation of neurofibrillary tangles and degeneration of the affected neurons and their similarities with the alterations observed in AD. Later, Vahidnia et al. [103] showed in HeLa-p35 cells that arsenic species (0–30 mM) increased p35-RNA expression levels and the cleavage of p35 protein to p25, the latter effect was associated with Ca2+-induced calpain activation. The authors suggested that an increase in internal Ca2+ levels and ­subsequent calpain activation might be the primary step in the mechanism of

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a­ rsenite-induced neurotoxicity, since p25 is a hyper-activator of CDK5. The p25/ CDK5 complex is responsible for MAPT and NF protein hyperphosphorylation, giving further support to the ideas advanced in previous papers from this group. These results are in agreement with recent studies in exposed animals showing that arsenite impaired the structural organization of the brain and altered the shape of fiber tracts and axons in the rat striatum [104]. Recently, Wang et al. [105] showed in the mouse neuroblastoma cell line Neuro-2a that arsenic trioxide (0–5  mM) induced ROS production and suppressed the activation of LKB1, a major AMPactivated kinase (AMPK) playing an important role in neuronal development and neurodegeneration, by inhibiting both LKB1 phosphorylation and its translocation to the cytoplasm. Arsenic also inhibited neurite outgrowth, an early process of neuronal differentiation. Simultaneous treatment with antioxidants antagonized the inhibitory effect of As on the LKB1-AMPK pathway, indicating that oxidative stress plays a critical role in blocking this pathway providing an insight into the underlying mechanisms of arsenic-induced developmental neurotoxicity. In summary, earlier studies using high concentrations of sodium arsenite showed that As activated both JNK/SAPK and p38 in fibroblasts. Further studies on ­cultured dorsal root ganglion neurons showed that arsenite-induced SAPKs were involved in the abnormal phosphorylation of NFH, a common feature of several neurological diseases. Arsenite increased the expression of MAPT in cell lines originating from the peripheral or central nervous system. Arsenite increased p35-RNA expression levels and the cleavage of p35 protein to p25, the latter effect was associated with Ca2+-induced calpain activation. p25 is a hyper-activator of CDK5 and the p25/CDK5 complex is responsible for MAPT and NF protein hyperphosphorylation. These findings suggest that arsenite may be involved in tau function deregulation associated with neurodegeneration. Arsenic induced apoptosis and activated p38 and JNK3 in primary cortical neurons, and also induced nuclear fragmentation, DNA degradation and reduced neuron viability in cerebellum. Arsenic trioxide induced ROS production, inhibited neurite outgrowth and suppressed the activation of LKB1, a major AMPK-kinase involved in neuronal development and neurodegeneration. Treatment with antioxidants indicated that oxidative stress played a critical role in blocking this pathway. Taken together these findings may explain some of the CNS effects attributed to environmental As exposure and ­indicate the need for further studies on its role in neurodegenerative disorders.

5 Conclusions and Perspectives 1. Oxidative and nitrosative stress underlie alterations in multiple cellular pathways potentially leading to cardiovascular diseases, diabetes mellitus and neurodegenerative disorders. This is consistent with the notion that oxidative and nitrosative stress are the more documented mechanisms of As toxicity. However, it is unlikely that human exposure to As occurs without exposure to other xenobiotics

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that may influence its molecular effects, thus suggesting the need for further studies on the interactions between As and other toxic substances. 2. Endothelial and vascular smooth muscle cells and platelets are As targets. Arsenite exacerbates inflammation, alters the composition of the intima’s extracellular matrix, and induces endothelial cell death, potentially causing a loss of endothelial monolayer integrity. Arsenite enhanced the expression of genes regulating VSMC proliferation, particularly those increasing the attachment, penetration, and migration of monocytes. Arsenite enhanced thrombin-induced procoagulant activity, whereas MMAIII and DMAIII induced procoagulant activity and apoptosis in non-activated freshly isolated human platelets, thus providing an environment conducive to the onset and progression of atherosclerosis. 3. Arsenite and its pentavalent metabolites increased the contraction induced by phenylephrine, serotonin and endothelin-1 in isolated arteries and increased the ex vivo vasoconstrictor responses of rat isolated aorta and renal arteries to phenylephrine by inhibiting endothelium-dependent NO-mediated vasodilation. In addition, arsenite activated many of the same proteins as angiotensin II does, suggesting a role for As-induced oxidative stress and angiotensin II in the loss of blood pressure control. 4. Arsenite decreased insulin secretion, insulin mRNA expression and glucosestimulated insulin secretion in pancreatic b-cells. The inhibition of the PDK-1/ PKB/Akt-mediated transduction step is an important mechanism for the inhibition of insulin-stimulated glucose uptake. The enhanced Nrf2-mediated antioxidant response induced by arsenite apparently plays a paradoxical role in insulin secretion, reducing oxidative damage but also blunting the glucose triggered ROS signaling needed for insulin secretion. Therefore, glucose-dependent insulin secretion, ROS production, and oxidative stress in pancreatic b-cells may be tightly linked processes. 5. Arsenite increased MAPT expression in peripheral or central nervous system derived cell lines. In other systems, arsenite increased p35-RNA expression levels, potentially leading to an increased formation of the p25/CDK5 complex, responsible for MAPT and NF protein hyperphosphorylation. Arsenite also induced apoptosis in primary cortical and cerebellar neurons. These findings suggest that arsenite, may be involved in the cascade leading to deregulation of tau function and indicated the need for further studies exploring a link between arsenite exposure and late-onset neurodegenerative diseases.

References 1. Valko, M., et al., Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem Biol Interact, 2006. 160(1): p. 1–40. 2. Valko, M., et al., Role of oxygen radicals in DNA damage and cancer incidence. Mol Cell Biochem, 2004. 266(1-2): p. 37–56. 3. Inoue, M., et al., Mitochondrial generation of reactive oxygen species and its role in aerobic life. Curr Med Chem, 2003. 10(23): p. 2495–505.

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Index

A Ab neurotoxicity, 272 Acrolein, 569, 570 Activator protein–1 (AP–1), 347–348, 522 AD. See Alzheimer’s disease Adenosine 5’-diphosphate (ADP), 328–329 Adenosine 5’-monophosphate (AMP), 590 Adenosine 5’-triphosphate (ATP), 328–329, 434 Aguilera, C.M., 39 Aldehydes, 434 Alessi, D.M., 187 Allen, T.J., 3 Altman, D.G., 430 Alzheimer’s disease (AD) animal models APPswe mice, 240, 242 a and b-waves, 242 morphological and biochemical changes, 242 transgenic mice, 240, 241 Ab plaques, 212 cellular metabolic equilibrium, 212 etiology, 226–228 functional annotations with oxidative stress DAVID bioinformatics, 255, 258 GEO, 255 NAD/NADP, 259 oxidoreductase-mitochondrion, 258 response to stress, 258, 259 upregulated and downregulated genes classification, 255–257 Gpx4, lipid peroxidation, 271–272 neuropathology, 212–213 oxidative stress biomacromolecules, 213 mitochondrial DNA and cytochrome oxidase, 214 mitochondrial dysfunction, 215 NFT, 214, 215

8OHG, 214 role of metals, 215–216 ROS, 213–214 SOD, 214 participants and experimental designs demographics, 255 genetic and epigenetic risk factors, 254 GSTM3, 254 JGH-McGill University Memory Clinic, 253 NEC, 254, 255 selection criteria sample, 254 pathologic mechanisms, 228 PBMC amyloid-b, 252 gene expression, 252–253 gene ontology, 253 neuronal degeneration and gliosis, 251 NFT, 252 oxidative stress, 253 senile plaques and intraneuronal neurofibrillary, 212 visual disturbances Ab plaques, 231–232 cerebral amyloid angiopathy, 231 melatonin, 232 retinal pathologies, 232–233 visuo-spatial cognition, 231 working model, 259–261 AMD, 315 4-(2-Amino-ethyl)-benzenesulfonyl fluoride (AEBSF), 26 AMP-activated protein kinase (AMPK), 556 Amyloid-b (Ab) PBMC, 252 plaques, 212 Amyloid precursor protein (APP), 272 Anand-Srivastava, M.B., 627, 628 Angiotensin II (AngII) hormone, 627

S. Basu and L. Wiklund (eds.), Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice 6, DOI 10.1007/978-1-60761-956-7, © Springer Science+Business Media, LLC 2011

681

682 Angiotensin II receptor 1 (AT1), 628 Animal models baboons, 499 dog, 499 endotoxemic models, 504–505 endotoxin, 498–499, 503–504 ethical considerations, 500 free radicals, 498 host-endotoxin interactions, 504 juvenile pig, 499 live bacteria models, 505 pathological process, 499 patient mortality, 498 peritonitis models, 505–506 porcine endotoxin shock models, 499 rabbits, 499 sepsis models (see Sepsis models) sheep, 499 SIRS (see Systemic inflammatory response syndrome) Antimycin A, 556 Antioxidants, marine organisms animals and humans, 651, 654 CAA (see Cellular antioxidant activity) cell culture, CAA and CLPAA experimental procedure, 643–644 chemical vs. cellular assays antioxidant capacity, 650–651 identical aliquots comparison, 650, 653 semipurified marine extracts, 650, 652 CLPAA (see Cellular lipid peroxidation antioxidant activity) comet assay, 642, 650, 651 FRAP, 643 human diet phytochemicals, 638 Lc-PUFA, 638–639 natural compounds active fraction analysis, 646 crude organic and aqueous extracts, 645 dereplication, 646 homogenization, 645 taxonomic determination, 644 two-step extraction protocol, 644 omega–3 fatty acids, 639 ORAC, 642–643 Aoki, E., 196 Apolipoprotein E e4 (APOEe4), 254 Arachidonic acid metabolism and lipid peroxidation brain oxygen COX, 75–76 cP450, 77–78 DHA, 71 LOX, 76–77

Index nonenzymatic oxidative lipid metabolism, 73–74 PLA2, 72, 73 PLC and PLD, 71 PUFAs, 71 hemorrhagic stroke experimental models, 70–71 ICH, 69 oxidative stress patterns, 69–70 SAH, 69 hypermetabolic brain tissue, 78 ischemic stroke embolic stroke, 65 etymology, 64 experimental models (see Middle cerebral artery occlusion) hyperbaric oxygen therapy, 79 natural vitamin E, alpha-tocotrienol, 79–80 oxidative stress patterns, 65–66 thrombotic stroke, 65 potential neuroprotective agents, 79 Armstrong, D., 303 Arsenic-induced oxidative stress cardiovascular system atherosclerosis (see Atherosclerosis) endothelium dysfunction, 662–663 hypertension, 666–668 CNS degeneration, 672–674 diabetes mellitus type 2 cellular injury, 668 free radical production, 668–669 link with environment, 668 Nrf2, 669, 670 pancreatic b-cell line, 671, 672 PDX1 translocation, 669 PKB/Akt, 670–671 PPARg agonist, 670 ROS levels, 669 free radicals, 660–661 ROS and RNS, 661 superoxide anion, 661 vasomotor function, 662 Atherosclerosis atherogenesis, 663 cholesterol-rich macrophages, 663–664 MMAIII and DMAIII, 665–666 NADH oxidase, 664 pathogenic redox signaling, 664 plaque formation, inflammation, 664 proteoglycans, 665 sodium arsenite, 665 VSMCs, 664, 665 ATP. See Adenosine 5’-triphosphate

Index Augustin, W., 441, 451 Aust, S.D., 445 Autophagy, 378 B Bae, O.N., 665, 667 Bagchi, D., 481 Bagchi, M., 481 Bailey, E.L., 46 Balakumar, P., 663 Baltimore, D., 159 Basu, S., 279, 467, 497 Baumgartner-Jaeger CSM2072i, 405 Bazuine, M., 670 Biomarkers antioxidants/anti-inflammatory agents, 411 Bronchoaveolar lavage cells, 411–412 EPR measurement, 415–416 flow cytometry and fluorescent microscopy, 412 glutathione, 415 intracellular 4-hydroxy–2-nonenal levels and its adducts, 413 myeloperoxidase assay, 412 Nrf2, 414 8-OHdG immunohistochemistry, 413–414 Bland, J.M., 430 Blankenberg, S., 6, 23 Blomhoff, R., 157 Blood–brain barrier (BBB), 92 Bodwell, J.E., 670 Boudina, S., 54 Boveris, A., 448 Box, H.C., 588 Bradford, M.M., 444 Bringmann, A., 319 Brodsky, S.V., 23 Bronchoalveolar Lavage, 410 Bubici, C., 172 Buege, J.A., 445 Bunderson, M., 662 Burch, R.L., 500 Butterfield, D.A., 271 C CAA. See Cellular antioxidant activity Cadet, J., 579 Cadmium (Cd), 484–485 Cai, L., 18 cAMP. See Cyclic adenosine monophosphate

683 Carbon tetrachloride (CCl4)-induced hepatotoxicity cyclooxygenase-mediated lipid peroxidation, 472 free radical-mediated lipid peroxidation and oxidative injury, 471–472 hepatotoxin, 468 isoprostane formation, 469–470 malondialdehyde, 469 metabolism, 470–471 oxidative stress induction antioxidants, 475–476 15-keto-dihydro-PGF2a, 474 male rat, 472 PGF2a in liver tissue, 473 PGF2a in peripheral plasma and urine, 474 perchloromethane/tetrachloromethane, 468 physiological kinetics, 476 prostaglandin F2a formation, 470 Cardiolipin (CL), 267 Cardiolipin species, 449 Cardiopulmonary resuscitation (CPR), 280 Cardiovascular disease (CVD) electrical neuromodulation, 46 genetically modified animals, 45–46 G protein signaling cyclic AMP, 622–623, 632 hyperglycemia, 631–632 and oxidative stress in hypertension, 627–629 peroxynitrite, 629–630 Gpx4, lipid peroxidation, 272–273 guinea pigs, 46 inflammatory cells and mediators, 40 lacunar ischemic stroke, 46 lipid-rich diets, 41 murine models of atherosclerosis, 44 oxidative stress NPR-C, 630–631 and ROS, 623–625 oxygen radicals and ROS, 40 porcine models, 46–47 rabbit dietary-induced atherosclerosis atheromatous plaques, 42 cholesterol-rich diets, 41 gender differences, 42 intraplaque haemorrhage/calcification, 44 LDL susceptibility, 43 lumen stenosis, 44 plasma and lipoproteins concentrations, 43 saturated fat, 42

684 Cardiovascular disease (CVD) (Cont.) unsaturated fat-enriched diets, 43 WHHLMI rabbits, 43–44 Tangier disease, 46 Carlsen, H., 157 Carotenoids, 641 Casadesus, G., 211 Castellani, R.J., 211 Cebrián, M.E., 659 Cell killing apoptosis, 377 clonogenic assay, 375–376 colorimetric assays, 378–379 necrosis and autophagy, 377–378 Cellular antioxidant activity (CAA) ABAP vs. H2O2, 646, 647 cell density, 641 DCFH-DA, 640 DMSO, 640, 641 dose-response tests, 646, 648 peroxyl radicals formation, 640 principle and procedure, 640 strong concentration dependence, 647 Cellular hypoxia, 537 Cellular lipid peroxidation antioxidant activity (CLPAA) carotenoids, 641 C11-BODIPY, 641, 647, 649 prevention, hydroxytyrosol, 648, 649 Cerebral blood flow (CBF), 281 Cerebral metabolic rate of oxygen (CMRO2), 287, 288 c-GMP. See Cyclic guanosine monophosphate Chang, J., 211 Cheeseman, K.H., 445 Chemokines, 349–350 Chen, D., 49 Chertkow, H.M., 251 CHF. See Congestive heart failure Chinese hamster ovary, 388 Chromium (Cr), 484–485 Chronic kidney disease (CKD), 14 Chronic obstructive pulmonary disease (COPD) airway disease, 410, 416 bronchoaveolar lavage, 410 centrilobular emphysema, 407 EBC, 408 leukotrienes, 430 oxidative stress, 400 superoxide anion, 411 Cigarette smoke condensate, 400–402 Cigarette smoke extract, 400–401 Cigarette smoke-induced oxidative stress

Index biomarkers (see Biomarkers) chronic inflammatory lung diseases, 400 COPD, 400 in vivo models lung inflammation and emphysema, 407–408 mice, 405–406 rats, 406 subchronic and chronic, 405 smokers and COPD patients bronchoalveolar lavage, 410 EBC, 408 human lung tissue collection, 410–411 induced sputum, 409–410 treatment model air-liquid interface system, 403–404 CSC preparation (see Cigarette smoke condensate) CSE preparation (see Cigarette smoke extract) total particular matter concentration, 403 CLPAA. See Cellular lipid peroxidation antioxidant activity Clustered and tandem lesions DSBs, 588 ionizing radiation and heavy particles, 588 • OH-mediated hydrogen abstraction C4’, 589–590 C5’, 590–591 peroxyl pyrimidine radicals, guanine, 588–589 radical oxidative degradation, thymine, 588 CMRO2. See Cerebral metabolic rate of oxygen Comet assay antioxidants, marine organisms, 642, 650, 651 markers, oxidative stress, 613–614 Conditional hormesis, 174 Congestive heart failure (CHF), 21, 106, 107 Contag, C.H., 164 Contrast sensitivity (CS), 230 Cook, J.A., 371 COPD. See Chronic obstructive pulmonary disease Coughlan, M.T., 19 COXs. See Cyclooxygenases cP450. See Cytochrome P450 epoxygenases C-reactive protein (CRP), 162 Cuenca, N., 238 Curcuma longa, 43 CVD. See Cardiovascular disease

Index Cyclic adenosine monophosphate (cAMP) abnormal production, 622–623 adenylate cyclase, 622–623 inhibitory G-protein, 623, 632 intracellular concentration, 622 second messengers, 622 Cyclic guanosine monophosphate (c-GMP), 292–293 (5’R)–5,’8-Cyclo–2’-deoxyadenosine formation, 590–591 Cyclooxygenase–2 (COX–2), 663 Cyclooxygenases (COXs), 75–76, 267, 293, 350–351 Cysteines, 556 Cytochrome P450 epoxygenases (cP450), 77–78 D Dabkowski, E.R., 269 Das, D.K., 131 DCFHDA. See 2’,7’-Dichlorodihydrofluorescein diacetate DeGraff, W., 371 de Haan, J.B., 3 Dejongste, M.J., 46 Desai, V.G., 201 DeVries, R., 187 Diabetes db/db (C57BL/KsJ-db/db) mice, 53–54 glucolipotoxicity, 47 Goto-Kakizaki (GK) rats, 52–53 ob/ob (C57BL/6J-ob/ob) mice, 54 OLETF rats, 52 pancreatic b cells, 47 streptozotocin and alloxan murine models autoimmune insulinitis, 48 cytotoxicity, 49 diabetogenic chemicals, 48 fat-fed/streptozotocin-induced diabetic rodents, 49–50 glucose-induced insulin secretion, 48 neonatal streptozotocin-induced diabetes, 49 Streptomyces achromogenes, 48 streptozotocin-SHRs, 49 type 1 and type 2 diabetes, 47 Zucker diabetic fatty rats, 50–51 Diabetes mellitus definition, 4 diabetes-associated atherosclerosis AGEs, 5 GPx1 knockout mouse (see Glutathione peroxidase–1)

685 HCys, 6 NOX knockout mouse, 11–13 oxLDL, 6 Pro197Leu substitution, 7 RAGE knockout mouse model, 13–14 diabetic cardiomyopthy Cu/ZnSOD knockout mice, 18 GPx1, 19–20 histopathological changes, 17 LVH, 17–18 metallothionein, 18 mitochondrially-derived ROS, 19 MnSOD, 18 NADPH oxidase, 19 quantitative RT-PCR gene expression analysis, 20, 21 RAGE deficiency, 19 structural changes, 17 vitamin E therapy, 18 diabetic nephropathy ApoE/GPx1–/– dKO mouse, 16 CKD, 14 GPx activity, 15 intensive glucose control, 15 NADPH oxidase-mediated oxidative stress, 16–17 renin-angiotensin system, 15 ROS, 4–5 targeted antioxidant therapies cardiovascular disease, 20 CHF, 21 ebselen, 22–24 mitochondrially-targeted antioxidants, 24–25 NOX-inhibitors, 25–27 vitamins E and C, 21 Diabetes mellitus type 2 (T2DM) cellular injury, 668 free radical production, 668–669 link with environment, 668 Nrf2, 669, 670 pancreatic b-cell line, 671, 672 PDX1 translocation, 669 PKB/Akt, 670–671 PPARg agonist, 670 ROS levels, 669 Diabetic nephropathy (DN) ApoE/GPx1–/– dKO mouse, 16 CKD, 14 GPx activity, 15 intensive glucose control, 15 NADPH oxidase-mediated oxidative stress, 16–17 renin-angiotensin system, 15

686 Diabetic retinal edema, 320–321 Diabetic retinopathy retinal edema, 334 retinal inflammation, 325 visual acuity and acquired blindness, 320 4,6-Diamino–5-formamidopyrimidine, 587, 588 Diaz-Villaseñor, A., 671 Dibutyryl cAMP (dbcAMP), 629 2’,7’-Dichlorodihydrofluorescein diacetate (DCFHDA), 145–146, 515, 609, 640 2,6-Dichloroindophenol (DCIP), 566 Diclofenac (DCLF), 459–460 Dihydrorhodamine 123 (DHR), 609 3,4-Dihydroxyphenylacetaldehyde (DOPAL), 192 Dilution reference indicators, 429 Dimethylarsinous acid (DMAIII), 665–666 Dimethyl sulfoxide (DMSO), 640, 641 Dithiothretiol (DTT), 607 Dizdaroglu, M., 594 DN. See Diabetic nephropathy DNA apoptosis, 377 cell killing and mutations, 374 damage and repair, 379, 381–383 IR exposure, 383 Micrococcus radiodurans, 375 thymidine, 390 DNA damage 2’-deoxyguanosine oxidation, 610, 611 haem proteins, 606 hydroxyl radical reactions cations formation, 582 clustered and tandem lesions, 588–591 predominant indirect effect, ionizing radiation, 581 single-base lesions (see Single-base lesions) THP1 human monocytes, 582 markers, oxidative stress cellular antioxidant status, 609 cellular level analysis, 8-oxodG, 611–612 Comet assay, 613–614 intracellular ROS, 608–609 lipids and proteins, 610 non-cellular markers, 607–608 urine, 614 MYH, 610–611 OGG1, 610

Index oxidative measurement chromatographic methods, 593 5,6-dihydroxy–5,6-dihydrothymine, 593 enzymatic assays, 595–596 HPLC-based methods, 593–595 oxidative stress, definition, 606–607 peroxynitrite (ONOO¯) formation, 580 relationship with disease, 617 singlet oxygen oxidation, guanine, 592–593 validation ECVAG work, 615–616 ESCODD results, 615 ESCULA work, 616 Docosahexaenoic acid (DHA), 71 Dopamine (DA), 188, 193–194 Dose-modifying factor (DMF), 390 Double-strand breaks (DSBs), 588 Douki, T., 579 Doxorubicin (Dox), heart adriamycin, 106, 107, 121 cardiomyopathy and CHF, 106, 107 cardiotoxicity, 106 differential mechanisms anthracycline aglycones, 108 DNA binding and alkylation, 107 DNA cross-linking, 107 DNA intercalation and topoisomerase II inhibition, 106, 107 doxorubicinol, 108–109 mitochondria, microsomes, nucleus and cytosol, 108 redox reaction, 108 ROS generation, 107, 108 vitamin E, 107 Hsps (see Heat shock proteins) Streptomyces peucetius, 106 Druhan, L.J., 105 E Ebselen anti-atherogenic action, 23–24 diabetes-associated atherosclerosis, 22–23 GPx1-mimetic ebselen, 22 EcoScreen® condenser, 422–424 EETs. See Epoxyeicosatrienoic acids Eicosanoids, 430 Electron paramagnetic resonance (EPR), 415–416 Electron spin resonance (ESR) decay rate constants, 95 3D ESR-CT images, 97, 99 2D ESR projection images, 98, 100

Index in vivo L-band ESR, 94, 96 nitroxyl spin probes, 94–95 noninvasive L-band ESR, 94, 95 oxidative stress measurement, 148 propofol anesthesia, 97 propofol MCT/LCT, 97, 98 ROS generation, 95 ROS scavengers, 97 Electron transport chain (ETC), 112 Electroretinograms (ERG), 306 Endogenous and exogenous antioxidants, 453–455 Endotoxemic pig model asphyxia, 506 atelectasis, 507 baseline arterial oxygen tension, 506 intramuscular injection, 506 morphine I.V, 507 oral tracheal intubation, 507 tracheostomy, 507 Environmental Cancer Risk, Nutrition and Individual Susceptibility (ECNIS), 615–616 Epoxyeicosatrienoic acids (EETs), 77–78 Erdogan, S., 622 Eriksen, T.E., 637 Eriksson, M., 497 Erlich, J., 174 ESR. See Electron spin resonance Esterbauer, H., 445 5-Ethyl–5,6-dihydro–6-phenyl–3,8diaminophenanthridine, hydroethidine (DHE), 388 The European Standards Committee on Oxidative Damage on DNA (ESCODD), 594, 615 European Standards Committee Urinary Lesion Analysis (ESCULA), 616 Exercise model antioxidants angiogenesis, 535 catalase, 533 contractile losses, 534 definition, 533 glutathione peroxidase, 533 oxypurinol treatment, 534 skeletal muscle, 534 superoxide dismutase, 533 free radical generation bio-oxidation, 531 eccentric exhaustive exercise, 532 muscular contractile activity, 532 NAD(P)H oxidase enzymes, 532–533

687 O2?- and H2O2 generation, 532 xanthine oxidase and dehydrogenase, 533 hypoxia, exercise and oxidative stress, 537 muscle adaptation, redox control, 535–537 muscle health/disease, RONS, 537–538 Exhaled breath condensate (EBC) biomarkers advantages and limitations, 435 airway lining fluid, 422 COPD and healthy smoker aldehyde, 434 ATP, 434 eicosanoids, 430 hydrogen peroxide, 432–433 isoprostanes, 431–432 leukotrienes, 430–431 metabolomics, 435 NO-derived product, 433 pH, 433–434 prostanoids, 431 TNF, 434 mass spectrometry, 436 methodology analytical technique, 424–426 dilution reference indicator, 429 experimental setup, 422–424 flow-dependence of biomolecule, 427–428 nasal and saliva contamination, 429 organ specificity, 428–429 origin of biomolecules, 426–427 time-dependence of biomolecule, 428 within-day and between-day variability of biomolecule, 429–430 pharmacological therapy, 436 volatile biomolecules, 421 Extracellular matrix (ECM) proteins, 15 Extracorporeal photoimmunotherapy (ECPI), 455–456 F Fabre, K., 371 fa/fa rats, 50 Federiuk, I.F., 48 Fernandez, M.L., 46 Ferric reducing antioxidant power (FRAP) assay, 643 Figueiredo, C., 356 F2-isoprostanes and 15-keto-dihydro-PGF2a biosynthesis, 469–470 and prostaglandins, 450 Floyd, R.A., 594 Fluorescein angiography (FAG), 305

688 Fluorescence plus Giemsa, 385 Folch, J., 444 Formamidopyrimidine glycosylase (FPG) enzyme, 613, 614 Fuchs, J., 93 Fu, J., 671 Fujiwara, Y., 665 G Gardemann, A., 441 Gastric epithelial cells apoptosis, 352 COX–2, 350 ERK and JNK signaling, 349 IL–8 production, 349–350 inflammatory signaling, 346 LPS, 344 NADPH oxidase complex, 345 nuclear factor-k B, 347 Ras activation and Elk-phosphorylation, 349 ROS and inflammatory gene expression, 345 Geletin zymography, 312 Gene expression omnibus (GEO), 255 Gene ontology (GO), 253 Genomic instability/bystander effects carcinogenesis and mutation, 383–384 CHO (see Chinese hamster ovary) chromosomal aberrations, 384–385 cytochalasin B, 386, 387 DHE, 388 DNA damage, 383–384 FPG, 385 HGRPT, 386–387 IR exposure, 383 micronuclei, 386 sister chromatid exchanges (SCEs), 385–386 Ghosh, S., 161 Giasson, B.I., 673 Gil, A., 39 Glial cells, rat retina clinical implications, 334–335 diabetic retinal edema, 320–321 functional implications adenosine, 332–333 hypoosmotic environment, 333 neuron-to-glia signaling, 333–334 NTPDase1, 332–333 osmotic swelling (see Osmotic swelling, retinal glial cells) purinergic inhibition ATP, 328–329 autocrine signaling mechanism, 328

Index diabetes alters glial expression, 331–332 ecto-nucleotidases, 330–331 glutamate activates, 328–329 Kir channels, 328 retinal edema, diabetics, 320–321 retinal fluid clearance aquaporin water channels, 321 gliosis, 322 ion and water transport, 321 osmotic glial cell swelling mechanisms, 323 potassium, 321 Glucose–6-phosphate dehydrogenase (G6PD)-derived NADPH, 18 Glutathione (GSH) biomarkers, 415 concentration-dependent induction, D3T, 567, 568 content assay, 566 intracellular redox state, 626 reaction with aldehydes, 573 ROS, 574 Glutathione peroxidase–1 (GPx1) diabetes-associated atherosclerosis antioxidant pathway, 7–8 ApoE/GPx1 dKO mice, 9–11 high fat diets, pro-atherogenic mechanisms, 9 polymorphisms, 6 diabetic cardiomyopthy, 19–20 mimetic ebselen, 22 Glutathione peroxidase 4 (Gpx4) cardiolipin, 267 COXs and LOXs, 267 hydroperoxides, 266–267 isoforms, 267–268 knockout mice, 268–269 lipid peroxidation aging, 270–271 Alzheimer’s disease, 271–272 CVD, 272–273 4-HNE and MDA, 266 PHGPx, 266 ROS, 266 transgenic mice, 269–270 Glutathione S-transferase M3 (GSTM3), 254 Gollnick, H., 441 Gorin, Y., 17 GPx1. See Glutathione peroxidase–1 Gpx4. See Glutathione peroxidase 4 Granada, J.F., 46 Grill, M., 352 Guo, Z., 273

Index H Hamanishi, T., 6 Hammer-Andersen, J., 637 Hansen, E., 637 HD. See Huntington’s disease Heat shock factors (HSFs), 110 Heat shock proteins (Hsps) antioxidant enzymes, 120–122 cardioprotectants, 123 cardioprotection cardiac H9c2 cells, 113, 115 cutaneous tumerogenesis, 118 DMPO spin adducts, 118 gene deletion/mutations, 112 Hsp25, 113, 115–117 Hsp27 expression, 113–115 in vitro and in vivo models, 112 mTOR, 119 PFT-a, 115 p53 immunoprecipitation, 115 p53 regulation, 115, 116 proapototic Bax, 116 protein carbonylation, 118 TLRs, 119 chemotherapy, 121 ETC, 112 genetic programs, 109 genotoxic and proteotoxic stresses, 110 HSF-HSE DNA, 111 Hsp90 and Hsp27, 112 immunoprecipitation and immunoblotting, 123 30 kD stress protein, 109 NOS, 112 phosphorylation and MAP kinase, 119–120 and sumoylation, 111 redundancy and feedback control, 110 serine/proline motifs, 111 thermotolerance and cytoprotection, 109 transcription factors, 110 transgenic animal models, 111 vincristine and vinblastine, 112 Helicobacter pylori AP–1, 347–348 cagA PAI genes, 355 chemokines, 349–350 COX, 350–351 iceA, 356 integrins, 354–355 MAPK, 348–349 NF-kB, iNOS, and apoptosis, 352 nuclear factor-k B, 347

689 oxygen radicals, NADPH oxidase and Cag A gastric epithelial cells, 346 inflammatory gene expression, 344–345 NADPH oxidase, 344–345 PMNs, 346 ROS, 344 PAR (see Protease-activated receptors) proinflammation COX, 350–351 nitric oxide synthase, 351 vacuolating toxin, 355 Hemolytic anemia, 484 HETEs. See Hydroxyeicosatetraenoic acids Hofer, T., 637 Homocysteine (HCys), 6 Houstis, N., 669 HPETE. See Hydroperoxyeicosatetraenoic acid HpODE. See Hydroperoxide isomer of linoleic acid Hsps. See Heat shock proteins Huntington’s disease (HD) animal models, 233–236 etiology, 226–228 pathologic mechanisms, 228 visual disturbances, 229 Hydroethidine oxidation, 549 Hydrogen peroxide, 432–433 Hydroperoxide isomer of linoleic acid (HpODE), 304–305 Hydroperoxyeicosatetraenoic acid (HPETE), 76–77 8-Hydroxy–2’-deoxyguanosine (8-OHdG), 413–414 6-Hydroxy-dopamine (6-OHDA), 189–190, 193 Hydroxyeicosatetraenoic acids (HETEs), 76–77 8-Hydroxyguanosine (8OHG), 214 4-Hydroxyl nonenal (4-HNE), 266, 271 Hydroxynonenal (HNE), 135 Hyperbaric oxygen (HBO), 79 Hyperinsulinemia, 51 Hypertension MMAIII, 667 MMAV and DMAV, 667, 668 smooth muscle contraction, 666 vasoconstriction, 666, 667 Hypoxanthine-guanine phosphoribosyltransferase (HGPRT), 386–387

690 I Iandiev, I., 322 Ilangovan, G., 105 Induction of oxidative stress, CCl4 antioxidants, 475–476 15-keto-dihydro-PGF2a, 474 male rat, 472 PGF2a in liver tissue, 473 in peripheral plasma and urine, 474 Inflammation acute exercise, muscle damage and anti-oxidative vitamins, 522 AP–1, 522 chronic inflammation and disease, 525 endurance exercise, 521 exercise, cytokines and oxidative vitamins antioxidative vitamins, 524–525 chronic systemic inflammation, 523 muscle damage, 523–524 TNF-a, 523 gene transcription and protein synthesis, 522 insulin sensitivity, 527 low grade inflammation model, 526 metabolic syndrome, 525 myokine IL–6, 526 neuroinflammation, 526 nuclear factor kB, 522 six-week aerobic exercise training program, 527 Integrins, 354–355 Intercellular adhesion molecule –1 (ICAM –1), 162 Intracerebral hemorrhage (ICH), 69 Ionizing radiation (IR) cancer treatment, 373 electromagnetic spectrum, 373–374 radiation-induced damage cell killing (see Cell killing) DNA damage repair measurement, 381–383 DNA Repair, 379 GI/BSE (see Genomic instability/ bystander effects) homologous recombination, 381 molecular target, 374–375 NHEJ, 380–381 radiation response modifiers IR protectors, 392 radiosensitizers, 390–391 vivo models, 388–389

Index Ischemia and reperfusion circulatory arrest, 279–280 experimental cardiac arrest and resuscitation AC electric shock, 281 analytical methods, 281–282 aortic occlusion catheter, 287 blood flow measurement, 280 brain stem reticular formation, 288 CBF, 281, 282 cerebral cortical blood flow, 287, 288 cerebral perfusion pressure, 287 c-GMP, 292–293 CMRO2, 287, 288 coronal section, pig brain, 288, 289 free radical oxidation, 289 G-protein coupled adenylyl cyclase, 293 8-iso-PGF2a, 289, 290 15-keto-dihydro-PGF2a, 289, 291, 292 linear correlation, 292, 295 lipid peroxidation, 289 mean arterial blood pressure and coronary perfusion pressure, 292, 293 mean jugular bulb plasma levels, 292, 294 mean parametric maps, 282, 286 methylene blue, 292–293 mid-thalamic nuclei, 288 MPAF, 282, 284 neurological deficit score, 292, 294 “no-reflow” phenomenon, 284 OEF, 287–288 open-chest and closed-chest CPR, 282 PBN, 292 plasma eicosanoids, 292, 295 radical scavengers, 292 ROSC, 281, 282 SPP and PPP, 282, 283, 285 ventilation, 280–281 ventricular fibrillation, 279 Isoprostanes, 431–432 Iwai, S., 303 J Jackson-Lewis, V., 187, 192 Jandeleit-Dahm, K.A., 3 Jensen, I.-J., 637 Jentzsch, A.M., 445 Jia, Z., 563, 621 Juzwiak, S., 42

Index K Kagan, V.E., 267 Kajikawa, H., 353 Kanagasabai, R., 105 Karin, M., 161, 173 Karlsson, H.L., 605, 606, 608, 611–613 Kaur, J., 663 Khanna, S., 63 Kirk, E.A., 12 Klei, L.R., 665 Kobayashi, K., 91 Kohler, J.J., 25 Koizumi, J., 66 Krishna, M.C., 371 Krishnamurthy, K., 105 Kühn, H., 447 L Lamensdorf, I., 192 Lang, J.K., 446 Lassila, M., 15 Lau, F.C., 481 Lawrence, T., 161 Lee, M.C., 91 Lee, M.Y., 666 Lee, P.C., 665 Left ventricular hypertrophy (LVH), 17–18 Leukotrienes, 77, 430–431 Levine, R.I., 414 Levine, R.L., 447 Liang, H., 265 Lim, S., 354 Lipcsey, M., 497 Lipid hydroperoxides (LHP) biomarkers, 306 corneal model, 307 immunohistochemical reaction, 310 leukocyte–endothelium interaction, 313 retinal model, 305–306 synthesis, 305 time curves, 308, 309 Lipid peroxidation cyclooxygenase-mediated, 472 doxorubicin, 107 free radical-mediated, 471–472 Lipid peroxidation and protein oxidation, 451–452 Lipohydroperoxides, 445 Lipopolysaccharide (LPS), 344 Lipoxygenases (LOXs), 76–77, 267 Liu, D., 621 Liu, L., 118 Liu, Y., 672

691 Li, Y., 563 Longa, E.Z., 66 Long-chain polyunsaturated fatty acids (Lc-PUFA), 638–639 Lontz, W., 458 Lorenz, P., 449 Low-density lipoprotein (LDL), 141 Lowry, O.H., 444, 448 LOXs. See Lipoxygenases Lu, L., 269 LVH. See Left ventricular hypertrophy Lynn, S., 664 M Macrophage antigen complex–1 (MAC–1), 198 Maes, O.C., 251 Malondialdehyde (MDA), 135, 266, 469, 610 Mannick, E.E., 351 Marcil, J., 629 Mariappan, N., 54 Marty, C., 630 MCAO. See Middle cerebral artery occlusion Mean pulmonary arterial blood flow (MPAF), 282, 284 Medium-chain triglyceride/long-chain triglyceride (MCT/LCT), 97, 98 Mellor, K.M., 17 Mesa, M.D., 39 Metabolomics, 435 Methylene blue (MB), 292–293 1-Methyl–4-phenyl–1,2,3,6-tetrahydropyridine (MPTP) cytokines astrocytes, 194 IL–1b, IL–1a, IL–6 and MMP–9, 195, 196 microglia, 194–195 NF-kappaB, 196 TNF-a, 195–196 DA neurons and terminals, 193–194 l-DOPA, 191 MAO-B astrocytes, 192 DOPAL, 192 H2O2, 192–193 nigrostriatal system, 191 6-OHDA, 193 mitochondria, 201–202 NO, 199–200 Parkinson’s disease, 237, 239 peroxynitrite, 200–201 SOD

692 1-Methyl–4-phenyl–1,2,3,6-tetrahydropyridine (MPTP) (Cont.) copper/zinc, manganese and extra-cellular SOD, 197, 198 COX–2, 197, 199 MAC–1, 198 M40401 and tempol, 199 NADPH oxidase, 197, 198 TH neurons, 199 Micrococcus radiodurans, 375 Microdialysis technique, 450 Microdialysis techniques, 513 Middle cerebral artery occlusion (MCAO) canine model, 67, 68 c-arm fluoroscopy, 67, 68 craniectomy approach, 68 intraluminal suture model, 66–67 intraluminal thread model, 67 laser Doppler flowmetry, 67 minimally-invasive small animal technique, 66 molecular biology tools, 69 rodent models, 66–67 transgenic lines, 69 Mild cognitive impairment (MCI), 271 Misra, H.P., 563, 621 Mitchell, J.B., 371 Mitochondria and submitochondrial membranes, 448 Mitochondrial biogenesis, 536–537 Mitochondrial oxidative stress cellular H2O2 cysteines, 556 glutathione S-transferase-Pi, 556 HeLa cells, 556–557 MAPK trafficking, 557 mitochondrial thioredoxin system, 555 phagocytic cells, 555 reactive oxygen species, 555 redox status, 554 rotenone and succinate, 557 O2?-/H2O2 formation, 550–551 outer and inner UQ pools antimycin A, 551 cytochromes bc1 segment, 551 H2O2 index concept, 553 mitochondrial O2?- production, 552 solid state model, 553 succinate dehydrogenase, 554 superoxide anion and hydrogen peroxide cellular steady-state concentration, 547 Fenton reaction, 546 fluorescent hydrogen donors, 548 H2O2 production rate, 547

Index horseradish peroxidase, 548 hydroethidine oxidation, 549 mitochondrial matrix, 546 monoamine oxidase activity, 546 spin probe DEPMPO, 549 superoxide dismutase-sensitive spectrophotometric assay, 548 Mitochondrial thioredoxin system, 555 Mitogen-activated protein kinases (MAPK), 348–349, 628 Möller, L., 605, 606, 608, 611–613 Monoamine oxidase-B (MAO-B), 191–193 Monomethylarsonous acid (MMAIII), 665–666 Montuschi, P., 421 Moreira, P., 211 Mouse embryonic fibroblasts (MEFs), 268 MPTP. See 1-Methyl–4-phenyl–1,2,3, 6-tetrahydropyridine Mukherjee, S., 131 Müller cells, 222–224 Murry, C.E., 174 Muscat, S., 170 N Naderi-Hachtroudi, L., 458 NAD(P)H:quinone oxidoreductase 1 (NQO1) NADPH oxidases (NOX), 5 diabetes-associated atherosclerosis ApoE–/– mice, 11–12 calcium-dependent isoform, 12 Poldip2, 13 p22phox, 12–13 p47phox KO mice, 12 redox signaling, 11 diabetic nephropathy (DN), 16–17 inhibitors, 25–27 Nakanishi, T., 303 Namgung, U., 673 Nam, S.M., 26 Naphthalene, 484 Natriuretic peptide receptors (NPRs), 623 Navarro, A., 448 Navas-Acien, A., 668 Necrosis, 377–378 Nelson, G.A., 233 Nemoto, M., 6 Neovascular rabbit eye models AOX, 304–305 biochemical and molecular biology results, 311–313 biomarkers, 306

Index choroidal model Bruch’s membrane, 310, 311 CNV, 306 retinal hemorrhage, 311 subretinal injections, HpODE, 306 corneal model adult male rabbits, 305 edema and superficial erosion, 307 electrophysiology, 306 histological results, 313 LHP (see Lipid hydroperoxides) ocular disease, 314 retinal model intravitreal injection, 305, 308, 309 LHP transport, 308, 309 streptozotocin animal model, 304 tissue culture cell migration, 311 endothelial, epithelial and stromal cells, 306 retinal ex vivo model, 313 VEGF (see Vascular endothelial growth factor) Zamboni’s paraformaldehyde, 306 Neurofibrillary tangles (NFT), 252 NF-kappaB activity antioxidants dietary plants extracts, 163–164 hypothesis, 176–177 Nrf2/EpRE, 167, 168 nutrition, 163 phytochemicals, 176 plant-based diets, 163 transgenic reporter mice, 164–167 dietary plants as modulators berry extracts, 169 cancer chemoprevention, 170–171 coffee consumption, 170 curcumin, 170 oregano, thyme, clove and turmeric extracts, 169–170 pomegranate extracts, 169 whole foods, 170 dietary treatments, 171 in disease carcinogenesis, 162–163 CRP and IL–6, 162 myocardial infarctions, 161 TAM, 162 targeted knock-out mice, 161 VCAM–1 and ICAM –1, 162 inflammation, cancer and cardiovascular diseases, 158 intestine, 171, 172

693 liver, 171–172 luciferase reporter, 168–169 organs ex vivo imaging, 172 preconditioning and hormesis, 174–176 conditional and stress-response hormesis, 174 dose–response relationship curves, 175, 176 LPS, 174, 175 nutritional hormesis, 175 U937 3xkB-LUC cells, 174 redox regulation, 172–174 spleen, 172 tissue distribution, phytochemicals, 171 transcription factors B-cell maturation, 161 delocalization, 161 IkBa, 159, 160 IKKa and IKKb, 159, 161 p65 phosphorylation and acetylation, 159 Rel/NF-kB, 159 signaling pathway, 159, 160 Nicotinamide adenine dinucleotide (NAD), 259 Ning, A., 242 Nitric oxide synthase (NOS), 112 Noack, H., 446 Nonhomologous end-joining (NHEJ), 380–381 Normal elderly controls (NEC), 254, 255 NOX. See NADPH oxidases Nuclear E2-related factor (Nrf2), 573 Nuclear erythroid-related factor 2 (Nrf2), 414 Nuclear factor kB (NF-kB), 535–536 Nunomura, A., 211 Nuntharatanapong, N., 663 Nutritional hormesis, 175 O 6-OHDA. See 6-Hydroxy-dopamine 6-OHDA-mediated cytotoxicity human primary astrocytes, 571, 572 human primary neurons, 569, 571 SH-SY5Y neuroblastoma cells, 569, 570 Olsen, R.L., 637 Osmotic swelling, retinal glial cells cytotoxic edema, 323 diabetic retinopathy, 325 hypoosmotic, 325 nucleoside transporters and ectonucleotidases, 323–325 oxidative stress and inflammatory lipids, 327–328

694 Osmotic swelling, retinal glial cells (Cont.) potassium conductance Kir channels, 326 retinal gliosis, 322 Ostrosky-Wegman, P., 659 Otsuka Long-Evans Tokushima fatty (OLETF) rats, 26, 52 Oxidant-induced neuronal cells injury antioxidant-rich fruits and vegetables consumption, 571–572 cell extract preparation, 565 cellular phase–2 enzyme NQO1, 567–569 cell viability assay, 566 chemicals and materials, 565 GSH concentration-dependent induction, D3T, 567, 568 content assay, 566 reaction with aldehydes, 573 ROS, 574 human primary astrocytes brain function maintenance, 569 cell culture, 565 neurocytotoxicity, MPP+, HNE or 6-OHDA, 570–572 human primary neurons cell culture, 565 D3T pretreatment, 569, 571 NQO1 activity assay, 566 Nrf2, 573 SH-SY5Y neuroblastoma cells cell culture, 565 D3T pretreatment, 569, 570 statistical analysis, 567 Oxidative stress atherosclerosis, 136–137 BBB, 92 definition, 132 diabetes mellitus, 139–140 free radicals production, 132–134 HNE, 135 human skin ECPI, 455–456 HaCaT keratinocyte and microdialysate, 456–460 iron/ascorbate induced peroxidation endogenous and exogenous antioxidant, 453–455 functional intactness of mitochondria, 452–453 lipid peroxidation and protein oxidation, 451–452 ischemia-reperfusion injury, 138–139

Index in isolated mitochondria, 442 L-band ESR/nitroxyl spin probe methods, 93–94 materials and methods antioxidants, 446 functionally intact mitochondria, 443–444 iron/ascorbate, 444 lipid peroxidation products, 445 phospholipid and fatty acid analysis, 444–445 protein content, 444 respiration and membrane potential, 444 UVA irradiation, 446–447 UVB irradiation and HaCaT keratinocytes, 447–449 UVB irradiation and microdialysis of human skin, 449–450 MC-PROXYL, 92 MDA, 135 measurement cytochrome c reduction, 146–147 DCFHDA, 145–146 ESR, 148 exhaled volatile alkanes, 143 8-hydroxyguanosine formation, 144–145 hydroxyl free radical formation, 149 isoprostanes, 143–144 LDL, 141 malonaldehyde detection, 141–142 pentane, 141 reduced glutathione method, 147 ROS generation, dihydroethidium, 145 nucleic acids, 134 oxidative DNA damage, 134–135 potential biomarkers, 140–141 protein oxidation, 136 PUFA, 135 redox homeostasis imbalance, 132 ROS, 92 cardiovascular disease, 136 RNS, 132, 134 SHR and SHRSP cerebral ischemia and reperfusion, 94 ESR (see Electron spin resonance) in vivo fluorescence microscopy, 94 by UV irradiation, 443 VSMC migration and proliferation, 136 Oxidized cardiolipin, 445 Oxidized low density lipoproteins (oxLDL), 6 8-Oxo–7,8-dihydroadenine, 587, 588

Index 8-Oxo–7,8-dihydro–2’-deoxyguanosine (8-oxodG) after 2’-deoxyguanosine, 610, 611 cellular level analysis, 611–612 urinary secretion, 614 Oxygen extraction fraction (OEF), 287–288 Oxygen radical absorbance capacity (ORAC) assays, 642–643 Oxyguanine glycosylase (OGG1), 610 P Pannicke, T., 322, 325 Paquet, C., 231 Paraquat (1,1’-dimethyl–4,4’-bipyridylium dichloride), 483–484 Parkinson’s disease (PD) animal models mouse models, 236 MPTP models, 237, 239 6-OHDA, 236–210 rotenone model, 239–240 toxin-based models, 236, 237 dopaminergic neurons, 187 etiology, 226–228 MPTP cytokines, 194–196 DA neurons and terminals, 193–194 l-DOPA, 191 MAO-B, 191–193 mitochondria, 201–202 NO, 199–200 peroxynitrite, 200–201 SOD, 197–199 6-OHDA, 189–190 oxidative stress, 188–189 paraquat, 190–191 pathologic mechanisms, 228 rotenone, 190 SNpc dopaminergic neurons, 187–188 visual disturbances CS loss, 230 dopamine and homovanillic acid levels, 229 electrical synapses modulation, 229–230 electroencephalography and electrooculography, 231 electrophysiological changes, 229 g-wave desynchronization, 231 PERG alterations, 230 VEP, 230 Pattern electroretinogram (PERG), 230 Paur, I., 157

695 PBMC. See Peripheral blood mononuclear cells Pereira, F.E., 665 Pérez de Lara, M., 221 Peripheral blood mononuclear cells (PBMC) amyloid-b, 252 gene expression, 252–253 gene ontology, 253 neuronal degeneration and gliosis, 251 NFT, 252 oxidative stress, 253 Peroxyl radicals formation, 640 Perry, G., 211 Petrasch-Parwez, L., 23 a-Phenyl-N-tert-butyl nitrone (PBN), 292 Phospholipase A2 (PLA2), 72, 73 Phospholipase C (PLC), 71 Phospholipase D (PLD), 71 Phospholipid and fatty acid analysis, 444–445 Phospholipid hydroperoxide glutathione peroxidase (PHGPx), 266 Pifithrin-a (PFT-a), 115 Pi, J., 670 Pintor, J., 221 Plasma triacylglycerols, 51 Polyenylphosphatidylcholine, 475–476 Polymorphonuclaer neutrophils (PMNs), 346 Polyunsaturated fatty acids (PUFAs), 71, 135 Pope, S., 445 Popov, T., 409 Porcine endotoxin shock models, 499 Pratico, D., 271 Programmed cell death (PCD), 173 Prostaglandin E2 (PGE2), 350–351 Prostanoids, 431 Protease-activated receptors coagulant protease thrombin, 353 leukocyte-endothelial cell interaction, 354 trypsin, 352–353 Przedborski, S., 187 Psammomys obesus, 47 Pulmonary perfusion pressure (PPP), 282, 283 Pysher, M.D., 667 Q Quist, S., 441 R Rahman, I., 399 Ran, Q., 265 Ravanat, J.-L., 579

696 Reactive nitrogen species (RNS), 132, 134, 482 Reactive oxygen species (ROS), 92 antioxidant role, 511 cardiovascular disease, 136 cell culture approaches, 514–515 DCFH-DA, 515 detoxification, 567 GSH, 567, 574 NQO1, 567 diabetes mellitus, 4–5 dichlorofluorescin, 515 dihydroethidium, 145 flexor digitorum brevis, 516 generation, 107, 108 generation, 6-OHDA neurotoxicity, 573 Gpx4, 266 Helicobacter pylori, 344 intracellular probes, 515 messenger action intracellular redox state, 626–627 superoxide anions, 627–629 target protein molecules, 626 microdialysis techniques, 513–514 muscle biopsy techniques, 512 needle biopsy approaches, 512, 513 and oxidative stress cell proliferation, 625 chronical elevation, 624 high and low levels, 625 H2O2 and nitric oxide, 625 • OH radical, 624 oxidative injury prevention, 623 OxyR protein, 624 phagocyte-type NAD(P)H oxidase, 624–625 renovascular hypertension, 625 RNS, 132, 134 semi-automated gun approach, 512 structurally diverse toxicants, 482 superoxide dismutases, 511 Regulatory domain (RD), 111 Reinheckel, T., 447, 452 Respiratory tract lining fluid (RTLF), 608 Restoration of spontaneous circulation (ROSC), 281 Richardson, J.R., 201 Rink, C., 63 ROS. See Reactive oxygen species RTube® condenser, 422–424 Ruíz-Ramos, R., 659 Russell, W.M.S., 500

Index S Saha, S., 563, 621 Santano, C., 221 Schaafsma, A., 282 Schaller, H., 444 Schipper, H.M., 251 Schlame, M., 449 Schmid, E., 626 Schnabel, R., 6 Schneider, M., 268 Seiler, A., 268 Sen, C.K., 63 Sen, R., 159 Sepsis models, 500, 501 Sevaskan, E., 232 SHRs. See Spontaneously hypertensive rats Siedlak, S., 211 Single-base lesions adenine, 587–588 guanine bi-photonic ionization, purine, 586–587 enzymic hydrolysate, DNA, 585 2,2,5-triamino(2H)-oxazolone formation, 586 thymine degradation products, thymidine, 582, 583 5,6-dihydroxy–5,6-dihydrothymine, 582, 583 5-formyluracil and 5-(hydroxymethyl) uracil fromation, 584 6-hydroperoxy–5-hydroxy–5,6-dihydrothymidine, 583, 584 • OH and one-electron oxidationinduced radicals, 584–585 six oxidized nucleosides, 582 Single cell gel-electrophoresis assay. See Comet assay Skeletal muscle contraction circulating markers measurement, 508 (Doubt) ROS (see Reactive oxygen species) Skin erythema, 449–450 Smith, K.R., 664 Smith, M.A., 211 Song, Y., 19 Sonta, T., 54 Southam, C.M., 174 Spontaneously hypertensive rats (SHRs) cerebral ischemia and reperfusion, 94 ESR decay rate constants, 95 3D ESR-CT images, 97, 99

Index 2D ESR projection images, 98, 100 in vivo L-band ESR, 94, 96 nitroxyl spin probes, 94–95 noninvasive L-band ESR, 94, 95 propofol anesthesia, 97 propofol MCT/LCT, 97, 98 ROS generation, 95 ROS scavengers, 97 in vivo fluorescence microscopy, 94 streptozotocin, 49 superoxide anions, 628 Sprague-Dawley rats, 406 Stress-response hormesis, 174 Stroke-prone spontaneously hypertensive rat (SHRSP). See Spontaneously hypertensive rats Structurally diverse toxicants experimental model Cd-induced toxicity, 489 cytotoxicity and mutagenesis, 488 hydrazone, 486 microsomal membrane fluidity, 488 peritoneal macrophage, 489 quinone metabolite, 487 TBA colorimetric method, 486 VES, 487 glutathione, 483 RNS, 482 ROS, 482 toxicant-induced oxidative stress chromium and cadmium, 484–485 endrin, 483 naphthalene, 484 paraquat, 483–484 TCDD, 483 toxicant poisoning, 490 urinary lipid metabolites, 485 Subarachnoid hemorrhage (SAH), 69 Substantia nigra pars compacta (SNpc), 187–188 Superoxide anions angiotensin II, 627–628 antioxidant treatment, 627 AT1 receptor, 628 cholera toxin, 629 MAP kinase, 628 SHR, 628 Superoxide dismutase (SOD), 197–199, 214, 312 Systemic inflammatory response syndrome (SIRS), 501–502 Systemic perfusion pressure (SPP), 282, 283

697 T Takahashi, H., 458 Tamai, K., 303 T2DM. See Diabetes mellitus type 2 Teague smoking machine, 405–406 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), 483 Thiobarbituric acid (TBA) colorimetric method, 486 Thiobarbituric acid-reactive substances (TBARS), 445 Thioredoxin (TRX), 626–627 Thomas, D.W., 447 Thymidine, 390 Tietze, F., 446 Tieu, K., 193 Tocilescu, M.A., 187 a-Tocopherol and ubiquinol, 446 Torreggiani, M., 14 Torzewski, M., 9 Toxicant-induced oxidative stress chromium and cadmium, 484–485 endrin, 483 naphthalene, 484 paraquat, 483–484 TCDD, 483 Transgenic NF-kB reporter mice apple, cherry and blackcurrant, 165–167 bioluminescence detection, 164 luciferase gene, 164 oregano, coffee, thyme, clove and walnuts extracts, 165, 166 Tsai, C.S., 231 Tsou, T.C., 663 Tumor cure dose (TCD50) assay, 389 Tumor necrosis factor (TNF), 434 U Ueda, T., 303 Urinary lipid metabolites, 485 UVA irradiation, 446–447 UVB irradiation HaCaT keratinocytes, 448–449 microdialysis of human skin, 449–450 UV irradiation and human skin ECPI, 455–457 HaCaT keratinocyte cardiolipin, 457 F2-isoprostane and prostaglandin, 458 potent antioxidant system, 456 superoxide dismutase, 458 microdialysis technique antiinflammatory drug, 460

698 UV irradiation and human skin (Cont.) DCLF, 459–460 F2-isoprostanes and prostaglandin, 459 U937 3xkB-LUC cells, 174 V Vahidnia, A., 673 Valianpoor, F., 445 Van Doorn, L.J., 356 Varmedal, I., 637 Vascular-cell adhesion molecule-1 (VCAM-1), 162 Vascular endothelial growth factor, 304, 311 Vascular smooth muscle cells (VSMCs), 136, 664, 665 Vatassery, G.T., 452 Vertebrate retina Alzheimer’s disease animal models, 240–242 etiology, 226–228 pathologic mechanisms, 228 visual disturbances, 231–233 Huntington’s disease animal models, 233–236 etiology, 226–228 pathologic mechanisms, 228 visual disturbances, 229 organization glial cells, 224 macula, 223–224 neurotransmission systems, 223 oligodendrocytes, 224 photoreceptor, 222, 223 retinal pigmented epithelium, 222 rod and cone distribution, 223 Parkinson’s disease animal models, 236–240 etiology, 226–228 pathologic mechanisms, 228 visual disturbances, 229–231 visual information transmission color vision, 226 modulation, 225

Index M, P and midget, 226 ON and OFF pathways, 224, 225 phototransduction, 224 receptive fields, 225–226 Visual-evoked potentials (VEP), 230 Vitamin E succinate (VES), 487 Volek, J.S., 46 W Walton, F.S., 670 Wang, E., 251 Wang, M.W., 49 Wang, X., 674 Watanabe heritable hyperlipidemic myocardial infarction-prone (WHHLMI) rabbits, 43–44 Wauson, E.M., 669 Wiesner, R., 447 Wiklund, L., 279 Williams, T.I, 271 Winter, J.P., 6 Wiswedel, I., 441, 445, 449, 451 Witting, P.K., 23 Wurm, A., 325, 331 X Xia, Z., 673 Y Yamawaki, H., 627 Yang, F., 160 Yao, H., 399 Yoshino, F., 91 Yuen, D.Y., 24 Z Zafra-Stone, S., 481 Zhang, G.X., 19 Zhu, H., 563 Zhu, X., 211 Zucker diabetic fatty rats, 50–51