Edited by Mohammed Amr El-Missiry

Antioxidant Enzyme Edited by Mohammed Amr El-Missiry
Contributors Praveen Krishnamurthy, Ashish Wadhwani, Janka Vašková, Ladislav Vaško, Ivan Kron, Ayşe Şen, Úrsula Muñoz Morón, Inma Castilla-Cortázar, Marion Seillier, Sylvain Peuget, Nelson J. Dusetti, Alice Carrier, Nelly Sapojnikova, Nino Asatiani, Tamar Kartvelishvili, Iagor Kalandadze, Alexander Tsiskaridze, Dawei Gao, Mirela Petrulea, Adriana Muresan, Ileana Duncea, ChihHung Guo, Pei-Chung Chen, Julita Kulbacka, Jolanta Saczko, Agnieszka Chwilkowska, Anna Choromańska, Nina Skołucka, Eqbal M. A. Dauqan, Aminah Abdullah, Halimah Abdullah Sani, Manju Singh, Divya Shrivastava, Raosaheb Kale, P. Ježek, P. Škarpa, T. Lošák, J. Hlušek, M. Jůzl, P. Elzner, Golandam Sharifi, Sujogya Kumar Panda

Published by InTech Janeza Trdine 9, 51000 Rijeka, Croatia
Copyright © 2012 InTech All chapters are Open Access distributed under the Creative Commons Attribution 3.0 license, which allows users to download, copy and build upon published articles even for commercial purposes, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications. After this work has been published by InTech, authors have the right to republish it, in whole or part, in any publication of which they are the author, and to make other personal use of the work. Any republication, referencing or personal use of the work must explicitly identify the original source. Notice Statements and opinions expressed in the chapters are these of the individual contributors and not necessarily those of the editors or publisher. No responsibility is accepted for the accuracy of information contained in the published chapters. The publisher assumes no responsibility for any damage or injury to persons or property arising out of the use of any materials, instructions, methods or ideas contained in the book.

Publishing Process Manager Sandra Bakic Typesetting InTech Prepress, Novi Sad Cover InTech Design Team First published September, 2012 Printed in Croatia A free online edition of this book is available at Additional hard copies can be obtained from Antioxidant Enzyme, Edited by Mohammed Amr El-Missiry p. cm. ISBN 978-953-51-0789-7



  Preface IX Section 1 Chapter 1 Basic Mechanisms of Protection 1 Antioxidant Enzymes and Human Health 3 Praveen Krishnamurthy and Ashish Wadhwani Oxidative Processes and Antioxidative Metaloenzymes 19 Janka Vašková, Ladislav Vaško and Ivan Kron Oxidative Stress Studies in Plant Tissue Culture 59 Ayşe Şen Protection Against Oxidative Stress and “IGF-I Deficiency Conditions” 89 Úrsula Muñoz Morón and Inma Castilla-Cortázar Antioxidant Role of p53 and of Its Target TP53INP1 117 Marion Seillier, Sylvain Peuget, Nelson J. Dusetti and Alice Carrier Biomedical Therapies 139 Plasma Antioxidant Activity as a Marker for a Favourable Outcome in Acute Ischemic Stroke 141 Nelly Sapojnikova, Nino Asatiani, Tamar Kartvelishvili, Iagor Kalandadze and Alexander Tsiskaridze Antioxidant Therapies for Hypolipidemia and Hyperglycemia 169 Dawei Gao Oxidative Stress and Antioxidant Status in Hypo- and Hyperthyroidism 197 Mirela Petrulea, Adriana Muresan and Ileana Duncea

Chapter 2

Chapter 3

Chapter 4

Chapter 5

Section 2 Chapter 6

Chapter 7

Chapter 8



Chapter 9

Mitochondrial Free Radicals, Antioxidants, Nutrient Substances, and Chronic Hepatitis C 237 Chih-Hung Guo and Pei-Chung Chen Apoptosis, Free Radicals and Antioxidant Defense in Antitumor Therapy 265 Julita Kulbacka, Jolanta Saczko, Agnieszka Chwilkowska, Anna Choromańska and Nina Skołucka Effect of Different Concentrations of Red Palm Olein and Different Vegetable Oils on Antioxidant Enzymes in Normal and Stressed Rat 303 Eqbal M. A. Dauqan, Aminah Abdullah and Halimah Abdullah Sani Antioxidants from Plants 321 Antioxidant Potential of Asparagus adscendens 323 Manju Singh, Divya Shrivastava and Raosaheb Kale Selenium – An Important Antioxidant in Crops Biofortification 343 P. Ježek, P. Škarpa, T. Lošák, J. Hlušek, M. Jůzl and P. Elzner Plant Antioxidative Enzymes – Case Study: In Vitro Organogenesis of Saffron (Crocus sativus L.) 369 Golandam Sharifi Assay Guided Comparison for Enzymatic and Non-Enzymatic Antioxidant Activities with Special Reference to Medicinal Plants 381 Sujogya Kumar Panda

Chapter 10

Chapter 11

Section 3 Chapter 12

Chapter 13

Chapter 14

Chapter 15


and stressed condition. chronic hepatitis C. Mansoura University. In the third part. This includes stroke. most books on antioxidants fail to stimulate the cross-fertilization of ideas between workers interested in basic aspects of antioxidants function and medically oriented scientists. Mohammed Amr El-Missiry Faculty of Sciences. and will be of interest to biologists. Prof. However. In the first part antioxidant enzymes and human health are discussed and recent developments in oxidative processes in animal and plant tissue culture are reviewed. physicians and so forth. Egypt   . “Biomedical Treatment “and “Antioxidants from Plants“. biochemists. The second part of the book is devoted to biomedical therapies and treatments using antioxidant enzymes. hypo. The multidisciplinary approach of this area and the permanent need for information regarding the recent advances have resulted in the need for new books on antioxidant enzymes. The objective of the present book is to contribute to such a cross-fertilization of ideas by selecting topics that illustrate the interconnection between basic and applied antioxidant biology and medical fields where multidisciplinary research is required. antioxidants from plants sources are reviewed and different methods for testing antioxidant activity of natural products are presented. Thus. This is followed by presentation of mechanisms for protection against oxidative stress followed by the role of p53 in oxidative stress and cancer control.and hyperthyroidism. The book is divided into the following parts: “Basic Mechanisms of Protection”. hypolipidemia and hyperglycemia. the book provides contributions for reference purposes at the professional level. It could be of great help to teachers and students at both the undergraduate and post graduate levels. It has become clear in recent years that the study of antioxidant enzymes is of great importance not only to interpret cellular defenses but also to understand the mechanisms leading to drug action.    Preface   The topic of antioxidant enzymes occupies a central position in cell biology and medicine.


Section 1 Basic Mechanisms of Protection             .


including the aging process. they may also interrupt with the oxidizing chain reaction to minimize the damage caused by free strokes. Free radicals and their scavengers Free radicals are electrically charged molecules. For the past decade. however. vital cellular structures and functions are lost and ultimately resulting in various pathological conditions. and denature them. i. Alzheimer’s disease. Introduction During normal metabolic functions. This chapter reviews the pathophysiological role of some of the important enzymes involved in free radical scavenging with their clinical applications. therefore. or deactivating free radicals before they attack cellular components. lipids and carbohydrates. cancer. In addition. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons. . They react with cellular molecules such as proteins. countless studies have been devoted to the beneficial effects of antioxidant enzymes.Chapter 1 Antioxidant Enzymes and Human Health Praveen Krishnamurthy and Ashish Wadhwani Additional information is available at the end of the chapter http://dx. absolutely critical for maintaining optimal cellular and systemic health and well being.5772/48109 1. licensee InTech.doi. provided the original work is properly cited. heart attacks and atherosclerosis.0). By reducing exposure to free radicals and increasing the intake of antioxidant enzyme rich foods or antioxidant enzyme supplements. Although the initial attack causes the free radical to become neutralized. and reproduction in any they have an unpaired electron. distribution.. These molecules are inherently unstable as they possess lone pair of electrons and hence become highly reactive. © 2012 Krishnamurthy and Wadhwani. thereby causing it to become stable. It has been found that a substantial link exists between free radicals and more than sixty different health conditions. which causes them to seek out and capture electrons from other substances in order to neutralize themselves. diabetes. highly reactive compounds called free radicals are generated in the body. Antioxidant enzymes are capable of stabilizing. our body’s potential to reducing the risk of free radical related health problems is made more palpable [1]. 2. they may also be introduced from the environment.e. which permits unrestricted use. As a result of this. Antioxidant enzymes are. They act by reducing the energy of the free radicals or by giving up some of their electrons for its use.

exposure to allergens and the presence of “leaky gut” syndrome.. The ability of the cell to utilize oxygen has provided humans with the benefit of metabolizing fats. glutathione and lipoic acid). Reactive Oxygen Species Reactive oxygen species (ROS) is a term that encompasses all highly reactive.g. and other illnesses.g. and by which foreign proteins (antigens) are denatured. tocopherols and tocotrienols.1. Xenobiotic metabolism. thousands of free radical reactions can occur within seconds of the initial reaction..4] 3. Consequently.4 Antioxidant Enzyme another free radical is formed in the process. Whenever the balance between ROS production and antioxidant defence is lost. i. GPx and reductase. hydrogen peroxide. metal binding proteins (e. pollution. oxygen containing molecules. Oxidative burst from phagocytes (white blood cells) as part of the mechanism by which bacteria and viruses are killed. hydrogen peroxide. ROS are generated by a number of pathways.[3. SOD. including free radicals. however. And until subsequent free radicals are deactivated. causing a chain reaction to occur. the superoxide anion radical. nitric oxide radical.. chronic inflammation. and carbohydrates for energy. it does not exhibit extreme reactivity due to quantum mechanical .[2] Thus cells under aerobic condition are always threatened with the insult of ROS. things like vigorous exercise. proteins. resulting in cellular damage. Most of the oxidants produced by cells occur as:    A consequence of normal aerobic metabolism: approximately 90% of the oxygen utilized by the cell is consumed by the mitochondrial electron transport system. ferritin. which however are efficiently taken care of by the highly powerful antioxidant systems of the cell without any untoward effect. which accelerates cellular metabolism. it does not come without cost. CAT. and various lipid peroxides. Consequences of generation of ROS Although O2 can behave like a radical (a diradical) owing to presence of two unpaired electrons of parallel spin. and insecticides may all contribute to an increase in the body’s oxidant load. Types of ROS include the hydroxyl radical.e. This antioxidant system includes.. nutrient-derived antioxidants (e. nucleic acids. and ceruloplasmin) and numerous other antioxidant phytonutrients present in a wide variety of plant foods.g. antioxidant enzymes (e. ‘oxidative stress’ results which through a series of events deregulates the cellular functions leading to various pathological conditions. and other small molecules. All are capable of reacting with membrane lipids. Oxygen is a highly reactive atom that is capable of becoming part of potentially damaging molecules commonly called free radical or reactive oxygen species (ROS).). and hydroxyl radicals by univalent reduction of O2. ascorbic acid. hypochlorite radical. proteins and enzymes. carotenoids. and exposure to drugs or toxins such as cigarette smoke. 3. infections. About 5% or more of the inhaled O2 is converted to ROS such as superoxide. albumin. singlet oxygen. etc. detoxification of toxic substances. lactoferrin. pesticides.

However. which reduces O2 by four electrons to H2O without releasing either invariably produced in respiring cells. To a lesser extent. Down’s syndrome.or H2O2.e. likewise. proteins.and H2O2 in a number of different tissue injuries. O2. except during abnormal exposure to ionization radiation. For the production of H2O2. and ischemic reperfusion injury in different tissues including . Cytochrome P-450. cardiovascular disease. P450 reductase and cytochrome b-5 reductase in the endoplasmic reticulum under certain conditions generate O2-. When the electron transport chain is highly reduced. During their catalytic cycles. Some specific examples of ROS mediated disease are Alzheimer’s disease. and AIDS. hydrogen peroxide (H2O2). loss of enzyme activity. as well as D-amino acid oxidase. gastrointestinal ulcerogenesis. as given following Fenton reaction: [2] Fe 2   H 2 O 2  Fe 2   OH  OH  Reactive oxygen species can attack vital cell components like polyunsaturated fatty acids. generation of ºOH in vivo requires the presence of trace amount of H2O2 and Fe2+ salt forms ºOH. leakage of electrons at the ubisemiquinone and ubiquinone sites increases so as to result in production of O2. metabolic disorders.01). carbohydrates are also the targets of ROS. cystic fibrosis. Its electronic structure result in formation of water by reduction with four electrons. for the production of ºOH. resulting in the appropriate single electron reduction of oxygen to O2-. and nucleic acids. These reactions can alter intrinsic membrane properties like fluidity. normally the tendency of univalent reduction of O2 in respiring cells is restricted by cytochrome oxidase of the mitochondrial electron transport chain. DNA damage. and the respiratory rate is dependent on ADP availability. neurodegenerative disease. This is due to the probable leak of single electron at the specific site of the mitochondrial electron transport chain. oxidative modification of low-density lipoprotein in atherosclerosis. ion transport. protein synthesis. several reactive intermediates are formed. such as superoxide (O2-). the process can be represented as: e e e e O 2  O 2   H 2O 2  OH  H 2O         For the production of O2-. and fatty acyl oxidase participate.: O 2  4H  4e   2H 2 O In the sequential univalent process by which O2 undergoes reduction. and H2O2.and H2O2. Parkinson’s disease. the catalytic cycle of xanthine oxidase has emerged as important source of O2.Antioxidant Enzymes and Human Health 5 restrictions. Finally. and the extremely reactive hydroxy radical (°OH): collectively termed as the reactive oxygen species. ultimately resulting in cell death (fig. i.[2] Damage to cells caused by free radicals is believed to play a central role in various human disorders like rheumatoid arthritis. cancer. hemorrhagic shock. peroxisomal oxidases and flavoprotein. L-hydroxy acid oxidase.

g. kidney. Endogenous Antioxidants  Bilirubin  Thiols. Among these.6 Antioxidant Enzyme heart.3] Ischemic acidosis and tissue damage Mitochondria Peroxisomes oxidases P-450 Neutrophil Antioxidants & Scavengers Enzymes Small compounds SOD α-tocopherol GSHAscorbate GPx CAT β-carotene O2 + H2O2 Fe2+/Cu2+  OH Lipid Protein Lipid peroxidation Site specific Oxidative damage Modification of base. Antioxidant protection system To protect the cells and organ systems of the body against reactive oxygen species (ROS). It involves a variety of components. sugar and fragmentation DNA Oxidative damaged cell constituents Pathological condition Figure 1. glutathione. humans have evolved a highly sophisticated and complex antioxidant protection system. N-acetyl cysteine  NADPH and NADH  Ubiquinone (coenzyme Q10)  Uric acid . that function interactively and synergistically to neutralize free radicals (Table 1)[5] These components include: a. lipoic acid.. An overall picture of the metabolism of ROS and the mechanism of oxidative tissue damage leading to pathological conditions 4. both endogenous and exogenous in origin. and gastrointestinal tract. brain. [2. liver. role of ROS in atherosclerosis and ischemic injury in heart and brain studied extensively. e.

e. Glutathione Flavonoids. GSH-Px removes H2O2 by coupling its reduction with the oxidation of GSH.. ii. Vitamin-E. flavonoids. haptoglobins. transferrin.. Enzymes: copper/zinc and manganese-dependent superoxide dismutase iron-dependent catalase selenium-dependent glutathione peroxidase Dietary Antioxidants  Vitamin C  Vitamin E  Beta carotene and other carotenoids and oxycarotenoids. protection against macromolecular damage by proteins such as stress or heat shock proteins.Antioxidant Enzymes and Human Health 7 b.g. Vitamin-E. iii. SOD Vitamin C. and Proanthocyanidins Metal Binding Proteins  Albumin (copper)  Ceruloplasmin (copper)  Metallothionein (copper)  Ferritin (iron)  Myoglobin (iron)  Transferrin (iron)  NEUTRALIZING ANTIOXIDANTS Vitamin C. intracellular H2O2 cannot be eliminated unless it diffuses to the peroxisomes [6]. lipoic acid Beta-carotene. metallothionein. Various ROS and corresponding neutralizing antioxidants Defence mechanisms against free radical-induced oxidative damage include the following: catalytic removal of free radicals and reactive species by factors such as CAT. vitamin C (ascorbic acid). such as GSH. flavonoids. bilirubin. and uric acid [6] Animal CAT are heme-containing enzymes that convert hydrogen peroxide (H2O2) to water and O2. and they are largely localized in subcellular organelles such as peroxisomes.g. vitamin E (α. c. Ubiquinone. caeroplasmin) to pro-oxidant metal ions. flavonol’s. SOD. GSH-Px can also reduce other peroxides. and iv.. . Mitochondria and the endoplasmic reticulum contain little CAT. These enzymes are present in the cytoplasm at i. Glutathione. Lipoic acid Vitamin C. beta carotene. flavones. GPx and thiol-specific antioxidants.tocopherol). binding of proteins (e. Glutathione peroxidase ROS Hydroxyl radical Superoxide radical Hydrogen peroxide Lipid peroxides Table 1. e. Glutathione. Flavonoids. reduction of free radicals by electron donors. lycopene and lutein  Polyphenols. such as iron and copper. Thus. such as fatty acid hydro peroxides. flavonoids.g.

vitamin C. The repair enzymes that can regrate some antioxidants are SOD. such as vitamin C. GSH(l-L-γ-glutamyl-l-cysteinyl glycine). SOD 2 O 2   2H   H 2O 2  O 2  GSH-Peroxidase ROOH / H 2O 2  ROH / H 2O  GSSG  GSH Peroxidase H 2 O 2  AH 2  2H 2 O  A  . nucleus. act as antioxidants within lipid environments. glutathione reductase (GR). whereas lipid soluble forms. Selenium is considered particularly important in protecting the lipid environment against oxidative injury. and then GSH synthetase adds glycine. and plasma. Dietary micronutrients also contribute to the antioxidant defence system.carotene. and manganese are also important elements. Selenium.tocopherol being the predominant and most active form). Watersoluble molecules. 5. GPx. and GPx constitute a mutually supportive team of defence against ROS.8 Antioxidant Enzyme millimolar concentrations and also present in the mitochondrial matrix. beta-carotene. catalase and peroxidases do the same for H2O2. either directly by reacting with reactive species or indirectly through glutathione transferases [6-8]. The copper-zinc SOD isoform is present in the cytoplasm. These include β carotene. such as vitamin E and β. CAT. C. CAT and the other metalloenzymes. the manganese SOD isoform is primarily located in mitochondria. one must continually produce more of the antioxidants in the body or ingest them either in diet or by supply mentation. with α. GSH prevents the oxidation of protein thiol groups. are potent radical scavenging agents in the aqueous phase of the cytoplasm. GSH is synthesized in two steps. etc. While SOD lowers the steady-state level of O2-. generating water peroxide as a final product of the dismutation. Antioxidant enzymes in health Antioxidants are of different types so that they might be available for action when and where they are needed. They are natural (enzymes antioxidants and metal carrier proteins in the body). and vitamin E (the vitamin E family comprises both tocopherols and tocotrienols. zinc. γ-glutamyl cysteine synthetase (γ-GCS) forms a γ-peptide bond between glutamic acid and cysteine. pharmacologic antioxidants and others. Most animal tissues contain both CAT and GSH-Px activity. as it serves as a cofactor for GSH-Px [6–8]. On the other hand. SODs are metal-containing proteins that catalyze the removal of superoxide. Three isoforms have been identified. since they act as cofactors for antioxidant enzymes. and they all are present in all eukaryotic cells. Therefore. Antioxidant compounds must be up’’ (converted) in the process of neutralizing free radicals. SOD. The most abundant cellular antioxidant is the tripeptide.). copper. First. scavenging or chain breaking (like vitamin A.

heme peroxidase.” Lipoic acid may also exert its antioxidant effect by cheating with pro-oxidant metals. and successive oxidation and . zinc. When an individual is exposed to high levels of xenobiotics. mitochondrial Mn-SOD. cytochrome P-450 mixed-function oxidase. and manganese for optimum catalytic activity. is synthesized from the amino acids glycine. Exposure of the liver to xenobiotic substances induces oxidative reactions through the up regulation of detoxification enzymes. an important water-soluble antioxidant. categorized as a “thiol” or “biothiol. the body relies on several endogenous defence mechanisms to help protect against free radical-induced cell damage. This breakthrough caused medical scientists to begin to look seriously at free radicals. Peroxide can be destroyed by CAT or GPX reactions [9-11]. the body has. Research suggests that glutathione and vitamin C work interactively to quench free radicals and that they have a sparing effect upon each other. iron. CAT. dihydrolipoic acid (DHLA). copper. and also plays a major role in xenobiotic metabolism. such as pyruvate and alphaketoglutarate.13]. more glutathione is utilized for conjugation (a key step in the body’s detoxification process) making it less available to serve as an antioxidant. Lipoic acid. glutamate. and SOD – metabolize oxidative toxic intermediates and require micronutrient cofactors such as selenium. Animal studies have demonstrated supplemental lipoic acid to protect against the symptoms of vitamin E or vitamin C deficiency. throughout the course of millions of years of evaluation become accustomed to coping with free radicals and has evolved various schemes for doing this [3]. Glutathione. SOD O 2   O 2   2H   H 2O 2  O 2  In humans. SOD (EC 1. In most cases the process is automatically controlled and the number of free radicals does not become dangerously high. in the Krebs cycle.Antioxidant Enzymes and Human Health 9 Catalase H 2 O 2  2H 2O  O 2  Catalytic removal of ROS by antioxidant enzyme Endogenous Antioxidants In addition to dietary antioxidants.. Glutathione directly quenches ROS such as lipid peroxides. i.e.1. are capable of quenching free radicals in both lipid and aqueous domains and as such has been called a “universal antioxidant. yet another important endogenous antioxidant. Lipoic acid and its reduced form.1) is the antioxidant enzyme that catalysed the dismutation of the highly reactive superoxide anion to O2 and to the less reactive species H2O2. there are three forms of SOD: cytosolic Cu/Zn-SOD. The antioxidant enzymes – GPx. which provides an important means of cellular defence against free radical damage. Superoxide dismutase In 1967 biochemist Irwin Fridovitch of Duke University and Joe McCord discovered the antioxidant enzyme SOD. and extracellular SOD (EC-SOD) [12. Research further suggests that lipoic acid has a sparing effect on other antioxidants. SOD destroys O2.” is a sulphur-containing molecule that is known for its involvement in the reaction that catalyzes the oxidative decarboxylation of alpha-keto acids. Fortunately.15.

. accounting for the majority of the SOD activity in plasma.SOD is essential for life whereas Cu/Zn-SOD is not. Inactivation of recombinant human mitochondrial Mn. lymph. coli [26]. (b) elimination of the gene in Saccharomyces cerevisiae increases its sensitivity to oxygen [20]. Cu/Zn-SOD is competitively inhibited by N3.SOD.23]. Extracellular superoxide dismutase (EC-SOD) is a secretory. f) expression of human Mn-SOD genes in transgenic mice protects against oxygen induced pulmonary injury and Adriamycin-induced cardiac toxicity [25]. EC-SOD is not induced by its substrate or by other oxidants and its regulation in mammalian tissues primarily occurs in a manner coordinated by cytokines.or Cu/Zn-SODs. Other recent reports involving SOD knockouts have revealed that Mn. tetrameric. (c) lack of expression in MnSOD knockout mice results in dilated cardiomyopathy and neonatal lethality [21]. but not Cu/Zn. but distinct differences have been noted in the susceptibilities of Fe-. (e) transection of Mn. (d) tumor necrosis factor (TNF) selectively induces Mn-SOD.SOD cDNA into cultured cells rendered the cells resistant to parquet. rather than as a response of individual cells to oxidants [32]. but is only moderately influenced by oxidants [17]. Mn. Cu/Zn-SOD knock-out mice appear normal and exhibit differences only after traumatic injury. EC-SOD was found in the interstitial spaces of tissues and also in extracellular fluids. The respiratory chain in mitochondria is a major source of oxygen radicals. the active site. with a high affinity for certain glycosaminoglycans such as heparin and heparin sulphate. These enzymes have two identical subunits of about 32 kDa. .29]. Mn-SOD is a homotetramer (96 kDa) containing one manganese atom per subunit those cycles from Mn (III) to Mn (II) and back to Mn (III) during the two step dismutation of superoxide [17]. and by F. All types of SOD bind single charged anions such as azide and fluoride. Each subunit contains a metal cluster. Calves that were fed milk supplemented with 25 ppm Cu and 100 ppm Zn showed a stronger immune response and a higher SOD activity [30].SOD by peroxynitrite is caused by nitration of a specific tyrosine residue [18]. CN. Cu/Zn-SOD is believed to play a major role in the first line of antioxidant defence.10 Antioxidant Enzyme reduction of the transition metal ion at the active site in a Ping Pong type mechanism with remarkably high reaction rates [14]. CAT or GPX mRNA in various mouse tissues and cultured cells [22. copper and zinc containing glycoprotein. The biological importance of Mn-SOD is demonstrated among others by the following observations: (a) inactivation of Mn-SOD genes in Escherichia coli increases mutation frequency when grown under aerobic conditions [19]. constituted by a copper and a zinc atom bridged by a histamine residue [27. Mn-SOD has been shown to be greatly induced and depressed by cytokines. whereas Mn-SOD knockouts do not survive past 3 weeks of age [31]. Cu/Zn-SOD (SOD-1) is another type of enzymes that has been conserved throughout evolution. TNF and Adriamycin-induced cytotoxicity.[15].28. Among various human tissues Mn-SOD contents were roughly one-half as large as the Cu/Zn-SOD contents [31]. although a monomeric structure can be found in a high protein concentration from E.[16]. and radiation induced-neoplastic transformation [24]. and synovial fluid.

Because this enzyme is a very potent antioxidant. and with H donors (methanol. 2H 2 O 2  2H 2O  O 2  ROOH  AH 2  H 2O  ROH  A  CAT (EC 1. and lighten skin pigmentation that has been caused by UV rays. This kind of illness can lead to death because it affects the nerve cells in the spinal cord and the brain. Taking dietary supplements that provide an adequate supply of Superoxide dismutase will be helpful in maintaining overall well being and health because it protects our entire body from the harmful effects of free radicals. . Brussels sprouts. SOD plays a significant role in preventing the development of the Lou Gehrig’s disease. corneal ulcer. and reversing the long term effects of radiation and smoke exposure. Apart from that. this enzyme is also used for treatment of inflammatory diseases. This is beneficial for people who are experiencing premature hair loss due to a genetic predisposition or free radicals. SOD is also known to help carry nitric oxide into our hair follicles. Catalase Catalase (CAT) is an enzyme responsible for the degradation of hydrogen peroxide. A molecule of hydrogen peroxide oxidizes the heme to an oxyferryl species. A second hydrogen peroxide molecule acts as a reducing agent to regenerate the resting state enzyme. It will also heal wounds.1. SOD combats the effects of free radicals that are causing hair follicles to die. ethanol.11. Since nitric oxide relaxes the blood vessels and allows more blood to circulate to the hair follicles and SOD helps to remove the free radicals. SOD is found in our skin and it is essential in order for our body to generate adequate amounts of skin-building cells called fibroblasts. and has a molecular mass of about 240 kDa [33]. reduce the appearance of scars. Additionally. Specificity The reaction of CAT occurs in two steps. wheat grass. if superoxide dismutase is made into a lotion and applied to the skin. arthritis. burn injuries. CAT reacts very efficiently with H2O2 to form water and molecular oxygen.6) is a tetrameric enzyme consisting of four identical tetrahedrally arranged subunits of 60 kDa that contains a single ferriprotoporphyrin group per subunit. A porphyrin cation radical is generated when one oxidation equivalent is removed from iron and one from the porphyrin ring.Antioxidant Enzymes and Human Health 11 Application This enzyme has been known to promote the rejuvenation and repair of cells. It is a protective enzyme present in nearly all animal cells. barley grass and broccoli. hair loss can be prevented and even reversed. Among the common natural sources of SOD are cabbage. while reducing the damages caused by free radicals. prostate problems. producing a molecule of oxygen and water. it will prevent the formation of wrinkles. also known as Amyotrophic Lateral Sclerosis (ALS). or phenols) with peroxidase activity. formic acid.

Recently. and cholesterol hydroperoxides that are produced in peroxidized membranes and oxidized lipoproteins [37].a few lens-cleaning products disinfect the lens using a hydrogen peroxide solution. removing hydrogen peroxide from fabrics to make sure the material is peroxide-free. a new member. thereby protecting mammalian cells against oxidative damage. ROOH  2GSH  ROH  GSSG  H 2O  There are five GPx isoenzymes found in mammals. GPX (80 kDa) catalyses the reduction of hydro peroxides using GSH. Glutathione peroxidase Glutathione peroxidase (GPx) is an enzyme that is responsible for protecting cells from damage due to free radicals like hydrogen and lipid peroxides. a solution containing CAT is then used to decompose the hydrogen peroxide before the lens is used again. respectively. GPX1 and the phospholipid hydroperoxide glutathione peroxidase (PHGPX or GPX4) are found in most tissues.11. fatty acid hydroperoxides. Several mask treatments combine the enzyme with hydrogen peroxide on the face with the intent of increasing cellular oxygenation in the upper layers of the epidermis. GPX5. CAT protects cells from hydrogen peroxide generated within them.12 Antioxidant Enzyme In animals. Survival of rats exposed to 100% oxygen was increased when liposome’s containing SOD and CAT were injected intravenously before and during the exposure [34]. and GPX4 is highly expressed in renal epithelial cells and testes. it plays an important role in the acquisition of tolerance to oxidative stress in the adaptive response of cells. the levels of each isoform vary depending on the tissue type. A minor use is in contact lens hygiene .1. Even though CAT is not essential for some cell types under normal conditions. and extracellular GPX3 or GPX-P is poorly detected in most tissues except for the gastrointestinal tract and kidney. GPX4 is located in both the cytosol and the membrane fraction. Another use is in food wrappers where it prevents food from oxidizing CAT is also used in the textile industry. Recently. Application CAT is used in the food industry for removing hydrogen peroxide from milk prior to cheese production. . and liver. kidney.19) contains a single selenocysteine selenocysteine (Sec) residue in each of the four identical subunits. The GPx (EC 1. In fact. GPX1 is predominantly present in erythrocytes. Cytosolic GPX2 or GPX-G1. glutathione metabolism is one of the most essential antioxidative defence mechanisms. hydrogen peroxide is detoxified by CAT and by GPX. Although their expression is ubiquitous. The increased sensitivity of transfected CAT-enriched cells to some drugs and oxidants is attributed to the property of CAT in cells to prevent the drug-induced consumption of O2 either for destroying H2O2 to oxygen or for direct interaction with the drug [35]. PHGPX can directly reduce the phospholipid hydroperoxides. which is essential for enzyme activity [36]. Cytosolic and mitochondrial glutathione peroxidase (cGPX or GPX1) reduces fatty acid hydroperoxides and H202 at the expense of glutathione. CAT has also begun to be used in the aesthetics industry.

Both corticosteroids and non-steroids anti inflammatory drugs interfere with formation of free radicals and interrupt the disease process. Acute Inflammation: At the inflammatory site. Cigarette smoking enhances the emphysema in alpha-1 protease inhibitor deficiency. there is more elastase and less protease inhibitor. the master antioxidant. activated macrophages produce free radicals. It is present in high concentrations in the cells and plays a pivotal role in maintaining them in reduced state lest they suffer damage by oxidation (from free radicals). selenium and glutathione peroxidase are very powerful in helping the body fight against the free radicals. In premature newborn infants. Levels of GPx in the body are closely linked with that of glutathione. Glutathione is found in vegetables and fruit. Respiratory burst and increased activity of NADPH oxidase are seen in macrophages and neutrophils. Cigarette smoke contains free radicals. it alone can react effectively with lipid and other organic hydroperoxides. It is caused by free 2. vitamin C and E. it is also acts as your body’s immune system boosters. Clinical applications of antioxidant enzymes 1. with CAT. Diseases of the Eye: Retrolental fibroplasia or retinopathy of prematurity is a condition seen in premature infants treated with pure oxygen for a long time. Respiratory Diseases: Breathing of 100 % oxygen for more than 24 hr produces destruction of endothelium and lung edema. GSH ensures that the red blood cells remain intact and protect the white blood cells (which are responsible for immunity). . Chronic Inflammation: Chronic inflammatory diseases such as rheumatoid arthritis are self-perpetuated by the free radicals released by neutrophils. leading to lung damage. This is due to the release of free radicals by activated neutrophils [39]. 6. ARDS is produced when neutrophils are recruited to lungs which subsequently release free radicals. Taking it as a supplement is a good idea. but cooking will significantly reduce its potency. Combination of certain antioxidants like glutathione. 4.Antioxidant Enzymes and Human Health 13 expressed specifically in mouse epididymis. 3. The role as antioxidant is particularly important for brain as it is very sensitive to presence of free radicals. Application This is one of the most important enzymes in the body with antioxidant properties. being the major source of protection against low levels of oxidant stress. Glutathione (GHS for short) is a tripeptide that not only protects the cells against ill effects of pollution. Although GPX shares the substrate. Thus. prolonged exposure to high oxygen concentration is responsible for bronchopulmonary dysplasia. is interestingly selenium-independent [38]. Adult respiratory distress syndrome (ARDS) is characterized by pulmonary edema. H2O2. Soot attracts neutrophils to the site which releases more free radicals.

To increase the therapeutic effect of radiation. Anti-oxidants have a protective effect. Tissues of the eye. Cancer Treatment [39]: Free radicals contribute to cancer development because of their mutagenic property. a major lipid-soluble antioxidant. called psoralens are administered in the treatment of psoriasis and leukoderma. Certain plant products. Free radicals produce DNA damage. Cataract is partly due to photochemical generation of free radicals. Peptic Ulcer: Peptic ulcer is produced by erosion of gastric mucosa by hydrochloric acid. radicals.14 Antioxidant Enzyme 5. Cataract formation is related with ageing process. sustained vascular contracture and cellular injury. Sunlight acting on porphyrins produces singlet oxygen. have high concentration of free radical scavenging enzymes. 7. which trigger inflammatory reaction. Vitamin C is considered the most important water-soluble antioxidant in extracellular fluids. Macrophages are them converted into foam cells. 6. Vitamin E. porphyrins accumulate in the skin. 8. This attracts macrophages. . Alpha tocopherol offers some protective effect. Vitamin C has been cited as being capable of regenerating vitamin E. Other antioxidants Dietary Antioxidants Vitamin C. Arthrosclerosis and Myocardial Infraction: Low density lipoproteins (LDL) promote atherosclerosis. Exposure of sunlight will lead to erythema and eruptions in the patients. They release leucotrienes from platelets and proteases from macrophages. All these factors cause increased vascular permeability. When the drugs is applied over the affected skin and then irradiated by UV light. 9. This infection potentiates the macrophage oxidative burst leading to tissue destruction. Skin Diseases: due to inborn defects. leading to the above symptoms. Cancer is treated by radiotherapy. This initiates the atherosclerotic plaque formation. It is capable of neutralizing ROS in the aqueous phase before lipid peroxidation is initiated. resulting in tissue edema. They are deposited under the endothelial cells. is the most effective chain-breaking antioxidant within the cell membrane where it protects membrane fatty acids from lipid peroxidation. Helicobacter pylori infection perpetuates the disease. vitamin E. 7. Shock Related Injury: Release of free radicals from phagocytes damage membranes by lipid peroxidation. singlet oxygen produced with clinical benefit. including the lens. and accumulated damages lead to somatic mutations and malignancy. It is shown that superoxide anions are involved in the formation of ulcer. causing thromboxane release. which increase the production of ROS. Irrational produces reactive oxygen species in the cells which trigger the cell death. Beta-carotene and other carotenoids are also believed to provide antioxidant protection to lipid-rich tissues. radio-sensitisers are administered. and beta-carotene are among the most widely studied dietary antioxidants. which undergo oxidation by free radicals released from endothelial cells. Research suggests beta-carotene may work synergistically with vitamin E.

” are becoming increasingly known for their antioxidant activity. Worthington Biochemical Corporation. collectively termed “phytonutrients. Antioxidant enzyme plays an important role in protecting oxidative injury to the body. in humans.000 flavonoid substances have been described. strokes. anti-viral. viral infections (that cause airway epithelial inflammation). anti-aging. flavonoids appear to function as “biological response modifiers.S. et al. . Curr. [5] 8. 77: 658-666. Fruits and vegetables are major sources of vitamin C and carotenoids. while whole grains and high quality.” Flavonoids have been demonstrated to have anti-inflammatory. alzheimer’s.” or “phytochemicals. Many plant-derived substances. 1999 “ROS: oxidative damage and pathogenesis”.Pillai. motor neuron diseases and axonal injury) and infraction. and anti-carcinogenic activity. antiallergenic. cancer. References [1] Worthington Enzyme Manual. [2] Uday Bandyopudya. Physiol. In plants. The broad therapeutic effects of flavonoids can be largely attributed to their antioxidant properties. flavonoid compounds may exert protection against heart disease through the inhibition of cyclooxygenase and lipoxygenase activities in platelets and macrophages.. and brain edema. Pharmacol.Antioxidant Enzymes and Human Health 15 A diet that is excessively low in fat may negatively affect beta carotene and vitamin E absorption. Phenolic compounds such as flavonoids are ubiquitous within the plant kingdom: approximately 3.. Ind. [3] Chitra K.) in the body by interventions which may include increases intake of dietary supplements rich in antioxidants/antioxidant enzymes and regular exercise. 46 (1): 01-05. J. India 9. Author details Praveen Krishnamurthy and Ashish Wadhwani TIFAC CORE HD JSS College of Pharmacy Ootacamund. Conclusion Oxidative stress plays a major role in the pathogenic of many disorders including aging. K. In addition to an antioxidant effect.P. Sci. diabetes. One of the therapeutic approach by which these disorders can be prevented is to increase the levels of these enzymes (SOD. properly extracted and protected vegetable oils are major sources of vitamin E. 2002 “Antioxidants in Health”. neurodegenerative processes (including cell death. GPx etc. [5] Phytonutrients A number of other dietary antioxidant substances exist beyond the traditional vitamins discussed above. flavonoids serve as protectors against a wide variety of environmental stresses while. CAT.. Retrieved 2009-0301. as well as other fat-soluble nutrients.

Superoxide radical and superoxide dismutases. 37: 1613–22. Fourier Transform infrared analysis of the interaction of azide with the active site of oxidized and reduced bovine Cu. [15] Leone M. Bueno P. Desideri A. Karlsson K. [10] Teixeira HD. Biochemistry 1998. 48: 231–8. Oxygen-dependent mutagenesis in Escherichia coli lacking superoxide dismutase. Physiol. 222: 1-15. Scherk C. D’ari R. Biol Trace Elem Res 1995. Hepatology 4 (1984) 586–590. Lauterburg. 64: 97–112. Peroxynitrite. Hepatic glutathione homeostasis in the rat: efflux accounts for glutathione turnover. 26: 187–94. [18] Stralin P. Lower. Biochem. 37: 4459–64. Crow JP. Fanburg. Cupane A. Deneke. Free Radic Res 1997. Lo´pez-Huertas E.D. Free Radicals in Biology and Medicine. Biochem J 1998. CuZn-superoxide dismutase and Mn-superoxide dismutase in human dermal fibroblasts. [16] Vance CK. J Biol Chem 1998. 257 (1989) L163–L173. 31: 01-04. J Biol Chem 1994. 273: 14085–9.).Zn superoxide dismutase. [8] B. Clinical Nutrition Insights. [14] Meier B. Oxford University Press. Am. 298: 347–52. Marklund SL. 1999. Biochemistry 1998. Liu Q. zinc superoxide dismutase in peroxisomes from watermelon (Citrullus vulgaris Schrad. B. Nilsson P. [12] Sandstro´m J. Taka H. Regulation of cellular glutathione. The mechanism for the effect of selenium supplementation on immunity. [5] Mark Percival. [6] B. Militello V. Adams..R. Intracellular hydrogen peroxide levels in cells overexpressing CuZn-superoxide dismutase. [9] Fridovich I. Parak F. J. Schmidt M. 83: 8268–72. J. Stroppolo ME.H. Miller AF. Fujimura T. Halliwell. 331: 403–7.L. 269: 19163–6. Gutteridge (Eds. Proc Natl Acad Sci 1998. J. 105–245. Murayama K. Xu H. Biochemistry 1998. Zuo P. pp. Proc Natl Acad Sci 1986. [20] Farr SB. [17] MacMillan-Crow LA. Effects of oxidative stress on expression of extracellular superoxide dismutase. Spectroscopic comparisons of the pH dependence of FeSubstituted (Mn) superoxide dismutase and Fe-superoxide dismutase. 37: 5518–27. Del R´o LA. [19] Yamakura F. Inactivation of human manganesesuperoxide dismutase by peroxynitrite is caused by exclusive nitration of tyrosine 34 to 3-nitrotyrosine. Immunocytochemical localization of copper. Biochem J 1994. Mitchell. [7] S. Meneghini R. Zhou J.16 Antioxidant Enzyme [4] Trevor F. J. 95: 7872–5. Schumacher RI. Annu Rev Biochem 1995. J.) cotyledons. 1998 “Antioxidants”. Thompson JA. [13] Sun E. [11] Sandalio LM. Slater.mediated inactivation of manganese superoxide dismutase involves nitration and oxidation of critical tyrosine residues. .M. Wang J. Marklund SL. Touati D. 1984 “Free radical mechanism in tissue injury”. New York. pH-dependent inhibition by azide and fluoride of the iron superoxide dismutase from Propionibacterium shermanii. 10-fold increase in human plasma extracellular superoxide dismutase content caused by a mutation in heparin-binding domain.

Sette M. Acta Physiol Scand Suppl 1980. J Biol Chem 1992. Polizio F. In: Bergmeyer H. Crapo JD. Blood 1993. Oberley TD. Warner BB. Cancer Res 1997. [26] Wispe´ JR. [33] Buschfort C. Rajewsky MF. Enzymes 1: oxidoreductases. Serum IgG and IgM responses to sheep red blood cells (SRBC) in weaned calves fed milk supplemented with Zn and Cu. [27] Battistoni A. [35] Turrens JF. 492: 19–23. Seeber S. Overproduction of human Mn-superoxide dismutase modulates para. Akashi M. Capo C. Role of the dimeric structure in Cu. Lin CW. [29] Leah RB. O’Neill P. et al. Pp. Dilated cardiomyopathy and neonatal lethality in mutant mice lacking manganese superoxide dismutase. [25] St. The copper chaperone CCS directly interacts with copper/zinc superoxide dismutase. Cu.Zn superoxide dismutase from Escherichia coli retains monomeric structure at high protein concentration. 11: 376–81. 57: 651–8. VOLUME 32. Casareno DW. FL: Verlag Chemie. Karmakar A. Kundu MS. Thomale J. Cervoni L. Huang TT.Antioxidant Enzymes and Human Health 17 [21] Van Loon APGM. Ho YS. J Biol Chem 1998. FEBS Lett 1991. 83: 3820–4. Methods of enzymatic analysis. Ed. vol. A yeast mutant lacking mitochondrial manganese-superoxide dismutase is hypersensitive to oxygen. [31] Prasad T. [22] Li Y.Zn superoxide dismutase. et al. J Clin Invest 1984. Biochem J 1996. Endogenous production of tumour necrosis factor is required for manganese superoxide dismutase expression by irradiation in the human monocytic cell line THP-1. Clair DK. Sakashita A. Osawa Y. Clark JC. Gabbianelli R. III. Biochem J 1997. 82: 1142–50. DNA excision repair profiles of normal and leukemic human lymphocytes: functional analysis at the singlecell level. Folcarelli S. NOVEMBER 1999 quat-mediated toxicity in mammalian cells. reversible denaturation of the monomeric enzyme from Escherichia coli. Pesold-Hurt B. Human Mn-superoxide dismutase in pulmonary epithelial cells of transgenic mice confers protection from oxygen injury. [23] Hachiya M. Regulation of manganese superoxide dismutase and other antioxidant genes in normal and leukemic hematopoietic cells and their relationship to cytotoxicity by tumor necrosis factor. 11: 712–15. pH-Dependent. 293: 199– 203. Schatz G. [34] Aebi HE. 273: 23625–8. 273–82. Biochemistry 1998.MATE´ S ET AL. Deerfield Beach. Freeman BA. 600 CLINICAL BIOCHEMISTRY. Gitlin JD. Proc Natl Acad Sci 1986. J Biol Chem 1998. 267: 23937–41. . Nat Genet 1995. [30] Stroppolo ME. Protection against oxygen toxicity by intravenous injection of liposome-entrapped catalase and superoxide dismutase. [24] Kizaki M. Rotilio G. Shimizu S. transferases. Carlson EJ. [32] Marklund S. The Cu. Evidence for altered subunit interaction in all the bacteriocupreins. Nutrition 1995. 320: 713–16. et al. 328: 615–23. Mu¨ ller MR. 37: 12287– 92. Distribution of Cu/Zn superoxide dismutaseand Mn superoxide dismutase in human tissues and extracellular fluids. 73: 87–95.Zn superoxide dismutase from Photobacterium leignathi is an hyperefficient enzyme. [28] Battistoni A. 273: 5655–61. 1980. Koeffler HP. Folcarelli S. Cambria MT.

Bagley AC. 52: 506– [38] Imai H. J Biol Chem 1993.18 Antioxidant Enzyme [36] Speranza MJ. . Arai M. 268: 19039–43. Glutathione peroxidase and hydroperoxides. Nakagawa Y. Ltd. New Delhi. 273: 1990–7. Cells enrichedfor catalase are sensitized to the toxicities of bleomycin. Narashima K. Pp 212-215 Jaypee Brothers Publications pvt. Suppression of leukotriene formation in RBL-2H3 cells that overexpressed phospholipid hydroperoxide glutathione peroxidase. Lynch RE.M. Methods Enzymol 1978. Text book of Biochemistry.Vasudevan et. [39] D. J Biol Chem 1998. Chapter 24. Free radicals and antioxidants. Sakamoto H. [37] Tappel AL. and paraquat. Chiba N. adriamycin..

 A series of oxidation processes take place in  the  peroxisomes. purines to form uric acid by xantine oxidase reaction. ROS and reactive nitrogen species (RNS) may also be of exogenous origin.doi.  or  sometimes  due  to  ionising  radiation. where hydrogen peroxide is  also  produced.  NADPH  is  needed  for  the  reduction  of  glutathione.  They  provide  the  energy  necessary  for  many  cellular functions. It also        © 2012 Vašková et al. One‐electron transmission leads to  the formation of reactive oxygen species (ROS).  Given that highly reactive substances could damage the cells and the whole organism. Most chemical energy in the body exists as ATP. and biotransformation of xenobiotics. provided the original work is properly cited. which are necessary for enzyme activity.  The  whole  system is often referred as the glutathione defence system. and reproduction in any medium.g. produced during aerobic  respiration.  Nutrient  oxidation  is  carried  out  by  reduced  coenzymes  in  the amino acids.  long and branched‐chain fatty acids.  Reduced  glutathione  is  the  most  important  of  these  endogenous  substances and is functional both as a cofactor of other enzymes and for its reducing effects  on  oxidized  molecules. they  must  be  inactivated  by  an  antioxidative  defence  system.  which  contain  transition  metals.  Oxidation reactions are also used for the degradation of unneeded molecules to excrement  form. e.  The  electrons  are  transferred  to  the  oxygen  created proton gradient that allows for ATP  They  can  be  taken  up  through  diet  or  ventilation.0). Ladislav Vaško and Ivan Kron Additional information is available at the end of the chapter http://dx. neurotransmitters. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.Chapter 2 Oxidative Processes and Antioxidative Metaloenzymes Janka Vašková.). distribution.  Hydrogen  peroxide  arises  as  a  by‐product  of  the  oxidation  of  very  long. by both  synthesis of endogenous and by intake of exogenous anti‐ or pro‐oxidant substances.   . It is influenced by a number of other factors and circumstances.  as  neutrophils  produce  hypochloric  acid  from  superoxide  radicals  via  NADPH oxidase. licensee InTech. synthesis and deamination of biologically  active molecules (hormones. form part of the  antioxidant  defence. Introduction  Oxidative  processes  are  necessary  for  life. etc. The antioxidant defence system  is very complicated.  Metaloenzymes.5772/50995 1. which permits unrestricted use.  The  body  also  makes  use  of  ROS  also  against  the  invasion  of  microorganisms.  and  other  antioxidant  enzymes  have  an  important  role  in  this  stage.  Various endogenous substances.  which  are  oxidized  in  the  respiratory  chain.

  lipopolysaccharides. The aim of this is to describe the  important aspects of this system that are mediated via metaloproteins. The isoform iNOS is inducibly expressed in macrophages after  stimulation  by  cytokines. Nitric oxide synthase (NOS)   Nitric oxide (NO•) is produced from a guanidine nitrogen of L‐arginine via electron transfer  from NADPH in two successive steps.   The  rate  of  NO•  synthesis  is  affected  to  some  extent  by  the  availability  of  the  substrate  L‐ arginine  and  by  the  cofactor  tetrahydrobiopterin  (BH4). Sb. such as Hg.  NOS‐I  or  NOS‐1).  Although  it  possesses  very  little  structural  resemblance  to  P450. peptides with high content of cysteine (approximately 1/3 of amino acids).  but  may  also  be  disposed  to  integration  into  the  metalotioneins.  nNOS  and  eNOS  are  constitutively  expressed. In physiological concentrations.  there  are  many  sources  within  the  cells  that  are  only  mentioned. These can  affect  the  whole  defence  system. and other.  The  five  mechanisms  described  produce  ROS  in  a  non‐regulated  mode. e.  The enzyme responsible this exists in three isoforms:  neuronal  (nNOS.  NOS‐III  or  NOS‐3)  and  inducible  (iNOS.  2.1.  the  most  common  RNS  . NO• is a reactive and unstable  free radical gas that can cross cell membranes easily by diffusion independent of any release  or uptake mechanism [86].  those which show a high affinity for sulphur. which carries out “P450‐like” mono‐ oxygenation reactions [130]. bi‐ domain enzymes.  the  oxygenase  domain  of  NOS  is  referred  to  as  being  “P450‐like”  due  to  the  presence  of  iron  protoporphyrin  IX  (heme). Expression of iNOS is regulated at the transcriptional and post‐transcriptional level by  signalling  pathways  that  involve  agents  such  as  the  redox‐responsive  transcription  factor  NF‐κB or mitogen‐activated protein kinases (MAPKs) [120].  transport  and  binding  of  metals  into  organic  compounds  of  the  organism.g.  endothelial  (eNOS. Sources of reactive oxygen species and free radicals in an organism  All  aerobic  organisms  produce  reactive  oxygen  species  physiologically.   It is necessary to realize that the whole system is inducible.  nNOS  exhibits NADPH‐diaphorase (NADPH‐d) activity.  but  their  activity  is  regulated  by  the  intracellular  Ca2+  concentration. The NOS isoforms are homodimeric.20 Antioxidant Enzyme includes  the  receipt.  The  physiological  function  of  NO•  varies widely due to the diverse localization of isoforms within different cell populations of  the body.  The  five  most  productive  pathways  are  involved  in  regulating  the  production  of  ROS/RNS  and  the  resulting  effects  on  signalling  cascades.1.  and  other  immunologically  relevant  agents  [21].  However.  type  II.1.  linked axially by a cysteine residue to the NOS protein. Cd. As.  NOS‐II  or  NOS‐2). Regulated production of reactive oxygen and nitrogen species  2.  type  I.  2. NO•  functions as an intracellular messenger [88].  In  pathophysiological  situations  where  iNOS  is  upregulated. Each monomer consists of a flavin‐containing reductase domain linked to  a  heme‐containing  oxygenase  domain  by  a  calmodulin‐binding  sequence.  type  III. not just those that are part of the redox phenomena but also toxic elements.

l‐1.1.  the  plasma membrane is internalized as the wall of the phygocytic vesicle. each consisting of two polypeptide chains of 108 and 466 amino  acids.  tyrosyl  radical  (Tyr•).  Activated  neutrophils  and  macrophages  also  generate  singlet  oxygen  (1O2)  by  reactions  that  involve  either  MPO  or  NADPH  oxidase  [23.  which  can  react  with  superoxide  (O2•‐)  to  produce  ONOO‐.  This  multicomponent  enzyme  catalyzes  the  one‐electron  reduction  of  O2  to  superoxide  (O2•‐).  is  produced.  MPO combines with hydrogen peroxide and in the presence of halide (chloride.  The  transfer  of  electrons  occurs  from  NADPH  on  the  inner  side  of  the  plasma  membrane  to  O2  on  the  outer  side. one of the strongest physiological oxidants and a powerful antimicrobial agent [76]. The combined activities of NADPH oxidase and  myeloperoxidase  (MPO)  in  phagocytes  leads  to  the  production  of  hypochlorous  acid  (HClO). MPO also catalyzes the oxidation  of  tyrosine  in  organisms  to  form  the  toxic  amino  acid  residue. with what was once  the outer membrane surface now facing the interior of the vesicle.2.    The  massive  production  of  antimicrobial  and  tumoricidal  ROS  in  an  inflammatory  environment is called the “oxidative burst” and plays an important role as the first line of  defence against environmental pathogens.  Neutrophils  also  produce  RNS.cell‐1 and may reach a concentration of 10‐100 μM in the  vicinity  of  these  cells  [46.  2. It is a heme‐containing protein complex.  Like  the  other  heme  peroxidases.  The activation of phagocytic NADPH oxidase can be induced by microbial products such as  .  During  phagocytosis.1. both of which are able  to induce nitrosative and oxidative stress [194].  MPO is a heterodimeric.Oxidative Processes and Antioxidative Metaloenzymes 21 generated are dinitrogen trioxide (N2O3) and peroxinitrite (ONOO‐). NADPH oxidase in phagocytic cells Activated neutrophils and macrophages produce superoxide and its derivatives as cytotoxic  agents  forming  part  of  the  respiratory  burst  via  the  action  of  membrane  bound  NADPH  oxidase on molecular oxygen. This targets the delivery  of O2•‐ and its reactive metabolites internally for localized microbicidal activity [11].1.      HOCl ↔ H+ +OCl‐  HOCl + Cl‐ → Cl2 + HO‐  (1)  (2)  The  oxidation  of  iron‐sulfur  centres  in  micro‐organisms  by  the  myeloperoxidase‐H2O2‐ halide system may contribute to the death of an organism.  at  an  estimated rate of 2‐6 x 10‐14 mol. bromide.  using  NADPH  as  the  electron  donor  through  the  transmembrane  protein  cytochrome  b558.46]. itself a powerful oxidant.  this  can  be  oxidized  by  the  effect  of  myeloperoxidase  (MPO)  to  form  either  nitryl ion (NO2+) or nitrogen dioxide (NO2•) [30]. cationic and glycosylated heme enzyme.  Each  half  contains  a  covalently  attached  heme  [7]. or  iodide) to form the highly reactive redox intermediate in the phagosomes of neutrophils.  the  major  oxidation  product  from  NO.  In  activated  neutrophils. The enzyme is a 140‐kDa  dimer of identical halves. Hydrogen peroxide  (H2O2)  is  produced  by  activated  macrophages  in  an  inflammatory  environment. Upon NOS activation in many inflammatory  diseases  nitrite  (NO2‐).2.  involved  in  the  activity  of  neutrophils. which may decompose to form a hydroxyl radical (•OH). NADPH oxidase  2.106].

 hemodynamic forces.1. or 15‐LOX refers to the arachidonic acid site  .  endothelial  cells.  Muscle cells  and  fibroblasts  account  for  the  majority  of  O2•‐  produced  in  the  normal  vessel  wall.   2.  where  changes  in  oxygen  tension  regulate  vascular  relaxation  through  changes  in  O2•‐  production  and  cGMP  formation  [198].  O2•‐  and  H2O2  are  mainly  produced  intracellularly  in  vascular  smooth  muscle  cells.   A  similar  group  of  proteins  was  suggested  to  be  involved  as  oxygen  sensors  in  the  regulation  of  erythropoietin  production  in  human  hepatoma  cells  [209].  a  mitochondrial  protein.  and  possibly a third heme protein [105.  Lipoxygenases  in  plants  and  animals  are  heme  containing  dioxygenases  that  oxidize  polyunsaturated  fatty  acids  at  specific  carbon  sites  to  give  enantiomers  of  hydroperoxide  derivatives  with  conjugated  double  bonds.208].1. vascular smooth muscle cells.2.  The  oxidative  burst  in  plants is an effective bactericidal mechanism.  and  fibroblasts.  The  cardiovascular  NAD(P)H  oxidase  isoforms  are  induced by hormones.2.  The  rate  of  O2•‐  production  in  non‐ phagocytic  cells  is  only  about  one‐third  that  of  neutrophils.  The  NAD(P)H oxidase isoforms of the cardiovascular system are membrane‐associated enzymes  that  appear  to  utilize  both  NADH  and  NADPH  [66]. or by local metabolic changes [66].  Increased  aortic  adventitial  O2•‐  production  contributes  to  hypertension  by  blocking  the  vasodilatatory  effects  of  NO•  [189]. Enzyme activation is mainly controlled by rac2 in neutrophils and  rac1 in macrophages and monocytes [46.  In  most  of  these. Arachidonate cascade enzymes  2.  There  are  several  lipoxygenases  which  differ  by  substrate  specificity  and  optimum  reaction  conditions.  or  by  the  cytokines  interferon‐γ.  where  they  may  be  involved  in  the  induction  of  NAD(P)H  oxidase‐like  enzymes  [167].118. cardiac monocytes and thyroid  tissue  nonphagocytic  NAD(P)H  oxidase  (similar  but  not  identical  to  phagocytic  NADPH  oxidase)  produce  O2•‐  and  to  regulate  intracellular  signalling  cascades  [66. endothelial cells.22 Antioxidant Enzyme bacterial  lipopolysaccharide. Mechanical  forces stimulate NAD(P)H oxidase activity in endothelial cells and reoxygenation in cardiac  myocytes. NADPH oxidase in nonphagocytic cells  Fibroblasts.  rac1  is involved in  the  induction  of NAD(P)H oxidase  activity  [91.1.  A  microsomal  NADH  oxidase  was  implicated  as  an  oxygen  sensor  in  bovine  pulmonary  and  coronary  arteries.193]. 12‐LOX.210].3.  in  contrast  to  neutrophils.126]. controlling the  rate  of  ventilation  [2].209]. 5‐lipoxygenase (5‐LOX)   The  enzyme  5‐LOX  has  been  identified  as  an  inducible  source  of  ROS  production  in  lymphocytes  [23. An NAD(P)H oxidase with low affinity for oxygen and high affinity for cyanide  is believed to act as one of the sensors for oxygen tension in the carotid body.1.  by  lipoproteins.  2.  There  is  a  strong  possibility  that  rac‐like  proteins  also  occur  in  plants  [1.  interleukin‐ 1β.  The  function  of  oxygen  sensing  is  apparently  shared  by  several  proteins.  but  the  evidence  for  its  physiological  role  in  redox  signalling  is  still  scarce. and interleukin‐8 [23].  The  number  in  specific enzyme names such as 5‐LOX.  including  a  nonmitochondrial  cytochrome  b558.112].3.

  interleukin‐1.  bacterial  lipopolysaccharide. It is also hypothesized that ROS production from complex I  during  RET  occurs  from  FMN  as  well  [103.  Complex  I  also  generates  ROS  after  the  oxidation  of  succinate  at  complex  II  via  a  process  referred  to  as  reverse electron transport (RET).3.1. B4.1.   .173]. The flavin mononucleotide (FMN) of complex I  accepts  the  electrons  from  NADH  and  passes  them  through  a  series  of  eight  iron‐sulfur  clusters  to  ubiquionone  [84]  to  generate  O2•‐  in  the  presence  of  NADH.  Two  electrons  from  semiquinones  in  Qo  are  required  for  the  reduction of ubiquinone to ubiquinol in the Qi site.  2.2. one electron from ubiquinol is  transferred  through  the  Rieske  Fe‐S  cluster  protein  to  the  electron  acceptor. Non‐regulated production of reactive oxygen species  2.  cytochrome  c. C4.  Cyclooxygenase  participation  in  redox  signalling  remains scarce. which are susceptible to scavenging by some antioxidants in cells stimulated  with  TNF‐α. Upon binding with the Qo site. The oxidized metabolites generated by 5‐LOX were found  to  change  the  intracellular  redox  balance  and  to  induce  signal  transduction  pathways  and  gene  expression.  It  is  well  documented  that  mitochondria  are  a  source  of  H2O2.   2.122].Oxidative Processes and Antioxidative Metaloenzymes 23 that is predominantly oxidized [202].  yielding  ROS  [173]  and  1‐5%  leads  to  H2O2  production  [134].  5‐LOX  was  shown  to  be  involved  in  the  production  of  H2O2  by  T  lymphocytes  after  ligation  of  the  CD28  costimulatory  receptor  [118]  and  in  response  to  interleukin‐1β [23]. NADH‐ubiquinone oxidoreductase (complex I) is composed of  ̴  45  subunits and is the site of NADH oxidation. A lipid metabolizing enzyme in fibroblasts similar to 15‐LOX has been  shown to generate large amounts of extracellular O2.  however. Complex III  plays an intricate role in passaging electrons from the ubiquinol generated by complexes I  and II to cytochrome c [116].  the release of O2•‐ from mitochondria into the cytosol has yet to be definitively established  [77].  Ubiquinol:cytochrome  c  oxidoreductase  (complex III) has 11 subunits and contains 3 hemes and an Fe‐S cluster center.  while  complex III is capable of producing ROS on both sides of the mitochondrial inner membrane  [135. It is estimated that 2‐3% of O2  consumed  by  mitochondria  is  incompletely  reduced. Cyclooxygenase (COX‐1)   Cyclooxygenase‐1  has  been  implicated  in  ROS  production  through  formation  of  endoperoxides.2.  ROS  are  only  produced  at  complexes  I  and  II  in  the  mitochondrial  matrix. D4 and E4. Mitochondrial respiration  The  four‐electron  reduction  of  oxygen  occurs  within  the  mitochondrial  electron  transport  system of all cells undergoing aerobic respiration. The electron in cytochrome b is then used to re‐reduce ubiquinone at the  Qi  site  to  produce  ubiquinol. It is generally thought that the two major sites of mitochondrial ROS production  are complexes I and III. 5‐LOX is best known for its role in biosynthesis of the  leukotrienes A4.  or  the  tumor  promoter  4‐O‐ tetradecanoylphorbol‐13‐acetate  [48]. This process is referred to as the Q‐cycle  because lone electrons remaining in semiquinone are reused to reduce ubiquinone back to  ubiquinol [35].  The resulting unstable semiquinone then donates the remaining electron to the heme groups  on cytochrome b.‐ [168].2.

2.  PS  II  is  a  multisubunit  protein  complex  also  present  in  cyanobacteria  that  use  light  energy  for  oxidation  of  water  and  reduction  of  plastoquinone  [146].  Firstly.  which  is  reduced  to  NADPH.  salt  stress  and  CO2  limiting  conditions.  and  ETC  in  PS  I  and  PS  II  make  chloroplasts a major site of ROS production in plants and algae [145]. Second. in particular the voltage‐dependent anion channel [77].   Oxygen  generated  in  chloroplasts  during  photosynthesis  can  accept  electrons  passing  through  the  photosystems  (PS).  such  as  cytochrome  c  [179]  as  well  as  pores  for  O2•‐  diffusion  across  the  outer  membrane into cytosol. The presence  of  ROS  producing  centres  such  as  triplet  chlorophyll.  and  the  Qo  site  in  the  vicinity  of  the  intermembrane space [154].  except  in  the  protonated  form.  Various  abiotic  stresses  such  as  excess  light.  It  then  enters  the  Calvin  cycle  and  reduces  the  final  electron  acceptor.  It  has  been  estimated  that  1‐2  %  of  O2  consumed  by  plants  is  sidetracked to produce ROS in various subcellular loci [19]. the release of O2.   2.  enhance  the  production  of  ROS. Another important decay pathway of O2•‐  at a diffusion‐controlled  rate may involve the reaction with NO• to yield ONOO‐ in the intermembrane compartment. cytoplasmic  aconitase and other cytoplasmic enzymes susceptible to O2•‐ may be targets of O2•‐  released  from mitochondria [61].  The  presence  of  O2  in  the  atmosphere enables respiratory metabolism and efficient energy generation systems which  use O2 as final electron acceptor.   This may be of some significance. as nitrosation of cytochrome c and proapoptotic caspases  occurs prior to apoptosis [123].  The  mechanism  underlying  the  release  of  O2•‐  into  the  intermembrane  space  covers  the  formation  of  ubisemiquinone  at  two  sites  in  the  ubiquinone  pool:  the  Q1 site  that  lies  near  the  matrix.  part  of  O2•‐  generated  during  mitochondrial  electron  transfer  is  vectorially  released  into  the  intermembrane  space  [78].   O2•‐  released  into  the  cytoplasm  from  mitochondria  could  play  an  important  role  in  cell  signalling.  In  cases  of  ETC‐ .‐ into the intermembrane space would be in a  functional  relationship  to  the  localization  of  a  superoxide  dismutase  (SOD)  activity  in  this  compartment. Atmospheric oxygen  is  relatively  non‐reactive.  Taken  together.  which  represents  only  a  small  fraction  of  the  O2•‐  pool  at  physiological  pH. leading to the formation of ROS in cells [166]. Chloroplasts  The  ability  of  phototrophs  to  convert  light  into  biological  energy  is  critical  for  life  and  therefore  organisms  capable  of  photosynthesis  are  especially  at  risk  of  oxidative  damage. O2•‐  cannot  cross  membranes. Autooxidation of ubisemiqiunone at the Qo site (UQo•‐) results  in the release of O2•‐ through the cytosolic side of the mitochondrial inner membrane. as O2•‐ has been implicated in several signalling events.  CO2.  the  electron  flow  from  the  excited  PS  centres  is  directed  to  NADP+. In addition. The intermembrane space contains several O2•‐  scavenging pathways besides  SOD.24 Antioxidant Enzyme The mechanism of mitochondrial production and release of H2O2 and O2•‐ takes place in two  steps.  due  to  their  bioenergetic  lifestyle  and  the  abundance  of  photosenzitizers  and  oxidable  polyunsaturated  fatty  acids  in  the  chloroplast  envelope.2.  H2O2  is  formed  both  at  the  intermembrane  space  and  the  matrix  from  O2•‐  generated  towards  the  respective  compartments [77].  drought.  Under  normal  conditions.

  and  two  clusters  with  iron  and  sulfur  compounds of FAD cofactor in both subunits.2.  The  physiological  substrates.  mainly formed at PS II even under low‐light conditions [29].  it  has  been  observed  in  TNF‐treated  endothelial  cells [58] and has been implicated as a major source of oxidative stress under ischemia and  reperfusion [46]. The  enzyme  is  derived  from  xanthine  dehydrogenase  by  proteolytic  cleavage. or as a  dehydrogenase (XDH) that utilizes NAD or oxygen as the final electron acceptor [17. Xanthine oxidoreductase (XOR)  XOR exists as either an oxidase (XO) which transfers reducing equivalents to oxygen. Dopamine (DA)  As a neurotransmitter. reducing it to O2•‐  via the Mehler reaction. O2•‐ is  enzymatically dismutated to H2O2 [50.Oxidative Processes and Antioxidative Metaloenzymes 25 overloading.  Under  normal  conditions.  xanthine  and  hypoxanthine. DA is stable in the synaptic vesicle.2.  bind  with  the  oxidized  enzyme  and  donate  two  electrons  into  the  molybdenum  cofactor  reducing it from Mo6+ to Mo4+.  Spontaneously  oxidized  cytosolic  DA  produces  O2•‐  and  reactive  quinones such as DA quinones or DOPA quinones. During the oxidation of DA by MAO. the chlorophyll triplet state becomes able to react with 3O2 to give up 1O2 [79].  Therefore.  or  univalently  reoxidized  in  two  steps  to  generate two  equivalents  of  superoxide  O2•‐  [17.  It  contains  molybdenum  in  the  form  of  molybdopterine.  DA  is  easily  metabolized  via  monoamino  oxidase  (MAO)  or  by  autooxidation  to  produce  ROS.   2. stimulating formation of XOR in the endothelium  with  further  O2•‐  production. When an excess of cytosolic DA  exists  outside  of  the  synaptic  vesicle. DA quinones are also generated in the  enzymatic  oxidation  of  DA  by  COX  in  the  form  of  prostaglandin  H  synthase. On the external “stromal” membrane surface.4.  1O2 is a natural byproduct of photosynthesis. QB) with  electron leakage to O2 producing O2•‐. and are then finally polymerized to form melanin. The acceptor side of ETC in PS II also provides sides (QA.   2.  highly  reactive  DA  quinone  or  DOPA  quinone  exert  cytotoxicity  predominantly  in  DA  neurons  and  .82].  XOR  accounts  for  only  a  minor  proportion  of  total  ROS  production [46].  subsequently  leading  to  the  formation  of  neuromelanin  [162].  Reduced  FAD  can  be  divalently  reoxidized  by  oxygen  to  produce  hydrogen  peroxide.  In  cases  of  insufficient  energy  dissipation. as reviewed  in  Miyazaki  &  Asanuma  [132].163].3. Generation takes place due to  the  excitation  energy  transfer  from  triplet  chlorophyll  formed  by  the  intersystem  crossing  from  singlet  chlorophyll  and  the  charge  recombination  of  separated  charges  in  the  PS  II  antenna  complex  and  reaction  center  of  PS  II  [146]. The release of O2•‐ results in the recruitment and activation of neutrophils  and their adherence to endothelial cells. H2O2 and dihydroxyphenylacetic  acid  are  generated  [67]. Substrates are hydroxylated by H2O at the molybdenum site  as the electrons travel via two iron‐sulfide residues to flavine–adenine dinucleotide (FAD).  Although  ROS  from  the  autooxidation  of  DA  show  widespread  toxicity  not  only  in  DA  neurons  but  also  in  other  regions.59].  The enzyme catalyzes the production of uric  acid  with  co‐production  of  O2•‐.  tyrosinase and XOR.65.  LOX. These quinones are easily oxidized to the cyclized aminochromes: DA‐ chrome and DOPA‐chrome. a part of the electron flow is diverted from ferredoxin to O2.

  flavoprotein  dehydrogenase  and  tryptofan  dioxygenase  can  all  generate  ROS  during  catalytic  cycling.   3. Less than 1% of triplet oxygen is converted in parallel to superoxide anion  (O2‐).  contributing  to  the  pathology  of  neurodegenerative  disorders  and  ischemia‐induced  damage in the striatum [24.    Electrons  leaking  from  nuclear  membrane cytochrome oxidases and electron transport systems may give rise to ROS [75].99].  If  not.  plastoquinones  and  vitamin  C.  as  it  is  not  related  to  electron transfer to O2. flavin oxidase.201]. Other cellular ROS sources  The most studied producers of O2. Chemistry of reactive oxygen and nitrogen species  During  plant  photosynthesis  and  in  analogous  reactions  of  the  respiratory  chain. Photosensitization reactions  Photosensitization  reactions  involve  the  oxidation  of  organic  compounds  by  atmospheric  oxygen upon exposure to visible light.  1O2  can  lead  to  gene  upregulation.  toxic  forms  of  oxygen. most often the triplet state  of  the  sensitizer.    It  is  thought  that  DA  acts  as  an  endogenous  neurotoxin. It is formed in photosensing reactions and is effectively quenched by  β‐carotene.  and  hydroquinones  can  also  be  an  important  source  of  intracellular  ROS production [57].  aldehyde  oxidase.  flavins.  triplet  oxygen  is  reduced  to  water  (reaction  3). Glycolate oxidase.‐ by oxidizing unsaturated fatty acids and xenobiotics are  cytochrome  P450  and  the  b5  family  of  enzymes  [168].  2.  pH‐dependent  cell  wall  peroxidases.  and  is  an  unusual  ROS.     1 3 O2 + 4ē → 2H2O  (3)  O2  is  the  first  excited  electronic  state  of  O2.2.  Auto‐oxidation  of  small  molecules  such  as  epinephrine.124.  is  the  key  photoreactive  intermediate  and  exerts  photodamage  through  direct  reaction  with  substrate  molecules  (type  I  photosensitization)  or  activation  of  molecular  oxygen  by  energy  transfer  reactions  (type  II  photosensitization)  [199].3.  dihydroorotate  dehydrogenase. The formation of O2‐ as a precursor of H2O2 occurs via electron transfer via production  of  a  sensitizer  radical  cation.  and  fatty  acyl‐CoA  oxidase  are  important  sources  of  total  cellular  H2O2  production  in  peroxisomes  [168].  or  after  an  intermediate  reduction  of  the  sensitizer  with  a  substrate followed by the single electron reduction of O2 [38. L‐α‐ hydroxy  acid  oxidase.  tocoferols.  involved  in  the  molecular  defence  responses  against  photooxidative  stress  .  two‐  and  three‐  electron  reduction.   2. The photoexcitated state.26 Antioxidant Enzyme surrounding  neural  cells.  free  radicals  and  covalent  compounds  are  produced  as  side products and oxidize additional biomolecules [181].  germin‐like  oxalate  oxidases  and  amine  oxidases  have  been  proposed  as  a  source  of  H2O2  in  the  apoplast  of  plant cells [22].  1O2  is  an  excited  state  molecule  formed  by  direct  energy  tranfer  between  the  excited  sensitizer  and  ground state 3O2. urate oxidase. In  addition  to  intracellular  membrane‐associated  oxidases.5.  As  a  result  of  one‐. D‐amino acid oxidase.

  O2•‐ has an approximate half‐life of 2‐4  μs and undergoes fast.  a  fraction  of  1O2  may  be  able  to  diffuse  over  considerable  distances  of  several  hundred nanometers.  with  one  O2•‐  giving  up  its  added  electron  to  another  O2•‐. one‐electron reduction or dismutation in the Haber‐ Weiss reaction (reaction 5).  generating  H2O2    following  protonation  (reaction 11) [181].Oxidative Processes and Antioxidative Metaloenzymes 27 [102]. the one‐electron reduction of  3O2 catalyzed  by NADPH oxidases.+ HO‐  .  In  cases  where  the  speed  of  its  decomposition  is  not  sufficient.  The  hydrogen  donor  for  the  reduction  of  PUFAs may  well  be  ascorbic acid. Enzymatic  dismutation to H2O2 is the most effective quenching mechanism (reactions 7. The lifetime of  1O2 in a cell has been measured to be approximately 3 μs [79] and in  this  time.  forming  H2O2  and  a  radical  of  ascorbic  acid.  it  may  lead  to  its  one‐electron  reduction  (reaction 13). It  is broken down partially enzymatically  by catalase  or  glutathione  peroxidase  to  water  or  in  case  of  substrate  peroxides  to  corresponding  alcohols  and  water.  At  low  pH.     3 O2 + ē → O2•‐  (4)  (5)    2•‐ O2•‐ + H2O2 → HO• + HO‐ + 3O2  It  has  been  noted  that  O   can  undergo  protonation  to  give  up  a  strong  oxidizing  agent  HO2•  (reaction  6)  which  directly  attacks  the  polyunsaturated  fatty  acids  (PUFAs)  in  negatively  charged  membrane  surfaces  [65].         Mn+ + O2 → M(n+1)+1 + O2•‐  O2•‐ + Fe3+ → 3O2 + Fe2+  O2•‐ + H+ + HO2• → H2O2 + O2  (9)  (10)  (11)  H2O2  is  produced  by  the  two‐electron  reduction  of  3O2  (reaction  12)  and  the  univalent  reduction of O2•‐.     3 O2 +  2H + ē → H2O2  (12)  (13)    H2O2 + ē → HO.  dismutation  of  O2•‐  is  unavoidable. gives rise to O2•‐ (reaction 4).   The monovalent reduction of molecular oxygen.         O2•‐ + H+ → HO2•  O2•‐ + ē → O22‐  O22‐ + 2H+ → H2O2  (6)  (7)  (8)  The  interaction  of  O2  with  trace  concentrations  of  redox‐active  transition  metals  leads  to  O2•‐ production (reaction 9) and the non‐enzymatic reduction of O2•‐  in the presence of Fe  forms  3O2  (reaction  10). H2O2 is moderately reactive and has a relatively long half‐life (1 ms) [19]. non‐enzymatic. Other studies have also found that 1O2 can last for nearly 4 μs in H2O  and 100 μs in polar solvent [102]. 8).

 In the presence of suitable transition metals.  producing  the  very  reactive  HO•  and  HO‐  (reaction  14)  [181].  lipid  . that the upper limit of free pools of Cu is far  less  than  a  single  atom  per  cell  casts  serious  doubt  on  the  role  of  Cu  in  Fenton‐like  generation of free radicals [178]. generation of the O2•‐ and HO• appear  to  be  involved  for  Fe.  which  makes  them  available  substrates for peroxidases.     3 O2 + 3ē + 3H+ → HO• + H2O  (17)  NO•  is  generated  by  specific  NOSs.s‐1)  [32].  V.  Oxidation  products  of  lipids. It predominantly  attacks the unsaturated fatty acids of membranes. especially Fe.  16).  Co  primarily  associated  with  mitochondria.  many  effects  of  NO•  are  exerted  as  a  result  of  its initial  binding  to Fe2+  heme  groups. However. taking the form of tocopherol radicals.  Cr. NO• binds certain transition metal ions.  particularly  (2E)‐4‐ hydroxyalk‐2‐enals  and  aldehydes  such  as  malondialdehyde.28 Antioxidant Enzyme The reaction takes place similarly to the Haber‐Weiss reaction in the presence of transition  metals  (Fenton  reaction).  which  is  actually  the  H2O2  radical  (reactions  15.      NO• + O2•‐ → ONOO‐  ONOO‐ + H+ → ONOOH → HO• + NO2• → NO3‐ + H+  (18)  (19)  4. Formation of radicals in biological systems and consequences of  oxidation of biological molecules  4.  NO•  reacts  with  O2•‐  (reaction  18)  in  a  reaction  with  the  highest  rate  constants  known  (7. a recent discovery.0  x  109  m‐1. for which  the  retroactive  reduction  of  the  reaction  requires  the  oxidation  of  ascorbic  acid. Oxidation of lipids  This is considered to be the most damaging process known to occur in living organisms [62]. The most effective protective mechanisms  include reduction of HO2•  by tocopherols.  HO2•  may  arise.  which  metabolise  arginine  to  citrulline  via  a  five  electron  oxidative  reaction  [63].  It  includes  a  number  of  reactions  leading  to  the  development  of  oxidized  lipids  and  fatty  acids  that  give  rise  to  free  radicals.  Common mechanisms involving the Fenton reaction.  The most commonly seen product of such a reaction is [Fe3+ NO‐] [177].1. HO•  can  also  be  produced  from  O2•‐  and  H2O2  at  neutral  pH  and  ambient  temperatures  by  the  iron‐catalyzed Fenton reaction [187].  Cu.  microsomes  and peroxisomes.         H2O2 + Fe2+ → HO•+ HO‐+ Fe3+  Fe2++ O2 + H2O → Fe3++ HO2• + HO‐  Fe2++ HO2• + H2O → Fe3++ HO‐  + H2O2  (14)  (15)  (16)  HO• is also generated by the three‐electron reduction of  3O2  (reaction 17).  as  well  as  alkanes.  Resulting  hydroperoxides  (R‐O‐OH)  are  released  by  phospholipase  A2.  Instead  of  O2•‐  .    ONOO‐  can  be  transformed  into  peroxynitrite acid and then to HO•  (reaction 19). in  fact.

 H+  splits  from  the  methylene  group  near  the  double  bond  producing  mainly  R‐O‐OH. the reaction takes place spontaneously. free  hydrogen H•  and fatty acid R• emerge as the C‐H covalent bond of the hydrocarbon chain is  split.    While  R•  is  prevalent  in  the  reaction  system.  react  with  proteins  and  nucleic  acids.  which  terminates  the  chain  reaction.  22)  or  HO•.  When  the  concentration  of  free  radicals  is  high.  however. As long as  there is enough oxygen.  The  rate  of  this  reaction  then  slows  and  starts  to  be  overtaken  by  the  degradation  of  R‐O‐OH.  This  reaction becomes easier as the number of double bonds increases.           •  R‐H + R‐O‐O• → R• + R‐O‐OH  R‐O‐OH → R‐O‐O• + H•  R‐O‐OH → R‐O‐O• + H2O  R‐O‐OH → R‐O• + HO•  (20)  (21)  (22)  (23)  The  reaction  of  R with  O2  is  much  faster  than  with  a  hydrocarbon  lipid  chain.  propagation  stage  the  reactive  R•  quickly  merges  with  O2. However. followed by the creation of other radicals.  another  molecule  of  unsaturated  fatty  acid  forms  hydroperoxide  (R‐O‐OH)  and  another  R•  (reaction  20). Oxidation of unsaturated  fatty acids only occurs in three stages at normal temperatures.   4.  there  is  a  preponderance  of  R‐O‐O•. The energy required to split bonds can come from ultraviolet radiation.  If.    The  overall  effects  of  lipid  oxidation are a decrease in membrane fluidity.  in  which  reactive  groups  are  diminished.  a  double  bond  in  the  cis  configuration is changed to more stable trans configuration.  the  termination  reaction  leads  either  to  recombination  of  R•  with  R‐O‐O•  forming  peroxide  bridged  dimers  (reaction  24)  or  to  the  reciprocal recombination of R‐O‐O• (reaction 25).1.  As  the  hydrogen  atom  splits  from  the  hydrocarbon  chain.  and also visible light.Oxidative Processes and Antioxidative Metaloenzymes 29 epoxides  and  alcohols. enzymes.1. In the case of unsaturated fatty acids. radioactivity.  The  initiation rate of oxidation for the production of R‐O‐OH is slow (induction period) leading  to a gradual accumulation of R‐O‐OH. it is a two‐electron oxidation of 1O2. and ion channels.  According to the current knowledge.    . During the second.  A reaction also  exists to break any binding with other free radicals or transition metals.  and  produces  a  peroxyl  radical  (R‐O‐O•).  R‐O‐OH degradation with conjugated double bonds  leads preferentially to formation of the alkoxyl radical (R‐O•) (reaction 23) [36]. Oxidation by 3O2  The most common oxidation of fatty acids is by  3O2  from the air.  hydrocarbon  radical  recombination  is  the  major  termination  reaction.  In the latter case. the double bond moves one carbon closer to the carboxyl or  methylene  end  of  the  chain.  By  moving  the  double  bonds. and inactivation of receptors. an increase in the leakiness of the membrane  to substances that do not normally cross it except through specific channels and damage to  membrane proteins. if the number  of double bonds is unchanged.  In the initiation stage.  it  is  likely  that  these  will  react  together  to  form  a  nonradical  product.  R‐O‐OH  is  very  fragile  and  H•  splits  from  the  molecule. sharply rising to reach the  maximum  speed  of  reaction.  leaving  R‐O‐O•  (reactions  21.

  R‐O‐OH  is  oxidized  by  the  nonradical  mechanism  and  the  resulting  epoxide  is  immediately  hydrolyzed  to  dihydroxyderivatives.  and  oxo  acids  are  formed.30 Antioxidant Enzyme     R• + R‐O‐O• → ROOR  R‐O‐O•+ R‐O‐O• → ROOR  (24)  (25)  4.  the  majority  of  radicals  combine  through  ether‐like  ‐C‐O‐C‐  or  peroxide‐like  ‐C‐O‐O‐C‐  bonds.2  dioxolane‐type  peroxohydroperoxides.  saturated  and  unsaturated  aldehydes.4 cyclization  to  the  six‐member  peroxides  derived  from  1. Oxidation by R‐O‐OH  R‐O‐OH of fatty acids and their radicals may react in three ways.  which  are  further  oxidized  and  react  with  the  proteins.  The  third  mechanism  is  oxypolymerization.  The  main  malondialdehyde  precursors  emerge  from  1. The addition of R‐O‐O• across  a  double  bond  can  take  place  intermolecularly.  The  most  reactive  compounds  formed  are  aldehydes.2‐dioxanes.  R‐O‐OH  molecules  by  1.    Breaking  the  molecule  takes  place  both  due  to  the  R‐O•  created  (reaction  26)  and  depending  on  the  position  of  the  double  bond  in  relation  to  the  hydroperoxide  group.    Malondialdehyde  is  an  important  product  of  this  oxidation  [125].  and  they  tend  to  pass  in  1.  in  which  the  number  of  carbons  in  the  molecule  is  increased  due  to  the  reduction  of  two  radicals.  R‐O•.  R‐O‐OH  species  from  polyunsaturated fatty acids (PUFAs) containing three or more double bonds in a molecule  are  unstable.  from  which  R‐O‐OH  is  formed  and  subsequently. because R‐O• is less available.  Concerning  R‐O•.  a  competent  hydroxyl  acid  or  oxo  acid  arises  by  elimination  of  H+.    Unstable  cyclic  peroxide  compounds  moloxides  with  four  or  six‐member  rings  are  .3  cyclisation  pass  to  five‐member  peroxides.1.3.    which  are  also  unstable  compounds  and  decompose  to  low  molecular  active  products.  The rate of reaction between common unsaturated acids and  1O2  is  at least 1450‐fold higher  in comparison to the reaction with triplet oxygen.     R‐O‐O•+ R‐O‐O• → ROOR  (26)  4. An  accrued  radical  of  epoxy  acid  reacts  with  oxygen  to  give  HO2•. Therefore.  In  the  second  case.  R‐O‐OH  and  R‐O‐O•.  Epoxides can arise even with the addition of R‐O• to PUFA by intermolecular reactions.  By  the  recombination  of  R‐O•  with  H•. which is not frequent.    From  this.  1.  radicals  are  condensed by a ‐C‐C‐ bond.  saturated  and  unsaturated  hydrocarbons. It reacts with the listed compounds  because they are rich in electrons and are therefore able to fill its free molecular orbital [158].1.2‐ dioxolanes  and  endoperoxides.  the  molecule  breaks  and  gives  volatile  and  sensory  active  substances  with  less  carbon  atoms. there is no  change  in  the  number  of  carbon  atoms  in  the  molecule. Oxidation by 1O2  Excitation of the common  3O2 leads to a reactive  1O2 which may react with the double bond  of unsaturated lipids and other unsaturated compounds. It has been found that the PUFAs (linoleic  acid  18:2  and  linolenic  acid  18:3)  are  particularly  susceptible  to  attack  from  1O2  and  HO•  [134].  react  very  easily  with  the  double bond of unsaturated fatty acids to generate epoxides. In the first case.2.

   Metals in a lower oxidation state.  The addition of an iron complex to biological samples encouraged peroxidation by peroxide  .   4.Oxidative Processes and Antioxidative Metaloenzymes 31 formed by adduction across double bonds. such as Fe and Cu catalyze decomposition of R‐O‐OH to  R‐O• (reaction 33) and. R‐O‐ OH  arises  in  a  similar  process  to  peroxide  oxidation  by  3O2. especially Fe and Cu.  The  reaction  of  HO2•  with  unsaturated  fatty  acids  is  slow.  which  are  present  in  tissues  that  are  reduced  by  accepting  an  electron. catalyze decomposition of R‐O‐OH to  R‐O‐O•  (reaction  34).  the  oxidation  reaction  is  catalyzed  by  the  ROS  produced.  They  are  involved  directly or indirectly in initiation.      R‐O‐OH + Mn+ → R‐O• + M(n+1)+  R‐O‐OH + M(n+1)+ → R‐O‐O• + Mn+  (33)  (34)  Metals bound in complexes might or might not be effective depending on the environment.  producing  a  transient  complex  with  the  metal.  HO•  is  more  reactive (R‐H + HO• → R• + H2O). propagation and termination reactions of radicals [181]. in their higher oxidation state.  However.  The electron transfer in the reactions leads to the formation of R• (reaction 27)    M(n+1)+ + R‐H → Mn+ + R• + H+  (27)  The  initial  reaction  is  also  indirectly  catalyzed  by  metals  in  the  lower  oxidation  state  Mn+.  as  the  metal‐catalyzed  R‐O‐OH  disintegration  is  faster  than  the  emergence of new radicals.   Metals in their higher oxidation state M(n+1)+ are responsible for initiating oxidation reactions.181].1.            Mn+ + O2 + R‐H → [Mn+ O2 (R‐H)] → R• + M(n+1)+ + HO2‐  Mn+ + O2 + R‐H → [Mn+ O2(R‐H)] → R• + M(n+1)+ + HO2•  Mn+ + O2 + R‐H → [Mn+ O2 (R‐H)] → RO• + M(n+1)+ + HO‐  Mn+ + O2+ R‐H → [Mn+ O2(R‐H)] → HO2• + [M(n+1)+ R‐]  Mn+ + O2 → [Mn+O2] → M(n+1)+ + O2•‐  (28)  (29)  (30)  (31)  (32)  Subsequently. Intermediate products of the reaction decompose  rapidly and give rise to respective hydroperoxides.  therefore producing a different ratio of constitutional isomers. Oxidation catalyzed by metals  This type of oxidation is catalyzed by compounds of transition metals.  while  O2•‐  does  not  react  at  all.  the  mechanism  of  primary  production  of  hydroperoxides  differs  from  the  mechanisms  of  3O2  oxidation. and is generated by the Fenton reaction.4.  These  emerging  radicals  increase  the  reaction  rate  by  increasing  the  propagation  phase  rate.  metals with higher oxidation state and  ROS (reactions 28‐32) [156.  oxygen  and  R‐H  before  decaying  to  R•.   By the reaction with an atom in methylene groups on the carboxyl end of fatty acids.

  Fe3+‐Px)  peroxidases  mediate  one‐electron  oxidation  of  organic  compounds  with  the  concomitant  reduction  of  H2O2  to  H2O.141]. The  enzyme  intermediate  reacts  with  reductants  (R‐H)  to  generate  substrate  free  radicals  and  another  redox  intermediate  (II).  has  been  given  as  1.    High  concentrations of free radicals may outweigh termination reactions.  however.  This  intermediate  consists  of  an  oxoferryl  protein  cation  radical. Other complexes are formed with Co (reactions 40‐42).  can  be  very  powerful  oxidants  capable  of  abstracting  hydrogen  atoms  in  lipid  peroxidation  [20].  in  which  oxoferryl  species  remain  intact  but  the  cation  radical  is  reduced.  it  is  known  that  (ferric.  peroxidase  donates  two  electrons  to  H2O2  resulting  in  cleavage  of  H2O2  and  formation  of  a  redox  intermediate  of  enzyme  (I). Inhibition of oxidation may occur with higher concentrations of metal ions.  which  is  higher  than  the  rate constant for the reaction of ferrous ions with H2O2 in the Fenton reaction 76 mol‐1.  It  is  supposed  that  Fe  and  Cu  ions  oxidize  and  reduce  hydrocarbon  free  radicals  to  their  corresponding anions (reaction 38) and cations (reaction 39) together with the emergence of  free radical complexes (reaction 40).  Ca2+.  In  fats.        Fe3+‐Px + H2O2 → CI + H2O  CI + R‐H → CII + R  CII + RH → Fe3+‐Px + R• + H2O  (35)  (36)  (37)  Also  the  perferryl  [Fe5+]  radicals  are  catalytically  active  in  numerous  biological  processes.    Exposure  to  heavy  metals  can  change  the  composition  of  the  reaction  products.l‐1.  e.s‐1  [73].  catalases  and  cytochrome  P450. where the metals inhibit  the oxidation.  and  these  ferryl/perferryl  moieties.s‐1.g.  Al3+.  g. It  is  catalytically  inactive  and  exists  as  a  resonance  form  between  the  Fe2+‐O2  and  Fe3+‐O2‐  complexes [49. It is not yet known whether the oxidation of lipids can also be catalyzed by complexes  of  Fe  with  oxygen  (Fe3+‐O2‐Fe2+)  and  hypervalent  iron  as  ferryl  cations  FeO2+  and  ferrate  anions  FeO42.l‐1.32 Antioxidant Enzyme decomposition.  generating  R‐O•  and  R‐O‐O•.  In  this  mechanism.  in  which  one  of  the  oxidation  equivalents  exists as the ferryl ion and the other as a porphyrin‐centred cation radical (reaction 35).  The  rate  constant  for  this  reaction  when  ferrous  ions  are  involved.  they  can  catalyze the decomposition of R‐O‐OH through the transient hydroperoxide complexes to R‐ O‐O•.  whether  as  components  of  enzymes  or  simple  iron  complexes.  Some  metal  ions  with  a  fixed  oxidation  number  can  affect  the  rate  of  peroxidation.5  x  103  mol‐1.  All of them break the radical chain reaction.  which  are  the  active  forms  in  the  enzymes  containing  heme  cofactors. The redox potentials of the metals Mn and Co are low and are therefore incapable of  catalyzing  the  breakdown  of  R‐O‐OH  in  aqueous  systems.  A  one‐electron  reduction  of  II  by  a  second  molecule  of  reductant  regenerates  the  ferric  enzyme  and  forms  a  second  equivalent  of  R  (reaction  36).  Another  redox intermediate (III) is formed in the course of peroxidase catalytic cycle (reaction 37).  However.       R• + Fe2+ → Fe3+ + R‐  (38)  .  e.  and  Pb2+  ions  can  accelerate  peroxidation  stimulated  by  iron  salts under certain conditions [72].

  xenobiotics. Cys (‐SH) are also quite  oxidabile in proteins.  A  protein  radical  arises  most  frequently  when  the  more labile hydrogen atom on Cα  splits from the protein. however. tetraoxides) [181].  activated  neutrophils  and  oxidoreductase  enzymes  [153].  HO•. sulfur‐containing amino acids.  γ‐irradiation  in  the  presence  of  O2.  sulfoxide  and  sulfone  miety  (sulfoxido  sulfone.   Besides Trp and Tyr.  47).  and 2 sulfone groups (disulfonates. such as the oxidation of Cys to cysteine. O2 oxidation of thiol groups (–SH) leads to disulfide formation (–S‐S‐)  and  vice  versa.94]. The  second  stage  is  the  reactions  with  thiols  and  their  emerging  radicals  (reaction  46.Oxidative Processes and Antioxidative Metaloenzymes 33           R• + Fe3+ → Fe2+ + R+  R• + Fe3+ → [Fe3+ R•]  R• + CoA3 → [R‐CoA2]  R‐O• + CoA3 → [R‐O‐CoA2]  R‐O‐O• + CoA3 → [R‐O‐O‐CoA2]  (39)  (40)  (41)  (42)  (43)  4.  trioxides).  O2•‐.2. for example.  or  with  free  lipid  radicals  to  form  copolymers. Oxidation of proteins  The  principal agents  for  protein  oxidation  are  atmospheric  O2.            RSH + HO‐ ↔ RS‐ + H2O  RS‐ + O2 ↔ RS• + O2•‐  RSH + O2•‐ → RS• + HO2‐  RSH + HO• → RS• + H2O  (44)  (45)  (46)  (47)  H2O2 and R‐O‐OH are.  As  their quantity increases.  thiosulfonate).  which  can  be  further  .  Under  normal  conditions.  dehydrogenases  have  the  same  effect  in  organisms.  and  hydroperoxide  from  a  peroxyl  radical.  1O2. In response to the reaction  of  protein  thiols  (PrS)  with  R‐O‐OH.    atoms  of  sulfur  are  simultaneously  oxidized  (frequently  those  in  Cys). more efficient oxidizing agents.  R‐O‐OH and  H2O2. Met (‐S‐CH3).  Other  agents  that  lead  to  protein  oxidation  include  HOCl. Thiolate reacts with oxygen and produces a thyil radical (RS•) (reaction 45) [85.  The first stage of oxidation  is the emergence of alkylthiolate (RS‐) in the presence of the hydroxyl anion (HO‐)  (reaction  44).  forming  corresponding  monoxides  (thiosulfinates)  and.  where  appropriate. further oxidized products containing 2 sulfoxide groups (disulfoxide). Reactions with hydro and hydrogen  peroxides  convert  thiol  proteins  also  into  sulfenic  acids  (RSOH). sulfone  moiety  (dioxide.  which  then  react  with  other  free  protein  radicals  to  form  dimers.  Recombination  of  protein  radicals leads subsequently to protein oligomeres. the probability of them reacting to form a non‐radical product also  grows (2 RS → RSSR).  Free  peroxyl  radicals  react  with  proteins  and  produce  protein  radicals.  reduced  transition  metals. A hydroxyl acid is obtained from  an  alkoxyl  radical.

  Oxidative  modifications  of  critical  amino  acids  within  the  functional  domain  of  proteins may also occur by S‐glutathionylation.  Further  methioninsulfoxide  oxidation  produces  methioninsulfone.  propagation  (forming  2‐  and  3‐adduct  peroxy  radicals)  in  reactions  53‐56  and termination reactions (57) of Trp autooxidation:               • Trp + HO• → Trp‐OH  Trp‐OH + O2 → Trp(OH)OO•  (51)  (52)  (53)  (54)  (55)  (56)  Trp + Trp(OH)OO• + O2 → Trp(OH)OO‐Trp•  Trp(OH)OO‐Trp•+ O2 → Trp(OH)OO‐TrpOO•  TrpH(OH)OO‐TrpOO• → RR”O2 + HO•  Trp(OH)OO‐TrpOO• → RR”O2 + HO2•‐  .  H2O2.169].  Met  is  oxidized  to  methioninsulfoxide.  Autooxidation  propagated  by  peroxy  radicals  is  a  chain  reaction.  peroxyacids.  H•  reacts  with  Trp  yielding  the  corresponding  Trp‐H  adducts.  Exposure  to  NO  during pathological conditions can lead to the formation of ONOO‐.  The  following  set  of  reactions  according  to  Janković  &  Josimović  [90]  demonstrates  initiation  (reaction  51. RS• then  reacts with a glutathionylate anion (GS‐) to form a radical mixed disulfide (RSSG•‐). which  can lose an electron to oxygen to form O2•‐.55]. in a photooxidation reaction catalyzed by riboflavine.  which  will  then  react  with  a  thiolate  anion  to  displace  HO‐  (reaction  49). especially in an acidic environment. yielding Trp‐OH adducts [93].34 Antioxidant Enzyme oxidized  to  higher  oxidation  states  such  as  sulfinic  (RSO2H)  and  sulfonic  (RSO3H)  acids  [85.  It  is  also  possible  that  S‐ nitrosylation  of  PrSH  to  form  PrSNO  can  lead  to  protein  glutathionylation  by  the  displacement  of  the  NO‐  by  glutathione  (reaction  50)  [26.195].  while  a  small  amount  of  the  H‐atoms  react  with  oxygen  yielding  HO2•‐. It is easily oxidized  by O2 on exposure to light.  which is unexploitable.        PrS• + GS‐ → RS•‐‐SG + O2 → O2•‐ + PrS‐SG  PrSOH + GS‐ → PrS‐SG + HO‐  PrSNO + GS‐ → PrS‐SG + NO‐  (48)  (49)  (50)  Trp is a very oxylabile compound. Oxidation  occurs  due  to  the  action  of  sulfoxides.  52). leaving a mixed disulfide (reaction 48) [155. Such alterations may alter the activity of an  enzyme  if  the  critical  cysteine  is  located  within  its  catalytic  domain  or  the  ability  of  a  transcription factor to bind DNA if it is located within its DNA binding motif [14].  R‐O‐OH. These adducts react with oxygen to produce the  corresponding  peroxy  radicals. which can oxidize thiols  to  either  RS•  or  RSOH  and  lead  to  protein  glutathionylation.  The  initial  phase  is  the  reaction  of  HO•  with  tryptophan  across  the  double  C=C bonds.  but  also  undergoes  autooxidation  under  γ‐irradiation  [90].  A  study  from  Thannickal  &  Fanburg  [168]  confirms  that  cysteine  modification  involving  S‐glutathionylation  is  readily  reversed  to  the  active  sulfhydryl  group  by  thioltransferases.  Another route to mixed disulfides is through the two electron oxidation of a thiol to RSOH.

  Glutathiolation of Cys residues similarly. are most susceptible to oxidative attack  due  to  their  proximity  to  the  radicals  formed  by  binding  transition  metals  [160]. aldehydes and methionine sulfoxide [80. Trp. Arg. Cd and Ni. other mechanisms.  and  finally  enolization.  The  mechanism  of  dityrosine  formation  begins  with  the  generation  of  a  Tyr•. Lys and Met residues leads to the formation of  chlorotyrosine.  Metal‐ catalyzed oxidation of histidine generally causes formation of oxo‐His or Asp [16.97]. incur formation of carbonyl groups  (aldehydes and ketones) on the side chains [6. Pro and Thr. The interaction of HOCl with Tyr. Other  amino acyl moieties.  the  presence  of  HO•  during  radiolysis  leads  predominantly  to  extensive  protein‐protein  crosslinkage via tyrosine‐tyrosine (dityrosine) bonding and possibly other amino acid cross‐ links  as  well  [71]. especially Lys. leading to the addition of aldehyde moieties to the  protein [149.  Phe. Tyr• may dissipate  by  pathways  other  than  those  involving  intermolecular  diradical  crosslinking  of  Tyr. such as His.    Whilst  Fe.   The chelating amino acids in proteins.   ONOO‐ causes nitrosylation of Tyr residues and oxidative modification of other amino acid  residues including Cys.  Formation of Tyr oxidation products might involve cyclization.  Cu. α‐β unsaturated alkenals may react with sulfhydryl groups of proteins  to form stable covalent thioether adducts also containing carbonyl groups [69]. the primary route for  their toxicity is depletion of glutathione and bonding to sulfhydryl groups of proteins. amino acids and sugars.  Indirect  oxidative  modification  of  protein  amino  acyl  side  chains  occurs  through  the  formation of adducts with products of oxidatively modified lipids. yield ketoamine protein conjugates [71].  The  overall rate constant for this process was reported to be 4 x 108 M‐1s‐1 [69].  Val  and  Ile. Trp. have been proposed [178].  V  and  cobalt  Co  undergo  redox‐cycling reactions.   4.Oxidative Processes and Antioxidative Metaloenzymes 35   2Trp(OH)OO• →R2O2 + O2  (57)  Similarly.  Lipid  peroxidation  products  such  as  hydroxynonenal. for a second group of metals. Products of  free  amino  acid  oxidation  can  also  form  covalent  attachments  to  proteins  [81].3.  upon  Amadori  rearrangement. obtained by the reaction of reducing  sugars  with  an  ε‐amino  group  of  lysyl  residues  in  proteins  may.114]. Met and Phe [89] but it is a poor inducer of protein carbonyls  [171]. As is  thought to bind directly to critical thiols.153. decarboxylation. and further  oxidation steps on either the protein or fragments released from the protein [70].  malondialdehyd  and  acrolein  bind  covalently to Lys.   Metal‐mediated formation of free radicals causes also various modifications to DNA bases. however.  the  preferred  targets  of  radicals  produced  during  γ‐radiolysis  in  proteins  are  other  hydrophobic  amino  acids  such  as  Tyr.  Cr.  altered  calcium  and  sulfhydryl  homeostasis. His and Cys residues. involving formation  of hydrogen peroxide under physiological conditions. Oxidation of DNA  Reactions  that  alter  DNA  and  other  macromolecules  in  living  systems  are  induced  by  oxidizing  conditions  resulting  from  normal  metabolism  or  ionizing  and  ultraviolet  . chloramines.  radical  isomerisation  followed  by  diradical  reaction. Hg.174].159].  In  biological  systems. Schiff bases.

    The  dominant  mechanism  for  radical  cation  migration  in  DNA  is  multi‐step  hopping  [113.  resulting  in  the  formation  of  7‐hydro‐8‐hydroxypurines. In particular.  which  may  be  converted  into  formamidopyrimidines.  the  reactivity  of  the  3´‐G  is  reduced  when  flanked  by  pyrimidines.  The  rate  constants  of  the  dehydration  of  C4‐OH  adduct  radicals  of  purines  at  neutral  pH  amount  to  1.  with  the  5´‐G  being  especially  reactive  [147].  C4‐OH and C5‐OH adduct radicals of purines dehydrate and are converted to an oxidizing  purine(‐H)• radical. the reaction of O2 with C8‐OH adduct radicals of purines  is  diffusion‐controlled  [182].  The  relative  reactivity of the guanines in a GG step is influenced by the surrounding bases.6‐diamino‐4‐hydroxy‐5‐formamidopyrimidine  from  guanine  and  4. different mesomeric structures of these  radicals  may  have  ambivalent  redox  states  [182].  whereas  C5‐OH  and  C8‐OH  adduct  radicals are primarily reductants. the result of which is essentially  independent  of  the  process  by  which  it  is  oxidized  [110].  and  C8  positions  of  purines  generates  OH  adduct  radicals. The formation of 8‐hydroxypurines is preferred in the presence of O2. An incoherent.  In  contrast to C4‐OH adduct radicals.  which  migrates  reversibly  through  duplex  DNA  by  hopping  until  it  is  trapped  in  an  irreversible  chemical  reaction  to  form  a  structurally  modified  base  [95]. HO•  reacts with DNA by addition  across double bonds of DNA bases at or near diffusion‐controlled rates with rate constants  of  3  to  10  x  109  M‐1.  The  one‐electron  reduction  of  the  ring‐opened  radical  leads  to  the  formation  of  2.  Guanine•+  does  not  hydrate  to  form  the  C8‐OH  adduct radical or go on to form 8‐hydroxyguanine (8‐oxoguanine. it was found that GG steps are the  preferred  sites  for  reaction.  The  one‐electron  oxidation  leads  to  the  formation  of  8‐ hydroxypurines  (7. which may be reduced and protonated to reconstitute the purine [142].  hemiorthoamides.  The  oxidation  of  C8‐OH  adduct  radicals competes with the unimolecular opening of the imidazole ring by scission of the C8‐ N9  bond  at  a  rate  constant  of  2  x  105  s‐1.157]  from  a  donor  to  an  acceptor through intervening bridging nucleobases [92].  which  has  also  been  attributed  to  electronic  effects  [33].  Superexchange  is  possible. multi‐step.  8‐hydroxypurines  are  also  formed  in  the  absence  of  O2.  but  less  effective  for  long  distances. HO• oxidation is most prevalent.s‐1  [186]. 8‐OH‐Gua) by oxidation. The most basic reaction is one‐electron oxidation. On the other hand.  The  loss  of  an  electron  converts  DNA  to  its  radical  cation  (an  electron  “hole”).  but  to  a  lesser  extent.    The observation that DNA oxidation occurs predominantly at guanines has been attributed  to the fact that this base has the lowest Eox [31].s‐1. Similarly.6‐diamino‐5‐formamidopyrimidine  from  adenine  [25].  The  one‐electron  reduction  of  C8‐OH  adduct  radicals  without  ring‐opening  may  also  occur.   With respect to DNA.  However.  the  rate  constant  of  H  atom  abstraction  is  2  x  109  M‐1.5  x  105  s‐1  and  6  x  103  s‐1.172]  where  charge  resides  on  a  single  base  or  on  small  number of adjacent bases and thermal fluctuations precipitate its movement from one base  to  another  [96].8‐dihydro‐8‐oxopurines)  in  DNA  [25].115].  .  The  addition  of  HO•  to  the  C4.  C4‐OH  adduct  radicals  possess  oxidizing  properties. random  passage from donor to acceptor consists of short‐distance tunnelling intervals linked by base  sequences that serve as resting sites for charges [64.  whereby  charge  is  transported  coherently  in  one  step  by  tunnelling  [13.  C5.36 Antioxidant Enzyme radiation.  The  guanine  radical  cation  (guanine•+)  is  formed  by  elimination  of  HO‐  from  the  C4‐OH  adduct  radical  of  guanine  and  may  deprotonate  depending  on  pH  to  give  guanine(‐H)•.

Oxidative Processes and Antioxidative Metaloenzymes 37 however.  Oxygen  reacts  with  the  allyl  radical. Dialuric acid is oxidized in the presence of O2 to alloxan [44]. resulting in the formation of the allyl  radical. such as HO• attack at the C2‐position of adenine.5(1H. C6‐OH adduct radicals of pyrimidines may lead to the  production  of  6‐hydroxy‐5‐hydropyrimidines.5‐dihydroxyimidazolidine as a major  product of cytosine [34.  Further  reactions  of  C5‐OH‐6‐peroxyl  and  C6‐OH‐5‐peroxyl  radicals  of  cytosine result in formation of 4‐amino‐5‐hydroxy‐2. C5‐OH‐6‐peroxyl radicals  are formed by addition of O2 to C5‐OH adduct radicals at diffusion‐controlled rates.85]. The  C4‐OH  adduct  radical  of  adenine  reacts  with  O2  with  a  rate  constant  of  1.44.  respectively. 5‐formyluracil. Oxidation of saccharides  The  functional  group  of  carbohydrates  is  subject  to  oxidation. C5‐OH‐ 6‐hydroperoxide gives rise to trans‐1‐carbamoyl‐2‐oxo‐4. and subsequently protonated  to give 5‐hydroxy‐6‐hydropyrimidines.47].   4.  and  then  protonated  to  give  hydroxyperoxides  [189].0  x  109  M‐1. O2 adds to guanine‐(‐H)• with a  rate constant of 3 x 109 M‐1. and 5‐hydroxy‐5‐methylhydantoin [189]. however.  Auto‐oxidation  of  carbohydrates  is  slow  in  neutral  and  faster  in  an  acidic  environments. C5‐OH adduct radicals may be reduced.   In the case of adenine. The reaction of guanine(‐H)• with O2 leads to imidazolone and  oxazolone derivatives [34. C5‐OH adduct radicals are reducing  while C6‐OH adduct radicals are oxidizing [161]. at least two OH adduct radicals are formed: C4‐OH and C8‐OH.  which  break  down  to  yield  thymine  glycol.  Cytosine  glycol deaminates to give uracil glycol.  followed  by  a  reaction  with  water  by  HO‐  addition  to  yield  thymine  and  cytosine  glycols  [34. Similarly.  Thymine  peroxyl  radicals  are  reduced.s‐1.6H)‐pyrimidinedione.  giving  rise  to  as  yet  unknown  products  [182]. The redox properties of adduct radicals differ.  These  products  are  typical  of  anoxic  conditions  because  O2  inhibits  their  formation  by  reacting  with  OH  adduct  radicals. On the other hand.  which  gives  rise  to  8‐OH‐Gua  upon  oxidation  [45].  By  contrast. and 5‐hydroxyuracil [25.188].  The  C4‐OH  adduct radical of guanine barely reacts with O2. C5‐OH‐6‐ peroxyl  radicals  eliminate  O2•‐. In the absence of O2.85].  causing  DNA  strand  breaks [129].  The  products  of  cytosine  oxidation  undergo  deamination  and  dehydratation. the hydration of guanine•+ in double stranded DNA forms  the  C8‐OH  adduct  radical. 5‐hydroxycytosine. leads to cytosine glycol and thymine glycol [25.  D‐glucose  and  D‐ .188] but as a minor product from DNA [43].6(1H.  producing  5‐ hydroxymethyluracil  and  5‐formyluracil.  followed by oxidation [136].  pyrimidine  glycols  and  5‐hydroxymethyluracil  are  formed  under  both  oxic  and  anoxic  conditions.4.  2‐hydroxyadenine  is  also  formed  from  adenine in DNA by a possible mechanism.  followed  by  addition  of  HO‐  (or  addition  of  water  followed  by  deprotonation). 5‐hydroxy‐6‐hydrocytosine readily deaminates into  5‐hydroxy‐6‐hydrouracil.   Addition of HO• across the C5‐C6 double bond of pyrimidines leads to C5‐OH and C6‐OH  adduct radicals and H atom abstraction from thymine.  it  may  react  with  2ʹ‐deoxyribose  in  DNA  by  H  abstraction.  5‐  hydroxymethyluracil.5H)‐pyrimidinedione and 4‐amino‐ 6‐hydroxy‐2.  In the absence of O2.s‐1. the oxidation of C5‐ OH  adduct  radicals.  which  may  deaminate  to  give  dialuric  acid and isodialuric acid.

  Similar  products  are  also  formed  by  oxidation  of  H2O2  alone.2‐diol  (enediol)  →  enediol  radical  anion  →  α‐dicarbonyl  +  O2•‐. O2 inhibits their formation by reacting  with C5ʹ‐centered sugar radical before cyclization is possible [34]. α‐dicarbonyl compounds  and α‐ketoaldehydes.  compounds  with  α‐hydroxyaldehyde.  which  break  down  to  form  D‐ arabinonic and formic acids [181].  which  becomes  a  hydroxyalkyl  radical  (or  alkyl  radical.  These  compounds  cause  concomitant damage to both base and sugar moieties. This reaction leads to  intramolecular cyclization.  H2O2. The radicals that develop as intermediate products. The mechanism of HO2•  formation.   Reactions  of  HO•  with  the  sugar  moiety  of  DNA  by  H  abstraction  give  rise  to  sugar  modification  and  strand  breaks. forming α‐ketoaldehydes as major  products  in  the  following  reaction.  thus  the  alkyl/alkoxyl  radicals  they  produce  would  have  different  properties  [119].  Hydroxyalkyl  radicals  in  the  presence  of  O2  give  rise  to  dicarbonyls  and  HO2•.  such  as  glucose  [196]  and  fructose  [181]  enolize and reduce transition metals and O2 sequentially. In the presence of HO•.  giving  rise  to  α‐hydroxyacids  and  further  ketoaldehydes  [196].  In  the  presence  of  Fe2+  ions. Both 5ʹR‐ and 5ʹS‐diastereomeres of 8. The alternative is to induce the transfer of H+ in  the  six‐member  structure  of  the  intermediate.  regenerates  the  catalytic metal oxidation state and produces HO•.42].  the  decay  of  H2O2  generates  free  radicals  and  also  oxidizes sugars to the glycosuloses. it is expected that the extraction of hydrogen  from  the  two  kinds  of  saccharides  by  HO•  would  take  place  at  different  sites  of  the  two  kinds  of  saccharides. hydroxyaldehyde  hydrate  is  formed.  R•).   .  The  resulting  radical  reacts  with  O2  and  the  resulting  peroxyl  radical  spontaneously decays to the corresponding glyculosis and HO2•.  This  creates  aldonic  acid.42].  formed  by  O2•‐  dismutation.  and  glyoxal  [181].  Hydroxyaldehyde  →  1‐en  1. N‐substituted 1‐amino‐1‐deoxyfructose (Amadori product) appears  as  one  of  the  early  products  of  protein  glycosylation  [197]. followed by 8. which is subject to further  disproportionation to H2O2 and O2 (reaction 58).  Alternativelly.   The formation of R‐O‐O• from lipid oxidation involves C‐H bond cleavage at the C2 carbon  of  the  carbohydrate.  Other  important  processes  in  these  reactions  are  glycation.    HO2• → H2O2 + O2  (58)  α‐dicarbonyl compounds with the original number of carbon atoms are further oxidised and  decomposed.5‐cyclopurine‐2ʹ‐deoxynucleosides after oxidation  [41.5ʹ‐ cyclo‐2ʹ‐deoxyadenosine  (cyclo‐dA)  are  formed  in  DNA  [41.  In  the  presence  of  transition  metals. react further with Lys and Arg in proteins and are involved in early  glycosylation reactions.  Because  glucose is an aldose and fructose is a ketose.2‐diols.  glycoxidation  and  the  formation  of  advanced  glycation  by‐ products [191].  A  unique  reaction  of  the  C5ʹ‐centered  sugar  radical  is  addition to the C8‐position of the purine ring of the same nucleoside. which  causes the transfer of H+ in five members structures of the intermediate structure is not the  only like that of carbohydrates by R‐O‐O•.  H2O2.38 Antioxidant Enzyme fructose  form  unstable  hydroperoxides  via  1‐en‐1.  peroxyl  radicals  and  HO2•  are  formed.   Monosaccharide  autooxidation  is  a  metal‐catalysed  process.5ʹ‐cyclo‐2ʹ‐deoxyguanosine (cyclo‐dG) and 8.

  FeSOD  and  eukaryotic  Cu/ZnSOD  are  dimers. solvent‐exposed.2) is  the  second  enzyme  responsible for  the detoxification of O2•‐. Therefore.87].  presumably  a  Fe3+‐ peroxo  species. The free.  FeSOD  isozymes.  and catalytic functions [200]. Antioxidant metaloenzymes  5. respectively. metal ligand  environment.  The  reaction  of  SOR  with  O2•‐  may  proceed  through  two  reaction  intermediates  [117. the catalytic dismutation activity  of  NiSOD  occurs  through  the  oxidative  and  the  reductive  half‐reactions.  SODs  are  classified  by  their  metal  cofactors  into  known  types:  the  Cu/ZnSOD  and  MnSOD. and spectroscopic properties.  The  first.s‐1.  SOR  is  a  non‐heme  iron  enzyme. Cu/Zn SOD is mainly extracellular and cytosolic.  is  formed  by  the  almost  diffussion‐limited  binding  of  O2•‐  to  the  ferrous  . takes place with a dismutation rate k = 105‐ 106 M‐1.      SOD‐Ni2+ + O2‐ + 2H+ → SOD‐Ni3+ + H2O2  SOD‐Ni3+ + O2‐ → SOD‐Ni2+ + O2  (60)  (61)  Formation of H2O2 in the absence of the enzyme.  Both  types  are  also  present  in  plants  [131].15.1. Superoxide reductase (SOR)  (EC  1.2. The active site consists of a mononuclear ferrous ion in an unusual [Fe2+ (N‐His)4(S‐ Cys)] square pyramidal pentacoordination complex [3.  which  are  localized  in  different  cellular compartments. while MnSOD is a  mitochondrial  enzyme.   5.  which  can  be  described  by  the  following  two  equations. like its counterparts.Oxidative Processes and Antioxidative Metaloenzymes 39 5. pH dependence.  However. The active site of the enzyme contains one or two different atoms  of  a  transition  metal  in  a  certain  oxidation  state  [148].1.  whereas  MnSOD  of  mitochondria are tetramers. NiSOD is distinct from other known SODs [28. Superoxide dismutase (SOD)  Superoxide  dismutase  (EC  1.203].  are  usually  associated  with  the  chloroplast  compartment  [5]. O2•‐  is capable of reacting with another molecule of O2•‐  (dismutation) or  is  also  able  to  react  with  another  radical.  Formation  of  HO•  from  O2•‐  via  the  metal‐catalyzed  Haber‐Weiss  reaction  has  a  reaction  rate  10  000  times  faster  than  that  of  spontaneous dismutation.1.  allowing  them  to  survive  in  the  presence  of  O2.  The  prokaryotic  MnSOD. On the basis of amino acid sequence.  found  only  in  prokaryotic  cells.    SOD  contributes  significantly  to  protecting  the  organism  from  the  toxic  effects  of  O2•‐  [152]. sixth  coordination  position  is  the  site  of  O2•‐  reduction  [104.  often  not  detected  in  plants.  In  living systems. in all  subcellular compartments.15.139].  such  as  NO•. all SODs are  known to have very similar catalytic rate constants. so SOD provides the first line of defence against ROS [65]. NiSOD is the most recent class of SOD.140].    2O2•‐ + 2 H+ → H2O2 + O2  (59)  These enzymes are present in almost all aerobic cells as well as in anaerobic organisms. This reaction is accelerated 104‐times by SOD [65].1)  belongs  to  the  group  of  oxido‐reductases.  where  SOD‐Ni2+  and  SOD‐Ni3+  represent  the  reduced and oxidized states of the metal center in the enzyme. which was discovered in  Streptomyces [205] and cyanobacteria [144].

 Since IDO is expressed both in the  periphery  and  in  the  central  nervous  system.  in  particular  plasma‐cytoid  cells.  IDO  does  not  show  substrate specificity exhibited by TDO.11.  After  conversion  into  3‐hydroxykynurenine.  possibly  a  Fe3+‐hydroperoxo  species. it has been  established  that  IDO  regulates  maternal  tolerance  and  possibly  more  general  aspects  of  T‐ . The complex has both reduced and oxidized forms of iron in the  active  site  [2].  and  microglial  cells  within  the  brain  parenchyma.  In  liver  hepatocytes.  [133]  proposed  a  mechanism  for  the  reaction  of  SOR‐ Fe(CN)6 complex with O2•‐.  most  of  the  kynurenine  formed  via  IDO  is  metabolized  into  xanthurenic  acid.  Macrophages  and  dendritic  cells. as  they cannot be involved in the formation of HO•.127].3‐dioxygenase (IDO)  Indoleamine  2.  IDO  is  a  heme‐dependent  cytosol  enzyme  present  predominantly  in  monocytes. catalyzing the oxygenative ring cleavage of various  indoleamine  derivatives. forming weakly reactive components in comparison to H2O2. first yielding  a  second  intermediate.  and  immunogenicity  [18].  IDO  is  stimulated  by  pro‐inflammatory  cytokines.  have  been implicated in the IDO‐mediated suppression of T‐cells [10].  macrophages.3‐ dioxygenase  (TDO)  and  indoleamine‐2.  especially  IFN‐γ  [150.3‐dioxygenase  (decyclizing)  or  indole:oxygen  2.  Molina‐Heredia  et  al.  Trp  is  essential  for  the  growth  of  bacteria  and  the  growth  of  bacteria  is  suppressed  by  actively  depleting  Trp  within  infected  cells  and  surrounding  milieu  [68. Indole‐2.  This  step  involves  two  different  enzymes. This intermediate undergoes two sequential protonation reactions.13.  The  active  site  of  SOR  binds  ferrocyanide.  and  then  the  final  reaction  products.  Even  though  it  catalyzes  the  same  dioxygenation  reaction  as  classical  hepatic  TDO.3‐oxidoreductase  (EC  1.  which  increases  only  when  SOD  is  inhibited.  it  differs  from  the  latter  with  respect  to  molecular  size  substrate  specificit  y.  TDO  is  predominantly  expressed  (for  more  details  see  [37])  and  as  a  proenzyme  [51.3.  it  represents  a  possible  link  between  the  immune  system  and  serotogenic  pathway.165]. The induction of IDO causes a marked  increase  in  Trp  catabolism  in  the  body  [164]  causing  kynurenine  production  and  overall  depletion  of  Trp  in  the  cell.  as  Trp  availability  controls  the  synthesis  of  serotonin  [111].52].  rather  than  complete  oxidation  along  the  glutarate  pathway  or  conversion  into  NAD  [18]. H2O2 and the ferric active site [140].  tryptophan‐2. More recently.  The  enzyme  scavenges  O2•‐.  IDO  is  down‐regulated  by  NO  as  a  consequence  of  the  L‐Arg  metabolic  pathway activation.40 Antioxidant Enzyme active site.3‐dioxygenase  (IDO).  virus  infection  [204]  and the administration of bacterial endotoxin [175].17) uses O2•‐ as cofactor in the initial step during the degradation of the indole ring of  Trp  to  form  kynurenine.  also  referred  as  hexacyanoferrate  (II)  or  K4Fe(CN)6.    SOR‐Fe2+‐NC‐Fe2+(CN)5 + O2•‐ → SOR‐Fe2+‐Fe3+(CN)5 + O22‐  (62)    SOR‐Fe2+‐NC‐Fe3+(CN)5 + HCOO‐ + 2H+ → SOR‐Fe2+‐NC‐Fe3+(CN)5 + HCOOO‐ + H2O (63)    SOR‐Fe2+‐NC‐Fe3+(CN)5 → SOR‐Fe3+‐NC‐Fe2+(CN)5  (64)  5. which is also affected by IFN‐γ [170].  at  its  sixth  coordination  position  through  a  cyano  bridge  between  the  iron  and  the ferrocyanide molecule.  however.  cofactor  requirements.

1. CAT has the highest turnover rate  among all enzymes.108].  by  eliminating  H2O2  with  oxidizing  alcohols.  generating  the  oxoferryl  species.  which  does  not  seem  to  be  essential  for  the  enzymatic  conversion  of  H2O2  to  H2O  and  O2.  In the first.Oxidative Processes and Antioxidative Metaloenzymes 41 cell  tolerance  [128].6)  is  a  heme‐containing  enzyme  that  is  present  in  virtually  all  aerobic  organisms  tested  to  date  [4.  respiration.  and  the  other  from  the  porphyrin  ring.  The  findings  of  Scott  et  al.  The  reaction  takes  place  in  two  two‐electron  reactions. compound II (CII). thereby releasing O2•‐ and  the natural enzyme.  66).  [151]  suggest  that  IDO  modulates  inflammatory responses. which can be subsequently converted  to another inactive form.5.  breaking  H2O2  down  into  H2O  and  O2  without  the  production  of  free  radicals. in particular those driven by B‐cells.  The  second  H2O2  then  reduces CI to regenerate the resting (ferric) enzyme while releasing H2O and molecular O2. one molecule of CAT can convert approximately 6 million molecules of  H2O2 to H2O and O2 per minute [62] and the pH optimum obtained from different sources is  6.   The  α‐phase  works  catalytically  (reactions  65.  EC  1. [4].         CI + HN3 (or RH2 + H2O2) → CII + N3• (or R + 2H2O)  CII + H2O2 → CIII + H2O  CIII → CAT‐Fe3+ + O2•‐  (69)  (70)  (71)  . Catalase (CAT)  Catalase  (H2O2:H2O2  oxidoreductase.  where  it  is  important  in  the  removal  of  H2O2  generated  by  oxidases  involved  in  β‐oxidation  of  fatty  acids.  generating  a  porphyrin  cation  radical. catalases may undergo a one‐electron reduction (reactions  67.  and  purine  catabolism  [9]. Each  tetrameric molecule of mammalian CATs contains four molecules of tightly bound NADPH.  it  is  localized  predominantly  in  the  peroxisomes  [15].   5. The enzyme can function in 2 ways: α and β phases [107. a H2O2 molecule oxidizes the heme to compound I (CI).8‐7. formate (RH2) or nitrate as described in Aksoy et al.11.  but rather protects CAT against inactivation by H2O2 [98].  CATs  from  many  species  are  known  to  be  tetramers  of  60‐65  kDa  subunits  with each subunit containing 1 Fe‐protoheme IX moiety (4 heme groups per tetramer).       CAT(Por‐Fe3+) + H2O2 → CI(Por+• ‐Fe4+=O) + H2O  CI(Por+• ‐Fe4+=O) + H2O2 → CAT(Por‐Fe3+) + H2O + O2  (65)  (66)  At limiting H2O2 concentrations.      CI(Por+• ‐Fe4+=O) + HA → CII(Por‐Fe4+‐OH) + A•  CII(Por‐Fe4+‐OH) + H2O2 → CIII(Por‐Fe2+=OOH) + H2O  (67)  (68)  The  β  phase  works  peroxidatively  (reactions  69‐71).4.12]. compound III (CIII) [138].  In  the  cell. removing one  oxidation  equivalent  from  the  ferric  iron. 68) to an inactive intermediate.

 In a catalase cycle. GPx scavenges various peroxides.9)  has  eight  known  izoenzymes  that.  The variable response of CAT activity has been observed under metal stress [65].  It  has  been  proposed  that  KatG  is  responsible  for  the  catalytic  oxidation  of  H2O2  in  a  two‐electron  oxidation  step  with  both  oxygen  atoms  being  derived  from  the  same  H2O2  molecule.  KatGs  are  the  only  peroxidases  known  with  both  catalase  activity  comparable  with  catalases  and  typical  peroxidase activity with broad specificity. and hydrogen atom transfer from H2O2  to  the  ferryl  species  to  yield  a  radical  intermediate  [185]. The Km  .101. The main diference in the  enzymatic mechanism between CAT and peroxidases is CI reduction.  Until  now.  This  non‐scrambling  mechanism  is  independent  of  pH  and  is  not  affected  by  manipulation  of  highly‐conserved  and  important  catalatic  residues.11. EC 1.1.  Principally.  It  was  shown  phylogenetically  that  the  closest  neighbours  of  KatGs  are  eukaryotic  ascorbate  peroxidases  and  yeast  cytochrome  c  peroxidase  [192]. a  second  H2O2  molecule  is  used  as  a  reducing  agent  for  CI.l‐1 [39].  Some  studies  have  shown  that  CAT  is  effective  in  the  degradation  of  H2O2  present  only  in  mmol.7).  the  pathophysiological  role  of  these  isoenzymes  in  antioxidant  defence  is  of  substantial  importance  [100. thereby oxidizing it.  Selenocysteine  participates directly in the transfer of electrons to a peroxide substrate.  there  are  two  possible  mechanisms  for  the  formation  of  O2  following  this  retention  mechanism:  an  ionic  mechanism.1.  in  active  positions.5.  and the non‐heme.  So  far.42 Antioxidant Enzyme The CAT reaction has evolved in at least three phylogenetically unrelated protein types: the  monofunctional or “classical” CAT.  which  contain  the  non‐metal  selenium  [27].l‐1. Mn‐containing catalase [138].  may contain co‐factors.  This  two‐electron  reduction  completes the cycle forming ferric‐CAT and O2 (for details see [207]). such as heme and residues of cysteine or selenocysteine. A distal His‐Asn pair has been shown to be essential for CI formation in classical CATs.  while a distal His‐Arg has the same funtion in peroxidases [48. rate constants for the formation  of CI from peroxidases and catalases were calculated to be in the range of 106 to 108 M‐1 s‐1  [48]. the bifunctional catalase‐peroxidase (KatG. The expression of CAT in most  cells is lower than that of GPx. while all  ions of heavy metals are non‐competitive inhibitors of CAT. Cyanides are strong inhibitors  of  CAT  as  they  form  a  strong  bond  with  the  heme  of  CAT  and  stop  its  catalytic  activity  [184]. With most substrates  in  a  peroxidase  cycle. with the exception of hepatocytes and erythrocytes.11. Generally.180].  KatGs  can  be  viewed  as  a molecular  fossil  revealing  the  common  phylogeny  of  catalytic  and  per‐oxidative  activity  during  evolution  [206].  the  complete  gene  sequences of KatGs were characterised only from prokaryotes (both from archaebacteria and  eubacteria)  although  several  reports  describe  the  presence  of  KatGs  in  lower  eukaryotes  [56]. via initial proton abstraction with the help of an acid–base catalyst followed by  a hydride‐ion removal from H2O2 and release of O2. GPx1‐4 and  GPx  6  are  selenoenzymes.  while  glutathione  peroxidase  is  effective  in  peroxide  degradation  at  concentrations lower than 100 μmol. Glutathione peroxidase (GPx)  Glutathione  peroxidise  (EC  1.  5.  CAT  is  found  in  many  types  of  cells  and  scavenges  H2O2 as its sole substrate.  However.54].  CI  is  reduced  back  the  the  ferric  enzyme  in  two  consecutive  one‐ electron  steps  via  CII.

  Thus.  without  G‐S‐T  activity  [53]. it binds to lipid peroxides. [137].  The  activity  of  GPx  is  affected  by  the  presence  of  another  important  antioxidant  enzyme.  HO  catalyzes  the  first.  glutathione  reductase.4  and  5. Instead.  Kinetic  analysis of  GPx  activity  indicated  a  tert‐uni  ping‐pong  mechanism similar to that described for other GSH peroxidases [176].  three  isoforms  (HO‐1.        GPxr + H2O2 + H+ → GPxo + H2O  GPxo + GSH → [GS‐SG] + H2O  [GS‐SG] + GSH → GPxr + GSSG + H+  (73)  (74)  (75)  Hall  et  al. while the free iron is promptly  sequestered  into  ferritin.  Non‐selenium dependent GPx also has the ability to reduce phospholipid hydroperoxides.14.  Under  physiological  conditions. implying the primary importance of GPx  in most tissues [8].6.  which  continuously  recycles  the  oxidised  glutathione  to  the  reduced  state.99.   Like  all  peroxidases.  an  enzyme  bound  to  GSH  may  attack  the  electrophilic  oxygen  of  the  peroxide  and  a  second  molecule of GSH may react in a non‐enzymatic fashion similar to the reaction with organic  nitrates.  thereby  generating  HO•.3)  can  be  legitimately  considered  a  part  of  the  phase  2  response  [40].    Lawrence  et  al.  or  by  another  enzyme  catalyzed  step  to  yield  the  glutathione  disulphide  (GSSG). Heme oxygenase (HO)  An  iron‐containing  decyclizing  oxygenase  (EC  1.     2GSH + H2O2 → GSSG + 2H2O  (72)  Kinetic behaviour of the overall reaction is discussed in detail in Ng et al. CO and free iron. This highly reactive species has the propensity to attack the heme porphyrin ring and  lead to irreversible inactivation [60].  GSH)  with  a  concomitant  reduction  of  H2O2  (for  more  detailed  mechanisms  of  peroxidase  action  see  4.  This  substrate  inactivation  leads  to  modification  of  the  heme prosthetic group and the formation of a verdohemoprotein as the final product. Biliverdin is subsequently  converted to bilirubin via the action of biliverdin reductase.  [74]  showed  that  an  epididymis‐specific.  they  mediate  the  one‐electron  oxidation  of  organic  compounds  (reduced  glutathione.  HO‐2  and  HO‐3)  have  been  identified.   5.  To  date.  [109]  described  that  non‐selenium  dependent  GPx  activity  contributes  to  glutathione‐S‐ transferase  B  (G‐S‐T)  activity  in  mechanisms  analogous  to  the  G‐S‐T  mechanism. The  existence of CIII as the peroxy iron prophyrin free radical resonance form can facilitate the  transfer  of  electrons  from  the  ferrous  state  to  an  extra  H2O2  molecule.4).1.  Virtually  all  known  peroxidases  are  inactivated  by  H2O2  and  other  hydroperoxides  at  relatively  high  concentrations  [83].  rate‐limiting  step  of  heme  degradation.  HO cleaves the α‐meso carbon bridge of b‐type heme molecules via oxidation  to yield equimolar quantities of biliverdin IXa.  secretory  GPx  has  very  little  activity  towards H2O2 or organic hydroperoxides.  HO  activity  is  highest  in  the  spleen  where  .Oxidative Processes and Antioxidative Metaloenzymes 43 value of CAT for H2O2 is higher than that of GPx.

 256(1‐2) 75‐78.  Oxidation  and  reduction  processes  are  inseparable.  but  they  also  serve  as  a  source  of  ROS.  In  fact  metalloenzymes  participate  significantly  in  the  antioxidant  protection  of  the  body  as  phase  I  and  II  antioxidants.  Activation  of  the  NADPH  oxidase  involves  the  small  GTP‐binding  protein  p21rac1.  Author details  Janka Vašková.   University of Pavol Jozef Šafárik in Košice. 28 65‐70. in a variety of cells and tissues. References  [1] Abo  A.  Dufau  E.  can  be  induced  by  a  variety  of  non‐heme  products  including  ultraviolet  irradiation. Conclusion  Oxidative  processes  are  essential  to  life.    HO‐1.  particularly  for  obtaining  the  energy  needed  for  various  metabolic  processes.  7.  353(6345)  668‐ 670. Journal of Experimental Botany 2002. Košice. Role of superoxide dismutases (SODs) in controlling  oxidative stress in plants.  Molina‐Heredia  FP  &  Bourgeois  D. they play an important role in the oxidoreduction processes and are constituents  of  various  proteins  and  enzymes.  Hall  A. Slovak Republic  Acknowledgement  This work was supported by Slovak Grant Agency for Science VEGA 1/1236/12.  Nivière  V.44 Antioxidant Enzyme senescent  erythrocytes  are  sequestered  and  destroyed  [143].  Pick  E. FEBS Letters 1989. Journal of Biology 2004.   .  6.  Totty  N. Ertuk N & Heath LS.  Given  that  transit  metals  readily  accept  or  give  away  electrons. Ladislav Vaško and Ivan Kron  Department of Medical and Clinical Biochemistry.  It  is  important  to  understand and study the antioxidant defence system of the organism so that one can  use  this knowledge to prevent and treat diseases in which it has been proven to participate. Faculty of Medicine.  Nature  1991.183].  endotoxins. 53(372) 1331‐1341.  and  oxidants  as  well  as  H2O2  [121.  heavy  metals.  Öğüş  H  &  Özer  N.  Royant  A.  Structure  of  superoxide  reductase  bound  to  ferrocyanide  and  active  site  expansion  upon  X‐ray‐ induced photo‐reduction. inducible oxidative stress represents part  of an adaptive cellular response to inflammation.  [2] Acker  H.  Teahan  CG  &  Segal  AW.  The  Mechanism  of  Inhibition  of  Human  Erythrocyte Catalase by Azide.  &  Sylvester  D.  Indications  to  an  NADPH  oxidase  as  a possible pO2 sensor in the rat carotid body.  Huber  J.  The  production  of  bilirubin/biliverdin  and  carbon  monoxide from heme catabolism is capable of exerting protection against toxic compounds  in the cell. Turk.  Balk  M. Structure 2004. 12(9) 1729–1740.  Indeed.  [4] Aksoy  Y.  [5] Alscher RG.  [3] Adam  V.

 Journal of Physiology 2004.  Biochimica  et  Biophysica Acta 2001.  Journal  of  Biological  Chemistry  1981. in leaves nad roots  of  wild‐type  and  catalase‐deficient  mutant  of  barley.  [9] Azevedo  RA.Oxidative Processes and Antioxidative Metaloenzymes 45 [6] Amici  A.  [12] Babior  BM.  American Journal of Physiology 1999. Hori M. Maryland.  WH  Freeman  and  Company.  Curnutte  JT  &  Okamura  N. pp.  Journal of Biological Chemistry 1995.  Oxygen  Radicals  and  Tissue Injury. senescence  and signal transduction in plant. 271(8) 4177‐4182.  Halliwell  (ed. Enzyme activities involved in  tryptophan  metabolism  along  the  kynurenine  pathway  in  rabbits.  5th  Edition. 1988.  [19] Bhattachrjee S. Fujii J. 17(7) 909‐919. Studies of hypervalent iron. 43‐48.  [16] Berlett  BS.  [14] Barrett  EG. NADPH oxidase: an update. Kahler DJ.  Implication  for  cytotoxicity. Ragazzi E. Tada M.  Response  of  antioxidant  enzymes  to  transfer from elevatde carbon dioxide to air and ozone fumigation. Science 2001.  [13] Barnett  RN.  Biochemistry. Biasiolo M. 555(Pt 3)  589‐606.  Physiologia  Plantarum  1991.  [7] Andrews  PC  &  Krinsky  NI.  Charge  migration  in  DNA: Ion‐gated transport.  Levine  RL. 93(5) 1464‐1476. Journal of Biological Chemistry 1989.). Blood 1999.  [18] Bertazzo A.  Cleveland  CL.  Tsai  L  &  Stadtman  ER.  [11] Babior BM. Seo HG.  Joy  A. 264(6) 3341‐3346.  In:  B. 1527(3) 167‐175. Suzuki K. Chandler PR.  producing  enzymically  active  hemi‐myeloperoxidase.  Levine  RL  &  Stadtman  ER. Costa CV & Allegri.  Alas  RM. Free radical research communications 1991. Kuzuya T.  Oberdörster  G  &  Finkelstein  JN.  Conversion  of  amino  acid  residues  in  proteins  and  amino  acid  homopolymers  to  carbonyl  derivatives  by  metal‐catalyzed  oxidation reactions.  104  280‐292.  Smith  RJ  &  Lea  PA. 2002.  Inactivation  of  glutathione  peroxidase  by  nitric  oxide. 270(36) 21035‐21039. 256(9) 4211‐4218.  [15] Berg  JM. New York.  Silica‐induced  chemokine  expression  in  alveolar  type  II  cells  is  mediated  by  THF‐α‐induced  oxidant  stress. 12‐ 13(Pt 2) 469‐477.  [8] Asahi M. Fujii S & Taniguchi N. 276(6 Pt 1) L979‐L988. 89 1113‐1121.  International Immunology 2005.  Comparison  of  the  effect  of  ozone  on  the  modification  of  amino  acid  residues  in  glutamine  synthetase  and  bovine  serum  albumin. 614.  Landman  U  &  Schuster  GB. Munn DH &  Mellor  AL. Current Science 2005.  [17] Berry  CE  &  Hare  JM. Reactive oxygen species and oxidative burst: roles in stress.  The  reductive  cleavage  of  myeloperoxidase  in  half. Bingaman A.  Xanthine  oxidoreductase  and  cardiovascular  disease:  molecular  mechanisms and pathophysiological implications.   [10] Baban B. 294(5542) 567‐571. 506. Hansen AM.  A minor  population  of  splenic  dendritic  cells  expressing  CD19  mediates  IDO‐dependent  T  cell  suppression  via  type  I IFN  signaling  following  B7  ligation. G.  Johnston  C. Upjohn Company.  Tymoczko  JL  &  Stryer  L.  .  The  Respiratory  Burst  Oxidase  of  the  Human  Neutrophil  Radicals  and  Tissue  Injury. pp.  [20] Bielski BH. Journal of Biological Chemistry 1996. Manlapat A.

 Merville MP & Bours V. 38(12) 3744‐3752. Accounts of Chemical Research 2008.  [30] Burner U.  Pinkham  JL. Kettle A.  Röllinghoff  M  &  Diefenbach  A.  [29] Buchert  F  &  Forreiter  C.  Arteriosclerosis.  [22] Bolwell GP & Woftastek P.  Journal  of  Biological  Chemistry  2004. 18(6) 1033‐1077.  [27] Brigelius‐Flohé R.  [36] Cleveland  CL. 59(1) 7‐11. 51  347‐349. Estimation of individual types of glutathione  peroxidases. 20(7) 1716‐1723.  Schuster  GB  &  Landman  U. and mutation of recombinant Streptomyces coelicolor NiSOD.  Joseph  J.  Arobo  SE. and Vascular Biology 2000. Mechanism for the generation of reactive oxygne species in  plant defense‐broad perspective.  [32] Carr  A.  McCall  MR  &  Frei  B. Biochemistry 1999. 29(3) 167‐174. 126(2) 460‐461. Koppenol WH & Obinger C. Yim YI.  [25] Breen AP & Murphy JA.  Examination  of  the  nickel  site  structure  and  reaction  mechanism  in  Streptomyces seoulensis superoxide dismutase.  Reactive  oxygen  and  reactive  nitrogen  intermadiates in innate and specific immunity. FEBS Letters 2010. 347 101–112. Piette J.  Dopamine  in  neurotoxicity  and  neuroprotection:  what  do  D2  receptors have to do with it? Trends in Neurosciences 2006. Barnett  RN.  41(8) 1075‐1083. Triggering and modulation of apoptosis by oxidative  stress.  reconstitution.  [31] Cadet J.  [23] Bonizzi G.  Begum  S. Davidson G.  Bongiorno  A.  [26] Brennan  JP. Journal of the American Chemical Society 2007.  Steric  Effects  on  Water  Accessability  Control  Sequence‐Selectivity  of  Radical  Cation  Reactions in DNA.  [24] Bozzi  Y  &  Borrelli  E.  Liu  C.  275(27)  20597‐ 20601. Douki T & Ravanat JL. Samali A & Orrenius S.  [33] Čeković  Ž.  Cabelli  DE  &  Maroney  MJ. Oxidatively generated damage to the guanine moiety of  DNA: Mechanistic aspects and formation in cell.  Singlet  oxygen  inhibits  ATPase  and  proton  translocation  activity of the thylakoid ATP synthase CF1CFo.  Wait R. Bose K.  Thrombosis.  Journal  of  Biological  Chemistry  2000. Furmüller PG. Free Radical Biology and  Medicine 1995.  Expression. 2000. Methods in Enzymology 2002. Tetrahedron 2003. Wingler R & Müller C. 59 8073‐8090. Kang SO.  12(1) 64‐76.  Bell  JR.  . Current Opinion in Immunology 2000. Cabelli DE  &  Maroney  MJ.  279(40) 41352‐41360.  Reactions  of  sigma‐carbon  radicals  generated  by  1. Journal of  the American Chemical Society 2004. Reactions of oxyl radicals with DNA.  [35] Choudhury SB.  Oxidation  of  LDL  by  myeloperoxidase  and  reactive  nitrogen  species:  reaction  pathways  and  antioxidant  protection.  Biochemical  Pharmacology.  Dunn  MJ  &  Eaton  P. 29(3‐4) 323‐333. 584(1)147‐152. Cell type‐specific role for reactive oxygen  species  in  nuclear  factor‐kappaB  activation  by  interleukin‐1.  [34] Chandra J. Physiological and Molecular Plant Pathology 1997. 129(27) 8408‐8409.5‐hydrogen  transfer  to  alkoxyl radicals.46 Antioxidant Enzyme [21] Bogdan  C. Mechanism of reaction  of  myeloperoxidase  with  nitrite. Sharma ML.Detection  and  mapping  of  widespread  intermolecular  protein  disulfide  formation  during  cardiac  oxidative  stress  using  proteomics  with  diagonal  electrophoresis. Lee JW.  [28] Bryngelson  PA. Free Radical Biology and Medicine 2000.

 34(3) 737‐742.  New  York.  [49] Duarte.  [46] Dizdaroglu  M.  [50] Dunford HB.  Photochemical and Photobiological Sciences 2004.  Free  radicals  in  the  physiological  control  of  cell  function. 32(45) 12105‐12111.  Tryptophan  metabolism  through  the  kynurenine  patway in rat brain and liver slices. pp. Kolling DR. 54(2) 195‐204.  Nucleic Acids Research 28(7): 1555‐1563. 98(6) 3404‐3409. Selenium has a protective role in  caspase‐3‐dependent  apoptosis  induced  by  H2O2  in  primary  cultured  pig  thyrocytes. Karlsson JO. Free Radical Biology and Medicine 2000.  Wiley‐VCH. Biochemical Journal 1986.  Holwitt  E  &  Dizdaroglu  M.. Dikanov SA. 1777(7‐8) 1001‐1019.  D..  guanine.  Monomeric  base  damage  products  from  adenine. Evans MD.5ʹ‐cyclopurine‐2ʹ‐deoxyribonucleoside  residues in DNA.   [41] Demelash A. Biochemistry 1995. Lhee S. Novel substrates of Escherichia coli  nth protein and its kinetics for excision of modified bases from DNA damaged by free  radicals.  European Journal of Endocrinology 2004.  M..  [38] Crofts AR.  Free‐radical  induced  formation  of  an  8. International Journal of Radiation Biology 1988.  in  Heme  Peroxidases. Nilsson M & Björkman U. Bauche C.  [47] Doetsch  PW.5ʹ‐cyclo‐2ʹ‐deoxyguanosine  moiety in deoxyribonucleic acid. Hicks RJ & Talalay P.  [48] Dröge.  Blakely  WF. 150(6) 841–849. and disease. 1999.  Martin  AM  &  Dizdaroglu  M.  The  Q‐cycle  reviewed:  How  well  does  a monomeric  mechanism  of  the  bc(1)  complex  account  for  the  function  of  dimeric  complex?  Biochimica  et  Biophysica  Acta 2008. Kuras R  &  Kuras  MG. Potency of Michael  reaction acceptors as inducers of enzymes that protect against carcinogenesis depends  on  their  reactivity  with  sulfhydryl  groups. formation of  its  compounds. Rodriguez H & Laval J. Bozak RE.  Physiological Reviews 82(1): 47‐95. Victoria D. 39(18) 5586‐5592. 6(8) 819‐822. Holland JT.  Substrate  specificity  of  the  Escherichia  coli  endonuclease III: excision of thymine‐ and cytosine‐derived lessions in DNA produced  by radiation‐generated free radicals.  In  vitro  DNA  synthesis  opposite  oxazolone  repair  of  this  DNA  damage  using  modified  oligonucleotides.  V.  and  thymine  induced  by  exposure  of  DNA  to  ultraviolet radiation. 238(1) 247‐254.  Effect  of  DNA conformation  on  the  hydroxyl  radical‐induced  formation  of  8.  Dang  Y  &  Brown  OR.  Laval  J  &  Boiteux  S. 17(10) 1195‐1214. 3(1) 17–25. Horseradish peroxidase I: Ligand binding.  [43] Dirksen  ML. FASEB Journal 2003.  Reactive  species  formed  on  proteins  exposed  to  singlet  oxygen.  Proceedings  of  the  National  Academy  of  Sciences of the United States of America 2001.Oxidative Processes and Antioxidative Metaloenzymes 47 [37] Cooke MS.  [45] Dizdaroglu M. 29(2) 191‐ 198.  .  Gasparutto.  W.  &  Cadet. Massiah MA.  J.  (2002).  mutation.  [42] Dinkova‐Kostova AT. Biochemistry 1993.  (2000). Dizdaroglu M & Lunec J.  Zasatawny  TH. Oxidative DNA damage: mechanisms.  [44] Dizdaroglu  M. 58‐91.  and  some  of  their  reaction. How does enzyme work? Effect of electron circuits on transition state acid  dissociation constant. redox potentials.  Jaquinod.  [39] Dale  WE.  [51] Dunford HB.  [40] Davies  MJ. Biochemistry 2000. Gilbreth R. Journal of Biological Inorganic Chemistry 2001.

  [57] Foster  MW  &  Stamler  JS. Spormann M & Wessely S.  Fioretti  MC.  Gizzi  S.  Hagen  WR  &  Van  Berkel  WJ.  Purification  and  characterization  of  an  intracellular  catalase‐peroxidase  from  Penicillium  simplicissimum. Kohler AK. Journal of Biological Chemistry  1999. 26(4) 190‐195.  Murine  plasmacytoid  dendritic  cells  initiate  the  immunosuppressive pathway of tryptophan catabolism in response to CD200 receptor  engagement.  Asselin‐Paturel  C. Amaudrut J. Fuctional expression of indoleamine  2. Grohmann U & Puccetti P. The active center of catalase.  [63] Gardner PR.  [62] García‐Arellano  H.13‐25.  [66] Giese B. FASEB Journal 1989.  Fedida  D  &  Spector  T.  [53] Fallarino  F. 1991. Journal of Biological Chemistry 2004.  Keskin  DB. 279(24) 25891‐25897. Plant Physiology and Biochemistry 2010. Dodia C. Journal of Molecular Biology 1985. Chen JW & Feinstein SI. Fioretti MC.  Marshall  B. In: E.  270(22)  13399‐13405. Biotechnology  and Bioengineering 2004.  New  insights  into  protein  S‐nitrosylation.  [60] Friedl  HP. Roseville.  [64] Garg  N  &  Manchanda  G.  Nature  2001.  Bianchi  R.  Mellor AL. 235(1‐2) 192‐198.  185(1) 21‐37.  [67] Gill SS & Tuteja N.  [56] Fita I & Rossmann MG.).  Mediator‐induced  activation  of  xanthine  oxidase in endothelial cells.  1989.3‐dioxygenase  by  murine  CD8  alpha(+)  dendritic  cells.  International  Immunology  2002. Biochemical Pharmacology 2006.J.  Ryan  US  &  Ward  PA. Direct observation of hole  transfer  through  DNA  by  hopping  between  adenine  bases  and  by  tunnelling. Phospholipid hydroperoxides  are substrates for non‐selenium glutathione peroxidase.  Buenrostro‐Gonzalez  E  &  Vazquez‐Duhalt  R. 412(6844) 318‐320.  Superoxide  radical  production  by  allopurinol and xanthine oxidase. Active Oxygen/Oxidative Stress and Plant Metabolism. Raineri I. 143 8‐96. American Society  of Plant Physiologists.   [54] Fallarino  F.  [55] Fisher AB. pp.  Mitochondrial  nitric  oxide  synthase. Mechanism of oxygen activation in different compartments.  [65] Ghafourifar  P  &  Cadenas  E. 274(30) 21326‐21334.  Grohmann  U  &  Puccetti  P. Superoxide radical and iron modulate  aconitase  activity  in  mammalian  cells.  Trends  in  Pharmacological Sciences 2005.  Bianchi  R. 173(6) 3748‐3754. 47(5) 412‐426. 71(12) 1747‐1752. Biology of disease: free radicals and tissue injury. Invest. Manevich Y.  ROS  generation  in  plants:  boon  or  bane?  Plant  Biosystems  2009.  . Pell.  Orabona  C. Lab. 48(12) 909‐930.  Biocatalytic  transformation of petroporphyrins by chemical modified cytochrome C.L. 14(1) 65‐68. 3(13) 2512‐2518.  Trinchieri  G. K.  Mitochondria  as  a model system.  [58] Fraaije  MW.  Vacca  C. 85(7) 790‐798.  Journal  of  Biological  Chemistry  1995.   [59] Freeman BA & Crapo JD.  Vacca  C.  Orth  P.  Steffen (ed. Epstein LB & White CW. European Journal of Biochemistry 1996.  Belladonna  ML.  Till  GO.48 Antioxidant Enzyme [52] Elstner EF.  [61] Galbusera  C. Journal of Immunology 2004. Reactive oxygen species and antioxidant machinery in abiotic stress  tolerance in crop plants.  Roubroeks  HP.

  [76] Hall L. 62(3) 213‐224.  [78] Hampton  MB. Frayne J & Jury JA.  [74] Gutteridge.  Gaut  JP. The majority of human glutathione  peroxidase type 5 (GPX5) transcripts are incorrectly spliced: implications for the role of  GPX5 in the male reproductive tract.  Clinical Chemistry 1995. Amino Acids 2003. Antunes F. Lambert JDC & Ogilby PR. Mitochondrial respiratory chain‐dependent generation  of superoxide anion and its release into the intermembrane space.  J.  Free  radicals  in  disease  processes:  a compilation  of  cause  and  consequences [Review].  Tyrosine  oxidation  products:  analysis  and  biological relevance.  [71] Guilivi  C  &  Davies  KJA.  dʹAvignon  A  &  Heinecke  JW. 276(26) 24129‐24136. Circulation Research 2000.  NADPH  oxidase:  role  in  cardiovascular  biology and disease. Tolerance. 25(3‐4) 227‐232.  [72] Guilivi  C  &  Davies  KJA. Williams K. Biochemical Journal 1998.  [82] Hazell  LJ.  [75] Gutteridge  JM.  Dityrosine:  a marker  for  oxidatively  modified  proteins  and  selective proteolysis. Methods in Enzymology 1994. Rettori D & Cadenas E.  Inside  the  neutrophil  phagosome:  oxidants.  Free  Radicals  in  Biology  and  Medicine.  Lipid  Peroxidation  andAntioxidants  as  Biomarkers  of  Tissue  Damage.  Journal  of  Biological  Chemistry  1997.  Traaseth  NJ  &  Davies  KJA. Benz B. Dopamine‐induced oxidative stress  in  neurons  with  glutathione  deficit:  implication  for  schizophrenia.  van  den  Berg  JJM  &  Stocker  R.  (1993).  the  major  product  of  L‐tyrosine  oxidation  by  the  myeloperoxidase‐H2O2‐chloride  system  of  phygocytes. myeloperoxidase. Blood 1998.  p‐ Hydroxyphenylacetaldehyde.  [77] Halliwell  B  &  Gutteridge  JMC.  . 1989.  [83] Hazen  SL. Measuring the lifetime of singlet oxygen in a single  cell:  addressing  the  issue  of  cell  viability. 233 363‐371. Voltage‐dependent anion channels  control  the  release  of  the  superoxide  anion  from  mitochondria  to  cytosol. 278(8) 5557‐5563. 353(Pt 2) 411‐416. Perry AC. 333(Pt 1) 5–9.  [80] Han D. p.  [79] Han D. Biochemical Journal 1994.  Hsu  FF. Williams E & Cadenas E.  Kettle  AJ  &  Winterbourn  CC. Parpura V. Cuénod M & Do KQ.  Photochemical  and  Photobiological  Sciences  2007.  covalently  modifies  epsilon‐ amino  groups  of  protein  lysine  residue. 41(12 Pt 2) 1819‐1828.Oxidative Processes and Antioxidative Metaloenzymes 49 [68] Griendling  K.  Mechanism  of  the  formation  and  proteolytic  release  of  H2O‐ induced  dityrosine  and  tyrosine  oxidation  products  in  hemoglobin  and  red  cells. DCs and tryptophan: much ado about  IDO. Fallarino F & Puccetti P.M. and bacterial killing.  [70] Grohmann U. 92(9) 3007‐3017. 24(5) 242‐248. 22‐85. 6(10) 1106‐1116.  New  York:  Oxford University Press.  Schizophrenia  Research 2003.  [81] Hatz S.  Journal of Biological Chemistry 2001.  Oxidation  of  low‐density  lipoprotein  by  hypochlorite  causes  aggregation  that  is  mediated  by  modification  of  lysine  residues  rather than lipid oxidation. 86(5) 494‐501. 302(1) 297‐304. Trends in Immunology 2003.  Journal  of  Biological Chemistry 2003. Canali R.  [69] Grima G.  Crowley  JR. Free radical research communications 19(3): 141‐158.  [73] Guilivi  C. Biochemical Journal  2001.  272(27)  16990‐16998.  Sorescu  D  &  Ushio‐Fukai  M.

  [96] Kawai  K. FEBS Letters 1995.  [90] Janković IA & Josimović LR. OʹDonnell VB. 6(5‐6) 504‐516. Wood JD. Kröncke KD & Sies H. Joseph J. Account of Chemical reserch 2010. Biochemical Society Transactions 2005.  41  835‐ 841. Rozhledy v Chirurgii 2010. Pharmacology and Toxicology 1990.  [93] Josimović  LR.  Molybdenum‐containing  hydroxylases.  [95] Kanvah S. Broughton JP.  Selenium  supplementation  in  patients with severe acute pancreatitis. Varón R.  274(20)  13908‐13914. Bixon M. 364(3) 279‐282. Hernández‐Ruiz J.  Peroxynitrite‐mediated  oxidative  protein  modifications.  [94] Jung  CH  &  Thomas  JA. Schuster GB. Disulphide formation on  mitochondrial protein thiols.  Kodera  H.  [85] Hiner AN. Journal of Physical Chemistry A 2002. Neutrofils convert tyrosyl residues in albumin to chlorotyrosine. 433(1) 107‐116. 1(2) 156‐159. Cánovas FG &  Acosta  M. 335(1) 61‐72.  S‐glutathiolated  hepatocyte  proteins  and  insulin  disulfides  as  substrates for reduction by glutaredoxin.  [97] Kettle AJ. and  glutathione.  Journal  of  Biological  Chemistry  1999. Ferraris AM & Gaetani GF. Carroll J. Oxidation  of DNA: Damage to Nucleobases. 33(6) 1390‐1393.  [100] Kocan  L. Heam‐dependent activation of guanylate cyclase and cyclic GMP formation  by  endogenous  nitric  oxide:  a unique  transduction  mechanism  for  transcellular  signaling. Superexchange mediated charge hopping in  DNA. Arnao MB.  [87] Hurd TR.  Sequence‐independent  and  rapid  long‐ range charge transfer through DNA. 43(2) 280‐287.  Guzy  J. Fearnley IM. Journal of Biological Inorganic Chemistry 2001. 271(4 Pt 2) H1626‐H1634. Dahm CC & Murphy MP.  Kinetics  and  stoichiometry. Cleveland CHL & Landman U. 66(9) 571‐580. 2(2) 88–94.  Simonová  J. Hughes EJ & Jones OT. Nature Chemistry 2009.  [92] Jortner J. 89(8) 518‐521. Rodríguez‐López J.  Biochimica  et  Biophysica  Acta  2003.  Archives  of  Biochemistry  and  Biophysics 2005.  Vasková  J.  Janković  IA  &  Jovanović  SV. 379(1) 103‐106. The nuclear encoded subunits  of  complex  I from  bovine  heart  mitochondria.  Osakada  Y  &  Majima  T.  .  [99] Klotz LO.  [91] Jones SA. Voityuk AA & Rosch N. Rolfo M.  [86] Hirst J. Shannon RJ & Walker JE. FEBS Letters  1996.  Radiation  induced  decomposition  of  tryptophan  in  the  presence  of  oxygen. protein disulfide isomerase. Filipovska A. 67(1) 1‐7. Archives of Biochemistry and Biophysics 1996. Journal  of the Serbian Chemical Society 2001. Expression of  phagocyte NADPH oxidase components in human endothelial cells.50 Antioxidant Enzyme [84] Hille  R.  [88] Ignarro LJ.  1604(3) 135‐150.  The  inactivation  of  horseradish  peroxidase  isoenzyme  A2  by  hydrogen  peroxide:  an  example  of  partial  resistance  due  to  the  formation  of  a stable  enzyme  intermediate.  Firment  J. Mechanisms of protection of catalase  by  NADPH. Costa NJ. American Journal  of Physiology 1996. Barnett RN. Autooxidation of tryptophan in aqueous solution. thioredoxin.  [89] Ischiropoulos  H  &  al‐Mehdi  AB. 106(33) 7599‐7606. Singlet oxygen‐induced signaling effects inmammalian  cells. Photochemical and Photobiological Sciences 2003.  [98] Kirkman HN.  Radiation  Physics  and  Chemistry  1993.

 231(2) 440‐446. Palin K & Dantzer R. 1298(2) 180‐190.  Behavior. 22(6) 337‐342.  37(11)  902–908.  Analytical Biochemistry 1995. Chait BT. 279(6) 4127‐4135.  GG.  Hepatic  cytosolic  non  selenium‐dependent  glutathione peroxidase activity: its nature and the effect of selenium deficiency.  [108] Lardinois  OM.   [105] Lahiri S & Acker H.  Journal  of  Experimental  Medicine  1996.  Parkhill  LK  &  Burk  RF. Characterization of  superoxide‐producing  sites  in  isolated  brain  mitochondria. Wang Q. Durandin A. Shafirovich  V.  [112] Leusen JH.3‐dioxygenase is  induced  in  the  mouse  brain  in  response  to  peripheral  administration  of  lipopolysaccharide  and  superantigen.   [109] Lawrence  RA. Geacintov NE & Sharirovich V. Diekmann D. Singlet oxygen production in photosystem  II and related protection mechanism. Vielhaber S.  [107] Lardinois  OM  &  Rouxhet  PG.  Determination  of  2‐oxohistidine  by  amino  acid  analysis. Sigmund K.  Getting  to  guanine:  mechanism  and  Dynamics  of  charge  separation  and  charge  recombination in DNA revisited. 272(7) 4323‐4326. Raytchev M.  [102] Krieger‐Liszkay A.  [106] Lander HM. Oxidation of guanine  in  G.  Disturbed  interaction  of  p21‐rac  with  mutated  p67‐phox  causes  chronic  granulomatous  disease.  and  GGG  sequence  contexts  by  aromatic  pyrenyl  radical  cations  and  carbonate radical anions: Relationship between kinetics and distribution of alkali‐labile  lesions.  [110] Lee YA. Structural basis for the nitric oxide‐p21(ras)  interaction. Journal  of Nutrition 1978. Zhu H. A molecular redox switch on p21(ras). Hall  A. Photosynthesis Research 2008.  184(4)  1243‐1249. Bimpong‐Buta NY.  [111] Lestage J.  [114] Lewisch  SA  &  Levine  RL.  [104] Kurtz  DM  Jr.  Journal  of  Biological  Chemistry 2004. Hajjar DP. Biochimica et Biophysica Acta 1996. Smith CI. Elger CE & Kunz WS.  Vaško  L. Daublain P. 112(6) 1834‐1844. Journal of Biological Chemistry 1997.  Krištofová  B. Dedon PC. Mirza UA. 108(6) 981‐987.  Microbial  detoxification  of  superoxide:  the  non‐heme  iron  reductive  paradigm  for  combating  oxidative  stress.  and  Immunity  2002. Photochemical and Photobiological Sciences 2008.  Verhoeven  AJ  &  Roos  D. Ahlin A.  Mestdagh  MM  &  Rouxhet  PG.  Biochimica  et  Biophysica  Acta 1996. Redox‐dependent binding of CO to heme protein controls P(O2)‐ sensitive chemoreceptor discharge of the rat carotid body. 7(5)  534‐539.  Vašková  J. 1295(2) 222‐238.  Accounts  of  Chemical  Research  2004.  Hoková  H.  Šimonová  J.  [113] Lewis FD. Campbell S & Quilliam  LA.Oxidative Processes and Antioxidative Metaloenzymes 51 [101] Kocan  L. Hempstead BL.  115(2) 169‐177. The enzyme indoleamine 2. Fiebig T. Fufezan C & Trebst A.  Peroxidatic  degradation  of  azide  by  catalase  and  irreversible enzyme inactivation. Journal of Physical Chemistry B 2008. Verrier D.  16(5)  596‐ 601. 98(1‐3) 551‐564.  Brain.  Majerník  M.  Firment J. Palmblad J. Hilarius PM. Anesteziologie a Intenzivní Medicína 2011.  Reversible  inhibition  and  irreversible  inactivation  of  catalase  in  presence  of  hydrogen  peroxide.  [103] Kudin AP. Respiratory Physiology 1999.  . de Klein A. Severe course of Still΄s disease with multiple organ failure with predominant  liver failure.

  Current  Drug  Metabolism  2004. Johnson T. Journal of Physical Chemistry A 2001. Schonhoff C. 492(1‐2) 29‐32. FEBS Letters 2001. Res.  Young  KL.  Free  Radical  Biology  and  Medicine  2011.  Superoxide  reductase from Desulfoarchus baarsii: reaction mechanism and role of glutamate 47 and  lysine 48 in catalysis. Lipid peroxidation‐DNA damage by malondialdehyde.  Houée‐Levin  C. Striatal damage and oxidative stress induced by  the  mitochondrial  toxin  malonate  are  reduced  in  clorgyline‐treated  rats  and  MAO‐A  deficient mice. S‐ Nitrosylation of mitochondrial caspases.  [128] Mellor AL.52 Antioxidant Enzyme [115] Li  XQ.  Uncoupling  proteins  and  the  control  of  mitochondrial  reactive  oxygen  species  production.  Advances  in  Experimental  Medicine  and Biology 2003.  Holschneider DP.  [125] Marnett LJ. Keskin D. Hypertension 1999. Journal of the American Chemical Society 1996. 527 27‐35. Nature Reviews Immunology 2004.  Altman  CS.  Sato  M. Journal of Cell Biology 2001. Induction of  strand  breaks  in  single‐ stranded  polyribonucleotides  and  DNA  by  photosensitisation:  one  electron  oxidised  nucleobase radicals as precursors.  Pocernich  CB. 15 323‐350.  [117] Lombard  M. Neurochemical Research 2004. 1201 84‐95.  Drake  J.  Nitric  oxide  and  macrophage  function.  Zhang  H  &  Yan  Y. Papeta N.  Xie  QW  &  Nathan  C.  Mu  Y. 34(4 Pt 1) 539‐545.  Qi  D‐h.  51(6)  1106‐1115. Fang K & Gaston B. Endothelial function in hypertension: the role  of superoxide anion.  Ishikawa  K.  Annual  Review of Immunology 1997.  IDO  expression  by  dendrtic  cells:  tolerance  and  tryptophan  catabolism.  ESR  studies  on  reaction  of  sacharide  the  free  radicals  generated  from  the  xanthine  oxidase/hypoxanthine system containing iron.  Tryptophan  catabolism  and  T  cell  responses.  Hexel  K.  Yan  G‐l. 14(15) 3731‐3740. 24(15) 2959‐2956. Mutat.  IL‐2  gene  expression  and  NK‐kappa  B  activation  through  CD28  requires  reactive  oxygen  production by 5‐lipoxygenase.  [118] Los  M. Chen K & Shih JC. Szibor M.  Baeuerle  PA.  Yang  T‐s  &  Shen  J‐C. 154(6) 1111‐1116.  Seif  I. 105 9563‐9567.   [127] Mellor  AL  &  Munn  DH. 40(16) 5032–5040. Ghafourifar P.  Butterfield  DA.  Touati  D.  424(1‐2) 83‐95.  [123] Mannick JB.   [116] Liu  SS. EMBO Journal 1995.  Mitochondrial  Q  cycle‐derived  superoxide  and  chemiosmotic  bioenergetics.  [122] Mailloux  RJ  &  Harper  ME. Botchway  S.   [120] MacMicking  J.  A superexchange‐mediated  sequential  hopping  theory  for  charge transfer in DNA. Jhaver K & Baban  B.  [126] McIntyre M.  Annals of the New York Academy of Sciences 2010. Munn D. 4(10) 762‐774.  Participation  of  altered  upstream  stimulatory  factor  in  the  induction  of  rat  heme  oxygenase‐1  by  cadmium. Bohr DF & Dominiczak AF. 118  10031‐10036.  Fontecave  M  &  Nivière  V. Chandler P.  [121] Maeshima  H.  [129] Melvin  T. Marshall B.  Schenk  H.  Dröge  W  &  Schulze‐Osthoff  K. 5(6) 535‐542. 29(4) 741‐746.  [119] Luo  G‐m. 1999.  Nucleic Acids Research 1996.  [130] Miller  RT.  Zheng  Y‐g.  Katagata  Y  &  Yoshida  T.  .  NOx  and  R‐NOx:  effects  on  drug  metabolism. Biochemistry 2001.  [124] Maragos  WF.  Parker  AW  &  OʹNeill P.

 51 51‐106.  Schafer  FQ.   [140] Nivière V.  [139] Nivière  V  &  Fontecave  M. Trends in Plant Science 2003. Larimer FW.  Chloroplast  redox  signals:  how  photosynthesis  controls  its  own  genes.  American Journal of Physiology ‐ Lung Cellular and Molecular Physiology 2000. Allen EE. Journal of Biological Chemistry 2004. Potential repair of free radical adducts of dGMP and dG by  series of reductants.  Buettner  GR  &  Rodgers  VG. 8(1) 33‐41.  Discovery  of  superoxide  reductase:  an  historical  perspective. Dufresne A. Brahamsha B. Touati D. Chemistry & Medicine 1985. 47(1) 71‐80. McCarren J. 424(6952) 1037‐1042. Partensky F.  Proceedings  of  the  National  Academy of Sciences of the United States of America 2002.  Adebayo  JO. Houveé‐Levin C.  Malomo  SO  &  Adediran  SA.  [143] Otterbein  LE  &  Choi  AM. Oxidative modifications to cellular components in  plants. Lamerdin J.  [137] Ng  CF. Guigliarelli B. 103(40) 14750‐14755.  Kasprzak  KS. International Journal of Radiation Biology  & Related Studies in Physics. Liu Y & Van Remmen H.  .  [133] Molina‐Heredia FP.  [146] Pospíšil  P.  [144] Palenik B. 43(3) 808–818. Lombard M.  [142] OʹNeill P & Chapmen PW. Paulsen I.  [145] Pfannschmidt  T. Tremey VF & Nivière  V. 7(9) 405‐410. Hauser L. Cancer Research 1991. Open Enzyme Inhibition Journal 2009. Nature 2003. Okayama 2008.  [138] Nicholls P. Jensen PE & Hansson A.  Mechanism‐ Based  Inhibition  of  Myeloperoxidase  by  Hydrogen  Peroxide:  Enhancement  of  Inactivation Rate by Organic Donor Substrates. Free Radical Research 2007.  Oxidative  stress. 279(47) 49064‐ 49073. 279(6)  L1029‐1037. Regala  W. Biochemistry 2004. Land M. Asso M.  [134] Moller IM.  Molecular  mechanisms  of  production  and  scavenging  of  reactive  oxygen  species by photosystem II. Webb EA & Waterbury J.  Detoxification  of  superoxide  without  production  of  H2O2:  antioxidant  acitivty  of  superoxide  reductase  complexed  with  ferrocyanide. Acta Med. Chain P.  [132] Miyazaki I & Asanuma M.   [136] Nackerdien  Z.  Nickel(II)‐  and  cobalt(II)‐dependent damage by hydrogen peroxide the DNA bases in isolated human  chromatin. Superoxide reductase from Desulfoarculus baarsii: identification of ptotonation steps  in the enzymatic mechanism. A pulse radiolytic study. Dopaminergic Neuron‐Specific Oxidative Stress Caused by  Dopamine Itself.    The  rate  of  cellular  hydrogen  peroxide removal shows dependency on GSH: mathematical insight into in vivo H2O2  and GPx concentrations.  Iniaghe  MO. 1878(1) 218‐231.  Halliwell  B  &  Dizdaroglu  M. 9(2) 119–123. 51(21) 5837‐5842.  Heme  oxygenase:  colors  of  defense  against  cellular  stress. Berthomieu C.  The genome of a motile marine Synechococcus. Enzymology and structure of catalases. Journal of Biological Inorganic Chemistry 2004.  [135] Muller FL. Weill CO.Oxidative Processes and Antioxidative Metaloenzymes 53 [131] Mittler  R. Complex III releases superoxide to both sides of  the inner mitochondrial membrane. 58 459‐481.  antioxidants  and  stress  tolerance. Biochimica et Biophysica Acta 2012. 2  28‐35.   [141] Olorunniji  FJ. Favaudon V & Houée‐Levin  C. 62(3) 141‐150. Annual Review of Plant Physiology 2007. Fita I & Loewen PC. Advances in  Inorganic Chemistry 2001.  Trends  in Plant Science  2002.  Rao  G. 41(11) 1201–1211.

 Journal of the Chemical Society. 27(3) 209‐ 218.  [161] Steenken S.  [154] Scott  NG.  Baynes  JW  &  Thorpe  SR. Lipids 1992.  Jenkins  AJ.  Lyons  TJ.  [149] Requena  JR.  Markey  SP  &  Heyes  MP.   Phenotypes  of  mice  lacking  extracellular  superoxide  dismutase  and  copper‐  and  zinc‐ containing  superoxide  dismutase.  Long‐range  charge  transfer  in  DNA:  Transient  structural  distortions  control the distance dependence.  [155] Sentman  ML.  Granström  M.  Gigney  BR  &  Dutton  PL.  Drug Metabolism Reviews 2000. Reactive oxygen‐mediated protein oxidation in aging and  disease.  [156] Shacter  E.3‐dioxygenase  in  brain  of  C57BL6  mice. Chemical Research in Toxicology 1997.  Greene  LA  &  Zecca  L.  Neuromelanin  biosynthesis  driven  by  excess  cytosolic  catecholamines  not  accumulated  by  synaptic  vesicles.  Effect  of  guanine  stacking  on  the  oxidation  of  8‐ oxoguanine in B‐DNA. Journal of the American Chemical Society 1998.  Larsen  KE. Biochemical Journal 1997.  Moser  CC.  Quantification  and  significance  of  protein  oxidation  in  biological  samples.  Aldehydic  lipid  peroxidation  products  derived from linoleic acid.  Ridge  N.  [158] Spiteller  P. 32(3‐4) 307‐326. Bhardwaj V.  Basu  S    &  Marklund  SL.  Fu  MX. Ankola DD. Journal of Immunology 2009. 113(3) 189‐207.54 Antioxidant Enzyme [147] Prat  F. Contemporary issues. Role of antioxidants  in  prophylaxis  and  therapy:  A  pharmaceutical  perspective.   [157] Sharp  RE.  Journal  of  Biological  Chemistry  2006.  Primary  Steps  in  the  Energy  Conversion Reactions of the Cytochrome bc1 Complex Qo Site.  Proceedings  of  . Accounts of Chemical Research 2000.  Reiner  J  &  Spiteller  G.The  Immunoregulatory  enzyme  IDO  paradoxically  drives  B  cell‐mediated  autoimmunity. Metal‐catalyzed oxidation of proteins.  Jakobson  H.  Prendergast  GC.  Muller  AJ  &  Mandik‐ Nayak  L.  281(11)  6904‐ 6909.  Kleinman  MH.  546(1) 151‐154.  Brain  Research  1991.  Edwards  RH. 31(3) 225‐233.   [153] Schuster  GB. 266(4) 2005‐2008. 30(11) 1191‐1212.  Ahmed  MU. Biochimica et Biophysica Acta 2001. Metals and lipid oxidation. 120 845‐846.  Bogulavsky  J. Faraday Transactions I 1987. Journal of Biological  Chemistry 1991. 10(5) 485‐494.  Free  Radical  Biology  and  Medicine 2001.  Turro  N.  Chronic  effects  of  gamma‐interferon  on  quinolinic  acid  and  indoleamine‐2. Addition‐elimination paths in electron‐transfer reactions between radicals  and molecules.  [148] Ratnam DV.  [151] Schafer FQ & Buettner GR. 83 113‐124. 182(12) 7509‐7517.  Journal of Bioenergetics  and Biomembranes 1999.  Krantz  D.  Journal  of  Controlled  Release 2006.  Pigott  E.  Quantification of malondialdehyde and 4‐hydroxynonenal adducts to lysine residues in  native and oxidized human low‐density lipoprotein.  Houk  KN  &  Foote  CS. 322(Pt 1)  317‐325. Sahana DK & Kumar MN.  [159] Stadtman ER & Berlett BS.  Karatekin  E.  [150] Saito  K.  [160] Stadtman ER & Oliver NC. 33(4) 253‐260.  [152] Schaich KM.  [162] Sulzer  D.  DuHadaway  J. Redox environment of the cell as viewed through the redox  state  of  the  glutathione  disulphide/glutathione  couple. 1531(3) 188‐208.  Behr  G.  Reaume  A.  Kern  W.

3‐ dioxygenase in alveolar interstitial cells of mouse lung by bacterial lipopolysaccharide.  Archives  of  Biochemistry  and  Biophysics 1995. Perrone GG & Dawes IW. Biochimica et Biophysica Acta 1985.  [176] Ursini  F. 92(10) 4158‐4163.  [175] Urade  Y. Trends in Cell Biology 2005.  Journal of Biological Chemistry 1983.  Relationship  between  interferon‐gamma.  indoleamine  2. 319(1) 1‐9.  Morimitsu  Y.  [171] Tien  M.  Peroxynitrite‐mediated  modification of proteins at physiological carbon dioxide concentration: pH dependence  of carbonyl formation.  [168] Thannickal  VJ  &  Fanburg  BL.  Journal  of  Biological  Chemistry 1998.  Yoshida  R. and tryptophan catabolism.  Journal  of  Biological  Chemistry 1994.  [173] Turrens JF.  Journal  of  Biological  Chemistry  1986. Proceedings of the  National Academy of Sciences of the United States of America 1999.  Yoshida  R.  [163] Takahashi  M  &  Asada  K. 552(Pt 2) 335‐344.  Superoxide  production  in  a aprotic  interior  of  chloroplast  thylakoids.    Correlated  small  polaron  hopping  transport  in  1Ddisordered  systems  u  at  high  temperatures:  a possible  charge  transport  mechanism  in DNA.  American  Journal  of  Physiology  ‐  Lung  Cellular  and  Molecular  Physiology  2000.  Kido  R  &  Hayaishi  O. 5(11) 2516‐2522. 839(1) 62‐70.  Levine  A.3‐dioxygenase  activity  in  interferon‐gamma  primed  mononuclear  phagocytes.  [172] Triberis  GP  &  Dimakogianni  M. Complex cellular responses to reactive oxygen  species.  Brisson  LF. Mitochondrial formation of reactive oxygen species. and methionine oxidation. 273(26) 16058‐16066.  Berlett  BS.3‐dioxygenase. 97(22)  11869‐ 11874.  Induction  of  indoleamine  2.  Formation  of  free  acrolein  and  its  conjugate  with  lysine  residues  in  oxidized  low  density  lipoproteins.  Nitric  oxide  inhibits  indoleamine  2. 258(10) 6621‐6627. 267(2) 714‐722.  Kanematsu  M.  261(8)  3648‐3653.  Kitamura  H  &  Hayaishi  O. 21(3) 035114. Archives of Biochemistry and Biophysics 1988.  [174] Uchida  K.  Chock  PB  &  Stadtman  ER. 96(14) 7809‐7814.  Osawa  T.  [167] Tenhaken  R.  [164] Takikawa  O.   [169] Thomas JA.  Proceedings  of  the  National  Academy  of  Sciences of the United States of America 1995.3‐ dioxygenase. FASEB Journal 1991.  [165] Taylor  MW  &  Feng  GS.  The  selenoenzyme  phospholipid  hydroperoxide  glutathione peroxidase. tyrosine nitration.  Function  of  the  oxidative  burst  in  hypersensitive  disease  resistance.  Levine  RL.  Noguchi  N  &  Niki  E.  Reactive  oxygen  species  in  cell  signaling. Perspectives: Protein sulfhydryls and their role in  the  antioxidant  function  of  protein  S‐thiolation. Journal of Physiology  2003. 15(6) 319‐326.  Mohr  D  &  Stocker  R.  Maiorino  M  &  Gregolin  C.  [166] Temple MD.  279(6)  L1005‐ 1028. Journal of Physics: Condensed Matter 2009.  Tryptophan  degradation  in  mice  initiated  by  indoleamine‐2.  [170] Thomas  SR. 269(10) 14457‐14464. Poland B & Honzatko R.  Acrolein  is  a product  of  lipid  peroxidation  reaction.Oxidative Processes and Antioxidative Metaloenzymes 55 the  National  Academy  of  Sciences  of  the  United  States  of America  2000.  Dixon  RA  &  Lamb  C.  .

  Journal  of  the American Chemical Society 1994. 112(36) 6986‐6994. Chemie potravin. Mazur M & Telser J.  Superoxide  anion  from  the  adventitia  of  the  rat  thoracic  aorta  inactivates  nitric  oxide. Jakopitsch C. 268(20) 14678‐14681.  conformational  features  and  thermal  decomposition  in  water.  PS1. 97(9) 4666‐4671.  Current  Medicinal Chemistry 2005. 3rd edition. Schumacker PT. Proceedings of the National Academy of Sciences of the United States  of America 2002.  Bernroitner  M. 1987.  Circulation Research 1998.  Oxidative  stress  resulting  from  ultraviolet  A irradiation  of  human  skin  fibroblasts  leads  to  a heme  oxygenase‐dependent  increase  in  ferritin.  Current  Opinion  in  Cellular  Host‐Pathogen  Interactions.  Journal of Biological Chemistry 1993.  Morris  H  &  Cronin  MT. 500(1) 74‐81.  [184] Vlasits  J.  Pattern  of  OH  radical  reaction  with  adenine  and  its  nucleosides and nucleotides. Proceedings of the National Academy of Sciences of the United States of  America 2000.  Firment  J  &  Vaško  L.  Van  Lier  JE.  [179] Vander Heiden MG. 602. Schwanninger M.  Du  Y.  Quinn  MT.  [178] Valko  M.  Jakopitsch  C. 91 1280‐ 1286.  . Journal of the American Chemical Society  1990.  Analysis  of  oxidative  cytosine  products  in  DNA  exposed  to  ionizing  radiation. 581(2) 320–324.56 Antioxidant Enzyme [177] Valko M. New York‐Philadelphia. Inzé D & Van Camp W. Ossis.  [182] Vieira  AJSC  &  Steenken  S. 429‐457. pp.  [185] Vlasits J.  Berger  M  &  Cadet  J. Li XX.  Zamocky  M. 2010. 12(10) 1161‐1208.  [189] Wagner  JR.  Kocan  L. Outer mitochondrial membrane permeability can regulate coupled respiration and  cell survival.  Thymidine  hydroperoxides:  structural  assignments.  Mechanisms  of  catalase  activity  of  heme  peroxidases. Moncol J.  [181] Velíšek J & Hajšlová J.  in  Enzymes. Characterisation of two types of isomeric OH adduct and  their unimolecular transformation reactions.  [186] Von  Sonntag  C.  Furtmüller  PG  &  Obinger  C. Hydrogen peroxide  oxidation  by  catalase‐peroxidase  follows  a  non‐scrambling  mechanism. Villarroel R.   [188] Wagner  JR.  FEBS  Letters  2007.  Pagano  PJ.. Cronin MTD. Tábor. Free radicals and  antioxidants  in  normal  physiological  functions  and  human  disease. Chandel NS. Holubar P & Obinger C. 2009.18.  toxicity  and  oxidative  stress. 82(7) 810‐818. Colombini M & Thompson  CB.  The  Chemical  Basis  of  Radiation  Biology. pp.  Taylor  and  Francis Ltd.  A Current  Opinion  in  Cell  Biology  Conference. 99(16) 10870‐10875.  [180] Vašková  J.  [183] Vile  GF  &  Tyrrell  RM.  Cayatte  AJ. Atichartpongkul S.  Brecher  P  &  Cohen  RA.  International  Journal of Biochemistry and Cell Biology 2007.  Metals.  Positive  correlation  of  selenium  supplementation  and  principal  antioxidant  defenders  at  sepsis. 39(1) 44‐84. Journal de Chimie Physique et de Physico‐Chimie Biologique 1994.  [190] Wang  HD.  Comprehensive analysis of gene expression in Nicotiana tabacum leaves acclimated to  oxidative stress. Van Montagu M.  [187] Vranová E. 116 2235‐2242. Leibfritz D.  Archives  of  Biochemistry  and  Biophysics 2010.

  Jacobson  MK  &  Jacobson  EL. Advanced glycation and products. Crystal structure  of  nickel‐containing  superoxide  dismutase  reveals  another  type  of  active  site.  Brembu  T  &  Bones  AM.  Proceedings  of  the  National  Academy  of  Sciences of the United States of America 1979.  Biochimica et Biophysica Acta 1992.  [201] Xia  XG.  [205] Youn HD.  Free  Radical  Biology  and  Medicine 1998.  Burke‐Wolin  TM  &  Mohazzab‐H  KM.  Proceedings of the National Academy of Sciences of the United States of America 2004.  Photochemical and Photobiological Sciences 2006. Lee JK.  Ferger  B  &  Schulz  JB. Yim YI. Biochemical Journal Letters 1987.  101(23) 8569‐8574. 1128(2‐3) 117‐131.  Dopamine  mediates  striatal  malonate  toxicity  via  dopamine  transporter‐dependent  generation  of  reactive  oxygen  species  and  D2  but  not  D1  receptor  activation. Kang SO & Djinovic Carugo K.  [202] Yamamoto  S.  Teismann  P.  Mauro  JK  &  Nørskov‐Lauritsen  L.  Common  phylogeny  of  catalase‐peroxidases  and  ascorbate peroxidases.  Schmidt  N. 248‐249.  Tokuda  M  &  Hayaishi  O.  [203] Yeh  AP.  Urade  Y. Lee JW.3‐ dioxygenase  in  mouse  lung  virus  infection.  Respiratory  Physiology 1999.  [198] Wolin  MS.  Janecek  Š  &  Koller  F. 245(1) 243‐250.  Structure  of  plant  and  fungal  peroxidases.  Jenney  FE  Jr.  Glucose  autooxidation  and  protein  modification. 256(1‐2) 169‐182.  Cloning  and  characterization  of  rac‐like  cDNAs  from Arabidopsis thaliana.   [199] Wondrak  GT.   [204] Yoshida  R.  [206] Zámocký  M. Lee JW.  Biochemistry  2000.  Endogenous  UVA‐photosensitizers:  mediatorors  of  skin  photodamage  and  novel  targets  for  skin  photoreception. Diabetes and Metabolism 2001. 76(8) 4084‐4086.  [196] Wolf  SP  &  Dean  RT.  Biochemical  Journal 1987.  [197] Wolf  SP  &  Dean  RT.  79(1)  63‐70. Yim YI.  cytotoxic  and  cytoprotective  mechanisms  of  nitric  oxide.  Archives  of  Biochemistry  and  Biophysics 1996.   . Yim HS. Gene 2000. Biochemical Society Transactions 1992.  [195] Winterbourn CC.  Mammalian  lipoxygenases:  molecular  structures  and  functions. 334(2) 341‐348. Plant Molecular Biology 1997. 14(1) 85‐90.  Induction  of  indoleamine  2.  Journal  of  Neurochemistry  2001.Oxidative Processes and Antioxidative Metaloenzymes 57 [191] Waultier JL & Guillausseau PJ.  Hu  Y. Free Radical Biology and  Medicine 1993. Youn H.  [200] Wuerges J. Superoxide as an intracellular radical sink.  Aldehydes  and  dicarbonyls  in  non‐enzymic  glycosylation  of  proteins. Hah YC & Kang SO. 39(10) 2499–2508. 20(2) 337‐340.  Roles  of  NAD(P)H  oxidases  and  reactive  oxygen  species  in  vascular  oxygen  sensing  mechanisms. their receptors and  diabetic angiopathy.  [193] Winge  P.  Adams  MW  &  Rees  DC. 25(4‐5) 434‐456.  [194] Wink  DA  &  Mitchell  JB. 5(2) 215‐237. Unique isoenzymes  of  superoxide  dismutase  in  Streptomyces  griseus. 35(4) 483‐495. 27 535‐542. 115(2) 229‐238.  Structure  of  the  superoxide  reductase  from  Pyrococcus  furiosus  in  the  oxidized  and  reduced  states.  Chemical  biology  of  nitric  oxide:  insight  into  regulatory.  [192] Welinder  KG.

  269(39): 24156‐24162.  Kuppusamy  P.  Journal  of  Biological  Chemistry  1994.58 Antioxidant Enzyme [207] Zámocký M.  Diz  DI  &  Robbins  ME.  Thompson‐Gorman  S  &  Lutty  GA.  [208] Zhao  W. Obinger C.  Oxidative  damage  pathways  in  relation  to  normal  tissue injury. Regelsberger G. Respiratory Physiology 1999.  . British Institute of Radiology 2007. 80(Spec 1) S23‐S31.   [210] Zweier  JL.  Broderick  R.  Oxygen  sensing  and  signaling:  impact  on  the  regulation  of  physiologically important genes. Jakopitsch C. FEBS Letters 2001.  Determination of the mechanism of free radical generation in human aortic endothelial  cells  exposed  to  anoxia  and  reoxygenation. 492(3) 177‐182. 115(2) 239‐247. The molecular peculiarities of  catalase‐peroxidases.  [209] Zhu  H  &  Bunn  HF.

. including the superoxide radical (O2¯). lipids.doi. besides their own specific effects (Rao. These advantages of plant tissue culture allow various opportunities for researcher to study the unique and complex responses of plants against environmental stresses (Sakthivelu et al. glutathione reductase. mitochondria. 2008. 2006). ROS can simply be described highly reactive and partially reduced-oxygen forms. heavy metals. which permits unrestricted use. provided the original work is properly cited. ascorbate peroxidase. peroxisomes.. When exposing of environmental stress factors. licensee InTech. plasma membrane. including proteins. Lokhande et al. Investigating these responses is difficult under field conditions. hydrogen peroxide (H2O2) like that. carbohydrates and DNA (Mittler et al. Gill and Tuteja. apoplast. nutritional disorders. distribution. and reproduction in any medium. APX. like induced oxidative stress by overproduction of reactive oxygen species (ROS).   . the ROS are scavenged by various antioxidant defense systems: both enzymatic antioxidant (superoxide dismutase.. Under steady-state conditions. Thus. hydroperoxyl radical (HO2). Oxidative stress is defined a serious imbalance between the production of ROS and antioxidant defense and this situation can cause damage to cellular macromolecules. including chloroplasts. ROS have inevitably been factors for aerobic life since the introduction of molecular oxygen (O2) into our atmosphere by O2-evolving photosynthetic organisms. Introduction Higher plants are sessile therefore are continuously exposed to different environmental stress factors. radiation without any protection. 2004.5772/48292 1. singlet oxygen (1O2). and cell-wall but also as a result of induced environmental stress factors. salinity. 2010). ROS. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons. such as drought. in the later stage. SOD. 2011). hydroxyl radical (OH). endoplasmic reticulum. Most of these stresses produce certain common effects on plants. CAT.0). leads to oxidative stress. are produced not only during metabolic pathway in several compartments of plants. plants have developed their own specific response(s) against each of these stresses as well as cross-stress response(s). but plant tissue culture techniques are performed under aseptic and controlled environmental conditions. ROS levels can dramatically increase and this increase.Chapter 3 Oxidative Stress Studies in Plant Tissue Culture Ayşe Şen Additional information is available at the end of the chapter http://dx. monodehydroascorbate       © 2012 Şen.

The first one of these techniques is used as a model to induce oxidative stress under controlled conditions via different stressor agents for researching in vitro screening in plants against abiotic stress. 2.e. Patada et al.. including mitochondria. 2008. POX and glutathione-S. Desikan et al. salinity. and genetic) studies (www. they become the sequential reduction of molecular oxygen. as a result of ROS-attack. Gill and Tuteja. 2010. ROS are highly reactive due to the presence of unpaired valence shell electrons and high concentration of ROS can result in non-controlled oxidation in cells. sugar. is also sometimes called Active Oxygen Species (AOS). chloroplast. endoplasmic reticulum. studying and observing morphological. Insufficient energy dissipations during the photosynthesis. shoot tip. Cui et al. . Desikan et al.kitchenculturekit. such as superoxide anion (O2·-). perhydroxyl radical (HO2·) and hydrogen peroxide (H2O2). and herbicides (Desikan et al. 2005).is the term used to describe highly reactive and partially reduced-oxygen forms (Desikan et al. during normal metabolic processes and due to induction of environmental perturbations. GST) and non-enzymatic (ascorbate. mature embryo. carotenoids.. the effects of induced-oxidative stress on antioxidant defense system in plant tissue culture and antioxidant defense systems of in vitro selected-plant against abiotic stresses.. suspension cultures and callus cultures) and organized tissue (i.transferase.. cellular compartments. 2005.e. plant breeding. 2005). plasma membrane and apoplast. Singlet oxygen (1O2). or Reactive Oxygen Intermediates (ROI). how to maintain ROS homeostasis in plants. 2008. protein. ROS include a wide range of oxygen-radicals. peroxisomes. glycine betain. whole plant) levels (Sivritepe et al. guaiacol peroxidase. DHAR. which is defined as oxidative stress. This is the major formation mechanism of 1O2 in plant cells. 2010). Ahmad et al. glutathione.. phenolic compounds. hydroxyl radical (OH·).. 2001). the chlorophylls are excited. 2005). Oxidative stress and Reactive Oxygen Species (ROS) Reactive Oxygen Species (ROS). physiological and biochemical changes in both unorganized cellular (i.. The purpose of this study is to compile the recent studies about ROS and oxidative stress. dehydroascorbate reductase. Additionally. proline. axillary shoot. 2010. glutathione peroxidase. such as drought. MDHAR. GPX. 2012). another form of ROS. Two of these application areas are important to study ROS homeostasis in plants. including DNA. radiation. membrane lipids may damage (Cassells and Curry.. cytoplasm. plant tissue culture. heavy metals. ROS are produced in many ways in several cellular compartments. and polyamines) defense systems (Foyer and Noctor. physiological. Plant tissue culture techniques are used to grow plants under aseptic and controlled environment for the purpose of both commercial (like mass production) and scientific (like germplasm preservation..60 Antioxidant Enzyme reductase. plant tissue culture techniques also allow opportunities for the researcher to improve plants against abiotic stress factors with the in vitro selection method (Jain. 2005. Shehab et al. or Reactive Oxygen Derivatives (ROD). can be produced by excited-chlorophyll formation in the photosystem II (PSII) reaction center and in the antennae systems.

2011). H2O2 is formed in the peroxisomes as part of photorespiratory. HO2 is formed from O2¯ by protonation in aqueous solutions. Yang et al. further reduction of H2O2 take place OH. Gill and Tuteja.. OH· is extremely reactive and will potentially react with all biological molecules. 2010). 1O2 has powerful damaging effect on the whole photosynthetic machinery..and OH·. 2006).can also reduce to H2O2 by SOD (2). afterwards increase generation of O2·. 2010).. Xanthine oxidase generates O2·. If productions of hydroxyl radicals are not eliminated by any enzymatic and non-enzymatic defense mechanisms. Azevedo et al. H2O2 is produced as a result of dismutation reaction of O2·. Gill and Tuteja. This reaction mostly catalyzed by SOD (Arora et al. 2002. including chloroplast membrane lipids. By means of transition metals. H2O2 is not a free radical. Shehab et al..and the other ROS (Arora et al. usually generate with the single electron reduction of O2. and an increasing production of O2·. such as Fe and Cu. 2005. and lipids. Hossain et al. Gill and Tuteja. and also produced from βoxidation of fatty acids as a by-product. proteins. O2·-. As a result of the measurement of ROS using spectrophotometric. Chl triplet state can react with 3O2 to give up the very reactive 1O2 (Arora et al. The reaction of O2·. proteins and nucleic acids. fluorescent dye probe and electron spin resonance (ESR) methods showed that various abiotic stress factors induced ROS formation in a wide range of plant species under in vitro conditions (Mohamed and Aly. The generation of O2·. the other major ROS (H2O2 and O2·-) producing sites in cells (Reddy and Raghavendra. 2005.. The primary means of defense within the chloroplast are the carotenolds (CARs) and a-tocopherol (vitamin E). such as DNA. complex I. ubiquinone.. 1985). like paraquat. 2002). 4). 2000). 2010). but is participates as an oxidant or a reductant in several cellular metabolic pathways (Reddy and Raghavendra. 2009. El-Beltagi et al. Gill and Tuteja. Gallego et al. 2010. HO2 can cross biological membranes and subtract hydrogen atoms from polyunsaturated fatty acids (PUFAs) and lipid hydroperoxides. thus initiating lipid autooxidation (Halliwell and Gutteridge. which are located within the thylakold membranes. caused certain herbicides. The major site of O2·. 2002. which is known photosynthetic inhibitors. which are mentioned below as Haber-Weiss/Fenton Reaction (3. They are a quencher against damages of 1O2 (Knox and Dodge. Gill and Tuteja. Paraquat (also called methyl violeng) prevents the transfer of electrons from ferredoxin (Fd) in PSI. O 2    Fe 3  Fe 2  1 O 2 SOD 2O 2  2H   H 2O 2  O 2  (1) (2) . Reddy and Raghavendra. 2010).. Additionally.with the transfer of electrons from molecular oxygen (Peixoto et al. 2006... 2010. and reduced-form of Fe+2.Oxidative Stress Studies in Plant Tissue Culture 61 which then can lead the formation of chlorophyll (Chl) triplet state. 2007. Chakrabarty et al. Reddy and Raghavendra. Reddy and Raghavendra.with Fe+3 may become 1O2 (1). O2·.. (2006) and Helaley and El-Hosieny (2011) reported that carotenoid contents increase under salinity stress in various plant species. 2010). 2006.may lead to formation of OH· and 1O2. It is also clear that environmental stress induced the production of O2·. 2006.during the catabolism of purines in the peroxisomes. and complex III in mitochondrial electron transfer chain (ETC). which is generally known as the first ROS to be generated. 2006. overproduction of its ultimately leads to cell death (Desikan et al.production is in the photosystem I (PSI) by Mehler Reaction. 2005.

2005). Ahmad et al. 2010.. Shri et al. Cui et al. including plants. Gill and Tuteja. 2007. such as chromosomal rearrangement... The reactions of MDA with thiobarbituric acid (TBA) produces color product. Many researchers reported that MDA content increased under several abiotic stress factors.12) activity. (2010) reported that LOX activities and MDA contents increased in Euphorbia millii and all rice varieties under hypehydric conditions and PEG induced drought stress in tissue culture. and aggregation of cross linked reaction products occur in plants as consequence of protein oxidations induced by ROS or by-products of oxidative stress. with the further degradation reactions of these reactions produce free radicals and thus initiating the chain reactions of LPO (Blokhina et.. resulting in ROS-attack. strand breaks. such as lipid membranes. (2006) and Basu et al. 2005. Dewir et al. (2010) reported that an increasing ratio of protein oxidations were measured in all rice varieties induced drought conditions in tissue culture. Another way to detect LPO is determination of Lipoxygenase (LOX. 2001. Various mechanisms can cause protein oxidation. ROS-induced genotoxic damage can induce structural changes in DNA.. which may damage several cellular macromolecules. These reactions are mostly irreversible (Ahmad et al. Shehab et al. prymidine dimers. Another result of ROS-attack in cells is an increase in protein oxidations. 2010).11. 2007). Ghanaya et al.. 2001.. OH  HO   Fenton Reaction  (4) As I mentioned above. which were induced in vitro conditions (Gallego et al. 2010. which is called thiobarbituric acid reactive substances (TBARS). mutations and other lethal genetic effects (Cassells and Curry. 2009. cross-links and base modifications. such as the formation of disulfide cross-links and glycoxidation adducts nitration of tyrosine residues. Site specific amino acid modifications. base deletions. 2003). It is also clear that all LPO-products are highly cytotoxic and as a result of reaction in biological molecules. including proteins.. 2008. Gill and Tuteja. 2009. 2008. proteins and DNA (Cassells and Curry. and carbonylation of specific amino acid residues (Oracz et al. fragmentation of the peptide chain. The spectrophotometric measurement of TBARS or MDA generally used as oxidative stress biomarker and also to assess the degree of LPO.13. al. One of them is malondialdehyde (MDA).. Erturk et al. 2011. Sivritepe et al.. El-Beltagi et al. 2011). respectively. 2010). The peroxidation of membrane lipids both cellular and organelles are known as the most damaging factors in all living organisms.. EC 1. When DNA-lesions are endogenously generated . 2008. an overproduction of ROS can result in non-controlled oxidation in cells... OH  OH   O 2 and H2O2 · OH  H2O  O2    H H 2 O 2  Fe 2 Cu   Fe 3 Cu 2  Haber  Wiess Reaction  (3)     . The spectrophotometric measurement of protein carbonyl with dinitrophenylhydrazine (DNPH) method is widely used marker for detection of protein oxidation in biological organisms. 2010).62 Antioxidant Enzyme O 2    H 2 O 2 . LOX catalyze the hydroperoxidation of PUFAs. Basu et al.. Azevedo et al. and DNA damage to them (Gill and Tuteja. As a result of lipid peroxidation (LPO) some products are formed by PUFAs.. Desikan et al...

2011). Israr et al. DHAR and GR and non-enzymatic antioxidant defense systems. 2008. Xu et al. Additionally. 2011. salinity. and drought (Gallego et H2O2 and O2. Del Rio and Puppo. Dasgupta et al. POX. Superoxide dismutase (SOD. 2010. 2007. Mn-SOD (localized in mitochondria). 2009) published in recent years. 2006. 2010. low concentrations of ROS are key factors to maintain intercellular signal transductions in plants. spontaneous mutations are one of the key factors of plant breeding. The Mn-SOD is resistant to both inhibitors.. and cytosol). as a metalloenzyme. APX.. 3. also has a great potential creating variability in the plant genome by activating transposons. 2008.. 2010). 2011. Cr. Sen and Alikamanoglu. 2005. Shehab et al.. Gill and Tuteja. There have been many reports of the increased activities of SOD under abiotic stresses induced with tissue culture techniques in a wide range of plant species. Cd.. and Cu/Zn-SOD (localized in chloroplasts. MDHAR. Gaspar et al. Yang et al. 2004. Dewir et al...1. Erturk et al. and base mutation and these situations are one of the main reasons of spontaneous mutations in cells (Cassells and Curry.. 2010. El-Beltagi et al. herbicides. SODs are classified into three types based on their metal cofactor: Fe-SOD (localized in chloroplasts). Fe-deficiency stress reduced activity of SOD (Lombardi et al. 2008. 2011. gamma radiation.1. Their productions are controlled by various enzymatic and non-enzymatic antioxidant defense systems. Enzymatic antioxidants 3. Rao et al. SOD.. which catalyzes O2·.. 2008. 3. and Cu. 2006. inducing chromosome breakage/rearrangement. 2003). As I mentioned below. Gill and Tuteja.. glutathione. 2011) about this subjects books (Smirnoff.. 2012) on the other hand. peroxisomes. Antioxidant defence system ROS are generated in plant cells by normal cellular metabolism or due to unfavorable environmental conditions such as drought. Karuppanapandian et al. 2011.. salinity.. Gupta and Prasad. there is an excellent review (Ahmad et al. sugar. including CAT. 2008. 2010. 2001. 2002). drought. Enzymatic antioxidant defense systems. phenolic compounds. nutrient deficiency. or radiation.. glycine betain.Oxidative Stress Studies in Plant Tissue Culture 63 mostly via ROS. as well as effects of damaging which were referred above.1) Superoxide dismutase. and polyamines (Ahmad et al. proline. Saher et al. Gill and Tuteja. Further information about ROS. 2008. Gill and Tuteja. EC 1. heavy metals. Karuppanapandian et al. Oxidative stress. 2010. The activity of SOD isozymes can be detected by negative staining and can be identified on the basis of their sensitivity to KCN and H2O2... it is called spontaneous DNA damage (Ahmad et al.. including heavy metals. Sivritepe et al.. 2006. Cu/Zn-SOD is sensitive to both inhibitors whereas. 2011. such as Al.1. Patada et al..1.. is the first enzyme of the detoxification processes. Lokhande et al. carotenoids. Karuppanapandian et al.15. 2010. Helaly and El-Hosieny . 2002... Fe-SOD is resistant to KCN and sensitive to H2O2 (Ahmad et al. 2011). Advanced-antioxidant defense . hyperhydricity. including ascorbate.

1.. 60. 2006 Niknam et al. CAT is also important in the removal of H2O2 generated in peroxisomes during the β-oxidation of fatty acids. respectively (Rahnama and Ebrahimzadeh. 2006.7.. Karuppanapandian et al. Additionally..11.. Sen and Alikamanoglu.. 2008. 3. 2004). 2005. 2008.64 Antioxidant Enzyme systems play an important role in plants not only to tolerate environmental stress but also to improve plants against these stresses. EC 1. Sivritepe et al. 2005. rootstock MM 106. and CAT-3) isoenzyme bands were observed on the gels. POX prefers aromatic electron donors such as guaiacol and pyragallol to catalyze H2O2. 2010. Zamora et al.. 2004).3. Shri et al. In Malus domestica Borkh.7) POX is a heme-containing enzyme. Gill and Tuteja. Erturk et al.. photorespiration. 2011. 2004) using in vitro selection method. Catalase (CAT. 2011). Chakrabarty et al. (2006) detected that Mn-SOD and Cu/Zn-SOD isoenzymes seem to play a major role in response to hyperhydricity. 2006)... 2011. CAT activities were detected with native–PAGE analysis besides spectrophotometric measurements. Two of them (CAT-1 and CAT-3) were strongly induced in hyperhydric apple leaves compared healthy leaves. 2003. 2012) in contrast. As a result of native polyacrylamide gel electrophoresis (native–PAGE). (2005) and Dewir et al.1. Shehab et al.. 2011. and 80 mM).. CAT activities also induced in Medicago sative clones. Additionally.. (2005) reported that as a result of native–PAGE analysis. Guaiacol Peroxidase (POX. EC 1.6) CAT is a tetrameric heme-containing enzyme that catalyzes dismutation reactions of H2O2 into H2O and O2 and is indispensable for ROS detoxification during stress conditions.1. drought. Chen et al.. Sen and Alikamanoglu. 2010. and purine catabolism (Ahmad et al. under PEG-treatment (Safarnejad. Helaly and El-Hosieny . CAT-2. 3. salinity. 2010. 2010.. 2008. Pehlivan and Flamura-85 wheat varieties tissue cultures. 2011).. NaCl and KCl treatment induced Mn-SOD isoenzyme form in leaves (Molassiotis et al. and S-(2-aminoethy)-cysteine AEC (Kim et al. Patade et al. (2009) observed that during the As-stress Cu/Zn-SOD isoenzyme band induced. 2006... Rahnama and Ebrahimzadeh (2006) and Roy et al. Fe-deficiency stress reduced activity of CAT (Lombardi et al. Various abiotic stresses induced CAT activities under in vitro conditions in different plants. El-Beltagi et al. including hyperhydricity..11.. and many researchers reported that excess POX . 2007. Chakrabarty et al... which were improved with in vitro selection method... and gamma radiation (Saher et al. Dasgupta et al. respectively. one and two CAT isoenzyme bands were visualized on the native-PAGE in Tekirdag. three CAT (CAT-1. 2011. 2008). 2007. Rahnama and Ebrahimzadeh.2. like CAT. Kumar et al. (2011) reported that under NaCl stress conditions one. Yang et al. Hossain et al.. Chakrabarty et al. Enhanced activities of SOD were observed in various plants to improve tolerance against salinity (Hossain et al.. Dewir et al. (2006) also reported that against salinity and gamma radiation Mn-SOD and Cu/Zn-SOD seem to play a major role in the potato and Vigna radiate calli. Helaly and El-Hosieny. Mohamed and Aly.. 2004. 2005. NaCl stress induced new SOD isoenzyme bands in Agria and Kennebec potato cultivar (50 mM) and in Jatropha curcas callus (40.

. The cycle involves the antioxidant metabolites: ascorbate.. EC 1. vacuole. There have been many reports of the changes in POX isoenzymes depending considerably upon plant species and abiotic stresses under tissue culture conditions. 2004).1) and Glutathione reductase (GR.8. Sivritepe et al.1. 2004. five. CAB-6P rootstock leaves. Dehydroascorbate reductase (DHAR..4. 2011. EC 1. Chakrabarty et al. 2006). 2006). Dewir et al. Niknam et al. A new POX isoenzyme band (Rf 0... MDHAR. POX-3 isoenzyme band appeared under different concentrations of NaCl and CaCl2. and 60 mM). 2010. Helaly and El-Hosieny. 2008). NaCl stress stimulated new POX isoenzyme band in Agria and Kennebec potato cultivar (50 mM) and in Jatropha curcas callus (40. (2006) reported that in Centaurea regusina L..11. (2011) reported that under NaCl stress conditions two. On the other hand.6.5. Radić et al.. sometimes called Halliwell-Asada Cycle. mild Fe deficiency was caused to disappearance of one POX band with Rf value 0. involving APX. which was improved using in vitro selection method (Hossain et al. is another metabolic pathway that detoxifies H2O2. EC 1. 2010)... DHAR and GR. Dasgupta et al.. Kumar et al. Erturk et al. chloroplasts and peroxisomes in plants and it may have a more crucial role in the management of ROS during stress (Noctor and Foyer. 2010). 2008. 1998). Sen and Alikamanoglu. 2008.. glutathione and NADPH and the enzymes linking these metabolites.4). two and five POX isoenzyme bands were detected on the native-PAGE in Tekirdag. five and three POX isoenzyme bands were visualized. at the highest Zn concentration induced new POX isoenzyme bands in Jatropha curcas cotyledons (POX IV). Rahnama and Ebrahimzadeh. 2005. Pehlivan and Flamura-85 wheat varieties tissue cultures. 2010).2)) The Ascorbate-Glutathione Cycle. hypocotyls and radicles under Pb-induced oxidative stress (Jiang et al. Additionally. and cell wall as well as in extracellular space (Gill and Tuteja. hypocotyls (POX V) and radicles (POX IV) (Luo et al. 2011).. four and five POX isoenzymes were detected in Luffa cylindrica cotyledons. In Malus domestica Borkh. 2008. and both 30 and 60 mM CaCl2 concentrations (Chatzissavvidis et al.. 2006. rootstock MM 106. 2006. EC 1.. Similar results were obtained under Cd-stress in Glycyrrhiza uralensis cotyledons. 2010.4. 2005. hypocotyls and radicles. Monodehydroascorbate reductase (MDHAR. H2O2 . In the first step of this pathway. 3.34) was also detected in Chrysanthemum salt-tolerant strain.1). NaCl and KCl treatment induced new POX isoenzyme form in leaves and stems (Molassiotis et al. 2007. Sen and Alikamanoglu... respectively (Zheng et al.Oxidative Stress Studies in Plant Tissue Culture 65 activities were measured in a wide range of plant varieties under abiotic stress conditions induced with in vitro culture techniques (Saher et al.1. but POX-4 isoenzyme band were detected highest in NaCl concentration (60 mM). Zamora et al.. four. Karuppanapandian et al.. 2011). 2005. Halliwell-Asada Cycles’ Enzymes (Ascorbate peroxidase (APX.. Kumar et al. In Prunus cerasus cv.85 (Mohamed and Aly. mitochondria. POX also decomposes indole-3acetic acid (IAA) and has a role in the biosynthesis of lignin and defense against biotic stresses by consuming H2O2 in the cytosol.5. 2008).6. This is located in the cytosol. respectively (Rahnama and Ebrahimzadeh. . all NaCl and mannitol treatments induced POX-3 and POX-4 isoenzymes but POX-9 appeared only in response to high NaCl concentration. After the electrophoretic analysis.

Kim et al.. A further study by hyperhydration. Shehab et al. 2004. (2005) reported that APX. Chakrabarty et al. 2011) were observed in various plants improved tolerance against abiotic stresses with in vitro selection method... (2004) reported that Fe-deficiency stress reduced activity of APX in Borage officinalis tissue culture. and dehydroascorbate (DHA).. Hossain et al. 2011). Millar et al. besides this situation GPX also has more crucial role for lipid peroxidation process. Karuppanapandian et al. 2007. Generally known that APX has a higher affinity for H2O2 (μM range) than CAT and POX (mM range) and it may have a more crucial role in the management of ROS during stress (Gill and Tuteja.. in rice tissue culture... APX-4 and APX-5) only appeared in hyperhydric apple leaves. Erturk et al. 2008.. 2011). chloroplast. .. Finally GSSG is reduced to GSH by glutathione reductase (GR) using NADPH as electron donor (Noctor and Foyer. 1998. 2011.. for APX. Three of them (APX-1.1. DHAR and GR) in Dianthus caryophyllus. 2004. Hossain et al. 2009). After. (2011) observed that under NaCl-induced oxidative stress conditions. respectively (Shri et al. Enhanced expression of Halliwell-Asada Cycles’ enzymes in plants has been demonstrated during different stress conditions.. New APX and GR isoenzyme bands were also induced during the As-stress both shoots and roots. (2007) reported that different types of herbicides (paraquat.. DHA is reduced to ascorbate by dehydroascorbate reductase (DHAR) using of glutathione (GSH) as the electron donor. 2010). 2003. and therefore helps plant cells from oxidative stress (Gill and Tuteja. 2006. (2005) reported that after the native–PAGE analysis. MDHAR is a flavin adenin dinucleotide (FAD) enzyme which uses NAD(P)H directly to recycle ascorbate.... (2004) reported that hyperhydric stress increased Halliwell-Asada Cycle’s enzyme activities (APX. EC 1. five APX isoenzyme bands were observed on the gels in hyperhydric apple leaves. 2010. ElBeltagi et al.. Peixoto et al. Chakrabarty et al. Sivritepe et al. Helaly and El-Hosieny. MDHAR. 2010. and DHAR (Kopyra and Gwozdz. Bittsanszky et al. MDHAR and GR activities increased but DHAR activity decreased in apple. The oxidized ascorbate (MDHA) is regenerated by MDHAR.11. such as APX. Mohamed and Aly. 2006. 2010.. Glutathione Peroxidases (GPX.4-D and dicamba) induced GR activities in potato tuber calli. In increase activities of some enzymes belonging to Halliwell-Asada Cycle’s. 2007. 3. Ahmad et al.. Hyperhydric stress increased GPX activity in Prunus avium and apple. Zamora et al. Karuppanapandian et al. Lokhande et al.5.9) GPXs are a large family of diverse isozymes that use GSH to reduce H2O2. and only roots. 2. 2008. GR. for GR. Helaly and El-Hosieny. Chakrabarty et al. (2003) reported that GPX includes a family of seven related proteins in cytosol.. 2010. 2005). In a wide range of plant species were observed increase in GR and APX activities under different abiotic stress conditions-induced with tissue culture (Israr et al.1..66 Antioxidant Enzyme is reduced to H2O and monodehydroascorbate (MDHA) by APX using ascorbate as the electron donor. 2008. As a result of this reaction oxidized glutathione (GSSG) occur. APX enzyme activities increased but CAT enzyme activities decreased in Sesuvium portulacastrum tissue cultures. respectively (Franck et al. 2011). mitochondria and endoplasmic reticulum. Saher et al... Gill and Tuteja.

Glutathione. O2·. tyrosine metabolism. which were improved using in vitro selection technique (Bittsanszky et al.5. respectively. vacuolar sequestration of anthocyanin. absorb light at wavelength between 400 and 550 nm and transfer it to the Chl. hormone homeostasis. 3. Shehab et al..2.. they act as energetic antenna. proline. Additionally. GSTs are generally cytoplasmic proteins. conjugation of metabolites. including regulation of sulfate transport. increasing ascorbate and glutathione contents were observed in salt tolerant Chrysanthemum morifolium strain and paraquat-tolerant poplar clones. 2008). signal transduction.. Carotenoids have several major functions such as preventing membranes for lipid peroxidation. Enhanced activities of GST in potato tuber callus was demonstrated during 2. carotenoids. Non-enzymatic antioxidants Apart from the enzymatic defense system. which are considered as potential scavengers of ROS and lipid radicals. glycinebetain.. γ-glu-cys-gly). Bittsanszky et al. including quenching or scavenging ROS like 1O2. Two of them. Third. such as ascorbate. (2010) and El-Beltagi et al. they are important for . EC 2. regulation of apoptosis and in plant responses to biotic and abiotic stresses. hydroxyperoxide detoxification. They are known major antioxidants in biological membranes for protection of membrane stability against lipid peroxidation. several non-enzymatic antioxidant defense mechanisms also play an important role in the response of plant stress tolerance. Glutathione contents increased in Sesbania drummondii callus under Cd-induced oxidative stress (Israr et al. One of them. 2006). It also acts as co-factor of violaxanthin de-epoxidase. like GPXs. which were improved using in vitro selection technique (Hossain et al.1. like ascorbate. but microsomal.4-D and Dicamba treatments (Peixoto et al.18) GSTs catalyse the conjugation of electrophilic xenobiotic substrates with the tripeptide glutathione (GSH. plastidic. Both of them are also main components of the Halliwell-Asada Cycle (Gill and Tuteja. (2011) reported that ascorbate and glutathione contents were increased under PEG-induced drought stress and low doses gamma radiation in rice and Rosmarinus officinalis L callus culture.. Carotenoids are a lipid soluble antioxidant. 1O2 and other harmful free radicals which are naturally formed during photosynthesis. Glutathione S-transferases (GST.Oxidative Stress Studies in Plant Tissue Culture 67 3. nuclear and apoplastic isoforms has also been reported. plays a pivotal role in several physiological processes.. Ascorbate can directly scavenge 1O2. Plant GST gene families are large and highly diverse. sugar. Second.tolerant poplar clones. ascorbate and glutathione are crucial metabolites in plants which are considered as most important intracellular defense against ROS induced oxidative damage. glutathione.and ·OH and by regenerate a-tocopherol from tocopheroxyl radical. 2010). RNA and proteins (Gill and Tuteja. 2010). and polyamines. 2008). GSTs have the potential to remove cytotoxic or genotoxic compounds.1.6. phenolic compounds. 2007) and also in paraquat.. they protect the photosynthetic apparatus by quenching a triplet sensitizer (Chl3). detoxification of xenobiotics and the expression of stress-responsive genes. They are known to function in herbicide detoxification. thus sustaining dissipation of excess excitation energy. 2006. which can react or damage the DNA.

2004). Phenylalanine ammonia lyase (PAL) activity is one of the main enzymes in the synthesis of phenolic compounds. are abundant in plant tissues... (2010) observed that glycinebetain. europaea callus culture the increasing amounts of proline were observed under Mannitol and NaCl induced stresses (Torabi and Niknam. which was improved using in vitro selection technique (Hossain et al.. sometimes is called osmoprotectant.. Patada et al. Helaly and El-Hosieny.. In another study.. 2000. respectively (Mohamed et al. in Salicarnia persica and S. including flavonoids. 2011). D-ononitil. Enhanced osmoprotectant contents have been demonstrated in plants during different stress conditions by many researchers. 2004. in their structures is another crucial mechanism in many plant species in response to environmental stress. Lokhande et al. (2012) reported that glycinebetain. Carotenoid content also increased in salt tolerant Chrysanthemum morifolium strain. 2006). reduced-sugar and disaccharidesugar contents were observed in drought-tolerant callus line of sunflower (Hassan et al. 2010). total sugar. PAL activities also increased in Glycyrrhiza uralensis and Luffa cylindrica cotyledons under Cd and Pb treatments in tissue culture conditions. 2010).2010). sucrose. 2006). Many secondary metabolites play widely important role from as defensive agents against pathogens to general protection against oxidative stress using as electron donors for free radical scavenging (Grace. Proline contents also increased in hyperhydric Prunus avium shoots (Franck et al. Increasing ratios of proline and soluble sugar contents were observed in drought tolerant Tagetes minuta clones and salt tolerant sugarcane (Saccharum sp.) callus. proline and soluble sugar contents enhansed in Sesuvium portulacastrum callus under NaCl treatment. an inhibitor of LPO. proline and reduced sugar contents increased in embryonic sugarcane callus under PEG and NaCl treatment. Accumulating osmotic adjustment. respectively (Zheng et al. 2011).. Phenolic compounds. Also. (2011) reported that carotenoid contents increased in Citrus lemon shoots under different oxidative stress conditions. and phenolic contents were increased under PEG-induced drought stress in rice callus culture (Shehab et al. including proline (amino acids). which are often referred to as secondary metabolites and functions of most of them have still poorly understood. Hassan et al. 2004). 2004).. NaCl and gamma radiation-induced oxidative stress conditions increased proline. glycinebetain and total soluble phenol contents in Citrus lemon shoots (Helaly and El-Hosieny. increasing proline. Jiang et al.) callus were improved using in vitro selection technique (Mohamed et al. anthocyanins. . 2005). trehalose. 2004).68 Antioxidant Enzyme the PSI assembly and the stability of light harvesting complex proteins as well as thylakoid membrane stabilization (Gill and Tuteja. glycinebetain (quaternary ammonium compounds) and sugars (mannitol. and OH· and 1O2 scavenger (Arshaf and Harris. fructan).. 2006). Proline and glycinebetain act as osmoprotectants by stabilizing both the quaternary structure of proteins and the structure of membranes. Cui et al... It was observed that under hyperhydric conditions PAL and lignin-concentrations reduced (Saher et al. and lignin. 2000. hydroxycinnamate esters. Gandonou et al. tannins. Gandonou et al. Additionally. and salt tolerant sugarcane (Saccharum sp.. 2010. Drought tolerant Tagetes minuta clones. sunflower callus lines. (2010) reported that proline and glucose contends were increased under sucrose-induced osmotic stress in Hypericum perfortum root suspension cultures. Proline also acts a metal chelator. In another study with rice cultivars were detected that under PEG induced drought stress conditions..

. measuring free radical scavenging or quenching capacities in cells are the other techniques the detection of total non-enzymatic antioxidant activity. 2003). but are sometimes placed directly into a liquid medium. 2010). tissues and organs of plant outside of an intact plant on solid or into liquid media under aseptic and controlled environment.. shoot tips. . nodes. 2010. 2010). respectively.e. 4. nodal or axillary bud cultures.Oxidative Stress Studies in Plant Tissue Culture 69 anthocyanins. (2004) and Ghnaya et al. 1968). (2011) reported that polyamine contents increased in hyperhydric Prunus avium shoots and Brasica napus cv. axillary buds. such as shoot-tip and meristem-tip cultures.. Therefore. putresine and spermine) are among the important non-enzymatic antioxidants... Gamborg B5 (Gamborg et al. 2010. Under sucroseinduced osmotic stress total flavonoids and phenolics contends were increased in Hypericum perfortum root suspension cultures (Cui et al. Zamora et al. Solid and liquid media are generally composed of inorganic salts. (El-Beltagi et al. LS (Linsmaier and Skoog. i. 2006. Chatzissavvidis et al. after the understanding the totipotency nature of plant cells. 1962). Low doses gamma radiation induced total phenol. 2006). maltose and raffinose). SH (Schenk and Hilderbrandt. 2008) methods. especially. it also includes a few organic nutrients. as an alternatively known cell. Plant tissue culture Plant tissue culture.2-diphenyl-1-picrylhydrazyl (DPPH) (Hossain et al. 2006. Basu et al. Phenol oxidases (PPO) activities. For this reason. Additionally. immature or mature embryos and generally can be obtained from the environment. which act to protect nucleic acids against enzymatic or oxidative denaturation and to prevent lipid peroxidation (Kaur-Sawhney et al.2-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) (Cui et al. energy sources (such as sucrose. another important enzyme which plays important role for oxidation of phenolic compounds. respectively. 2010).. Franck et al. Polyamines (spermidine. flavonoids and phenolics contents increased (Basu et al. cell suspension and callus cultures. soluble sugar and PAL activity in Rosmarinus officinalis L. Jumbo under Zn-induced oxidative stress. which are commonly called explant. It has also been used to describe various pathways of cells and tissue in culture depending on starting plant materials. 2011). was changed under NaCl induced stress conditions in callus and seedlings of Trigonella species (Niknam et al. refers to growing and multiplication of cells.. tissue and organ culture or in vitro culture. can be taken from any part of a plant.. Various methods have been used for measuring total antioxidant activities in biological systems. 2. 2010) and ferric reducing antioxidant power (FRAP) (Sotiropoulos et al. 1965). The increasing ratios of total antioxidant capacity were measured under different abiotic stress conditions induced with tissue culture techniques using 2. Explants are then usually placed on a solid culture medium. glucose. they are naturally contaminated on their surfaces (and sometimes interiors) with microorganisms.. Starting plant materials of this techniques. particularly when cell suspension cultures are desired.. surface sterilization of explants in chemical solutions (usually sodium or calcium hypochlorite or mercuric chloride) is required. This technique is one of the key tools of plant biotechnology. The most well-known of these inorganic salts is MS (Murashige and Skoog.. Synthetic media do not include only inorganic salts... 1972). flavonoid. Cui et al.

producing secondary products in liquid cultures. In recent years.70 Antioxidant Enzyme vitamins and plant growth regulators (i. plant growth. 4. Nuemann et al.4-Dichlorophenoxyacetic acid (2. Thus. George at al. A balance of both auxin and cytokinin will frequently produce an unorganized growth of cells.. As the name suggests. Synthetic medium compositions are generally prepared to be based on purpose and explant-source (IAEA.4. For example. As I previously mentioned plant tissue culture techniques are performed under aseptic and controlled environmental conditions.5-T). physiological and biochemical changes of both unorganized cellular (such as suspension cultures and callus cultures) and organized tissue (such as axillary shoot. particularly the plant growth regulators and the nitrogen source (nitrate versus ammonium salts or amino acids) have profound effects on the morphology of the tissues that grow from the initial explant. mature embryo. Solid medium is prepared from liquid medium with the addition of a gelling agent. because of these advantages.. one of the most serious problems is the influence of environmental stress factors on plants which are exacerbated day-by-day through anthropogenic effects. shoot tip. In addition. in vitro tissue culture techniques is performed under aseptic and controlled environment using artificial solid or liquid media for growing and multiplication of explants. auxins such as 2. The composition of the medium. oxidative stress is secondary . These negative results forced humans to find new solutions to minimize these problems. Indole-3acetic acid (IAA). while an excess of cytokinin may yield shoots.. crossing distantly related species by protoplast fusion and regeneration of the novel hybrid.1. 2008).4. and/or cytokinins such as 6-benzylaminopurine (BAP). Because of these characteristics in vitro techniques are suitable for researching both specific and common response to stress factors in plants. There are also several excellent books about plant tissue culture techniques for those who want further information (Jha and Ghosha. 2004. in vitro techniques have been extensively used not only in vitro screening in plants against abiotic stress but also creating in vitro models for studying and observing morphological.. this technique allows for the study of large plant population. production of dihaploid plants from haploid cultures to achieve homozygous lines more rapidly in breeding programs. Kumar and 6Furfurylaminopurine (Kinetin). 2009).e.liv. 2011). 2008. 2009. stress treatment of large population in a limited space and short period of time. such as producing large numbers of identical individuals via micropropagation using meristem and shoot cultures. Induced-oxidative stress conditions in plant tissue culture and antioxidant defense systems Nowadays. and homogeneity of stressor application (Sakthivelu et al. using tissue cultures as a model for inducing oxidative stress via different stressor agents and improving plants against abiotic stresses using in vitro selection techniques (www. 2006. which is called callus. As it is known. Lokhande et al. Yadav and Tyagi.5-Trichlorophenoxyacetic acid (2. and whole plant) levels against abiotic stresses. Naphtaleneacetic acid (NAA). 2. have vast potential for various applications both plant science and commercially. Plant tissue culture techniques. development and the yield performance of plants is adversely affected. usually purified agar.4-D). an excess of auxin will often result in a proliferation of roots.

polyethylene glycol (PEG). a rapid and a low cost breeding method (Lu et al. vegetables. 4. CaCO3) in artificial medium to imply calcareous conditions. Zn.. After the creating genetically stable variations. In addition. Cd (for Cd-tolerance). Ni. which is widely used in plant tissue culture. NaHCO3. various systems. These genetic variations may occur spontaneously or may be induced by any kind of agents (such as physical (i. and sucrose generally used as osmotic stress agents in in vitro culture conditions to stimulate drought stress in plants. X-ray) or chemical agents (i. paraquat or atrazine (for herbicide-resistance). shoot cultures. KHCO3.2. they are called somaclonal variations which are useful in crop improvement (Joyse et al. If these variations are created in somatic cells or tissues. Rai et al. such as NaCl (for salttolerance). Antioxidant activities of in vitro selected plants under abiotic stress conditions In vitro selection is another technique. . Cu. Adding NaCl or any kind of specific metals. Oxidative stress is one of the main reasons for spontaneous mutations in genome because of hyperactive ROS. But the main effects of these mutagens directly trigger to DNA-lesions. nodal cultures. which were published in recent years. as referred to some studies. This technique conventionally defines selection of desired genotypes after the induction of genetic variation among cells. polyploidy. 2003). 2007..e. it will be seen that investigated-oxidative stress parameters. embryonic callus. for selection of desired genotypes. Generating nutritional disorders. inducing chromosome breakage and/or rearrangement.If these studies are also carefully examined. mannitol. these selective agents added to the culture media. Al. varied from species to species and also in plant organs with respect to different stressor treatments. for researching hyperhydricity. including cereals. In plants screening for variations depending on the ability to tolerate relatively high levels of stressors in media. fruits and other commercially important plant. 2011). If I summarize in a few sentences of these studies which are referred in these tables. Ethyl methanesulfonate (EMS)) which are called mutagen) in culture conditions (Rai et al. PEG or mannitol (for drought-tolerance). researching-material is irradiated with different doses of gamma radiation. gamma radiation. which were detected various methods. by inducing under stress conditions. in the table 5... tissues and/or organs in cultured and regenerated plants. have been used in tissue culture conditions. such as Cd. The general method for investigating the biological effects of gamma radiation. It is assumed that in vitro selection is an efficient. explants are exposed to various kinds of selective agents. Pb. Hereby. such as callus. Cr and As in MS culture media also widely used techniques to induced salt or heavy metal stress conditions. Oxidative stress also induced to be indirect effects of physical or chemical mutagens. Between table 1 and 4 were summarized in recent studies by screening against abiotic stresses in a wide range of plants. sometimes researchers prepared missing media contents or adding some chemicals (such as. point mutations.e. genotypic and phenotypic variations created in the progeny of plants regenerated from plant tissue culture. cell suspensions.Oxidative Stress Studies in Plant Tissue Culture 71 effect of these stress factors and several techniques have been used to induce oxidative stress under tissue culture conditions. epigenetic variations. such as transposons activation. Hg. researchers generally prefer changing agar concentrations or gelrit agents in media compositions and/or using bioreactors.

GB: Glycinebetaine. and 576 mM Mannitol 0. Pro. (2008) Molassiotis et al. DPPH.. MDA. Co 86032 Salicornia persica and S. LOX. callus 0. 5. PP. (2010) Sivritepe et al. cv.and 1000 mM Mannitol 0. GSH. SOD. Oryza stiva L. (2012) Torabi and Niknam (2011) Hypericum perforum L. H2O2.. and PB) Deschampia antarctica adventiti ous roots spectrophot ometric Cui et al.(2006) Chai et al. and 40% PEG 6000 spectrophot ometric spectrophot ometric spectrophot ometric and isoenzyme variations spectrophot ometric and isoenzyme variations spectrophot ometric Shehab et al. FRAP. Pro. Pro. and GB SOD. SOD. APX. CAT. Flavonoid. Phenol and chlorogenic acid. 7. POX. and 300 mM Mannitol 0. Hypericin and Residual Sugars SOD. and 20% PEG 0. (2010) seedlings 0. TBARS.. GR. APX. 2. H2O2. (2010) shoots PEG-8000 spectrophot ometric Zamora et al. PP. CAT.: Proline. MDA. and 4% PEG 8000 0. MDA.72 Antioxidant Enzyme Antioxidant Enzymes. H2O2.europaea spectrophot ometric spectrophot ometric Patada et al. and 20% (w/v) PEG-6000 spectrophot ometric Basu et al. MDA. and MDA Abbreviations: AA: Amino Acid. APX. 10. 1. and 9% (w/v) Sucrose Detection Methods References Saccharum officinarum L. MDA. MDA. and MDA Plant Species Type of Explants embryog enic callus callus Stressors and Concentration 0. TSS and TRS. ABTS. PAL. Pro. AsA. POX. (2005) shoot tips SOD. and Phenol DPPH. H2O2. rootstock MM 106 Centaurea ragusina L. CARs: Carotenoids. POX. and Pro. 1. PC. In vitro studies concerning drought stress . and Pro. PP. 15. CAT GR. and Phenol SOD. TSS and AA. Pokkali. PP PO. CAT.5 mM Sorbitol 0. DPPH. 5. Musa AAA ‘Berangan’ and Musa AA ‘Mas’ shoot tips shoot tips shoots POX. PC: Protein Content. and H2O2 Radić et al. Ascorbate. PC. canecens Malus domestica Borkh. (2010) Oryza sativa L. (2006) Prunus cerasus x P. and Phenol H2O2. CAT. 3. GR. GR. APX. TRS: Total Reducing Sugar. MDA. and 20% (w/v)PEG 8000 0. Flavonoid. PPO. Antocyanin Flavonoid. (cv. PP: Photosynthetic Pigments TSS: Total Soluble Sugar. CAT. POX. CAT. POX. Proline.500. CAT. H2O2. APX. IR-29. and 562. Table 1. CARs. None-antioxidant Components and Oxidative Stress Biomarkers SOD.

MDA. 100. Sesuvium portulacastrum L. Pro. spectropho tometric . (2011) Lokhande et al. 100. and GB SOD. etc. and CAT spectropho tometric spectropho tometric spectropho tometric and isoenzyme variations spectropho tometric spectropho tometric spectropho tometric Patada et al. MDA. CARs. (cv. several methods have been used. and 600 mM 0. and CAT spectropho tometric Kusvuran et al. TSS. 200. Pehlivan and Flamura-85) Sesuvium portulacastrum L.) as well as the other oxidative stress biomarkers (H2O2. As also shown in the table 5. Additionally. Besni. All of them also agreed with advancedantioxidant capacity increase tolerance against stress factors in plants. fortune (Seemann and Hemsley) callus 100 mM SOD. (2012) callus 150 mM 0. and GB SOD. TSS and TRS. 100 and 200 mM SOD. CAT. glycinebetaine etc. POX. H2O2. 300.). TBARS. Midyat. PC. PPO. (2010) Ayala Astorga and AlcarezMelendez. GR etc. APX. 80 and 160 mM MDA. Tekirdag. PC. CAT. Paulownia imperialis (Seibold and Zuccarini) and P. POX. in vitro selected abiotic stress tolerant plantlets have been characterized by detections of enzymatic antioxidants (SOD. and MDA SOD. Co 86032 Salicornia persica and S. PC. APX. and NADPHoxidase callus seedlings 0. POX. PP. None-antioxidant Components and Oxidative Stress Biomarkers Plant Species Type of Explants NaCl Concentrations Detection Methods References Cucumis melo L. CAT. 150. 100. (2011) Sen and Alikaman oglu. 200 and 400 mM 0. Antioxidant Enzymes.. APX. PP. cv. As for the characterizations of selected abiotic stress tolerant plants. One of them is based on antioxidant defense systems. (2010) callus mature embryos axillary shoots callus SOD. there is known to be a strong correlation between stress tolerance and antioxidant capacity in plant species. and Proline. CAT. GB. H2O2.europaea Triticum aestivum L. and 250mM 0. (2012) Torabi and Niknam. 200. SOD. Nitraria tangutorum Bobr. Pro. TSS. 20. CARs. and 600 mM 0. 50. 50. 60.. APX.Oxidative Stress Studies in Plant Tissue Culture 73 2011). CAT. (2011) Lokhande et al. POX. Pro. (2010) Yang et al. 400. CAT. ascorbate. (cv. 40.) and/or non-enzymatic antioxidant (proline. Yuva. and Pro.. Semame and Galia C8) Saccharum officinarum L. APX.

APX. and 250mM 0. Total Flavonoid and GB Zhao et al. (2008) Dasguptan et al. NADPICDH. MDA. 100 mM SOD. 30. (2007) shoot tips SOD. Azevedo et al. GR. and PP shoots Sugar. CAT. MDA and Proline H2O2. and 200 mM Valderrama et al. munofluor es. and ROS mes. 35. Ascorbate. (2009) Thellungiella halophila and Arabidopsis thaliana Solanum tuberosum L.74 Antioxidant Enzyme Antioxidant Enzymes. CAT. 40.. (2006) . and Proline Sotropoulos . and Pro. and Sugar Plant Species Type of Explants NaCl Concentrations Detection Methods References Catharantus reseus L. Pro. Prunus cerasus L. Rosea and Alba shoots 0. SOD. and 200 mM (NaCl) 0. 5. G6PHD. POX and CAT shoot tips FRAP. PC. 100. and 1% 0. Manzanillo seedlings 0. 0.. (2008) Chatzissavv idis et al. 80. POX.. POX. 100. POX. and transcript. 75. None-antioxidant Components and Oxidative Stress Biomarkers SOD. 60. PP. and 150 mM SOD. CAT. 50. SOD.(2009 ) shoot apices and callus SOD. isoenz. 60. CAT. PP. Cardinal and Desiree callus 0. 100. and PC spectropho tometric spectropho tometric and isoenzyme variations spectropho tometric spectropho tometric and isoenzyme variations spectropho tometric Jatropha curcas callus 0. POX.canescens shoot apexes 0. 20. 100. 25. (2010) Pinus pinaster suspension cells 0. (2007) Olea europea L. POX. 50. 200. 100. 45. Kumar et al. CAT. GSH. 50. 50. Treholase Pro. 40. Erturk et al. and 60 mM (NaCl and CaCl2) 0. Rootstock CAB-6P Malus domestica Borkh. 60.5. 150. and 150 mM MDA. GR. 120 and 140 mM Sucrose. cv. (2008) Impomoea batatas L. 80. and FNR spectropho tometric spectropho tometric. 20. Rootstock M 4 Sweet chery rootstock Gisela 5 Prunus cerasus x P. 30. 15. and 100 mM spectropho tometric isoenzyme variations and transcriptio n analysis spectropho tometric Garg. cv. 100. and 10 mM (CaCl2) 0. Phenol. (2009) Sajid and Aftab.

‘Valencia late Centaurea ragusina L. sinensis cv. PC: Protein Content. 50. CAT. Proline. CAT. Pro.: Fluorescent dying. Solanum tuberosum L. 150.: Proline. 100 and 150 mM SOD and POX Rahnama and Ebrahimza deh (2006) Ferreira and LimaCosta (2006) Radić et al. 300 and 400 mM SOD. FRAP. and 200 mM CAT. 50. Rahnama and Ebrahimza deh (2005) Woodward and Bennett (2005) shoots 0. 50. None-antioxidant Components and Oxidative Stress Biomarkers SOD. 150. PP.f. 300. Trascript. PP Abbreviations: AA: Amino Acid. TRS: Total Reducing Sugar FNR: ferredoxin-NADP reductase. GB: Glycinebetaine. Flour. 50. 50. POX. Kennebec. 200. (2006) seeds and callus 0. and MDA Plant Species Type of Explants NaCl Concentrations Detection Methods spectropho tometric and isoenyme variations spectropho tometric and isoenyme variations spectropho tometric and isoenyme variations spectropho tometric spectropho tometric and isoenyme variations spectropho tometric and isoenyme variations spectropho tometric References Malus domestica Borkh.Oxidative Stress Studies in Plant Tissue Culture 75 Antioxidant Enzymes. NADP-ICDH: NADP-isocitrate dehydrogenase.: Transcription analysis Table 2. and 240 mM NaCl 0. POX. Agria. Diamant and Ajax) Citrus hybrid ‘Carvalhal’ and C. 150. H2O2. Kennebec. PC. and H2O2 nodes 0. 100. and Proline Niknam et al. PC: Protein Content. POX. PP: Photosynthetic Pigments TSS: Total Soluble Sugar. Proline and MDA shoots 0. and Trigonella aphanoneura Rech. G6PHD: glucose-6-phosphate dehydrogenase. FRAP. (2006) cell suspension 0. (2006) internodes 0. POX and APX. 100. CAT. Diamant and Ajax) Eucalyptus camadulensis Dehnh. CARs: Carotenoids. In vitro studies concerning NaCl stress . 450 and 600 mM POX. 100 mM Proline. clones shoot tips 0. PPO. (cv. MDA. and 220 mM KaCl Molassiotis et al. PP: Photosynthetic Pigments: Isoenz: Isoenzyme variation. Agria. (cv. Solanum tuberosum L. 75 and 100 mM SOD. rootstock MM 106 Trigonella foenum-graecum L.

POX. POX. (2008) Arachis hypogaea L. POX. 0. CAT. 100. 0. (2010) Oryza stiva L. 0.05. cv. PP. 200 and 300 μM 0. POX. 0.25. (2010) Jatropha curcas L. Thiols. 150 and 250μM 0-1 mM 0. and MDA Shri et al. cv. CAT. embryos Pb SOD.(2011) Zn Alternanthera philoxeroides callus Cu spectroph otometric spectroph otometric and isoenzyme variations spectroph otometric and isoenyme variations spectroph otometric and isoenyme variations spectroph otometric and isoenyme variations spectroph otometric and isoenyme variations spectroph otometric spectroph otometric Xu et al. embryos Zn SOD. 200. (2009) Jatropha curcas L.6.1. 0. PPO. and 1 mM 0. and PAL Yan et al. CAT.1. POX. (2011) Glycyrrhiza uralensis L. CAT. 400 and 800μM 0. 100. MDA. None-antioxidant Components and Oxidative Stress Biomarkers APX.(2010) Luffa cylindrical L.2. (H2O2 and O2-• ) Plant Species Pinus nigra L. seeds suspensio n cultures Cd CAT.8. and PAL Jiang et al. POX. (2012) Ghnaya et al. JL-24 Picea rubens Sarg. 200. and Polyamines . MDA. 1.76 Antioxidant Enzyme Antioxidant Enzymes. POX and MDA Kumar et al. 100 and 500μM As(V) 0. PP. and Phytochelatins POX. 50. 0. and PAL Luo et al. CAT. 400 and 800 μM 0. GSSG. seeds Cd SOD. 0. and PAL Zheng et al.(2008) Cd and Zn Thiol. APX. Lalat seeds As(III) and As(V) SOD. 50.5. 0. (clone Poli and 58-861) Brassica napus L.05. AA. 50 and 100μM As(III) 0. 0.5. 0. cv. GR.2 and 0. Jumbo Type of Explants Stressors Concentr ations 0. POX. 100 and Detection Methods References callus thin cell layers Cd spectroph otometric spectroph otometric Iori et al. 2 and 3 mM 0. CARs. embryos Ni SOD. ROS mes. 12. and Polyamines SOD.4 mM 0. 25. 100. CAT.

0. 10. cv. 400 and 800μM (Zn) Detection Methods References Thangavel et al. 0. APX..1. CAT. 25.0. Al+3. Table 3. isoenz. GSSG.(2007) Medicago sativa L. cv. POX. ROS mes. Flour. 0. GSSG. (2002) Abbreviations: AA: Amino Acid. GSH. 200. 50. and transcrit. fluor. (2005) Helianthus annuus L. GSSG. Mycosol callus Cd SOD. Phytochelatins Gallego et al. Ascorbate. 0. None-antioxidant Components and Oxidative Stress Biomarkers Plant Species Type of Explants Stressors Concentr ations 200μM (Cd) 0. 0.1.: Fluorescent dying..: Transcription analysis.. APX GR. spectroph otometric Villasante et al. SOD.02. PP: Photosynthetic Pigments. 3. and ROS mes. CAT. 100. Isoenz: Isoenzyme variation. and FRAP spectroph otometric spectroph otometric and fluorescei n dye Sotiropoul os et al. and GSH Israr et al. Phytochelatins. In vitro studies concerning heavy metals stress . (2006) callus Cd 150 μM MDA. (2002) Saccharum officinarum L.(2007) Sesbania drummondii Malus domestica Borkh. 50.Oxidative Stress Studies in Plant Tissue Culture 77 Antioxidant Enzymes. PP.5. callus Cd 0.01. 100 and 250μM 0. GSH. rootstock MM 111 Helianthus annuus L. 10 and 30μM 0. 0. and 6 mM H2O2 . APX. 1. and SOD. and Dehydroascorbate. Ascorbate. 3. callus Cd+3. POX . (2006) shoots B SOD. Cr+3 150 μM spectroph otometric Gallego et al.2.5 and 1 mM SOD and CAT spectroph otometric and isoenyme variations Fornazier et al. TBARS. Aragon Cd and Hg 0. Trascript.05. GR. spectroph otometric. GSH.

mes. Vigna radiate L. and isoenyme variations spectroph otometric and isoenyme variations Gupta and Prasad. Phenol. AsA. GST. 2. (2007) Roy et al.5 gl-1 CaCO3 ) (pH 7. infloresce nces hyperhydri city bioreactor culture MS (+Fe) control. (2006) calli herbicides gamma radiation (60Co) hyperhydri city SOD. GSH. 15 and 20Gy spectroph otometric El-Beltagi et al. (2011) Gladiolus hybridus Hort. (2006) Chakrabart y et al. GR and GST SOD. LOX. SOD. APX. DHAR. TSS.We dding Bouquet Solanum tuberosum L. APX. POX. (2005) . CAT. Noneantioxidant Components and Oxidative Stress Biomarkers MDA. 100 and 200 Gy bioreactor culture spectroph otometric and isoenyme variations spectroph otometric isoenyme variations spectroph otometric. GSSG. 10. CAT. AA. AsA. PAL. and POX MDA.78 Antioxidant Enzyme Plant Species Type of Explants Stressors Treatments Antioxidant Enzymes. GR. GPX. and POX callus nodal segments SOD. Cadaman. PAL. H2O2 and O2-. 20. APX. H2O2 and FRAP spectroph otometric and isoenyme variations Molassiotis et al. 5. Saint Julien 655/2 and GF-677) shoots Fedeficiency CAT. callus gamma radiation (60Co) 0. MDHAR.9) and MS (10 mM NaHCO3 + 0. SOD. APX. SOD. and MDA Euphorbia millii L. flour.3) Dewir et al.5 gl-1 CaCO3) (pH 6. and POX Detection Methods References Rosmarinus officinalis L.4-D and dicamba 0. MDHAR.DHAR. and ROS mes. Wilczek Apple “M9 EMLA” callus hyperhydri city different culture systems paraquat. GSH. POX. MS (-Fe). (2010) Peixoto et al. 50. MS (5 mM NaHCO3 + 0. Flavonoid. (2006) Prunus rootstocks ( CAT. GR. GPX. CAT.

8% to 0. APX. POX. Felicita Citrus limon L. and Polyamines Plant Species Type of Explants Stressors Treatments Detection Methods References Dianthus caryophyllus L. APX. cv. Ethylen. In vitro studies concerning the other abiotic stresses Plant Species Culture Techniques Mutagens Stressors Antioxidant Enzymes. Agat and Konsul drought (PEG) . CAT. Pro. Feminello gamma radiation (137Cs) gamma radiation (60Co) gamma radiation (137Cs) drought (PEG) spectrophoto Sen and metric and Alikamanogl isoenyme u. Noneantioxidant Components and Oxidative Stress Biomarkers SOD. MDHAR.8% agar to 0. (2012) variations Helaly and spectrophoto El-Hosieny. and EPR Mohamed and Aly. AsA.: Fluorescent dying. Isoenz: Isoenzyme variation. 13 and 27. and MDA Proline. MS (+Fe) and MS (+ 1mM KHCO3) spectroph otometric Saher et al. GB. Flour.CARs. H2O2. PP. (cv. (2004) shoots Fedeficiency PP. CAT. and SOD Lombardi et al. Prunus cerasifera rootstocks Mr. LOX. PP. Burm. MDA. Franck et al. GR. (2003) Abbreviations: AA: Amino Acid.Oxidative Stress Studies in Plant Tissue Culture 79 Antioxidant Enzymes. POX. TS. PP: Photosynthetic Pigments. Trascript. Ethylene. and Phenols SOD. APX. CAT and POX SOD. and Alister) Prunus avium L. nods cv. PAL.PP. cv. DHAR. CAT and POX Detection Methods References Beta vulgaris shoot tips L. GR.: Transcription analysis Table 4. (2004) Borage officinalis L. GSH. Killer.8 mgl-1FeSO4 control. CAT. H2O2 lignin. metric (2011) protoplasts NaCl Solanum tuberosum L. CAT. metric (2011) Alikamanogl spectrophoto u et al.25% gelrit) 0. APX. MDA. shoots hyperhydri city changing concentrati on of agar from 0. Noneantioxidant Components and Oxidative Stress Biomarkers SOD.S2/5 seeds Fedeficiency APX. Oslo.. (2004) shoots hyperhydri city spectroph otometric spectroph otometric and isoenyme variations spectroph otometric and transcript.58% changing from 0.f. GPX.

metric (2004) shoot EMS NaCl callus NaCl drought (PEG) gamma radiation (60Co) callus callus AEC res. DHAR. CAT. O2. Noneantioxidant Components and Oxidative Stress Biomarkers SOD. cv. H2O2.80 Antioxidant Enzyme Antioxidant Enzymes. spectrophoto GR. Donganbyeo Medicago sativa L. APX. and Pro. and Pro. fluorescent Ramel et al. AsA PP.] Merr. and metric and Bittsánszky LOX transcriptom et al. Regal Time Saccharum sp. Solanum tuberosum L. DPPH. metric (2011) NaCl plantlets sucroseinduced atrazine leaf petioles paraquat Alikamanoğl spectrophoto u et al. APX and AA seeds drought (PEG) CAT. GR. MDAR. Co10) Poplar clones (Populus X Canescens) Chrysanthem um morifolium Ramat. (2006) variations spectrophoto Gandonou et metric al. PP. Maghi Yellow Cynodon transvaalensis x C. . (2004) spectrophoto metric and Kim et al.. CP65357 Helianthus annuus L. CUF 101 embryogenic callus nodes gamma radiation (60Co) gamma radiation (137Cs) NaCl spectrophoto Chen et al.. CAT.. GR. cv. APX. APX. GR and Pro. metric (2009) SOD. CARs. spectrophoto Hossain et metric al. 1O2..-. cv. Myak Oriza japonica L. CAT and POX Plant Species Culture Techniques Mutagens Stressors Detection Methods References Zoysia matrella [L. and dying. SOD. MDAR. cv.. SOD. GST. POX and Pro. and Charbonhydrates spectrophoto Lu et al. and TSS Pro. spectrophoto DHAR. Pro.. SOD. dactylon cv. and Pro. cv.. callus NaCl SOD. PP. Granola Arabidopsis thaliana (ecotype Colombia. metric. APX. and (2009) ROS-scavenging transcriptom systems ic analy. (2007) callus NaCl and drought SOD. Tifeagle Chrysanthem um morifolium Ramat. H2O2. (2006) spectrophoto Hassan metric et al.. CARs. (2008) ic analy. proteomic (2004) analysis spectrophoto Safarnejad. metric (2007) spectrophoto metric and Hossain isoenzyme et al. GR.

Improving plants via in vitro selection methods generally based on spontaneous or induced mutations. Plant tissue culture techniques are performed under aseptic and controllable environmental conditions for this reason allows various opportunities to study details of this balance.. Despite all these knowledge about ROS. Noneantioxidant Components and Oxidative Stress Biomarkers SOD. Tagetes minuta cell suspension paraquat and Cd drought (Mannitol) callus spectrophoto metric and isoenzyme variations spectrophoto metric Kopyra and Gwozdz. including antioxidant enzymes and molecules that can protect cells from oxidative damage and maintain ROS homeostasis.: analysis. TSS: Total Soluble Sugar. Additionally. Besides causing damage. Conclusion The overproduction of ROS in plants is stimulated by environmental stressors as well as many metabolic reactions. Faculty of Science. In vitro selected examples of against abiotic stresses. Vezneciler. CAT and POX Pro. it should not be forgotten that these mutants need to be tested under field conditions to maintain genetic stability for desirable character/characters. improving crops against abiotic stress factors and isolating of cell/callus lines or plantlets using in vitro techniques are the other main usage of plant tissue cultures. and the activities of antioxidants and oxidative stress indicators 5. Controlled stress in in vitro may help to overcome the cross tolerance/cross responses. PP: Photosynthetic Pigments TS: Total Sugar.. such as photosynthesis. how to maintain balance between these oxidant and antioxidant properties in plants have still poorly been understood. 34459. and oxidative stress is one of the main reasons of both spontaneous and induced mutations which are caused DNA damages in various ways. var. Although. Author details Ayşe Şen* Istanbul University. Department of Biology. protein oxidation or DNA-lesions. Isoenz: Isoenzyme variation. (2000) Abbreviations: GB: Glycinebetaine. Turkey * Corresponding Author . as suggested by Jain (2001). Table 5. analy. APX. Istanbul.: variation. All of these ROS are toxic to biological molecules and generally lead to non-controlled oxidation in cellular macromolecules. Pro. CARs: Carotenoids. These irreversible damages of cellular macromolecules cause many cases in plants from mutations to cell death. ROS can also participate in signal transduction. (2003) Mohamed et al. and respiration. in vitro selections will save time to improve crops. photorespiration. Plants possess sophisticated-antioxidant defense mechanisms. AA: Amino Acid. and TSS Plant Species Culture Techniques Mutagens Stressors Detection Methods References Armoracia rusticana Geart.Oxidative Stress Studies in Plant Tissue Culture 81 Antioxidant Enzymes. Therefore.: Proline. such as lipid autocatalytic peroxidation. it is a driving force for improving crops.

. Plant Cell Tiss.. Therios. antioxidants and signaling in plants. Differential antioxidative responses of indica rice cultivars to drought stress.. Sen. Water stress-induced oxidative damage and antioxidant responses in micropropagated banana plantlets.. Ashraf. Ann. 2011. 49(1): 153-156.. I. M.. P. Organ Cult. Ayan... Blokhina.... 2004.. References Ahmad. R... Curr. 95: 37-45. Mingliang Chai. 103: 7-14. Veneti. Gama radyasyonu ile teşvik edilmiş tuza toleransl patates (Solanum tuberosum L.. X. 64: 145-157. C. Gu. Chakrabarty. Plant Biotech. 60:51–59..S. Effect of salt on ROS homeostasis. J. Antioxidants. Virolainen. K.. Effect of NaCl and CaCl2 on the antioxidant mechanism of leaves and stems of the rootstock CAB-6P (Prunus cerasus L. O. Fagerstedt. A...P. H.V. Zhang.. Peak.-Y.. 2011. 51(3): 167-173.C. Kiss. 9-12. Kusnan.. Komives. 2008.. D.. Plant Cell Tiss.. Annals of Botany.. M.] Merr. R. 2005. P.. G.. Alikamanoglu. O.. metabolite. Chen. Katay. K. Sairam.. A. E. Alikamanoglu. Yaycili. Oxidative stress and physiological.M.K. Azevedo.. and antioxidant levels in root suspension cultures of Hypericum performatum L. 2003. R. p:312. N. G.. Sharma..... S.. Plant Sci.M. Y. Z. 2008... A.. Sengupta. Plant Physiol.. Mahmood. Salinity effects on protein content. Yaycili..) under in vitro conditions. 13(5): 1-11.. 2010. J. Fadzillah. pigments. B. Cui.. L.-H. Plant Cell Tiss..I. For. G. D. Srivastava. and proline in Paulownia imperialis (Siebold & Zuccarini) and Paulownia fortunei (Seemann & Hemsley) grown in vitro.. 2002. In vitro selection of salt tolerant variants following 60Co gamma irradiation of long-term callus cultures of Zoysia matrella [L. Hyperhydricity in apple: ultrastuctural and physiological aspects. Bittsanszky. November.. C. Paraquat-tolerant poplar clones (Populus x Canescens) selected in vitro. M. 2001. G.-T. Sen. S. Murthy. O. A. and Alcaraz-Melendez. Ali. 2010.. K....Y. Plant.. Scie... Chatzissavvidis. L.. Ayala-Astorga. X.. Arora. Potential biochemical indicators of salinity tolerance in plants. 166: 3–16. 2008.. epigenetic and genetic variability in plant tissue culture: implications for micropropagators and genetic engineers. 2010.. K. 66: 211. Wu. H.. 107:493–500. Plant Growth Regul. Gullner. and oxygen deprivations stress: a review. Roychoudhury. 26: 377–388. M. S. Serwat. Chai. Tree Physiol. Organ Cult. oxidative damage. Medicinal and Aromatic Plants in Generating of New Values in 21st century. L. V. Cassells.82 Antioxidant Enzyme 6. Gao. T. 82(10): 1227-1237. Biochemical analysis of potato (Solanum tuberosum L.. Elc. Ulusa Nükleer Bilimler ve Teknolojileri Kongresi. A. and Curry. P. Basu. 4thBioremediation Conference.. Tavares..F. I.. S... and Harris.N.2009.) mutantlarnda biyokimyasal değişmelerin incelenmesi. Papadakis. p: 116. M. lipid peroxidation and antioxidant mechanisms in Pinus pinaster suspension cells. 2011..N. Shin. G.) mutants induced by gamma radiation. A. Amorim-Silva. Saha. Sci. Jia....C. Organ Cult. Plant Cell Tiss Organ Cult. A. Oxidative stress and antioxidative system in plants.. Reactive oxygen species.. M. S. Gyulai.M. Heszky. Abstract Book. Paek. Sucrose-induced osmotic stress affects biomass. Park. 2005. S. .2009. J.-H. 91: 179-194. Biol.Y. lipid peroxidation. A.J.C. T. Bildirim Tam Metinleri Cilt-1.

Kevers. 51(3): 597-600. 37: 263–285. E... J.. N. 71: 125-131.. 2002. Yerlikaya.. Selection of callus cultures of sugarcane (Saccharum sp. SpringerVerlag. M. Deby. Del Rio. B. N. and Noctor. Berlin. F. Environ Sci. 58: 93–99. C.L. C.F.A. 2004. Ferreira. Reactive oxygen species as signaling molecules. C. 2006.. Plant Growth Reg. Serteyn.... Cadmium stress in sugar cane callus cultures: Effect on antioxidant enzymes..) genotypes for salt tolerance through shoot apex culture under in vitro NaCl mediated salinity stress conditions.E. M. Plant. Heidelber.J. M. Azpilicueta. H.. Metabolic responses to salt stress in cell suspension cultures of sensitive and resistant Citrus. O...M. L. Dewir.. C. Radiation Physics and Chemistry. Pp: 169-196.H. . J. S.. Haussman.. C...H.. Gallego.. Neill. Horticul.2010. Kole. Y.. and Lima-Costa. 2005. 2006. B. Biol. A. Plant Growth Regul.Oxidative Stress Studies in Plant Tissue Culture 83 Dasgupta. Gamborg. Senhaji. R. Hancock. Gaspar. Turkan. cells.. Kogan. 81 (6): 983– 988. 28:1056-1071. Glutathionemediated antioxidative mechanism in sanflower (Helianthus annuus L. Bor. 42: 519-527.. 46: 267-276.. Pereira. Bioch. Azevedo. Plant.J.. O. M. Effect of low doses γ-irradiation on oxidative stress and secondary metabolites production of rosemary (Rosmarinus officinalis L. Garg. 2006...S. Desikan. B. Develop. 2002.. Abrini.R.. El-Desouky. I. Plant Physiol. Tomaro. D. Plant Cell Tissue Org. 50(1): 151-158. Dommes. Exp.. Evaluation of orange-fleshed sweet potato (Impomoea batatas L. Int.... Nutrient requirement suspensions cultures of soybean root cells.. Ozdemir.. Plant Cell Tiss. Jouve. in: Smirnoff... 2008.. 94: 161-170. DebyDupont. Exp. Bisbis. Miller.M. Germany. Molina. M. Ltd. Oxidant and antioxidant signaling in plants: a reevaluation of the concept of oxidative stress in a physiological contex. T. Hyperhydricity of Prunus avium shoots cultured on gelrite: a controlled stress response.. Foyer. J. D.. Fornazier.. W. Paek K..R. N. K. U.. T. Sahoo. R. Blackwell Pub. In vitro Screening of Catharanthus roseus L. T... 1(1): 24-30.C. Ojima. Organ Cult. Plant Cell Tiss.A.) cells in response to cadmium stress. Greimers. Lipid peroxidation and antioxidant enzyme activities of Euphorbia millii hyperhydric shoots. Gaspar. G.... L. Cell Environ. Sivritepe. 87: 9-16.. J.. Dommes. 2007. Benavides.L. R. 2011.. Sci.. J. R.) callus culture... T. El-Beltagi.) tolerant to NaCl and their response to salt stress. Botany.. M. C. Responses of the cherry rootstock to salinity in vitro. eds.... J.. cultivars for salt tolerance using physiological parameters. G. Mukherjee..J. Antioxidants and Reactive Oxygen Species in Plants. S. Ali. Cell Res. T. Gandonou. Organ Cult. P.. Smith. ISBN 978-3-642-00390-5.J.E. Concepts in plant stress physiology: Application to plant tissue cultures...G. R. Gallego. 1968.... Ferreira.. Pena.A. Biotech. 2005.S. Hahna. S.... Chakrabarty.B. Reactive Oxygen Species in Plant Signaling. C. Cult.F.. S. Environ. Erturk.. Ahmeda... M. M. R.G.. Idaomar.Y. M.. Franck. 2002. J. Lea. 36: 267-273. and Puppo. A. P. Kevers. Tomaro. G.. 2005. J. C.. 2009. 80: 968–976. Plant Growth Reg. ed.K. R. Franck. Involvement of an antioxidant defense system in the adaptive response to heavy metal ions in Helianthus annuus L.. Errabii..

74: 103-121 Karuppanapandian.f. Kim. L. Massacci. Kim.K. Int.D. Stress and aberrant phenotypes in in vitro culture.C. Cult. Modulation of Zn-induced oxidative stress...-J. The background (3rd ed. B. C..K. 2004... Res. Burm. 2012.. A. 4:85–94. Sahi.M. Zheng.V. In vitro selection for water stress tolerant callus line of Helianthus annus L. Moon.S. ISBN 81-7371-488-6. J. X. Reactive oxygen species in plants: their generation. Hourmant. . Grace S. Cadmium accumulation and antioxidative responses in the Sesbania drummondii callus..E. and Ghosha. S. Z.A. 12(2): 205-210. S. through shoot organogenesis of selected callus line. 2011. Datta. and Prasad. Ghnaya.A. Hossain. A.K.. Biswas. 2007.K..derived variation in crop improvement. Oxford New York. Agri. E..K.K.. F. eds. Plant Cell Tissue Org. Dordrecht. Tissue culture.. Israr. Datta. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. signal transduction.. Biotech. Zeng. A. Halliwell. in Antioxidants and Reactive Oxygen Species in Plants. Universities Press.H. Biotech. (ed. A. Manoharan.S.. Yang. Gupta. Euphytica.84 Antioxidant Enzyme George.D. 2005. S.. 118:153-166... J.. Mandal..M. N. Gill. Z. cv. 50: 121-127. 2010. V. Development of NaCl tolerant line in Chrysanthemum morifolium Ramat.M. Luo. S. Contam.. Toxicol.K. Plant Biol. Biochem.. L.S.) Blackwell Scientific Publishers. S. Int.. 2006.B. Volume 1.. J...C. Cassells.A.. S.C. Wang. Effectiveness of gamma irradiated protoplast on improving salt tolerance of lemon (Citrus limon L. S. 8: 450-461.. Zacchini... Pietrini.. and Tuteja. and El-Hosieny.Y. 2006. Oxford. M. Organ Cult. 6(1): 13-18. M. Crop Scie. Jha. Springer. Hossain.S.S. 108: 17-26. Plant Cell Tiss. Low cost options for tissue culture technology in developing countries. 2008. E. 3.. N. and scavenging mechanisms. J.. pp.. S. Jain. India. J. Biswas. N. M.. Joyce.. A.. Jiang... Plant propagation by tissue culture. 2010. Hashem. J. Int. N.F.ed..N. A. E.. T.. nigra) clones with different tolerance to cadmium. Jumbo) regenerated from transversal thin cell layers in the presence of zinc... 2003... 2011. Lead toxicity induced growth and antioxidant responses in Luffa cylindrical seedlings. Couderchet. Hall.. Austria..S. Australian J. Biol.). Plant Physiol. A. M.). 2(3): 62-71. K. U. polyamine content and metal accumulation in rapeseed (Brassica napus cv. J. 5(6): 709-725. A. and Gutteridge. Hassan.. T. Myak. Plant Biotechnol. Biol. Arch. Branchard. Mandal. Am. A. M..H. Hydarabat. Vienna. ISBN 978-1-4020-5004-6. Induction of metal binding compounds and atioxidative defence in callus cultures of two black poplar (P...B... G. Environ. 2005. Shaaban. G... Charles. 2011. W. ISSN 1011–4289 Iori.. IAEA.M.. 129: 658-667. Shoot multiplication kinetics and hyperhydric status of regenerated shoots of gladiolus in agar-solidified and matrix-supported liquid cultures. IAEATECDOC-1384. 2001.R. Rep.M. Z.... 2004. J. Seleem.. Smirnoff.. M. B. Free Radicals in Biology and Medicine. through in vitro mutagenesis. M. Jain. De Klerk. A. Helaly.R. J. 2010. 141-168. 2000. V. 6(4): 190-208. Jain. Development of NaCl tolerant strain in Chrysanthemum morifolium Ramat. Phenolics as antioxidants.. Plant Tissue Culture: Basic and Applied. 48: 909-930.T... Agric. Plant Physiol.

... E.. Peng. 2010... Plant Cell Rep.L... Foyer. C.. Vitagliano.H. Review article number 7.Oxidative Stress Studies in Plant Tissue Culture 85 Kaur-Sawhney.... Z.. Biol. S.. M.. Kusvuran. Altabella. Jang. 2003...M. P.. Bartoli..H. Biol.. Mittova... Reactive oxygen gene network of plants. Singlet oxygen and plants. Cell Mol. Lee. KCl. antioxidant activity and Fe(III)-chelate reductase activity of five Prunus rootstocks explants in response to Fe deficiency. 2012. M. S... Plant Growth Reg. Luo. Lokhande. Z. He. Physiological.G.N.W. Breusegem. 133: 443-447. C.. 2011. 46: 69-78. Wang. Therios. J.. 26(10): 2149-2163. Kiddle. Abak.V. 2003. Wang. Kumar. 2009. India.H. F. Guo. 171: 440–447. H.. I.D.. K. biochemical. physiological and growth changes in response to salinity in callus cultures of Sesuvium portulacastrum L.127. Plant Cell Tiss. Kofidis. Antioxidant and anatomical responses in shoot culture of the apple rootstock MM 106 treated with NaCl. AEC resistant rice mutants induced by gamma-ray irradiation may include both elevated lysine production and increased activity of stress related enzymes..S. C.P. Sebastiani. Plant Cell Tiss. Nikam. C. T. S..J.P.. Plant... J.. A.. 102:17–25. 24(5): 889-896.. 17: 305-316.. S... Plant Physiol. Wang.D.. Plant Nutr. TRENDS in Plant Sci.W.. Mittler. Chen. G. U. Pang. 2003.J. Nikam. 18:100 . G... M. osmolytes and in vitro growth responses in Sesuvium portulacastrum L. and Dodge.S.. and Skoog.. I.. 104:41–49. Effects of zinc on growth and antioxidant responses in Jatropha curcas seedlings.. Biol. C.S. Antioxidative enzyme activities in the leaves and callus tissues of salt-tolerant and salt-susceptible melon varieties under salinity.. Biol.. Gao.. F. Plant Nutr.L.Y. Lee... Plant.. Gollery. T. A. 2004. Hazra... Chen. APH Publ.D. 2003... A. Linsmaier. Diamantidis. Effects of optimal and supra-optimal salinity stress on antioxidative defence. Biotech.. Song. Oxidative stress. Physiol. and Singh.. . 2008. V.N. Molassiotis. Mehta. Z.) seedlings—Its effect on lipid peroxidation and on the antioxidative enzymes catalase and guaiacol peroxidase. Heazlewood. Diamantidis. Lett. Phytochemrstry.S. Millar. Polyamines in plants: An overview. Tsirakoglou. Lu. M. Accumulation of cadmium in growing peanut (Arachis hypogaea L. L.. X.J.. 9(10): 490-498. G. Z. Y. J.. Fan.. A. 1965. African J... Zhang. Y. V.. V. S. and Gwozdz. L. and molecular effects of in vitro induced iron deficiency in peach rootstock Mr.... Kopyra. A. F. Organ Cult. Galston... Tang. X. F.B. Lombardi. Knox. Plant Sci. J. Kumar. Lokhande. R. mannitol or sorbitol..... 2007. G. Sebnem Ellialtioglu.. R.. Tiburcio.I. eds.L.H. Suprasanna.N. Plant Tissue Culture. J. V. Molassiotis.C. J. 1985. Antioxidant enzymes in paraquat and cadmium resistant cell lines of horseradish.S 2/5. 12(1): 119-124.C. Dimassi. Seo.. A. Vanderauwera. S. Sotiropoulos. S. A.2005. 50 (1): 61-68. 26: 1413-1420... New-Delhy. 40(1): 61-69. dactylon) and their physiological responses to salt and drought stress. 11(3): 635-641. Patade. 2010. F.N. L. Organ Cult. Ahire. 2004.. Tanou.. Lee. S..Control of ascorbate synthesis by respiration and its implication for stress responses. Soil Sci.S.. In vitro selection of salinity tolerant variants from tripkoid bermudagras (Cynodon transvaalensis x C.. Int. L. V. Therios..F. Organic growth factor requirements of tobacco tissue culture..A. I. Agri. S.. 2006. ISBN: 978-81-313-0439-6. Kim. T. D. 2: 1-12. G. Fikret Yasar. J. G. F.. Biochemical. Penna S. Theodoulou.

A. C. H. 2006. Physiol.. M.. Niknam. A. F. 159: 213-222. Gouesbet. 1998. roots and shoots.A Tool in Biotechnology: Basics and Application. J... Farrant. and Foyer. Gomes-Laranjoa. Noctor. 50(4): 591-596. Int. 71: 89-98... V... pp. Ascorbate and Glutathone: keeping active oxygen under control. Corbineau. 9(28): 1-18. Plant Cell Tiss Organ Cult.P.. in Rao. Pevalek-Kozlina.. R. Springer-Netherlands. Plant. K..4-D and paraquat on non-green potato tuber cali.M. eds. Physiology and Molecular Biology of Stress Tolerance in Plants.. Agri.. and Ebrahimzadeh. Springer-Netherlands.M. M. 2005. 2006. H. Ebrahimzadeh. 15:473-497. A. Influence of NaCl and mannitol on peroxidase activity and lipid peroxidation in Centaurea ragusina L. Madeirac. The Plant Journal.. Peixoto.. 2009. Singh. Radic´.. 163: 1284—1292.. K. lipid peroxidation and free radicals production in Borage officinalis induced in vitro. Bailly. Ramel. 2012. The effect of NaCl on antioxidant enzyme activities in potato seedlings. 2006. M.J.. J. 2009.A. Springer-Netherlands. in Rao. Expr.. A. R. protein and proline contents. Bouteau.V. Mohamed. A revised. 2006.. J. K. Introduction. Gangola.K. Couee. and Ebrahimzadeh. I. M. Sci..M. Plant Physiol. Effect of NaCl on biomass. H.K. Patade. F. eds. T. Radic´-Stojkovic´. C. A..V. J. P. Raghavendra. C. 2006. Sharifizadeh.. ISBN 978-3-540-93882-8. M... Plant Mol. 2. K. Dhawan. and Skoog. 2008.A. . Rahnama.R. 2006..P. H.. Raghavendra. antioxidant enzymes in seedlings and calli of two Trigonella species. C... Plantarum. Berlin.. K. Rao. In vitro selection and characterization of a drought tolerant clone of Tagetes minuta... J. Biol.S. and Raghavendra. K. of Iran.. Harris. Rev. Biol. Razavi. Plant.. J. 2007..Y. K. Sulmon. G.. A.. J.. 108:279–286.. 17(3): 225-230.-H. BMC Plant Biol. Physiology and Molecular Biology of Stress Tolerance in Plants. Suprasanna..medium for rapid growth and bioassays tobacco cultures. K.-M. Bhargava... Rahnama. H. 49:249–79. E.. J..J. B. 6(1): 179-184. 49(1): 93-97. G. Plant Cell and Tissue Culture .. Biol.) under salt stress... F..... ISBN 10-1-4020-4224-8. Belghazi. Kumar.C. A. Plant Physiol. V. Antioxidant isozymes activities in potato plants (Solanum tuberosum L.. 50: 452–465. B.J... Reddy. Reddy. Developing stress tolerant plants through in vitro selection-an overview of the recent progress.C. Botany. Cooper.. Henderson.. Job.. Islamic Rep... Oracz. S. 1962. Germany. Raghavendra. M.. Springer-Verlag.S.. P. ROS production and protein oxidation as a novel mechanism for seed dormancy alleviation.A. H. D. A.. Bogard. Effects of NaCl and iso-osmotic PEG stress on growth. Rao. 2004.. Reddy.. Kalia.M. Plant Sci..S. J. N..M. A.. Vicenteb. pp.J.A. K.. Diffrentia pattern of reactive oxygen species and antioxidative mechanisms during atrazine injury and sucrose-induced tolerance in Arabidopsis thaliana plantlets. F.V. Photooxidative stress. Physiology and Molecular Biology of Stress Tolerance in Plants. Env. 2011.86 Antioxidant Enzyme Mohamed.S. Annu. 165:1125—1133. eds. 157-186. 2000. Murashige. Iron deficiency stimulated some enzymes activity.. and Aly. Heidelber. 1-14. Reddy.H. Plant Physiol.. osmolytes accumulation and antioxidant defense in cultured sugarcane cells. Biol. S. V.. Comparative effects of the herbicides dicamba.V. Rai..K. Job. K. Imani.-H. Neuman. eds.M.

J. 51 (1): 177-180.. T. Ecotoxicol... G. Shri.... R. 2004. S. Hyperhydricity in micropropagated carnation shoots: the role of oxidative stress. Effects of Iso-osmotic Concentrations of NaCl and Mannitol on some Metabolic Activity in Calluses of Two Salicornia species. J. G.H. and Hildebrandt.. Saf. A. S. S. Effects of various chemical agents for alleviation of drought stress in rice plants (Oryza sativa L. Biol. Ozdemir.. D. Characterization of Somaclones of Medicago sativa L. Fernandez-Ocana. Ravishankar. U. Hort... J. Trivedi. S. Biotech. Biol. Ltd. Appl.. 120: 152–161.) mutants induced with gamma radiation. Mallick. Growth... A. 103-112.(Abstract-in press). Valderrama. Giridhar.S.. 2010. G. J. J.E. S. Plant.. Biochemical analysis of drought tolerant sugar beet (Beta vulgaris L. R... 20: 489-495. J. Pedrajas. ISBN 10-1-4051-2529-2.. Y.. Sen. E.... Safarnejad. A. Yerlikaya. 2008.R.. 2005. Therios.. 38 (1): 139-148. O.S. Chakraborty.) Wilczek. Saher. and antioxidant system in rice seedlings. Mouhtaridou. Thangavel.. Dimassi. and Niknam... Carreras. 2007.. A. Environ..Z.. Chaki. 45:540–549.. Int.K. Environ. 47:734–742. Diamantidis. S. Nedev. Changes in phytochelatins and their biosynthetic intermediates in red spruce (Picea rubens Sarg. Effect of salt stress on growth parameters and antioxidant enzymes of different wheat (Triticum aestivum L.. Molassiotis.A. Gomez-Rodriguez. 1972. V. L. Corpas.Biol. Sivritepe. Schenk. Canadian J. A. El-Beltagi. Srivastava. Sajid. S.. Rajasekaran.... Piqueras. 2012. H. 72: 1102-1110. S.B. Hellin.. and antioxidant responses of the apple rootstock MM111 shoots cultured under high boron concentrations in vitro. Barrosa. Torabi. by exogenous application of ascorbic acid.) cell suspension cultures under cadmium and zinc stress.. Blackwell Pub...K. 2009... Sci. M. F. N....C. R.. M. 2006. chlorophyll. N. Plant Physiol. Effect of arsenic on growth.—Plant.G. A..).—Plant. R. Ahmed. osmotic potential and in vitro regeneration of soybean cultivars.. Del Rio. Shukla. In Vitro Cell.. The dehydrogenase-mediated . Cluj... M. Bot. P. ed. G.Dev.. F... for Drought Tolerance. 50:199-204. T. 2007. Physiol. 34 (1-2). S.. Sen.D. Antioxidants and Reactive Oxygen Species in Plants. Agric.. Chakrabarty.A. M. nutritional status. C. Sotiropoulos. Technol.. 6: 121-127.. Gen. I.. P. Tripathi.. Plant. S. Misra. G.. G... 2008. Sotiropoulos. Radiation-induced phenotypic alterations in relation to isoenzymes and RAPD markers in Vigna radiate (L. I. proline and sugars in the apple rootstock M 4 cultured in vitro. Olmos... Amelioration of salinity tolerance in Solanum tuberosum L. Kumar. Kosturkova. Shehab. Fress.Oxidative Stress Studies in Plant Tissue Culture 87 Roy. Bor. Sakthivelu.. In Vitro Cell. Long. A. Begum. Radiat. Drought-induced alterations in growth. Effect of NaCl and CaCl2 on growth and contents of minerals. Plant Cell Tiss Organ Cult.. E. Raychaudhuri. oxidative stress. D. 82(11):823-832.. Tuli.. F. M. J. T. 88:201–216.. Minocha. and Alikamanoglu.V. A. chlorophyll content. Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. and Alikamanoglu. P. A. Turkan. 2011... 2009.) varieties on in vitro tissue culture. Smirnoff.Bot.K. Mishra.E. 2006.. 2006. Response of the cherry rootstock to water stress induced in vitro. 29: 575-583. D. 52(3): 573-576. Not. A... P. S. Erturk. 2004.. Agrobot. Biol. and Aftab. T. R. Devi. Plant.Biol. Bull. K. 2011. Almaliotis..Dev.. Plant Nutrition. A.

Lv. The effect of salt stress and abscisic acid on proline production. cotyledons. Nickel toxicity induced antioxidant enzyme and phenylalanine ammonia-lyase activities in Jatropha curcas L. 29: 1449-1459.. 102:387–395. Organ Cult. Plant Soil Environ.. Biotechnology of Plant Tissue. Tan . Yang. Effects of cadmium on growth and antioxidant responses in Glycyrrhiza uralensis seedlings. Zhang . and Tyagi . X. 2005.J. chlorophyll content and growth of in vitro propagated shoots of Eucalyptus camaldulensis.. S.E.. Rasmussen.. Carpena-Ruiz.R. Zuniga.. Plant Cell Tiss Organ Cult. Fan . S. Villasante. A. Discovery Publ. 56(11): 508515. F. Effect of salinity on antioxidant enzymes in calli of the halophyte Nitraria tangutorum Bobr. S.. Gao. Plant Cell Tiss Organ Cult.F. Chen. R. Zamora. www. 2007... S.. Yadav.. 176: 96-107.kitchenculturekit. Affordable plant tissue culture for the hobbyist. Li.J.. Q. New Phytol..P.. I.. P.. L. Yan. Effect of salt stress on growth and osmotic regulation in Thellungiella and Arabidopsis callus. Wang.. . ISBN 81-8356-073-3. Xu...R.O. Wang. F.. G. www. X. Del Campo. NewDelhi. Antioxidant responses of in vitro shoots of Deschampsia antarctica to polyethylene glycol treatment.. L. A.. April..J. Rapid alteration of cellular redox homeostasis upon exposure to cadmium and mercury in alfalfa seedlings. Yang.. Cao. Y. Kang.E.. 2010... April. An. H.. Plant Cell Tiss. R. [Accessed.88 Antioxidant Enzyme recycling of NADPH is a key antioxidant system against salt-induced oxidative stress in olive plants..X. Prieto. G. 2010.. Rellan-Alvarez.L. G. C..X.. Shi. 54(7): 294-300. 22(2):163–169. 7th 2012]. India. H. M.. J.... Woodward. P.B. 2006. Gao.. 82: 189–200. Hernandez. Shi. Use of plant tissue culture.. R. Plant Soil Environ.. 7th 2012]..htm. Y. R. and Bennett.N. Liu . Copper-induced oxidative stress in Alternanthera philoxeroides callus. Plant Cell Environ. 2009... Wei. 106:243–251. Zhao...R.O.. Y. Chen. Wang..liv. 2008. H.. Pardo. R. Plant Cell Tiss Organ Cult. 98:97–103. [Accessed. Y... W. Antarctic Scie. S. 2011. eds. 2010. X. G..

1. because it is a continuous process during the life span of every cell in the organism. The main sources of ROS/RNS are: exogenous ( irradiation. mitochondrial respiration. This detoxification pathway is the result of several enzymes. rheumatoid arthritis. cancer.1) catalyzing the first step O2 into H2O2 and Catalase (CAT.1. Parkinson. cardiovascular and liver diseases several of them are related with low levels of IGFs such as degenerative and aging disorders [1-8]. Although the exposure of the organism to ROS/RNS is extremely high from exogenous sources. Amyotrophic Lateral Sclerosis…). enzyme activities.         © 2012 Muñoz Morón and Castilla-Cortázar. aging and diseases). 1. These oxidative damages lead to the cellular death. The mechanisms of oxidative cellular damage are summarized in the exposure to endogenous sources is much more important and extensive. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.9) removing H2O2 into H2O by two different pathways. drugs. 1. . xenobiotics.Chapter 4 Protection Against Oxidative Stress and “IGF-I Deficiency Conditions” Úrsula Muñoz Morón and Inma Castilla-Cortázar Additional information is available at the end of the chapter http://dx. licensee InTech. proteins or membrane lipids (including mitochondrial lipids). which permits unrestricted use. with Superoxide Dismutase (SOD.15.11.11. Oxidative stress represents an imbalance between the production of ROS/RNS and a biological system's ability to detoxify the reactive intermediates or to repair the resulting damage. and reproduction in any medium. EC 1. UV irradiation. fagocytosis. distribution. Introduction Oxidative stress is thought to contribute to the development of a wide range of diseases including neurodegenerative (Alzheimer. In normal conditions ROS are reduced into water.5772/51047 1.0). For these reason cells are protected against oxidative stress by an interacting network of antioxidant enzymes. and toxin metabolism) and endogenous (metabolic pathways. EC. Disturbances in the normal redox state of tissues can cause toxic effects through the production of peroxides and free radicals exert deleterious effects on cell through direct attack on EC 1.doi. diabetes. oxidative burst. provided the original work is properly cited.6) and Glutathione Peroxidase (GSH-Px.1.

By these pathways. which is an endpoint to initiate cell death. a large number of studies have associated mitochondrial dysfunction caused by ROS/RNS to both accidental cell death (necrosis) and programmed cell death (apoptosis). Intramitochondrial ROS production increases after peroxidation of intramitochondrial membrane lipids. Recently. Continuous exposure to various types of oxidative stress from numerous sources has led the cell and the entire organism to develop defense mechanisms for protection against reactive metabolites. Catalase. and GSHPx.90 Antioxidant Enzyme Figure 1. because 2% to 3% of the O2 consumed is converted to O2-. ROS/RNS can also induce mitochondrial DNA deletions and mitochondrial membrane permeability transition (MMPT). oxidative damage leads to cellular death. Furthermore mitochondria are particularly sensitive to ROS/RNS-induced injury in the pathogenesis of disease. Mitochondrial like a source of ROS are summarized in Fig. The generation of hydroxyl radicals from hydroperoxide produces the development of oxidative cell injury: DNA damage. carboxylation of proteins. Free radicals are reduced into water with the cooperation of the three main antioxidant enzymes: SOD. Mechanisms of oxidative cellular damage. MMP pore opening activates caspases. . including lipids of mitochondrial membranes. and lipid peroxidation. 2. [5] Mitochondria are the major endogenous source of ROS underphysiologycal conditions. Oxidative stress exerts deleterious effects on mitochondria function by directly impairing oxidative phosphorylation through direct attack of proteins or membrane lipids.

cell motility. 7] and it is the main intracellular source of ROS our results show a novel mechanism for oxidative stress regulation through mitochondrial protection and normalization of antioxidants enzymes activities by IGFs signaling pathways.1. and iron. heme synthesis. Mitochondria contain their own DNA (mtDNA). cerevisiae identified at least 750 mitochondrial proteins that perform mitochondrial function . The mtDNA occurs in small clusters called nucleoids or chondriolites. Mitochondrial damage produces mitochondrial dysfunction decreasing MMP and ATP synthesis and increasing ROS production. While nuclear DNA encodes the majority of the mitochondrial proteins only a few of these proteins are encoded by mitochondrial DNA. A recent mitochondrial proteomic study in S. Mitochondrial damage Mitochondria play a central role in many cellular functions including energy production. 6. Mitochondrial damage is an intracellular source of ROS. Oxidative stress exerts deleterious effects on mitochondria function by directly impairing oxidative phosphorylation through direct attack of proteins or membrane lipids. The number of mtDNA molecules in nucleoids varies in size and numbers in response to physiological conditions. 2.Protection Against Oxidative Stress and “IGF-I Deficiency Conditions” 91 Figure 2. nucleotides. metabolism of amino acids. Understanding that mitochondria are the most important cellular targets of IGF-I [5. and lipids synthesis. Oxidative Stress: Mitochondrial damage and antioxidant denfences 2. 9]. respiration. cell proliferation and apoptosis [8. and maintenance of intracellular homeostasis of inorganic ions.

The electrochemical potential across mitochondrial membrane. Injured mitochondria can release cytochrome-c into the cytoplasm when cells are treated with . rhodamine 123 (Rh123). in most mammalian cells. Given that mitochondria are the major producer of ATP. These DNA lesions cause mutations in mtDNA that can lead to impairment of mitochondrial function [23]. e.. As an index of mitochondrial function in living cells. ROS also produce more than 20 types of mutagenic base modifications in DNA [22]. frame-shift mutation [25] sister chromatid exchange.92 Antioxidant Enzyme [10]. It is conceivable that mitochondrial damage contributes to muta-genesis of the nuclear genome in part due to impaired nucleotide biosynthesis. it is well established that an imbalance in the dNTP pool is mutagenic to cells [25]. 16]. recombination and doublestrand break [26]. is known to be highly sensitive to apoptotic stimulation. mitochondria are the principal organelles for ROS production. can generate ROS. Mitochondrial DNA (mtDNA) seems to be highly vulnerable to oxidative challenges compared to nuclear DNA for three main reason: 1) its close proximity to the electron transport chain (ETC).11]. which fluoresces in direct proportion to MMP [31]. such as H2O2 in primary neuronal cultures [32]. including ischemia. xanthine oxidase. Mitochondria are also a major site for the accumulation of low molecular weight Fe2+ complexes. not only because mitochondria were recognized to play a central role in apoptosis but also since mitochondrial genetic defects were found to be involved in the pathogenesis of a number of human diseases [9. hypoxia. The production of mitochondrial ROS is a consequence of oxidative phosphorylation at the respiratory chain complexes I and III where electrons derived from NADH and FADH can directly react with oxygen or other electron acceptors and generate free radicals [12-14]. Taken together. The last decade has witnessed an increased interest in mitochondria. and DNA recombination. DNA repair. uncoupled eNOS (endothelial NO synthase) and cytochrome P450 enzymes. Mitochondria do not only represent the major source of ROS production but they are also the major targets for their damaging effects. but. and myocardial infarction [27-30]. which promote the oxidative damage of membrane lipids [17-19]. A variety of cellular systems. DNA replication. it is also likely that mitochondrial dysfunction leads to the reduction in ATP level that may affect ATP-dependent pathways involved in transcription. Decreased MMP occurs in cells undergoing apoptosis induced by oxidative agents. Studies demonstrate that a dNTP pool imbalance can induce nucleotide insertion. Mitochondria are intimately involved in deoxyribose nucleoside triphosphate (dNTP) biosynthesis [24]. 2) it is continuously exposed to ROS generated during oxidative phosphorylation (it is estimated that up to 4% of the oxygen consumed by cells is converted to ROS under physiological conditions) [20] and 3) its limited capacity of DNA repair strategies and the lack of protection by histones [21]. this makes clear that mtDNA is extremely susceptible to mutation by ROS-induced damage. the increase of the redox potential at complex I and complex III induces ROS generation [15. MMP can be measured with an indicator dye. Indeed. Mitochondria also play a key role in regulation of apoptosis under a variety of pathological conditions. including NADPH oxidase. In fact.g. MMP.

and altered signal transduction during the aging process. ROS and RNS may react with fatty acids. Examples of ROS and RNS are superoxide. antioxidant defenses include the enzymatic and non-enzymatic defense systems regulate the ROS and RNS produced. Under physiological conditions.2. Recent studies show that treatment of aged rodents with IGF-I confer mitochondrial protection. including an attenuation of mitochondrial ROS generation in the liver [40-42]. 5. further studies are necessary to determine the role of mitochondrial mechanisms in beneficial effects of IGF-I treatment. In excess. Autophagy is a catabolic process that contributes to the maintenance of cellular homeostasis through the degradation of damaged mitochondria in lysosomes. . or in situations where defenses are compromised. dysfunction of the proteasomes [36] may be also contributed to the accumulation of damaged mitochondrial proteins in the age diseases. 5-7. oxidative stress. Mitochondria are highly dynamic organelles. these are counterbalanced by an array of defense pathways. which likely contributes to the accumulation of damaged non-functional mitochondria. Antioxidant defenses ROS and RNS consist of radicals and other reactive oxygen/nitrogen factors that can react with other substrates. thereby causing damage to these substrates. including the effects of IGFI on autophagy of dysfunctional mitochondria and apoptosis. different groups have described that IGF-I decreases mitochondrial superoxide production [37] and low levels of IGF-I have been linked to increase in oxidative stress damage [3. 40-42]. and its anti-apoptotic effects occur through engagement with IGFI receptor (IGF-IR) and thought to activate an intracellular signal transduction pathway that may modulate the mitochondria. proteins and DNA. peroxynitrite and hydrogen peroxide. Mitochondria are important cellular targets of IGF-I [5. thus preventing the damage caused by ROS and RNS. 42]. IGF-I also acts as an anti-apoptosis factor of multiple cell types. Under normal conditions. nitric oxide. which contributes to deregulation of cell metabolism. and deregulation of mitochondrial turnover is likely one of the intrinsic causes of mitochondrial dysfunction. In addition. and the promotion of healthy aging [35]. The available evidence suggests that there is an age-dependent decline in autophagic function. normal energy production. and it needs to be emphasized that ROS and RNS have many physiological roles that include signaling. Thus. 39].Protection Against Oxidative Stress and “IGF-I Deficiency Conditions” 93 proapoptotic stimuli [33]. Cytochrome-c activates the apoptosome containing the caspaseactivating protein Apaf-1 and subsequently the caspase cascade that induce apoptosis [34]. cytochrome c and caspase pathway [38. 6]. 2. Because macromolecules in mitochondria (including mtDNA) are particulary susceptible to oxidative damage of mitochondrial turnover is critical for the maintenance of a healthy mitochondrial phenotype. decreasing the production of ROS and RNS. Antioxidants regulate oxidative and nitrosative reactions in the body and may remove ROS and RNS through scavenging radicals. The available data suggest that treatments that increase circulating IGF-I levels exert citoprotective effects in aging and degenerative diseases [1-3.

lung disease. SOD3 is the primary enzymatic antioxidant defense of the vascular wall. and hydroperoxides. Glutathione Peroxidases (GPx). hypertension. Enzymatic defense systems such as Superoxide Dismutases (SOD). has been implicated as the source of some cases of familial Amyotrophic Lateral Sclerosis (ALS) [47. these are normally related with the development of mental disorders [57]. and placenta. formic acid. ischemia-reperfusion injury. Cu-Zn SODs exist as homodimers found in the periplasm. lymph. carbohydrate. rendering the potentially harmful superoxide anion less hazardous.1. CAT is a homotetramer encoded by ctt1 gene mapping in chromosome 11 [55]. The eukaryotic extracellular SOD is a subset of the Cu-Zn variety but functions as a tetramer rather than the usual dimmer found in eukaryotes. SODs require a metal cofactor for function and can be grouped by the bound metal ion [44-46]. Human Cu-Zn SOD. lungs. EC 1. It has been proposed that polymorphisms in these enzymes are associated with DNA damage and subsequently the individual’s risk of wide range of diseases susceptibility [42. which require both copper and zinc as cofactors (Cu-Zn SODs). and neurological diseases.1. Iron containing SODs have been found in prokaryotes and some plants. 48].5. SOD3 protein can be measured in plasma. Human extracellular Cu-Zn SOD. encoded by the SOD1 gene mapping to chromosome 21.18) protect mitochondria and DNA from oxidative stress. 53]. Genetic polymorphisms in catalase and its altered expression and activity are associated with oxidative DNA damage and subsequently the individual’s risk of cancer susceptibility [56]. The physiopathological role of SOD 3 has been examined in vascular-related H2O2 and O2. atherosclerosis. homocysteine metabolism . Almost all human tissues contain measurable levels of SOD3 and at least eight different tissues. EC 2. Glutation Reductases (GSR or GR. CAT is a common antioxidant enzymes found in nearly all-living organisms that are exposed to oxygen and they can also remove organic H2O2 to oxidize toxins including phenols. except rat heart mitochondria [54]. Catalases (CAT). the only enzymatic defense system against hydrogen peroxide in mitochondria is the glutathione redox cycle system. Catalase catalyzes the reduction of H2O2 to water using either an iron or manganese cofactor [52. Humans with low catalase levels (acatalasemia) have an increased risk for diabetes mellitus. CAT is present only or primarily in the peroxisome fraction and is absent in mitochondria of mammalian cells. Superoxide Dismutases catalyze the dismutation of O2. including heart. while the clinical features of acatalasemia are oral gangrene. encoded by the SOD3 gene mapping to chromosome 4 [49]. altered lipid. cerebrospinal. Cu-Zn SODs are homodimers in eukaryotes and are located predominantly in the cytoplasm. 43].7) and Glutathione Transferases (GST. and synovial fluids [50. synthesize SOD3 mRNA. diabetes. MnSOD is encoded by the SOD2 gene mapping to chromosome 6 in human.8. Enzymatic defense systems.94 Antioxidant Enzyme a. 51]. In most prokaryotes. A few polymorphisms have been described for the catalase-encoding gene. The third class represents SODs. Manganese SODs (MnSOD) are found in both prokaryotes and eukaryotes and are localized primarily to the mitochondria. Therefore. various inflammatory conditions.

GST. The enzyme forms a FAD-bound homodimer. These enzymes catalyze nucleophilic attack by reduced glutathione (GSH) on nonpolar compounds that contain an electrophilic carbon. 6. There are at least eight GPx enzymes: GPx1–GPx8 [60. Three major families of proteins that are widely distributed in nature exhibit glutathione . Glutathione Peroxidase (GPx) are critical intracellular enzymes involved in the reduction of hydrogen peroxide H2O2 to water and lipid peroxides to their corresponding alcohols using selenium cofactor in the majority of the cases. Glutathione Transferases have also been called glutathione S-transferases. and α. The GR is conserved between all kingdoms. in plant genomes. if the PPP is non-functional. like red blood cells. 61]. GPx6 from rodents and GPx5 from both humans and rodents do not contain Sec or Se [69]. seem to be asymptomatic. 67] and has a peroxidase independent structural role after sperm maturation [68]. 78]. Expression of GPx3 is greatest in the kidney. Lower GPx activity predispose towards an impaired antioxidant protection and consequently stress oxidative damage to membrane fatty acids and functional proteins and. 61]. by inference. For every mole of oxidized glutathione (GSSG). although this enzyme is expressed in various tissues and is secreted into extracellular fluids as a glycoprotein [63. The GPx 1-8 genes mapping to chromosomes 3.β-unsaturated carbonyls [8084]. one mole of NADPH is required to reduce GSSG to GSH. 14. GPx are present both in the cytosol and in mitochondria [59]. Recently. arene oxides. GPx4. one gr gene is found.Protection Against Oxidative Stress and “IGF-I Deficiency Conditions” 95 and the increased risk of diabetes mellitus [58] and lower levels of catalase activity in other tissues. and hence the process of neuroprogression that accompanies severe or persistent illness [74]. In bacteria. GPx6 was identified as a selenoprotein in the human genome by homology search [69]. Human GSR gene mapping to chromosome 8. In these organisms. or sulphur atom. GSR is a homodimer found are present both in the cytosol and in mitochondria. is an enzyme that reduces glutathione disulfide (GSSG) to the sulfhydryl form GSH. but rather a monomer. glutathione reduction is performed by either the thioredoxin or the trypanothione system. 5. yeasts. respectively [77. Their substrates include halogenonitrobenzenes. GPx7 and GPx8 were also identified as seleniumindependent GPx can act as true H2O2 scavengers. 64]. In addition. GPx4 contains a mitochondrial isoform that mediates the apoptotic response to oxidative stress [66. however. to neurotoxic damage. In cells exposed to high levels of oxidative stress. GPx2 expression is most prominent in the gastrointestinal tract [62]. up to 10% of the glucose consumption may be directed to the pentose phosphate pathway (PPP) for production of the NADPH needed for this reaction. which is an important cellular antioxidant [75. 6. and is the only GPx enzyme that reduces phospholipid hydroperoxides [65]. However. Drosophila and Trypanosomes do not have any GR at all [76]. or phospholipid hydroperoxide GPx. as expected of the selenium-dependent members [70-73]. two gr genes are encoded. 1 and 5 respectively. is not a tetramer. then the oxidative stress in the cell will lead to cell lysis and anemia [79]. Glutathione Reductase. Different from other glutathione peroxidases. 76]. quinones. Whereas GPx1 is the most abundant selenoperoxidase and is ubiquitously expressed in almost all tissues [60. In the case of erythrocytes. nitrogen. also known as GSR or GR. 19. and animals.

These mitochondrial-targeted drugs can achieved concentrations in the mitochondrial matrix 100. Neurodegenerative diseases such as PD and schizophrenia are characterized by the degeneration of dopaminergic neurons. A significant decrease of glutathione transferase activity was described in amygdala. The Mu class of GSTs has five genes (GSTM1–5) [90] that are found in a gene cluster on chromosome 1 [91]. Pi. The chromosomal locations of the genes that encoded for the different types of GST and the related diseases are summarized in table 1. and larynx [94. thioredoxin (Trx). Hence. CoQ10 is a strong anti-oxidant that confers resistance to mitochondrial damage by decreasing ROS/RNS production and that may suppress the production of proinflammatory substances. These antioxidant systems thus protect the tissues against ROS and RNS. Cytosolic GSTs of mammals have been particularly well characterized. The GSTM1 gene contains four alleles that have been associated with a decreased risk of bladder cancer [92]. is essential to electron transport and ATP production via the respiratory chain. Examples of non-enzymatic defense systems are scavenger antioxidants (coenzyme Q10. a ROS generated in the redox cycling of orthoquinones within dopaminergic neurons [93]. transferrin. like nuclear factor κ B (NFκB) gene expression and the production of pro-inflammatory cytokines [95-100]. play an important role in cellular protection against oxidative stress and they are the most abundantly expressed glutathione S-transferases in liver. located in a cluster mapped to chromosome 1000-fold higher than those in the cytosol because of their strong positive charge. comprise soluble enzymes that are only distantly related [85. Coenzyme Q10 (CoQ10) is an endogenous compound found in the inner mitochondrial membrane. b.96 Antioxidant Enzyme transferase activity. 86]. pharynx. GSTM2-2 has been proposed to play a protective role against neurodegenerative diseases. The alpha class genes (GSTA1-5). Mu. The null phenotype is associated with an increased risk of tumors of the head and neck. an inability to detoxify carcinogens is associated with an increased risk toward a variety of cancers. Zeta and Omega classes on the basis of a substrate/inhibitor specificity. The third family comprises microsomal GST and is now referred to as membrane-associated proteins in eicosanoid and glutathione (MAPEG) metabolism [87]. Decreased in alpha class GSTs has been observed in stomach and liver tumors. 95]. SS-peptides and acute phase proteins such as albumin. Non-enzymatic defense systems. and were originally classified into Alpha. The lack of enzyme activity. vitamin C and E. Two of these. The Theta class of GST has a null phenotype whereby individuals do not express catalytically active protein. and therefore. and glutathione) and some proteins which act as antioxidants by binding ROS and RNS. CoQ10 and vitamin E are lipophylic antioxidants that are target to mitochondrial matrix by Tripenylphoshonium ion (TPP+) (mitoQ and mitovitamin E). the cytosolic and mitochondrial GST. primary and tertiary structure similarities and immunological identity [88]. Theta. hippocampus and inferior parietal lobule in patients with AD [89]. e. as mitochondria have a highly negative . GSTM2-2 has been shown to catalyze the conjugation of GSH to aminochrome.g. oral cavity. haptoglobin and ceruloplasmin.

Protection Against Oxidative Stress and “IGF-I Deficiency Conditions” 97 Antioxidant Enzymes Subcellular Location Cytosol Chromosomal Location Chromosome 21 E.1.9 GPx4 GPx5 GPx6 GPx7 GPx8 Cytosol/Mitochondrial Chromosome 19 Chromosome 6 Chromosome 6 Chromosome 1 Chromosome 5 Glutathione Reductase GSR or GR. diabetes.11.7 Alpha.1 SOD2 (MnSOD) SOD3 (Cu-Zn SODs) Mitochondrial Chromosome 6 Extracellular Chromosome 4 Catalase CAT. M3-3 Cytosol/Mitochondrial Chromosome 8 Chromosome 6 Chromosome 6 Chromosome 6 Chromosome 6 Chromosome 6 Red blood diseases Cytosol Chromosome 1 Chromosome 1 Chromosome 1 .1. asthma and degenerative disease Vascular-related diseases. A1-1 Alpha. hypertension. A3-3 Alpha.15. Diabetes mellitus. atherosclerosis. EC.C. EC 1. A2-2 Alpha. ss EC 1. Subtypes SOD1 (Cu-Zn SODs) Related Diseases Familiar Amyotrophic Lateral Sclerosis (ALS) Cancer. M2-2 Mu. ischemiareperfusion injury. and neurological diseases Mental disorders. Superoxide Dismutases SOD. A5-5 Mu.11.1.lung disease. various inflammatory conditions. A4-4 Alpha. EC 1. M1-1 Mu. GPx1 GPx2 GPx3 Peroxisome fraction Chromosome 11 Chromosome 3 Chromosome 14 Chromosome 5 Mental disorders Glutathione Peroxidases GSH-Px.

T1-1 Theta. Several other studies have used MitoQ10 in a variety of animal models of disease [102] and the results indicate that MitoQ10 protects against liver damage in an animal model of sepsis [103]. decreasing blood glucose.98 Antioxidant Enzyme Antioxidant Enzymes Glutathione Transferases Subcellular Location Chromosomal Location Chromosome 1 E. O2-2 Kappa. K1-1 gp I. MGST3 gp IV. EC 2.1. enhancing endothelial function and improving cognitive function in both Alzheimer’s and Parkinson’s disease patients [101]. However. LTC4S gp II. Z1-1 Omega. the first clinical evidence of a potential benefit of MitoQ10 in humans comes from a study that MitoQ10 reduces liver damage induced by hepatitis virus infection [107]. liver diseases and cancer Mu.18 Subtypes Mu. one of the major limiting factors in the use of CoQ10 as a supplement is its bioavailability and delivery to the source of ROS generation. limiting tumor growth.5. Enzymatic defense systems membrane potential between -150 mV and -180 mV. . P1-1 Theta. PGES1 Microsomal Chromosome 1 Chromosome 11 Chromosome 22 Chromosome 22 Chromosome 14 Chromosome 10 Chromosome 10 Mitochondrial/peroxisome Chromosome 7 Chromosome 4 Chromosome 4 Chromosome 1 Chromosome 1 Chromosome 12 Chromosome 9 Table 1. Both clinical and rodent studies have reported moderately beneficial actions of CoQ10 in reducing blood pressure. forestalling myocardial damage secondary to chemotherapeutic administration. O1-1 Omega.C. M4-4 Related Diseases Neurodegenerative. M5-5 Pi. contributes to the aetiology of the metabolic syndrome and atherosclerosis in a mouse model [104] protects pancreatic β-cells against oxidative stress and improves insulin secretion in glucotoxicity and glucolipotoxicity [105] and even protects against oxidative stress and cell death in the brain of rats exposed to the insecticide dichlorvos [106]. Importantly. T2-2 Zeta. FLAP gp I. MGST1 gp IV. MGST2 gp I. GST.

cytosolic (TrxR1) and mitochondrial (TrxR2). namely glycine. 92].Lys) contains two active Cys residues (Cys32 and Cys35 in human Trx1 and Cys90 and Cys93 in human Trx2) that are oxidized into corresponding disulphides due to the transfer of two reducing equivalents from Trx to a disulphide-containing substrate. Although the positive charge might explain the mitochondrial-targing effect. Glutathione has three major functions: 1) it is a strong antioxidant that protects cells against damage caused by free radicals and it recycles vitamin C and E. . Importantly. The promoter of the Trx gene contains a series of stress responsive elements. as they are concentrated even in depolarized mitochondria [91. Thioredoxin (Trx) is a multifunctional low-molecular weight protein containing an active thiol/disulphide site and possessing oxido reductase activity. glutathione may help increase the resistance to bacterial and viral infections. the active form of glutathione. Trx and GSH systems are involved in a variety of redox-dependent pathways such as supplying reducing equivalents for ribonucleotide reductase and peptide methionine sulphoxide reductase. Cysteine is the rate-limiting step in the synthesis of reduced glutathione (GSH). Originally discovered in E. TrxR is an NADPH-dependent homodimer of oxidoreductase which reduces the active centre of disulphide in oxidized Trx and has two major isoforms. such as SP1. The major Trx isoforms are cytosolic Trx1 and mitochondrial Trx2. SS-Peptides the Szeto-Schiller (SS) compounds are tetrapeptides with an alternating aromaticcationic amino acids motif. so that they again become active as antioxidants after been used in antioxidant processes. Thioredoxins evolved similarly to chaperone-like proteins. AP-1. NFkB and antioxidants response elements (ARE) [118].Protection Against Oxidative Stress and “IGF-I Deficiency Conditions” 99 Glutathione is formed in the liver from three amino acids. the mitochondrial uptake of these SS peptides appears to be on mitochondrial potencial. Cellular redox regulation of many processes is provided by the cooperation between the Trx and glutathione systems [115. various transcription factor binding sites. whose function is maintenance of the dithiol/disulphide structure of proteins. A highly conservative amino acid sequence of the active centre (Trp-Cys-Gly-Pro-Cys. Using these peptides in an animal model of ischaemia/reperfusion injury improves cardiac function [112]. By reducing mitochondrial ROS production. c) Glutathione is a natural purifier and therefore high concentrations are found in the liver. pre-clinical studies support the use of these peptides during ischaemia/reperfusion injury and neurodegenerative disorders [114]. SS peptides are capable of scavenging H2O2 and ONOO− and inhibiting lipid peroxidation. coli. Whereas treatment with SS peptides attenuated mitochondrial H2O2 release induced by a high-fat diet and preserved insulin sensitivity in skeletal muscle [113]. b) Glutathione is employed by the white blood cells as a source of energy used for lymphoproliferation. 116]. The disulphides formed in the active centres of Trx1 and Trx2 are reduced by thioredoxin reductase (TrxR). Trx was later found in many prokaryotic and eukaryotic cells. glutamine and cysteine. and demonstrated in the inner mitochondrial membrane more than 1000-fold in comparison with the cytosolic concentration [108-110]. The Trx system plays a key role in cell function by limiting oxidative stress directly via antioxidant effects and indirectly by protein – protein interactions [115]. Therefore. the latter being involved in antioxidant defense and regulation of the cellular redox state [117]. these molecules inhibit MMPT and cytochrome c release and so prevent oxidant-induced cell death [111].

100 Antioxidant Enzyme c. As the regeneration of GSH and reduced Trx2 depends on the NADPH/NADP+ redox state. an efficient mitochondrial bioenergetic function is required to maintain antioxidant activity. Mitochondrial and cell cytosolic antioxidant systems can neutralize excess mitochondrial ROS under most conditions. Antioxidant defense systems against mitochondrial ROS formation. but may be modulated by either mitochondrial ROS themselves or changes in antioxidant defenses. Matrix ROS can also pass through the MMTP. Thus the efflux of H2O2 from the mitochondria is relatively modest. Complex III generates ROS on both sides of the mitochondrial inner membrane and in the intermembrane space. where CuZn SOD converts O2− into H2O2. The mitochondrial respiratory chain. With the exception of generation at complex III. which uses four electrons to reduce molecular oxygen to water (Fig. located in the inner mitochondrial membrane (IM). and cytochrome c (cyt c). Electrons then move down an electrochemical gradient through CoQ to complex III. the increase of the redox potential at complex I and complex III induces ROS generation [15. cyclophilin D (cyp D) and the adenine nucleotide translocator (ANT). Antioxidant defense systems against mitochondrial ROS formation . The production of mitochondrial ROS is a consequence of oxidative phosphorylation at the respiratory chain complexes I and III where electrons derived from NADH can directly react with oxygen or other electron acceptors and generate free radicals [12-14]. 16]. 3). Figure 3. which diffuses in the cytosol where catalase reduces it into H2O. directly to the cytosol where Cu-Zn SOD catalyses dismutation to H2O2 [119] which is then reduced to H2O by catalase. and from cyt c to complex IV. Complex I accepts electrons from NADH and complex II accepts electrons from succinate. ROS production in mitochondria is exclusively directed towards the matrix where MnSOD catalyses dismutation to H2O2 [119] which is then reduced to H2O by GSH and Trx systems (Trx2) [120]. coencymeQ (CoQ). is composed of four multimeric integral membrane proteins complexes (complexes I-IV). from complex III to cyt c. Indeed. formed by voltage-dependent anion channel (VDAC).

is tighly coupled with RTKs activated by various growth factors such as IGFs. These PH domain proteins. Regulation of enzymatic antioxidant defense systems. Through PTEN. This leads to antioxidant gene expression that protects the cell. 126].5 bisphosphate (PIP2). wherein the membrane bound PIP3 serves as a signaling molecule to recruit proteins containing the pleckstrin homology (PH) domain. The most studied transcription factors are: Nrf2 transcription factor: Phosphorilated Nrf2 translocates to the nucleus and binds the ARE. PI3K pathway. In order to prevent oxidative stress. the PI3K pathway is subject to reversible redox regulation by ROS generated by growth factor stimulation.122]. Platelet-Derived Growth Factor (PDGF). drug transport. a cytoplasmic peroxiredoxin isoform that eliminates H2O2 generated in response to growth factors [125]. It was also demonstrated that endogenously generated ROS following treatment with peptide growth factors such as IGFs. EGF. H2O2 was shown to oxidize and inactivate human PTEN through disulfide bond formation between the catalytic domain Cys-124 and Cys-71 residues [125. therefore. However. it is not known whether these oxidants induce PTEN oxidation and inhibition of phosphatase activity leading to gene activation. It is noteworthy that various oxidants and ROS-producing chemicals activate transcription of a battery of antioxidant genes through a 2 (Nrf2)antioxidant response element (ARE) mechanism. consisting of p110 cataytic subunit and p85 regulatory subunit. or PDGF causes oxidation of PTEN leading to the activation of the PI3K pathway [127]. and antioxidant enzymes.Protection Against Oxidative Stress and “IGF-I Deficiency Conditions” 101 d. The synthesis of PIP3 is negatively regulated primarily by the phosphatase and tensin homology (PTEN) phosphatase. ARE driven expression of detoxifying and antioxidant enzymes and the cystine/glutamate transporter involved in GSH biosynthesis [121.4. Nerve Growth Factor (NGF). Epidermal Growth Factor (EGF). PTEN oxidation is reversed by peroxiredoxin II. . PI3K catalyzes the synthesis of the second messenger phosphatidylinositol 3. Antioxidant enzymes play a major role in reducing ROS levels. A role for NRF2 in drug resistance is suggested based on its property to induce detoxifying. redox regulation of transcription factors is significant in determining gene expression profile and cellular response to oxidative stress. There are different transcription factors involved in regulation of antioxidant enzymes and they can be regulated though IGFs signaling and others pathways related with receptors tyrosine kinases (RTKs). which dephosphorylates PIP3 back to PIP2 [124]. where PTEN knockdown enhances transcription of ARE regulated antioxidant genes [128]. PI3K is recruited to activate RTK dimers through a SH2 domain in the PI3K p85 regulatory subunit. The PI3K/Akt/PP2A/GSK3ß and PKC/GSK3 play a role in regulation of Nrf2 and antioxidant gen regulation. the cell must respond to ROS by mounting an antioxidant defense system. such as the phosphoinositide-dependent protein kinase (PDK) and protein kinase B (AKT) serine/threonine kinases are thus activated and mediate further downstream signaling events [123]. and Vascular Endothelial Growth Factor (VEGF).5 triphosphate (PIP3) from phosphatidylinositol 4.

High ROS levels increase antioxidant enzymes expression via Nfr2/ARE and low ROS levels decrease antioxidant enzymes expression via FOXO. lipids and proteins. H2O2 treatment increases FOXO transcriptional activity and translocation of FOXO from the cytoplasm to the nucleus and activation of the transcription factor. Activation of PI3K/PKB signaling decreases FOXO activity and thus the levels of FOXO target genes like MnSOD and catalase [130]. Their regulation via PI-3K/PKB/FOXO signaling therefore implies that insulin. may modulate the cellular ROS level. 6.102 Antioxidant Enzyme FOXO transcription factors: FOXO-mediated upregulation of MnSOD expression results in considerable lowering of cellular ROS [129]. Active JNK induces the phosphorylation of on FOXO. Increase in ROS enhances FOXO transcriptional activity. This regulation may contribute to the citoprotective effects of treatment with low doses of IGF-I in experimental “IGF-I deficiency” conditions [2. leading to a decrease in ROS levels. 5. . This model is summarized in Fig. IGF-I decreases mitochondrial ROS production and IGF-I/Akt pathway is involved in Nrf2 and FOXO activity. which will in turn lead to the phosphorylation and activation of the stress kinase JNK. Phosphorylation of these residues is essential for FOXO transcriptional activity as shown by mutational analysis. through this signaling cascade. Thus. activation of FOXO by oxidative stress is part of a negative feedback loop to reduce the levels of oxidative stress in a cell. preventing damage to DNA. Activation of FOXO through can now induce transcription of MnSOD and CAT. 40-42]. Figure 4. both transcription factors involved in the antioxidative enzymes regulation. 4. and thus functions as a feedback mechanism. IGF-I exerts a dual role depending on ROS concentration. We propose that IGF-I can exert direct effects on cells and can alter in opposite ways the expression of antioxidant enzymes depending on the ROS levels. Based on oxidative stress is an imbalance between the production of ROS and antioxidative defenses systems. Regulation model of antioxidant enzymes mediated by IGF-I. An increase in ROS levels induces activation of the small GTPase Ral. Consistent with this.

131] and in others “IGF-I deficiency” conditions such as liver cirrhosis where there are a diminution in IGF-I levels but not in GH levels followed of a decrease in liver biosynthetic capacity. From these results we suggested that aging seems to be an unrecognized condition of “IGF-I deficiency.139. It is known. including mitochondrial protection [2. IGF-I. In cirrhosis the reduction of receptors for GH in hepatocytes and the diminished ability of the hepatic parenchyma to synthesize cause a progressive decrease in serum IGF-I levels [135]. 6. Our team results show that exogenous administration of low doses of IGF-I restores IGF-I circulating levels and some agerelated changes. Circulating IGF-I serum levels decline by more than 50% in healthy older adults [132. In order to reproduce of “IGF-I deficiency” condition and the possible benefices of treatment with low doses of IGF-I.” The best-known condition of “IGF-I deficiency” is Laron’s dwarfism [134]. 32. and showed hepatoprotective and antioxidant properties. liver contents of pro-oxidative metals [138] (iron and copper).140]. Experimental model of cirrhosis: Male Wistar rats in which liver cirrhosis was inducted using CCl4. Old animals were randomly assigned to receive either saline or human IGF-I [5. however. 137-139]. The identification of physiological regulators of antioxidative processes is critical to the understanding of degenerative diseases and aging processes. IGF-II concentrations decline with age. In these experimental groups we measured the oxidative stress by determination of: Total serum antioxidant status [40]. characterized by an absence of GH receptors in the liver. improving glucose and lipid metabolisms.Protection Against Oxidative Stress and “IGF-I Deficiency Conditions” 103 3. 41]. . We have also shown previously that short courses of treatment with low doses of IGF-I in rats with carbon tetrachloride-induced cirrhosis had many systemic beneficial effects. parameters of oxidative damage such as lipid peroxidation (MDA). Aging and others conditions of “IGF-I deficiency” and oxidative stress Mechanisms that cellular protection against oxidative injure are not well understood. increasing testosterone levels and serum total antioxidant capability. Experimental model of aging: Healthy male Wistar rats were divided into two groups according to age: young control of 17 weeks. and aging control rats of 103 weeks. we used two different experimental models: a. The IGF-I is an anabolic hormone produced mainly in the liver in response to GH stimulation [131]. b. Another condition of IGF-I deficiency is liver cirrhosis. GH. 41. Oxidative Stress is one of the most important mechanisms of the cellular damage in aging [42. protein carboxyl content (PCC) and activities of antioxidant enzymes in homogenates of brain and liver. that factors that promote the generation of ROS and/or impair antioxidative processes contribute to oxidative damage. Oxidative damage accumulates with aging and is likely responsible for the progressive decline in physiological systems. IGF-I therapy or saline was administrated the last 4 weeks [2. 6. and reducing oxidative damage in the brain and liver associated with a normalization of antioxidant enzyme activities and mitochondrial protection [5]. 133]. 3.

activities capase-3 processing and apoptosis in liver homogenates and isolated liver mitochondria [5.104 Antioxidant Enzyme Understanding that mitochondria are one of the most important cellular targets of IGF-I [5. 6. Recently. leading to reduced apoptosis [5. Recently. hepatoprotective and neuoroprotective effects. 41. a correlation between the SODs levels and MDA as shown in Fig. The IGF-II is a peptide hormone that belongs to the family of IGFs. All these data together provide evidence of the beneficial effect of IGF-I replacement therapy inducing anabolic [139] and antioxidant [5. 42] and intramitochondrial antioxidant capability of isolated liver mitochondria. 42] . we have also shown that IGF-II exerts similar effects [139. a decrease of oxidative cell damage reducing MDA and PCC and improving antioxidant enzyme activities and a mitochondrial protection improving MMP.139] actions in both experimental “IGF-I deficiency” conditions: cirrhosis and aging and experimental basis for further studies at exploring the potential IGF-I like a bioprotector due to its antioxidant. we have also shown that low doses of IGF-II in aging rats exerts similar hepatoprotector and neuroprotector effects than IGF-I low doses therapy. A) Correlation between TAS and IGF-I levels B) Correlation between SOD activity and MDA concentration . In these conditions of “IGF-I deficiency” low doses of IGF-I induced: a increase in the total serum antioxidant capacity closely related with serum IGF-I levels [40]. 5. ATP synthesis. 6). proton leak and reducing intramitochondrial ROS production and increasing ATP synthesis (Fig. It plays an important role in the embryology development but the physiological function of IGF-II in the adult life are not fully understood [139.140]. 41. Figure 5. 7] and they are the main intracellular ROS sources we studied the mitochondrial function by MMP. 41.140]. 40. IGF-II concentration decline with the age.

Our work provides new evidence of beneficial effect of IGF-I replacement therapy in degenerative diseases including aging. Different mitochondrial parameters were measure by cytometry in isolated liver mitochondria from healthy young control animals.Protection Against Oxidative Stress and “IGF-I Deficiency Conditions” 105 Figure 6. Conclusions Our results show that the cytoprotective effect of IGFs is closely related to a mitochondrial protection. In agreement with these results. 4. which could contribute to the described mitochondrial protection. B) ATP synthesis. leading to the reduction of intramitocondrial free radical production. . and apoptosis. Previously. we reported that low doses of IGF-I restored the expression of several protease inhibitors such us the serine protease inhibitor 2 in cirrotic rats [137]. including the effects of IGF-I on autophagy of dysfunctional mitochondria and apoptosis. C) Proton leak and D) ROS intramitochondrial production. old rats and old rats treated with low doses of IGF-I: A) MMP is considered a good marker of mitochondrial function. Further studies are necessary to elucidate all mechanisms involved in the IGFs mitochondrial protection. it has been reported that IGF-I differentially regulates Bcl-xL and Bax. increased ATP production and a normalization of antioxidant enzyme activities. oxidative damage.

Mitochondrial Membrane Permeability Transition (MMPT) Nerve Growth Factor (NGF) Nuclear Factor κ B (NFκB) Pentose phosphate pathway (PPP) Phosphatidylinositol 3. 5 triphosphate (PIP3) Phosphatidylinositol 4. 5 bisphosphate (PIP2) Platelet-Derived Growth Factor (PDGF) Protein Carboxyl Content (PCC) Reactive Nitrogen Species (RNS) . Insulin-like growth factor I (IGF-I) Manganese SOD (MnSOD) Mitochondrial DNA (mtDNA).106 Antioxidant Enzyme Abbreviations Adenine Nucleotide Translocator (ANT) Amyotrophic Lateral Sclerosis (ALS) Antioxidants response elements (ARE) Catalase (CAT) Coenzyme Q10 (CoQ10) Copper and Zinc SOD (Cu-Zn SODs) Cytochrome c (cyt c) Deoxyribose nucleoside triphosphate (dNTP) Electron Transport Chain (ETC) Glutathione (GSH) Glutathione Peroxidase (GSP) Glutathione Ttransferases (GST). IGF-I receptor (IGF-1R) Inner mitochondrial membrane (IM). 4. Glutation Reductases (GSR) Growth factor hormone (GH).

Santidrián S. Prieto “Effect of insulin-like growth factor I on in vivo intestinal absorption of D-galactose in cirrhotic rats. González-Barón S. 2012 [5] García-Fernández M. Muguerza B. 1999. Perez R. 149(5):2433-42. 26(1):191202. García M. ”Low doses of insulin-like growth factor I improve insulin resistance. Nuñez M. Quiroga J. 113(4): 1180-7. ”Impaired intestinal sugar transport in cirrhotic rats: correction by low doses of insulin. Endocrinology. Quiroga J. “Antifibrogenic effect in vivo of low doses of insulin-like growth factor-I in cirrhotic rats”. Kim SJ. Institute of Applied Molecular Medicine (IMMA). 1997 Jan. 1536(2-3):185-95. 1997 Nov. and oxidative damage in aging rats”. Ainzua J. 1997 Oct. Puche JE. Gastroenterology ”Hepatoprotective effects of insulin-like growth factor I in rats with carbon tetrachloride-induced cirrosis”. growth factor I”. Zudaire E. Am J Physiol. Biochim Biophys Acta. Gastroenterology. 276: 37–42 . 2001 May 31. Garcia M. Santidrian S. [2] Castilla-Cortázar I. Castilla-Cortázar I. Quiroga J. J Hepatol. [6] Muguerza B. Journal of Neural Transmission. References [1] Picardi A. Muguerza B. Pascual M. de Oliveira AC. 113(5): 1682-91. Prieto J. Spain 5. [4] Ramalingam M.” J. García M. Santidrian S.”Low doses of insulin-like growth factor-I improve nitrogen retention and food efficiency in rats with early cirrosis”. Pascual M. Prieto J. "Reactive oxygen/nitrogen species and their functional correlations in neurodegenerative diseases". 2008 May. School of Medicine. Prieto J. Picardi A. CEU San Pablo University. Castilla-Cortázar I. [3] Castilla-Cortázar I. Quiroga J. Delgado G. Santidrián S. Urdaneta E. Pascual M. Tosar A. [7] Castilla-Cortázar I. lipid metabolism. Urdaneta E. García M. Prieto J. Castilla Cortázar I. Quiroga J.Protection Against Oxidative Stress and “IGF-I Deficiency Conditions” 107 Reactive oxygen species (ROS) Rhodamine 123 (Rh123) Superoxide Dismutase (SOD) Szeto-Schiller (SS) Thioredoxin (Trx) Tripenylphoshonium ion (TPP+) Tyrosine Kinases (RTKs) Vascular Endothelial Growth Factor (VEGF) Voltage-Dependent Anion Channel (VDAC) Author details Úrsula Muñoz Morón and Inma Castilla-Cortázar Department of Medical Physiology.

S. “Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stressProc.. coenzyme Q10. H. Kontush A. Rehling. Van Houten. Rehling. [22] Arlt S. L. Andreyev “Complex I-mediated reactive oxygen species generation: modulation by cytochrome c and NAD (P)+ oxidation– reduction state. Yakes.. 1997.J. [23] Barbaro G. H. DNA. Biochem.E. Schonfisch. Reinders. Andreyev. 1: 1–2. Acad. the killer organelles and their weapons.E. Wagner. . I. Sci. B. Natl. Nat.. R. J. Guiard. Y. . Muller. USA. A. and Singh. A. and Meisinger. K. R. P. B. 2004.. Bellomo G. Biochemistry (Mosc. [10] Sickmann. 278: 36027–36031.”.. Beisiegel U..) 2005. 2002. NY. A. and Kroemer. “The proteome of Saccharomyces cerevisiae mitochondria” Proc. 2003. “Quercetin.K. 2003. Chem.L. B. Grisorio B. C “The proteome of Saccharomyces cerevisiae mitochondria. “Mitochondria in homeostasis of reactive oxygen species in cell. “Manipulations of the mitochondrial germ line must be openly debated and followed up”. and Cancer”. J..K. “Lipid peroxidation in neurodegeneration: new insights into Alzheimer's disease”. Wagner. H lavata. 100: 13207–13212. Rev. and organism”. J.”. Natl Acad.108 Antioxidant Enzyme [8] In: “Mitochondrial DNA Mutations in Aging.. “Vagal system impairment in human immunodeficiency virus-positive patients with chronic hepatitis C: does hepatic glutathione deficiency have a pathogenetic role?” Scand J Gastroenterol. B. [11] Singh... Perschil. T.. Belloni G..E. [15] Y... Soldini M.N. 279: 49064–49073. E. N. C. Curr Opin Lipidol 2002. 2001. Khalifa AE. [14] F. “Mitochondrial metabolism of reactive oxygen species”.Y. E. Y. 13(3): 289–94.. Singh. 368: 545–553. [19] Naviaux. Meyer. Ed. R. “Mitochondria. Nature. Lesnefsky “Production of reactive oxygen species by mitochondria: central role of complex III.. Roumier. [9] Ravagnan.D. 2001.. Liu. Genet. Y. Murphy. 70: 200–214. Springer.3:475– 481.. 2003. Cell Biol. et al. Mitochondrion. G. J. L. 2002. Joppich. 100: 13207–13212.“The curious history of yeast mitochondrial”. Chacinska. Schonfisch. USA 1997. I. Chacinska. [13] P. Zahedi. Pharmacol Res. A. Journal. and Meisinger. 192: 131–137. tissues. 347. Guiard. B. A. Zahedi. 413. “Mitochondrial me and the Mitochondrion”. Sci. 2005. 2002.L. [20] Abd El-Gawad HM. and L-canavanine as protective agents against lipid peroxidation and nitric oxide generation in endotoxininduced shock in rat brain”. Kushnareva. Sci. Jezek.. [12] A. J. Proc. A. Y. J. Perschil. Reinders. Joppich. Chem.M. Pfanner... 94: 514–519. New York. 43 (3): 257–63 [21] F. Moghaddas. Di Lorenzo G... K.K. N. Van Remmen.” Cell. K. Natl Acad. Hoppel.A. Biochem. Int. C. “Complex III releases superoxide to both sides of the inner mitochondrial membrane” J.J. Meyer. [17] Williamson. C. [16] Q.. 32(12): 1261–6. Biol. 37:2478–2503. Kushnareva. Vazquez. C. Pfanner. 2000. Physiol. Biol. [18] Sickmann. Starkov. H. A. P. Chen. Disease.

and function of IGF-1. Perez V. Labinskyy N. Tasaki H. Li Y. Mech Ageing Dev. Song YH. [32] Li Y. [38] Delafontaine P. L. Bruce-Keller AJ. Wheeler. “Expression. [26] Popescu. 174:159–165 [30] Li Y. 24:435–444 . Tsutsui M. 1997. “Aging and dietary restriction effects on ubiquitination. Millard WJ. Biochem Biophys Res Commun 2009. Wu H. Qian T.. J Neurochem 81:853–858. 2008. [28] Beal MF. Zhang L. Neuron 21:695–705 [35] Lopez-Lluch G. “Deoxyribonucleotide pool imbalance stimulates deletions in HeLa cell mitochondrial DNA”. Walker DW. Geng YJ.. Chem. 91:559–562 [34] Deshmukh M. 2008. “Sister chromatid exchange formation in mammalian cells is modulated by deoxyribonucleotide pool imbalance” Somat. Meyer EM. 43: 813– 819. 106:1729–1735. cytochrome c release and neurotoxicity in primary rat hippocampal neuronal cultures”. regulation. “Mitochondrial dysfunction in neurodegenerative diseases”. C. Dasuri K. 384: 259–264 [31] Satoh T. Uchiyama Y. 2004. [36] Li F. 129:515–521. King MA “ Alpha7 nicotinic receptor activation inhibits ethanolinduced mitochondrial dysfunction. Navas P. Morishita T. [27] Suda O. Mol Cell Biochem 1997. Podlutsky A. Exp Gerontol. Tanimoto A. ”Long-term treatment with N(omega)-nitro-Larginine methyl ester causes arteriosclerotic coronary lesions in endothelial nitric oxide synthase-deficient mice”. Genet. Mukhopadhyay P. Arterioscler Thromb Vasc Biol . N. He YJ. Am J Physiol Heart Circ Physiol. Cell Biochem. hypoxic and reperfusion injury”.K. 1998. [25] Song. Recchia FA. J. sumoylation. 25: 101–108.” Mol. Hatanaka H “Changes in mitochondrial membrane potential during oxidative stress-induced apoptosis in PC12 cells”. “Dihydroorotat-ubiquinone oxidoreductase links mitochondria in the biosynthesis of pyrimidine nucleotides. Enokido Y. 1999. Jockel. Song YH. Nieminen AL. “Insulin-like growth factor-1 receptor activation prevents high glucose-induced mitochondrial dysfunction. Circulation 2002. Sasaguri Y. [33] Reed JC “Cytochrome c: can’t live with it–can’t live without it”. J Neurosci Res 1997. Ungvari Z “Endothelial function and vascular oxidative stress in long-lived gh/igfdeficient ames dwarf mice ”. M. and the proteasome in the heart”. S. Horiuchi M. J. Nguyen A. 174: 125–129. 2003. 2002. 50:413–420. Nakashima Y. 1366:211–223 [29] Lemasters JJ. Biochim Biophys Acta 1998. Biol. C. Yanagihara N. Aoshima H. Pacher P. and Mathews. Huang PL. Johnson EM “Evidence of a novel event during neuronal death: development of competence-to-die in response to cytoplasmic cytochrome c”. Austad SN. Khardori R. 295: H1882–H1894. Lu YW. Craddock J. “The mitochondrial permeability transition in toxic. IGF-1R.C. Keller JN. Losonczy G. Schuster. 2008.. and Becker. G. Irusta PM. 278: 43893– 43896. Cell Mol. [37] Csiszar A.J. Herman B . Trost LC. Bartke A. and IGF-1 binding proteins in blood vessels”.Protection Against Oxidative Stress and “IGF-I Deficiency Conditions” 109 [24] Loffler. de Cabo R “Mitochondrial biogenesis and healthy aging”. cytochrome-c release and apoptosis”. Cell 1997.

D. and extracellular SOD (SOD3) gene structures. 7: 1223–1233. Koller F.” Antioxid Redox Signal 2005. 32:1990–2001. 64:97–112. Bot 2002. Roos. Crapo. Exp. D. [44] Dringen R. . Smyth. Varela-Nieto I. Conchillo M. A. Jones C. R. J. W. C. 8:736–738. T. [45] Alscher. S. Getzoff. J. Ahmed. Med. Fernandes E. and expression”. [43] Naziroglu M. 33:337–349. Parge. Nature 1993. “New molecular mechanisms on the activation of TRPM2 channels by oxidative stress and ADP-ribose. Soriano. J Clin Invest 1984. Roses. Prog Biophys Mol Biol.”Heparin-induced release of extracellular superoxide dismutase to human blood plasma”. 14:2731-9 [42] Puche JE.. Deng. Bush. J. and T. 65:45–80. Muntané J. A. A. K.. “Amyotrophic-lateral-sclerosis and structural defects in Cu. “Low doses of insulin-like growth factor-I induce mitochondrial protection in aging rats”. Castilla Cortazar I. “Oxidative and antioxidative potential of brain microglial cells. Endocrinology. [55] Radi. [47] Zelko. Folz. N. Loewen P "Diversity of structures and properties among catalases". I. Erturk. 2002. P.” Neurochem Res 2007. X. Hu. Siddique. [48] Deng. “Detection of catalase in rat heart mitochondria”.. P. Díez Caballero F. Heath. González-Barón S. 2008. 59: 145-6. [41] Pérez R. Annu. Tainer. Herzfeldt.. 242:55–59. R. Lima JL “Fluorescence probes used for detection of reactive oxygen species”.Fernández M. A. R. García.. 1995. N. J. [52] Marklund S. 261:1047–1051 [49] Rosen. A. Superoxide radical and superoxide dismutases. Fita I. Mariani. “Effect of IGF-I on total serum antioxidant status in cirrhotic rats”. Y. M. 266: 22028–22034. May. 1991. “Superoxide dismutase multigene family: a comparison of the CuZn-SOD (SOD1). B. H. J.I in experimental cirrosis”. I. E. J. [53] Chelikani P. J Biochem Biophys Methods 2005. Hallewell. Chem. extracellular superoxide dismutase gene to 4pter→ [51] Karlsson K. Pericakvance. García-Fernández M. J. D. Muntané J. and L. 61 (2): 192–208. Science 1993. M. Biochem. Turrens. Fisher JH. Marklund SL. 362:59–62. [50] Hendrickson DJ. R. Biol.. R. G. Warner. Castilla A. E. et al. 2004. Díaz-Sánchez M.Zn superoxide-dismutase”.. Castilla-Cortázar I.110 Antioxidant Enzyme [39] Gomes A.J Physiol Biochem. [40] Fernández M. F. 53: 1331–1341 [46] Fridovich. I. World J Gastroenterol. 2008. Biochem J. 1999. Free Radic.. Hung. Cayabyab. Z. and Freeman. evolution. D. [54] Zámocký M. Genomics 1990. Mn-SOD (SOD2). Delgado G. 2003. A. Puche JE. D. “Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis”. A. T.. “Mitochondrial protection by low doses of insulin-like growth factor. Cell Mol Life Sci.”Extracellular superoxide dismutase in human tissues and human cell lines”. González-Barón S. "Understanding the structure and function of catalases: clues from molecular evolution and in vitro mutagenesis". Siddique. P. 1987. Rev. Biol. Díaz-Sánchez M. and R. 72 (1): 19–66. H. D. G. C. E. Ho YS: “Regional localization of human q21”. Y. Rioja J. A. Chang. Sapp. L. Castilla-Cortázar. García. Iqbal. B. Figlewicz. 74:1398–1403. 149. A. Díaz Casares A. Patterson. Hentati. “Role of superoxide dismutases (SODs) in controlling oxidative stress in plants”.

Biophys. 2003. 2004. Biochem Cell Biol. Biochem. 256:677–86. 155: 741–750. Mahtani MM. 274:29294–302. Machado A. Korneluk. [65] Yoshimura S. R. Esworthy RS. [63] Chu FF.. 2006. [64] Takahashi K. Mizoguchi J. Arai M. “Tissue specific expression of the plasma glutathione peroxidase gene in rat kidney”. [62] Drevet JR. Environ. 1986. Science 300:1439–43. Francavilla F. 1987. 274:4924–33. Mol Diagn. GPX-GI”. Ursini F. [66] Thomas JP. 1999. J. [71] Vernet P. “Expression and chromosomal mapping of mouse Gpx2 gene encoding the gastrointestinal form of glutathione peroxidase. [57] Khan MA. Watanabe K.Protection Against Oxidative Stress and “IGF-I Deficiency Conditions” 111 [56] Quan. [68] Nomura K. Chem. Yoshida M. Drevet JR. Forti G. Swiderek K. “Mitochondrial phospholipid hydroperoxide glutathione peroxidase suppresses apoptosis mediated by a mitochondrial death pathway” . 10:156–62. Emoto K. 250: 70–79. Rock E. 1984. Koumura T. “The antioxidant glutathione peroxidase family and spermatozoa: a complex story”. J. Nancarrow DJ. Nucleic Acids Res. 2000:427–461. Kirby A et al “Genome scan of schizophrenia” Am J Psychiatry.. Chen H "Antioxidant enzymes and cancer". [58] Levinson DF. Chem. Avissar N. M. Cohen H. Biol. Whitin J. 14. Dufaure JP. Imai H.. 1991109:918–23. Biol. Purification and characterization of human plasma glutathione peroxidase: a selenoglycoprotein distinct from the known cellular enzyme. Roveri A. J. [60] Vitorica J. Maiorino M. “Isolation and characterization of the human catalase gene”. Tropak. Rayssiguier Y.. 285:1393–96 [70] Kryukov GV. et al. 1999. et al. Biochem. "Characterization of mammalian selenoproteomes”. Elliott RW. Biomed. 1996. Koumura T. Bermeister M. Zhang D. Girotti AW. Gravel. Mazur A. “Glutathione peroxidases expression in the mammalian epididymis”. Dufaure JP. 5321-5335. Imai H. Dual function of the selenoprotein PHGpx during sperm maturation. “Mitochondrial phospholipid hydroperoxide glutathione peroxidase plays a major role in preventing oxidative injury to cells”. 74:125–131. 1990. Andrology 2000. Lobanov AV. Drevet JR. “In vitro expression of a mouse tissue specific glutathione-peroxidaselike protein lacking the selenocysteine can protect stably transfected mammalian cells against oxidative damage”. 2010. Kruglyak L. Heim S. “Catalase enzyme mutations and their association with Diseases”. & Páy A. Arch. B. G. “Seleniumindependent epididymis-restricted glutathione peroxidase5 protein (GPX5) can back up . Mol Cell Endocrinol. Brown DM. F. [69] Ursini F. Suemizu H. et al. 265:454–61. 42:351–357. J. Ghyselinck NB. Rass P. Science 1999. Ho YS. [61] Drevet JR. 1998. [59] Góth L. Chem. Kiess M. Biol. Satrustegui J “Age-dependent variations in peroxide-utilizing enzymes from rat brain mitochondria and cytoplasm”. Nakagawa Y. Novoselov SV. Sci. A. “Protective action of phospholipid hydroperoxide glutathione peroxidase against membrane-damaging lipid peroxidation. Rigaudiere N. J Neurochem. 8(3):141-9. Zehtab O. Castellano S. Maiorino M. Tania M. In situ reduction of phospholipid and cholesterol hydroperoxides”. [67] Arai M. 1997. et al. Francavilla S. R. Chin J Cancer Res 22 (2): 87–92. Onozawa T. [72] Vernet P.

119: 2074–2085. 1997. J Clin Invest. Epididymis selenoindependent glutathione peroxidase 5 (Gpx5) contributes to the maintenance of sperm DNA integrity. Int J Neuropsychopharmacol 2009. 2001. Saez F. Jakoby WB. 10:2–18 Hayes JD. Persson B. “The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance”. Biochem. 360:1–16 Ladner JE. Bauer H. Henry-Berger J. Toxicol. 43:352–61. Gilliland GL. 2007. Comini MA "Redox control in trypanosomatids. Armstrong RN. Kanzok SM. Drevet JR. 1988. function and evolution of glutathione transferases: implications for classification of nonmammalian members of an ancient enzyme superfamily”. 54: 362–370. 15 (4): 717–8. and evolution of the glutathione transferases”. Schoor M. “Structure. Botella-Munoz J. 274:2163–2180. “Common structural features of MAPEG—a widespread superfamily of membrane associated . Keen JH. 1999. Mannervik B "The enzymes of glutathione metabolism: an overview". Schneuwly S. Parallel evolutionary pathways for glutathione transferases structure and mechanism of the mitochondrial class Kappa enzyme rGSTK1-1. Herbette S. “Neuroprogression: pathways to progressive brain changes in bipolar disorder”. Rev. J. Meister A "Glutathione metabolism and its selective modification". Berk M. 263 (33): 17205–8. 253:5654–57. Schirmer R. Rife CL. Biochem. Biochem. Vernet P. 30:445–600 Armstrong RN. McLellan LI. Science 291 (5504): 643–6. “Structure. Pulford DJ. J. Soc. 1780 (11): 1236–48. Chem. Lenoir A. 379:541–52. Biol. 1999. Trans. Parsons JF. “Seleno-independent glutathione peroxidases. “Modelling and bioinformatics studies of the human Kappa class glutathione transferase predict a novel third transferase family with homology to prokaryotic 2-hydroxychromene-2-carboxylate isomerases”. “Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defence against oxidative stress” Free Radic. 12(4):441–5. Biochem J. Zevnik B. 2004. Catalysis of nucleophilic reactions of glutathione”. Biol. Mancini J. Booth HS. 1995. “Glutathione transferases. Cadet R. Mol. Dowd CA. Ulschmid JK. More than simple antioxidant scavengers”. Krauth-Siegel RL. Biochim Biophys Acta 2008. Ford-Hutchinson A. J. Huttley GA. Crit. 2001. Müller HM. Drevet JR. 31:273– 300 Sheehan D. Meade G. Damon C. FEBS J. Morgenstern R. Foley VM. Chem. Res. 2009. Mol Reprod Dev. Biol. Chem. Res. Gottwald U. Jakobsson P-J. Chabory E.112 Antioxidant Enzyme [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] [86] [87] failing Se-dependent GPXs in mice subjected to selenium deficiency”. parasitic protozoa with trypanothione-based thiol metabolism". 1978. Kauselmann G. Kern H. Garrel C. Board PG. catalytic mechanism. Becker K "Substitution of the thioredoxin system for glutathione reductase in Drosophila melanogaster". Habenicht U. Biochemistry 2004. Hayes JD. 1987. Roeckel-Drevet P. Fechner A. Robinson A.

23:283–337 [89] Lovell. Menke T. [93] Baez S. A. “Decreased glutathione transferase activity in brain and ventricular fluid in Alzheimer’s disease”. Sci. “Functions of coenzyme Q10 in inflammation and gene expression”. [99] Schmelzer C. 32(1–4):179–83. Rev.Pharmacol.. L. Lindner I. 8:689–92 [88] Mannervik. Acad. P. P. J. Rimbach G. 2008. 51:1562–1566. Sim E. Segura-Aguilar J. N. Xie. M. Berger R.. 2010. B. M. [94] Strange RC. Mannervik B. “Identification of class-mu glutathione transferase genes GSTM1. Figg. [92] Smith G.53: 220–33. Vidal-Puig. Carcinogenesis 1995.. [102] Barbato. R. Hypertension 2009.. Biofactors 2008. “Glutathione S-transferase M1 and T1 polymorphisms: susceptibility to colon cancer and age of onset”. Lindner I. and Murphy. Animal and human studies with the mitochondriatargeted antioxidant MitoQ.180:15– 22. Hart I. Beal MF. Board P. Biofactors 2007. Cheng. 1998. Rimbach G. Prime. Biofactors 2007. 43: 257-63 [101] Chaturvedi RK. A.. Protein Sci 1999. et al. H. “Mitochondrial approaches for neuroprotection”. Strange RC. C. “The mitochondria targeted . A. J. coenzyme Q10. et al. E. Ann. 31(1):35–41. Fourth Edition. Johansson AS. Khalifa AE.. Döring F.” Cancer Surv 1995. R.16:1655–7. Lippincott Williams and Wilkins. Wolf CR. “Glutathione transferases— structure and catalytic activity”. M. T. “Quercetin. and Danielson.. Am J Hum Genet 1993. et al. and Markesbery.Vannais D. Menke T. MitoQ10 is here”. [104] Mercer. Rimbach G. IARC Sci Publ 1999:231–49 [95] Chenevix-Trench G. Lorenz G. R. Stanley LA. C. W. 1201: 96–10386. M..Protection Against Oxidative Stress and “IGF-I Deficiency Conditions” 113 proteins with highly divergent functions in eicosanoid and glutathione metabolism”. and Bennett. “Have no fear. Tu CP. “Glutathione transferases catalyse the detoxication of oxidized metabolites (o-quinones) of catecholamines and may serve as an antioxidant system preventing degenerative cellular processes”. [91] DeJong JL. Neurology. [98] Schmelzer C. [90] Pearson WR. 25: 27–65. U. “Effects of Coenzyme Q10 on TNF-alpha secretion in human and murine monocytic cell lines”. J.Widersten M. [96] Champe. N. Young J.GSTM5 on human chromosome 1p13”. 31(3–4):211–7. Fryer AA.Res 2001. Lorenz G. [97] Schmelzer C. Coggan M. K. Ann N Y Acad Sci 2008. “The glutathione S-transferases: influence of polymorphism on cancer susceptibility”. “The human Hb (mu) class glutathione S-transferases are encoded by a dispersed gene family”.Vorachek WR. Crit.. [100] Abd El-Gawad HM. M. Niklowitz P. Niklowitz P. and L-canavanine as protective agents against lipid peroxidation and nitric oxide generation in endotoxininduced shock in rat brain”.. K. Biochemistry. Biochem Biophys Res Commun 1991. “Influence of Coenzyme Q_{10} on release of pro-inflammatory chemokines in the human monocytic cell line THP-1”.Mohandas T.Y. Biochem J 1997. 1147:395–412. Murphy. 1988. 54: 222–223 [103] Smith.324(Pt 1):25–8. Xu SJ. Masoodi. Döring F. Griffin. R. Yu. A. Biochem. “Metabolic polymorphisms and cancer susceptibility.

S.. Y. Y.. M.. 18: 215–220. A. 2004. K. and reperfusion injury”. M. K. K. 2010. S.. Cell. Zhao.. J. 9:1825– 1836. Keogh. Lustig. Gibson. H. Rabinovitch. Lockhart. Kim. R. “Protective efficacy of mitochondrial targeted antioxidant MitoQ against dichlorvos induced oxidative stress and cell death in rat brain”. Sunkaria. M.. Orr. "Mitochondria-targeted antioxidant peptides”. Bal. Frampton. 30: 1019–1026 Zhao K.. D. G.Weilert. Y. Y. III. “Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program: 2.Biochem. 2011. Doughan AK.. Des 16.Won. D. K.W.. 279:34682– 34690. “Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling. P. H. M. 2012. Wu D. Antioxid Redox Signal.A. M. E.. M. J.. P. J Biol Chem. Soong Y. and Szeto.. S. M. R. H.H. V. V.. M. Hernandez-Mijares. R.. and Ali. L. Gibson. Pharm. Coron. Egorov MV.. Biol. M. Liver Int. F.. L.. Liver Int. M. M.. J. and Gill. Schiller. E. Keogh. Chem. D. P. Price. 2007. Dikalov SI. K. and Victor. Woo. M. C. E.Weilert. Physiol . K.. Kang. M. C.. Zhao.. and Hong. oxidative cell death. H. P. J. Mitochondrial H2O2 emission and cellular redox state link excess fat intake to insulin resistance in both rodents and humans. Kandimalla.. Singh. et al.M. “Mitochondrial redox cycling of mitoquinone leads to superoxide production and cellular apoptosis”. and Murphy. P. F.. Barskov IV. M. 61: 1193–1201. J... Potent mitochondria-targeted peptides reduce myocardial infarction in rats. Bellod. Curr. M. M... and reperfusion injury”.. Garcia-Malpartida.. kidney ischemia.. J. Rocha. “The mitochondriatargeted anti-oxidant mitoquinone decreases liver damage in a phase II study of hepatitis C patients ”. A. Szeto. K. Rashid. 119: 573–58.. Neuropharmacology. Clin. 30: 1019–1026 Gane. Schiller PW. Birk AV. Lin. A. Jang. Soong. Artery Dis.Wu. Orr. 2011. C.. Treatment of some ros. and Murphy. Bakeeva LE.W. Lim. Taylor.. 2008.. A. F. Med. 73:1288 –1299... P. Birk.. J. S. Y.. Soong. T. D... 2004. 3124–3131 . J..W. A.. G.. F. oxidative cell death.. Biochemistry. P. R. Szeto. Frampton.Won. Lee.. heart infarctions.W. Anderson. D.. 279: 34682–34690 Cho. 2007. Gane.114 Antioxidant Enzyme [105] [106] [107] [108] [109] [110] [111] [112] [113] [114] [115] antioxidant MitoQ decreases features of the metabolic syndrome in ATM+ /− /ApoE− /− mice” Radical Biol. C. M.Murphy. A. K. 2010. 2010. 52: 841–849. Wu. J. and stroke)”. Kane. T. Invest. Szeto HH. W. D. “Mitochondria-targeted antioxidants protect pancreatic β-cells against oxidative stress and improve insulin secretion in glucotoxicity and glucolipotoxicity”. A. Liu.. Kim. M. D. Gudup. E. Boyle... L.M.and age-related diseases (heart arrhythmia. “Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling... Zhao GM. E. Banuls. Smith. H. Sharma. G. J. et al.. T.. 2009. Taylor. Lockhart. Smith. “The mitochondriatargeted anti-oxidant mitoquinone decreases liver damage in a phase II study of hepatitis C patients”. S. L. 28: 873–886 Wani. H. Woodlief.

“Thioredoxin: a key regulator of cardiovascular homeostasis”. Kondo N. Lee C. H. Ballabh P.. Circ Res 2003. Vascular oxidative stress in aging: a homeostatic failure due to dysregulation of nrf2-mediated antioxidant response Am J Physiol. Burgering BM (2002) “Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress” Nature 2002. Cell 2009. S. Acad. Rhee S. Yodoi J... Cell Sci. “Modulation of glutathione and thioredoxin systems by calorie restriction during the aging process”. “Physiological functions of thioredoxin and thioredoxin reductase”.. D.. catalase. A. Medema RH. Holmgren A. Gautam T. 20: 1606–1617.. Koncz P..S. Chung HY. S. and Chang. A.. Bailey-Downs L. [127] Lee S.A. ..A. Biochim Biophys Acta 2010. Telljohann R. Oncogene 2003. 277: 20336–20342. Sosnowska D. Biol.. “PTEN: The down side of PI 3-kinase signaling”. de Cabo R.R. Sonntag WE. [125] Leslie N. 419: 316–321. Pinto JT. S. 101: 16419–16424. Jung KJ. J. T.S. Proc. Saarloos I.P. Monticone RE. [117] Cho CG. Chem. 14: 285–295. J.... Cell 2005. Tsuji Y. Redox Signaling 2005. Exp Gerontol 2003. Yeol Yeo C. 38:539 – 548.. “Peroxiredoxin as a Peroxidase for as well as a Regulator and Sensor of Local Peroxides”. 2002. Yang. 2011. Stadtman E. Haendeler J. “Reversible Inactivation of the Tumor Suppressor PTEN by H2O2” Biol. “Phosphoinositide 3-kinase signalling pathways” J.. Shim KH. and glutathione peroxidase via post-translational modification” Antioxid.. and Tainer. Mol. 267: 6102 – 6109. Downes C. Lee S.J.R.. Kwon J.. J. [129] Kops GJ. Polderman PE. [128] Seo J. [130] Sakamoto K. Yang K. 7: 619–626 [122] Ungvari Z. Dansen TB.Protection Against Oxidative Stress and “IGF-I Deficiency Conditions” 115 [116] Yamawaki H. Ahn Y.. Mol. E. Rhee S. 2002. Wirtz KW. [118] Arn é r ES.R. K. 114: 1439–1445. “Thioredoxin-dependent redox regulation of the antioxidant responsive element (ARE) in electrophile response”. Iwasaki K. Bailey-Downs L.S. 1804: 245–262 [121] Rhee. Kim HJ. J. Yu BP. J Gerontol Biol Med Sci. Kim Y. 2001. 2004. Wang M. Biol. Huang TT. Yang K. [124] Cantrell... Berk BC. W. Shin. 16: 348–357.R.G. “The major target of the endogenously generated reactive oxygen species in response to insulin stimulation is phosphatase and tensin homolog and not phosphoinositide-3 kinase (PI-3 kinase) in the PI-3 kinase/Akt pathway”. Csiszar A. D. 66:866 – 875. Kang. 2011. S. Masutani H. Losonczy G. [119] Kim YC.301: H363–H372. Sonntag WE. 22:1860 – 1865. Sugiyama H.R. Ahn Y. [123] Ungvari Z. “Controlled elimination of intracellular H2O2: regulation of peroxiredoxin. Sosnowska D.G. nrf2 dysfunction and nf-kb activation in the non-human primate macaca mulatta. [126] Kwon J.. Chung Hur K.. [120] Perry. 93: 1029 – 1033..Woo.. Eur J Biochem 2000. Role of the tumor suppressor PTEN in antioxidant responsive element-mediated transcription and associated histone modifications. Chung SW. D. U. Age-associated vascular oxidative stress. Natl.. Lakatta E. Yamaguchi Y. Sci. Csiszar A. Gautam T. Coffer PJ. The structural biochemistry of the superoxide dismutases. G. Cell. Signal. Lee S..H. S. de Cabo R. Jeong W.. Yodoi J. Getzoff. Bos JL.

Jansen PL. Abbott R. Delgado G. Casares AD. Berasain C. Sangro B. Clemmons DA. Puche JE. Yoshizawa C. Hall K. Johnson ML. N Engl J 1996. Straume M.. “Antioxidant effects of insulinlike growth factor-I (IGF-I) in rats with advanced liver cirrosis”. [141] Garcia-Fernandez M. Castilla-Cortázar I.116 Antioxidant Enzyme [131] Sara VR. “Insulin-like growth factor I (IGF-I) replacement therapy increases albumin concentration in liver cirrhosis: results of a pilot randomized controlled clinical trial”. J Endocrinol Invest 2005. Int J Biochem Cell Biol. Díaz-Sanchez M. Weltman A. Sierra I. González-Barón S. J Clin Endocrinol Metab 1995. . Chang J. 2005. Prieto J. 2005. 366:461–464. Ferrucci L. Clin Sci Mol Med 1974. Quiroga J. “C. Grimaldi W. Lauretani F.” Nature. 2002. Mulligan T. González-Barón S “Hepatoprotection and neuroprotection induced by low doses of IGF-II in aging rats”. 34:242-52. Bandinelli S. RodriguezOrtigosa C. BMC Gastroenterol. Sierra I. 28:96–100 [134] Laron Z Short stature due to genetic defects affecting growth hormone activity. Castilla A. Hoffman AR Clinical implications of the reduced activity of the GH-IGF-I axis in older men. [135] Wu A. Grant DB. Payeras M. 80:3209–3222 [133] Ceda GP. A. Puche JE. Castilla-Cortazar I. Ceresini G. Med 334:463–465.. García-Trevijano ER. [137] Mirpuri E. Iranmanesh A. 43:630-6. Gensch E. Hambley J. [138] García-Fernández M. Valenti G. Frystyk J. Guerra L. Barhoum R. Quiroga J. 1993. Weltman J. Navarro I. Maggio M. Dall’Aglio E. [139] Conchillo M. Falzoi C. “Differential impact of age. Castilla-Cortazar I. Puche JE. Clavijo E. Levi AJ. and obesity on basal versus pulsatile growth hormone secretion in men as assessed in an ultrasensitive chemiluminescence assay”. Physiol Rev 1990. de Knegt RJ. Prieto J. Scharschmidt B. 2011 28:9:123. elegans mutant that lives twice as long as wild type. "Reduced serum somatomedin activity in patients with chronic liver disease". J Transl Med. sex steroid hormones. J Hepatol. South S. Avila MA “Altered liver gene expression in CCl4cirrhotic rats is partially normalized by insulin-like growth factor-I”. CastillaCortazar I. “Liver mitochondrial dysfunction is reverted by insulin-like growth factor II (IGF-II) in aging rats”. Herrero JI. Liem AY. 47:359–366 [136] Kenyon C. 3:5:7. 70:591–613 [132] Veldhuis JD. [140] Castilla-Cortázar I. Mato JM. J Transl Med. Pincus S. 2011 6:9:103. Corradi F. Flyvbjerg A.“Insulin-like growth factors and their binding proteins”. García-Fernández M.

This was shown for immune cells. which participates in cancer progression as well as in the selection of resistant cells that are unable to die by apoptosis. which permits unrestricted use. licensee InTech. clinical and epidemiological investigations have provided evidence supporting the role of RNOS in the etiology of cancer due to both endogenous and exogenous factors. Dusetti and Alice Carrier Additional information is available at the end of the chapter http://dx. cancer cells favor angiogenesis which is necessary for tumor survival. dysfunction of cancer cells is both due to events intrinsic to these cells and to their response to signals generated by normal cells from their environment. cell death. Genetic events leading to genome instability enable those cell deregulations. provided the original work is properly cited. which is paradoxical since they are known to play a crucial anti-tumoral role.doi. Introduction Cancer is a complex pathology characterized by aberrant cell proliferation. To summarize. Inflammatory immune cells secrete proinflammatory cytokines and chemokines. DNA lesions form either directly when RNOS modify bases or indirectly as a consequence of lipid peroxidation. and reproduction in any medium. distribution. and fight against infection. normal cells can even collaborate to neoplasia. In addition. Although RNOS actively participate in a diverse array of biological processes including cell proliferation. matrix-remodelling proteins. Nelson J. cancer cells are frequently under persistent oxidative stress.Chapter 5 Antioxidant Role of p53 and of Its Target TP53INP1 Marion Seillier. and tumoral cell capacity to leave initial tissue and form distant tumors (metastasis). This is an open access chapter distributed under the terms of the Creative Commons Attribution License ( as well as reactive oxygen species (ROS) and reactive nitrogen species (RNS) (collectively called RNOS). excessive RNOS levels damage cell macromolecular components therefore promoting oncogenesis [1-4]. resistance to induced cell death. Sylvain Peuget. DNA lesions may be genotoxic when error-free repair mechanisms fail to remove them leading to mutations. growth factors. Interestingly.       © 2012 Carrier et al.5772/50790 1.. In addition.   . these mutations leading to loss of its tumor suppressive function. In some circumstances. in particular gain of oncogenes and loss of tumor suppressors functions observed in all cancer cells. the resulting products reacting with DNA.0).org/licenses/by/3. progression and dissemination. The tumor protein p53 is encoded by the tumor suppressor gene TP53 which is mutated in more than fifty percent of human tumors.

besides a central player in the redox field. even if the antioxidant role of TP53INP1 at the molecular level is still speculative and remains to decipher. an expression that resumes its main physiological function. p53 has been the focus of a huge number of investigations. Complexity of the p53 world p53 is complex at many levels. 7]. distinctly from conditions driving rapid and acute p53 induction in response to high levels of DNA damage. 2. we will describe the current knowledge on the relationship between p53 and redox. Interestingly. p53 is shown to be involved in embryonic development and energetic metabolism. Since its discovery. Loss of p53 function promotes tumor development. alterations in TP53 are the most universal cancer-driving genetic defects. highly induced upon stress events. i. Nowadays. p53 was initially reported as a stress factor. p53 is a key actor in prevention of cancer development The p53 protein was discovered in 1979 by different research groups.1. p53 is a fascinating multifaceted protein. emphazing its complexity since on one hand p53 is regulated by redox and in the other hand p53 regulates cell redox status. p53 implication in cell redox control 2. which is grossly overestimated (p53 longest isoform is 393 aminoacids long) presumably owing to the presence of a prolinerich region that slows down the migration of the protein in SDS-polyacrylamide gels.118 Antioxidant Enzyme In this chapter. 6]).e. featuring p53 as a potent tumor suppressor. For this reason. in particular as interacting with oncogenic viral SV40 Large T antigen (for historical reviews. p53 is necessary for silencing of mutant thus potentially cancerous cells by all means of tumor suppression. We will then review the current knowledge on one of p53 target genes. growth arrest. Tumor Protein 53-Induced Nuclear Protein 1 (TP53INP1).e. it was shown to be induced in response to DNA damage then named “the guardian of the genome”. see [5. More recent reports emphasize additional role of p53 in basal or low stress (“everyday life” stress) conditions. senescence and apoptosis. Finally. the protein p53 is the most famous tumor suppressor in the field of oncology for basic research scientists as well as clinicians. In both settings however (acute stress or basal condition). In particular. In particular. which we have defined as a major actor in p53-driven oxidative stress response. The DNA-damage response mediated by p53 is also an oncogeneinduced barrier against progression of cancer beyond its early stages. (1) TP53 gene encodes different p53 isoforms by differential splicing [8]. we will describe some models of genetically engineered mutant mice and experimental inflammation settings which have provided important insights into the link between oxidative stress and cancer.2. Its name is related to its apparent molecular weight of 53 kDa. Thus. p53 is sensitive and responsive to redox conditions. (2) This gene is the first reported member of a family encompassing three . i. and participating in stress resolution thus elimination of potential protumoral events towards cell homeostasis. This protein is encoded by the TP53 gene which is mutated or lost in a large range of human cancers [5. 2.

These three members play both overlapping and non-overlapping functions [9-11]. in particular either cell survival or cell death. TP63. In the following sections. (4) Induction of p53 activity results from different processes. according to informations available in the literature. TP63 and TP73 encode also many different protein isoforms. This induction relies mostly on structural modifications that turn p53 from dormant to active state via modifications in protein level.3.intensity . p53 is endowed with multiple basal activities (Figure 1). 2. and TP73. p53 is regulated by redox p53 is induced by different kinds of stress. including genes involved in cell redox status regulation (Figure 2). and interaction with itself . Complexity of p53 at different levels. (5) p53 possesses both transcriptional and nontranscriptional activity (Figure 1).type . (7) p53 can differently influence cell behaviour upon stress. And finally. (6) Transcriptional target genes of p53 are numerous.duration MOLECULE Redox modifications Post-translational modifications Protein level p53 Subcellular localization: Cytoplasm. depending on stress duration and intensity (Figure 1). nucleus. CONTEXT Steady-state Stress: . in addition to its key role in stress.Antioxidant Role of p53 and of Its Target TP53INP1 119 members: TP53. either genotoxic (including oxidative lesions induced by ROS and RNS) or non-genotoxic (listed in Figure 2A). including oxidative stress (Figures 1 and 2). subcellular localisation. (3) p53 is induced by many different stress conditions. including oxidative modifications (Figures 1 and 2). mitochondria Interaction with other proteins FUNCTION Transcriptional activity Non-transcriptional activity OUTCOME Cell survival / Apoptosis DNA repair Autophagy Senescence Metabolism Redox control Cell-fate decisions Figure 1. we will focus on the relationship between p53 and the cell redox status at different levels of this complexity.

Cytotoxic drugs .Acetylation .Glycosylation . Therefore.Phosphorylation . and JNK [14]. Upon stress signal.Methylation .S-glutathionylation . They comprise also redox modifications on cysteine and tyrosine residues.Sumoylation . The potential candidates are ATM.4.Cys residues: .Tyrosine nitration Post-translational modifications p53 . p53 is post-translationally modified then stabilized by loss of interaction with MDM2 thus MDM2-driven degradation [6.120 Antioxidant Enzyme (homotetramer) and other proteins (Figure 1). p53 possesses two amino-terminus transactivation domains and a core DNA binding domain . gamma. p53 post-translational modifications are very diverse (listed in Figure 2B). LKB1. p53 structure can be redox-modified either directly or indirectly via redox-driven induction of kinases activity.Telomere loss .Activation of oncogenes . and multiple transcriptional targets involved in redox control (C). A Genotoxic Stress type Non-Genotoxic . In addition. Indeed p53 activity can be directly post-translationally modified via thiol redox modulation of critical cysteine residues in its DNA binding domain. 12]. p53 activity can be indirectly modified via thiol redox modulation of kinases which post-translationally affect p53 via phosphorylation.Nutrient deficiency . The core domain of p53 holds a zinc atom that protects p53 from oxidation and is critical for DNA binding [13]. AMPK. As proposed in this latter. p53 regulates redox state The first described molecular activity of p53 was its action as a transcription factor.Carcinogens . Complexity of p53 with regards to multiple stress inducers (A).Neddylation Figure 2. In summary. 2. p53 is a ROS sensor.S-oxidation . UV) Sestrin 1 Sestrin 2 GPX1 MnSOD (SOD2) Catalase TIGAR ALDH4 TP53INP1 GSL2 Puma Bax PIG3 B Redox modifications . p53 oxidative modifications were extensively discussed in a recent review [8]. Dormant state of p53 is mostly due to its interaction with the E3 ubiquitine ligase MDM2 targeting p53 to permanent proteosomal degradation. multiple post-translational modifications (B).Breakdown of cell adhesion C Transcriptional activity: Multiple target genes Including antioxidants and prooxidants .Radiations (X.ROS / RNS .Deprivation of survival factors . p53 is at the core of a complex network of redox-dependent reactions.Hypoxia .

As these data provided a clue to understand the dual prosurvival versus proapoptotic activities of p53. Interestingly. they were subsequently discussed in several reviews [1. The transcriptional response to p53 induction is highly heterogeneous since it depends on the tissue/cell type and stress context [15]. In our laboratory. Besides its direct impact on the regulation of gene expression in the nucleus. We recently demonstrated that TP53INP1 is able to mediate the antioxidant function of p53 (see part 2). and catalase. p53 was also found to function as a transcriptional repressor. Among p53 target genes. for which transcription is more often activated. Efforts have been undertaken in developing p53 gene therapy and restoring p53 activity [6]. There is no doubt that the future in the p53 and cancer field is restoration of p53 tumor suppressive activity.5. MnSOD (encoded by SOD2 gene). The proteins encoded by p53-target genes are involved in many different cellular processes. this activity can shift to prooxidant with a proapoptotic outcome. several play a role in redox control (Figure 2). we identified a new target of p53 involved in oxidative stress response named TP53INP1. 19]. [18]. This endeavor benefits from basic research on deciphering the diversity of p53 activities and regulation modes at the molecular level. p53 is known to directly activate the transcription of the antioxidant enzymes GPx1. p53 was found to possess non-transcriptional biochemical activities. p53 influences mitochondrial functions such as apoptosis and respiration which is the most prominent source of ROS. favoring tumor suppression (cell-cycle arrest. p53 can prevent the Warburg effect which is one of the features of cancer cells [24]. differentiation. by inhibiting glycolysis. The link between sestrins family and p53 in redox regulation has been reviewed recently [14]. In particular. Restoration of wild type p53 expression triggers elimination of tumors in vivo. This task is hindered by the fact that p53 is neither a cell surface protein nor an enzyme which are targetable by antibodies or inhibitors. The consequence of this promotion of oxidative phosphorylation is a decrease in oxidative stress and thus prevention of DNA damage. autophagy. Nevertheless. senescence. As such.Antioxidant Role of p53 and of Its Target TP53INP1 121 which can bind tightly to specific DNA sequences [8]. 2. One of the key functions of sestrins is the regeneration of the peroxiredoxins antioxidant enzymes [17]. Clinical issues The central role of p53 in human cancer makes it a target for cancer therapy development. …) [16]. In addition. Besides this indirect antioxidant action. These are very diverse and can be exerted both in the cytoplasm and the nucleus [20]. some of the small molecules which are able to reactivate mutant p53 and induce apoptosis share the ability to target thiols and affect the redox state of p53 [25]. in particular as a main ROS sensor and actor in the redox equilibrium. . apoptosis) or basal cell homeostasis (energy metabolism. More than one hundred targets of p53 have been well characterized. In parallel. in conditions of sustained or high intensity stress. Thus dual role of p53 depending on the context was initially demonstrated by Sablina et al. p53 is endowed with a potent antioxidant activity in parallel with a cell survival outcome. p53 was shown to indirectly promote mitochondrial functions and inhibit glycolysis [21-23].

any difference between the cellular effects of both isoforms has been identified. and autophagy (see below) [28-32]. in NHF (normal human fibroblasts) but not in cell lines where p53 is mutated or deleted: HeLa (derived from a cervix adenocarcinoma). serine and threonine residues. The sub-cellular localization of TP53INP1 is nucleo-cytoplasmic. heart and testis . like apoptosis. except the additional C-terminal part in TP53INP1β. as an acidic protein of unknown function in the mouse thymus. The two proteins are identical in sequence. BxPC-3 cells (derived from a pancreatic adenocarcinoma) and SW480 and HT29 cells (both derived from a colorectal adenocarcinoma) [34]. low in lung. but the addressing mechanism to the different cellular compartments remains to be elucidated. ROS regulation. More recently. in . we showed that TP53INP1 is also localized in autophagosomes into the cytoplasm [28]. In parallel. TP53INP1 was in parallel identified as a p53 target gene by Okamura et al. and is expressed ubiquitously in the whole organism.2. pancreas and stomach. resulting from the alternative splicing of the transcript [27. Characterization of TP53INP1 TP53INP1 (also known as TEAP. SIP. Afterward. but not by a mutated form of p53. It was first described by Carrier et al. TP53INP1 was shown to be involved in a large panel of cellular processes. apart from a sequence rich in proline. but with differences in the expression level between organs. H358 cells (derived from a lung adenocarcinoma). TP53INP1 gene encodes two isoforms. Moreover. suspected to be an important factor in thymocyte maturation [26]. but upon ectopic over-expression the protein accumulate in the nucleus of the cell. They don’t show any known motif. and p53DINP1) is a p53 target gene that encodes the TP53INP1 protein. TP53INP1 was identified by Tomasini et al. TP53INP1α and TP53INP1β (18 and 27 kDa. skeletal muscle. glutamic acid. and very low in brain [27. colon. 36]. more precisely in sub-nuclear structures called the promyelocytic leukaemia protein nuclear bodies (PMLNBs) [35].122 Antioxidant Enzyme 3. bone marrow. To date. TP53INP1 is a target gene of p53 Tomasini et al. cell cycle regulation.1. cellular adhesion and migration. 34]. as a stress response gene highly induced during acute pancreatitis in the mouse [27]. in 2001 [36]. spinal cord. which is characteristic of short half-lives proteins. 3. A p53-response element site is found at position -1329 of the TP53INP1 promoter. respectively). TP53INP1 gene is localized in the human chromosome 8q22 [33]. and a LIR (LC3-interacting region) which allows the interaction between TP53INP1 and LC3 within the autophagosomes [28]. showed induction of TP53INP1 expression in response to adriamycin or hydrogen peroxide (H2O2) treatment. Basal levels of TP53INP1 are high in thymus. kidney. These authors used a differential display approach to isolate p53-inducible transcripts. TP53INP1 antioxidant role 3. the PEST region. TP53INP1 expression is induced by wild type p53 expression.

This work also showed that PKCδ is able to modulate the expression level of TP53INP1. Flow cytometry and TUNEL experiments confirmed the cellular effect of this transcriptional regulation on cell cycle arrest in G1 phase and on apoptosis. During a cellular stress. This p53 binding site was confirmed by electrophoretic-mobility shift assay and luciferase reporter assays. Okamura et al. Finally. showed that co-expression of p53 and TP53INP1 enhances p53 Ser-46 phosphorylation. In response. It was well referenced that Ser-46 phosphorylation of p53 and induction of p53AIP1 are essential features to DNA damage response [37. TP53INP1 is able to activate the transcriptional activity of p53. 40]. as observed by flow cytometry and Terminal deoxynucleotidyltransferase-mediated dUTP Nick End-Labeling (TUNEL) [36]. Furthermore. confirming the implication of this kinase in p53 activation through TP53INP1. therefore being implicated in a positive feedback loop with p53. they found a p53 binding site of 20 nucleotides in the intron 2 of TP53INP1. Altogether. demonstrated a direct interaction between TP53INP1. the p38 MAPK [38]. which can phosphorylate p53 on Ser-46 in response to DNA damage [41]. the authors showed that TP53INP1 and HIPK2 can regulate the p53 activity on genes involved in cell cycle regulation (Mdm2 and p21) and apoptosis (Pig3 and Bax). How TP53INP1 activates the p53 response to stress? The phosphorylation of p53 on its serine 46 (Ser-46) seems to play a key role in the activation of p53-driven apoptosis by TP53INP1. 38]. TP53INP1 co-localizes with HIPK2 and p53 in PML-NB. Okamura et al. inhibition of TP53INP1 expression by antisens oligonucleotides represses p53AIP1 expression.MEF. and they identified TP53INP1 among other p53 targets. TP53INP1 is implicated in p53-driven response to stress In turn. suggested that TP53INP1 interacts with a specific p53 Ser-46 kinase. which are described to be the site where HIPK2 binds to p53 and phosphorylates its Ser-46. These observations suggest that TP53INP1 activates p53 protein toward activation of apoptosis by regulating phosphorylation at Ser-46. Tomasini et al. Another study demonstrated that TP53INP1 co-immunoprecipitates also with PKCδ. Our molecular model is summarized in Figure 3.3. 3. Several proteins were shown to have a kinase activity on the Ser-46 of p53 and to promotes p53-dependent apoptosis: the homeodomain-interacting protein kinase-2 (HIPK2) [39. TP53INP1 transcription is induced by p53. which matches the consensus p53 binding site by 85%. Using a kinase in-vitro assay with immunoprecipitated TP53INP1 and p53. and that modified version of p53 activates transcription of apoptosis-inducing genes such as p53AIP1. and the protein kinase C delta (PKCδ) [41]. these data clearly indicate that TP53INP1 is a target gene of p53. p53. induces p53AIP1 and strongly increases apoptotic cell death. and HIPK2 by GST-pulldown and co-immunoprecipitation assays [35]. Moreover. Using luciferase-reporter assays. they observed an induction of TP53INP1 following γ-irradiation in p53+/+ MEF but not in p53-/.Antioxidant Role of p53 and of Its Target TP53INP1 123 cell line expressing wild-type or mutated p53. Moreover. TP53INP1 is able to bind different kinases .

UV radiation. Those kinases will phosphorylate p53 on its Ser-46. p73 p53 E2F HIPK2 PKCδ TP53INP1 CELL CYCLE ARREST APOPTOSIS STRESS p53 Ser-46 P Transcription p53AIP1 Mdm2 p21 Bax Pig3 … Figure 3. changes in protein-protein interaction and sub-cellular relocalization. and this phosphorylation will trigger transcriptional activity of p53 on its targets: p53AIP1. Pig3. DNA damage-induced cell death and cell cycle arrest (upon γirradiation and adriamycin treatment) were strongly decreased after inhibition of TP53INP1 expression by oligonucleotide antisens. the transcriptional activity of p53 is highly dependent on the promoter context and on the type of stimulus. This activation leads to transcription of several genes which will trigger a large panel of cellular processes. In response to stress. to suggest that at least two different p53-dependent mechanisms are involved in TP53INP1 induction. or redox state regulation. p53 was induced similarly by both stresses. As described in the first part. forming a multiproteic complex which can recruit p53. Study of TP53INP1 induction in MCF7 cells (expressing wild type p53) treated with several stress (γ-irradiation. Those observations led Okamura and coll. whereas antisens had no effect on UV radiationinduced cell death. p21. DNA repair. All the presented data suggest that TP53INP1 is one of the p53 co-factors involved in such a regulation. This cascade will lead to G1 cell cycle arrest or apoptotic cell death in response to severe cellular stress. p53 is activated mainly by complex posttranslational modifications. like cell cycle arrest. senescence.124 Antioxidant Enzyme (HIPK2. Molecular model of p53-TP53INP1 functional interactions (positive feedback loop). adriamycin) showed that stress-triggered DNA doublestrand breaks strongly induced TP53INP1 within 4h. whereas TP53INP1 is induced more slowly and to a lesser extent by UV radiation [36]. apoptosis. p53 is a tightly regulated protein maintained at low levels under normal conditions. To control this broad variety of mechanisms. Moreover. autophagy. Mdm2. . By contrast. Bax. PKCδ) in PML-NB.

like other proapoptotic p53 co-factors (ASPP1. with a maximum at 9h. showed that p73α and β isoforms induce TP53INP1 [43]. as demonstrated by CAT-reporter assays. suggested that this induction occurs through the activation of p53-dependent mechanisms in response to cell transformation [27]. The mechanistic explanation was provided by the demonstration that TP53INP1 is a target of p73. ethanol. It also encodes a nuclear transcription factor which shares structural and functional homologies with p53. Many isoforms of p73 exist. and oxidants such H2O2 [27. UV irradiation. In addition to the role of TP53INP1 in the regulation of the p53-dependent response to stress. Tomasini et al. which directly binds to its promoter. TP53INP1 is a stress response protein. 43]. mRNA level then decreases to reach control values 15h after induction [27]. E2F1 induces phosphorylation of p53 on Ser-46 through TP53INP1 and this modification is important for E2F1-p53 cooperation in apoptosis. Induction of TP53INP1 in response to genotoxic and oxidative stress As described above. TP53INP1 expression is rapidly induced within 3h after induction.  In vivo. showed a p53-independent action of TP53INP1 [43].e. leading to . whole-body γ-irradiation or dexamethasome (corticoid analog) intraperitoneal injection [32]. JMY) [42]. The expression of this gene is induced by a large panel of cellular stresses. leading them to suggest different pathways of TP53INP1 activation. explain the E1A-induced expression of TP53INP1 by the disruption of RB/E2F complex by E1A. Moreover. This independency was initially suggested by the observation that TP53INP1 is induced in p53-/mice during acute pancreatitis.. Tomasini et al. TP53INP1 is described to be quickly and strongly induced by different cell stress agents: adriamycin. 3. Different levels and kinetic of TP53INP1 expression were observed by authors in response to each of these treatment. p73 binds directly to the promoter of TP53INP1.Antioxidant Role of p53 and of Its Target TP53INP1 125 TP53INP1 is also regulated by E2F. methyl methanesulfonate. Tomasini et al. 36. which result from alternative splicing and from differences in the initiation of transcription by different promoters. Similarly to its action on p53. In vitro. TP53INP1 expression is also induced during chronic pancreatitis [44]. the ability of TP53INP1 to stimulate the activity of p53 is slightly higher than that observed with p73.4. Hershko et al. ASPP2. γ-irradiation. p73 is also known to be able to activate p53 target genes and to induce cell cycle arrest and apoptotic cell death. TP53INP1 then modifies the transcriptional activity of p73 and stimulates G1 cell cycle arrest and proapoptotic functions. i. heat shock. TP53INP1 is induced in pancreatic acinar cells in a mouse model of acute pancreatitis (intraperitoneal injection of caerulein). and that TP53INP1 over-expression is able to trigger G1 cell cycle arrest in p53 deleted or mutated cell lines. Moreover TP53INP1 expression is highly increased in the thymus of mice upon in vivo treatment by inducers of thymocyte oxidative stress and death.  TP53INP1 is also induced by oncogenic stress (mutated RasV12D and viral E1A protein). cisplatin. Some isoforms share functional similarities with p53 [11]. Nevertheless. p73 belongs to the p53 family.

it became important to know which phenotype would be observed in an in vivo murine model. 3. induced both by genotoxic stress and oxidative stress. spleen) were strongly depleted in ascorbate and glutathione [32]. and lipid peroxide content as reflect of the total antioxidant capacity of the body. Chronic oxidative stress in TP53INP1-deficient mice First evidences of exacerbated oxidative stress in absence of TP53INP1 have been demonstrated in vivo thanks to TP53INP1-deficient mice [31]. ESR (Electron Spin Resonance) spectroscopy analyses demonstrated that TP53INP1 deficiency is associated with decreased ascorbate levels and increased lipid peroxide content in plasma [31]. Therefore. No difference was seen in . This was the first demonstration of a chronic oxidative stress in TP53INP1-deficient mice. in the absence of induced colitis. leading to an overload of ROS and RNS as described in the Introduction. as demonstrated previously in the colon [31]. To get further insights into the physiological role of TP53INP1 during oxidative stress. once a part of mechanistic implicating TP53INP1 had been elucidated. by the use of DCF-DA (2’.7’-dichlorofluorescein diacetate) which is a cell permeable dye oxidized and retained within cell in DCF fluorescent probe [32]. Once it was proven that there was a deregulated redox status in TP53INP1 KO mice. As altered ascorbic acid status has been reported in the mucosa [45] and plasma [46] in Inflammatory Bowel Diseases patients. As TP53INP1 is induced by stress including oxidative stress. The main phenotypes of TP53INP1deficient mice were shown to be independent of the genetic background (unpublished data). Staining on total thymocytes of mice challenged or not with whole-body γ-irradiation (6 Grays) showed that absence of TP53INP1 increased ROS levels in the latter. Others organs have also been tested but displayed different ascorbate and glutathione profiles.126 Antioxidant Enzyme deregulation of E2F activity. Mice with inactivated Trp53inp1 gene (Knock-out mice) were generated in our team by homologous recombination on a mixed 129/Sv x C57BL/6 background [31]. Data obtained on colons of mice during colitis further confirmed these results as TP53INP1 KO mice displayed more colonic ROS than their WT counterparts. This was further validated by ESR spectroscopy in thymocytes as well as in blood samples. intestine. We confirmed that thymocytes. TP53INP1 seems to be involved in all major stress pathways. Indeed. oxidative stress in the colon and plasma was also observed at basal state i. resulting in activation of TP53INP1 [42]. Interestingly. we postulated that this protein could be involved in cell redox homeostasis. Oxidative stress arises from an imbalance between oxidants and antioxidants in favor of the former. suggesting that this gene plays a central role in cellular response to damage. blood and different organs of TP53INP1-deficient mice (colon. it was important to assess whether this deregulation was linked with an overall deficit in antioxidant defenses. Additional proofs supporting this observation came just four years later by studies achieved in our lab by N’guessan et al.e.5. Knock-out mice were then backcrossed on the C57BL/6 parental genetic background for nine generations [32]. measurements were carried out in colon and plasma of mice. we first evaluated in TP53INP1 KO and WT mice the level of small anti-oxidant molecules such as plasmatic ascorbate (vitamin C).

in spite of this protective microenvironment.7. further suggesting a protection of thymus against oxidative stress. Interestingly. cell cycle arrest. By contrast with primary MEFs. in 2001 first demonstrated that over-expression of exogenous .Antioxidant Role of p53 and of Its Target TP53INP1 127 pancreas. other anti-oxidants such as Trolox (a water-soluble vitamin E derivative) and Ebselen (organoselenium compound possessing βantioxidant properties) were able to decrease ROS content in WT but not in TP53INP1-deficient cells. proliferation: redox-linked TP53INP1 tumor suppressor role 3. demonstrated that what was observed in vivo could be transposed in vitro: MEFs deficient for TP53INP1 exhibited higher DCF staining thus higher ROS level than WT cells when challenged during 1h with 50 μM H2O2 treatment (after 3 or 10h recovery) but also at basal state.7. Nevertheless. DCF is a general oxidant indicator rather than a specific marker for H2O2 [47]. and interestingly. a precursor of glutathione) significantly reduced ROS level in both genotypes. Same series of experiments was carried out on E1A-RasV12D transformed MEFs exposed to γirradiation (10 Grays) which is at the origin of a global oxidant stress [32]. Treatment with antioxidant NAC (N-acetylcysteine. As neither Trolox nor Ebselen can correct a defect in glutathione and regarding our in vivo results related to glutathione deficiency. 3. Whether this loss is the cause or consequence of chronic oxidative stress in TP53INP1-deficient animals and cells deserves further investigation. our data demonstrate a profound dysregulation of antioxidant balances in the absence of TP53INP1. Cano et al. which displays a different pattern of oxidative defenses compared to thymocytes. Our data suggest a higher de novo production of ascorbate in TP53INP1deficient liver that could be due to a higher need owing to higher ROS level in TP53INP1 -/mice. we suggest a higher provision of ascorbate in TP53INP1-deficient thymus. These results represented the first report of TP53INP1 cell-intrinsic antioxidant function. Apoptosis.cells is the important factor in sensitizing these cells to oxidative stress. Taken together. levels of vitamin C were higher at basal state in KO mice. Tumor suppressor role upon ectopic over-expression The elucidation of the TP53INP1 mechanistic led us to assess the role of this protein in the cellular context. 3. in liver and thymus.6. Chronic oxidative stress in TP53INP1-deficient MEFs in vitro In order to study more in depth and more easily the impact of TP53INP1 in the regulation of cellular redox status.1. no significant difference was seen at basal state. primary Mouse Embryonic Fibroblasts (MEFs) were prepared from TP53INP1 WT and KO mice. irradiation stress induces a higher production of ROS in deficient thymocytes compared to WT. Tomasini et al. Further experiments demonstrated that TP53INP1 deficiency provoked more particularly H2O2 accumulation linked with abnormal extracellular release of H2O2-derived free radicals after H2O2 challenge [30]. we can propose that loss of glutathione in TP53INP1-/. The fact that ROS content was different between TP53INP1 WT and KO MEFs 24h after irradiation underscored dysfunction of ROS regulation in deficient cells. Regarding thymus.

ROS have a promoting role in tumor initiation and promotion. as a cell cycle inhibitor. linking increased proliferation in absence of TP53INP1 with ROS.7. In TP53INP1-deficient mice. As ROS regulation is impaired in absence of TP53INP1.128 Antioxidant Enzyme TP53INP1 α and β in COS7 cells induced cell death via an apoptotic pathway [27]. but also on genes involved in cell cycle regulation (Mdm2 and p21). TP53INP1-deficient MEFs revealed more aggressive than WTs [48]. Cano et al. To evaluate this supposed link. Against all expectations. NAC treatment abolished differences observed between WT and KO cells. Further works in our lab demonstrated that TP53INP1s and HIPK2 regulate the p53 transcriptional activity on genes involved in apoptosis (Pig3 and Bax). 49]. N’Guessan et al.mice were far more sensitive to development of induced colorectal tumors compared to WT [31]. experiments have been performed in MEFs cells and thymocytes ex-vivo. These preliminary data on cell-death resistance and replicative potential are reminiscent of hallmarks of cancer depicted by Hanahan and Weinberg [24] and pinpointed first tracks of implication of TP53INP1 in tumor suppression. Flow cytometry analysis on HEK 293T cells transfected with TP53INP1 α or β did revealed a G1 cell cycle arrest in presence of TP53INP1. 3. known to promote cancer progression is to be put in correlation with G1 cell cycle arrest observed by Tomasini et al. p21. Altogether. we put in place three different models of induced tumorigenesis. in presence of TP53INP1. and oxidative stress-associated carcinogenesis in the colon was promoted (model iii). ROS and tumor suppression. ROS implication has been considered in the two last tumorigenesis mouse models. we developed a genetic model by crossing mice deficient for TP53INP1 with p53 KO mice: p53 heterozygous mice displayed an accelerated tumor development in absence of TP53INP1 and majority of tumor revealed to be lymphoma [30]. This feature. Our results clearly showed that TP53INP1 -/. As mentioned in the introduction. i/ First model consisted in injection of transformed E1A-RasV12D MEFs in nude mice. ii/ In parallel. could be one of the molecules involved in the increase in G1 phase arrest. investigations have been performed to try to validate this hypothesis. demonstrated that TP53INP1-deficient primary MEFs proliferated more rapidly than WT cells. oxidative stress-related lymphoma incidence was markedly increased in p53+/mice (model ii). All models strongly suggested an anti-tumoral role of TP53INP1. these data showed that chronic oxidative stress in the absence of TP53INP1 played a crucial role in facilitating tumorigenesis. consistent with 2001 Tomasini’s works. this could at least partially explain its tumor suppressor role. in the absence of TP53INP1.2. We first showed that TP53INP1 was lost in human pancreatic and gastric cancer and that its restoration inhibited tumor development [48. Tumor suppressor role assessed in TP53INP1-deficient models. in relation with redox status Then. showed that . iii/ Last model consisted in induction of colorectal tumors by injection of carcinogen AOM (Azoxymethane) followed by a chronic colonic inflammation provoked by 3 ingestion cycles of DSS (Dextran Sulfate Sodium) assuring promotion of tumoral cells initiated by AOM. To go more in depth in the link between TP53INP1. Notably.

p53 plays its tumor suppressor role mainly via transcriptional induction of target genes involved in cell cycle. Thus. Autophagy would then represent a protective process for cell against stress. We recently demonstrated that TP53INP1 is indeed involved in autophagy [28]. showing that oxidative stress. TP53INP1 could be a major actor in p53-driven oxidative stress response. although this observation is explained by a higher level of excess of ROS in those cells. Table 1 recapitulates the state of knowledge regarding the impact of TP53INP1 on cellular processes in the settings of gain of function (ectopic over-expression) and loss of function (deficient cells and mice). Role of TP53INP1 on redox status can be p53-dependent but also p53independent As mentioned above. these results seemed contradictory with what have been published previously demonstrating a proapoptotic role of TP53INP1 consistent with a tumor suppressor role.Antioxidant Role of p53 and of Its Target TP53INP1 129 TP53INP1-deficient cells were more sensitive to induced death than WT. Mechanisms regulated by TP53INP1 Proliferation Cell death by apoptosis Autophagy Intracellular ROS level Level of anti-oxidant small molecules Loss of function ↗ ↗ ↗ Gain of function ↗ ↘ ↘ ↗ ↘ Table 1. . These differences could be abolished by supplementing media with NAC. p53 antioxidant function is dependent on its transcriptional activity and proceeds by sequential induction of antioxidant targets. which is a feature of TP53INP1-deficient cells. Impact of TP53INP1 deficiency (Loss of function) or over-expression (Gain of function) in different cell processes.8. and regulation of cell redox status. We then hypothesized that this lack relies on a deficit of autophagy in TP53INP1 -/cells. but that its absence impairs stress resolution and sensitizes cells to induced cell death by a lack of a prosurvival activity. apoptosis. As a target of p53. is responsible for their sensitivity to induced apoptosis. ↘ 3. we clearly demonstrated at cellular level the anti-tumoral role of TP53INP1 related with its function as antioxidant regulator. To reconcile these apparently contradictory observations. none of the known p53 targets were able to fully recapitulate the p53-mediated antioxidant response in the p53-deficient cells. On the whole. However. we postulated that TP53INP1 is protective against cancer by a proapoptotic activity upon strong stress.

. these data show that ectopic expression of TP53INP1 in p53-deficient cells is sufficient to restore a normal redox status. defining TP53INP1 as a major actor in p53driven oxidative stress response. Cano et al. Altogether.) CELL SURVIVAL APOPTOSIS Figure 4. TP53INP1 antioxidant effect was even unchanged after cotransduction of p53 along with TP53INP1. For those reasons. and etoposide treatment). we demonstrated that TP53INP1 absence confers increased thymocyte death sensitivity both in a context of p53-dependent cell death (irradiation.130 Antioxidant Enzyme Interestingly. once TP53INP1 is induced upon oxidative stress. Puma. .. To test this hypothesis. In the same manner. Low stress High stress TP53INP1 TP53INP1 Cytoplasm Nucleus Interaction with p53 in PML-NB Mostly in cytoplasm Autophagy Transcription of proapoptotic p53 target genes (Bax. Noxa and Bim (pro-apoptotic target of p53) between TP53INP1 WT and KO thymocytes. at least in part. Hypotheses on TP53INP1 antioxidant function Figures 4 and 5 schematically recapitulate the state of knowledge regarding TP53INP1 activities. and/or p53 in p53 KO primary MEFs.. Model of p53-dependent and -independent TP53INP1 activities. 3. moderate amount of TP53INP1 located mostly in cytoplasm is involved in autophagy. TP53INP1 action over ROS could be. and in consequence favors cell survival. quantitative RT-PCR experiments did not show any difference in the induction of expression of Bax. and in a p53-independent cell death context (dexamethasone) [32]. Therefore. independent of p53.9. Figure 4 illustrates the dual (dependency/independency) relationship between TP53INP1 and p53. Consistent with this. we propose that death sensitivity in the absence of TP53INP1 does not exclusively depend on p53 transcriptional activity. In low stress conditions. in high stress conditions. high levels of TP53INP1 would induce apoptosis both by promoting autophagy-dependent cell death in the cytoplasm and p53-driven cell death in nucleus. TP53INP1 restoration induces a decrease of ROS level in p53deficient cells (Table 1). Level of ROS was even lower than after restoration of p53 alone. Pig3. By contrast. it seems to play its antioxidant function independently of p53. performed transduction experiments to reintroduce expression of TP53INP1 α or β.

Antioxidant Role of p53 and of Its Target TP53INP1 131 Figure 5 takes Figure 4 forward by adding the setting of TP53INP1 absence observed in tumors and in experimental TP53INP1-deficient mice. deficient cells lack the tumor suppressive proapoptotic activity of TP53INP1 which is induced during high stress situation (Figure 5. Tomasini et al. 51]. As mentioned above. Both isoforms could therefore be post-translationally redox-modified. demonstrated in 2005 that TP53INP1 gene is a transcriptional target of p73. Low stress High stress TP53INP1 X TP53INP1 ROS TP53INP1 Unpaired stress response Autophagy ROS CELL SURVIVAL APOPTOSIS CANCER INITIATION Figure 5. which would result in modifications of both their physical interaction with partners and their subcellular localization. right). Model recapitulating anti-tumor activities of TP53INP1 and the consequences of its absence in tumor cells. TP53INP1 modulates p73-induced cell cycle arrest and apoptosis by modulating p73 transcriptional activity. The question whether TP53INP1 would be a direct ROS-detoxifying enzyme. or a cotranscription factor of genes implicated in ROS elimination remains unclear for the moment. implying the p53 tumor suppressor gene homologue p73 notably. Several authors showed that p73 was induced in response to oxidative stress and was implicated in oxidative cellular response [50. Absence of TP53INP1 is associated with ROS increase which promotes cancer initiation and progression (Figure 5. . independently of p53. and that in turn. TP53INP1 could regulate redox status by activating p73 and thus transcription of target genes implicated against oxidative stress. We have other propositions. The possibility that TP53INP1 is a ROS-sensor is high since both TP53INP1 isoforms are rich in cysteine residues. Furthermore. left). Deficient cells lack the redox control activity of TP53INP1 which is schematically shown here as a direct activity but that can be an indirect effect.

while heterozygous p53-deficient mice (p53 +/-) develop cancers at later age and lower incidence. macroautophagy is a catabolic process removing malfunctioning organelles responsible for ROS generation and oxidative stress.132 Antioxidant Enzyme Finally. deficiency in TP53INP1.1. either an increase or a decrease [56]. These observations illustrate the role of p53 in regulating organismal aging. Bmi1.2. developing mainly T-cell type lymphomas. Deficiency . most of them showing an impact on life-span. 4. and HIF-2α. with a broader panel of tumor types than p53-null mice. 4. the implication of TP53INP1 in autophagy could indirectly be the way of its antioxidant activity. showing an antioxidant role for p53 [18]. However. such as JunD. decreases p53 +/. One hundred percent of null (p53 -/-) mice die during the first months of age. Interestingly. Mice in which Trp53 was inactivated by homologous recombination (p53-null mice) apparently develop normally. Additionally. different models of p53 transgenic mice have been generated. suggesting that their permanent oxidative stress is the primary cause of lymphoma carcinogenesis.mice viability by exacerbating chronic oxidative stress in those mice and favoring lymphoma development. Indeed. p53 mouse models Mouse models targeting the Trp53 gene (encoding p53 in mice) have provided a wealth of information regarding p53 function. Conversely. related to its impact on redox control either as an antioxidant or a pro-oxidant [52]. Reciprocally. independently of their genetic background. FoxOs. these observations show that p53 plays an important role during embryonic development. which must be kept under control by Mdm2. Thus TP53INP1 could be involved in redox level regulation via its participation in autophagy. Mouse models of oxidative stress and cancer 4. as mentioned above in Figure 4 and 5. Strikingly. Antioxidant enzymes mouse models Mouse models of oxidative stress were recently reviewed. 58]. which we have defined as a major actor in p53-driven oxidative stress response (see above). In addition. Altogether. p53 deficiency is associated with an increase in intracellular ROS and with excessive oxidation of DNA and linked genomic instability. also involved in the modulation of antioxidant enzymes expression. The Trp53-deficient mice are remarkable since they are prone to develop a variety of tumors during the first six months of life. long-term dietary supplementation with NAC completely prevents lymphoma development in p53-null mice. This emphases the crucial role of p53 as a tumor suppressor [53-55]. some reports showed that a fraction of p53-deficient embryos display exencephaly and die in utero [52]. absence of p53 in Mdm2-deficient mice rescues these latter from embryonic lethality which is probably related to the absence of p53 degradation. illustrating several cases where inactivation of one antioxidant enzyme promotes cancer development [57. these reviews underscore other transcription factors than p53.

which rely on different activities. RNOS are found at high levels in inflammatory sites. the murine AOM/DSS colitis-associated colorectal carcinogenesis protocol rely on a single injection of procarcinogen AOM inducing tumor initiation. For examples. this mouse model represents an excellent preclinical system to both characterize the molecular events required for tumor formation at inflammation sites. Inflammation and cancer As mentioned in the introduction. U1068. 5. Antioxidant activity of TP53INP1 at the molecular level is still elusive. 4. and can be used as preclinical models in cancer research. Helicobacter pilori chronic gastritis increases the risk of gastric cancer. France Corresponding Author * . Conclusion In this chapter. we recapitulate the state of knowledge regarding p53 antioxidant role. and assess the ability of agents to inhibit this process. We propose several hypotheses which deserve being studied further. mainly transcriptional induction of antioxidant molecules and control of energetic metabolism. followed by repeated cycles of DSS ingestion mimicking chronic colitis thus promoting colorectal tumors [63. we underscore the interest of mouse mutant mice endowed with a chronic oxidative stress. Indeed. a sensor of DNA damage and involved in the DNA damage response upstream from p53. Stress Cellulaire. France Aix-Marseille Université. CRCM. deficiency in ATM. Marseille. France CNRS. However. such as p53 and TP53INP1 deficient mice. Finally. These mice provide plenty of basic knowledge. Finally. France Institut Paoli-Calmettes. Marseille. we resume the identification of p53-target TP53INP1 as a main actor in p53-driven redox control. chronic inflammation was demonstrated to be a risk for cancer development. Hepatitis viruses infection favors liver cancer development. For example.3. Marseille. is also an oxidative stress-associated tumor prone mouse model. Furthermore. UMR7258. Marseille. participating in elimination of the inflammation cause (infection or wound). and Inflammatory Bowel Diseases increase the risk to develop colon cancer [59-62].Antioxidant Role of p53 and of Its Target TP53INP1 133 of one of these transcription factors also favors oxidative stress and redox-driven tumorigenesis. pancreatitis promotes pancreatic cancer. Stress Cellulaire. Nelson J. Experimental models of inflammation-associated cancer are widely used both in basic and applied research. 64]. RNOS can be harmful depending on duration or intensity of inflammation. Sylvain Peuget. Hence. Dusetti and Alice Carrier* Inserm. CRCM. Author details Marion Seillier.

Chem Biol Interact 160(1):1-40. [10] Pietsch EC. Antioxid Redox Signal 15(6):1655-67. Nat Rev Cancer 9(10):701-13. McMahon SB. References [1] Halliwell B (2007) Oxidative stress and cancer: have we moved forward? Biochem J 401(1):1-11. Neuzil J. Murphy ME (2008) The p53 family and programmed cell death. Caron de Fromentel C. [14] Budanov AV (2011) Stress-responsive sestrins link p53 with redox regulation and mammalian target of rapamycin signaling. [7] Brosh R. Nat Rev Mol Cell Biol 9(9):702-12. [3] Ralph SJ. Oncogene 27(50):6507-21. Mann K (2001) Zinc binding and redox control of p53 structure and function. Hainaut P (2011) Redox control and interplay between p53 isoforms: roles in the regulation of basal p53 levels. [13] Hainaut P. Rotter V (2009) When mutants gain new powers: news from the mutant p53 field. [8] Hafsi H. Nat Rev Cancer 9(10):749-58. Association pour la Recherche sur le Cancer. Slee EA. Acknowledgement The authors are supported by Institut National de la Santé et de la Recherche Médicale. is supported by Ministère de la Recherche et de la Technologie. and senescence. . cell fate. Antioxid Redox Signal 3(4):611-23. by La Ligue Nationale contre le Cancer. Oren M (2009) The first 30 years of p53: growing ever more complex. Centre National de la Recherche Scientifique. Hainaut P (2004) p53 protein variants: structural and functional similarities with p63 and p73 isoforms. [9] Stiewe T (2007) The p53 family in differentiation and tumorigenesis. M. [2] Klaunig JE. Antioxid Redox Signal 15(6):1679-90. 7. Mol Aspects Med 31(2):145-70. S. [4] Valko M.S. Nat Rev Cancer 7(3):165-8. Lu X (2008) A complex barcode underlies the heterogeneous response of p53 to stress.134 Antioxidant Enzyme 6. Gu W (2009) Modes of p53 regulation. Lancet Oncol 10(9):913-9. Mazur M (2006) Free radicals. [12] Kruse JP. Institut National du Cancer. [6] Levine AJ. metals and antioxidants in oxidative stress-induced cancer. Moreno-Sanchez R (2010) The causes of cancer revisited: "mitochondrial malignancy" and ROS-induced oncogenic transformation . Wiman KG (2009) 30 years and a long way into p53 research. Sykes SM. Annu Rev Pharmacol Toxicol 44:239-67. Oncogene 23(3):631-8. and La Ligue Nationale contre le Cancer. Rodriguez-Enriquez S.P.why mitochondria are targets for cancer therapy. Cell 137(4):609-22. Izakovic M. Rhodes CJ. [11] Courtois S. Saavedra E. Kamendulis LM (2004) The role of oxidative stress in carcinogenesis. Moncol J. [5] Hainaut P. [15] Murray-Zmijewski F.

Cano CE. [19] Vousden KH. Ryan KM (2009) p53 and metabolism. Rocha D. interacts with LC3 and ATG8-family proteins through the LC3-interacting region (LIR) and promotes autophagy-dependent cell death. [24] Hanahan D. Ilyinskaya GV. Pouyet L. Morselli E. et al. Science 304(5670):596-600.Antioxidant Role of p53 and of Its Target TP53INP1 135 [16] Vousden KH. et al. Antioxid Redox Signal 15(6):1715-27. Siret C. et al. Immunogenetics 50(56):255-70. Weinberg RA (2011) Hallmarks of cancer: the next generation. Nat Rev Cancer 9(10):691-700. Kravchenko JE. Wiman KG (2009) Mutant p53 rescue and modulation of p53 redox state. Cell 137(3):413-31. Lambert JM. Bernard K. Culcasi M. Sablina AA. et al.and RAG1-deficient thymuses: definition of a set of genes potentially involved in thymocyte maturation. [23] Galluzzi L. Hamlaoui S. Wang PY. (2007) Colitis and colitis-associated cancer are exacerbated in mice deficient for tumor protein 53-induced nuclear protein 1. Samir AA. [17] Budanov AV. Cancer Res 69(1):219-26. Pietri S. Mol Cell Biol 27(6):2215-28. et al. (2001) Molecular and functional characterization of the stress-induced protein (SIP) gene and its two transcripts generated by alternative splicing. J Biol Chem 276(47):44185-92. Antioxid Redox Signal 15(6):1691-714. [29] Seux M. Cell 144(5):646-74. [31] Gommeaux J. (2012) TP53INP1. Culcasi M. Cano C. [26] Carrier A. [28] Seillier M. [25] Bykov VJ. Victorero G. Antioxid Redox Signal 15(6):1739-48. Cell Death Differ In press. Monte M. Rigot V. Gayet O. Gommeaux J. Granjeaud S. Hainaut P. Chumakov PM (2005) The antioxidant function of the p53 tumor suppressor. Kepp O. Hwang PM (2011) p53. Clerc P. Pietri S. [30] Cano CE. and cancer. [18] Sablina AA. Cell Cycle 8(16):2509-17. Ma W. Agapova LS. Gosset G. Garcia S. Gauthier C. a tumor suppressor. SIP induced by stress and promotes cell death. Nguyen C. (2009) Tumor protein 53-induced nuclear protein 1 is a major mediator of p53 antioxidant function. Dagorn JC. et al. Vaccaro MI. Seux M. (1999) Differential gene expression in CD3epsilon. N'Guessan P. Prives C (2009) Blinded by the Light: The Growing Complexity of p53. [22] Lago CU. Koonin EV. [20] Green DR. Feinstein E. Kroemer G (2011) Mitochondrial liaisons of p53. Peuget S. [32] N'Guessan P. (2011) Absence of tumor suppressor tumor protein 53-induced nuclear protein 1 (TP53INP1) . Iovanna JL. Vitale I. [27] Tomasini R. Chumakov PM (2004) Regeneration of peroxiredoxins by p53-regulated sestrins. Nat Med 11(12):1306-13. homologs of bacterial AhpD. Seillier M. Pebusque MJ. Gironella M. Sung HJ. Peuget S. Galeotti T (2011) Role of MnSOD and p66shc in mitochondrial response to p53. Garcia S. Nature 458(7242):1127-30. (2011) TP53INP1 decreases pancreatic cancer cell migration by regulating SPARC expression. Pinti M. Oncogene 30(27):3049-61. aerobic metabolism. Kroemer G (2009) Cytoplasmic functions of the tumour suppressor p53. et al. Montero MP. Budanov AV. [21] Pani G.

Okada G. Samir AA. Nakanishi H. Antioxid Redox Signal 15(6):1639-53. a potential mediator of p53-dependent apoptosis. Saito S. Tomasini R. Carrier A.136 Antioxidant Enzyme [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] sensitizes mouse thymocytes and embryonic fibroblasts to redox-driven apoptosis. Appella E. Hollander MC. and its regulation by Ser-46phosphorylated p53. J Biol Chem 278(39):37722-9. Droge W. et al. Calvo EL. (2002) Regulation of p53 activity by its interaction with homeodomain-interacting protein kinase-2. Yoshida K. Pebusque MJ. Cecchinelli B. (2002) Homeodomain-interacting protein kinase-2 phosphorylates p53 at Ser 46 and mediates apoptosis. (2001) p53DINP1. Bontemps C. Tanikawa C. J Biol Chem 281(9):5734-40. Bulavin DV. Motoo Y. . Isnardon D. Taya Y. Hofmann TG. Pebusque MJ. Zentgraf H. et al. Moller A. Carrier A. (2003) TP53INP1s and homeodomain-interacting protein kinase-2 (HIPK2) are partners in regulating p53 activity. Tomasini R. Tanaka T. Free Radic Res 22(2):131-43. Sakaguchi K. Higashimoto Y. regulates p53-dependent apoptosis. Arakawa H. Cell 102(6):849-62. Karp SM. et al. (1999) Phosphorylation of human p53 by p38 kinase coordinates N-terminal phosphorylation and apoptosis in response to UV radiation. Nowak J. Arakawa H. Oncogene 24(55):8093-104. Anderson CW. Buffinton GD. Koch TR (2006) Oxidative stress and antioxidants in inflammatory bowel disease. Miki Y (2006) Protein kinase C delta regulates Ser46 phosphorylation of p53 tumor suppressor in the apoptotic response to DNA damage. Ginsberg D (2005) Novel link between E2F and p53: proapoptotic cofactors of p53 are transcriptionally upregulated by E2F. Oren M. Hershko T. Saito S. Tomasini R. Ng CC. J. Mol Cell 8(1):8594. Dagorn JC. Mori T. (2000) p53AIP1. Taya Y. Sirma H. Nowak J. Xie MJ. Seux M. Doe WF (1995) Altered ascorbic acid status in the mucosa from inflammatory bowel disease patients. Dagorn JC. Dis Mon 52(5):199-207. et al. (2002) Assignment of tumor protein p53 induced nuclear protein 1 (TP53INP1) gene to human chromosome band 8q22 by in situ hybridization. (2004) Tumor protein p53-induced nuclear protein 1 (TP53INP1) in spontaneous chronic pancreatitis in the WBN/Kob rat: drug effects on its expression in the pancreas. Tomasini R. (2005) TP53INP1 is a novel p73 target gene that induces cell cycle arrest and cell death by modulating p73 transcriptional activity. Chaussepied M. et al. Okamura S. Nat Cell Biol 4(1):1-10. Samir AA. a p53-inducible gene. et al. Cecchinelli B. Dusetti N. Soddu S. Cytogenet Genome Res 97(1-2):140E. Cell Death Differ 12(4):377-83. D'Orazi G. et al. Liu H. Jiang PH. Iovanna JL. Azizi Samir LA. Eur J Cell Biol 81(5):294-301. et al. Embo J 18(23):6845-54. Pancreas 5(4):20516. (2002) P53dependent expression of the stress-induced protein (SIP). Matsuda K. Nat Cell Biol 4(1):11-9. Bruno T. Totaro S. Oda K. et al. Tanaka T. et al. Mattei MG. Dagorn JC. Manni I.

Jin YJ. [59] Federico A. [56] Liu D. Xu Y (2011) p53. Jangi SM. Asumendi A. and its restoration inhibits pancreatic tumor development. Mori H (2003) A novel inflammation-related mouse colon carcinogenesis model induced by azoxymethane and dextran sodium sulfate. et al. Gastroenterology 140(6):1807-16. Whiteman M (2004) Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean? Br J Pharmacol 142(2):231-55.Antioxidant Role of p53 and of Its Target TP53INP1 137 [47] Halliwell B. World J Gastroenterol 12(5):691-6. and aging. Montgomery CA. Suzuki R. Sawabu N (2006) Downexpression of tumor protein p53-induced nuclear protein 1 in human gastric cancer. Nature 356(6366):215-21. Liu YX. [61] Hussain SP. [49] Jiang PH. Sugie S. (1994) Tumor spectrum analysis in p53-mutant mice. . Hofseth LJ. [58] Pouyet L. Remington L. Curr Biol 4(1):1-7. Mutat Res 690(1-2):3-11. Drug Discov Today Dis Models 4(2):67-73. Bernier G (2011) p53 pro-oxidant activity in the central nervous system: implication in aging and neurodegenerative diseases. et al. et al. Pebusque MJ. [63] Tanaka T. Alonso-Tejerina E. McArthur MJ. Gardeazabal J. [53] Donehower LA. Cancer Sci 94(11):965-73. [52] Chatoo W. Apoptosis 16(12):1253-67. J Biol Chem 282(40):29152-62. [51] Wang J. Int J Cancer 121(11):2381-6. [54] Donehower LA (1996) The p53-deficient mouse: a model for basic and applied cancer studies. Itzkowitz SH (2011) Intestinal inflammation and cancer. Halachmi S. Perez-Yarza G. Kohno H. [55] Jacks T. Butel JS. Antioxid Redox Signal 15(6):166978. Garcia S. Iovanna JL. Yamada Y. Morgillo F. Slagle BL. Terfenadine induces apoptosis and autophagy in melanoma cells through ROS-dependent and -independent mechanisms. Tomasini R. Motoo Y. oxidative stress. Ciardiello F. [62] Ullman TA. et al. Nat Rev Cancer 3(4):276-85. Schmitt EM. Transgenic Res 19(2):155-64. Wong AC. Seux M. Cano C. (1992) Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Harvey M. [50] Nicolau-Galmes F.. Bronson RT. Carrier A (2010) Mutant mouse models of oxidative stress. Yin Y (2007) TAp73 is a downstream target of p53 in controlling the cellular defense against stress. Abdouh M. Xie MJ. Tuccillo C. Loguercio C (2007) Chronic inflammation and oxidative stress in human carcinogenesis. (2007) Tumor protein 53-induced nuclear protein 1 expression is repressed by miR-155. Semin Cancer Biol 7(5):269-78. Proc Natl Acad Sci U S A 104(41):16170-5. Antioxid Redox Signal 15(6):1729-37. Huang P (2007) Models of reactive oxygen species in cancer. Jr. Williams BO. Ogasawara MA. Hande MP. [57] Lu W. Gommeaux J. [48] Gironella M. Harris CC (2003) Radical causes of cancer. [60] Ferguson LR (2010) Chronic inflammation and mutagenesis.

Kajiura K.138 Antioxidant Enzyme [64] Okayasu I. Sakamoto S (1996) Promotion of colorectal neoplasia in experimental murine ulcerative colitis. Ohkusa T. Kanno J. Gut 39(1):87-92. .

Section 2

Biomedical Therapies






Chapter 6

Plasma Antioxidant Activity as a Marker for a Favourable Outcome in Acute Ischemic Stroke
Nelly Sapojnikova, Nino Asatiani, Tamar Kartvelishvili, Iagor Kalandadze and Alexander Tsiskaridze
Additional information is available at the end of the chapter

1. Introduction
Ischemic stroke (IS) is a leading cause of mortality and disability in industrial countries, only overwhelmed by cardiac disease and cancer (Donnan et al., 2008; Doyle et al., 2008; Flynn et al., 2008). In Western countries stroke causes 10-12% of all deaths (Bonita, 1992). Stroke is also the leading cause of adult disability, because 76% of people survive their stroke. Of these survivors, 50% have hemiparesis, 26% are dependent in activities of daily living, and 26% are forced into a nursing home. Thus stroke is a lethal disease, but it disables more than it kills (Carmichael, 2005). This fact has led a recent effort to develop strategies for neural repair after stroke and to search for neuroprotective therapies to reduce cell death and infarct volume after stroke. Many studies have been directed to understanding the molecular events involved in cerebral ischemia and developing agents for neuroprotective therapies. These studies result in the concept that early injury due to the loss of energy substrates is followed by secondary inflammation, which produces tissue damage (del Zoppo et al., 2000). As the inflammatory process develops during a span of hours to days, there is a potential window for therapeutic treatment. There is very little treatment for stroke. At present it was shown, that treatment with tissue plasminogen activator (rt-PA) can improve outcome in patients with acute ischemic stroke (Clark et al., 1995). However due to a narrow time window and fear of hemorrhagic complications, this treatment is effective in the first hours of stroke and is only appropriate for a very limited number of patients (Clark et al., 1995; Grophen et al., 2006). Focal ischemia can be caused by systemic hypoperfusion or by occlusion of an artery in the brain by thrombosis or embolism from the heart. Other causes are abrupt occlusion of small penetrating arteries (at lacunar stroke), arterial dissection, and various genetical and haematological disorders (Hossman, 1994). Sudden decrease or loss of blood circulation to


© 2012 Sapojnikova et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


142 Antioxidant Enzyme

an area of the brain results in insufficient oxygen and glucose delivery to support cellular homeostasis. This produces complex series of events that lead to cell death: excitotoxicity, acidotoxicity and ionic imbalance, peri-infarct depolarization, oxidative and nutritive stress, inflammation (Doyle et al., 2008; Gonzalez et al., 2006; Sims & Muyderman, 2010). Each of these processes usually goes on for minutes, hours or days. Within the core of the ischemic area blood flow is more severely restricted, less than 20% of normal, necrotic death occurs within minutes. In the periphery of the ischemic area, where perfusion takes place, a lesser ischemia develops. The blood flow is reduced 20-40% of normal flow (Back et al., 2004 Belayeev et al, 1997; Hossman, 1994). In this area, which is potentially salvageable, called penumbra, the degree of ischemia and timing of reperfusion determine the outcome for individual cells. In ischemic penumbra cell death occurs less rapidly via apoptosis and inflammation (Gonzalez et al., 2006). Restoration of the blood circulation has a decisive importance for the reverse of an arterial occlusion. However, the restoration results in secondary damage, called reperfusion injury, which is a recognized complication of restoring blood flow to ischemic tissue (Hallenbeck & Dutka, 1990). One of the mechanisms of the secondary damage consists in the increased generation of reactive oxygen species (ROS) initiated during the reoxygenation from parenchymal and endothelial cells and from infiltrating leucocytes. There is considerable evidence that reactive oxygen and nitrogen species are important mediators of tissue injury in acute ischemic stroke (Cuzzocrea et al., 2001, Warner et al, 2004). Oxidative stress is defined as an imbalance between the production and removal of reactive oxygen species (Halliwell & Gutteridge, 1999). Oxygen is inevitable component of aerobic life. Incomplete reduction of oxygen to water during normal aerobic metabolism generates reactive oxygen species, which have one or more unpaired electrons. The main ROS such as superoxide anion, singlet oxygen, hydrogen peroxide and nitric oxide, which reacts with superoxide anion producing different types of reactive nitrogen species (RNS), are very transient species and play an important role in many physiological and pathological processes. Reactive oxygen and nitrogen species differ to each other, e.g. superoxide is a single electron oxidant of only moderate strength and crosses cell membrane via the anion channel (Kontos, 2001), and hydrogen peroxide is lipid soluble and easily crosses cell membrane via diffusion, as it is a neutral particle. Hydroxyl radical has only one unpaired electron and represents the most reactive oxygen radical, it cannot diffuse and causes its damaging effect in the vicinity of the biomacromolecules. Each of the reactive oxygen and nitrogen species has specific reactivity and properties and accordingly can activate different specific signalling pathways and biological responses. One of the most popular theories to explain oxygen toxicity has been the superoxide theory, which proposes that oxygen toxicity is due to overproduction of superoxide anions (Halliwell & Gutteridge, 1999). Mitochondria are the organelles in eukaryotic cells responsible for aerobic respiration, and they are the most common source of ROS. In normal cells, 1-2% of electrons carried by the mitochondrial transport chain leak from this pathway and pass directly to oxygen generating superoxide anion, which can be a source of the ROS by developing different type of chain reactions (Curtin et al., 2002). Abnormal electron

Plasma Antioxidant Activity as a Marker for a Favourable Outcome in Acute Ischemic Stroke 143

leakage is connected with perturbation of mitochondrial metabolism and inflammatory responses to injury (Halliwell & Gutteridge, 1999). Although mitochondria is a main source of superoxide, superoxide anions can be also produced by auto-oxidation of tissue components such as small molecules, haemoglobin and myoglobin or generated by intracellular oxidative enzymes such as oxidases, peroxidases, oxygenases, metal catalyzed reactions, inflammatory cell activation (neutrophils and macrophages) (Dalton et al., 1999). Superoxide rapidly dismutates to hydrogen peroxide or reduces Fe(III) to Fe(II) releasing the iron from storage sites. Although dismutation of superoxide is the main source of hydrogen peroxide in tissue, the later can be produced directly by several oxidases such as glycolate oxidase, urate oxidase, flavoprotein dehydrogenase, localized in peroxisomes (Halliwell & Gutteridge, 1999). Hydroxyl radical is generated from hydrogen peroxide in the presence of transition metals (e.g. Fe(II) or Cu(I) ions) via Fenton and Haber-Weiss reactions (Halliwell & Gutteridge, 1999). In this case superoxide is essential because it serves to reduce transition metal, which is then oxidized in the reaction that produces hydroxyl radical. As a result, the cycle can be repeated. Thus, superoxide and hydrogen peroxide are unavoidable by-products of aerobic metabolism. The most biomolecules resist univalent redox reactions and are nonreactive with superoxide. One way in which superoxide is believed to cause toxicity is through its participation in hydroxyl radical production representing an extremely powerful oxidant. Of particular importance at ischemic stroke is the interaction of superoxide with nitric oxide, a water and lipid soluble free radical, which is produced by nitric oxide synthases (NOS). Nitric oxide combines with superoxide anion generating very strong oxidant peroxinitrite anions (Beckman & Koppenol, 1996; Dugan & Choi, 1994). Excessive ROS are harmful because they react with and modify all classes of cellular macromolecules causing wide-ranging cellular effects such as lipid peroxidation, protein denaturation, inactivation of enzymes, nucleic acid and DNA damage, release of calcium ions from intracellular stores, damage of cytoskeleton, chemotaxis. Oxygen radicals have significant vascular effects. Superoxide, hydrogen peroxide and peroxinitrite are strong cerebral vasodilators. Cerebral vascular effects of these radicals include vasodilation, increased platelet aggregability, increased endothelial permeability, and focal destructive lesions of endothelial cell membranes. Vascular effects are very important for cerebral blood flow. The registration of these effects offers the convenient monitoring of ROS presence and action (Kontos, 2001). The effect of ROS is balanced by antioxidant systems, which provide either direct or indirect protection of cells against adverse effects on different biological sites. The cellular protective antiradical mechanisms consist of multiple interacting enzymatic such as superoxide dismutases (SODs), catalase, and glutathione peroxidases (GPx) and non-enzymatic antioxidants such as glutathione (GSH), vitamin A, vitamin C, vitamin E, uric acid etc. The brain contains 2% of total body, but utilizes 20% of the oxygen consumed by the body, indicating that the brain can be the source of many more free radicals than the other tissues (Dringen, 2000; Margail et al., 2005). However, the antioxidant level of the brain is low (Chan, 2001; Kelly et al., 2008; Polidori et al., 1998). As a result the brain can be very vulnerable to oxidative stress especially at ischemic stroke.

144 Antioxidant Enzyme

The techniques, which are usually used for detection of free radical generation, as spin trapping, electron paramagnetic resonance are not applicable for human brain. Because of the transient nature of oxygen radicals and technical difficulties in measuring their brain levels, experimental strategies have been focused on the use of pharmacological agents and antioxidants, seeking a correlation between an exogenous supply of specific free radical scavengers (e.g. SOD, catalase) and the subsequent protection of cerebral tissue from ischemic injury. Human studies evaluate the presence of either oxidized molecules or antioxidants in blood, urine or cerebrospinal fluids (CSF). Antioxidant activity is known to reflect the altered redox balance of affected fluids, tissues or organs in several pathological processes including brain ischemia (Cherubini et al., 2005). A biomarker of oxidative stress is classically defined as a biological molecule whose chemical structure has been modified by ROS. Additionally, any biological process influenced by ROS could be used as an oxidative stress biomarker. Therefore, antioxidant concentration or degree of antioxidant activity can be useful to estimate the extent of oxidative stress. The prediction of outcome in ischemic stroke is important for clinicians, patients, and researchers. The pathogenesis of ischemic stroke (IS) is highly complex. Oxidative stress is proposed as a fundamental mechanism of brain damage at ischemic stroke. Measurements of antioxidants in plasma can allow revealing a new pathological feature of formation the ischemic stroke seat and can be considered as noninvasive tools in the monitoring of the disease, as cellular changes may be reflected in body fluids. We studied a wide spectrum of components of antioxidant system in plasma of healthy volunteers (controls) and patients within the first 72 h of acute ischemic stroke onset, including enzymatic and non-enzymatic antioxidants, and discriminate of their activity and quantity for the establishment of possible correlation. The obtained correlations can be considered as biomarkers during the acute phase of ischemic stroke (IS) and corroborate the existing clinical prognostic models to predict the outcome in individual patients with stroke, which are not enough accurate (Counsell et al., 2004).

2. Experimental
2.1. Clinical study
Case subjects are selected from the all acute stroke patients admitted to the Sarajishvili Institute of Neurology and Neurosurgery (SINN). 42 eligible subjects (22 males and 20 females; 69±15 years of age) were selected from 70 patients with suspected acute stroke admitted to either Clinical or Critical Care departments of the SINN. Reasons for exclusion were: final diagnoses other than stroke (7 cases), admission after 72 hours of stroke onset (4 cases), hemorrhagic stroke (10 patients) and patients’ refusal to participate in the study (7 cases). All study subjects underwent the following investigations: detailed neurological examination (special stroke scales for evaluating the stroke severity and functional state were used according to the study protocol), CT, Extracranial Dopplerography, EKG and

Plasma Antioxidant Activity as a Marker for a Favourable Outcome in Acute Ischemic Stroke 145

detailed laboratory work-up including routine blood and urine analysis, coagulation tests, venous hematocrit, routine blood biochemistry (glucose and total cholesterol). Patients were clinically evaluated using GOS (Glasgow Outcome Scale), GCS (Glasgow Coma Scale), Barthlet-Rankin (Scale), Allen (Scale). Additionally, patients were stratified according to the NIHSS (National Institute of Health Stroke Scale) score and the Oxfordshire Community Stroke Project (OCSP) classification.  The Oxfordshire Community Stroke Project classification is widely used for stroke pathophysiology classification. This classification divides cerebral infarction into four categories: total anterior circulation infarction (TACI), partial anterior circulation infarction (PACI), lacunar infarction (LACI), and posterior circulation infarction (POCI). From all patients 14 patients were in the category TACI, 14 patients in the category PACI, and 14 patients in the category LACI. It was not sufficient cases in the category POCI for the statistical analysis. Healthy individuals without stroke are selected randomly from outpatients paying visits to the Polyclinic of the Sarajishvili Institute of Neurology and Neurosurgery. This study includes plasma samples from 15 healthy individuals (11 males and 4 females; 43±30 years of age). Controls were persons without acute stroke/history of stroke and without current acute or chronic inflammatory illness. Blood samples were drawn in sterile tubes and then centrifuged for the further analyses of the plasma. Besides, the plasma of 17 healthy donors from the Blood Bank of Jo Ann's Medical Center was used as the control subjects. The study protocol was approved by the local ethics committee, and written informed consent was obtained from each participant or their relatives before inclusion in the study.

2.2. ELISA method for the quantification of Cu, Zn-SOD in plasma
Cu,Zn-SOD assay ELISA kit (IBL International, Germany) based on the monoclonal antibodies to human Cu,Zn-SOD has been used to quantify Cu,Zn-SOD in plasma. We have followed the manufacturer’s instructions.

2.3. Quantification of SOD activity in plasma by the spectrophotometric method
Superoxide Dismutase Assay (IBL International, Germany) based on colorimetric superoxide radicals detection has been used to quantify total SOD activity in plasma. Superoxide radicals are generated by the xantine oxidase and hypoxanthine pair. One unit of SOD is defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radicals. The SOD assay measures total SODs (Cu,Zn-SOD, Mn-SOD, and extracellular SOD) activity in plasma.

2.4. Quantification of catalase activity in plasma
Hydrogen peroxide created after superoxide radical disproportionation by SOD can be neutralized in blood by catalase, which along with peroxidases may regulate hydrogen peroxide either generated in blood or coming from other tissues. Catalase spectrometric measurement at 240 nm based on the ability of catalase to oxidize hydrogen peroxide

146 Antioxidant Enzyme

proposed by the method of (Beers & Sizer, 1952). The conditions for catalase measurement in plasma were set. Briefly the method is as follows: 2.25 ml of potassium phosphate buffer (50 mM, pH 7.0 or 65 mM, pH 7.8) was added to 0.05 ml of plasma (diluted (1:10) by potassium phosphate buffer (50 mM, pH 7.0)) and incubated at 25ºC for 30 min. 650 μl of hydrogen peroxide (to get 7.5 mM final) were added to initiate the reaction. The change in absorbency was measured at 240 nm for 3 min. The catalase activity was expressed in IU. One international unit (IU) of catalase is the enzyme activity, which decomposes one μmol of hydrogen peroxide per minute at 25ºC. Western blots were prepared from total blood plasma protein samples diluted 1:25 times in Laemmli loading buffer and separated on 12% SDS-polyacrylamide gels (normalized to 75 μg per lane) and blotted on Hybond-C Extra membrane (Amersham, USA). Membranes were blocked in 3% (w/v) Ovalbumin (Sigma) in 1xPBS for 1 h, washed in 1xPBS and 0.02% Tween 20 and incubated with diluted antibodies (IgG) against human erythrocytes catalase (500 ng/ml) (Oxis, USA) overnight at 4°C and then secondary goat anti-rabbit IgG (1:2500) (Sigma, USA) for 2 h. After further washing with 1xPBS and 0.02% Tween 20, chromogenic detection was performed using the chromogen 4-cloro-1-naphtol.

2.5. Total glutathione quantity in plasma
The BIOXYTECH GSH/GSSG kit (OXIS, USA) was used to estimate the content of GSH and GSSG in plasma of healthy volunteers and patients with ischemic stroke. The kit procedure is based on the use of Elman’s reagent – DTNB (5,5’-Dithiobis-(2-nitrobenzoic acid)). The color developed was read at 412 nm.

2.6. Total thiols concentration in plasma
A 96-well plate method of thiols’ quantification using DTNB optimized for plasma was used to estimate the content of total thiols in plasma of healthy volunteers and patients with ischemic stroke (Hawkins et al., 2009). The procedure is based on the use of Elman’s reagent (DTNB), which interacts with SH groups producing chromogenic substrate. The range of GSH concentration 0 – 0.5 mM serves as the standards. The color developed was read at 412 nm.

2.7. Methods of analysis
All values are expressed as means and medians by using Origin for Windows, version OriginPro8, and were analyzed using the Mann-Whitney U test (two-tailed). Correlation between variables implies a statistical test carrying out under the null hypothesis. A null hypothesis is a precise statement relating to the research question to be tested, expressed in terms which assume no relationship (association) or difference between variables. Correlations between variables were determined by Spearman’s rank test and Pearson’s rank test. Spearman’s rank correlation coefficient (rs) provides a measure of how closely two sets of rankings agree with each other. Pearson's correlation coefficient (rp) is a measure of

05 was taken to be of statistical significance. the use of free unmodified SOD was not successful. tetrameric (130 kDa) Cu. Their sole function is to remove the superoxide and thus protect cells against oxygen toxicity. containing one cooper Cu(II) per subunit joined to buried Zn(II) by a bridging histidyl imidasolate group (Fridovich.05 was not taken to be of statistical significance. It is found in the cytosolic and lysosomal fractions. 1999).5 hours). 1984).Zn-SOD and Mn-SOD (Marklund. The EC-SOD content is about 100 times higher compared with other tissues such as muscle or fatty tissues. Okato-Matsumoto & Fridovich. Liposome-entrapped SOD has an increased half-life (4. SOD catalyzes the superoxide dismutation to hydrogen peroxide and oxygen by alternate reduction and reoxidation of the transition metal at the active site (Hsieh et al. The arterial wall contains exceptionally large amount of EC-SOD as a result of EC-SOD binding to the surface of endothelial cells and the extracellular matrix.Zn-SOD (6 min) in circulating blood and its failure to pass the blood-brain barrier (BBB) makes it difficult to use enzyme therapy in cerebral ischemia. 2001). Based on the metal ion requirement and the atomic distribution two main types of endogenous SOD exist. EC-SOD is secreted into extracellular fluids.01 was taken to be of significant difference. 1999).Zncontaining glycoprotein explaining SOD activity in extracellular fluids (Enghild et al. P value ≥0. The modified enzyme with an increased half-life. Cu.. which can be seriously damaged at the ischemic stroke. SOD plays a central role in protecting cells against harmful effect of superoxide radicals. The extremely short half-life of exogenous Cu. and binds with sulphated polysaccharides such as heparin and heparin sulphate (Marklund.Zn-SOD has been extensively used to reduce brain injury caused by ischemia and reperfusion by its exogenous supply and the subsequent protection of cerebral tissue from ischemic injury. but it exists also in the mitochondrial intermembrane space (Mates et al. 1998. P value <0. Mates.. Mn-SOD homotetramer (96 kDa). 1995). SOD activity The wide distribution of superoxide dismutase among aerobic organisms points to the special role of this enzyme (Fridovich. 3.. Results and discussion 3. Extracellular SOD (EC-SOD) is the secretory. 1989).1. is found in the mitochondrial matrix (Mates et al.. Enzymatic antioxidants at ischemic stroke in plasma 3. and it has also proved . However.1.Plasma Antioxidant Activity as a Marker for a Favourable Outcome in Acute Ischemic Stroke 147 the strength of the association between the two variables. containing one manganese per subunit. A P value <0. 1993). such as polyethylene glycolconjugated SOD has been successfully used to reduce infarct volume in rats that have been subjected to focal cerebral ischemia (He et al. BBB permeability.. suggesting a special function of EC-SOD within the vascular walls. 2000). by cells such as fibroblasts. 1999. and cellular uptake. endothelial cells and smooth muscles. but its concentration is substantially lower than Cu. 2003). 1984) as well as other matrix components (Fattman et al.1. EC-SOD also expressed in brain tissue. Cu. such as plasma and lymph..ZnSOD is a homodimeric enzyme (32 kDa).

in serum (Spranger et al.Zn-SOD overexpression. It was established that total SOD activities were significantly lower in patients compared to healthy controls (P=0. Sheng et al. In our study we used colorimetric method to detect the total SOD (including Cu. pharmacokinetic. We evaluated the changes of plasma SODs activity after stroke to determine their utility in predicting outcome in terms of survival and functional status. The alternate and more direct method for the study of oxidative stress in ischemia and reperfusion injury is to use transgenic/knockout technology to alter the levels of prooxidants.. 1992). Besides. Neither Cu.Zn-SOD. 1987. As to endogenous SOD level or activity in cerebrovascular ischemia the data are contradictory: SOD activity in brain tissue after ischemia/reperfusion has been found both to be decreased (Tokuda et al. 1999a..148 Antioxidant Enzyme to be an effective treatment in reducing severity of traumatic and focal ischemic brain injuries (Chan et al. and possible toxic effects of drugs.. 2001) or no modification (Alexandrova et al. In these studies SOD activity was inversely correlated with the size of infarction and the severity of neurological deficit.. Knockout and overexpressing mutants for both Cu. 1997) and in red blood cells (Demirkaya et al. 1994) or CSF (Strand. 1993) and increased (Sutherland et al.. 1). Some studies observed an augmentation of SOD concentration in plasma (Gruener et al.. In the human study the data are also contradictory: SOD concentration after stroke was unchanged in serum (Adachi et al. the SOD activity has been monitored in human erythrocytes at IS. 1996). and the decrease (Demirkaya et al..Zn-SOD targeted deletion alter the outcome from permanent focal ischemia (Chan et al. The reason of the low SOD activity in plasma can be related to the exhaustion of enzymes owing to ROS scavenging or the inhibition of enzymes caused by ROS (Escobar et al. . while EC-SOD knockout exhibits enhanced damage (Sheng et al... 2004) has been observed. 1994). indicating the requirement of reperfusion for this enzyme to play role. 2002. However. as well as their blood-brain barrier permeability properties... Experiments with transgenic mice overexpressing Cu.Zn-SOD and Mn-SOD isozymes have been created. 1998)... & Marklund. But in some instances. the lower SOD activity was associated with the worst outcome. nor Cu. 2000)... Imaizumi et al.0018) (Fig. 1994).Zn-SOD reveal reduction of ischemic damage resulting from ischemia/reperfusion at middle cerebral artery occlusion (Yang et al... EC-SOD overexpressing mice have increased tolerance to both local and global cerebral ischemia (Sheng et al. The decrease of SOD activity in stroke patients has been detected in plasma (Cherubini et al. 1993).Zn-SOD activity in CSF with the size of the infarct and functional impairment. Mn-SOD and EC-SOD) activity in plasma of healthy controls and patients at the early stage of ischemic stroke. and oxidant related enzymes or proteins and to study the role of a specific oxidant or antioxidant in ischemic brain injury. 1991).. 2000). Strand and Marklund (1992) reported good correlation between the increased Cu. 1990). antioxidants. Murakami et al. modified SOD has been used with conflicting results caused by hemodynamic.. 1999b). Mn-SOD targeted deletion worsens outcome from both temporary and permanent middle cerebral artery occlusion (Kim et al. 2001).

.Zn-SOD.Zn-SOD content in blood plasma of patients within the first 72 h of ischemic stroke onset in comparison with control samples has been detected. The reliable increase of Cu. Cu.Plasma Antioxidant Activity as a Marker for a Favourable Outcome in Acute Ischemic Stroke 149 To quantify Cu. Figure 1. The difference between the ischemic stroke and healthy groups was statistically significant (P=0. P=0. P=0.0079.0018. 2). Figure 2. SOD activity in plasma of healthy volunteers (Controls) and patients with ischemic stroke onset (IS).Zn-SOD level in plasma we used assay based on the monoclonal antibodies to human Cu.Zn-SOD activity in plasma of healthy volunteers (Controls) and patients with ischemic stroke onset (IS).0079) (Fig.

& Marklund. Enzymes of low molecular weight may passively leak from the intracellular space within hours from ischemically disturbed membranes..1. Heiss et al. the latter functions in the cytosol and mitochondria. such as depletion of ATP. Besides. . Necrosis is the predominant mechanism follows acute occlusion. whereas milder injury. Both catalase and GPx are present in the brain. 1984. When the Cu.. Safe disposal of hydrogen peroxide is carried out by catalase and glutathione peroxidase.150 Antioxidant Enzyme The increased Cu. 2000). & Marklund.. The differences in the severity of the ischemia in the core and penumbra result in the switching on the different mechanisms of the cell death – necrosis and apoptosis.Zn-SOD in apoptotic cells does not significantly differ from normal ones.Zn-SOD activity was detected in CSF (Strand. the improvement of the outcome points to sufficient concentration of catalase and GPx in the adult mice brain to defence brain against hydrogen peroxide produced owing to superoxide dismutation. and the damaged endothelial cells cannot be excluded as the source of intracellular SOD in plasma of IS patients. In developing brain catalase and GPx are poorly expressed. disrupting mitochondrial membrane potential that trigger the apoptotic pathway (Kroemer & Reed.ZnSOD overexpression in adult mice improves the outcome (Yang et al. the ischemic patients’ blood showed significantly higher catalase and GPx activity in comparison to the control group. although GPx activity is greater than that of catalase. 2004). Catalase is a tetrameric protein (240 kDa). which increase the rate of hydrogen peroxide production. Cu. 2004.Zn-SOD overexpression was studied in the neonatal mice. Although it is difficult to say which one (catalase or GPx) plays a central role for the brain defence? In animal models estimation of endogenous antioxidant system in brain tissue showed a significant decrease of catalase activity at the reperfusion stage. NADPH pools and induction of mitochondrial permeability. However. GSH. will lead to the accumulation of hydrogen peroxide in cytosol and mitochondria. produced by Cu.. The activity of Mn-SOD and Cu. 1996). if apoptosis is caused by oxidative conditions (Asatiani et al. which contains a ferric (Fe(III)) haem group per subunit bound to its active site (Mates. Elevation of hydrogen peroxide results in harmful consequences.2. and the outcome from ischemia/reperfusion was worsened (Fullerton et al. it was shown that excess of hydrogen peroxide. infarcts initially develop in the core tissue but continuous to penumbral regions (Back et al. 1998).. The former is located only in peroxisoms. Severe oxidative stress causes cell death through necrosis while moderate oxidation can trigger apoptosis (Evans &Cooke. the damages in endothelial cells may also accompany the IS. which may explain why the increase of SOD activity/concentration was frequently found within 8-36 hours after symptom onset in extracellular fluids (Marklund. 3. 1994). Strand. 2004). 1992) in stroke patients. Catalase activity Pathological conditions. as well as a 48 h delayed decline in GPx activity. In human total blood catalase and GPx activities did not reflect the severity of neurological deficit (Alexandrova et al. Thus. 2000).Zn-SOD cannot be scavenged neither by catalase... Liu et al. 1992). particularly within the ischemic penumbra often results in apoptosis. 1995. Following arterial occlusion. 1994). nor by GPx.

Western blot. The increased activity was detected (data not shown). and the difference between the ischemic stroke and healthy groups was statistically significant (P=0. The data are presented in Fig. Figure 4. P=0. Catalase activity in plasma of healthy volunteers (Controls) and patients with ischemic stroke onset (IS).Plasma Antioxidant Activity as a Marker for a Favourable Outcome in Acute Ischemic Stroke 151 In our study the increase of catalase activity was observed in the patients’ plasma that points to the oxidative stress developed under ischemic conditions. It was also estimated GPx activity in patients’ plasma. stained with anti-catalase. The catalase activity in plasma was expressed in IU/ml. 3. C-control. USA). Figure 3. St – IS patients. . catalase from bovine liver (Oxis.0089). M – marker.0089.

Severely disabled (conscious. EC-SOD is considered to serve for defence against superoxide.Good recovery (the patient has resumed most normal activities but may have minor residual problems.. The results demonstrate activation of catalase in case of IS 7 as the response to oxidative stress (low quantity but high activity) and activation of catalase in case of IS 3. but the patients requires others for daily support due to disability).Zn-SOD level reliably increased in all cases. In all three cases SOD activity decreased (with different degree) compared to control and was statistically different (P<0. Thus the both possibilities of the increase of the catalase activity take place at the ischemic stroke conditions: by the increase of quantity and by activation under oxidative stress conditions. The results are presented in Fig. or the increased catalase activity is the result of catalase activation under the oxidative stress conditions at IS. it may be important for ischemic event. 1982). It should be noted that whereas Cu. if in case of ischemic strokes.01) at moderate disability and recovery (Table 1). Plasma enzymatic antioxidant profile as the predictor of functional outcome A disturbance in the oxidant/antioxidant balance in favor of antioxidants may be implicated as a prognostic factor in human stroke. but alive). moderately disabled (4) and recovered (5).05). 1992). catalase and GPx) are activated in the first 72 h in plasma of patients diagnosed with acute ischemic stroke in our study.01) and moderate state (P<0. For this reason we have stratified the patients in accord with Glasgow Outcome Scale of ischemic stroke development. EC-SOD is a major one (Marklund et al. the observed activation of catalase is connected with quantitative increase of catalase in plasma.3.Dead.Zn-SOD and Mn-SOD are found in very small amounts in human extracellular fluids.Zn-SOD is overlapped by decrease of ECSOD level which predominates in extracellular plasma.152 Antioxidant Enzyme We have used Western blotting to estimate.1. (4)Moderately disabled (the patient is independent but disabled). Our patients were subdivided into three groups: dead and severely disabled (1+3).4. Cu. In spite of the relatively low EC-SOD concentration in whole brain. The extracellular compartment is small and thus EC-SOD concentration in the extracellular compartment may be sufficient to provide defence (Cherubini et al. Three investigated enzymatic antioxidants (Cu.05).. (2)Vegetative state (meaning the patient is unresponsive. augmentation of Cu. 4. (3). (5).Zn-SOD.9 as the result of the quantitative increase of catalase (high quantity and high activity) in blood plasma of IS patients in comparison with controls. . which can be produced by membrane– bound NADPH or secreted by inflammatory cells into the extracellular space (Oury. According to GOS ischemic patients are subdivided into five groups (GOS is a 5-level score): (1). but is significantly different (P<0. Perhaps in a case like that. 3. 2005). characterizing by the increased catalase activity.8. In all three cases catalase activity of patients increased compared to controls and correlates with recovery (P<0.

Control SOD activity (U/ml) Cu.2 97.3 tly different significant tly different ±24.62 P 0.1.5x rs 0. 0.71 P 0.0038 (are 219.7.0137 (are 11.significan20. IStroke Min.31 12.5 39.05).0038 (are 55.Zn-SOD at the different functional outcomes in accord to GOS.8) (69.5 139.5) 513 tly different tly different tly different 422 249 360 ±151.5 ±129.63 23.7) ±25.05) 0. 41.72 P < 0.01 ±81.marginally significan(48. Correlations of plasma antioxidants in the IS patients with different functional outcome. 42.13 ±14. Catalase (5) GOS y=a+bx y= 56.029 (are 0.27 (are not 0.01) P < 0.61 7.ZnSOD vs. P – significance levels) and Pearson rank correlation analysis (rp – coefficient of correlation.98 3.Zn-SOD in plasma has not been observed (P>0.84 42.85 P<0.26 0.ZnSOD vs.9 Min. significan. 0.01 rp 0. (173.significan. Correlation Cu.Zn-SOD and catalase) can be considered as adequate markers for the positive outcome in the range of Glasgow Outcome Scale within the early phase of ischemic stroke development.873.88 P≥0. The correlation between the enzymatic antioxidants We have considered the relation between catalase and Cu.5 significant tly different significant ±17.41 0.81+0.746 85.01) P<0.65) ( ±101. Correlations were determined by combining data from 42 patients and 32 controls using Spearman rank correlation analysis (rs – coefficient of correlation.05) P < 0.05) P<0.011 (are 0.5) (92.82 8. 41.21 (23.5) (83.8 51.38 0.1 130.35) ± 60.7.27 0.ZnSOD vs. P (Control P (Control P (Control Max (1+3) Max (4) Max (5) Max & IS (1+3)) & IS (4)) & IS (5)) 0.07 (are not 0.05) 0.4. Plasma levels of enzymatic antioxidants in control and ischemic stroke patients stratified according to GOS.5) (230.4) (4.6 +1.24. In case of moderate recovery and a poor functional outcome a correlation between catalase and Cu. 3. Catalase (4) GOS Cu.01) P≥0.3) ±19.51 0.00187 (are 0.21 0.1. significan.5 (36. 0. 28.63 x y=111.5 98.71.05) P<0.15 ±32. Catalase (1+3) GOS Cu.23 0. it cannot be considered as an adequate marker of IS outcome. Values (means) are analyzed using the Mann-Whitney U test (numbers in the parentheses are medians) ±SD (standard deviation).002 Table 2.4 51.25 66.92x y = 105. In patients with ischemic stroke the increased catalase activity and Cu. As total SOD activity in plasma of IS patients reliably decreases in all cases.6) 97. P – significance levels). IStroke Min.7 78.marginally (3.ZnSO D (ng/ml) Catalase activity (IU/ml) 25. The results are presented in Table 2. marginally significan.55+3.Plasma Antioxidant Activity as a Marker for a Favourable Outcome in Acute Ischemic Stroke 153 The activated antioxidants (Cu. 0.31 236.44 0.8 50.01) Table 1.Zn-SOD levels are associated with a positive functional outcome and are significantly correlated with each other only in case of recovery . IStroke Min.6 (41.66 ± 11.0031 (are 0.

1.6. significantly 28.9 97. Plasma enzymatic antioxidant profile in ischemic stroke patients stratified according to OCSP classification IS pathophysiology classification of the patients in accord with Oxfordshire Community Stroke Project has revealed. The tandem action of Cu.5 (36.19 (are not difference is 141. 0. Glutathione exists in two major forms: reduced (GSH) and oxidized (GSSG).05).3 97.5.4 Cu.3 113.2. significantly (48.29 (are not significantly different P≥0.5 different ±17. significantly highly (136.93 13.8 360 311.025 (are marginally significant P < 0.5.05) Table 3. 48.43 P≥0. and sulfhydryl-donating capacity.44 P < 0.3 P<0.154 Antioxidant Enzyme (P<0.05) 0. 1.7 ±15. Activity significantly (23.08 4.0052 (are not 50.1) (210.65) (69.20 ±19. electron-donating.21 11.5 83.7) (IU/ml) different ±25.48 73.05) P<0.059 (are 0.8 513 280 significant ±111. The ratio of GSH/GSSG plays an important role in regulating the cellular redox status.1 ±19.0056 (are significantly different P<0.24.01) 0.64 ± 16.8 ±137.00057 (the 0.6 60. 0. .7 ±3.05) 0.30 marginally 3. The reducing power of GSH is a measure of its free radical scavenging.7. I Stroke Min. 2.8.6 125.7) 23. P (Control & P (Control & Max (TACI) Max (PACI) Max (LACI) Max IS (TACI)) IS (PACI)) 0.ZnSOD 78. Values (means) are analyzed using the Mann-Whitney U test (numbers in the parentheses are medians) ±SD.8) (69.3) (3.85) (5.1.2. it increases only in patients with PACI and LACI (Table 3). GSH has a potent electron-donating capacity as indicated by the high negative redox potential of the GSH/GSSG redox couple.6 (41.05) Control P (Control & IS (LACI)) 0. 20. that Cu.2.03) significant 60.66 Catalase 50.001) 0.4 44.05) P≥0. 48.94 74. Non-enzymatic antioxidants at ischemic stroke in plasma 3. since it is the most abundant thiol-disulfide redox buffer in a cell.1.9) 0.036 (are SOD not 25.9 226.01) P≥0. For catalase activity.6.5. Plasma levels of enzymatic antioxidants in control and ischemic stroke patients stratified according to OCSP classification. I Stroke Min. 17.Zn-SOD and catalase is clearly elicited in plasma of IS patients and their activation is necessary for a recovery after IS.2) (89) (ng/ml) different ±101. 3. 3. I Stroke Min.402 (are 0. The exclusive role of Cu. Glutathione The major water soluble non-enzymatic antioxidant glutathione is localized in both the cytosol and the mitochondria of cells. Zn-SOD is elicited in case of such brain damage location as PACI.Zn-SOD only increased in plasma of patients with partial anterior circulation infarction. Min.7 10 (U/ml) different ±19.6 ±98.6) (5.

1980) in rats. 2008).275) at both studied conditions. astrocytes appear to contain higher GSH level than neurons both in vivo and in vitro (Cooper. In our study GSH and GSSG levels were estimated in plasma of healthy volunteers and patients within the first 72 h of ischemic stroke. forming GSSG (Dringen et al...g. 2011). 1983) in non-enzymatic reactions and is the electron donor in the reduction of peroxides by GPx. ischemic outcome is worsened by pharmacological depletion of GSH (Vanella et al. It was shown that concentration of GSH decreases early after ischemia (Cooper et al. Kussmaul et al. The data are presented in Fig. It is known. The product of the oxidation is GSSG. from oxidation. and to the reversibility of redox balance distortion in tissues and organs. that in a cell GSSG may form mixed disulfides with thiol-containing enzymes. Extracellular glutathione defenses SH-groups of proteins. disrupting their normal activity (Mieyal et al. 1993). Astrocytes in culture can decompose hydrogen peroxide with a rapid oxidation of GSH. The plasma soluble components and formed elements of blood do not destroy plasma circulating glutathione. GSH concentration does not differ (P=0. constituting the blood formed elements’ plasma membrane.. 2000.2001. We can suppose that the increased GSSG concentration in plasma is the result of GSSG export from neural tissue and blood formed elements into plasma for the maintenance of redox potential of a cell. 1999).. e..00098). it is very important to estimate not only changes in GSH and GSSG content in plasma at acute ischemic stroke conditions. . GSSG content increases highly significant at acute ischemic stroke (P=0. However. But these conditions are characterized by the different GSSG concentration. 2000).. Its content and localization varies in different regions of the brain. Glutathione system plays a very special role in the brain defense. The correlation between GSH and GSSG in plasma can testify to the extent of oxidative conditions. Park et al. 2000). plasma concentration of protein thiols is associated with the degree of neurological impairment (Leinonen et al. 1997). As it follows from the picture.. developing in tissues and organs. 5. Oxidation of the cysteine sulfhydryl groups joins two glutathione GSH (reduced form of glutathione) molecules with a disulfide bridge to form glutathione disulfide GSSG (oxidized form of glutathione). Rehncrona et al.. Thus. but also to estimate the correlation between these two forms.. Glutathione reacts directly with radicals (Wefers & Sies. which is reduced by glutathione reductase (GR). a cell effectively opposes the development of oxidative stress by getting rid of GSSG either by glutathione reductase reduction or by active export of the disulfide from a cell (Nur et al.. preventing the formation of S-S bonds. 1980.. 2000). Thus GSH is recycled during this process (Dringen et al. Plasma contains both forms of glutathione: reduced and oxidized. Depletion of the total GSH and decrease of GSH/GSSG ratio are markers for oxidative stress in ischemic brain and as long as 72 h may be required to restore concentrations to normal values following an ischemic insult according to (Namba et al.Plasma Antioxidant Activity as a Marker for a Favourable Outcome in Acute Ischemic Stroke 155 Glutathione is a tripeptide (γ-L-glutamyl-L-cysteinylglycine).

SH-groups of blood plasma proteins can quench up to 50% of peroxyl radicals and as the result inhibit the process of lipid peroxidation taking place under the oxidative conditions (Wayner et al. study of the concentration changes of glutathione and total thiols can provide an understanding of processes at the ischemic stroke The total thiols concentration in blood plasma at ischemic stroke can serve as an indirect indicator of the oxidative conditions developing under the stroke circumstances. and the values between the total thiols concentration in healthy subjects and IS patients are significantly different (P=0. The oxidized thiols make a valuable contribution to the neurodestruction mechanisms. GSH and GSSG levels in plasma of healthy volunteers (Controls) and patients with ischemic stroke onset (IS) 3. 1987). tissues and biological fluids in an organism. It was shown in our study. . oxidative modification of SH-groups in enzymes or in their coenzymes influences on enzymatic activity. leading to cellular and tissue hypoxia. Reversible modification of SH-groups is considered as nonspecific defense mechanism of organism in response to the extreme conditions. The data are presented in Fig. the total thiols concentration decreases at acute ischemic stroke.156 Antioxidant Enzyme Figure 5. As it follows from the analysis and the picture. that the total thiols level at ischemic stroke within the first 72 h of ischemic stroke onset was decreased.2.0083). is considered as the protective effect of thiol-containing compounds against irradiation and its accompanying oxidative stress. 6. Thus. SH-group modification of membrane proteins changes membrane permeability.. At the irreversible displacement of the thiol-disulfide ratio the expression of the pro-apoptotic proteins is also possible. SH-group containing compounds are the subject of oxidative stress in the first place and provide first line of defense by direct scavenging of hydroxyl radicals. Total thiols Thiol (SH-group)-containing compounds are the important components maintaining redox homeostasis in cells. that in turn decrease reducing potential of a cell.2. namely displaying thiol-disulfide system to the augmentation of the oxidized thiols concentration. Thus. Thiol’s autooxidation.

02 (2.36 (are not 0.05) < 0.05 P≥0. significantly marginally marginally (1.425 0.0133 (are 0.7) ±1.17 ± 0.56. 0.4) different significant P 0.57 0.211 ± 0.213) (2.05) GSH (μM) 0.12 3.315. I Strok Min.484) (0.476) (mM) ±0.46 P≥0. The total thiols (RSH) concentration in plasma of healthy volunteers (Controls) and patients with ischemic stroke onset (IS).49 0.467.3 9.47. The decrease of the total thiols and the increase of GSSG points to the necessity of the redox regulation of the intracellular and extracellular processes for the good outcome at IS (Table 4). 0.503 different significant P significant P ±0.36 (are not not not 2. 0.05) < 0.797 0.8 different different ±1. P (Control & P (Control & P (Control & IS (4)) IS (5)) Max (1+3) Max (4) Max (5) Max IS (1+3)) 0.194.69 ±1.05) 0.25 ±3. P=0.453.0133 (are 1.52 ±1.93 1.44 4.008 0. significantly significantly marginally (0.145 (are 0.05 < 0. IStroke Min. 0.05) P≥0.294.05 P≥0. 0.367) (μM) ±0.71) (1. 1.871 1. .51 Thiols (0.033 1.297 (are 0. Plasma non-enzymatic antioxidant profile as the predictor of functional outcome The behavior of the non-enzymatic antioxidants at acute IS correlates with the behavior of the main enzymatic antioxidants (Cu.476) (0.25 Min.38 GSSG (0.61 (are not 0.08) 0.329 3.2. 0.3.885 1.132.0083.72.252 1.196 1.7) (1.020 (are 0.088) different 4. IStroke Min. Plasma levels of non-enzymatic antioxidants in control and ischemic stroke patients stratified according to GOS.Plasma Antioxidant Activity as a Marker for a Favourable Outcome in Acute Ischemic Stroke 157 Figure 6.006) (1. significantly significantly (2.051(are not 0.11 different ± 0.759. Control Total 0.456 P≥0.255 P≥0.23 Table 4.187. Values (means) are analyzed using the Mann-Whitney U test (numbers in the parentheses are medians) ±SD.706 3.Zn-SOD and catalase) directed to the protection against the oxidative stress.05 0.32 ±0.09.213 1. 0. 3.76 significantly 1. 1.528 3.

38 GSSG (0.111 0. 2010). 0.4.885 1.1) different different different 4. Values (means) are analyzed using the Mann-Whitney U test (numbers in the parentheses are medians) ±SD. Biological half-life of this protein is 24 hours.45.768 3.24 ±1.2.17 ±0. marginally marginally significantly (0.44 4. of .536.158 Antioxidant Enzyme As it follows from the Table 4.05) 0.12 3.63 ±1.528 3.315.445) (0.343) 0.476) (mM) ±0.869) (2.262 3.871 1.367) (μM) ±0. 3. This may indicate to the defense reactions at the early stages of ischemic stroke. 0. P (Control P (Control P (Control Max (TACI) Max (PACI) Max (LACI) Max & IS (TACI)) & IS (PACI)) & IS (LACI)) 0.006 (are 0. IStroke Min.56. Control Total 0. widely used for stroke pathophysiology classification. 0.64 1.038 (are 0.72) (1. Plasma non-enzymatic antioxidant profile in ischemic stroke patients stratified according to OCSP classification It was not revealed the specific connection of the non-enzymatic antioxidants activation and the division of cerebral infarction into four categories in accord with the Oxfordshire Community Stroke project classification. 3.58 0.05) < 0. A CRP concentration in plasma is widely used by clinicians as a marker for acute inflammation and tissue necrosis. This could be an evidence of active export of GSSG from the cells in case of ischemic stroke and the possible restoring of the redox balance in the cells of damaged tissue areas. 0.987 1.704 2.9 P≥0.15 < 0. which is reflected in the participation to run multiple signaling pathways leading to neutralization impact. A reliable decrease of the thiols concentration in plasma of the IS patients with a favorable outcome (score 5) points to their oxidative modification.02 (2.28 (are not 2.375) 0.05) P<0.reactive protein (CRP) is an acute-phase protein.356 0. significantly significantly significantly (0.05) P≥0. IStroke Min. CRP is produced exclusively in the liver.25 ±0.765 ±3.01) GSH (μM) 0.08.66) (1.039 (are 0.194.7) ±1.797 0.05) P≥0. significantly significantly significantly 0. 0. and in case of irreparable damage to the elimination of cells (apoptosis) (Circu & Aw.05) P≥0.759. 1. Plasma levels of non-enzymatic antioxidants in control and ischemic stroke patients stratified according to OCSP classification.25 Min.132. 1.09.51) (0.8 ±1.47.096 P≥0.196 significant P significant P different ±0.23 Table 5.1 1.88 0.178 (are 0. IStroke Min.549 different different different ±0. and in case of acute inflammation it starts to rise within the 6 hours in plasma. in the case of a favorable outcome the concentration of the oxidized glutathione increased in plasma.476 0.26 ±1.3 9.22 (are not not not 0. Plasma CRP levels in ischemic stroke patients as a valuable diagnostic marker at acute IS C .852.29) (1.05) P≥0. 0.3.27 (are not 0.51 Thiols (0.231.43 (are not 0.05) 0. (1.044 2. Plasma concentration of CRP increases significantly in cases of both infectious and non-infectious inflammation.084 (are 0.

51 14. CRP is present in the acute stages of inflammatory disorders like rheumatoid arthritis.000002 CRP 0.99 different ±23. 0. IStroke Min. the patients with total anterior circulation infarction and partial anterior circulation infarction are characterized by the significantly elevated levels of CRP. Kuhlmann et al. the elevated level of CRP was observed in patients. difference is significantly significantly ≤10 (1. As among the pathological processes in acute ischemic stroke are inflammation.99 ±23.46) highly different different 19 98 77. Values (means) are analyzed using the Mann-Whitney U test (numbers in the parentheses are medians) ±SD.00034 (are 0..51) (20. difference significantly ≤10 significantly (1.001 norm P≥0. 3. IStroke Min.001) Control Table 6.16.4 36. 0.117 (are (the 0. our data are in accordance with the generally accepted view on the CRP as the predictor of the poor outcome in IS.90 ±8. We prospectively measured the CRP concentration in plasma of IS patients in our study (≤24 hours from symptom onset) and compared it with the CRP plasma level of healthy persons. P (Control P (Control & P (Control & Max (TACI) Max (PACI) Max (LACI) Max & IS (TACI)) IS (PACI)) IS (LACI)) 0. in whom the score 4 in GOS (moderate disability) was appropriated as well. inflammatory bowel disease. 2001. As it follows from the Tables 6 and 7. and only in plasma of patients with lacunar infarction CRP level is in the range of normal concentration.6) (4. neuronal and glial injury. Min.81 6. Min.84 0.42. 2009). When we stratified the IS patients in accord with the Oxfordshire Community Stroke project classification.8) (9. IStroke Min.59 significant P<0.05) P<0.84 not 25.51) (22. I Strok Min. but it is non-specific for the kind and place of inflammation. 0.65) (8.07 ±17. 0.001) Control Table 7. Plasma level of CRP in control and ischemic stroke patients stratified according to GOS. IStroke Min. The plasma CRP concentration.32) (5.091 (are not (μg/ml) 3. Plasma level of CRP in control and ischemic stroke patients stratified according to OCSP classification. . CRP concentration in plasma was estimated throughout to correlate with and predict infarct growth in acute ischemic stroke and stroke progression.. Thus. 3. IStroke Min.Plasma Antioxidant Activity as a Marker for a Favourable Outcome in Acute Ischemic Stroke 159 tissue damage and necrosis. Values (means) are analyzed using the Mann-Whitney U test (numbers in the parentheses are medians) ±SD.5 is highly μg/ml ±4.000002 CRP (the 0. The elevated plasma level is currently accepted as an outcome-predicting factor at IS (Di Napoli et al. polyarteritis nodosa. CRP is considered as a very specific inflammatory marker. P (Control P (Control & P (Control & Max (1+3) Max (4) Max (5) Max & IS (1+3)) IS (4)) IS (5)) 0.0002 (are (μg/ml) 3.71 μg/ml ±4.05) norm P<0. systemic lupus erythematosus.4 22.7.74 13.71) different 19 98 77.84 28. is considered as normal concentration.53 0.41 ±15.63 significant P<0. However.84 6. The data are presented in the Tables 6&7.19 ±5.001 P≥0. ranging up to 10 μg/ml.

The exclusive role of Cu. antioxidants in plasma can be not only markers of oxidative stress at IS. and Shota Rustaveli National Science Foundation. Georgia Acknowledgement The work was supported by the Science and Technology Centre in Ukraine (STCU). which switch on the neuronal death program. grant 546. the neurodestruction at ischemic stroke is accompanied by the complicated metabolic cascades in neurons. Javakhishvili Tbilisi State University. Possibly. The behavior of the non-enzymatic antioxidants (GSSG and total thiols) correlates with the behavior of the main enzymatic antioxidants (Cu. and the study of the antioxidant system is a key moment in the understanding of the correct therapeutic strategy at the ischemic stroke. 4. It was revealed. that among the studied spectra of antioxidants the tandem activation of Cu. developed in case of IS poor outcome. We observed the decrease of the total thiols concentration and the significant increase of the oxidized glutathione concentration at acute ischemic stroke. Andronikashvili Institute of Physics.Zn-SOD and catalase) in case of the IS positive outcome directed to the protection against the oxidative stress.. but also the markers of brain tissue damages.Zn-SOD and catalase is necessary for a recovery after IS. the unprotected by antioxidant defense system oxidative conditions. Vasodilatation from hydrogen peroxide could be under control of the catalase activity. . 2009). CRP induces activation of surface Fcγ receptors CD16/32 followed by p38-mitogen-activated protein kinase-dependent ROS formation by NAD(P)H-oxidase. participate in the CRP activation registered in these conditions. According to our results. All these observations corroborate strategies targeting antioxidants for the therapeutic intervention in clinical settings. Iagor Kalandadze and Alexander Tsiskaridze I. The activation of catalase at acute IS of the disease onset points to the development of the oxidative conditions. Conclusion According to the current conception. oxidized thiols and products of oxidized modification of proteins and nucleic acids. grant 1-6/97.160 Antioxidant Enzyme However. Author details Nelly Sapojnikova. Tamar Kartvelishvili. what could point to the displacement of the reduced/oxidized balance to the increased oxidized thiols concentration. participation of CRP in blood-brain barrier disruption and its mechanisms are specified by Kuhlman (Kuhlmann et al. Recently. It was shown that the clinically relevant concentrations 10 and 20 μg/ml cause a disruption of BBB in a cell coculture BBB model and in the guinea pig isolated whole brain preparation. The switching of the death program can be accomplished by ROS.Zn-SOD is elicited in case of such brain damage location as PACI. Nino Asatiani. the question whether the elevated CRP levels are induced by stroke or reflect preexisting inflammatory conditions is still open. The oxidative conditions activate the contractile machinery involving phosphorylation of myosin light chain and as the result the disruption of tight junctions takes place.

Vol.Plasma Antioxidant Activity as a Marker for a Favourable Outcome in Acute Ischemic Stroke 161 5. Nakamura. N. (1996a).J. Journal of Inorganic Biochemistry. V. NeuroRx. the Bad.396-409.339. No. Kamii.. & Holman. Markova. Superoxide and Peroxinitrite: The Good. Annals of Neurology.. ISSN0162-0134 Back. & Fishman.J. 342-344. Vol. (April 2004).. No. Vol.H.5.. Popova. Epidemiology of Stroke. pp. N. 566-577. K. (2004). M. 490-496.. T. ISSN 0271678x Back. V. Dynamics of Free Radical Processes in Acute Ischemic Stroke: Influence on Neurological Status and Outcome..251.. Kato.1-2. Busto. Vol. Journal of Neurology.1. Brain Infarction is Not Reduced in SOD-1 Transgenic Mice After a Permanent Focal . W. Yamada.. Mechanism. (1995). (1952). American Journal of Physiology. Lesion Evolution in Cerebral Ischemia.. No. S.271. ISSN 0140-6736 Carmichael. R. Journal of Biological Chemistry. ISSN 1531-8249 Chan. & Ginsberg. Carlson. No..15.. 6. (1993). pp. (June 1987). ISSN 0340-5354 Beckman.. ( December 1997).3. Vol. pp.... Abuladze. ISSN 0967-5868 Asatiani. Kulikova. H. Journal of Cerebral Blood Flow & Metabolism.G. pp. The Lancet. 1266-1280 . G. Effects of Cr(VI) Long-term and Low-dose Action on Mammalian Antioxidant Enzymes (an in Vitro Study). N. 123-131. Journal of Clinical Neuroscience. H-Y. M. 133-140. Kiziria.4 (July 1995). H. (1994).A.229.21.W. and Ugle. L. B. C1424-1437. (2004). & Reola.S.195. M. C. References Adachi. Bochev. Vol. Gafni. (2004). ISSN 0363-6143 Beers.D. (July 2005). Vol. E. & Hirano. W.. pp. and Purpose. Protective Effects of Liposome-entrapped Superoxide Dismutase on Post-traumatic Edema. E. Hemmen. Rodent Models of Focal Stroke: Size. Nitric Oxide. Zhao. S. Danovska.98. R. T.17. ISSN 0021-9258 Belayev. No.. Vol. (March 2004). W.. & Simeonova. Longar. pp. (2005).12. & Sizer. pp.2. No. T.F. pp. Journal of Cerebral Blood Flow & Metabolism. 388-397. ISSN 0009-8981 Alexandrova. (8 February 1992)... T. T.1.8789. P. Vol. 540-547. L. Epstein. & Schuler O. No. (March 1952). Transient Middle Cerebral Artery Occlusion by Intraluminal Suture. (June 2004).. A. (1997). No. (November 1996). & Koppenol. (1987). Bechev. J.T. Vol. Futenma.. Three-Dimensional Autoradiographic Image Analysis of Focal Cerebral Glucose Metabolism-Blood Flow Interrelationships during Ischemia and Early Recirculation. I. Kartvelishvili.5. ISSN 1545-5343 Chan. No. A Spectrophotometric Method for Measuring the Breakdown of Hydrogen Peroxide by Catalase. (September 1994). pp. K. M. E. 501-506. No. pp. Namchevadze.. P. R.3. M.H.D. 3-Dimentional Image Analysis of Brain Glucose Metabolism Blood Flow Uncoupling and Its Electrophysiological Correlates in the Acute Ischemic Penumbra Following Middle Cerebral Artery Occlusion.. ISSN 0271-678x Bonita R. Yang. Vol. pp. No.4. Zhao. (1992).H. & Ginsberg M.. Sapojnikova. M. Clinica Chimica Acta .11. P. Quantitative and Qualitative Changes of Extracellular-Superoxide Dismutase in Patients with Various Diseases.

. & Kunk. S.24. DiMauro. No.75. & Hamilton. Donovan. 49-72. R. Boston Counsell. ISSN 0891-5849 Clark... Vo. and Apoptosis..P. pp.W.3. (April 1999)..31. NeuroReport. Dennis. (2010). ISSN: 0362-1642Doyle. R. (November 1980). M.. D. T. Butterworth-Heinemann. K. & Duffy.L. 67-101. 1195-1230. Free Radical Biology and Medicine.5. A.3. (2004). C. (2005).. Tissue Plasminogen Activator for Acute Ischemic Stroke.. Vol. Vol. and Ischemia/ Reperfusion Injury. (2001). A. Glutathione and Ascorbate during Ischemia and Postischemic Reperfusion In Rat Brain. Neuropharmacology.. (1997). S.P.G. A. (January 2001). Ingnegni. J. Polidori. Antioxidant Therapy: A New Pharmacological Approach in Shock. (1999). pp. M.. In The Molecular and Genetic Basis of Neurological Disease (Rosenberg.M. Vol. 2-14. pp. P. G. (2000). No. Neurosurgery & Psychiatry. ISSN 0028-3908 .48. No. (2001).5.333. Vol. No. (2002). M. S. Vol.6. Senin. T. Potential Markers of Oxidative Stress in Stroke.E.1. 1581-1587.. 841-852. (15 March 2010). T. (2008). Vol.C. Pezzuto. Journal of Neurochemistry. & Mecocci. Stroke.. D. H. No. Bregnocchi. Glutathione in the Brain: Disorders of Glutathione Metabolism. Vol. Albers. U. Polidori. 1242-1245..Y.3. No.35. Pulsinelli.L.B. M. P. & Puga. pp. & Aw.P.55. & Stenzel-Poore. Vol. ISSN 1471-4159 Cooper. M. Reactive Oxygen Radicals in Signalling and Damage in the Ischemic Brain. Prusiner. ISSN 09594965 Chan. (October 2000). A. (June 2002). M. Regulation and Measurement of Oxidative Stress In Apoptosis. 135-159.L. Shertzer... C.F.T. M. L. pp. R.. ISSN 00316997 Dalton. Journal of Neurology.. No. W. ISSN 0022-3050 Curtin. Cellular Redox Systems. Journal of Cerebral Blood Flow & Metabolism.10. M. pp. Vol. pp.J.A. No. ISSN 0022-1759 Cuzzocrea. pp. (September 2008). 401-405.39. Free Radical Biology and Medicine. A. Vol.265. Di Iorio. Reactive Oxygen Species. pp. Antioxidant Profile and Early Outcome in Stroke Patients.A. R.C.21. T. Simon. Annual Review of Pharmacology and Toxicology. No. ISSN 0891-5849 Circu. pp. Regulation of Gene Expression by Reactive Oxygen. ISSN 0028-4793 Cooper. & Salvemini. (1995). A. Barch....H.J.C. & Cotter. pp.162 Antioxidant Enzyme Cerebral Ischemia. Riley. Caputy.53. pp. Ruggiero. (December 1993). 749-762. A.P. Vol. S.N. ISSN 0271-678x Cherubini. & Mecocci. 310318.P.1-2.1. Cecchetti. (October 2005). No.39. Pharmacological Reviews.. (March 2004). The New England Journal of Medicine. 293-296. W. Journal of Immunological Methods. Predicting Functional Outcome in Acute Stroke: Comparison of a Simple Six Variable Model with Other Predictive Systems and Informal Clinical Prediction. (March 2001). No. P. Eds). Mechanism of Ischemic Brain Damage. pp. 2295-2300..G.P. (1980).7. & McDowall. Inflammation.L. (14 December 1995). ISSN 00392499 Cherubini. S.

(January 2001). R. & Bocola. F. (1989). & Hirrlinger. (May 1989). Aydin.. Factors Contributing to the Outcome of Oxidative Damage to Nucleic Acids. R. E.3.26. Vol. (August 2003).. pp. ISSN 0021-9258 Fridovich. ISSN 0028-3908 Fridovich. S17-21. (2000). No. (May 1999).1. Ginis. Free Radical Biology and Medicine. ISSN 1750-3639 Demirkaya. Brain Pathology. Inflammation and Stroke: Putative Role fr Cytokines. I.. Progress in Neurobiology. G. A. Stroke. Oury.A. No. Glutathione Peroxidase and Superoxide Dismutase in Peripheral Blood Erythrocytes of Patients with Acute Cerebral Ischemia. Vol.3. Vol. T.M. D. Rubio. No. pp. ISSN 0039-2499 Donnan. Stroke.16.8. ISSN 0891-5849 Flynn. & Feurstein. No. J. Superoxide Dismutase. Ulas.10.. 43-51. M. No. pp.B. J. 97-112. Iadecola.S. ISSN 0066-4154 . M.1. pp. No. Vol. (1995). pp. Valnickova.S.S1. pp. MacWalter..L. Bioessays.264. No. 250-256.A. ISSN 0891-5849 Evans. Glutathione Metabolism in Brain. and Cell Membrane Changes. No. European Journal of Neurology. Wang.. S.371. 649-671. Adhesion Molecules and iNOS in Brain Response to Ischemia. & Vural. (2004).A.20. M. L.H. & Lissi. & Doney.& Davis. Hojrup.A. Journal of Biological Chemistry.276. (May 2004). (December 2000)... Journal of Biological Chemistry.C. Papa.J. 133-138. M. pp. 4912-4916. I. M. Superoxide Radical and Superoxide Dismutase. Vol. Fischer. Exitotoxicity. Extracellular Superoxide Dismutase in Biology and Medicine. Isimer.. J.D.D.Vol. pp. S. ISSN 15318249 Enghild. J. SOD and Catalase Inactivation by Singlet Oxygen and Peroxyl Radical. Thogersen. X. Vol. Vol. No. Hallenbeck. ISSN 0140-6736 Dringen.S. pp.V. Free Radicals. (2001).. The Lancet. & Crapo. Free Radical Biology and Medicine.55. (2008)..21. & Oury.533-542. ISSN 15211878 Fattman C. The Cost of Cerebral Ischemia. Annals of Neurology. M. (August 2000).M. Metabolism and Functions of Glutathione in Brain. A. (1996).6. (1994). No. Vol.W. pp. GA... 7761-7764.285-290. & Cooke. (1999). Malonaldehyde. ISSN 1351-5101 Di Napoli.274.9624.L. No. (January 2001). I. Annual Review of Biochemistry. (2001).D..5.M.F. Metabolic Interaction between Astrocytes and Neurons in the Defence against Reactive Oxygen Species.D. ISSN 1742-4658 Dugan.I. The Heparin-binding Domain of EC-SOD is Proteolitically Processed Intracellularly During Biosynthesis.3. (2000). Vol. 95-112.32. ISSN 0301-0082 Dringen. 14818-14822. Prognostic Influence of Increased C-Reactive Protein and Fibrinogen Levels in Ischemic Stroke. Vol.. I. 1612-1623. (January 2000). (September 2008)..1. J. Vo. O.35. pp. P. V.W. Neuropharmacology. Z. G.35. No. 236-256. pp. (May 1994).64.62.14.Plasma Antioxidant Activity as a Marker for a Favourable Outcome in Acute Ischemic Stroke 163 del Zoppo. pp. T.. R.R. (2003). Schaefer L. No.. Macleod. ISSN 0021-9258 Escobar. Gutterer.Z. European Journal of Biochemistry. (June 1995). M. pp. M.M. (2008). Topcouglu. U. (10 May 2008). Vol. Vol. A. & Choi. J. (2000).

54. Free Radicals in Biology and Medicine (3rd Edition).. P. pp.. Tainer. American Journal of Physiology.. Azhar. Vol. R. Graf.. Free Radical Biology and Medicine. 88-93. Gross.67. J...14. Richmond. ISBN 0198500459. Vol.46.W.1Pt2.J.. (1994).. Gozlan. Y.164 Antioxidant Enzyme Fullerton. Lee.L. T. Journal of Cerebral Blood Flow & Metabolism. Chen. Fujita.. Background Review and Current Concept of Reperfusion Injury. (Eds. (1998). Probing the Active Site of Human Mn-SOD: the Role of Glutamine 143. Annals of Neurology.L. R. C.. pp. O. (November 1994). (April 2009). Saito. M. (11 July 2006). V. Lo. & Chan. ISSN 1531-8249 Hsieh. C. (1994).L. M.H.47.14. (November 1990). S. M. C. 711713. No. ISSN 0024-3205 Hallenbeck... A.R. ISSN 0891-5849 He.D.. Woolworth. pp.F. Tu. P. S. No.S. Acute Ischmic Stroke. Blake. R. (October 1994). Increase of Superoxide Dismutase after Cerebrovascular Accident.. Hsu. (2006). A. Koroshetz. Ditelberg..A. pp. pp. J. 11. Terry. Sacco. W. Quality Improvement in Acute Stroke: The New York Stroke Center Designation Project. Oxford University Press. & Ferriero..36. D.M.B. Hirsch. Y.J. Vol. ISSN 0003-9942 Halliwell. (November 1994).G. G. J. W.Y. Imaging and Intervention (2nd Edition). 9780198500452 Hawkins.. R.11. Hickey.E. M. Lev..J.. 557-565. Lederer. T. H252-H256. M. M. Bratt. R.. & Gutteridge. & Furie.21.. (2008). Gagliano. Vol. Ning. ISBN 978-3-64212750-2 Grophen. pp.A. pp. No. Life Science. K.265. M.8. (1994).1. & Wagner. K.M. P. (1993). Y. P.A. Annals of Neurology. S. pp. & Dally. 12451254. Libman. D. No. & Silverman.. (1999).. (July 1993).3. D. Polyethylene Glycol-conjugated Superoxide Dismutase in Focal Cerebral Ischemia-Reperfusion. Liposome-entrapped Superoxide Dismutase on Post-traumatic Edema. Quantification of Protein Modification by Oxidants.. J. Kwiatkowski.) (2011). J.H. ISSN 0039-2499 Kelly. Epstein.R. L.H. (2009)... Stevenson. 44.. T. P. (April 1998). P. R. No. ISSN 1531-8249 Gonzalez. E. C. Guan.. Archives of Neurology.M. Angerhofer.357-364.J. Schalfer. ISSN 0002-9513 Heiss.J. Morgan..A.J.. Viability Thresholds and The Penumbra of Focal Ischemia.J.. D.. No. B.37. Biochemistry. ISSN 0028-3878 Gruener. Chan. P. Stroke..6.. pp. & Barak.. K. & Dutka. (1990).J.. No. G. Neurology. ISSN 0006-2960 Imaizumi. & Miller.M. H. Lepok..4.. B. E.S. Sarco. Dynamic Penumbra Demonstrated by Sequevential Multitracer PET After Middle Cerebral Artery Occlusion in Cats.. Vol. ISSN 0271678x Hossman.. Leifer. Oxidative Stress and Matrix .C. Vol. Morrow. 4731-4739. H. Nick.. pp. Hubbard. J. Milne. Springer. No. M. 892-902. Ezrin. (September 1990). No.H..N. C.P. & Davies.S.9.I.H.A.. H.. Vol.. (1990). N. (September 1998). No. Vol. (1998) Copper/Zinc Superoxide Dismutase Transgenic Brain Accumulates Hydrogen Peroxide After Prenatal Hypoxia Ischemia. Lottgen. & Schwamm. A. J. Vol. Wienhard. Fishman... Vol. R.N. 965-988. E. J. Rosner.1312-1317.Y.L.D. J.. J.

Stroke.. & Reed. (2008). J-K. Closhen. 100-104. ISSN 0270-6474 Margaill. ISSN 00392499 Kussmaul.B. pp. pp.L. H. S. 1398-1403. Molnar. Journal of Neuroscience.4. M. 1941-1988. Free Radical Biology and Medicine. C. No. R. T. ISSN 1523-0864 Marklund. Vol. N.10. & Dringen. Hamprecht. pp. S. Extracellular Superoxide Dismutase in Human Tissue and Human Cell Lines.11. (April 2009). & Heiko.K. L..3 (March 2002).M. M. Librizzi.4. T.. The Detoxification of Cumene Hydroperoxyde by The Glutathione System of Cultured Astroglial Cells Hinges of Hexose Availability For The Regeneration of NADPH.A. 429-443. 809-815.. Ahonen J-K. Kondo. Lessmann. M. (2009). & Heller. 33-39. Mitochondrial Control of Cell Death. Vol. Dizdaroglu. Antioxidants & Redox Signaling. Low Plasma Antioxidant Activity is Associated with High Lesion Volume and Neurological Impairment in Stroke.. 1246-1253. P. Hsu. Pietrzik. No. Antioxidant Strategies in the Treatment of Stroke. Vol. V. & Chan.. J. ISSN 0021-9738 .1458-1466... G.W.3 (September 1999). & Alho.L. 5. Rabou. No. Superoxide Dismutase in Extracellular Fluids. ISSN 0891-5849 Mieyal.. No. Vol. Dastidar.. L. 513-519.21.A. (August 2005). M. No. pp..C.. (January 2008).33.. (October 1984). S. pp. Damage. Vol.16. Stroke.W.1. L. (2002). P.6.. ISSN00098981 Marklund.4. Vol. 40.R.. Vol.A. ISSN 0039-2499 Liu...U. Qanungo.Plasma Antioxidant Activity as a Marker for a Favourable Outcome in Acute Ischemic Stroke 165 Metalloproteinase-9 in Acute Ischemic Stroke: The Biomarker Evaluation for Antioxidant Therapies in Stroke (BEAT-Stroke) Study. No. & Lerouet. M. ISSN 1471-4159 Leinonen.. J. pp. Vol. pp. & Cui. (1984). No.32. Journal of Clinical Investigation. pp.. 41-51. Gallogly. pp. (November 2001). Plotkine.. (1999). (2001)Oxygen Radical in Cerebral Ischemia: The 2001 Willis Lecture. J. (2000). Noshita. Manganese Superoxide Dismutase Deficiency Exacerbates Cerebral Infarction After Focal Cerebral Ischemia/ Reperfusion in Mice: Implications For the Production and Role of Superoxide Radicals. E. Journal of Neurochemistry..74.1. Karakaya A.. D. D. (2000). ISSN 0039-2499 Kim. Stroke. H.1. 2712-2716. G.73.39. No. J.H. Stroke. No.E. G. ISSN 1078-8956 Kuhlmann. (May 2000) pp.11.W. Vol. Vol. I. Floyd. Sabens. No.. pp.S. C. No.126. de Curtis. P. Lonnrot K. Repair and Mutagenesis in Nuclear Genes after Mouse Forebrain Ischemia-Reperfusion.39.J. Y. M.31. Mechanisms of C-Reactive Protein-Induced BloodBrain Barrier Disruption.D. (2005). Molecular Mechanisms and Clinical Implications of Reversible Protein SGlutathionylation. (1982b). ISSN 0039-2499 Kontos. R. Vol. C. (November 1982). pp. & Shelton. L. Vol.. Kow. Jehkonen. E. Stroke. No. ISSN 0039-2499 Kroemer. Holme. Nature Medicine. (1996). (November 1996). Clinica Chimica Acta. 6795-806.. Pflanzner. (January 2000).Y.

(September 1998). Folbergrova. R. G. and Central Nervous System O2 toxicity. (April 2001).F. Increased Efflux Of Oxidized Glutathione (GSSG) Causes Glutathione Depletion and Potentially Diminishes Antioxidant Defense in Sickle Erythrocytes.. Frei. No. Vol. Koroshetz. & Crapo. Takeda.42. 11.. Li. Kondo. 9715-9719. P. Pearlstein. J. Vol.8.89.. Bo. 561-567. 25-32. M.. Biochimica et Biophysica Acta.F.D. T. Sato... No. 477-486. Toxicology.1. J. Vol. Choi. Temporal Profiles of the Levels of Endogenous Antioxidants after Four-vessel Occlusion in Rats. S. D. J. Mecocci. ISSN 0270-6474 Namba. Park... Subcellular Distribution of Superoxide Dismutases (SOD) in Rat Liver: Cu. ISSN 1471-4159 Sheng. pp. (November 2000). Vol. de Waart.. & Hirakawa. (1998). J. 38388-38393.18. 131-137.S. K. No. Jr. Mitochondrial Susceptibility to Oxidative Stress Exacerbates Cerebral Infarction that Follows Permanent Focal Cerebral Ischemia in Mutant Mice With Manganese Superoxide Deficiency. H.M. pp. (March 1980). Journal of Neurosurgical Anaesthesiology. K. 205-213. pp. Otten. Ho. ISSN 0898-4921 Nur.M. pp. Journal of Biological Chemistry. P.D. Nitric Oxide.. Perz-Gomez.25. R. J. (January 1998). Sunami. Clinical Biochemistry. Antioxidant Enzymes and Human Diseases..J. H.R. C.S. G..M.. Vol. (May 2011). J. & Beal. Increased Plasma Levels of Lipid Hydroperoxidation in Patients with Ischemic Stroke. (October 2001).F.. I.N. D. M. ISSN 0027-8424 Park. No. No. S..1-3. & Fridovich. W. Vol. E. (2001). (1992). ISSN 1385-299x Polidori. (2011)...H.2’-deoxyguanosine Accumulation in the Gerbil Hypocampus Following Global Ischemia. Extracellular Superoxide Dismutase. Mice Overexpressing Extracellular Superoxide Dismutase Have Increased Resistance to . 83-104. & De Castro. Keaney J. pp.D. ISSN 0300-483x Murakami. M. R. No. K. No. Schong.. (2000).. Piantadosy. 1412-1417.166 Antioxidant Enzyme Mates. Free Radical Biology and Medicine. B. & Warner. Bart.J. ISSN 0891-5849 Rehncrona.. 595-603.. Han. No. Measurement of Glutathione Oxidation and 8-Hydroxy... Journal of Neurochemistry. T..34. pp. pp. ISSN: 0925-4439 Okato-Matsumoto. No. Y. Kawase.13. Vol. Crapo. Oury. Effects of Antioxidant Enzymes in the Molecular Control of Reactive Oxygen Species Toxicology.. Cherubini. B. (1999a). A.S. Biemond. Brandjes. (1980). & Siesjo.D.1-2. Y.32. Nelles. Proceedings of the National Academy of Sciences.. (2001).Y.4-5. I. (2000). Schwamm.D. Smith D. (November 2000). (15 October 1992). Influence of Complete and Pronounced Incomplete Cerebral Ischemia and Subsequent Recirculation on Cortical Concentration of Oxidized and Reduced Glutathione in the Rat.S. & Chan. T. Ekferink. & Park. S.153.. Vol. (1999).A.M. Vol. A. C .20.276. ( November 1999).J. pp.. USA.P. E. D. J. Y.. Y.2. Verwijs.M. (1998). J.. L.D.. Rordorf. ISSN 00219258 Oury. Journal of Neuroscience.H..3. M C. Chen.. 1812. No. M. pp. Brain Research Brain Research Protocols. pp..K. ISSN 00099120 Mates. 6.Zn-SOD IN MITOCHONDRIA. Vol.P. M.

Plasma Antioxidant Activity as a Marker for a Favourable Outcome in Acute Ischemic Stroke 167 Focal Cerebral Ischemia. Krempien. Mice Overexpressing Extracellular Superoxide Dismutase Have Increased Resistance to Global Cerebral Ischemia. Vol. Uozumi. & Sies. The Relative Contributions of Vitamin E.12 (December 1997). K. (1991). J. C. No. No. H. Vol. 169-172.U. Stroke.R.C. Sheng.12. G. & Hacke.. Donneberg. Ingold. H. 185-191. pp. The Superoxide Dismutase Activities of Cerebral Tissues.1. 29-36. & Warner. Russo. (1993). & Kawasaki. No. Pearlstein.M. pp. D. pp. G. Neuroscience Letters. Makensen. Assayed by the Chemiluminescence Method. (1987).207.23. S.C. R. (May 1999). 408-419. Release of Superoxide Dismutase into Cerebrospinal Fluid as a Marker of Brain Lesion in Acute Cerebral Infarction. I.D. T. Brody. (January 2010). ISSN 0039-2499 Sutherland G. (1983). M..D..128.1. Vol. ISSN 0014-2956 . & Muyderman. (August 2004).. (December 1983). 107-114. Vol. & Locke. pp.. Schwab. No.1-2.. S. Experimental Neurology. (December 1993).D.R. S..D. No. Pinsky C. pp. pp. No. pp.D. A. Louw D. & Batinic-Haberle. Neuroscience Letters. In The Gerbil Focal Ischemia/Reperfusion and Global Ischemia Models. Vol.L.. D. Mitochondria.S.W.163.R.267. H.18. No. Journal of Experimental Biology. Burton. pp. V. & Warner. (July 1991) pp. J. Antioxidants and the Ischemic Brain..18. Stroke. ISSN 0306-4522 Sheng.. C. Vol. Sorrenti.. (22 June 1987). Crapo. A. Oxidants. L.1802. 1337-1340. Vol. 392398.S. (1992).. (1997). European Journal of Biochemistry. ISSN 03043940 Sheng.. Pearlstein. Di Giacomo.2. N. (April 1992). Free Radical Scavenger Depletion in Post-Ischemic Reperfusion Brain Damage.. Bose R. T. Biochimica et Biophysica Acta. pp. No.3. Vol.S. H. ISSN: 0304-3940 Tokuda Y. Urate.924. Crapo. Ascorbate and Proteins to the Total Peroxyl Radical-Trapping Antioxidant Activity of Human Blood Plasma.. Biochimica et Biophysica Acta. ISSN 0197-0186 Vanella. Vol. Extracellular Superoxide Dismutase Deficient Mice Have Decreased Resistance to Focal Cerebral Ischemia.Vol. Renis M.2.. No. Oxidation of Glutathione by the Superoxide Radical to the Disulfide and the Sulfonate Yielding Singlet Oxygen. pp. T. Global Elevation of Brain Superoxide Dismutase Activity Following Forebrain Ischemia in the Rat. (January 1999).88. ISSN 0304-4165 Wefers.1. No.13-17. pp. (2010). M. ISSN 0039-2499 Spranger. Kudo..137..23.4. 515-518. Superoxide Dismutase Activity in Serum of Patients With Acute Cerebral Ischemic Injury. 80-91. 32213231. & Marklund.. Oxidative Metabolism and Cell Death in Stroke. Barclay.J. (1993). ISSN 0364-3190 Warner. Vol. (2004). 2425-2428. ISSN 0022-0949 Wayner. S. No. (June 2000). H.. D. (1999b). & Perez-Polo.2 (August 1993). W. H. Correlation with Clinical Course and Infarct Size. Campisi. ISSN 0006-3002 Strand.28. A. No. R.B. Castorina... D. J. T.. Neurochemical Research. Vol. (2000). S. ISSN 0014-4886 Sims. Neurochemistry International. Neuroscience.

(1994). Chen J. H. Stroke. Weinstein. (January 1994). E. P.J.H.G. P. Carlson. Vol. Chan.1. C.. Human Copper-Zinc Superoxide Dismutase Transgenic Mice are Highly Resistant to Reperfusion Injury After Focal Cerebral Ischemia.25. S. pp.. & Kamii. ISSN 0039-2499 .. Chen. Epstein. 165-170. No....168 Antioxidant Enzyme Yang.

. 2000). This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons. and arthritis have been correlated with oxidative damage (Brown and Bicknell..doi. atherosclerosis.. resulting in formation of toxic products such as MDA. Introduction High level fat. 2000). licensee InTech. Kawase et al. carbohydrates and proteins along with an increased risk of complications from vascular disease (Taskinen et al.5772/47432 1. 1987). Diabetes mellitus (DM) can be defined as a group of syndromes due to defects in pancreatic secretion of insulin or insulin action. 1985). For example. increasing free radical and reducing antioxidant levels (Aragno et al. obesity and metabolic syndrome may increase oxidative stress. provided the original work is properly cited. There are various reports indicating the benecial effects of antioxidant supplementation in preventing dyslipidemia. Momordica charantia fermented milk       © 2012 Gao. The antioxidant scavenging enzymes superoxide dismutase (SOD). 2003. and/or influence the levels of cellular homeostasis (Gao et al. 2001. Numerous studies have reported that lactic acid bacteria fermented food display hypolipidemic effects by inhibiting cholesterol biosynthesis and decreasing low-density lipoproteins (Haberer et al. oxidative damage and its consequences may result in many chronic health problems.. Diet modification may be one way to reduce serum lipid level. and reproduction in any medium. Increased free radical production exerts cytotoxic effects on the membrane phospholipid. 2004).. Hyperglycemia impairs the prooxidant/antioxidant balance.. altered metabolism of lipids. The level of lipid peroxidation in cells is controlled by various cellular defense mechanisms consisting of enzymatic and nonenzymatic scavenging systems. 1995).Chapter 7  Antioxidant Therapies for Hypolipidemia and Hyperglycemia Dawei Gao Additional information is available at the end of the chapter http://dx. 2002).0). 2011. Free radicals react with lipids and cause peroxidative changes that result in enhanced lipid peroxidation (Girotti. Thus. catalase (CAT) and glutathione peroxidase (GPX) offer protection to cells and tissues against oxidative injury (Bonnefont-Rousselot et al. Furukawa et al.   . 2004).. Alexander. cancer.. which permits unrestricted use. The efficiency of the antioxidant defense mechanism is altered in diabetes (Wohaieb and Godin. hyperglycemia. which characterized by hyperglycemia.

2007).170 Antioxidant Enzyme is effective in preventing and retarding hyperlipidemia and atherosclerosis in hamsters (Tsai et al.e. and widely used as a traditional Tibetan medicine with the reputations for excitement nervous system..2009 ). Ligustrum lucidum Ait (LLA) is a traditionally Chinese medicinal plant. Bor (Chinese Name: Hong jing tian) is a traditional Tibetan pharmacology. 1993).. glutathione peroxidase (GPx). we investigate the effect of OA on serum level of hepatic enzymes and tissue level of lipid peroxidation and antioxidant enzymes in alloxan-induced diabetic rats.. The hypoglycemic effect of oleanolic acid (OA) isolated from LLA was identified in streptozotocin-induced diabetic rats. Saccharomyces boulardii. known with a local name as “Nv zhenzi”.. Examples of phase II enzymes (p2Es) include the ratelimiting enzyme in the GSH synthesis pathway. live microorganisms as food supplement in order to benefit health. Bifidobacterium sp. lipids modulating and antioxidant efficacy in alloxan-induced diabetic rats. which distributed in Eastern Europe and Western Asia (altitude 3500-5000 meters). pilot and mountaineer to enhance the body’s ability surviving in adverse environments (MookJung. sachalinensis (RS) on diabetes and antioxidative ability in streptozotocin-induced diabetic rats. A number of Lactobacillus species. glutathione Stransferase. γ-gluta-mylcysteine ligase. hold a wide variety of LAB which may have . relieving fatigue. Li et al. Some strains and species of lactic acid bacteria (LAB) have the antioxidative activity (Gao et al.15% (Xiang and Gu. preventing high altitude sickness. Mook-Jung et al. and reduced nicotinamide adenine dinucleotide phosphate–quinone oxidoreductase (Matafome et al. As a drug of ‘‘source of adaptation to environment’’.. 2011. sachalinensis has been used in such special posts as diver. 2010). and some kinds of LAB could adjust blood lipid and lower cholesterol. 2007). Gao et al. In order to reveal the efficacy of LLA in alloxan-induced diabetic rats. 2009). 2009). 2002). decreasing depression.. In addition. and some other microbes have been proposed as and are used as probiotic strains.. i. R. sachalinensis is a precious perennial herbaceous plant. et al.. 1988. Sotiroudis et al. and possess antioxidative and antidiabetic potentials. the ability of LLA stimulating secretion of insulin was disclosed in our study (Gao et al. the effects of OA from LLA were estimated on hypoglycemia. and resisting sideeffects of anoxia and microwave radiation (Stancheva and Mosharrof. R. Rhodiola sachalinensis A. Nrf2 regulates the transcriptional activation of more than 200 antioxidant and protective genes that constitute the so-called phase II response. 1991). 2011. one of the most important foods in the traditional Chinese diet. at the same time. 2002).. We found that RS could significantly stimulate insulin secretion. and increased utilization of medicinal plants became a World Health Organization policy on 1970. heme oxygenase 1. It has been used to treat cancer whose tumor inhibitory rate was 46. 1992) and hepatoprotective properties (Yin and Yu. Our study was performed to investigate the therapeutic effects of R. as well as superoxide dismutase (SOD).. LLA was proven to have the abilities of antimutagenic (Wang et al. astronaut. 2007. enhancing work performance.. Nuclear factor erythroid 2-related factor 2 (Nrf2) controls the antioxidant response element (ARE)-dependent gene regulation in response to oxidative stress.. Meanwhile. Fermented cabbages.. which can prevent and treat some diseases by activating antioxidant enzymes (Jain et al. Some herbal drugs are a good source of natural antioxidants. antidiabetic (Hao et al. 1987. Ming et al.

and so on. by the activation of Nrf2. The two strains were given to the normal.Antioxidant Therapies for Hypolipidemia and Hyperglycemia 171 interesting features for application in health. 28 LAB strains were isolated from pickled cabbage. anti-lacking oxygen. We analysed the scavenging effects of superoxide anion radicals and hydrogen peroxide of the strains isolated from fermental cabbages in vitro. 2. and their effects of hypoglycemia and hypolipidemia were analyzed. 2. and refluxed three times (each time for 1 h) to remove lipids with chloroform: methanol solvent at 80°C (2:1) (v/v). including China. which possesses antifatigue. After filtering . which is an effective scavenger of free radicals to inhibit the lipid peroxidation. At the same time. In the study. The abilities of antioxidation and protecting pancreatic β cells might be the main mechanisms of RS on antidiabetic effect. its main compound was polysaccharide. In our study. Meanwhile. the Nrf2mediated antioxidant defense pathway and immune status of the lactic acid bacteria-treated mice were investigated. The experiment was designed to determine whether the antioxidative effects of lactic acid bacteria from fermented cabbage are mediated. anti-microwave radiation and anti-caducity potentials. oleanolic acid and lactic acid bacteria on hypolipidemia and hyperglycemia. especially with respect to lipids modulating. sachalinensis root was ground to fine powder. sachalinensis Dried R. hyperlipidemic mice by ig. 28d continually. RS in the streptozotocin-induced diabetic rats showed significant hypoglycemic activity. Furthermore. CAT and GSH-px activities of the liver and kidney of diabetic rats. Preparation and characterization of R. Antidiabetic and antioxidative potentials of Rhodiola sachalinensis polysaccharide Rhodiola sachalinensis has been used as one of Tibet traditional herbs. The present study was performed to investigate the antioxidant therapeutic effects and mechanism of R. Fermented cabbages have made up a significant part of food intake in Asia countries for several centuries. RS treatment decreased malondialdehyde (MDA) level. which were Lactobacillus plantarum and Lactobacillus brevis. Japan. Korea. A significant enhancement in the serum insulin levels of diabetic rats following RS treatment was also observed. The levels of serum total cholesterol (TC) and triglcerides (TG) of RS-treated diabetic rats were lower than control diabetic rats. We found that they could significantly stimulate lipid metabolizing. hypolipidemic and hypoglycemic potentials. RS has not expressed significant toxicity in LD50 test and single cell gel electrophoresis assay. while increased SOD. sachalinensis. we examined the antidiabetic effect and probable mechanisms of Rhodiola sachalinensis root extract (RS). and two strains with high acid tolerance and bile salt resistance were screened. parameter of blood lipid was examined. and evaluate liver antioxidative activities related to the elimination of reactive oxygen species in L. at least in part.plantarum-treated high-fat diet mice. These results indicate that RS has the hypoglycemic and hypolipidemic activities. and possess antioxidative.1. and antioxidative ability in diabetic rats. Activities of antioxidant enzymes in liver and kidney tissues were observed.

The results were shown on Table 1.248-0. The plates were heated at 105°C for 10 min.553 0. The precipitation was washed by 95% and 100% ethanol. Food and drinking water were provided. meanwhile including a little of flavone. After filtering and centrifuging.2. examined Components polysaccharide flavone organic acid saponin hydroxybenzene terpene and steroid anthraquinone alkaloide lignin Ratio of Flow(Rf) 0. respectively. respectively(Sezik et al. sachalinensis root extracts prepared using the above mentioned procedure. but no alkaloide... 2005). and nbutanol:acetic acid: H2O (20:9:1) was used as the solvent system. the precipitate was collected and vacuum-dried. The animals were housed under suitable lighting and temperature. named RS. eight rats were randomly assigned as normal control group. The rats with blood glucose levels above 15. Nine kinds of indicator system were used to identify the compounds of RS. 2009). then the different indicators were sprayed on the plates. The residue was extracted three times in dH2O (60°C) and then combined filtrate to concentrate through decompressing using a rotary evaporator. Compounds of RS Extracts by TLC Analysis 2. anthraquinone. became the diabetic model rats according to the standard method (Gao et al.564 0. hydroxybenzene. which indicated that the main compound in RS was polysaccharide.. terpene. obtaining light brown extracts.0 mM were . and different color spots were visualized (Gao et al. Preparation of STZ-induced diabetic rats Male wistar rats weighing 180-200 g were obtained from the animal department of Beijing institute of traditional medical and pharmaceutical sciences.278-0. and the rest rats. RS solution was dotted on the TLC plates. treated with STZ.193-0. The extract was examined by thin layer chromatography (TLC) analysis to identify the main compounds. after then the precipitate was added 80% ethanol and deposited for 12 h in 4°C. 2007). Following one week of acclimation. steroid and lignin were detected.217-0.463 0. saponin and organic acid. These results have indicated that polysaccharide is the main ingredient of the extract of R. Color brown yellow dark yellow dark blue - - - - - Table 1.493 - - - - - Indicator phenol / sulfuric acid 10% NaOH bromophenol blue / ethanol phosphomolybdic acid / ethanol ferric trichloride / water acetic anhydride / sulfuric acid 10%KOH iodine / potassic iodide sulfuric acid / ethanol Note: “-”means without any spot on the plates.172 Antioxidant Enzyme the residue was air-dried and then refluxed again with 80% ethanol to remove monosaccharide and oligosaccharide.

Antioxidant Therapies for Hypolipidemia and Hyperglycemia 173

defined as diabetic model rats. Thirty-two rats (eight normal rats, twenty-four diabetic rats) were chosen and randomly divided into four groups: normal control group (NC), DM control group (DC), DM + RS low dose group (DM+RS LD), and DM+RS high dose group (DM+RS HD).

2.3. Examination of effect of RS in the oral glucose tolerance test
On the first day, after overnight fasting with free access to water, the rats were administered RS solution by oral gavage at the following doses: 200 or 400 mg/kg·bw for the DM+RS LD & DM+RS HD groups, and with the same volume of dH2O alone for the NC and DC groups. Tail blood samples were drawn from each rat, then a glucose (2.0 g /kg·bw) solution was orally administered by oral gavage after 30 min following RS administration. Blood samples were taken at 30, 60, 90, and 120 min intervals following glucose administration, and blood glucose levels were measured at various time points. The supplement of RS improved the acute blood glucose levels in the rats (Figure 1). There was no significant difference at 0 h between the DC and RS-treated groups, but in the RS LD and HD groups, hypoglycemic effect of RS became significant 1 h following oral administration comparing with DC group (p < 0.01), then the decreased rates were 22.3% and 24.4% at 2 h after oral administration, respectively. However, the blood glucose levels in diabetic control rats declined after 90 min, but it was still high during the experiment. Moreover, the blood glucose level of DM + RS HD was not significantly different with that of DM + RS LD (p >0.05).


Bloood glucose level (mM)

25 20 15 10 5 0 20 40 60 80 100


Time (min)
The values represent the means ± SE. of eight rats per group.

Figure 1. Acute effect of RS treatments in OGTT for STZ-induced diabetic and control rats.

174 Antioxidant Enzyme

2.4. Determination of subacute effect of RS treatment on blood glucose level
In the DM+RS LD and HD groups, the rats were given RS solution (200 & 400 mg/kg·bw) daily by gavage for 40 days, respectively. The control rats (NC and DC groups) were given the same volume of dH2O. On days 0, 10, 20, 30 and 40, the blood samples were collected from rat’s tail veins and measured, followed by an overnight fast. Changes of plasma glucose level as a result of RS treatment for 40 days are shown in Figure 2. Before treatment of diabetic rats, there was no significant difference for the blood glucose levels among diabetic rats (p > 0.05). They were consistently staying at similar levels within the experimental course in NC & DC groups. However, in DM +RS treated groups significantly lower blood glucose levels were observed as compared to DC group at the same time points, from day 10 to day 40 (p < 0.01). The decreasing rates of the blood glucose were 45.51% (DM + RS LD group) and 55.11% (DM + RS HD group), respectively. The difference of dose-effect of RS was not significant according to the results of blood glucose levels of RS-treated groups (p >0.05).


Blood Glucose Level (mM)

18 16 14 12 10 8 6 4 0









Time (days)
The values represent the means ± SE. of eight rats per group; *p <0.01 vs. diabetes control group (DC) at the same time point.

Figure 2. Subacute effect of RS treatment on blood glucose level of STZ-induced diabetic rats.

The oral administration of RS for 40 days for the STZ-induced diabetic rats showed a significant reduction in blood glucose, which elucidate that R. sachalinensis has obvious hypoglycemic potential. In a healthy person, plasma glucose concentrations are maintained within a fairly narrow range in the fasting state, even if no food is ingested for many days. However, after glucose intake an acute increase in plasma glucose is seen, the concentration peaking generally within 1 h. Plasma glucose then decreases and fasting levels are regained by 2 h post ingestion (Choi, et al., 2008). The acute hypoglycemia of diabetic rats was seen after RS-treated 1 h in the oral glucose tolerance test, but it was still high in diabetic control rats, which imply that RS could improve the hyperglycemia in an acute course.

Antioxidant Therapies for Hypolipidemia and Hyperglycemia 175

2.5. Determination of effects of RS treatment on blood biochemical parameters
At the end of the experiment, the blood samples of fasted tested rats were collected from the eyes under ether anaesthesia, to determine the levels of TG, TC and insulin, according to commercial advice by the Automatic Biochemical Analyzer. The levels of TG and TC in DC rats were significantly higher compared with those of NC group (Table 2, p < 0.01). When diabetic rats were treated with RS for 40 days, the levels of TG and TC were significantly decreased as compared to DC rats (p <0.01), but which didn’t fall to the normal levels. Meanwhile, there was difference on TG and TC levels between high and low dose groups (p < 0.05), which indicated RS displaying dose-dependent ameliorative blood lipid effect within the range of 200~400 mg/kg·bw. The situation of insulin secretion of the diabetic rats has been ameliorated by RS treatment (Figure 3). The levels of insulin in DC rats were significantly lower as compared to those of NC group (p < 0.01). When diabetic rats were treated with RS (200, 400 mg/kg·bw) for 40 days, the serum insulin levels were significantly enhanced compared to DC group (p <0.01), the increased rates were 20.79% and 29.68%, and there are significant difference between low and high dose groups (p <0.05). The results indicated that RS could stimulate insulin secretion in vivo by the dose-dependent manner. Groups NC DC DM+RS LD DM +RS HD TC (mg/dl) 83.288±3.084* 135.375±4.203 106.913±4.491* 96.125±4.591*# TG (mg/dl) 72.513±3.169* 170.438±3.376 94.075±3.837* 84.188±4.121*#

Values are means ± SD for eight rats in each group, *p <0.01 vs. diabetic control group (DC), # p <0.05 vs. DM+ LD

Table 2. Effect of RS on TC and TG in normal and diabetic rats

Serum Insulin Level (uU/ml)

12 10 8 6 4 2 0








The values represent the means ± SE. of eight rats per group. *p <0.01 vs. diabetic control group (DC). #p<0.01 vs.RS LD group.

Figure 3. Effect of RS treatment on insulin level of STZ-induced diabetic rats.

176 Antioxidant Enzyme

Diabetes is a metabolic disorder affecting carbohydrate, lipid and protein metabolisms, complicated with multiorgans regression in the later period. The levels of serum lipids are usually raised in diabetes mellitus (Sakatani et al., 2005). The increase of blood glucose is accompanied with the rise of TC and TG (Sharma et al., 2003). The significant rise of blood glucose, TC and TG levels has been observed in STZ-induced diabetic rats, whereas those were significantly decreased by RS treatment. Our results suggested that RS not only possess significant hypoglycemic ability but also have remarkable hypolipidemic effect. RS also enhanced serum insulin release in STZ-diabetic rats by 40 days treatment, which was obviously different with the diabetic control rats. We presume that RS appears the hypoglycemic effect in diabetic rats is partly attributed to its stimulation of insulin secretion.

2.6. Determination of tissue antioxidative enzyme activities
The animals were sacrificed under ether anaesthesia. Their liver and kidney tissues were immediately removed, washed using chilled saline solution, homogenized in 4 volumes of Tris-HCl buffer (pH 7.4). The homogenates were centrifuged at 4,000 × g for 20 min at 4°C. The protein concentrations of the homogenates were determined by the Bradford method using bovine serum albumin as the standard (Bradford et al., 1976). The levels of SOD, MDA, CAT and GSH-px were measured by commercial suggestion of the kits, and the results are shown in Table 3. There was a significant increase in SOD, CAT and GSH-px Antioxidative level in liver and kidney tissues Liver MDA (nmol/ Kidney MDA (nmol/ Liver SOD (U/ Kidney SOD (U/ Liver GSH-px (U/ Kidney GSH-px (U/ Liver CAT (U/ml) Kidney CAT (U/ml)

NC 8.105± 0.329** 9.701±0.269** 46.05±1.129** 50.725±1.184** 24.849±0.944** 32.321±0.795** 20.176±0.551** 25.544±0.433**

DC 10.848± 0.455 12.315±0.352 39.913±1.045 44.188±0.834 16.959±0.734 24.438±0.600 14.786±0.390 20.410±0.549

DM+RS LD 9.773 ±0.375 11.239±0.441 43.45±1.018 47.263±0.927 20.973±0.925* 27.779±0.691* 17.398±0.416* 22.604±0.539*

DM+RS HD 9.132 ±0.374* 10.604±0.236* 44.375±0.958* 48.638±0.814* 21.848±1.136* 30.853±0.898** 18.776±0.597** 22.665±0.491*

The values are means ± SE. for eight rats per group. *p <0.05 vs. diabetic control group. **p < 0.01 vs. diabetic control group.

Table 3. Antioxidative effect of RS in the liver and kidney tissues of the tested rats

Antioxidant Therapies for Hypolipidemia and Hyperglycemia 177

activities in NC group compared to DC group (p < 0.01), but the level of MDA was significantly increased in diabetic control rats. The increase of MDA level was inhibited significantly by RS treatment in the diabetic rats, and the MDA level was decreased in RS HD group (p < 0.05), but the decrease of the level of MDA was not significant in the RS LD group as compared to DC group (p > 0.05). In RS treated groups, there was a partial elevation of antioxidant capacity including CAT, SOD and GSH-px in the liver and kidney tissues as compared to DC group (p < 0.05 or 0.01). It was reported that diabetic subjects are highly sensitive to oxidative stress (Pritchard et al., 1986). In STZ-diabetic animals, STZ generates nitric oxide, which is a powerful free radical oxidant (Kwon et al., 1994) resulting in an increase in blood glucose level. Several studies have documented the relationships between the increase of free radicals and blood glucose, lipid peroxidation as well as low-density lipoprotein in the progress of diabetes (Rabinovitch et al., 1996; Tanaka et al., 2002). Free radicals can diffuse intracellularly and result in mitochondrial enzyme damage and DNA break, impair cellular function and contributes to the pathophysiology of diabetes (Bonnefont-Rousselot et al., 2000). Increased free radical production exerts cytotoxic effects on the membrane phospholipid, resulting in formation of toxic products such as MDA. Several reports have shown the alterations in the antioxidant enzymes during diabetic condition (Preet et al., 2005). The antioxidative defense system like SOD and CAT showed lower activities in liver and kidney during diabetes. The decreased activities of SOD and CAT may be a response to increased production of H2O2 and O2– by the auto oxidation of excess glucose and non-enzymatic glycation of proteins (Argano et al 1997). Pigeolet et al (1990) have reported the partial inactivation of these enzyme activities by hydroxyl radicals and hydrogen peroxide. The decreased activity of SOD and CAT could also be due to their decreased protein expression levels in the diabetic condition as reported recently in liver (Sindhu et al 2004). GSH is often regarded as antioxidant agents, since they protect protein –SH groups against oxidation and can scavenge oxygen radicals and some other reactive species (Robertson, 2004). It reduces different oxidants after increasing of its hydrogen atom. In these reactions, two GSH molecules transform into one molecule of oxidized glutathione (GSSG). This reaction catalyzes the enzyme GSH-px in cells (Reiter, 1995). In our research, the level of SOD, GSHpx and CAT was increased and the concentration of MDA was decreased after RS treatment, which suggests that RS has effective antioxidative properties and could scavenge well excess free radicals, which may prevent the oxidative damage of the tissues and can increase a protective effect on improving diabetic complications.

2.7. Single cell gel electrophoresis experiments
SCGE does not require cell division, and under alkaline conditions, enables the assessment of DNA double-and single-strand breaks and alkali-labile sites (Singh et al., 1988; Belpaeme, et al., 1996). It is a rapid, simple, visual and sensitive technique for measuring DNA breakage in individual mammalian cells.Single cell gel electrophoresis (SCGE) experiment was made. The blood samples were collected from five rats, then lymphocytes were separated from whole blood using a Ficoll Paque lymphocytes separation medium, then

178 Antioxidant Enzyme

suspended in PBS (Collins and Dusinska, 2002). Cells were incubated in RPMI 1640 (10% fetal bovine serum) and exposed to dH2O control (same volume), RS (100, 200 μg/ml final concentration) or H2O2 (5μmol/L), cultured at 37°C in a 5% CO2, 95% air incubator for 1 h, then cells were centrifuged at 4°C, suspended in a small volume of PBS. Cells were mixed with 0.5% low melting temperature agarose at 37°C, and then placed on a precleaned microscope slides which were already covered with thin layer of 0.5% normal melting agarose. The slides were immersed in a lysing solution for 1 h to lyse the cells. Electrophoresis was conducted at 25 V for 20 min, and then the slides were washed gently to remove alkali and detergents with Tris-buffer, rinsed with dH2O, and stained with ethidium bromide. Four different cultures were analyses under a fluorescence microscope, the tail lengths of 300 cells per culture evaluated and categorized. Four different cultured lymphocytes were assayed by SCGE. The results were shown in Figure 4 and Table 4. The percentage of DNA in the tail was calculated to express the amount of DNA damage by [(2.5 × cells0 + 12.5 × cells1 + 30 × cells2 + 60 × cells3 + 90 × cells4)/∑cells] (Collins and Dusinska, 2002). The result showed that RS-treated lymphocytes (low and high doses) were not significantly affected, whose DNA images were similar with dH2O-treated cultures, whereas the H2O2-treated cells were heavily damaged. In our study, the results showed that RS had not toxic effect to the cultured lymphocytes’ DNA, which were similar with dH2O-treated.
Scores Percentage DNA in the tail Average Images Cell 0 <5 2.5 Cell 1 5-20 12.5 Cell 2 20-40 30 Cell 3 40-80 60 Cell 4 >80 90

Visual classification of DNA damage, accoding to the relative proportion of DNA in the tail (cells 0-4), obtained by single-cell gel electrophoresis. Cell 0 represents undamaged cells, and cell 4 represents the most heavily damaged cells.

Figure 4. Single Cell Gel Electrophoresis Images of Different Damaged Lymphocytes

Scores dH2O RS (100 μg/ml) RS (200 μg/ml) H2O2(5 μmol/L)

Cells 0 281 275 271 0

Cells 1 19 25 29 8

Cells 2 0 0 0 157

Cells 3 0 0 0 125

Cells 4 0 0 0 10

percentage DNA in the tail 3.13 3.33 3.47 44.03

Cells 0 represents the number of undamaged cells, and Cells 4 represents the most heavily damaged cells.

Table 4. Percentage DNA in the tail of different cultures in SCGE assay

Antioxidant Therapies for Hypolipidemia and Hyperglycemia 179

3. Antidiabetic and antioxidant effects of oleanolic acid in diabetic rats
Our study evaluates the antidiabetic and antioxidant effects of oleanolic acid (OA) from Ligustrum lucidum Ait in alloxan-induced diabetic rats. OA in the alloxan-induced diabetic rats showed significant hypoglycemic activity. The levels of serum TC, TG and low-density lipoprotein cholesterol (LDL-c) of OA-treated diabetic rats were lower, and the high-density lipoprotein cholesterol (HDL-c) level was higher than control diabetic rats. Furthermore, OA treatment decreased MDA level, while increased SOD and GSH-Px activities of the liver and kidney in diabetic rats. These results indicate that OA has the hypoglycemic, hypolipidemic and antioxidant efficacy for the diabetic rats and protects the liver function avoiding alloxan induced damage. The antioxidant ability of OA might be the main mechanism of hypoglycemic and hypolipidemic effects.

3.1. Sample preparation and characterization
The extraction of Ligustrum lucidum Ait was based on the multi-crystal method (Gao et al., 2007), and the white powder was obtained. The powder was detected by thin-layer chromatography analysis. The mass spectra and tandem mass spectra were obtained. The authenticity of the purified sample was confirmed. The OA sample was confirmed by electrospray mass spectrometry (Fig.5A, 5B), oleanolic acid is a pentacyclic triterpene (C30H48O3) and has a molecular weight of 456.71. The mass spectrum of the purified OA has indicated the presence of a major fragment at m/z 455.6 corresponding to the deprotonated molecule [M-H]- of OA. The low level of contaminants, observed in this spectrum (for instance at m/z 393 and at m/z 403) indicated that the sample purification procedure was efficient. To further confirm its structure, tandem mass spectrometry experiment (MS/MS) was performed on the molecular ion fragment at m/z 455.6 (Figure. 1B). Fragmentation of this molecular ion has led to the loss of neutral CO2 molecule detected at m/z 407.3. Other abundant fragment ions observed at m/z 391.4, m/z 389.4, m/z 375.4 and m/z 373.3, corresponded to demethylation and/or dehydration products of OA. The mass spectrometric analysis results were in agreement with the molecular characteristics of OA.

Figure 5. (A) Representative LC/MS chromatogram of OA. (B) Representative LC/MS/MS chromatogram of OA

180 Antioxidant Enzyme

3.2. Preparation of alloxan-induced diabetic rats
Wistar rats weighing 180-200 g were purchased and house as previous condition. Eight rats were randomly picked up as normal control (NC), and the rest were fed on high-fat diet. After exposure to the high-fat diet for 3 weeks, the rats were fasted overnight with free access to water, and injected intraperitoneally with alloxan that was dissolved in sterile normal saline solution, and used dosage of alloxan was 200 mg/kg bw. After injection 72 h, the fasting blood glucose level of the rats was determined according to glucose oxidase method (Trinder, 1969) using a Glucose Analyzer. The blood glucose level above 15 mM was defined as DM rats. Thirty-two rats (8 normal rats, 24 alloxan-induced diabetic rats) were chosen and divided into four groups: normal control group (NC), diabetic control group (DC), diabetes + low-dose OA group (DM + OA LD) and diabetes + high-dose OA (DM + OA HD).

3.3. Determination hypolipidemic effect of OA
The rats of DC, DM + OA LD and DM + OA HD groups were fasted overnight with free access to water, blood glucose level of each animal was determined as zero-time blood glucose. The animals of DC group were received 0.5% carboxymethylcellulose (CMC) solution by gavage. The rats were orally administered OA 60 mg/kg bw (for the DM + OA LD group) and 100 mg/kg bw (for the DM + OA HD group), OA was dissolved in 0.5% CMC solution. Blood samples of all the rats were taken at 0.5, 1, 2, 4 and 6 hour intervals following the administration and blood plasma glucose levels at various time points were measured. Whereafter, in the DM + OA LD and HD groups, the rats were given OA (60mg/kg bw, 100mg/kg bw) daily by gavage for 40 days, respectively. In contrast, the control rats (NC& DM groups) were given the same volume of 0.5% solution CMC only for 40 days. On day 0, 10, 20, 30 and 40, blood samples were collected from a tail vein, following overnight fasting, and measured. The supplement of OA improved the acute blood glucose levels in the rats (Figure 6). There was no significant difference at 0 h between the DC and OA-treated groups. But in the DM + OA LD and HD groups, hypoglycemic effect of OA became significant 1 h following oral administration, and reached the peak 2 h (p < 0.01), was still significant 6 h after oral administration. There was not marked difference for blood glucose levels in diabetic control rats (p > 0.05). The blood glucose level of DM + OA HD was lower than that of DM + OA LD, but there was no significant difference (p >0.05). In the long term test, changes of plasma glucose level as a result of OA treatment are shown in Figure 7. Fasting blood glucose levels were measured on day 0, 10, 20, 30 and day 40. Before treatment of diabetic rats, there was no significant difference for the blood glucose levels among diabetic rats (p > 0.05). They were consistently staying at similar levels within the time course of 0 to 40 days in NC group and DC group. However, in DM +OA treated groups significantly lower blood glucose levels were observed as compared to DC group at the same time points, from day 10 to day 40 (p < 0.01). The decreasing rates of the blood glucose levels at day 40 were 32.4% (in DM + OA LD group) and 46.4% (in DM + OA HD

The values represented the means ± S. diabetic control group (DC). Long term effect of OA on blood glucose level in diabetic rats .05). respectively. Figure 7.01 vs. for eight rats per group. for eight rats per group. Figure 6. *p <0. DM+OA HD group and diabetic control group (DC). DC DM+OA LD DM+OA HD 16 15 Blood glucose level(mM) 14 13 12 11 10 9 8 0 1 * * * * 2 3 * * 4 5 * * 6 Oral Administration Time(hour) Results of acute blood glucose test for DM+OA LD group.Antioxidant Therapies for Hypolipidemia and Hyperglycemia 181 group).01 vs. diabetic control group (DC).E. Acute effect of OA on blood glucose level in diabetic rats 16 14 NC DC DM +OA LD DM +OA HD Blood glucose level (mM) 12 * * * * 10 * 8 * 6 4 0 10 20 30 40 Oral Administration Time (days) The values represented means ± S.E. The difference of dose-effect of OA was significant according to the results of blood glucose levels of OA-treated groups (p < 0. *p <0.

032 0.895 ± 0.396 ± 0.05 vs. and blood samples were collected from eyepit of all rats under ether anaesthesia. Table 5. After OA treatment. Kidney and liver were homogenized in 4 volumes of Tris–HCl buffer (pH 7.039** 1.026 1. The liver and kidney were immediately removed. . but their HDL-c level significantly increased (p <0.014 ± 0. the rats were fasted overnight. MDA and GSH-px were measured by commercial suggestion of the kits. and p <0. **p <0. The lipid metabolic parameters were also affected in OA-treated (60 mg/kg) rats.039** TC (mmol/L) 1.05 or 0.951 ± 0.. DC.033* 1. but which were less effective than that of OA-treated (100 mg/kg) rats. TC and LDL-c in DM control rats were significantly higher (p<0.01 with HD group).048** 1. 1976). the levels of blood glucose.01) than those of NC group..983 ± 0. while their level of HDL-c was significantly lower (p < 0. weighed and washed using chilled saline solution.049** 2. The levels of SOD.036 ± 0.973± 0.182 Antioxidant Enzyme 3.031* 1.01 with HD group) as compared to DM control group. Groups NC DC DM+OA LD DM+OA HD TG (mmol/L ) 0. The blood samples were used for the measurement of TG.05 with LD group. Our results suggested that OA not only possess significant hypoglycemic ability but also have remarkable hypolipidemic effect in alloxaninduced diabetic rats with hyperlipidemia.4). HDL-c and LDL-c levels.031** LDL-c (mmol/L) 0.033 2.991 ± 0.279 ± 0. TC.034* HDL-c (mmol/L) 0.211 ± 0.028** 0. TC. Determination of blood lipid parameters On the day 41.E.078 ± 0.825 ± 0.946 ± 0.145 ± 0. *p <0.024* The values are means ± S.01). When diabetic rats were treated with OA (100 mg/kg) for 40 days. diabetic control group (DC). TG and LDL-c. 2002).01 vs.05 with LD group and p <0. The homogenate was centrifuged at 4000 × g for 20 min at 4°C and the protein concentration was determined using bovine serum albumin as the standard (Bradford et al. China). and the level of HDL-c in OA-treated rats was higher than those of diabetic rats.038 1.460 ± 0. TC. the animals were sacrificed under ether anaesthesia.025 0. The long term effect of OA treatment on blood lipid levels of tested rat groups is given in Table 5. Effect of OA on serum lipids Before OA treatment of alloxan-induced diabetic and hyperlipidemic rats.4. LDL-c were significantly decreased (p <0. Afterthat. TG and LDL-c were significantly decreased. the levels of serum TG.05 or 0. The results showed that the levels of TG. These findings indicate that OA might be beneficial to diabetic patients with atherosclerosis.0278 0. the significant rise in blood glucose was accompanied with increases in TC. for eight rats per group.971 ± 0. since elevated HDL-c level is associated with the reduced risk of the development of atherosclerosis in diabetes mellitus (Taskinen et al. according to commercial advice by the Automatic Biochemical Analyzer (Scientific and Technical Center of Beijing Hospital Clinic Medicine.

04** 30. and consists of lipid hydroperoxides.82 ± 0. while the activities of SOD and GSH-px were decreased comparing with NC group. diabetic control group. The results showed that the level of MDA was significantly increased in diabetic control rats. Group NC DC DM+OA LD DM+OA HD Tissue Liver kidney liver kidney liver kidney liver kidney MDA(nmol/mg.01).00** 32. The increase of SOD and GSH-px activities of kidney tissue between the DM+OA HD and DM+OA LD groups was shown in the dosage dependent manner (p < 0..02** 40.46 12. 2005).05).73 24.The cause was probably decreased activity of SOD because higher blood glucose could combine with SOD (Fuliang et al.05 or 0.05 vs.70* GSH-px(U/mg. diabetic control group. Analysis of antioxidative enzyme activities The effect of OA on MDA. for eight rats per group.43 ± 0.13 ± 0.54 42.25 ± 0.E.76 21.Antioxidant Therapies for Hypolipidemia and Hyperglycemia 183 3. *p <0. Oxidative stress was involved in the early diabetic dysfunction that led to reduced activities of antioxidant enzymes.35 ± 1.84 ± 0.32** 9. It is a three-carbon dialdehyde.35 11.27** 11.93** 16. SOD and GSH-Px activities Alloxan establish a redox cycle with the formation of superoxide radicals. which has important effects on the control of oxidation reactions in the body.5.18 ± 0. while the decrease of the level of MDA was not significant in the DM+OA LD group as compared to DC group (p > 0. Our research suggests that OA possesses antidiabetic potential in alloxan-induced diabetic 8.24 9. These radicals undergo dismutation to hydrogen peroxide.35 ± 1.73 27.27** SOD(U/mg. MDA would reflect the degree of oxidation in the body.6 ± 0.14** 50.32 ± 0.22 ± 0. As the secondary product of lipid peroxidation.06 ± 0.86 44.80 ± 1.95 ± 0.73 19.49 ± 0.70 ± 0.68 ± 0. The action of reactive oxygen species with a simultaneous massive increase in cytosolic calcium concentration causes rapid destruction of B cells.35 9. the results showed that the GSH-px activity was significantly increased in OA-treated diabetes group compared with diabetes control group.69 ± 0.67* 48. SOD is a scavenger of free radicals. The concentration of SOD in diabetes was significantly lower than that of normal (Wohaieb and Godin . Thereafter highly reactive hydroxyl radicals are formed.01 24. Oxygen free radicals exert their cytotoxic effects on membrane phospholipids resulting in the formation of MDA. there was a partial elevation of total antioxidant capacity including SOD and GSHpx in the liver and kidney as compared to DC group (p< 0.19 ± 0. OA treatment recovered activities of antioxidant enzymes . The MDA level was decreased in DM +OA HD group (p < 0.30 ± 0.77** The values are means ± S.52 ± 0.73 45. Effect of OA on tissue MDA.55 ± 0. **p < 0. Table 6. In our study. In OA treated groups. This suggests that OA has effective anti-oxidative properties and could scavenge well excess free radicals and reduce the production of MDA. SOD and GSH-px in the rats is given in Table 6.05).99 47.42* 10.05). 1987).16 ± 0.29 ± 1. The level of SOD was increased and the concentration of MDA was decreased after OA 45.

Bile salt tolerance test MRS broth was inoculated with 106 cfu mL-1 from overnight cultures. the strains can not decrease the blood glucose level of hyperglycemic mice.3% oxgall. Activities of SOD. and two strains with high acid tolerance and bile salt resistance were screened. Cells were serially diluted 10-fold in phosphate buffer (0. The initial bacterial concentration was 106 cfu mL-1 and was checked by viable count determination on MRS as described above. However. The residual viable count was determined by dilution and plate counting on MRS agar after 24-48 h of incubation.1.1 M.28 d continually. 4. Levels of serum TC. Strains isolation and identification LAB strains were isolated using MRS broth from Chinese pickled cabbage that was bought from the Shandongpu market. p<0. 4. 4.184 Antioxidant Enzyme and improved liver and kidney function resultantl. The survival rate was calculated as the percentage of colonies grown on MRS agar compared to the initial bacterial concentration.0 M). Determination of acid tolerance The 28 strains were grown in MRS broth at 37°C overnight. Compared with the high fat food control group. 28 LAB strains were isolated from pickled cabbage.2.01). Growth in control (no bile) and test cultures (0. The isolated LAB strains were stored in -80°C. while change in CAT was insignificant.plantarum (lab1) and L. and subcultured in 10 mL of fresh MRS broth adjusted to pH 3 with hydrochloric acid (3.1 . serum TC and TG levels were significant decrease (p<0. HDL-c level was significant increase (p<0.3. MO USA) was monitored and incubated for 4 h at 37°C. pH 6. St. 4. They were given to normal and hyperlipidemic mice by ig. Antioxidant therapies of lactic acid bacteria on hypolipidemia Pickled cabbage is popular Chinese traditional food. Louis. Sigma Chemical Co. which indicated that oleanolic acid was benefit to early diabetic rats due to its antioxidant property partly at least. Our study was explored to characterize effects of lactic acid bacteria (LAB) isolated from the pickled cabbages on activities of antioxidant enzymes and hypolipidemia in normal and high fat diet mice. The strains were identified to be L.05.. Samples were incubated for 4h at 37°C. The strains were serially diluted 10-fold in phosphate buffer (0.01).2) in order to neutralize the medium acidity. Differences of SOD levels between the lab2-treated normal diet group and the normal control group was significant (p<0. GSH-px in liver and kidney tissues of the LAB-treated mice were increased. The result indicates that the strains have the potentials of enhancing activities of antioxidant enzymes and relieving hyperlipidemia-induced oxidative stress.05) in lab2-treated and lab1+lab2-treated high fat diet groups. TG and LDL-c were decreased and HDL-c level was higher in the LAB-treated groups. and identified according to Gram stain positive and catalase test negative.brevis (lab2) by the API 50CHL identification kit.

France) (Gao et al. 2×10 9 Experimental animal Quantity 8 8 8 8 8 8 8 Feeding method Normal diet High fat diet Lab1 suspension gavage. lab2 + high fat diet group (lab2 + HFD) and mixed bacteria + high fat diet group (MB + HFD).Bacteria Number (cfu mL-1) 0. weighed and washed with cold physiological saline. The mice of the HFC. The .2.2) in order to dilute the medium bile salt. and stored in the Bioengineering laboratory of Yanshan University (Qinhuangdao. high fat diet Lab2 suspension gavage. high fat diet Lab1+Lab2 suspension gavage. Experimental design in vivo Male ICR mice weighing 18–22 g were purchased from the Experimental Animal Center of China Academy of Military Medical Science (Beijing. 2×109 0.Antioxidant Therapies for Hypolipidemia and Hyperglycemia 185 M. Group NC HFC lab1+DN lab2+ND lab1+HFD lab2+HFD MB+HFD Dosage(MI).2. lab1 + normal diet group (lab1 + ND). The survival rate was calculated as the percentage of colonies grown on MRS agar compared to the initial bacterial concentration. the mice were killed under ether anesthesia. On the day 31.2. two LAB strains showed high acid tolerance and bile salt resistance. 28 d continually. and the 10% tissue homogenate was prepared using physiological saline by a cold glass homogenizer. 2×109 0. normal diet Lab1 suspension gavage. high fat diet 0. and blood samples were collected from eyepit of all the mice. Livers and kidneys of the mice were removed immediately. In the acid and bile salt tolerance tests..2.2. 2×109 0. normal diet Lab2 suspension gavage. lab1+HFD. high fat diet control group (HFC). Determination of tissue enzyme activities and blood lipid levels On the day 59. the living lab1 and lab2 suspensions were fed to the mice by ig.plantarum (lab1) and L. The strains were identified to be L. Experimental groups and ways of feeding 4. China). 2×109 0. 2011) . pH 6. 4.2. Fifty-six mice were divided randomly into 7 groups: normal diet control group (NC). lab1 + high fat diet group (lab1 + HFD).5. China).brevis (lab2) by the API 50CHL identification kit (BioMerieux SA.4. 2×109 0.2. Experiment groups and feeding treatments are shown in Table 7. 2×109 Table 7. The residual viable count was determined by dilution and plate counting on MRS agar after 24﹣48 h of incubation. lab2+HFD and MB+HFD groups were fed with high fat diet 30 d continually to construct hyperlipidemic models. lab2 + normal diet group (lab2 + ND).

**p<0. GSH-px activities in liver and kidney tissues of lab1 + ND group has not significant difference compared with NC group.01). Meanwhile. It could eliminate superoxide free radicals and protect body cells against superoxide 50 * ** 40 30 20 10 0 NC HFC lab1+ND lab2+ND lab1+HFD lab2+HFD MB+HFD Groups The values are mean ± SD. the activities of SOD in lab1 + HFD.186 Antioxidant Enzyme homogenate was centrifuged at 4000×g for 15 min at 4°C. GSH-px is a key antioxidant enzyme catalyzing the reduction of peroxides to protect against oxidative tissue damage.01).. but the lab2 + ND group was significantly . *p<0. +p<0. SOD. 1951) using bovine serum albumin as standard. HDL-c and LDL-c were measured according to the commercial instructions. TG. SOD plays a very important role in the balance between oxidation and antioxidation. CA) based on the lowry colorimetric assay (Lowry et al. Richmond. The protein concentration of the tissues supernatant was determined by the DC Protein Assay Kit (Bio-Rad Labratories.05). Figure 8. the activities of SOD were significant in liver and kidney tissues of lab1 + ND and lab2 + ND groups compared with HFC group (p<0.05 vs high fat control group (HFC). The levels of CAT.05 vs normal control group (NC). lab2 + HFD and MB + HFD groups in kidney tissues were increased significantly compared with HFC group (p<0. and the levels of SOD in liver tissue were enhanced in lab2 + HFD and MB + HFD groups than that of HFC group (p <0. but were not significant. ++p<0. GSH-px. Figure8 indicates that the decrease in the level of SOD was significant in the HFC group compared with NC group (p<0. Liver Kidney + + ++ 60 ++ ++ ++ ++ + SOD activities (mmol/mg. Figure 9 shows effects of lactic acid bacteria on tissue GSH-px activities which demonstrate that there was significant difference between NC and HFC groups (p <0.05).01 vs high fat control group. TC. Effects of lactic acid bacteria on tissue SOD activities. The levels of SOD were increased in lab1 + ND and lab2 + ND groups compared with NC group. However.01 vs NC group.05).

+p<0.08 ± 0.15* 1.05 vs normal control group.05. lab2 + HFD and MB + HFD groups were different significantly compared with HFC group (p<0.99±0.21 1. The activities of CAT of liver and kidney tissues for the HFC group were different significantly compared with NC group (p<0. *p<0.94±0.18 0. However.07±0. Effects of lactic acid bacteria on tissue GSH-px activities CAT is a main enzy me in the microbody of cells. Liver Kidney + ++ 300 * ** * GSH-px activities (mmol/mg. CAT (mmol/mg·pro) Liver 1.15 Kidney 1.97±0.01).05). which can oxygenolysis toxic components (Holmes and Masters 1978).01± 250 + ++ ++ 200 ** 150 100 50 0 NC HFC lab1+ND lab2+ND lab1+HFD lab2+HFD MB+HFD Groups The values are mean ± SD.*p<0.26 0.14 0.01 vs NC group.27 Group NC HFC lab1+DN lab2+ND lab1+HFD lab2+HFD MB+HFD The value are mean ± SD.21 0.05).93±0.98± 0.15* 0. difference of the levels of CAT was insignificant between all LAB-treated groups and NC group (p>0. Table 8).94 ±0. **p<0.92± 0.01 vs high fat control group.12 0.05 vs high fat control group (HFC). ++p<0.17 0. GSH-px levels in the lab1 + HFD. Effect of lactic acid bacteria on tissue CAT activities of the tested mice . The result indicated that the two strains may have no effect on the CAT activities for the LAB-treated mice.01±0.85±0.14 0.14 0.99±0.05.05 vs normal control group (NC). Table 8.24 0. Figure 9.79± 0.Antioxidant Therapies for Hypolipidemia and Hyperglycemia 187 different compared to NC group (p<0. 0.

. Table 9.15++ 7.25 5.11 9. 106 cells were incubated with primary anti-Nrf2 antibodies for 30 min on ice.14+ 7.6.01 vs normal control group.04++ 3.1 M PBS with 0.01).15++ 2.10 3.14 6.1 M PBS with 0.16 3. TC and TG levels in lab1 + HFD. and the experiment was repeated three times.05 vs normal control group.93±0. +p<0.18 4. which may be contributed to the mixture of the two strains.10 4.22±0.01 with MB + HFD).16++ 2. 0.04++ 6. After two washes in 0. The software used was BD CellQuest Pro (Becton Dickinson Biosciences. LDLc is a main factor of the danger on atherosclerosis.45±0.01±0.1% sodium azide.188 Antioxidant Enzyme The results of TC and TG levels of the experimental mice are presented in Table 9.05** 2.76±0. Finally. USA) and the data were expressed as fluorescence intensity formula (I = Log (xmode) × 340) (Gao et al.1 M PBS with 0.08** 2.12±0.37±0.81±0. ++p<0.01++ HDL-c(mmol/L) 1.03++ LDL-c(mmol/L) 3. Table 9).59±0. USA). The result showed that the two strains have good effects on cholesterol-degrading activity. **p<0.32+ 3.5 μg of secondary FITCconjugated rabbit anti-mouse antibody were added and incubated on ice for 30 min.18 4. Nrf2 antibodies (Santa Cruze biotechnology Inc.81±0.16±0. Analysis of Nrf2 expression in liver tissues Single hepatocyte suspensions of the mice were prepared in ice-cold 0.1 NaN3.05 vs high fat control group (HFC).16 2.05 with lab2 + HFD group and p <0. and difference was extremely significant (p<0.19+ The values are mean ± SD.99±0.02** 2.31±0.1% sodium azide.02±0.05.19 4. but which were not significant compared with NC group.01).74±0. Effect of lactic acid bacteria on blood lipid levels 4. USA) were diluted in 0. and the MB + HFD group was the lowest.21±0.03±0. Group NC HFC lab1+DN lab2+ND lab1+HFD lab2+HFD MB+HFD TC(mmol/L) 2.19±0..1 M PBS with 0. and HDL-c plays an important role in protecting cardiovascular system.1% sodium azide. Nonspecific binding of secondary antibody was excluded by incubating the cells only with the FITC-labelled secondary antibody.35±0. In order to identify the effects of two strains on Nrf2 expresssion in .14 6.98±0.25±0. TC and TG levels of hyperlipidemic mice were higher than NC group.71±0.674±0. the cells were resuspended in 1 mL of 0.01 vs high fat control group.58±0. 2011).06±0. and fluorescence of Nrf2 positive cells was quantified. lab2 + HFD and MB + HFD groups were significantly lower compared to HFC group (p<0. which indicated that the hyperlipidemic models were established.18* 3.88±0. HDL-c levels in the LAB-treated hyperlipidemic groups were all higher than HFC group (p <0.27±0.09++ TG(mmol/L) 2. *p<0.24++ 3. and levels of LDL-c were lower significantly in lab2 + HFD and MB + HFD groups compared with that of high fat control group (p <0.86±0.26 3. The hepatocytes were scanned using a FACSCalibur (Becton-Dickinson. Nrf2 serves as master regular of a cellular defense system against oxidative stress. The levels of TC and TG in lab1 + ND and lab2 + ND groups were slightly lower.

E was high fat control group. et al. High acidity in the stomach and high concentration of bile components in the proximal intestine are the first host factors. P <0.. lysozyme.05).00+ 275. In our study. 2003. frequency of cells displaying certain fluorescence intensity. Group NC HFC lab1+ND lab2+ND lab1+HFD lab2+HFD MB+HFD Nrf2 expression fluorescence intensity 219. Nrf2 levels of the hepatocyte nuclear extract were measured by Flow cytometry technology. Effect of LAB on expression of Nrf2 protein of liver cells in the mice Single-colour histograms represent hepatocyte staining with anti-Nrf2 antibodies. untreated normal control.. two high acid tolerance and bile salt resistance strains were screened. in accordance with a decrease in untreated high fat diet mice (P <0. + Significantly different from high fat control group.67±6. G was Lab2 treated high fat mice. TG and glucose in the blood were higher than those of normal diet mice. the levels of TC. D was lab+lab2-treated normal mice.86±3.. DTAF flurescence intensity. However. probiotic bacteria must be resistant to the acidity of the stomach.16+ 286. bile. C was lab2 -treated normal mice. A was normal control group. Table 10). which were L.01).01. B was lab1 treated normal mice. P <0. et al.49* 200. F was lab1 treated high fat mice.88±3. 2010). Probiotics are commonly used as viable microbial feed supplements that affect the host animal by improving its intestinal microbial “balance” (Holzapfel. Figure 10. The experimental mice were divided into normal diet and high fat diet groups. Several studies demonstrated that some lactobacilli possess antioxidative activity. The concentration of blood lipids in the normal diet mice was under normal range. Activities of antioxidant enzymes and the function of reducing cholesterol and blood glucose of the two LAB strains were studied.brevis.42 144.62±2. and the results showed the two strains signicantly increased translocated nuclear expression of Nrf2 (vs.56±2. and could decrease the risk of accumulation of reactive oxygen species during the ingestion of food (Ito. y-axis.82 211. and activated expression of Nrf2 in the hepatic nucleus of HFD-induced hyperlipidemic mice. Kuda et al. Table 10. pancreatic enzymes. 1998). x-axis.Antioxidant Therapies for Hypolipidemia and Hyperglycemia 189 vivo. .15* 285. P <0. as evidenced by a significant elevation of Nrf2 in the nuclear fractions (P <0.27±4.plantarum and L.01. The data also indicated that supplementation of Lab strains promoted further translocation of Nrf2 into the nucleus. respectively. which affect strain selection and adhesion.93+ *Significantly different from normal control group. we analysed Nrf2 concentration in the hepatic cells for lab1and lab2-treated mice. When the mice have been fed high fat food for 30 d.25±2.01. H was lab1+lab2 treated high fat mice.

x-axis. F was lab1 treated high fat mice. H was lab1+lab2 treated high fat mice. A was normal control group. D was lab+lab2-treated normal mice. G was Lab2 treated high fat mice. Flow cytometric analysis of Nrf2 expression in the liver tissues of lactic acid bacteria treated mice and control mice.190 Antioxidant Enzyme (A) (B) (C) (D) (E) (F) (G) (H) Single-colour histograms represent hepatocyte staining with anti-Nrf2 antibodies. E was high fat control group. . C was lab2 -treated normal mice. y-axis. frequency of cells displaying certain fluorescence intensity. DTAF flurescence intensity. Figure 10. B was lab1 treated normal mice.

SOD is a scavenger of free radicals. Nrf2 serves as master regular of a cellular defense system against oxidative stress (Motohashi et al. Researchers showed that LAB could decrease the level of cholesterol but the mechanism has not been demonstrated clearly yet. the levels of antioxidant enzymes and the function of reducing blood lipid in MB + HFD groups were higher than the other two hyperlipidemic groups. since it protects protein –SH groups against oxidation and can scavenge oxygen radicals and some other reactive species. The liver plays a central role in the maintenance of systemic lipid homeostasis and it is especially susceptible to reactive oxygen species (ROSs) damage (Hamelet et al. and it also could fall significantly the cholesterol level in hyperlipidemic mice. Smet et al. which has important effects on control of oxidation reactions in the body. LDL-c in the mice. et al.. It reduces different oxidants after increasing its hydrogen atom. Further research is indispensable to clarify the exact mechanism. bile salt and cholesterol were coprecipitated. Levels of serum TC. TG and LDL-c were slightly decreased and HDL-c level was a little higher by the LAB suspension treating on the normal mice. Some predicted that LAB. Nrf2 is sequestered in the cytoplasm by Keap1. et al. 2009). Meanwhile. Some LAB may enhance SOD and GSH-px activities and prevent oxidative damage (Tsai.. 2000). (1994) suggested that the reason of LAB reducing cholesterol might be due to the activity of bile salt hydrolysis produced by the LAB. High fat diet could be used to induce signicant oxygen-centered free radicals and ROS generation in the mice. At the same time. the concentration of SOD and GSH-px in the high fat diet mice was significantly lower than those of the normal rats.Antioxidant Therapies for Hypolipidemia and Hyperglycemia 191 respectively. SOD is the most important survival protein and ubiquitously induced antioxidant by various stimulants.. After the male SD rats were fed high-fat diet with Lactobacillus ferment. GSH is often regarded as an antioxidant agent. and then expelled with feces. which facilitates its . compared to the HFC group. supplementation of Lab also promoted expression of Nrf2 in the liver tissues of the mice. This reaction is catalysed by enzyme GSH-px in cells (Reiter. lab2 + HFD and MB + HFD groups were all increased compared with HFC group... 2004. The finding indicates that the two strains might decrease the risks for cardiovascular and arteriosclerosis diseases in various degrees. In the research. 2004). Under physiological conditions.. 1995). (1997) found that consumption of acidophilus yogurt significantly lowered the values for plasma TC. so we hypothesized that the strains might play a more crucial role in a severe stressful condition. or the cholesterol was absorpted by lactic acid bacteria (Jeun. but not achieve the level of the NC group. there were significantly decrease on the levels of body weight. Otherwise. 2007).. However. Free radicals can diffuse intracellularly and result in mitochondrial enzyme damage and DNA breaks. Several studies have documented the relationships between increase of free radicals and blood glucose. levels of TC and TG were decreased extremely in lab2 + HFD and MB + HFD groups. Akalin et al. LDL-c and TC compared with the highfat diet control rats (Choi et al. 2010). 2006). the activities of SOD and GSH-px were increased in various degrees in Lab1 + ND and lab2 + ND groups compared with the normal control mice after the LAB administering. 2002). In this study. impair cellular function (Bonnefont-Rousselot et al. The levels of SOD and GSH-px in the lab1 + HFD. lipid peroxidation as well as low-density lipoprotein (Tanaka et al.. Nguyen et al.

Our Flow cytometry data clearly show that high fat diet-induced oxidative stress is associated with activation of Nrf2. Nrf2–ARE signaling is also known to be mainly responsible for the upregulation of SOD and GSH-px gene expression and hence constitutes a crucial cellular response to environmental stresses (Surh et al. Brignardello E. 11965152D). Boccuzzi G. Argano M. As expected. Danni O. Cell Biochem Funct. Hypertension and the Pathogenesis of Atherosclerosis Oxidative Stress and the Mediation of Arterial Inflammatory Response: A New Perspective. 2004)Upon exposure to oxidative stress.192 Antioxidant Enzyme ubiquitination and proteasomic degradation (Kang.Hypertension 1995. W..Gatto V. Vural H. as evidenced by a signicant elevation of Nrf2 in the nuclear fractions (P<0. L. Qinhuangdao. L. a grant from Hebei Province Natural Science Fund (No. 21:121-125. 20101333120011). plantarum isolated from fermented cabbage upregulates antioxidative enzymes in high fat diet mice via Nrf2-dependent transcriptional activation of ARE sites. Department of Biological Engineering. et al.plantarum could inhibit HFD-induced oxidative stress through the Nrf2Keap1 signaling pathway. Sabuncu T.plantarum is further capable of activation of Nrf2 and preventing HFD-induced inhibition of antioxidant enzymes. 1997.. Tamagno O.. J Endocrinol 1997.05). Thus.. Influence of yogurt and acidophilus yogurt on serum cholesterol levels in mice. J. Dehydroeppiandrosterone administration prevents the oxidative damage induced by acute hyperglycemia in rats. Vries et al. L. where it binds to cis-acting antioxidant response elements (AREs) and promotes the transcription of a large number of cytoprotective genes (Kensler et al. 2007.. C2011203137.. Supplementation of L. Yanshan University.. China Acknowledgement This work was financially supported by the research grant from the Chinese Ministry of Education Doctor Degree (No. References Akalin AS.. Author details Dawei Gao Key Laboratory of Applied Chemistry in Hebei Province. 80: 2721-2725. Dairy Sci.plantarum markedly promoted further translocation of Nrf2 into the nucleus. the sequestration complex breaks down and the dissociated Nrf2 translocates into the nucleus. Effects of melatonin on oxidative-antioxidative status of tissues in streptozotocin-induced diabetic rats. The upregulation of several antioxidative enzymes is associated with the reduced formation of ROS and enhanced survival of liver cells upon the induction of oxidative stress. and a Chinese Postdoctoral grant (480013). Aksoy S. Alexander R. 2003. 2008). 5. Duzel S. 25:155-161. Aksoy N. 2009). Gonc S.155: 233–240 .

1985. Zhu G. J. Phytother Res.R. Chen M. 51:147-152. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Food Microbiol.S. Furukawa S. Snel J. Effects of propolis on blood glucose.Antioxidant Therapies for Hypolipidemia and Hyperglycemia 193 Belpaeme K... Li Y.41:85-101 Ito M. Radloff SE. Haberer P. Dicks LMT. 2000. Ohishi K. 2003. 2004. Increased oxidative stress in obesity and its impact on metabolic syndrome. J Hepatology 2007.Antioxidative and hypolipidemic effects of lactic acid bacteria from pickled Chinese cabbage. 26:163-176 Choi YM. 2006. Hepburn HR. Mechanisms of lipid peroxidation.. J. et al. Hyperhomocysteinemia due to cystathionine beta synthase deficiency induces dysregulation of genes involved in hepatic lipid homeostasis in mice. J Clin Invest... Toit MD. Gao D. Diabetes Metab. Hang BQ. Hamelet J.S.. Shimabukuro M.87: 287–291. Kirsch-Volders M. Journal of Medicinal Plants Research 2011.5:1439-1446 Gao D. Food Microbiol. Methods Find Exp Clin Pharmacol 2009.31: 375381 Girotti MW. Iwaki M. Can J Physiol Pharmacol 2007. Oxidation of cellular DNA measured with the comet assay.J. Demuth K.M. 72: 248-254 Brown N. Dusinska M.Antioxidative effects of lactic acid bacteria on the colonic mucosa of Iron—overloaded mice. Bae SH.. 17: 429-431 Holmes R. Pharmacol Res. Antidiabetic potential of rhodiola Sachalinensis root extract in Streptozotocin-induced diabetic rats.. Yokoi W.114:1752–1761 Gao D. Antidiabetic potential of oleanolic acid from Ligustrum lucidum Ait. Suh HJ... 20:1056-1060 Collins A.. metastatic potential and response to therapy of breast cancer. Zhu L. Chinese Materia Medica. 2003. Daya S. 1998. and Masters C..Overview of gut flora and probiotics. Liu Z. blood lipid and free radicals in rats with diabetes mellitus. Schillinger U. Jaudon MC. Meth Mol Biol 2002. 1992. Hypoxia and oxidative stress in breast cancer Oxidative stress: its effects on the growth. Paul J. Food Chem. Li Q. Mutagenesis 1996. Hypolipidemic effect of lactobacillus ferment as a functional food supplement. Bastard JP. Xuan H. Agric.. 1:87–95.Genetic control and ontogeny of microbody enzymes: A review Biochemical genetics 1978. 3:323–327 Bonnefont-Rousselot D. Huis int Veld JHJ. Ahrens F. Free Radic Biol Med. Kang DH. 85:1076-83.46:151–159 Hao ZQ. high-cholesterol diet-a preliminary in vivo trial. 11:485–492 Bradford M. Int. Wang Y. Fujita T. Breast Cancer Res 2001. Sawada H. Study of Ligustrum lucidum Ait on decreasing blood glucose. et al. Holzapfel WH.51: 44564460 . J. Haberer P. Bicknell R. Li Q.16:171-190 Holzapfel WH. Consequences of the diabetic status on the oxidant/antioxidant balance. Anal Biochem 1976. Effect of potentially probiotic lactobacilli on faecal enzyme activity in minipigs on a high-fat. Yoshida Y. Int. 6:147–159 Fuliang HU. 2005... Delbeke K.Cytogenetic studies of PCB77 on brown trout (Salmo trutta fario) using the micronucleus test and the alkaline comet assay. Delattre J.

R.B. Protein measurement with the Folin phenol reagent. Efficacy of lower doses of vanadium in restoring altered glucose metabolism and antioxidant status in diabetic rat lenses. against beta-amyloid toxicity. Tezuka Y. Seo JG.. Biol. Randall RJ. T. Hypocholesterolemic effects of Lactobacillus plantarum KCTC3928 by increased bile acid excretion in C57BL/6 mice. Diabetes Metabolism Res Reviews.284:13291–13295 Pigeolet E. N. The Nrf2-antioxidant response element signaling pathway and its activation by oxidative stress..... 25:1101-1104 Motohashi. National Acad Sciences 2004. Nishijo H. superoxide dismutase and Catalase inactivation by peroxides and oxygen derived free radicals.S. Houbion A... Neuroprotective effects of constituents of the oriental crude drugs..76:385-391 Jeun J. Rosebrough NJ. Park HJ. Sun C..C. Annu. Traditional Herbal Drugs. . H. T. Gupta B.120:517–522 Kwon N. Cho SY. Morita H. Wu L. Mori M. Rev. 1988. J Biosci 2005. P. Li T. C. S. Hashimoto H... Corbisier P. Kobayashi A. Baquer N. Louro T. Kadota S.193: 265-275 Matafome P. J. Jun H.W. J.Z... Fan W.. Hosoda M.Biol Pharm Bull 2002.194 Antioxidant Enzyme Jain S. et al. Nitric oxide generation from streptozotocin. 8:529-33. Kim S.Antioxidant and cholesterol assimilation activities of selected lactobacilli and lactococci cultures. Farr AL. Nrf2-Keap1 denes a physiologically important stress response mechanism.. Biswal. 2000.10: 549–557 Nguyen. Nutrition. Yano T.. 19: 37–42..Scaffolding of Keap1 to the actin cytoskeleton controls the function of Nrf2 as key regulator of cytoprotective phase 2 genes.. 2009.. 2010. Kho T.. Kim H. Chin. 1951. Wakabayashi. Cell survival responses to environmental stress via the Keap1-Nrf2-ARE pathway.2007. Rodrigues L. 2004. Lee HS. Pharmacol. J. Induction of superoxide anion radical scavenging capacity in Japanese white radish juice and milk by Lactobacillus plantarum isolated from aji-narezushi and kaburazushi Food Chemistry 2010. Mech Aging Dev 1990..D. Med. Trends Mol. Chem..Q. 2011.26: 321-330 Kang M. sachalinensis and Tokaku-joki-to. M. Chung MJ. Choi C.47:89–116 Kuda T. 51:283–297 Preet A. Nioi. Biol.Yadav H. Pharmacological studies on the sedative and hypnotic effect of salidroside from the Chinese medicinal plant Rhodiola sachalinensis. Yamamoto M.S. FASEB J 1994. Dairy Sci. Chem. oxidative stress and apoptosis.K. J. Zhang R. Ming H. Dairy Res. Kaneko N.. Yamamoto... Sinha PR.14:601-604 Lowry OH. Lee SJ. 30: 221–230. Wakabayashi N.. Metformin and atorvastatin combination further protect the liver in type 2 diabetes with hyperlipidaemia..83: 255–263 Kensler. Hosono A.W. Xia G. Glutathione peroxidase. Yadava P. Lee S. Xu G.27:54–62.. Mook-Jung I. Effect of administration of fermented milk containing whey protein concentrate to rats and healthy men on serum lipids and blood pressure. Progress in Rhodiola rosea L. 2009.. Kim S. Pickett. 17: 2046-2051 Kawase M. Jung M.. Rhodiola sacra. Phytomedicine 2007.L.H. Toxicol..

Cha. J. Res. Diabetes 1986. 2005.P... 76: 1223-1238 Sharma SB. Suarez-Pinzon W. Suzaki Y. Lee CL..H. S. Woestyne M. Tice R.Schneider E. A. Karpen C. 1995.. A simple technique for quantitation of low levels of DNA damage in individual cells. Rabinovitch A.. Yamamoto T.57: 2065-2071 . J Ethnopharmacol. Mosharrof.. Pharm.lowering effect of dietary vitamin E in streptozotocin-induced diabetic rats. 2009. Chinou I. on the content of the brain biogenic monamines. 81: 3197–3202.. Sotiroudis T.Antioxidant Therapies for Hypolipidemia and Hyperglycemia 195 Pritchard J. Lakey J R. Nasir A. J Clin Endocrinol Metab 1996. 2009. Dysregulation of hepatic superoxide dismutase.. J Biol Chem.V. Harmon J.T.. Verstraete W. Reiter RJ.. The association between cholesterol and mortality in heart failure. 40: 85–87 Sotiroudis G. Aslan M. J. Yesilada E.K. H.. Singh N. Robertson RP. Koo J. Chemical analysis. Strynadka K. Exp Cell Res 1988...R. Mani H. Proc Natl Acad Sci USA 2002.T. Hypoglycaemic and hypolipidemic effect of ethanolic extract of seeds of Eugenia jambolana in alloxan-induced diabetic rabbits.. Med Physiol 1987.W.D. Ecol.P.O.. Patel S. Li. Chu LH.. Microb. Comparison between patients with and without coronary artery disease.N. 9:526-533. 26: 43–53. Arch. Roberts C. catalase and glutathione peroxidase in diabetes response to insulin and antioxidant therapies..L..L.. Effect of the extract of Rhodiola rosea L.. Prabhu KM. et al. Triglyceride.34: 61–78 Surh. J food biochemistry 2010. Rajotte R V. Health Dis.V. Vaziri M.. Role of Nrf2-mediated heme oxygenase-1 upregulation in adaptive survival response to nitrosative stress.. 99: 12363–12368 Tsai TU.K. 2005. Kundu. Melliou E.L.K. Int Heart J. Saeyer ND.32:1163–1176 Tanaka Y. Y. Kawasaki T. Hoorde L. Sugihara H.J. 1994. Pan TM. FASEB J. 85: 201-206 Sindhu R. M. Agric. Oxidative processes and antioxidative defense mechanisms in the aging brain. antioxidant and antimicrobial activity of three greek cucumber (cucumis sativus) cultivars. 2003.279: 42351-42354 Sakatani T.R. Nosratola D. Hypoglycaemic activity of Gentiana olivieri and isolation of the active constituent through bioassay-directed fractionation techniques. Matsubara H.R. Y.In vitro study of bile salt hydrolase (BSH) activity of BSH isogenic lactobacillus plantarum 80 strains and estimation of cholesterol lowering through enhanced BSH activity. Human pancreatic islet beta-cell destruction by cytokines involves oxygen free radicals and aldehyde production..175:184-191 Smet ID.Life Sci. A role for glutathione Peroxidase in protecting pancreatic β cells against oxidative stress in a model of glucose toxicity. Chronic oxidative stress as a central mechanism for glucose toxicity in pancreatic islet beta cells in diabetes.35: 278-281.. Robertson R. Ito S. Na. Atherosclerosis-preventing activity of lactic acid bacteria-fermented milk-soymilk supplemented with Momordica charantia. Increased lipoprotein lipase activity in livers of diabetic rats fed high dietary vitamin E.. Tran P. 7:315-329 Stancheva. 2004. Murthy PS. McCoy M. 46: 619-629 Sezik E. Shirayama T.K. Food Chem. Dev G. Clin Exp Hypertens 2004.

. Alleviations in free radical tissue defense mechanisms in streptozotocin-induced diabetes in rats: Effects of insulin treatment. D.45: 1375– 1383 Wang ZX..22: 50-51 Wohaieb SA. Nrf2-induced antioxidant protection: a promising target to counteract ROSmediated damage in neurodegenerative disease? Free Radic. M. Study on chemical components and hepatoprotective properties of Ligustrum lucidum Ait. 1969. A. 2002. Gu ZL. Diabetic dyslipidemia. Diabetes. Yu CS. Study on antimutagenic effect of Ligustrum lucidum Ait by drosophila test. van Horssen.. Clin. Fujian J. Res.J. Med. Hoozemans. Xu BY. 1987. Atheroscler Suppl. Huang GC. B..22: 158-161 Vries.3: 47-51 Trinder P. Biol. Godin DV. J.196 Antioxidant Enzyme Taskinen MR. Traditional Chinese Medicine 1991. J. Drukarch. 2002.. Rozemuller.36:1014–1018 Xiang M. Antitumor effect of Ligustrum lucidum Ait extract in vivo. J Clin Pathol. Hondius.E. Determination of blood glucose using an oxidase-peroxidase system with a noncarcinogenic chromogen. Gao BZ. 2008. H. Witte.. Jiangsu Medical J.10: 13-15 Yin YS. Chinese Traditional Patent Medicine 1993.15: 18-19 .

  . The most effective way to obtain energy is oxidation. catalase (CAT). Introduction Thyroid hormones are involved in the regulation of basal metabolic state and in oxidative metabolism [1]. ROS have a high reactivity potential. superoxide dismutase (SOD). In recent years.13.3].14]. distribution. Oxidative processes predominantly occur in mitochondria [8]. When ROS generation exceeds the antioxidant capacity of cells. mitochondria are the favorite targets of thyroid hormones. Adriana Muresan and Ileana Duncea Additional information is available at the end of the chapter http://dx.doi. protecting them from harmful effects. which is absolutely indispensable for iodine intrafollicular oxidation in the presence of thyroid peroxidase. provided the original work is properly cited.Chapter 8 Oxidative Stress and Antioxidant Status in Hypo.. which permits unrestricted use. the possible correlation between impaired thyroid gland function and reactive oxygen species has been increasingly taken into consideration [9].11]. which is required to fight against entropy. there is a constant production of oxygenated water. oxidative stress develops [7]. lipids and DNA [5].5772/51018 1. βcarotene. and reproduction in any medium. In hypothyroidism. are glutathione reductase (GR). On the other hand. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons. Experimental studies and epidemiological data suggest that hyperthyroidism is associated with increase in free radical production and lipid peroxide levels [10. They can cause many changes in the number and activity of mitochondrial respiratory chain components. Oxidative stress is a general term used to describe a state of damage caused by ROS [4]. licensee InTech. and flavonoids [6]. Among the enzymatic antioxidants. During thyroid hormone synthesis.       © 2012 Petrulea et al. vitamin E. 0). while the non-enzymatic antioxidants are glutathione (GSH). therefore they are toxic and can lead to oxidative damage in cellular macromolecules such as proteins. a decrease in free radical production is expected because of the metabolic supression brought about by the decrease in thyroid hormone levels [12. org/licenses/by/3. glutathione peroxydase (GPx). vitamin C. This may result in the increased generation of reactive oxygen species (ROS) [2.and Hyperthyroidism Mirela the cell contains a variety of substances capable of scavenging the free radicals. Life means a continuous struggle for energy. In fact.

It was shown that both methimazole and propylthiouracil abolished or reduced radical production by complement attacked thyroid cells and decreased cytokine production[18]. ROS also cause injury to the basic cell structures. Oxidative stress A major threat to homeostasis and therefore to the integrity of aerobic organisms arises from chemical species possessing one or more unpaired electrons in their outer orbital. this study aims at investigating oxidative stress parameters. or substrate limitations. glutathione [15] and coenzyme Q[16] and activities of antioxidant enzymes [11]were found to be imbalanced and often opposite. such as lipid. But when this increased synthesis cannot be achieved due to damage to enzymes. Disequilibrium between ROS production and inactivation leads to oxidative stress. 2.and hypothyroid rats. an imbalance persists and the result is oxidative stress [25]. reactions of oxidant enzymes and auto-oxidation reactions [20. Cells can tolerate moderate oxidative loads by increasing gene expression to up-regulate their reductive defense systems and restore the oxidant/antioxidant balance. They readily react with macromolecules. The ultimate effects of ROS activity include mutations.21. especially in activated neutrophils. their generation is inevitable for some metabolic processes. Reviewing the most recent data on the subject. and the antioxidant defense system. Antioxidants treatments might be helpful in reducing the oxidative damage due to hypothyroidism and hyperthyroidism. antioxidant status markers and their response to vitamin E supplementation in hyper. depending on the intensity of aerobic metabolism. metabolic dysfunction and cell . the microsomal membrane electron transport chain. called the oxidative load. Oxygen free radicals can develop during several steps of normal metabolic events.22]. Although free radicals (FR) have the potential to damage the organism. It is worth mentioning that some of the antithyroid drugs have antioxidant effects[17]. leading to potential damage”[24]. The main endogenous sources of free radicals are the mitochondrial electron transport chain. Oxidative stress is a term that was introduced by Sies in 1985 and refers to any situation where there is a serious imbalance between the production of FR or reactive oxygen species (ROS). smooth muscle cells and in endothelial cells [26]. or when the oxidative load is overwhelming. Superoxide and hydroxyl radicals. protein and DNA molecules. The available data concerning oxidative stress in both hypothyroidism and hyperthyroidism are scarce and controversial. Oxidative stress has been defined as ”a disturbance in the prooxidant-antioxidant balance in favour of the former. which results in degradation of cell membranes and excessive activation or inactivation of enzymes[27]. called free radicals [19]. The oxidative load is described as ”a measure of the steady-state level of reactive oxygen or oxygen radicals in a biological system”[23].198 Antioxidant Enzyme The changes in the levels of the scavengers α-tocopherol. monocytes. ROS are produced in all cells. along with non-radical oxygen species such as hydrogen peroxide (H2O2) are commonly termed reactive oxygen species (ROS) and have the highest biological activity.

lipids. and consequent mutagenesis and apoptosis [42]. In most cell cultures. and include enzymatic and non enzymatic antioxidant defenses produced in the body. an iron-containing hemoprotein. glutathione peroxydase (GPx) and catalase (CAT). or sequester transition metals that are the source of free radicals.37]. nucleic acids. oncogenesis and impaired organ functioning [28. tocopherols. Oxidative stress is considered to play a pivotal role in the pathogenesis of aging and several degenerative diseases. Positive Comet assays demonstrate these breaks. Among the most known enzymatic antioxidants. glutathione reductase (GR). 43]. H2O2 has been designated as “the enemy within” [44]. When H2O2 is applied to the exterior of cultured cells. A reduction in GPx activity results in increased H2O2 levels and hence severe cellular damage is observed [41]. They in turn are a cause of development of inflammatory processes. humans have developed sophisticated mechanisms in order to maintain redox homeostasis [33]. etc. and at high levels double-strand breaks [46]. such as atherosclerosis. Because there are great variations in the rate of H2O2 degradation in different cell types and models. Catalase. Antioxidant enzymes act to scavenge free radicals by converting them to less harmful molecules [38].Oxidative Stress and Antioxidant Status in Hypo. converts hydrogen peroxide to water and oxygen [39]. proteins. endogenous[34. such as glutathione. cardiovascular disease. 29]. GPx is an enzyme containing a selenium ion as a cofactor [40]. and for the catalyzed reaction it requires reduced glutathione (GSH). namely exogenous[36. Non-enzymatic antioxidants. which is provided by glutathione reductase. block their production.31. For the phagocytes. At higher concentrations than those that have a signaling role. 7 μM. retinols. The positive Comet assays . and ascorbate. play an important role in scavenging ROS. The accumulation of oxidatively damaged proteins accelerates chaperone-mediated autophagy. single-strand breaks.35]. more for the breaks) [47]. GPx is one of the most effective antioxidants in erythrocytes.32]. The half-life of these damages varies for the various lesions (from 9–62 min for the adducts. 3. the intracellular concentrations are approximately 10-fold lower than the extracellular concentrations [42. H2O2 in the medium disappears in less than 1 h. Oxidative damage to DNA produces adducts (including 8-oxo-deoxyguanosine and thymine glycol). Toxic effects of H2O2 The levels of H2O2 reached physiologically in cells vary from a low 0. 001 μM to a maximum of 0. In order to cope with an excess of free radicals produced upon oxidative stress. we notice superoxide dismutase (SOD). SOD catalyzes the dismutation of superoxide anion radical to peroxide (H2O2) and molecular oxygen (O2). which will degrade them [45]. type 2-diabetes and cancer [30. H2O2 induces oxidative stress. it is difficult to compare concentration-effect relations. and others supplied with the diet. namely. Oxidative stress involves the oxidation of various cellular components.and Hyperthyroidism 199 ageing. DNA oxidation and damage. These protective mechanisms either scavenge or detoxify ROS.

Beginning with active transport of dietary iodide (the rate limiting substrate) into the cell by sodium-iodide symporter [65]. catalyzed by a peroxidase enzyme. Chronic H2O2 administration at low levels induces senescence in cultured cells in vitro in human fibroblasts [57. which surrounds a lumen that contains colloid. This effect has been linked to a loss of GSH and reduced glutaredoxin and consequent activation of apoptosis signal-regulating kinase (ASK) and of an apoptosis program [55]. The luminal side of the follicular cell membrane contains microvilli. Transfection of an H2O2-generating system transforms epithelial cells [53]. H2O2 favors inflammation [59]. It contributes to the body’s energy output by regulation of cardiac rate and output. heat production. TH synthesis includes a radical intermediate. occurs on the endoplasmic reticulum of the thyroid gland cells. H2O2 is required by peroxidase.61]. lipid catabolism. 4. creating a need for a ROS reaction as part of the organ’s function to maintain homeostasis. the essential constituent of protective enzymes. Thyroid hormone synthesis The thyroid is a shield-shaped organ in the neck region composed of an outer layer of follicular cells and c-cells. High-level acute H2O2 treatment of various cells in vitro leads to apoptosis [54]. and skeletal growth [62]. Iodination of tyrosine residues. which by depriving lymphocytes of tryptophan is immunosuppressive. It is therefore not astonishing that even in relatively short-lived (7 h) neutrophils [60] and macrophages. Lack of protective systems in knockout mice such as lack of peroxiredoxin or glutathione (GSH) peroxidases indeed leads to malignant cancers [51. Thus. The colloid contains thyroglobulin which is converted into thyroid hormone (TH). T4. Several of these processes deal with direct or indirect collaboration between the thyroid. if it leads to constitutive activation of a protooncogene or to inactivation of tumor suppressor genes is carcinogenic.52]. and to a lesser extent T3. selenium. 64]. TH includes thyronine (T3). H2O2 is carcinogenic and has been found to play a role in several human cancers (7) even if it may not be sufficient [49]. is synthesized in the follicular cell and is propagated by thyroid stimulating hormone (TSH) secreted by the pituitary gland. TSH synthesis is propagated by thyrotropin releasing hormone (TRH) secreted by the hypothalamus. Coupling forms various THs [65]. and its inhibitory effect on indoleamine dioxygenase. which greatly increase the surface area of the cell to facilitate transfer of colloid into the follicular cell. especially if it is combined to a proliferative effect. Mutagenesis. would enhance immune reactions.200 Antioxidant Enzyme for thyroid cells incubated with 50 μM H2O2 disappear by 80% in 2 h [48]. These effects are stronger in actively proliferating cells [56].58]. H2O2 generation is tightly regulated by a synergic two-pronged mechanism involving both intracellular calcium and diacylglycerol protein kinase C[58. or pineal glands [63. iodide oxidation and hormone synthesis occur at the apical . Conversely. hypothalamus. and is formed by an enzyme from NADPH (nicotinamide adenine dinucleotide phosphate-oxidase) and Ca2+ ions. and thyroxine (T4). prevents tumor development in rats submitted to chemical carcinogenesis [50]. which explains the wide range of symptoms related to thyroid abnormalities. pituitary.

Following the addition of oxygenated water (H2O2) to thyroperoxidase (TPO). under the action of superoxide dismutase (SOD) will be transformed into oxygenated water. xanthine oxidase. which oxidizes iodine (I+). DNA oxidation and damage. H2O2 –generating system was detected in thyroid particulate fractions that appears to be distinct from cytochrome c reductase. compound I is spontaneously converted into a stable compound. produced inside the cytoplasm. These remain bound to thyroperoxidase. H2O2 is produced by an NADPH-dependent process on the external aspect of the apical plasma membrane of follicular cells. anything but optimal levels are linked to several thyroid diseases and disorders. The excess of oxygenated water (H2O2) determines the conversion of compound II to inactive compound III. Normal levels of H2O2 in the body vary from 0. Other enzymatic systems capable of generating oxygenated water have been evidenced: monoamine oxidase. which catalyzes the coupling reaction of iodotyrosines. Golgi complex. such as congenital hypothyroidism. 7 mM. Although various enzyme systems. which favors the organification of iodine with the formation of iodotyrosines: monoiodotyrosine (MIT) and diiodotyrosine (DIT). 001 mM to 0. an NADPH-dependent. The production of oxygenated water is stimulated by TSH-cAMP and phosphatidylinositolCa2+. Tyrosine residues also bind to thyroperoxidase. 71]. including cytochrome reductases. The activation of this NADPH oxidase requires Ca2+ ions. glucose oxidase [66]. Several selenoproteins act as a protective barrier for thyrocytes from endogenous H2O2 [72]. is absolutely indispensable for thyroperoxidase activity [67]. close to the apical membrane. The generation of oxygenated water. In the absence of iodine. compound I is formed. compound II. and the active iodine form results: the iodinium ion (I+) or the hypoiodite ion (IO). under the action of NADPH-oxidase. having extremely reactive free radicals as intermediate products. Various reports deal with thyroid disorders and H2O2.Oxidative Stress and Antioxidant Status in Hypo. Inactivation is prevented by iodine [66]. Iodination (organification) and the coupling reaction of iodotyrosines require the presence of thyroperoxidase (TPO). tumorigenesis. and is produced outside the thyroid follicular cell via the two-electron reduction of O2 [69]. but excess “induces oxidative stress. is released outside the thyroid follicular cell only after its transformation into oxygenated water [68]. as an electron acceptor. there are two theories: The superoxide anion is the primary product of the enzymatic conversion of oxygen which. myxedematous cretinism. The molecular mechanism of iodination consists of a series of successive stages. and consequent mutagenesis and apoptosis” [71]. nuclear envelope. resulting in the formation of thyroid hormones. can support H2O2 production in the thyroid. Other data suggest that oxygenated water is the primary product of the NADPH-oxidase system. thyroiditis. The mechanism of formation of oxygenated water (H2O2) is controversial.and Hyperthyroidism 201 membrane of the follicular cell. If DNA damage is . It is necessary to prevent excess or deficiency of H2O2. and cancer [70. a hemoprotein located in the apical plasma as well as in the adjacent cytoplasm. endoplasmic reticulum. The superoxide anion.

and are generally more easily reversed than are developmental inadequacies [77]. epidermis. Here. concomitantly with the increased concentration and the intensified activity of oxidative phosphorylation enzymes. Still. and removal is not uncommon. particularly of their cristae. This includes the diaphragm. more rapid in its reaction. any free iodotyrosine derivative left over is deiodinated quite rapidly due to excess iodotyrosine deiodinase. Coupling in this reaction is catalyzed by TPO. liver.202 Antioxidant Enzyme perpetuated. Hypothyroidism proved to have an effect on the rate and result of development. only T3 and T4 can be found in the thyroid vein’s blood supply [76]. tissue differentiation and reproduction. but mildly stimulated by low iodide levels [75]. Thus. First. ADP capture. The primary ligands of T3 are the nucleus and the mitochondrion. and may be the active form of excreted T4 that is deiodinized by the target cells [75]. yet these observations were described as quantitative rather than qualitative. gastric mucosa. altering the natural level by injection or thyroidectomy showed altered metabolism rates for several organs. heart. There are also effects of TH on development. Oxidative stress in experimental hyperthyroidism and hypothyroidism Thyroid hormones regulate several essential physiological processes such as energy metabolism. growth and formation of the central nervous system. respectively. it is not considered necessary to the survival of the organism. As an autoregulatory effect. Basal metabolism decreases in hypothyroidism and increases in hyperthyroidism. The molecular action of thyroid hormones is mediated via the thyroid hormone receptors which. they increase oxygen consumption and heat production. yields T3 and T4. After hormone synthesis. TH inhibits production of TSH and TRH. 5. Several genetic disorders have been shown to decrease H2O2 production by creating a partial iodide-organification defect and reducing or eliminating hormone production [74]. consequently. monoiodotyrosine (MIT) and diiodotyrosine (DIT) are released from the lumen into the follicular cell. Thyroid hormones control the intensity of basal metabolism. kidney. Also. This led to permanent congenital hypothyroidism in non-TH producing individuals. and recycled back into the thyroid. As stimulation by TSH permits. In fact. increased levels of H2O2 inhibit iodide uptake and organification [73]. and mild. Two general effects of TH are described. an increase in the number and size of mitochondria. activate genes by binding to the thyroid hormone response elements [79]. followed by coupling. it can lead to carcinogenesis. H2O2 production is diminished by high iodide concentration. ATP formation and oxygen consumption. They are calorigenic and. has been seen. an autoregulatory effect. suggesting the need for TH for energy metabolism. pancreas. thyroid hormones have . In the second case. transient hypothyroidism in low hormone level subjects. T3 is more potent than T4. after ligand binding. salivary gland. Oxidation of either MIT or another DIT. ferric TPO product (oxidized by H2O2) reacts with DIT to form a radical stabilized by the aromatic ring. although TH affects many of the body’s cells. and skeletal muscles. avoiding formation of other iodoamino acids. T3 and T4 have been found to stimulate in vitro protein synthesis in mitochondria.

through which metabolic energy required for nuclear transcription and posttranscription is released.Cluj-Napoca biobase. Superoxide formation is continuous in the respiratory chain. cytochrome b). representing the final step of oxygen transfer in the respiratory chain [67]. in a coordinated succession: binding to the cell membrane as a substrate. The aim of our study was to evaluate oxidative stress parameters. 00-20. The great majority of the reactive oxygen species (ROS) are generated at mitochondrial level. White. 00 h) and water ad libitum. approximately 1-2% of the electrons that participate in the chain form superoxide and its dismutation product – hydrogen peroxide [80].Oxidative Stress and Antioxidant Status in Hypo. group 2 – animals treated with L-thyroxine 10μg/animal/day for 30 days and group 3 – L-thyroxine treated rats . male. on the one hand. ubiquinones (Q). the possible correlation between impaired thyroid gland function and reactive oxygen species has been increasingly taken into consideration [9]. and to the incomplete reduction of the oxygen molecule (“trivalent” reduction occurs instead of “tetravalent” reduction). Data from in vivo and in vitro studies indicate that thyroid hormones have a considerable impact on oxidative stress [11]. via oxidative phosphorylation. significant amounts of hydrogen superoxide and peroxide radicals are formed. which allows for the formation of water. to mitochondria. They were divided into 5 groups of 10 animals each: group 1– controls. and mitochondria are a major source of intracellular free radicals [82. with an artificial lighting cycle (lights on 08. NADPH+H+-dehydrogenase. and certain cytochromes participate in the main oxidoreduction reactions of the respiratory chain [79]. NADH+H+-dehydrogenase. antioxidant status markers and their response to vitamin E supplementation in experimental hyperthyroidism and hypothyroidism. Thyroid hormones act on mitochondria by regulating energy metabolism. During thyroid hormone synthesis. flavoproteins (FMNH2/FADH2). Wistar rats weighing between 220 and 240 g were purchased from The Iuliu Hatieganu University of Medicine and Pharmacy.and Hyperthyroidism 203 primary actions in several cell organelles. as well as Na+ and K+ permeability. probably due to the auto-oxidable nature of the enzymatic system components (coenzyme Q. In mitochondrial respiration. on the other hand. which is absolutely indispensable for iodine intrafollicular oxidation in the presence of thyroid peroxidase. 15% to 40% of the basal energy used by the cell is used for the maintenance of an electrochemical gradient. non-porphyrin iron-sulfur proteins. Thyroid hormones increase the concentration and activity of Na+-K+ dependent ATP-ase. there is a constant production of oxygenated water.83]. and the specific synthesis of structures and functions is directed. determining in this way the intensification of energy consumption [81]. All animals were kept under the same environmental conditions. Mitochondria are particularly important for the action mechanism of thyroid hormones. In recent years. at a room temperature of 23±1ºC. Mitochondrial respiration is a complex metabolic process by which hydrogen from the reduced forms of dehydrogenases is oxidized to proton (H+) and molecular oxygen from air is reduced to anion. Thyroid hormones concomitantly stimulate the activity of cellular anabolic and catabolic enzymes.

Concentration values of MDA are expressed in nmol / ml based on specific calibration curves. The L-thyroxine and Propylthiouracil quantity dissolved in 2 ml of milk was administered by gavage in the morning on an empty stomach. One plasma volume was mixed with Tris (0. In the samples thus obtained the protein concentration was determined by measuring extinction at 280 nm. . 7 ml phosphate buffer pH 8 and 1 ml o-phthalaldehyde. The Ellman( DTNB)10mM reagent was added.5N. The carbonyl concentration was given by the formula C  Abs 355 x 45. the precipitate obtained by centrifugation was washed with a 1: 1 (v/v) mixture of ethyl acetate and absolute ethylic alcohol and dissolved in guanidine chlorhydrate 6M. Fluorescence was used to determine the glutathione (GSH) values [86]. Protein oxidation was determined through the estimation of carbonyl groups photometrically with dinitrophenylhydrazine according to the Reznick method[85] and expressed as nmol per mg of protein (nmol/mg protein). absorbance being read at 412 nm. according to the Hu method [86]. The lipid peroxides level was assessed by fluorescence according to the Conti and Moran method [84]. Thirty days into the experiment. and treated with 20% trichloracetic acid. Emission intensity was measured at 420 nm at an excitation of 350 nm.reduced glutathion (GSH) and superoxide dismutase (SOD) were determined from the serum. Serum samples were submitted to a reaction with 2. For the GSH dosage one plasma volume was mixed with TCA 10% and then centrifuged. carbonyl proteins.204 Antioxidant Enzyme protected with 10 mg/animal/day of vitamin E administered intramuscularly.4. SH groups.25M)EDTA20mM pH 8. Malondialdehyde (MDA). for 30 days. group 4 – animals treated with Propylthiouracil (5mg/100g animal /day). blood was taken from the retro orbital sinus and the rats were sacrificed by cervical dislocation following ether anaesthesia. Tissue homogenates were used for analytical procedures. the supernatant separated and additioned with 1. for 30 days and group 5 – Propylthiouracil treated rats protected with 10 mg/animal/day of vitamin E administered intramuscularly. the marker of lipid peroxidation. Plasma or tissue homogenates were boiled in 2-thiobarbituric acid solution 10mM in K2HPO4 75mM PH3 and extracted on n-butanol consecutively. and the absorption was determined again at the same wave length. Thyroid gland was immediately dissected out and placed into ice-cold isolation medium.dinitrophenylhydrazine 10 mM in HCL 2.45nmol / ml The thiol content of samples was determined with dithionitrobenzoic acid (DTNB). which produces a staining reaction. carbonyl proteins. for 30 days. SH groups and GSH were determined from the thyroid tissue homogenates. The results were expressed as nmol SH per milligram of protein (nmol/mg protein). the marker of lipid peroxidation and thiobarbituric acid. based on the reaction between malondialdehyde. while MDA.2 buffer. measured spectrophotometrically at 534nm.

the MDA levels did not differ significantly from euthyroid values (p>0. 31±0. 99±0. the levels of carbonyl proteins did not differ significantly from the control group. 33. 05) while in the thyroid tissue. Superoxide-dismutase (SOD) catalysed the superoxideradical (O2•‾) dismutation in peroxide (H2O2 ) and oxygen (O2 ). 05) of the Propylthiouracil treated rats. . FT4 values of the L-thyroxine and vitamin Eadministered group were significantly decreased in respect to those of the L-thyroxine only administered group. 70). We found that carbonyl proteins levels were significantly higher (0. as compared with the control group. The superoxide radical (O2•‾) reacts with C ferricytochrome. 05) in serum. p<0. 01) as compared with euthyroid values. We also investigated the relation between the mean values of FT4 and the mean values of MDA in the L-thyroxine-administered group. Serum free-thyroxine (FT4) concentrations were measured with an enzyme immunoassay kit (EIAgen Free T4 Kit. p<0. In serum and thyroid tissue of the hypothyroid rats.Oxidative Stress and Antioxidant Status in Hypo. 05).and Hyperthyroidism 205 Glutathione concentration was determined using a calibration curve made with known concentrations of glutathione processed in the same way. superoxide dismutase ( SOD) and reduced glutathione (GSH) were lower in the L -thyroxine-administered group in comparison to the control group (p<0. Dosage was performed on lysed erythrocytes at 250C. The results were expressed as micromoles per litre (μmol/l). and the thyroid tissue (1. Superoxide dismutase (SOD) activity of the samples was evaluated using the Flohe method [87] and expressed as U SOD per milligram of protein (U/mg protein). One unit of SOD activity is defined as the amount of enzyme able to inhibit the reduction rate of cytochrome C by 50%. which can be continuously monitored by recording the absorbance at 550 nm. A significantly high SH level and a significantly low GSH level were observed in the thyroids of the L-thyroxine-administered group in comparison to the control group (p<0. Superoxid-dismutase reduces the concentration of superoxide ions and thus inhibits the reduction of the C cytochrome and the SOD amount may be thus calculated from the degree of inhibition of the C cytochrome using a calibration curve achieved by the known SOD standards. the MDA levels did not differ significantly from euthyroid values (p>0. 5. 99±0. 001). 0001) in the serum of Thyroxin treated rats. Adaltis Italia). 001) values were observed in the Propylthiouracil administered group as compared with the control group. Significantly low FT4 (p<0. while in the thyroid homogenates. There was a significant positive correlation between hyperthyroidism and oxidative stress. Significantly higher FT4 (p<0. r2=0. 27. 001). In the hyperthyroid rats. (p>0. Thiol groups (SH). the MDA levels were significantly decreased (p<0. 001) values were observed in the L-thyroxine administered group as compared with the control group. 61. p=0. We found that carbonyl proteins levels were significantly higher (1.

95. exert a multitude of physiological effects affecting growth. such as lipids. which are mediated through nuclear thyroid hormone receptors [92]. proteins and nucleic acids [101]. However. SOD. it is conceivable that some of the biochemical changes favouring the establishment of the oxidative stress (increase in mitochondrial levels of electron carriers. On the other hand. over-production of free radicals and/or a decrease in antioxidant defence mechanisms [100]. i.99]. and nonenzymatic defense mechanisms. e. atrial arrhythmias. Thiol groups (SH). Lipid peroxidation is an . Indeed. Plasma membrane [89]. elevated circulating levels of thyroid hormones are associated with modifications in the whole organism (weight loss and increased metabolism and temperature) and in several body regions. such as ascorbic acid. superoxide dismutase (SOD) and reduced glutathione (GSH) levels in the hypothyroid group did not differ significantly from the control group.206 Antioxidant Enzyme Vitamin E supplementation increased significantly the carbonyl proteins levels as compared with the hypothyroid rats. Disturbances of the oxidant/antioxidant balance resulting from the increased production of ROS are causative factors in the oxidative damage of cellular structures and molecules. Among the mediators involved in the pathophysiology of hyperthyroidism and subsequent tissue injury in animal models. hydrogen peroxide and the hydroxyl radical are the major reactive oxygen species in our body. endoplasmic reticulum [90] and mitochondria [91] have been considered as potential cellular sites of action of thyroid hormone. GSH) levels as compared with the Propylthiouracil treated rats. it is now generally accepted that most of the actions of thyroid hormone results from influences on trascription of T3-responsive genes. free radical-mediated lipid peroxidation plays a pivotal role. Administration of Vitamin E to hypothyroid rats resulted in a significant decrease in serum antioxidant status parameters (SH. biological membranes rich in unsaturated fatty acids are cellular structures susceptible to free radical attack [102]. In particular. GPx and CAT.98. Free radicals are produced as a consequence of normal metabolism and their levels and activities are controlled by enzymatic defense mechanisms. NOS activity and the unsaturation degree of lipids) are due to stimulation of the expression of specific genes initiated through T3 binding to nuclear receptors. and it is conceivable that this also happens in other tissues in which T3-induced NO• overproduction has been shown [94. of which T3 is the major active form. heart failure. and GSH [97. tachycardia. low plasma lipid levels. Indeed. Vitamin E. Oxygen free radicals react with all biological substances. 96]. The superoxide anion. muscle weakness and wasting are commonly found in hyperthyroid animals. such as the SOD. It is worth noting that the idea that oxidative stress underlies dysfunctions produced by hyperthyroidism is not in contradiction with mediation of T3 action through nuclear events. Thyroid hormones. Thyroid hormone induces upregulation of NOS gene expression in rat hypothalamus [93]. development and metabolism of vertebrates [88]. Oxidative damage arises when an imbalance occurs in this system. so that they can be considered major regulators of their homeostasis.

[115] showed that the damaging effect of lipid peroxidation was increased in liver. Fernandez et al. [10] and Dumitriu et al. The up regulating of these hormones can result in a mitochondrial respiration perturbation and a consequent increase in ROS generation [107]. [10] Effects of thyroid hormones on lipid peroxidation have been subject of investigation in several laboratories but the results are rather contradictory. On the contrary. In some studies. it was demonstrated that the products of lipid peroxidation were decreased [111. impaired functions of the mitochondria and Golgi apparatus and inhibition of enzymes. It was reported that hypermetabolic condition in hyperthyroidism was associated with an increase in free radical formation and lipid peroxidation levels [10. [113] found high products of lipid peroxidation. Although reactive oxygen species play an important role in physiological mechanisms. These agents were reported to change the number and activity of the mitochondrial respiratory chain components. However. In previous studies. toxicity is found in biomembranes and lipid peroxidation occurs. If cellular mechanisms cannot scavenge these reactive oxygen species. Thyroid stimulating hormone (TSH) affect metabolism and may be affected by the thyroxine secretions. Similarly. while activating metabolic systems of the body in general.and Hyperthyroidism 207 autocatalytic mechanism leading to oxidative destruction of cellular membranes. extremely reactive oxygen radicals can cause severe oxidative damage to molecules [110]. 118. 5 years) but not from young (8 weeks) hyperthyroid rats was also reported [120]. Recently. there is a suppression of the metabolic rate and decline in ROS release [109]. Thyroid hormones cause oxidative stress as they increase ROS.Oxidative Stress and Antioxidant Status in Hypo. [114] found that lipid peroxidation was increased in hyperthyroid patients. in the case of hypothyroidism. heart and some skeletal muscles of rats. Iangalenko et al. 11. These ROS would lead to oxidative damage to biological macromolecules. increasing experimental and clinical studies have shown that free radicals play a key role in the etiology of many diseases. Triiodothyronine (T3) and thyroxin (T4) circulating hormones are involved in the modulation of the physiological mitochondrial respiration process [105]. Such destruction can lead to cell death and to the production of toxic and reactive aldehyde metabolites called free radicals [103]. Such effects were also found in heart homogenates [110. 119] from young rats. including lipids. Malondialdehyde (MDA) is an end-product of lipid peroxidation and is frequently measured as an index of these processes [104]. High concentrations of thyroid hormones stimulate free radical formation in mitochondria by affecting oxygen metabolism [18]. diminishing antioxidant enzymes in experimental hyperthyroidism.110]. Peroxidative effects elicited by thyroid hormones were found in the brain of newborn [116] and adult [117] rats. In contrast. including decreased membrane fluidity and function. Asayama et al. This damage is usually more evident in cellular membranes. Thyroid hormone treatment was found to increase lipid peroxidation in lymphoid organs such as mesenteric . 11. Lipid peroxidation is associated with a wide variety of toxic effects. proteins and DNA [108]. increased lipid peroxidation in hearts from old (1. there are conflicting results about oxidative stress in hyperthyroidism.112].

whose biological activity can differ from T3 in some tissues. recent studies have shown that T4. 123]. On the other hand. unchanged in heart [129] and decreased in liver [107] from hyperthyroid mice. 12] and cat [123]. and the thyroid hormone-induced increase in lipid peroxidation was found to be confined to some skeletal muscles. it is still to be determined whether the various target tissues of thyroid hormone undergo other biochemical changes that either predispose to free radical-mediated injury. a red muscle mainly composed of slow-twitch oxidative glycolitic fibres (type I).126]. levels of lipid peroxidation were found to be increased in hindlimb muscles [128]. but not white muscles. Although the pathophysiological consequences of the accelerated lipid peroxidation are not yet fully elucidated. a mixed fibre muscle also containing fast-twitch oxidative glycolytic fibers (type IIa). no change was found in the extensor digitorum longus (EDL) [11. Thus. This reaction occurs without release of any intermediate in the O2 reduction. such an increase was found in the soleus. instead of the next electron carrier in the chain. in which an electron is transferred to O2 directly. 125]. Despite some contradictory reports. However. However. The results concerning liver were attributed by the authors to the animal species or long-term (4-5 weeks) treatment they used. but was decreased in the white portion of such a muscle [12]. a white muscle mainly composed of fast-twitch glycolitic fibres (Type IIb). the aforementioned results provide strong evidence that thyroid hormones induce oxidative stress in target tissues. . it is interesting that in both mouse and rat hyperthyroidism was induced by T4. which can be turned into highly reactive hydroxyl radical (•OH). Conversely. or oppose it. Lipid peroxidation was also increased by thyroid hormone in rat gastrocnemious [110. are sensitive to thyroid hormones [124. Studies on the mouse showed lower susceptibility to thyroid hormone-induced lipid oxidative damage. This radical is then converted by spontaneous or catalysed dismutation into hydrogen peroxide (H2O2) [132]. Although this may be true. increases lipid peroxidation in rat interscapular brown adipose tissue [130]. although the tissue exhibits a calorigenic response to thyroid hormone similar to that elicited in liver [121]. because a laboratory study describing no increase in index of lipid peroxidation in hyperthyroid rat liver used the same long-term treatment [11]. despite the efficiency of the mitochondrial electron transport system. a thyroid hormoneunresponsive tissue [121]. In both rat [11. no significant change (TBARS) or decrease (HPs) were observed in lipid peroxidation level in the testis from adult hyperthyroid rats [122]. it is surprising that in kidney from hyperthyroid rats the lipid peroxidation level does not change [127]. Indeed. but not T3. this biochemical change is thought to be responsible for some complications of hyperthyroidism. These results are consistent with early observations that red. Indeed. Oxidative stress results from a disturbance of the normal cell balance between production of ROS and the capacity to neutralize their action. In aerobic cells O2 is mainly consumed through its four-electron reduction to water by cytochrome c oxidase. generating O2• [131]. without major effects in the spleen [12]. the nature of the alternating one-electron oxidation-reduction reactions it catalyses predisposes electron carriers to side reactions.208 Antioxidant Enzyme lymph nodes and thymus.

[10] : increased liver content in lipid peroxides induced by thyroid hormones. can induce cell proliferation in fibroblast cultures from patients with severe ophthalmopathy. [120] have investigated how thyroid function might influence the production of oxygen free radicals. propylthiouracil (synthesis antithyroid drugs). heart and skeletal muscle in experimental hyperthyroidism. R. generated by the xanthine oxidase/hypoxanthine system. but not in liver tissue. For this. The authors found that the superoxide radical determined fibroblast proliferation. thyroid. the intensity of this phenomenon depending on the concentration of reactive oxygen species. Shinohara et al. and nicotinamide (an antioxidant). Tapia et al. retroocular tissue was incubated with methimazole. endoplasmic reticulum and outer mitochondrial membrane also contribute to O2 consumption and lead to O2• and H2O2 generation [133]. Retroocular fibroblast proliferation is involved in the pathogenesis of ophthalmopathy in Basedow-Graves disease. These changes in the prooxidant/antioxidant balance. The conclusion was that hyperthyroidism increase Kupffer cells activity and the production of oxygen free radicals at this level. [135] studied the influence of thyroid hormones on Kupffer cells activity in isolated liver. Thyroid state-linked changes in the balance between ROS production and .Oxidative Stress and Antioxidant Status in Hypo. Mitochondria are particularly susceptible to ROS-induced damage because they are a major site of oxygen free radical production [138] and contain great amounts of high and low molecular weight Fe2+ complexes.140]. [134] revealed an increase of the lipid peroxides content and carbonyl proteins level in blood. Also the antioxidant enzyme activity changed: increased the xanthine oxidase and superoxiddismutase and decreased the glutathione peroxidase. Conflicting results obtained Fernandez et al. Major complications of hyperthyroidism are the myopathy and cardiothyreosis [81]. caused by thyroid hormones excess could be involved in myocardial dysfunction. liver. Zaiton et al. [136] studied the way in which the superoxide radical. allopurinol (a xanthine oxidase inhibitor). assessed by measuring substances that react with thiobarbituric acid. allopurinol and nicotinamide. which promote the oxidative damage of membrane lipids [139. The effectiveness of some pharmacological agents on retroocular fibroblast proliferation induced by the superoxide radical was also monitored. Burch et al. the lipid peroxidation process and antioxidant activity in muscle of rat myocardium. were methimazole. These results suggest the implication of reactive oxygen species in retroocular fibroblast proliferation in Basedow-Graves disease [137]. It was found that the degree of lipid peroxidation. The most effective regarding the inhibition of superoxide radical production and implicitly. Joanta et al. that of fibroblast proliferation. Therefore liver macrophages could be an alternative source of reactive species.and Hyperthyroidism 209 Numerous oxidases in the cytosol. suggesting that thyroid hyperfunction is accompanied by oxidative stress. significantly increases in animals with hyperthyroidism than euthyroidiene. as well as from control patients. perfused with colloidal carbon solution. H. in whom the excision of retroorbital tissue was performed. [123] revealed increased concentration of lipid peroxidation products in the myocardium and solear muscle in rat.

However our results are not in concordance with the findings of Seven et al. arginine. leading to an increase in electrons transfer from the respiratory chain through the acceleration of the cellular metabolism rate. injections for 10 days caused significantly increased MDA levels in liver. The latter can indirectly reflect the status of the metabolism of free radicals. including hydroxyl radicals (OH•). heart and skeletal muscle homogenates. which are undoubtedly related to any alteration in the thyroid function. the iodothyronine used and treatment duration. Superoxide radicals can lead to the formation of many other reactive species. resulting in the increased generation of superoxide (O2•−) at the site of ubiquinone [7]. and returns results which differ according to the assay conditions used [19].210 Antioxidant Enzyme antioxidant capacity should result in changes in the damage to mitochondrial components. Protein oxidation can lead to a loss of critical thiol groups (SH) in addition to modifications of amino acids leading to the formation of carbonyl and other oxidized moieties[143. [141] who found a significant increase in MDA levels in the plasma of rats rendered hyperthyroid by administration of T4 in their food for 24 days and Venditti et al. and via a variety of mechanisms. [11] who found no change of MDA in liver homogenates from hyperthyroidism induced rats rendered hyperthyroid by administration of T4 to their drinking water over a 4-week period. such as tissue. It is well known that MDA is a terminal product of lipid peroxidation. In our study in the plasma of L-thyroxine-treated rats. mitochondrial respiratory chain activity is altered. On the other hand. direct oxidation of lysine. For example the method for evaluating thiobarbituric acid reactive substances (TBARS) is inaccurate.144. So the content of MDA can be used to estimate the extent of lipid peroxidation. we investigated the effects of altered thyroid states on the extent of oxidative damage of mitochondrial lipids and proteins. the provoked hyperthyroidism. [110] who noticed that hyperthyroidism induced in rats by T3 daily i. the marker of lipid peroxidation (MDA) levels did not differ significantly from the euthyroid values. the degree to which the tissue cells are attacked by free radicals and the degree to which lipid is peroxidated. [2] and [28]. Oxidative cleavage of proteins by either the alpha-amidation pathway or by oxidation of glutamyl side chains leads to formation of a peptide in which the N-terminal amino acid is blocked by an alpha-ketoacyl derivative. These discrepancies among results seem to reflect a dependence of peroxidative processes on various factors. Proteins are also sensitive to oxidative damage which leads to alteration in their structure and ability to function [142]. it is not possible to exclude the fact that some conflicting results depend on the different accuracies of the methods used for lipid peroxidation determination. The high increase in the level of MDA and hydroperoxides in hyperthyroidism might be due to the possible changes in the cellular respiration of target tissues. which can readily start the free-radical process of lipid peroxidation [3] and [6]. This result of unchanged lipid peroxidation level can be correlated with the observations of Asayama et al.145]. . knowing the major role of the thyroid hormones in the control (acceleration) of the mitochondrial respiration rate [108]. However. Therefore. From a biochemical point of view. species. p.

nonenal. the increased levels of protein-bound carbonyls in serum of L-thyroxinetreated rats is in agreement with the earlier reports [150.and Hyperthyroidism 211 proline and threonine residues may also yield carbonyl derivatives. Antioxidant status Substances that neutralize the potential ill effects of free radicals are generally grouped in the so-called antioxidant defence system. such as GPx. Enhanced myocardial protein oxidation was also shown in the study of [148] by means of carbonyl group measurement.hydroxi-2. Such an effect may be related to the enhanced metabolic rate generated by thyroid hormone administration. deoxyosones) generated as a consequence of the reaction of reducing sugars or their oxidation products with lysine residues of proteins (glycation and glycoxidation reactions). superoxide dismutases. and peroxiredoxins that contribute to limit cellular injuries when H2O2 or other ROS are produced in excess [155. malondialdehyde) produced during lipid peroxidation or with reactive carbonyl derivatives( ketoamine. In our study.151] suggesting the role of free radicals in the pathogenesis.157]. The presence of carbonyl groups in proteins has therefore been used as a marker of ROS-mediated protein oxidation [134].141]. The synthesis of thyroid hormones crucially depends on H2O2. It is therefore possible that decreased oxidative stress observed in thyrocytes. In addition. Such a system includes both low molecular weight . H2O2 synthesis must always remain in adequation with the hormonal synthesis and strictly contained at the apical pole of the cell. leading to an accelerated ROS production [153. the MDA values were significantly decreased and carbonyl proteins levels did not show significant changes. which works as a donor of oxidative equivalents for thyroperoxidase [154]. 6.147]. which demand the need for studies assessing the therapeutic role of antioxidants in hyperthyroidism.Oxidative Stress and Antioxidant Status in Hypo. ketoaldehydes. In the thyroid homogenates of the L-thyroxine administered rats. There are not many data regarding the effect of the thyroid state on protein oxidation. carbonyl groups may be introduced into proteins by reactions with aldehydes (4.156. A recent study[152] found a positive association between thyroid hormones in excess and lipid peroxides correlated by linear regression which clearly suggest induction of oxidative stress. Because of its great toxicity. Thyrocytes possess various enzymatic systems. In experimental hyperthyroidism increased protein oxidation was demonstrated in different tissues [146. Our findings may be explained by the fact that the external administration of thyroid hormones usually inhibits pituitary secretion of TSH and indirectly hormonal synthesis [158]. These results show that hyperthyroid state is not accompanied by oxidative stress in the thyroid gland and contradict the results of [134] who observed an increase in lipid peroxides and carbonyl proteins in the same tissue in experimental hyperthyroidism. is due in part to the absence of H2O2.147]. catalase. An elevation of this protein oxidation marker was demonstrated in the plasma of hyperthyroid patients [149.

During catalysis the oxidation state of the . They are of enormous importance in limiting ROS-mediated damages to biological macromolecules. these substances are strategically compartmentalized in subcellular organelles within the cell and act in concert. Collectively. the major portion of this regulator is in its reduced form and is distributed in nucleus. which is cleaved by a second GSH molecule to yield the reduced GPx. This second line of defense against ROS is provided by enzymes such as GPx. while a few data concerning other species are available [159]. peroxiredoxin and glutathione peroxidase (GPx) [160. better referred to as glutathione disulfide. Thus. terminating chain reactions.166. it needs to be underscored that although thyroid hormone can directly control levels of enzymes with antioxidant activity or regulate scavenger content. This cysteine-containing tripeptide exists either in reduced (GSH) or oxidized (GSSG) form.212 Antioxidant Enzyme free-radical scavengers and a complex enzyme array involved in scavenging free radicals. GSH may be covalently bound to proteins through a process called glutathionylation and acts as a coenzyme of numerous enzymes involved in cell defense [170]. selenium is oxidized by the hydroperoxide to a selenic acid derivative. the central role of reduced GSH appears clear in intracellular endogenous antioxidant defenses as it is involved in all the lines of protection against ROS [35]. endoplasmic reticulum and mitochondria. ubiquitously present in all cell types at millimolar concentration [168]. There are several tissue-specific GPx's that exhibit also tissuespecific functions [171]. Several antioxidant enzymes exist that convert ROS into less noxious compounds. Glutathione peroxidases constitute a family of enzymes. All of them are selenoproteins and their primary function is to counteract oxidative attack. a seleno-disulfide is formed. thioredoxin reductase. Under normal cellular redox conditions. This intermediate is subsequently reduced by the electron donor. for example.167]. The tripeptide γ-glutamylcysteinylglycine or GSH is the major nonenzymatic regulator of intracellular redox homeostasis. aldo-keto reductase and aldheyde dehydrogenase [165. During the catalytic cycle. In addition. and participates in redox reactions by the reversible oxidation of its active thiol [169]. To provide maximum protection. but the consequence of the oxidative stress. which are capable of reducing a variety of organic and inorganic hydroperoxides to the corresponding hydroxy compounds. superoxide dismutase (SOD). In examining antioxidant changes found in hyperthyroid tissues. and removing or repairing damaged cell constituents. utilizing GSH and/or other reducing equivalents. It is then mandatory to detoxify these secondary products in order to prevent further intracellular damage. When GSH is used.164]. catalase.162.163. lipid hydroperoxides and electrophilic compounds. glutathione S-transferase (GST). but they are not able to be 100% effective because certain compounds generated by the interaction of ROS with macromolecules are highly reactive. degradation of cell components and eventual cell death. Glutathione can thus directly scavenge free radicals or act as a substrate for GPx and GST during the detoxification of hydrogen peroxide. The effects of thyroid hormone on antioxidant status have been extensively investigated in rat tissues. these enzymes provide a first line of defense against superoxide and hydrogen peroxides.161. antioxidant depletion could not be the cause.

which can be opposed by a de novo synthesis or by reducing the formed GSSG. the presence of GSH is essential. or to the release of GSSG excess by the cell. programmed cell death. environmental pollutants and antitumor agents. to prevent the cytotoxicity of ROS. such as chemical carcinogens. . This has two important consequences: (1) the thiol redox status of the cell will shift and activate certain oxidant response transcriptional elements. In general. The recycling pathway for GSH formation is thus fundamental in the metabolism of GSH-dependent defense reactions [175]. cytokine production. epoxides and hydroperoxides. It has been observed that exposure to agents that lead to increased oxidative stress also leads to an increase in its mRNA content. but not in itself sufficient. Further experimental data have shown the importance of GRed activity in GSH metabolism. Glutathione Stransferases exert those protective effects because they are able to catalyze the conjugation of GSH with oxidation end products and represent a second line of defense against the highly toxic spectrum of substances produced by ROS-mediated reaction. increasing the cellular requirement for de novo GSH synthesis. Mixed disulfide formation together with GSSG or GS-conjugated efflux can result in the depletion of cellular GSH. Glutathione reductase is a flavoenzyme and is represented by a single-copy gene in humans. to maintain the intracellular GSH/GSSG ratio. and so on [172]. proliferation. During the course of the reaction catalyzed by GPx. they protect against reactive compounds produced in vivo during oxidative stress by inactivating endogenous unsaturated aldehydes. or consumption of overcooked or mycotoxin-contaminated food. being of fundamental importance the functionality of the glutathione-dependent enzymes. Glutathione disulfide can also be reduced back to GSH by the action of glutathione reductase (GRed) utilizing NADPH as a reductant [174]. During the GST-mediated reactions. GSH is conjugated with various electrophiles and the GSH adducts are actively secreted by the cell. or polluted water [173]. demonstrating that the enzymatic activity is regulated in response to stress. quinones. GSH concentration rapidly decreases while GSSG — potentially highly cytotoxic — increases because of the reduction of peroxides or as a result of free radical scavenging. Both GPx and GST activities can eventually lower the level of total intracellular GSH. these isoenzymes may have a role in the regulation of the delicate regional redox balance. In the presence of oxidative stress. The phospholipid hydroperoxide GPx — discovered as a factor preventing lipid peroxidation — is considered to be involved in the protection of biomembranes against oxidative stress. Moreover. in particular the regulation of the appropriate tone of hydroperoxides known to be involved in cellular signaling. and that mutations affecting GRed activity would have deleterious consequences. which participate in the first and second lines of defense. all of which are produced intracellularly after the exposure to pollutants.and Hyperthyroidism 213 enzyme depends on the relative concentration of the reducing (GSH) and oxidized (hydroperoxides) substrates. for example. mitochondrial and microsomal — that detoxify noxious electrophilic xenobiotics.Oxidative Stress and Antioxidant Status in Hypo. Glutathione S-transferases are three enzyme families — cytosolic. and to evoke several cellular responses. the exaggerated production of GSSG can lead to the formation of mixed disulfides in cellular proteins. In conclusion. and (2) GSSG may be preferentially secreted from the cell and degraded extracellularly.

Therefore. and (ii) to provide H2O2 for hormone synthesis [190]. One study correlates several thyroid disorders to levels of CuZn-SOD and Mn-SOD. One is Cu-Zn SOD. It has been demonstrated that hyperthyroidism leads to accelerated free radical formation [183]. several authors reported that SOD activity were reduced in patients with hyperthyroidism [180. In hyperthyroidism caused by thyroxine or triiodothyronine administration. which are very high in malignant tumors [189]. mainly found in the cytoplasm of cells. The observed diminution of SOD activity in rats. these diseases. increased free radical formation enhances intracellular scavenging enzymes. This is a natural occurrence in the body to prevent and eliminate excess ROS that might result from. CuZn-SOD) present in the thyroid are the first line of defense in neutralizing ROS [188]. [187] found decreased SOD activity in the blood samples of patients with hyperthyroidism.181]. Erdamar et al. Such a discrepancy between our and their results may be due to different experimental conditions and different methods used to assay SOD activities. or have caused. following L-thyroxine treatment can be correlated with the observations of [184]. in experimentally induced hyperthyroidism [141].179. Effects of thyroid hormones on SOD activity have been evaluated by others.179] and remain unchanged [118. There are two types of SOD enzymes reported in higher vertebrates. The increase of SOD has been shown in the blood of patients with hyperthyroidism [6]. our results are not in good agreement with the findings of [141] and [185]. Varying forms of SOD (Mn-SOD.120.192]. There is no difference in SOD activity between hyperthyroid patients and controls or between hypothyroid patients and controls in the studies of both [6] and [186].193]. On the contrary. Superoxide dismutase is an important intracellular oxygen radical-scavenging enzyme. Conversely. However.214 Antioxidant Enzyme Thyroid hormones increase oxygen consumption via a thermogenetic effect. Mn–SOD activity in cardiac tissue was reported to both increase [11. Regarding the way in which thyroid gland hyperfunction influences antioxidant defense capacity. but we observed a decreased SOD activity in our study. who noticed that hyperthyroidism induced in rats by T3 caused an elevation of SOD activity in liver. There are conflicting results about an increase or decrease in the activities of antioxidant enzymes in hyperthyroidism [12. On the contrary. 176-182]. SOD in the thyroid may involve two roles: (i) to serve as an antioxidant enzyme to protect the thyroid from oxidative stress. The organism can defend itself against the effects of oxidative stress by increasing SOD activity as a protection mechanism. 16. it has been reported that SOD activity was significantly increased [12. In some studies. while the other one is mitochondrial in nature and is known as Mn-SOD[191. the results are different from one study to another. the increase in metabolic rate together with the increase in oxygen consumption enhances microsomal oxidative capacity and free radical formation. like SOD. Mn–SOD was also found to increase in the soleus and white portion of gastrocnemious muscle from rats made . even though in all cases hyperthyroidism was elicited by long-term treatment with T4. but results are rather contradictory.182].

characterised by an overproduction of superoxide anion. it was found that T3 treatment increased GPX activity in gastrocnemious [110]. in turn. which increases CAT activity until the dismutation of hydrogen peroxide [196]. whereas lack of change [120. but both increased [16] and remain unchanged [110. while T4 and T3+T4 treatments decreased such activity in gastrocnemious [194] and in its white portion [12]. has been postulated that hyperthyroidism might be accompanied by the induction of either SOD or GPx or both [140].and Hyperthyroidism 215 hyperthyroid by combined T3 and T4 administration [12] and in soleus[11] and gastrocnemious [194] from T4.156. 179] and decrease [11] were found in heart. . The SOD is also known for its role in transforming O2•− into inorganic hydroperoxide (H2O2). T4 administration also decreased GPX activity in both thyroid hormone responsive (soleus) [11] and unresponsive (EDL) [11] muscles. Enzyme activity was found increased in brain from hyperthyroid newborn rats [116]. Decreases in CAT activities were found in brown adipose tissue after T3 or T4 treatment [130] and in liver [11. which will. 156] and old [120] rats.199] after T3 treatment. despite the same prolonged treatment with T4. The latter is known for its harmfulness to the cell membrane. whereas it was reported to both decrease [179] and remain unchanged [11. Indeed. The relationship between hyperthyroidism and glutathione peroxidase (GPX) activity also appears not well defined. it was reported that cardiac activity decreased after longterm T4 treatment of both young [11.120] in cardiac muscle. GPx may be inactivated by the superoxide radical excess. whereby the excess of hydrogen peroxide may serve as a factor of SOD inactivation. Liver GPX activity was found to decrease after T4 treatment [11]. Moreover.16]. One enzyme activity leads to the formation of a substrate for another one.treated rats. Cu-Zn SOD activity increased in gastrocnemious [194] and in its white portion [12]. be reduced by both CAT and GPx enzymes [108]. in agreement with insensitivity of such muscle to thyroid hormone. GPx is protected from its inactivation via superoxide radical just by the enhanced SOD activity [198]. This accelerates the speed of the formation of superoxides and the renewal of H2O2 quantity (substrate of CAT). and their activities are adjusted by their variation in the thyroid gland's activity. Both SOD and CAT function together in a way linked to the dissociation and formation of H2O2. For catalase (CAT) activity an increase in the white portion of gastrocnemious [12] and both decrease [11] and increase [12] were found in soleus from hyperthyroid rats. and remained unchanged after short-term T3 treatment of young rats [110].Oxidative Stress and Antioxidant Status in Hypo. [197] and [147]. increased [118] and remained unchanged [120] after long-term T4 treatment of young rats. an increase in CAT activity in the homogenates of hyperthyroid rats is noted. Accordingly.157] but not from old [120] hyperthyroid rats. On the other hand. Based on such a sequence of events. Total SOD was found to decrease in liver [180] and increase in heart from young [120. respectively. [120] and [195]. The increase in SOD activity in hyperthyroidism indicates the presence of oxidative stress due to the increasing mitochondrial oxidation rate.

Another explanation could be that at cellular level. deiodination is given preference over GPx in selenium supply. Other selenoproteins such as selenoprotein P mediate the transfer of selenium between the two enzymes. In contrast. It is interesting that in brain of newborn hyperthyroid rats the activities of antioxidant enzymes (Cu. According to hypothesis proposed by Seven et al. The decrease in GPx activity could in part be ascriebed to the fact that it is a selenoenzymelike D1(5′-deiodinase I). there are other antioxidant systems [201]. the dose and the duration of treatment are also of a major influence on antioxidant enzymes. giving rise to hydrogen peroxide (H2O2) concentrations [108]. Since the body stores of selenium are limited. Morini et al. deiodination of T4 is also increased. The physiological state of the thyroid gland. An explanation could be related to the amount of thyroid hormones administered to the animals. These differences have multiple causes. thyroid. 0012% in the drinking water.-SOD.correction . Köhrle described GPx as a sort of selenium store easily available for D1 activity [202]. the above-mentioned effects might involve an accumulation of superoxide anion that inhibits CAT activity. It was reported in previous studies that the level of lipid peroxidation in the heart was affected by both the age and the state of the thyroid gland.199] and heart [110]. Joanta [200] evidenced an increase in the concentrations of total peroxidase and catalase in the liver. From another point of view. selenium deficit might be the cause of reduced GPx activity [203]. in hyperthyroid rats [120]. where an icrease in GPx activity in hyperthyroid rats was observed. CAT and GPX) exhibited compensatory increase that did not prevent oxidative stress [116]. [11] found a low glutathione peroxidase concentration in the liver tissue taken from rats with experimental hyperthyroidism. it can be suspected that these cells contain high reserves of enzymatic protein levels. In 1994. increased ROS production may lead to elevated GPx activity [205]. [110] 10 μg T3/100 g body weight/day to the rats previously treated with methimazole. but is also in agreement with that of [16]and[6]. Asayama et al.110. Function of intracellular GPx is degradation of H2O2 and hydroperoxides of free fatty acids. brain and blood. and on the other hand. This does not only confirm the main role of the thyroid hormones in regulating the oxidative stress in target cells. whose activity has not been evaluated by the mentioned investigations.which is involved in T4 transformation into active T3. whereas in plasma GPx catalyses degradation of H2O2 and hydroperoxides of phospholipids. a decrease in the activity of these enzymes in the myocardium and skeletal muscle.216 Antioxidant Enzyme The changes induced by T3 treatment in both liver [16. The difference in GPx enzyme activity was probably due to the age (eight weeks) of the rats used in the investigation of [6]. therefore on one hand it is possible to activate antioxidant enzymes in response to ROS activity. In addition GPx exert a protective effect on membrane phospholipids by inhibiting their peroxidation processes [204]. [16] 30 μg T3/100 g body weight/day and Venditti et al. but not in muscle glutathione reductase (GR) activities shown in the various laboratories were consistent with those found for GPX activities. Thus. [11] administered thyroxine in a dose of 0. Asayama et al. As the enhanced hormone production is very pronounced in hyperthyroidism. Zn. Because of the fact that proteins are not synthesized de novo in erythrocytes.

GSH is a nucleophilic “scavenger” of numerous compounds and their metabolites. This not only confirms the main role of the thyroid hormones in regulating the oxidative stress in target cells. Glutathione is a tripeptide. we noted important reduction in GSH levels in hyperthyroid rats. grade of hyperthyroidism. the increase of some antioxidant enzymes activities such as SOD. Thus.212]. in the plasma of hyperthyroidism-induced rats in comparison to the control group (p<0. superoxide dismutase (SOD) and glutathione (GSH) were significantly decreased in the present study.210]. Significantly high levels of the SH groups (p=0. which protects the cell membrane against oxidative damage by regulating the redox status of protein in the cell membrane [215.and Hyperthyroidism 217 of losses caused by oxidative stress. SOD catalyzes the conversion of superoxide anion radical to H2O2. methods of determination and result expression (enzyme activity or concentration. and CAT may be an indication of the failure of compensating the induced oxidative stress. reflecting reduced oxidative stress and low antioxidant capacity. detoxification of xenobiotics. γ-L-glutamyl-L-cysteinyl-glycine. and is found in all mammalian tissues and it is especially concentrated highly in the liver [214]. it has been suggested by[140] that free-radical scavenging enzyme activity can be induced by excessive formation of ROS in experimental hyperthyroidism was previously reported. Results of the studies analyzing the indicators of SOD. We suggest that the mentioned alterations are given of functional changes induced by radical over-production and an increase in the biosynthesis of antioxidant enzymes. by Western blot in a recent paper [213] where in T4 treated rats there was a decrease in the level of oxidative stress and in the level of GPx. a major hepatic alteration induced by hyperthyroidism . These enzymes may scavenge excess O2‾ and H2O2. The resulting hydrogen peroxide in turn is decomposed by the enzymes GPx and CAT [209. and peroxides ROOH produced by free radicals. 0001) were found in thyroid homogenates of the L-Thyroxin treated group as compared with the control group. For example. GPx and catalase enzymes in thyroid tissue are quite contradictory [11. but also is in agreement with previous data. In this study. GSH depletion. and a cofactor in the GPxmediated destruction of hydroperoxides. which are the main antioxidants in the body may be indicative of the failure of compensating the induced oxidative stress [207.208]. 0006) and low levels of GSH (p=0.12.Oxidative Stress and Antioxidant Status in Hypo. It is widely distributed and involved in many biological activities including neutralisation of ROS. namely thiol groups (SH). Antioxidant status parameters. Also. which reflects its consumption through the oxidative stress. The increase of some antioxidant enzymes activities such as SOD. expression of enzyme concentration or activity per protein or tissue mass). GPx. The discrepancy may be due to variation in the samples analyzed. Reduction of antioxidant potential of red blood cells occurring in thyrotoxicosis is explained by more rapid degradation of enzymatic proteins [206]. 211.216]. 001). GPx and CAT. Similar results were described at the level of expression. and maintenance of –SH levels in proteins [108].

including: (i) an increased oxidation rate. extremely reactive. High levels of GSH in the erythrocytes of hyperthyroid rats are open to various interpretations. and (iii) decreased glucose-6-phosphate dehydrogenase activity. These differences in antioxidant enzyme activity may be caused by various mechanisms. which causes diminished production of GSH. lowered blood GSH levels may also be explained by some other possibilities. which requires the presence of oxygenated water (H2O2) as . the decreased level of GSH may be due to the overproduction of free radicals and increased lipid peroxidation in hyperthyroidism [115]. There are a number of factors that may influence antioxidant system activity: the physiological state of the thyroid gland. It has been reported that GSH plays an important role in the detoxification of hydroperoxides and prevents the effect of lipid peroxidation [220]. In contrast with our results. Iodine is oxidized by an enzymatic complex termed thyroperoxidase (TPO). The great majority of the energy released under basal conditions is used by the cell for the maintenance of the Na+-K+ dependent ATP-ase activity. the dose and the duration of treatment. which will bind to tyrosine residues from the structure of thyroglobulin. Therefore. In experimental studies. is transported in increased amounts from the liver to blood to meet the needs of increased peripheral T4. On the basis of the suggestion by Morini et al.[16] and [221] have demonstrated increased levels of GSH in blood from hyperthyroid rats. However. inorganic iodine. is determined by both loss of tripepetide into the blood and higher intracellular catabolism. At the level of the thyroid follicular cell. Thyroid hormones enhance the function of this pump by intensifying its activity at cellular level. Seven et al [141] suppose a change in GSH concentration due to altered transport hyperthyroid state. According to Visser [222]. introduced in the body through diet. antioxidant enzyme activity was affected by the age of the animals with induced hyperthyroidism[120].218 Antioxidant Enzyme in experimental animals [199] and [180] and man [217]. is oxidized to the iodinium ion (I+). GSH. Activities of oxygen radical scavenging enzymes are expected to increase in response to sustained oxidative stress such as that in hyperthyroidism [115]. Insufficiency of GSH is one of the primary factors that permits lipid peroxidation. The GSH-dependent defence system plays an important role against lipid peroxidation in cells. A decreased activity of antioxidant enzymes or a decreased non-enzymatic antioxidant concentration may be caused by their intensified utilization in protection against oxidative tissue damage [181. [16] that thyroid hormones alter the membrane fluidity. a required endogenous cofactor in the conversion of T4 to T3. despite the enhancement in the rate of GSH synthesis and in the GSH turnover rate triggered in the liver [199. 223].T3 conversion. This increased use of ATP associated with the intensification of oxygen consumption by the oxidative phosphorylation pathway generates reactive oxygen species [224]. [140]. (ii) increased utilization of GSH during the removal of lipid and other peroxides.218]. The reactive oxygen species contribute to an intensified synthesis of antioxidant enzymes in tissues and hence their elevated activity may be a manifestation of adaptation mechanisms in response to oxidative stress. Enhanced production of free radicals and the increase of antioxidant enzymes activities have been suggested as possible mechanisms to explain hyperthyroid-induced oxidative damage [219].

heart and . Our results show a general lack of significant changes in levels of lipid peroxidation (MDA) in serum and thyroid tissue of hypothyroid rats. hypothyroidism is a disease because of a diminished thyroid hormone synthesis. This is in line with the results of Venditti et al. Further on. the malondialdehyde (MDA) levels did not differ significantly from euthyroid values. [15] found that the concentration of lipid peroxides. The results of Yilmaz et al. did not change in hypothyroid rats when compared with the euthyroid animals. such as NADPH-cytochrome P-450 reductase. These different results were explained in terms of tissue variation in haemoprotein content and/or of antioxidant capacity by Venditti et al. [229] observed increased lipid peroxidation in plasma. liver. the process of oxidative condensation of iodotyrosines also involves thyroperoxidase (TPO) and oxygenated water (H2O2). Dumitriu et al. Recent studies have shown an increased production of reactive oxygen species in hypothyroidism. TSH stimulates the organification of iodine by the increase in the production of oxygenated water (H2O2). Sarandol et al. Under normal conditions. On the other hand. The depression of basal metabolism is associated with decreased mitochondrial oxygen consumption and less ROS generation. Dariyerli et al.and Hyperthyroidism 219 an oxidizing agent. Mano et al. accompanied by a fall in peroxide levels [112]. it is supposed that NADPH-dependent cytochrome c reductase is involved in the intrafollicular generation of oxygenated water (H2O2) [225]. the higher the production of oxygenated water (H2O2) in the thyroid follicle [9]. [12] suggested that hypothyroidism tended to diminish lipid peroxidation in lymphoid organs. determined indirectly by the measurement of thiobarbituric acid reactants.Oxidative Stress and Antioxidant Status in Hypo. Although the exact mechanism of the generation of oxygenated water (H2O2) is uncertain. The activity of some hepatic enzymes. the excess of thyroid hormones followed by the intensification of the cytochrome P-450 reductase activity is responsible for the increased production of superoxide and hydroperoxide anion at hepatic level [108]. It has been also reported that antioxidant enzyme levels are decreased in hypothyroid stage. Physiologic alterations generally occur because of the hypometabolic state induced by hypothyroidism [226]. While Pereira et al. The higher the synthesis of thyroid hormones. resulting from thyroid gland dysfunction. is regulated by thyroid hormones. and reduction of free-radical formation. liver and muscle MDA levels in hypothyroid rats contradict our findings. [228] who reported increased plasma. [110]. [113] observed the high levels of blood lipid peroxidation in hypothyroidism. [227] showed that there is no statistically significant difference found between hypothyroid and control groups in the lipid peroxidation indicator MDA. Hypothyroidism is known to induce metabolic suppression and lower respiration rate. In hyperthyroidism. resulting in decreased lipid peroxidation and protein oxidation [210]. [110] who showed that in all tissues of hypothyroid rats. So. There is disagreement on the effect of hypothyroidism on tissue oxidative stress. TSH anti-receptor antibodies induce a sustained and continuous secretion of thyroid hormones.

On the other hand. In fact. [234] and[235] who observed no difference in SOD levels between hypothyroid rats and controls. This antioxidant molecule is one of the main parts of the cellular endogenous antioxidant systems. The decrease in its ratio and the restoration to its normal value by T3 administration confirms the critical role of thyroid hormone in regulating mitochondrial oxidative stress [13]. This is in line with the results of Messarah et al. Das et al. Venditti et al. Although most of the studies did not suggest it.220 Antioxidant Enzyme muscle of Propylthiouracil treated rats reflecting an enhanced oxidative status in hypothyroidism. [230] who found significantly higher carbonyl proteins levels in plasma of hypothyroid patients compared to their respective controls. This is in agreement with Nanda et al. The mechanism of increased oxidative stress in hypothyroidism is controversial. . superoxide dismutase ( SOD) and reduced glutathione (GSH) levels did not differ significantly in serum. In our study we found that carbonyl proteins levels were significantly increased in serum. The increase in GSH content in liver under the hypothyroid state may be an adaptive response to protect the mitochondria from the elevated level of H2O2. [229] who didn’t observed any significant changes in GSH levels in the liver and kidney tissues of hypothyroid rats agree with our findings. GSH: GSSG in tissue is now considered one of the important markers of oxidative stress. but we did not observe any alteration in the serum and thyroid tissue of the hypothyroid rats. the level of the increase in protein-SH groups in the hypothyroid state corroborates the above statement. ion homeostasis and mitochondrial redox state activity of numerous. suggesting the presence of oxidative stress in hypothyroidism. GSH is reported to be involved in numerous mitochondrial functions including mitochondrial membrane structure and integrity. In contrast with our results. [210] reported significantly decreased levels of hydroperoxides and protein-bound carbonyls in hypothyroid tissues. [108] have reported increased GSH levels in the mitochondria of hypothyroid rat liver. Antioxidant status parameters. GSH is endogenously synthesized in the liver and is the first line of defence against prooxidant stress [231]. The organism can defend itself against the effects of oxidative stress by increasing SOD activity as a protection mechanism. and the thyroid tissue of the hypothyroidism-induced rats in comparison to the control group. This conflicting findings are thought to be due to different study materials in several animal models [110]. and the thyroid tissue of the Propylthiouracil treated rats. while the results of Sarandol et al. The increase in the GSH level in mitochondria of hypothyroid rats may give protection to –SHdependent proteins. namely thiol groups (SH).SH. an insufficient antioxidant defence system is thought to be a factor. which is subsequently reduced by the enzyme glutathione reductase [232]. It exerts its antioxidant function by donating electrons to radicals and changing to its oxidized form.dependent enzymes [233].

it does not have any effect on the SOD activity. An increase in SOD activity in the hypothyroid state will accelerate the production of hydrogen peroxide while a decrease in catalase activity will slow down its moval. Antioxidant capacity markers in serum of group 3 were decreased compared with group 1. Das et al. which might be due to an adaptation against the oxidative stress provoked by the thyroid hyperactivity which could be the answer to our results. some of them increase. [141] who found decreased T4 and T3 levels in vitamin E-supplemented euthyroid rats and suggested that vitamin E supplementation in the euthyroid state decreases either T4/T3 synthesis or T4-T3 conversion. In the study of [229] and [11]. It is reported that production of superoxide radicals leads to the inactivation of catalase activity and the consequent accumulation of hydrogen peroxide causes inactivation of SOD [236]. the two principal enzymes responsible for the metabolism of hydrogen peroxide in liver. Data on the effects of vitamin E supplementation on thyroid hormone levels are limited. vitamin E supplementation caused a decrease in FT4 levels (p=0. . Venditti et al [110] have showed that antioxidants are not affected in the same manner in different tissues of hypothyroid rats. showing the possible effect of thyroid hormones in the determination of the antioxidant enzyme levels. one might expect a fall in cellular respiration and.196]. 04). [108] found increased SOD activity in the liver of hypothyroid rats which is accompanied with a decrease in catalase activity. Vitamin E is a potent lipid soluble antioxidant in biological systems with the ability to directly quench free radicals and function as membrane stabilizer [237]. by analogy. are under the regulatory influence of the thyroid status of the body. Vitamin E supplementation significantly increased serum MDA levels in the Thyroxin treated group compared with the control group and with the only Thyroxin treated animals (p=0. It protects and prohibits the propagation of lipid peroxidation. Similar assumptions have already been made by other authors [120. catalase activity levels were found to be decreased in the liver tissue of hypothyroid rats. Messarah et al [234] observed an increase in vitamin E concentrations in rats suffering from hyperthyroidism.000). The physiological state of the thyroid gland. These results show that Vitamin E has a thyroid function suppressing action. It is apparent that SOD and CAT.Oxidative Stress and Antioxidant Status in Hypo. This is in line with the report of Seven et al.and Hyperthyroidism 221 On the contrary. This could be explained by the relative doses of vitamin E administered as compared with other studies [141. As far as the impact of vitamin E on thyroid status in L-thyroxine-treated rats is concerned. Further studies on deiodinase activity in liver tissue of hyperthyroidism-induced vitamin E-supplemented rats will clarify the crucial impact of vitamin E on T4-T3 conversion. arising from oxidative stress. the dose and the duration of treatment are also of a major influence on antioxidants enzymes. Carbonyl proteins levels in serum of the hyperthyroid supplemented rats were also increased compared with the controls (p=0. SOD activity reduced and CAT activity increased following T3 administration to PTU-treated rats.205] which were not enough to suppress the oxidative stress in hyperthyroidism. 0002). while several decrease or remain unchanged. In the case of the thyroid gland inhibition.

route of administration and timing of antioxidant therapy should be determined.222 Antioxidant Enzyme In our study. in order to evaluate its role on antioxidant mechanisms to defend the organism from oxidative stress. Cluj-Napoca. optimal dosage. Erdamar et al [187] showed that the level of vitamin E was significantly increased in patients with hypothyroidism. vitamin E supplementation significantly increased serum and thyroid tissue protein carbonyls levels and decreased the levels of serum antioxidant markers SH. For the first time in the literature. Romania Adriana Muresan Department of Physiology. Author details Mirela Petrulea and Ileana Duncea Department of Endocrinology. Vitamin E supplementation in hyperthyroidism could exert beneficial effects in favour of the diminution of thyroid hormone levels. revealing an individuality of antioxidant status in relation to tissue properties and responsiveness. These findings indicate that thyroid hormones have a strong impact on oxidative stress and the antioxidant system. Any alteration in the thyroid state of the body will considerably influence the antioxidative status of mitochondria and can lead to a pathophysiological state. Therefore further studies have to be carried out on patients. This could be explained by the relative doses of vitamin E administered. Romania . Iuliu Hatieganu University of Medicine and Pharmacy. Although it has been suggested that the hypometabolic state is associated with a decrease in oxidative stress. Significantly low levels of the SH groups (p<0. which is under the subtle control of thyroid hormone. literature data are controversial. Antioxidants treatments might be helpful in reducing the oxidative damage due to hyperthyroidism. Conclusion Our results suggest that thyroid hormones in excess are accompanied by increased oxidative stress and impairment of the antioxidant system. The present study confirmed an increased oxidative stress in hypothyroid state. as compared with the study of Sarandol [229] which were not enough to suppress the oxidative stress in hypothyroid rats. Cluj-Napoca. Also. Iuliu Hatieganu University of Medicine and Pharmacy. GSH and SOD in the Propylthiouracil treated group compared with the only Propylthiouracil treated rats. 05) were found in thyroid homogenates of the Propylthiouracil supplemented group as compared with the only Propylthiouracil treated rats. Under normal conditions there exists a delicate balance between the rate of formation of ROS and the rate of breakdown of ROS in mitochondria. 7. which might be due to an adaptation against oxidative stress provoked by hypothyroidism.

(2000) Am J Clin Nutr. Partata WA. Effects of thyroid hormone on coenzyme Q and other free radical scavengers in rat heart muscle. (1999) Effect of thyroid status on lipid composition and peroxidation in the mouse liver. Academic Press. Sawai Y(1995). Fernandez TRG. [9] Vitale M. R. 26 : 73-80. [7] Sies H. [6] Messarah M. Mol and Cell Endocrinol. 330: 107-112. (2000). Free Rad Biol Med. Megata Y. 226: 403–408. Endocrinology. Valenzuela. NADPH oxidase activity and cytochrome P-450 content of rat liver microsomal fractions in an experimental hyperthyroid state: relation to lipid peroxidation. Myocardial antioxidant enzyme activities and concentration and glutathione metabolism in experimental hyperthyroidism. Biologies. 8. (2007)Thyroid disease and the heart. (1994) Control of superoxide dismutase. . catalase and glutathione peroxidase activities in rat lymphoid organs by thyroid hormones. Endocrinology. Hayashibe. A. Bello-Klein A(2006). Videla(1985) Superoxide radical generation. [11] Asayama K. Bechara EJ. Sinohara R. Dobashi K. Barja G. References [1] Klein I. [3] Guerrero A. Enzveiler A. J Endocrinol. Lipid peroxidation and free radical scavengers in thyroid dysfunction in the rat: a possible mechanism of injury to heart and skeletal muscle in hyperthyroidism. Curi R. (2008)Oxidative stress of chronic kidney disease. Di Matola T. Kato K (1987). [14] Paller MS(1986). [12] Pereira B. Portero-Otin M. Abdennour C. Kidney Int. 145: 131-136. Schenkel P.and Hyperthyroidism 223 Acknowledgement This study was supported by a research grant for young PhD students. [2] Mano T. [8] Mircescu G. Boumendjel A. Biochem J. Lopez-Torres M. Irigoyen MC. K. 4(4): 433-446. Iodide excess induces apoptosis in thyroid cells trough a p53-independent mechanism involving oxidative stress. 116: 1725-1735. 141: 598-605. Hypothyroidism protects against free radical damage in ischemic acute renal failure. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work. Pamplona R. Barrientos. [5] Araujo ASR. [4] Jenkins R.Oxidative Stress and Antioxidant Status in Hypo. El Feki A. Circulation. Ribeiro MFM. Kiperos. 121: 2112-2118. (2007) The impact of thyroid activity variations on some oxidizing-stress parameters in rats. 72(2): 670S-674s. [13] Swaroop A. Acta Endocrinologica (Buc). X. 140: 73–77. Safi DA. Rosa LF. J Endocrinol. A. 249: 133-139. L. offered by The National Board For Scientific Research In Higher Education (CNCSIS). (1991) Oxidative Stress: Oxidants and Antioxidants. London. D’ascoli F. Boulakoud M. 117: 496–501. [10] Fernandez. 29: 1162–1166. Endocrinology Soc. C. Ramasarma T(1985) Heat exposure and hypothyroid conditions decrease hydrogen peroxide generation in liver mitochondria. Danzi S.

Hamide A. 40: 405-412. (2002) A diet high in cholesterol and deficient in vitamin E induces lipid peroxidation but does not enhance antioxidant enzyme expression in rat liver. (1995) Changes in lipid peroxidation and free radical scavengers in the brain of hyper-and hypothyroid aged rats. Oxford University Press.28: 970-978. Oda N. 47: 412-426. [18] Weetman radicals and tissue injury. [32] Storz P. Crapo JD. Am J Clin Nutr. ed. Bartke A(2000). Feb 4. (1995)Interaction among vitamin C. (1993) Free radicals as mediators of tissue injury and disease.23(10): 1025-30. 56: 1350-1355. Free Rad Biol Med. Rouanet JM. [24] Sies. Castro IN(1999).32: 595-603. London. and beta-carotene. [22] Hauck JS. Cases J. 12. et al. Besancon P. . Diabetes.340(8820): 633-6 [19] Halliwell B. Mokumo T. Sblano C. . Perspectives in diabetes: Role of oxidative stress in development of complications in diabetes. antioxidant enzyme activities and glutathione concentration to the thyroid hormone. Antioxidant enzymes and human disease. Endocrinol. Perez-Gomez C. Morgan BP. Clin Biochem. Tandon N. 147: 361-365. Kotake M. Landriscina C. Bobby Z. Science. (1993) Free radicals in disease processes: a compilation of cause and consequence. 1-8. (1992).WongLS. (1982) Biology of disease. Lab Invest. Giovannini C. 3rd ed.Tsuchihashi H. [27] Niki E. (2005)Novel mechanisms of natural antioxidant compounds in biological systems: involvement of glutathione and glutathione-related enzymes. (1991) The response of rat liver lipid peroxidation. [33] Masella R. Free Radicals in Biology and Medicine. Effects of growth hormone on hypothalamic catalase and Cu/Zn superoxide dismutase. [31] Kehrer JP. [30] Gutteridge JM. [17] HicksM. Free Radic Res Commun. Koner B. London. . Noguchi N. [25] Halliwel B.201: 875-880. Sridhar M. H(1985). . [20] Freeman BA. Antioxidant activity of propylthiouracil. 13: 296-301. Oxidative stress: introductory remarks. Crit Rev Toxicol. J. J Nutr Biochem. Lancet. [28] Maggi-Capeyron MF.19(3): 141-58. Cristol JP. Asano Kito Y. 16: 577-586. Sies H.224 Antioxidant Enzyme [15] Mano T. Metabolism. (2005) Free radicals and other reactive species in disease. Biochem Pharmacol. vitamin E.62 : S1322-S1326. 43(3): 439-44. [21] Mates JM. 23: 21-48. Academic Press. Sawai Y. J Nutr Biochem. Casalino E. (1978)The biology of oxygen radicals. Di Benedetto R. [23] Baynes J(1991). . et al. Varì R.DayRO(1992). C. Gutteridge JMC(1997). Front Biosci. Badia E. G(2007) Association between oxidative stress and coronary lipid risk factors in hypothyroid women is independent of body mass index. (2005) Reactive oxygen species in tumor progression. Int J Biochem. . 10: 1881-96. [16] Morini P. John Wiley and Sons [26] Nanda N. In Oxidative Stress. Antithyroid drugs and release of inflammatory mediators by complement-attacked thyroid cells. Filesi C. Shinohara R. [29] Fridovich I.

[51] Lee DH. [36] BenzieIFF(1999). Biochim Biophys Acta 1014: 17 [47] Beckman KB. 49: 481-493. Christian C. . Singanusong R. Schuff-Werner P( 2002) Oxidative stress in phagocytes: “the enemy within”. Kojima Y. McLellan LI. Meyn RE. . [50] Bjorkhem-Bergman L. Science. Saito K. . Chu C. 215: 213-219. Cattabeni F. [37] Yao LH. Cancer Res 66: 9845–9851. an in vivo thyroid carcinogenic agent. Slater TF. Chen SS(2004)Flavonoids in food and their health benefits.Oxidative Stress and Antioxidant Status in Hypo. [41] Cheeseman KH. Esworthy RS. Larsen EH. Yang S (2006) Hydrogen peroxide: a signaling messenger. Strategies of antioxidant defense. [39] Kurasaki M. Saito T. Datta N. 179: 588-590. Mol Biol Cell 15: 4829–4840. Carcinogenesis 26: 125–131. 59: 11322. induces DNA damage in rat thyroid cell lines and primary cultures. [45] Kiffin R. In: Sadler MJ. Ganther HE. Andry G. Murray D(1989) Hydrogen peroxide insult in cultured mammalian cells: relationships between DNA single-strand breakage. Kaji H. Capitanio A. Jiang YM. [42] Stone JR (2004) An assessment of proposed mechanisms for sensing hydrogen peroxide in mammalian systems. Free Radic Biol Med. [35] Sies H. NewYork: Academic Press. V. Jin L. J Biol Chem 272: 19633–19636. Horm Metabol Res. [49] Halliwell B (2007)Oxidative stress and cancer: have we moved forward? Biochem J 401: 1–11. Mol Cell Endocrinol 257–258: 6–14.Cabellero B. Arch Biochem Biophys 422: 119–124. Eken S. (1986) Increased erythrocyte catalase activity in patients with hyperthyroidism. . Tomás-Barberán FA. [48] Chico G. Vanvooren V. 31: 273-300. [40] Rotruck JT.27: 916-21. Caillet-Fauquet P. Lothaire P. H(1993). Nystrom C. Cerutti P. Stocchi V. Ames BN(1997) Oxidative decay of DNA.and Hyperthyroidism 225 [34] Hayes JD. [44] Splettstoesser WD. [38] Sies. Knecht E. Van Sande J( 2006 )Acrylamide. Friedman M. Plant Foods Hum Nutr. Torndal UB.106-115. editors. (1993) An introduction to free radical biochemistry. poly(ADP-ribose) metabolism and cell killing. (1999) Glutathione and its role in cellular functions. [43] Stone JR. Eur J Bichem. et al. Bjornstedt M. Antioxid Redox Signal 8: 243–270. Dequanter D. Br Med Bull. Shi J.Strain JJ. Pfeifer GP. Eriksson LC( 2005) Selenium prevents tumor development in a rat model for chemical carcinogenesis. Pope AL. The encyclopedia of human nutrition. Microsc Res Tech 57: 441–455. (1973) Selenium: biochemical role as a component of glutathione peroxidase. 18: 56-59. Cuervo AM (2004) Activation of chaperone-mediated autophagy during oxidative stress. Free Radic Res. Antioxidants: observational epidemiology. [46] Cantoni O. Massart C. Chu FF( 2006) Mutation accumulation in the intestine and colon of mice deficient in two intracellular glutathione peroxidases. (1999) Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defence against oxidative stress.

Spitz DR. [63] Zoeller RT. Truscott RJ. Sigal N. Fujiwara Y. reactive oxygen species. Cancer Res 56: 4846–4852. Rittinger K( 2005) Activation and assembly of the NADPH oxidase: a structural perspective. [60] Zhang B. 3: 83–95. Reddy KC. . in Wilson J. Willows RD. Textbook of Endocrinology. . Becker S. Philadelphia. Hirahashi J. Sachdev P.. J Biol Chem 278: 28443–28454. Philadelphia. Cullere X. [53] Chu R. Eds. Tyl RW. Yeldandi AV( 1996) Transformation of epithelial cells stably transfected with H2O2-generating peroxisomal urate oxidase. B Saunder Co. 17: 539– 562.. . Itan M. [56] Rancourt RC. Pick E( 2000) Targeting of Rac1 to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly. and MAPK/ERK activation. The thyroid. E. [66] Werner And Ingbar’s(1996). [58] Groemping Y. Keng PC. Pituitary. Takikawa O. Grant R. Jamie JF.7th Edition. pp. [61] Gorzalczany Y. (2007) General background on the hypothalamicpituitary-thyroid (HPT) axis.Foster D. WB Sounders Company. [57] Duan J. [68] Williams T(1998).226 Antioxidant Enzyme [52] Neumann CA. Glutaredoxin as a sensor of oxidative stress mediated by H2O2. Suntharalingam M. Pan J. Reddy JK. Van Etten RA(2003) Essential role for the peroxiredoxin Prdx1 in erythrocyte antioxidant defence and tumour suppression. D. [67] Ingbar S(1985). J Biol Chem 277: 46566–46575. Lippincot-Raven Publishers. Haussinger D(2005) Involvement of NADPH oxidase isoforms and Src family kinases in CD95-dependent hepatocyte apoptosis. Duan J. [54] Reinehr R. Crit Rev Toxicol. [64] Toni R(2000). Lippincott Williams & Wilkins: Philadelphia. The thyroid. L. Braverman. Mayadas TN( 2003) Elucidation of molecular events leading to neutrophil apoptosis following phagocytosis: cross-talk between caspase 8. [65] Carrasco N. R. Lee YJ( 2002) Role of glutaredoxin in metabolic oxidative stress. a Fundamental and Clinical Text. Ancient views on the hypothalamic-pituitary-thyroid axis: an historical and epistemological perspective. Das S. Arch Biochem Biophys 450: 9–19. Nature 424: 561–565. Grether-Beck S. Chess PR. Bronson RT.3 dioxygenase activity by H2O2. Biochem J 386(Pt 3): 401–416 [59] Poljak A. J Cell Physiol 193: 26–36. In Werner and Ingbar’s The Thyroid: a Fundamental and Clinical Text. Rhee JG. [55] Song JJ. Carman CV. 37: 11–53. Abraham JL. Semin Ultrasound CT MR. Smythe GA( 2006) Inhibition of indoleamine 2. Int J Biochem Cell Biol 37: 1407–1420. Orkin SH. Textbook of endocrinology. J Biol Chem 275: 40073–40081. Dubey DP. Tan SW. Rao MS. Tong T( 2005) Irreversible cellular senescence induced by prolonged exposure to H2O2 involves DNA-damage-and-repair genes and telomere shortening. Walker MJ. Lin Y. O’Reilly MA( 2002) Growth arrest in G1 protects against oxygen-induced DNA damage and cell death. D. Hayes DD. Walsh SA. J Biol Chem 280: 27179–27194. Lotan O. (2005) Thyroid Iodine Transport. 37–52. Zhang Z. 9th Ed.9th Edition. (1996) Imaging of the thyroid gland. Utiger. Krause DS. [62] Loevner LA. Littlejohn TK. Austin CJ. Eberle A.

D. 108: 431–439. Editura Tehnica. N Engl J Med. 347: 95–102. [81] Orasan R(2001). [87] Flohe L. VA(1997). [80] Olinescu R. Utiger. Corvilain B. De Deken X.26: 944-984. Driessens N. Becker R. [74] Rom-Boguslavskaia ES. . 92: 3764-73 [72] Köhrle J. Edwards C(2010) Integrated approach to the mechanisms of thyroid toxins: electron transfer. Braverman. and antioxidants. Metab. Bikker H. Trends Endocrinol. Dumont JE. L. (2002) Inactivating mutations in the gene for thyroid oxidase 2 (THOX2) and congenital hypothyroidism.Virion A. [71] Song Y. Diageleva EA. In: Methods Enzymol : 93-104. 53–76. Van Sande J. Gene regulation by thyroid hormone. Costa M. Lippincott Williams & Wilkins: Philadelphia.266: 37393743. . Roles of hydrogen peroxide in thyroid physiology and disease. In Werner and Ingbar’s The Thyroid: a Fundamental and Clinical Text. (2005)Genomic and Nongenomic Actions of Thyroid Hormones. [85] Reznick AZ. Somova EV. Mechanism of hydrogen peroxide formation catalyzed by NADPH oxidaze in thyroidplasmamembrane. [78] Wu Y. R. Ovsiannikova TN. . Lippincott Williams & Wilkins: Philadelphia.. and oxidative stress: functional and cytotoxic consequences. [73] Moreno JC.Koenig RJ(2000). (1994). E. Cluj-Napoca. (1984) Superoxide dismutase assay. . and the endocrine system. Karachentsev IuI Asaula. [82] Goglia F. Eds. reactive oxygen species. Editura Albastra. oxidative stress. Chem.. Kempers MJ.and Hyperthyroidism 227 [69] Dupery C. de Vijlder JJ. thyroid calorigenesis. [70] Landex NL. Lengfelder E. Silvestri E. van Trotsenburg AS. [76] Kovacic P. Baas F. Miot F. 69 : 111–114. Braverman. D. Packer L. Editura Intelcredo. Ris-Stalpers C. [75] Kopp P. Acta Histochem. Otting F. [86] Hu ML: Measurement of protein thiol groups and glutathione in plasma. [77] Yen PM. Biochimia proceselor metabolice in organisme animale.Seria Medicina. [79] Ciurdaru V(1997). the thyroid. Moran PC. Many MC. Thyroid Hormone Synthesis. [83] Videla LA. Utiger. 37(2): 1273-1275. (2002)Thyroid hormones and mitochondria. Lipid peroxidation in thyroid tissue of people with diffuse toxic goiter. Clin Chem. 30: 133-42. Dumont JE(2007). Biol. E. . [84] Conti M. Selenium.Ohayon R. cell signaling. Jakob F. (2006) Methimazole increases H2O2 toxicity in human thyroid epithelial cells. (2005).. Radicalii liberi in fiziopatologia umana. . Redox Rep. Biosci Rep.Oxidative Stress and Antioxidant Status in Hypo. (2005). (2000) Energy metabolism.22(1): 17-32. Brigeluis R. R. In: Methods Enzymol Academic Press Inc : 380-384. J.. Thomsen J. 2000. . Improved fluorimetric determination of malondialdehyde. Detours V. Vulsma T. Eds. . Kayser L.Deva. 11 : 207–211. 135–150. receptors. In Werner and Ingbar’s The Thyroid: a Fundamental and Clinical Text. 5(5): 265-75. Lanni A. Fiziologia sistemului endocrin. (1991). J Clin Endocrinol Metab. Levillain P(1991). Contempré B. J Recept Signal Transduct Res. Maenhaut C. 233: 357-363. L. Bucuresti. Endocr Rev. (1994) Oxidative damage to proteins : spectrophotometric method for carbonyl assay In: Methods in enzymology 1994. Ukr Biokhim Zh.

Matarazzo M. (1987) Advances in our understanding of thyroid hormone action at the cellular levels. H. Dasa K.. 8: 288-308. Appella E. cell damage and antioxidant therapy. Gutteridge JM. N.. A. Bhanja S. G.. et al. [105] Taleux N. Methods Enzymol. Rev. A. Clin. Appleton and Lange. Kinlaw WB.228 Antioxidant Enzyme [88] Greenspan FS. and Baxter J. Interact. [90] Cheng S. 47: 233–261. 300: 117-123. and Ingbar S.. -Y. Merlino G.173: 105-114. measurement and dietary influences..ChainyGBN. Erkanl. (1996)Lipid peroxidation: a review of causes. et al. Cell. [100] Favier A. J. High expression of thyroid hormone receptors and mitochondrial . [104] Draper HH. The thyroid gland. Agnisola C. Nováková V. F. Biol. Angelini V. Med. Chem. Biondi B. (2004) Role of nitric oxide in the functional response to ischemia-reperfusion of heart mitochondria from hyperthyroid rats. [101] Kehrer JP. Pardo F.. [92] Oppenheimer JH. Chem. [99] Subudhi U.. 186: 421–31. [95] Napoli R. Gedik. oxygen radicals. (1993) Free radicals as mediators of tissue injury and disease. Goglia F. [98] Benzie IF. Blahosová I. (2001) Impact of hyperthyroidism and its correction on vascular reactivity in humans. Mariash CN. Perkinson C. In: Basic and clinical endocrinology Greenspan F. D. Toxicol. A. (1994). Leverve XM(2008). Cornejo P. Lancet. Endocrinology 136: 4182-4187.. 104: 3076-3080. Mol. [93] Ueta Y. and Lightman SL. -Y. Crit. Paital B. Robinson E. Chowdrey HS. End. Guigas B. De Rosa R. consequences. Ö.. pp 160-223. [94] Fernández V. Circulation.. Schwartz HL. 108–115. Nitric Oxide.. and Di Meo S. Biol. Moreno M. Cigliano L. (1990) Malodialdehyde determination as index of lipid peroxidation. J. Norwalk. Thyroid hormone action at the cell level. B.. Yeğen(2006). Tapia G.. (2008) Alleviation of enhanced oxidative stress and oxygen consumption of L-thyroxine induced hyperthyroid rat liver mitochondria by vitamin E and curcumin. Wong NCW.Dubouchaud H. (1979). and Freake HC.. 6: 463-468. i: 1396–7. Ercan. and Videla L.. Ç. (2003) Le stress oxydant: intérêt conceptuel et expérimental dans la compréhension des mécanismes des maladies et potentiel thérapeutique. 23: 21–48. 70: 919-926. [97] Neradilová M Hurbá F. Engl J. Gong Q. [102] Halliwel B. Int J Vitam Nutr Res. 262: 11221-11227. [89] Segal J. 32: 728–736. (1982) Specific binding sites for triiodothyronine in the plasma membrane of rat tymocytes: correlation with biochemical responses. Weitzel JM. T. Velioğlu-Öğünç. (1997) Influence of hyperthyroidism on the activity of liver nitric oxide synthase in the rat. L’actualité Chim. [96] Venditti P. Şener. 43: 283–290. S. (1995) Hypotalamic nitric oxide synthase gene expression is regulated by thyroid hormones. (1984) Lipid peroxidation.. Şehirli. Life Sci. (1973) Investigations of the relationship between thyroid function and α-tocopherol concentration of serum and in some organs of the rat. (1987) The nucletide sequence of a human cellular thyroid hormone binding protein present in endoplasmic reticulum. Guardasole V. N. Int J Food Sci Nutr. Rew. Hadley M. Invest. [103] G. [91] Sterling K. Propylthiouracil (PTU)-induced hypothyroidism alleviates burn-induced multiple organ injury Burns. 61: 2244-2252. FavierR.

Mol. Bartoc R. 37: 2478–2503. [116] AdamoAM. Biol. De Leo T(1997). [108] Das K. Health A. Neurochem. . Med. LlesuySF. Endocrinologie. Nagata N. tissues. Biol.. 26: 35–38. Yigit G. Seymen O. [114] Iangolenko VV. Hlavatá L (2005). Biol.. Biol.. 263: 273-277. 156 : 13–19. Seven A. (1990) Oxidative muscular injury and its relevance to hyperthyroidism. Biull..Oxidative Stress and Antioxidant Status in Hypo. andBoverisA. Biochem. PasquiniJ. and organism. Free Radic. Res. (2001) Oxidative stress in heart tissue of hyperthyroid and iron supplemented rats..and hypothyroidism. Watanabe F. J. Portero-Otin M. (1989)Brain chemiluminescence and oxidative stress in hyperthyroid rats. and Burçak G. and Di Meo S. Free Radic. Biol. (1999) Effect of thyroid status on lipid composition and peroxidation in the mouse liver. 1573: 1–13. J. (1991) Blood levels of medium molecular weight peptides and lipid peroxidation activity in the differential diagnosis of diffuse toxic guatr. Med. [113] Dumitriu L. J. Endocrinol. Di Meo S. Interact. [111] Bozhko AP. Ursu H. 8(3): 293–303. antioxidant defences. [107] Guerrero A. 164: 97–102. (2004)Thyroid hormone influences antioxidant defense system in adult rat brain.Kato K.Iwase K. Ishizuki Y. Lissi EA.. Endocrinol. [110] Venditti P.. [109] Duntas LH. Balestrieri M. J. 142: 15-23. [119] Venditti P. Med. (2001) Modulation of liver mitochondrial antioxidant defense system by thyroid hormone. J. a rat strain resistant to obesity J.Videla LA(1991). Probl. Tsugawa T. [106] Ježek P. 26: 73–80. Biophys. Lipid peroxidation levels in rat cardiac muscle are affected by age and thyroid status. 29: 1755-1766. Itoh M(2000). 37: 10–12. Barja G. Biondi B. Biochem. Cell. Gorodetskaia IV. 77: 173–185. (1988) Significance of high levels of serum malonyl dialdehyde (MDA) and ceruloplasmin (CP) in hyper. [115] Asayama K. Environ. Endocrinol.64: 499-506. Evaluation of the antioxidant properties of thyroid hormones and propylthiouracil in the brain-homogenate autoxidation system and in the free radical-mediated oxidation of erythrocyte membranes. 109: 539–541. Biol. Int. 284 : 4308–4316. 155: 151–157. and Chainy GBN. Kakizawa H. J. (1998) Antioxidant-sensitive shortening of ventricular action potential in hyperthyroid rats is independent of lipid peroxidation. M. Uchimura K. [120] Shinohara R. Eksp. Mitochondria in homeostasis of reactive oxygen species in cell. Pamplona R.. Toxicol. Chem. Chem. and susceptibility to oxidative stress in rat tissues.. Acta. Mano T. (1990) Restriction of stress-induced activation of lipid peroxidation by small doses of thyroid hormones. Biochem. Cell. Endokrinol (Mosk). [112] Faure M. Solodkov AP.Lopez-TorresM. MakinoM. Effect of thyroid state on lipid peroxidation. Hatemi H. [117] Das K. (2007) Short-term hypothyroidism after Levothyroxinewithdrawal in patients with differentiated thyroid cancer: clinical and quality of life consequence. De Leo T. Nakano I. [118] Civelek S. Eur. Okorokov AN.and Hyperthyroidism 229 glycerol-3-phosphate dehydrogenase in the liver is linked to enhanced fatty acid oxidation in Lou/C. Nayashi R. Endocrinol. Chainy GBN.

(2001) Influence of hyper. Mol. (1993) The effects of propanolol on skeletal muscle contraction. Koutras DA. Karadimas P. Khalid BAK. 47: 412-426. Gen. (1978) Evidence for a different response of red and white skeletal muscle of the rat in different thyroid states. [129] Gredilla R. Hatemi H. [122] Choudhury S. and Kassenaar AAK. Endocrinol. Am.. [135] Tapia G. Acta Endocrinol. and Burçak G. glutathione system and oxidative damage to nuclear and mitochondrial DNA in mice skeletal muscle. Joseph LJ.Smok G. Yonsei Med. Cell. [136] Burch HB. Merican Z. 57-58: 610-618. and Klitgaard HM. Res. [130] Petrović N. and López-Torres M. Chainy GBN. [123] Zaiton Z. (1977) Response of mitochondria of different types of skeletal muscle to thyrotoxicosis. 35: 417-425.and hypothyroidism on lipid peroxidation. J. Buletin USAMV-CN. Pamplona R. Am. 41: 1334-1337. 45: 413-418. Invest. Filip A. lipid peroxidation products and antioxidant activity in experimental hyperthyroidism. 24: 195-199. Free Rad. [133] Freeman BC. and Crapo JD.. Videla LA(1997). Mohamed JB. [131] Turrens JF and Boveris A. 176: 31-38. Yigit G. FEBS Lett. Civelek S. Cvijić G. and Davidović V. Pharmacol. 65(2): 311-6. Physiol.26: 267-79. Andrologia 35: 131-140. Biol. [134] Joanta A. and Mishro M M. Lahiri S. Kupffer cell function in thyroid hormoneinduced liver oxidative stress in the rat. Biochem. Bahn RS. 87: 768-775. and Baharom S. . J. 42: 68-72.. (2002) Changes in lipid peroxidation and free radical scavengers in kidney of hypothyroid and hyperthyroid rats. [127] Sawant BU. Am J Ophthalmol. (2003) Experimentally induced hypoand hyper-thyroidism influence on the antioxidant defence system in adult rat testis. J. . Portero-Otín M. [137] Bouzas EA. (1997) Superoxide radical production stimulates retroocular fibroblast proliferation in Graves' ophthalmopathy. Barja G. Krausz T. [124] Winder WW. and Rajan M G.. Free Radical Res.. Richter C and Flohé L. J. (2002) Oxidative stress evidence in rats treated with thyroxin. J.. Seven. 191: 421-427. 232: C180-C184. (2003) Thyroxine and tri-iodothyronine differently affect uncoupling protein-1 content and antioxidant enzyme activities in rat interscapular brown adipose tissue. [128] Gredilla R. glutathione and DNA in the mouse heart. (2000) Antioxidant agents in the treatment of Graves' ophthalmopathy. Andrei S. 221: 41-48.230 Antioxidant Enzyme [121] Barker SB. Biochem. . and Barja G.. van Handerveld C. Physiol. [132] Loschen G. (1980) Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria. [126] Seymen HO.129(5): 618-22. Barnes S. Suciu S.. unsaturation of phospholipids. Mastorakos G. Exp. Free radicals and tissue injury.. (2001) Thyroid hormone-induced oxidative damage on lipids. [125] Janssen J W. López-Torres M. Indian. Lab. Thakare UR. J. Exp Eye Res. (2004) Iron supplementation in experimental hyperthyroidism: effects on oxidative stress in skeletal muscle tissue.. (1952) Metabolism of tissues excised from thyroxineinjected rats. Nadkarni GD.. and Holloszy JQ. 170: 81-86. (1974) Superoxide radicals as precursors of mitochondrial hydrogen peroxide. Azzi A. (1982) Biology of disease. Pepper I.

Chim. Methods Enzymol.266: 4244-50. Peroxynitrite oxidation of sulfhydryls. [140] Kowaltowski AJ. Ribeiro MF. [148] Araujo AS. [143] Kehrer JP(1993). Hydroperoxide metabolism in mammalian organs. Mantle D.Meerman JHN. [152] Petrulea MS. Enzveiler A. Partata WA. 100: 84-90. [144] Radi R. Rev. Dumont JE(1991) The H2O2-generating system modulates protein iodination and the activity of the pentose phosphate pathway in dog thyroid. Boveris A(1979). Basic Clin Pharmacol Toxicol. Free Radic Biol Med. Vercesi AE (1999). Clin. (2009) Thyroid hormones in excess induce oxidative stress in rats. Mitochondrial damage induced by conditions of oxidative stress. Mckillop. Med. (1999). Clin. Role of free radicals and catalytic metal ions in human disease: an overview. Biomarkers of free radical damage applications in experimental animals and in humans. Mol Cell Endocrinol. Endocrinology. Chim. 205: 185–192. Oxidative damage in liver disease. Free Radic. Irigoyen MC. Toxicol. [149] Venditti P. [151] Mohamadin AM. Bradley. (2006) Myocardial antioxidant enzyme activities and concentration and glutathione metabolism in experimental hyperthyroidism. A. 5(2): 155-164. Reilly ME. 256: 65–73. Bush KM. Koner BC(2003)Oxidative changes and desialylation of serum proteins in hyperthyroidism. Acta. Hammad LNA. Hatemi S. 26: 463–471. Endocrinol.30: 429–433. 59: 527–605. Free Rad.. Videla L(1993).and Hyperthyroidism 231 [138] Chance B. J. Res. Crit. LaurentE. Llesuy S. Effect of thyroid state on H2O2 production by rat liver mitochondria. Muresan A. (1991). Influence of hyperthyroidism on superoxide radical and hydrogen peroxide production by rat liver submitochondrial particles. Yigit G. Duncea I. Smith. Di Meo S (2003).Van SJ. [146] Fernández V.Chopra H. De Rosa R. [150] Subudhi U. Belló-Klein A. 186: 1–85. Free radicals as mediators of tissue injury and disease. Peters TJ(1998). Nandakumar DN. Candan G. [142] De ZwartLL.Oxidative Stress and Antioxidant Status in Hypo. Biol.10: 16-20. Paital B. 26: 202-26. Physiol.. Fernandes TR. Alleviation of enhanced oxidative stress and oxygen consumption of L-thyroxine induced hyperthyroid rat liver mitochondria by vitamin E and curcumin. Gutteridege JMC(1990). [147] Goswami K. Hatemi H. [141] Seven A. Acta Endocrinologica (Buc). 173(2): 105151. Vermeulen NPE. [153] Wilson M. Clin. J Biol Chem. (1989) Thomson. We. . [154] Corvilain B. Free radicals and Graves’ disease: effect of therapy. Comm. (1996) Antioxidant status in experimental hyperthyroidism: effect of Vitamin E supplementation. 337 : 163–168. . 128: 779–785. 23: 21–48. El-Bab MF. 18: 329–335. Chainy GBN(2008).249(1-2): 133-9. Acta. Bhanja S. Freeman BA. [139] Halliwell B. Schenkel P. Commandeur JNM. Chemico-Biological Interactions. Das K. JIFCC. Gawad HAS (2007)Attenuation of oxidative stress in plasma and tissues of rats with experimentally induced hyperthyroidism by caffeic acid phenylethyl ester. Sies H. Rev. Endocrinol. J. [145] Preedy VR. Cell. Mol. Beckman JS. Seymen O.

[168] Meister A. Takeda K. Biochem Pharmacol. van den Hove MF(2002) Cell necrosis and apoptosis are differentially regulated during goitre development and iodine-induced involution. [159] Venditti P. Arch Biochem Biophys. Shinohara R. 241: 75–81. Many MC. Aumann K. F(2003). 31 : 273–300. Paolicchi A. Casini A. Okuno Y. Annu Rev Bichem. Detection of thiyl radical adducts formed during hydroxyl radical. 27: 951–965. Hauser S. Afectiuni ale tiroidei. [156] Mano T. Pérez-Gomez C. [166] Brigelius-Flohè R(1999). Maiorino M. Nakai A. Hayashi R. In: Fauci A. McLellan LI(1999)Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defense against oxidative stress. [162] Arnér ES. Free Radic Biol Med.and peroxynitrite-mediated oxidation of thiols . Kato M. [172] Nakashima I. [165] Armstrong RN(1997). J Endocrinol. Free Radic Biol Med. Iwase K. Denef JF. Anderson ME (1983). Suzuki H (2005). 33: 154–169. The diversity of glutathione peroxidases. Kotake M. 12: 5–11. Food Chem Toxicol. 29: 351–354. [161] Talalay P (2000). Anderson ME. Meister A. Glutathione. Karoui H. Biofactors. [160] Hayes JD. Regulation of enzymatic lipid peroxidation: the interplay of peroxidizing and peroxide reducing enzymes. Schomburg D et al. Poma JF. (1995). Di MeoS (2006)Thyroid hormone. Intracellular antioxidants: from chemical to biochemical mechanisms. Annu Rev Biochem. Ed. Cell. Borchert A (2002). Singh RJ. Tissue-specific functions of individual glutathione peroxidases. Eur J Biochem.232 Antioxidant Enzyme [155] Mutaku JF. Mol. D.a high resolution ESR spin-trapping study at Q-band. Daumerie C. 252B: 38–53. Wilson J. Many MC. Ishizuki Y. (1983).52: 711-60.induced oxidative stress. Teora : 2211. Kawamoto Y. Isselbacher K. [170] Pompella A. Braunwald E. Structure. Holmgren A. [163] Matés J. [169] Kalyanarama B. Kasper D. 434: 3–10. Brigelius-Flohé R. 66 : 1499–1503. Felix CC (1996). 267 : 6102–6109. 10 : 2–18.2217. Life Sci. 63: 414-434. The changing faces of glutathione. Hamada M. [158] Leonard Wartofsky(2001). Visvikis A. 32: 595–603. Thyroid. [164] Chaudière J. Redox control of catalytic activities of membrane-associated protein tyrosine kinases. 37 : 949–962. 15: 205–209. Martin J. Knoops B. a cellular protagonist. Physiological functions of thioredoxin and thioredoxin reductase. catalytic mechanism and evolution of the glutathione transferase. Clin Biochem. Hayakawa N. [171] Ursini F. Glutathione. Antioxidant enzymes and human diseases. Nagasaka A(1997) Changes in free radical scavengers and lipid peroxide in thyroid glands of various thyroid disorders. Colin IM(2005) Peroxiredoxin 5 expression in the human thyroid gland. Chemoprotection against cancer by induction of phase II enzymes. Anal Biochem. Roveri A. Ferrari-Iliou R(1999).52: 711–760. Chem Res Toxicol. 172: 375–386. Free Radic Biol Med. [157] Gerard AC. Longo D Harrison Principiile medicinei interne. Horm Metab Res. (2000). . Methods Enzymol. Nùnez De Castro I (1999). [167] Kuhn H. Uchimuro K. de Tata V.

hyperthyroidism. [177] Asayama K. Free Radic. 34(3): 497–500. Med.Smith CV (2004). Seven A. Hayashibe H. Inal-Erden M. (1999) Effects of propylthiouracil. 36(5): 687–694. Clin. Res.Kucharz EJ.5(2): 77–84. Acta. Bradley H. Demirci H. 78 : 89–142. Annu Rev Pharmacol Toxicol. and their treatment on parameters of oxidative stress and antioxidant status. Burcak GJ (1999). Clin. [183] Venditti P. [187] Erdamar H. Radic..Welty SE. [176] Halliwell B(1994) Free radicals. . and vitamin E on lipid peroxidation and antioxidant status in hyperthyroid patients.Aschner M. Chopra M. 6: 283–95. Yaman H. Smith We. 10: 315–325. [185] Seymen HO.Biberoğlu G. Junqueira VB. Rodrigues L. Res. Flavoprotein disulfide reductases: advances in chemistry and function. Clin Biochem. [178] Sun Y. Endocrinol. Yakar T. Mckillop J. Prog Nucleic Acid Res Mol Biol. Kato K (1989). Clin Chem Lab Med. [174] Argyrou A. Kipreos K. Chem. (2008) The effect of hypothyroidism. (2007)Effects of manganese on thyroid hormone homeostasis: potential links.28: 951-956 . Oxidative muscular injury and its relevance to hyperthyroidism. Pharmacol. Effects of beta-adrenergic blockers with different ancillary properties on lipid peroxidation in hyperthyroid rat cardiac muscle. De Leo T.Olczyk K. and human disease: curiosity. Marcisz C.46(7): 100410.Oxidative Stress and Antioxidant Status in Hypo.and Hyperthyroidism 233 [173] Hayes JD. Boveris A(1988). Videla LA (1998). [182] Wilson R. Free. [186] Adali M. Rodrigues T. Hatemi H. Cell. . [181] Komosinska-VassevK.A Kotulska A(2000). or consequence? Lancet. DiMeo S. [188] Soldin OP. Tamura T. Biochem. Flanagan JU. Sancak B.32(5): 363-7. Thomson JA (1989). (1996). [180] Fernandez V.Winsz-Szczotka K. Efe B. Oberley LW. Blanchard JS (2004). Dobashi K. A simple method for clinical assay of superoxide dismutase. Free radical activity and antioxidant defence mechanisms in patients with hyperthyroidism due to Graves’ disease during therapy. 300: 107–117. Endocrinol. Elbeg S. Glutathione transferases. 8(3): 293–303. [179] Asayama K. Analyses of glutathione reductase hypomorphic mice indicate a genetic knockout.. Toxicol Sci. antioxidants. Free Radic. Chim. [184] Simon Giavarotti KA.30: 429–433. Chemiluminescent and respiratory responses related to thyroid hormone-induced liver oxidative stress. Clin. Physiol. Rogers BJ. 82 : 367–373. Kato K(1990). Physiol. Hansen TN. Evaluation of antioxidant status in liver tissues: effect of iron supplementation in experimental hyperthyroidismJ.45: 51–88. Civelek S. [175] Rogers LK. Biol. Commun. Neurotoxicology. Llesuy S. Liver microsomal parameters related to oxidative stress and antioxidant systems in hyperthyroid rats subjected to acute lindane treatment. Akalin A. Free radicals and Graves’ disease: effect of therapy. propranolol. Yetkin I. Li Y (1988).29 : 35–42. Videla LA. Jowsey IR(2005). Solari L. Jpn. Effects of thyroid state on characteristics determining the susceptibility to oxidative stress of mitochondrial fractions from rat liver. 344: 721–724. Basic Clin. cause. Erbil MK. Yigit G.

Archives of Physiology andBiochemistry. J. Otani S. Biochem Biophys Res Commun.94: 1112-1117. Determination by enhanced luminescence technique of liver antioxidantcapacity. Shinohara R. Am J Clin Nutr. 102(2): 63-89. Tache S. Sawai Y. [197] Varghese S. Yigit G. [199] Fernández V. et al. Oommen OV. [205] Seven A. Inagaki A. Brochem. Clin Chem. Yonsei Med. activities. and turnover. 239: 212-216. [192] I Fridovich(1985). Seven A. Shameena B.234 Antioxidant Enzyme [189] Iwase K. Fridovich I. [201] Venditti P. Clin Chem Lab Med. Mokuno T. Yur F. Editura Todesco. Assoc. Burçak G. Oommen OV (1999).. Tsuchihashi H. Seymen O. Tsujimura T. . Superoxide dismutase: organelle specificity. vitamin E. Helsel G. Superoxide dismutase: regularities and irregularities.240: 500-8. Oda N.. Interaction among vitamin C. [204] Niki E. [190] Singh PP. [202] Köhrle J. glucose 6 phosphate dehydrogenase and glutathione Isr.. Thyroid hormones regulate lipid metabolism in teleost Anabas testudinens (Bloch). Azzalis LA. Chem. [194] Seymen HO. (1995). Oxidative stress in physiological and pathological processes. Belge F. Arch Biochem Biophys. Tatzber F.. [198] Blum J. HatemiH. Vet. et al. Comp.103: 484-491. 45: 413-418. 128: 165–171. .40(11): 1132-4.Di Meo S. J. Barros SBM. [191] R. 248 : 3582–3592. Pimentel R. 79 : 51–75. [193] Mano T. Noguchi N. In: Muresan A.Orasan R. Civelek S. [196] Varghese S. I Fridovich(1973). 62: S1322-S1326. [203] Resch U. Alkan M. 129: 85–91. . Endocrinol. Miura K.57 (2). (1985) Inactivation of glutathione peroxidase by superoxide radical. Harvey Lect. Simizu K. Physiol. (1996) Lipid peroxidation and vitamin E supplementation in experimental hyperthyroidism. Nippon Geka Gakkai Zasshi. (2004) Iron supplementation in experimental hyperthyroidism: effects on oxidative stress in skeletal muscle tissue. Med. (1994) Thyroid hormone deiodination in target tissues--a regulatory role for the trace element selenium? Exp Clin Endocrinol. Sinzinger H. Hatemi S. Biochem.Martino R(1993). (1995) Effects of thyroid hormone on coenzyme Q and other free radical scavengers in rat heart muscle. Hatemi H. (1997) Regulation of superoxide anion radicalsuperoxide dismutase system in the avian thyroid by TSH with reference to thyroid hormonogenesis.124B : 445–450. and beta-carotene. 42(7): 1118-9. Junqueira VBC et al. J. (2001)Thyroid hormones regulate lipid peroxidation and antioxidant enzyme activities in Anabas testudinens (Bloch).10: 162-171. Nishida Y. Comp. Kumar P. Kilil PK (2002) The effect of hyperthyroidism on the level of Na+ K+ ATPase. Effects of hyperthyroidism on rat liver glutathione metabolism: related enzymes. Candan G. Endocrinology. Yiğit G. [195] Bildik A. (2002) Antioxidant status in thyroid dysfunction. Physiol. 145: 131-136. Kato K. efflux. Biol. (1991). A Weisiger. (1993) Study of the localization and the concentration of superoxide dismutase in various thyroid disorders. Laloraya M. [200] Joanta A(2006) Oxidative stress induced by thyroid gland hyperfunction.

Regulation of glutathione synthesis: a review. H. Thyroid hormone. 149(1): 424-433.146 : 383–391. FL. Cancer Lett. Cell. and lipid peroxidation J. The interaction of oxidative stress response with cytokines in the thyrotoxic rat: is there a link? Mediators Inflamm. Comp Biochem Physiol C. Subudhi U. [222] Visser TJ(1980). Knoops B. Gérard AC. [210] Venditti P. Weber (Eds. 30 : 42–59. Effect of thyroid state on H2O2 production by rat liver mitochondria. Trends Bichem Sci.OommenOV(2001). A. Kowaltowski AJ. Senou M. R. Nadkarni GD(1996).5: 222-224. Toxicol Lett. 109: 231-5. [209] Fernández V. Makay O. Tapia G. [214] Shelly C. Armas-Merino(1987). [220] Maddaiah VT (1990). Boca Raton. 142 : 213– 239. Mol Aspects Med. Handbook of free radicals and antioxidants in biomedicine. Triiodothyronine-induced hepatic respiration: effects of desferrioxamine and allopurinol in the isolated perfused rat liver. Toxicol Lett.128 : 165–171. 3. Comp Biochem Physiol. Akyildiz M. Wolff J. Colin IM(2008) Oxidative stress in the thyroid gland: from harmlessness to hazard depending on the iodine content.and Hyperthyroidism 235 [206] Bednarek J.Videla LA (2005). Vercesi AE. Clin Sci. Icoz G. Wysocki H. Sowiński J. 124B : 445–450. Differential expression profiles of antioxidant enzymes and glutathione redox status in hyperthyroid rats: a temporal analysis. Endocrinology. Deiodination of thyroid hormone and the role of glutathione. 2009: 391682.Oxidative Stress and Antioxidant Status in Hypo. ). Thyroid hormone-induced oxidative stress in rodents and humans: a comparative view and relation to redox regulation of gene expression. Oommen OV(1999). (1998). Przegl Lek. Videla LA(1996). [212] Fernandez V. Quintanilha. [219] Castitho RF. [207] Fernandez V. 61(8): 841-4. 205: 185–192. Videla LA(1993). Mol. . CRC Press Inc.5. Lengelé B. Varela P. [215] Varghese S. Relationship between hepatic levels of glutathione and sulphobromophtalein retention in hyperthyroidism. Comp Biochem Physiol. Chainy GB(2007). [216] VargheseS. Soto R. Miquel. Ozgen G. [211] Sadani GR. [218] FernándezV.. (2004) The effect of one-month antithyroid therapy on peripheral metabolism of reactive oxygen species in Graves' disease with infiltrative ophthalmopathy. De Rosa R. Unsal E.ShameenaB. Comp Biochem Physiol. T.. Cartier-Ugarte D. Endocrinol. active oxygen.105–115. Di Meo S (2003).. Sahoo DK. Lu (2008). 73: 235–237. FASEB J. Yenisey C. Hepatic glutathione biosynthetic capacity in hyperthyroid rats. Calderon PB.3-Triodothyronine induces mitochondrial permeability transition mediated by reactive oxygen species and membrane protein thiol oxidation. Romanque P. Arch Biochem Biophys. 69: 205-10. 4: 1513– 18. [217] Sir C. [213] Poncin S. Role of tissue antioxidant defence in thyroid cancers. (2009). 345: 151–157. Boucquey M. Thyroid hormones regulate lipid peroxidation and antioxidant enzyme activities in Anabas testudinens (Bloch). Many MC. Yetkin E. . [208] Chattopadhyay S. Glutathione correlates with lipid peroxidaiton in liver mitochondria of triiodothyronine-injected hypophysectomised rats. 89 : 205–363. Thyroid hormones regulate lipid metabolism in teleost Anabas testudinens (Bloch). Videla LA (1989). [221] Makay B..

Bhanja S. Endocrine. J. [226] Gravina FS. Chainy GBN (2008). Acta. [237] Subudhi U. Oxidants and antioxidants. R Prohaska (1980). Male/female difference. [231] Nicotera P. (2001) Influence of propylthiouracil treatment on oxidative stress and nitric oxide in Basedow disease patients. (2007). Hatemi H. (2007)Experimental hypothyroidism inhibits delta-aminolevulinate dehydratase activity in neonatal rat blood and liver. Superoxide radical inhibits catalase. Erbil Y. . Cell Biochem Funct. Bozbora A. Benzer F. 21: 325–330. and oxidative stress. Seibel FE. [225] Berlett BS. Oxidative damage and antioxidant enzyme activities in experimental hypothyroidism. Boumendjel A. I Fridovich(1982). Experim. [236] Y Kono. Biochim.82: 291-295. [233] J. 173(2): 105114.. Burdon R. et al. Burcak G. Cell Biochem Funct. 25: 1-5. [227] Dariyerli N. Adv Exp Med Biol. [234] Messarah M. 62: 495-503. Gelisgen R. The glutathione peroxidase activity of glutathione S-transferase. Alleviation of enhanced oxidative stress and oxygen consumption of L-thyroxine induced hyperthyroid rat liver mitochondria by vitamin E and curcumin. El Feki A. (2004) Erythrocyte osmotic fragility and oxidative stress in experimental hypothyroidism. Serdar Z(2005). . Oxidative stress and serum paraoxonase activity in experimental hypothyroidism: effect of vitamin E supplementation. (1993) Free radical-lipid interactions and their pathological consequences.8: 101-108. 257: 5751–5754. 330(2): 107-12.232: 1021-1026. Dirican M. Protein oxidation in aging. [228] Yilmaz S. Belló-Klein A. Biol. 611: 87–98. 187: 41-51. 272(33): 20313-6. [232] Rice-Evans C. Paital B. Seven A. Stadtman ER. Orrenius S.29(5): 408-13. Toplan S. J Biol Chem. Da Silveira CK. Exp Biol Med. Akyolcu MC. 32: 71-110. Yigit G. Chem. de Assis AM. Fernandes T. Tas S. Das K. [224] SiesH(1997). Oxidativestress. Boulakoud MS. [235] Araujo AS. (2008). . Thyroid hormone-induced haemoglobin changes and antioxidant enzymes response in erythrocytes. (1986) Role of thiols in protection against biological reactive intermediates. disease. Physiol.236 Antioxidant Enzyme [223] Seven R. Biophys. Canatan H(2003). [229] Sarandol E. ( 1997). (2011). Cell Biochem Funct. Prog Lipid Res. 23: 1-8. Oliveira UO. CR Biol. The impact of thyroid activity variations on some oxidizing-stress parameters in rats. Abdennour C.. Chemico-Biological Interactions. Llesuy S. Clin Exp Med. Oxidative stress and protein glycation in primary hypothyroidism. Bobby Z. Kucharski L. Ozan S. [230] Nanda N. Hamide A. J Toxicol Environ Health A.

0). Mitochondrial oxidative phosphorylation is regarded as the main source of free radicals (Naoi and Maruyama. licensee InTech. including oxygen free radicals (superoxide [O2*-]. this article discusses recent advances in the antioxidant therapeutic intervention. and hypochlorous acid (HOCl). 2009)..doi. which permits unrestricted use. peroxyl [RO2*]. 2009).Chapter 9 Mitochondrial Free Radicals. lipoxygenase.5772/51315 1. provided the original work is properly cited. and reproduction in any medium. ROS can be further converted to RNS such as nitric oxide (NO*). and Chronic Hepatitis C Chih-Hung Guo and Pei-Chung Chen Additional information is available at the end of the chapter http://dx. distribution. The excessive generation of ROS and/or RNS can be attributable to the action of nicotinamide adenine dinucleotide phosphate (NADPH) Introduction Clinical evidence shows that oxidative stress plays vital roles in a wide variety of pathological processes. as well as oxidative stress and antioxidant defense in patients with chronic viral hepatitis C. monoamine oxidase. p450 monooxygenase. 1996). which overwhelms the body’s endogenous antioxidant defense capacity. mitochondrial oxidative phosphorylation. singlet oxygen (1O2). and other oxides of nitrogen (Wiseman and Halliwell. 2006.       © 2012 Guo and Chen. free radicals can directly impair mitochondrial structure and function.. free radical molecules are representative of both reactive oxygen species (ROS) and reactive nitrogen species (RNS). This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons. Once generated. xanthine oxidase. peroxynitrite (ONOO-). A decline in mitochondrial respiratory function along with an insufficient supply of energy can significantly increase mitochondrial free radical production (Van Houten et al. Lee et al. 2007). cyclooxygenase. The term ROS refers to several products that result from the partial reduction of oxygen.   . Nutrient Substances. In general. Oxidative stress can arise as result of the production of free radicals. In addition. endothelial NOS (eNOS) uncoupling. and alkoxyl [RO*]). highly reactive molecules containing one or more unpaired electrons. nitrogen dioxide (NO2*). Antioxidants. and some non-radical derivatives of oxygen such as hydrogen peroxide (H2O2). This review article focuses on the production of free radicals from the mitochondria. hydroxyl [OH*]. Increased oxidative damage may enhance inflammatory responses and alter immune function and appear to be involved in the pathologic mechanisms of many diseases. and myeloperoxidase (Muller and Morawietz.

West et al. HCV may escapes innate immune sensing by Toll-like receptors and acerbates HCV infection and replication (Zhang et al.. IFN-a in combination with ribavirin is generally not well tolerated. The combination of pegylated interferon (IFN)-a and ribavirin is the only treatment for chronic HCV infections with proven efficacy.. Insufficient helper (CD4) and cytotoxic (CD8) T-lymphocytes have been shown significantly linked to HCV persistence (Grüngreiff and Reinhold. 2009). and macrophages) and adaptive immunity (Tand B-lymphocytes) have influences in the development and progression of HCV infection. 2008).. neutrophils. hair loss. leading to chronic hepatitis with increasing risk of developing hepatic fibrosis. monocytes.. Chronic Hepatitis C Hepatitis C virus (HCV) infection is a major cause of chronic liver disease. this sometimes makes it difficult for the immune response to suppress or eliminate HCV. The imbalance between cell-mediated and humoral immunity in chronic HCV-infected patients was also observed. nonalcoholic fatty liver disease (NAFLD). and fibrosis stage (Yamada et al. Altered innate immunity (i. Montero Vega and de Andrés Martín. and extrahepatic diseases (Choi and Ou. Unfortunately. steatosis.. 2006). The presence of hepatic steatosis correlates directly with serum and intra-hepatic titers of HCV-RNA (Younossi et al. insomnia. nausea.238 Antioxidant Enzyme 2.. Recent evidence has shown that damaging ROS and mitochondrial injury play a vital role in immune responses (Kohchi et al. fatigue.. Thus. Pillai et al. Thus. Polyak et al. this therapeutic strategy results in a low sustained virologic response (SVR).. but also affects the immune response to HCV infection and decreases SVR (Onoda et al. insulin resistance (IR).. HCV infection frequently does not resolve. obesity. 2009. 2008. 2004.. 3. cough.. nasal congestion. 2006). potential risk factors associated with SVR in HCV-infected patients include baseline HCV-RNA and aminotransferase levels. Pearlman and Traub. depression. dendritic cells. and growth delay (Ko et al. 2008). 2011. 2011). However. NAFLD is not only strongly associated with IR and metabolic syndrome. 2005a). liver cirrhosis. 2007).e. hepatocellular carcinoma. 2010). NK cells. 2010). The major adverse effects are anemia. dyspnea. pruritus. and the adverse side effects may lead to interruption or cessation of therapy. Hepatic stellate cells can be activated by pro-inflammatory cytokines thus contributed . In particular. vertigo. 2004. further advances in effective antiviral treatments against chronic hepatitis C are necessary. There is evidence indicating that SVR is associated with long-term clearance of HCV infection and lower HCV-related complications (Ghany et al. Further. defined as an absence of detectable serum HCV-RNA at six months after completion of antiviral therapy. 2006.. but also with chronic HCV infection. 2011). Hübscher. anorexia.. SVR is achieved in less than 50% of treated patients that have HCV genotype 1 and a high viral load (Ghany et al. alcohol. Although innate immunity can regulate adaptive immune response. Oxidative stress and related risk factors in chronic Hepatitis C Recent studies indicate that oxidative stress not only accelerates the progression of liver damage (Vidali et al.

2012). serum protein carbonyl (De Maria et al..Mitochondrial Free Radicals.. but also the target of potentially damaging free radicals (Orrenius et al. erythrocyte... Recent evidence has demonstrated that this oxidative stress induced during HCV infection via mitochondrial dysfunction generates ROS (Choi and Ou. Gomez-Cabrera et al. 2000. However. 2004. 2006. 2006. few studies have elucidated clinical importance of mitochondrial oxidative damage in chronic hepatitis C.. Lin and Yin. Mahmood et al. 2006). 2009). Oxygen (O2) serves as the terminal electron acceptor for cytochrome c oxidase of complex IV in the mitochondrial electron transport chain (ETC) that catalyzes the four electrons reduction of O2 to H2O (Thannickal and Fanburg. and Chronic Hepatitis C 239 to liver fibrosis.. Mahmood et al. Furthermore.. High serum. Nutrient Substances. plasma. also referred to as NADH: ubiquinone oxidoreductase). Sahach et al. 2007).. Mitochondria-driven free radical propagation Not only are mitochondria the source of adenosine triphosphate (ATP) through oxidative phosphorylation on the inner mitochondrial membrane. Guo et al. and PBMC concentrations of oxidative stress markers serves as an indirect index of mitochondrial oxidative stress in pathologic conditions (ModicaNapolitano et al.. as well as hepatic inducible nitric oxide synthase (iNOS) expression and nitrotyrosine production (i. 2004.. nicotinamide adenine dinucleotide (NADH) and reduced flavin adenine dinucleotide (FADH2). 2000). 2006). Evidence have shown that the involvement of oxidative stress and inflammation in the progression of NAFLD and IR (Reiman et al. 4. plasma.e. Wen et al. 2008.. 2008. Cardin et al.. 1995. ubiquinone) behaves as an electron pool and a mediator of the electron transport between complex II (succinate dehydrogenase. The determination of serum. plasma F2-iso. plasma.prostanes levels (Konishi et al.. 1996... 4-HNE. hepatic and leukocyte 8-oxo-7-hydrodeoxyguanosine (8-oxo-dG)(Farinati et al. and serum malondialdehyde (MDA)(Farinati et al. Mitochondrial energy generation is first accomplished by tricarboxylic acid (TCA) cycle and represented in the form of ATP. Chuma et al. hepatic and serum 4-hydroxy-2-nonenal (4-HNE)(Kageyama et al. 2004). 1996). 1999. including increased hepatic. 2010). oxidative phosphorylation is the primary energy process by which the oxidoreduction energy of mitochondrial electron transport is converted to the highenergy phosphate bond of ATP.. . and decreased glutathione (GSH) and ascorbic acid (vitamin C) levels could reflect mitochondrial dysfunction (Wiswedel et al.position of aromatic amino acids)(Garcia-Monzón et al. 2002. Narasimhan et al. erythrocyte and PBMC concentrations of MDA... lymphocyte. 2000). Mahmood et al. HCV-infected patients have significantly higher oxidative stress status. 2008). Barbaro et al.. superoxide dismutase (SOD) and glutathione peroxidase (GPx). 1999. nitration on the ortho..... and F2-isoprostanes. Antioxidants. urine. plasma. in combination with decreased levels of the antioxidant enzymes catalase. Coenzyme Q (CoQ. erythrocyte. De Maria et al. 2007). 1999a. also referred to as FADH2: succinate CoQ reductase) and complex III (ubiquinone-cytochrome c reductase) with complex I (NADH dehydrogenase. 2001. Farinati et al..

whereas complex III-derived O2*is produced both towards the inner-membrane space and the matrix (Matsuzaki et al.anions can damage heme moieties or enzymes with iron-sulfur centers such as aconitase ([4Fe-4S]→[3Fe-4S]+) to release ferrous ion (Fe+2)(Ott et al. and membrane lipid peroxidation and nitration of proteins on tyrosine residues are promoted (Beckman and Crow.. 2006).is reportedly complexes I and III. a Fenton process). II. It appears that mitochondria are the organelle responsible for the majority of ROS production.. 2003).itself is not particularly reactive in biological systems. 2005). damaged mitochondria produce increasingly more ROS in a process known as ROS-induced ROS release (RIRR) activation..+ NO* (in mitochondria) → ONOO– ONOO– + H+ → OH* + NO2* . II. 2006). Mitochondrial oxidant production O2*. Those superoxide radical anions can also react with NO* to form the damaging oxidant ONOO-. 4. The O2*. 1993). activated reverse electron transfer. the matrix contains the components of the TCA cycle and fatty acid βoxidation pathway. It is also noteworthy that self-amplification of the mitochondrial ROS generation can occur following ROS activation of mitochondrial permeability transition pore (MPTP).1. The major production site of O2*. cytosolic ROS released from the mitochondria could potentially function as second messengers to activate RIRR in neighboring mitochondria (Zorov et al. In turn. The above-described reactions are summarized in the following equations: O2 (in mitochondria) + e. The mtDNA is also a critical target for oxidative damage. ONOOfurther damages complex I.. GPx. 2007). which is more reactive than either precursor (Barber et al.240 Antioxidant Enzyme A decrease in CoQ concentrations. however.(Lenaz et al.. O2*. decline in the electron transport rate. In turn. 2011).. In particular. Leakage of electrons from the mitochondrial ETC can result in incomplete reduction of molecular oxygen to produce O2*-. In addition. or inhibition of electron flow can result in high-energy electrons leaking from the ETC at complexes I... 2006). as well as mitochondrial deoxyribonucleic acid (mtDNA). III.→ O2*O2*. 2007). mDNA can amplify the secondary ROS generation (Van Houten et al. and aconitase (Holley et al. and V as well as mitochondrial SOD. 2009). and IV to produce O2*. Complex I produces O2*predominantly on the matrix side of the inner membrane. Once MPTP opening is triggered. hydroxyl radical and nitric dioxide can be produced from ONOO-. Once the initial ROS generated in mitochondria during oxidative phosphorylation. The Fe+2 can subsequently react with H2O2 to generate hydroxyl radicals (i. Dym) and a further increase in ROS generation by the ETC (Andreyev et al. ROS can induce the simultaneous collapse of the mitochondrial membrane potential (Δѱ.. A growing body of evidence demonstrates that NO diffuses easily along its gradient into mitochondria and that NO is also produced by mitochondria (Alvarez et al.e.

In animal models. 2 O2* – + 2H+ → H2O2 + O2 (i. 2012). superoxide can react with the radical OH* to form highly reactive single oxygen. increase the mitochondrial membrane potential. H2O2 also decomposes to form the highly reactive hydroxyl radical. deplete cytochrome c. ROS-induced mitochondrial damage that is considered an important mechanism involved in the onset and development of a diverse series of pathologies. and thus . Because the mitochondrial membrane is permeable to H2O2. impair the flow of electrons along the ETC. 2009).. In particular. and produce high levels of unwanted oxidants (Mecocci et al. fatty acids of the inner membrane are highly unsaturated (Berdanier.can be catalyzed by antioxidant enzymes. (Cu+ → Cu+2) O2* – + OH* → 1O2* + OH- 4. whose interdependence is crucial for mitochondrial function (Gohil and Greenberg. Mitochondrial membranes are primarily composed of protein and phospholipids. The inevitable by-products of oxidative phosphorylation can modify and damage mtDNA. ROS attack to the mitochondrial membrane lipid components result in lipid peroxidation.. Among these antioxidants. 2003. proteins. 2012. Petrosillo et al.2. lipid. 5. induce cellular calcium (Ca+2) dyshomeostasis. O2*. Nutrient Substances. Consequences of mitochondrial oxidative stress Increased free radicals generated by damaged mitochondria can cause oxidative damage and a significant decline in metabolic processes. and Chronic Hepatitis C 241 As illustrated in the equations below.. Nowak et al.Mitochondrial Free Radicals.. Antioxidants. which alter the membrane potential (Paradies et al.. administration of antioxidant vitamins increases mitochondrial SOD.e. Mei et al. as well as deplete cellular antioxidants. GPx and catalase activity and significantly decreases MDA and carbonyl group levels. oxidate cardiolipin (a phospholipid and located at both the inner and outer membranes). the dismutation reaction of superoxide) O2* – + Fe+3 → O2 + Fe+2 Fe+2 + H2O2 → Fe+3 + OH* + OH-.. Moreover. 2012). decrease respiratory control ratios and cellular oxygen consumption. 1988). and matrix components in the mitochondria. decrease mitochondrial membrane fluidity. 1997. 2004). hydrogen peroxide can diffuse into the cytoplasm. the non-enzymatic antioxidant systems are the second line of defense against free radical damage. Enzymatic antioxidants in mitochondria A network of specific non-enzymatic and enzymatic antioxidants can counteract mitochondrial ROS generation.can either spontaneously dismute to H2O2 by reacting with itself or O2*. Therefore. and this decomposition is accelerated in the presence of either ferrous or cuprous ions (Cu+). It has been known that non-enzymatic antioxidants can act synergistically with enzymatic antioxidants.. which all lead to cell death (Marchi et al.

and therefore mimics anti-inflammatory agent. Additionally. Clinical Mn deficiency is not common. 2009). Rosa et al... thioredoxin. many patients have decreased Mn levels and marked impairments in insulin sensitivity. and catalase. 2011). Mn-SOD can scavenge O2*. vitamin C and E are the non-enzymatic components of the antioxidant defense system in mitochondria (Ott et al. The essential trace element Mn principally supports Mn-SOD activity and is required for a variety of physiological processes.production and tyrosine residue nitration. Mn-SOD participates in the mitochondrial repair processes and has a role along with p53 in preventing mitochondrial DNA damage (Bakthavatchalu et al.2.. higher oxidative stress..formation. nuclei.has a pro-inflammatory role and induces ONOO. and liver diseases (Kitada et al. In the equations that follow. Mn+3 -SOD + O2*. Liu. GPx. Manganese-dependent superoxide dismutase Mn-SOD is highly restricted and located in the mitochondrial matrix. Thus.Zn-SOD.encoded primary antioxidant and plays a vital role in the modulation of redox states. 5. Zang et al. Decreased activity of mitochondrial SOD and GPx were associated with mitochondrial oxidative stress (Zang et al. In this review. Although the dismutation reaction of O2*. catalase. Mn-SOD activity is positively related to the nutritional status of Mn (Luk et al..+ 2H+ → Mn+3 -SOD + H2O2 Mn-SOD not only suppresses ONOO. This enzyme is a nuclear. 2007). and high mitochondrial abnormalities (Han et al.. and recruitment of neutrophils to sites of inflammation. glutathione reductase (GR). CoQ. metabolic diseases. and peroxiredoxin (PRx). 2007. The GSH.. that contains Cu and Zn.→ Mn+2 -SOD + O2 Mn+2 -SOD +O2*.. 2009).can take place spontaneously. 2005). however. similar to cytoplasmic Cu. both the +2 and +3 states of manganese (Mn) are involved in the course of Mn-SOD turnover and the dismutation cycle. but also inhibits membrane lipid peroxidation and mtDNA damage (Stojanović et al. GPx. glutaredoxin. Mn dys-homeostasis may be inactive or decrease Mn-SOD levels. lysosomes.. resulting in decreased Mn-SOD and GPx levels. 2005. Copper. The enzymatic antioxidant systems in mitochondria involve SOD. 5. lipid peroxidation. 2011). and lipoprotein metabolism. Mn-SOD can accelerate the reaction and rapidly convert O2*. 2005). 2002). Altered Mn-SOD levels and chronic inflammation have been associated with neurodegenerative diseases (Li and Zhou.1. glucose H2O2. RodríguezRodríguez et al.. thioredoxin reductase (TrxR). leading to mitochondrial oxidative damage. 2007. zinc-dependent superoxide dismutase A SOD isozyme. 2012). 2005. O2*. is also found localized in the mitochondrial inter-membrane space (Kira et al.242 Antioxidant Enzyme prevents rupture of mitochondrial membrane (Siler-Marsiglio et al.. . 2011). lipoic acid. we discuss the characteristics and functions of SOD.

Cytochrome c also scavenges H2O2 and significantly decreases H2O2 production in vitro (Wang et al. GPx-1 levels in mitochondria are very low. have significant associations with increased levels of MDA. 2003). 2009. 2009). Therefore. Guo et al. Russo.. Both trace elements Cu and Zn participate in the SOD enzymatic mechanisms that play an important role in oxidative balance. 2010. 2009.. Patients with low GPx activity. oxidizes O2*. DNA repair.production in disease conditions.. alterations in immune regulation. catalase. Further. prevent transport of lipid peroxides and oxidative damage. viral infection.Zn-SOD. 2003).ZnSOD deletion and the loss of cytochrome c from the mitochondrial inter. 2006). 2007) that metabolized H2O2 to O2 and H2O. Se deficiency is associated with marked decreases in GPx activity and expression and the inhibition of ATP production. 2012). 5. 2006. GPx-2. Sagdic et al.3. deficiencies of Cu and Zn can result in impairment of the oxidant defense system (i.Zn-SOD gene result in SOD that are highly susceptible to glycation and are linked to elevated ROS production (Takamiya et al.. have significantly decreased levels of inner mitochondrial membrane-associated cytochrome c and increased mitochondrial lipid peroxidation (Kirkinezos et al.Zn-SOD.. and maintain the mitochondrial oxidative-phosphorylation (Brigelius-Flohé. GPx. Nutrient Substances... 2005. and cytochrome c oxidase activities). lower Cu. including GPx-1. 2011).. Song et al. 2011). Cole-Ezea et al. 2009.. Glutathione peroxidase Selenium (Se)-containing GPx is a selenocysteine-containing enzyme. GPx-4 has also been shown to repair mitochondrial oxidative damage (Liang et al. lipoproteins synthesis and secretions have been shown to decline by lipid peroxides (Murthy et al. Thus. and retroviral therapy (Stephensen et al. GPx-4 is membrane-associated that is found in the inter-membrane space of mitochondria. 2003). Recent evidence has shown that transgenic mice with overexpressing mutant Cu. while some O2*. Cu. 2007). The electron carrier cytochrome c. Antioxidants. which is also located in the mitochondrial intermembrane space. and GPx-6.Zn-SOD. GPx-5. and fatty acid hydroperoxides with protect mitochondrial ATP generation. Mutations in the mitochondrial Cu. and Chronic Hepatitis C 243 and peroxisomes (Culotta et al. Apparently.e. Significantly lower serum and erythrocyte Cu. 2005).Zn-SOD activity and higher lipid peroxidation compared to controls have also been observed in patients with mitochondria injury-related disease confitions (Pawlak et al. alkyl peroxides. it can be partially catalyzed to H2O2 by Cu.. and increased oxidative stress (Ho and Song. . GPx can interfere with nuclear factor-kB (NF-kB) activation by IL-1 and TNF-a. and is capable of reducing lipid hydroperoxides.membrane space can lead to reduced ETC and increased O2*.. inhibit cyclooxygenase-2 (COX-2) expression along with reduce production of arachidonic acid (AA) metabolites. indicating that activated GPx can attenuate hepatic triglyceride accumulation. GPx-4. 1998). However. GPx-1 is a major isoform localized in the cytoplasm and mitochondrial matrix (Orrenius et al..back to O2 (Pereverzev et al.. of which multiple isoforms have been identified.escapes into the intermembrane space from the matrix side of the inner mitochondrial membrane. compared with those in the cytoplasma.. GPx-3..Mitochondrial Free Radicals.

disruption of mitochondrial membrane potential.. 2005).244 Antioxidant Enzyme 5. overproduction of mitochondrial ROS and 8-oxo-dG. altered mitochondrial function has shown that not only results in hepatic fat accumulation but also leads to increased ROS that induces inflammatory response. and NADPH dehydrogenase (Moriya et al.4.. 2001). Bäuerle et al. including the following: induction of ROS. Korenaga et al. 2005). thereby activating stellate cells and . Hara et al.. Simula and de Re. Cu. Tinahones et al.. 2006. Quarato et al.. cleavage of DNA repair enzyme poly (ADP-ribose) polymerase. In fact. Various studies have reported lower plasma and erythrocyte catalase activity and increased oxidative stress in patients suffering from mitochondriarelated diseases (Wang et al. 2005.. 2007. 2005a. Decreased mtDNA levels have also been found in these patients (Barbaro et al. Fe overload in vitro were observed to cause further ROS augmentation and amplify the expression of catalase. 2009.. and increased intrahepatic lipid peroxidation in response to CCl4 have been observed (Okuda et al. catalase along with GPx becomes the most important scavenger in the cytosol. 2005). increased mitochondrial permeability transition in response to exogenous oxidants and TNF-a. loss of complex I activity.. Guo et al. decreased GSH.. incorporation of core proteins into the mitochondrial outer membranes and endoplasmic reticulum via its COOHterminal region.. a peptide which regulate Fe metabolism by decreasing Fe absorption). 2010). HCV-induced ROS generation suppresses the expression of hepcidin (i. in turn.. aggravates HCV-induced mitochondrial damage. On the other hand. 2012). Further. decreased GSH and NADPH levels in liver mitochondria. in the presence of large amounts of H2O2 and thereby diffusing to the cytosol from the mitochondria. and enhanced release of cytochrome c from the mitochondrial to the cytosolic fraction (Okuda et al. whereas hepcidin expression was restored by antioxidants (Miura et al. Catalase consists of four subunits. Mitochondrial injury in chronic Hepatitis C Aberrant production of mitochondrial ROS and decrease GSH is though to be caused by HCV core proteins and possibly contributes to oxidative stress in HCV-infected patients (Thorén et al. Choi and Ou. 2010). 2004. b. MPTP was shown to prevent a range of pathological changes included by HCV core proteins. Catalase is found primarily in peroxisomes and is also present in heart mitochondria (Bai and Cederbaum. These observations presented that increased intracellular Fe and oxidative stress. 2006. reduction of respiration. In infectious cell system.... Korenaga et al.. 2011). facilitating the Fe overload. 1999.. 2008). b). The mitochondrial membrane is impermeable to catalase.e.. 6. but has not been found in mitochondria from other tissues (Phung et al. 2005a. In animal models of HCV infection. 2002. Ca+2 overload.Zn-SOD. Catalase Catalase is also an important antioxidant enzyme that catalyzes the conversion of H2O2 to H2O. each of which contains a ferric (Fe+3) heme group bound to its active site (Bras et al. however. HCV core protein has been shown to induced IR (Cheng et al. Fe deficiency causes a significant decrease of catalase activity.. increased ROS. 1994).. Piccoli et al. 2002.

B3. Fe overload induced hepatic 8-oxo-dG and eventually increased mitochondrial injury and the risk of hepatocellular carcinoma development (Furutani et al. biotin. plasma.. B2.Mitochondrial Free Radicals. 2012). 2006).2. B6. B2. serum.. 2005b. and increased oxidative stress and inflammatory responses (Depeint et al. Patients with chronic HCV infection have significantly lowered plasma vitamin B1. Inadequate vitamins and glutathione status The evidence regarding antioxidants. act as cofactors or substrates to protect mitochondrial enzymes. and folic acid) concentrations and lower concentrations of folic acid and vitamin B12 (Roca et al. 2009). 1997... these components can enter cells and mitochondria following exogenous treatment (Liu et al. 2012). or unchanged catalase levels (Ko et al. improve mitochondrial function. and erythrocyte Zn and Se concentrations (Czuczejko et al. 2005b.. SOD. 2005b. some nutrients along with substances play an important role in mitochondrial resuscitation (Liu and Ames. 2005). 2004. 2012). GSH and vitamin B complex (B1. Levent et al. There were significant negative relationships between MDA and HCV-RNA levels with Zn contents in erythrocytes and whole blood. plasma. 2010).. Alterations in enzymatic antioxidants and cofactors Chronic HCV-infected patients were observed to have an increase or decrease in plasma and erythrocyte SOD and GPx activity. pantothenic acid. Anti-HCV therapy causes further decrease in vitamin B1. 6. 6. On the other hand.. 2006. B6 and E concentrations and reduces SOD and GPx activity (Lin and Yin. Antioxidants. Associations have been observed between plasma MDA. increased oxidative stress and altered mitochondrial function both in vitro and in vivo is proven to be involved in chronic hepatitis C infection and is thought to contribute to its progression.. 2005b. B6. Ko et al. and erythrocyte levels of Fe and Cu were significantly higher in hepatitis C patients. and folic acid) protect mitochondria from oxidative damage. 2006). and IR in HCV-infected patients (Ko et al. Moriya et al.. Further.. Guo et al. Chen et al. C. 2003. 2012). and Chronic Hepatitis C 245 fibrogenesis (Fromenty et al. SVR patients have been observed to have lower . Thus. 2009). and decreases in serum... Ko et al. 2006). 6... The plasma homocysteine levels were inversely correlated with the concentrations of folic acid in HCV-infected patients (our unpublished observation). which is influence by vitamin B2. 2012). HCV core proteins induced ROS generation leads to a decreased hepcidin expression also contribute to Fe accumulation (Nagashima et al. B2. 2011.. 12.. higher.. Nutrient Substances. Positive correlations were also noted between plasma Cu and hepatic Fe levels with HCV-RNA in these patients (Fargion et al. Se deficiency has been observed to be inversely associated with HCV-RNA loads. the severity of hepatic fibrosis. and GPx levels with viral loads (Ko et al. 2006. Deficiency in vitamin B complex and GSH leads to decreased mitochondrial membrane potential.. and restore GSH content.1. and folic acid levels.. These patients were also observed to have significantly higher plasma homocysteine (a sulfur-containing amino acid. Khan et al. decreased ATP synthesis.. Rolo et al. lower. Himoto et al. Kaya et al. increased serum and plasma Fe. 2011. Himoto et al.. 2005b)..

.. and mild anemia were significantly decreased (Ko et al. whereas Zn concentrations were remediable by daily administration of 50 mg elemental Zn from Zn gluconate for six months. 2008). However. serum vitamin B12 levels are positively correlated to end-of-treatment response (Rosenberg and Hagen.. 2009). and the lymphatic system. 2007. the ratio of GSSG to GSH increases. 2011). 2012).. 2005a. Guo et al. Thus. Serum Zn level was also found to be significantly higher in complete responders to IFN-a therapy than in non-responders. Besides the above-noted findings. some of the adverse side effects seen during antiviral treatment were similar to the symptoms of Zn deficiency (Saper and Rash.. The non-structural protein NS5A is an active component of HCV replicase and is a Zn metalloprotein. Effects of antioxidants and nutrient substances in Hepatitis C The supplementation of antioxidants or cofactors may show greater benefits in mitochondrial function and antiviral therapy in patients infected with HCV. however. . but it may also reduce hepatic inflammation in chronic hepatitis C patients through induction of Zn metallothionein. 2007. 2009). 2009). suggesting complex interaction between Zn and NS5A activation (Tellinghuisen et al. indicating a high GSH turnover and oxidative stress (Seronello et al. HCV replication enhances activation of the NF-kB-signal pathway triggered by TNF-a (Kanda et al.1. Recently. weight loss. but adverse side effects including gastrointestinal disturbance. these observations suggest that the antioxidant defense is clearly depleted in patients suffering from hepatitis C. Prasad. plasma. Zn supplementation Zn as a cofactor of Cu..246 Antioxidant Enzyme plasma homocysteine levels than non-SVR patients (Borgia et al. The concentrations of serum Zn were declined further in hepatitis C patients receiving treatment with IFN-a and ribavirin. Disturbances in Zn homeostasis can lead to a shift in the Th1/Th2 balance towards a Th2 response (Rink and Haase. 2009). 1998). which functions as a free radical scavenger and immune-modulator (Ko et al. The effects of Zn administration on these side effects. similar to CRP. Zn has been shown to influence antigen-specific immune response and unspecific immune mechanisms (Grüngreiff and Reinhold. 7. Pre-treatment with IFN-a and ribavirin in chronic HCV-infected patients. 2004). 2006). Zn inhibits NF-kB activation results in decreasing inflammatory cytokine levels (Prasad. 7. No apparent difference was seen in virologic response. oxidative stress. Decreased serum and plasma Zn may serve as a potential inflammatory marker. 2010).Zn-SOD and thus is a potential modulator of mitochondrial oxidative phosphorylation. GSH depletion might be one reason for the low rate of patient response to treatment (Bernhard et al.. In addition. the combination of antioxidant with antiviral therapy is recommended for hepatitis C.. Taken together. HCV-infected patients have lower GSH and higher GSSG concentrations in blood. 2005a). and inflammatory responses remain to be determined. liver. Lin and Yin.

2001. 2007). Vitamin E administered in the diet predominantly localizes in the mitochondrial inner. Matsuoka et al. monocytes.and outer-membranes (Lauridsen and Jensen. 2000. Chang et al. 2009). 7. 2012). leukocyte. Zn responders were observed to have a clearly lower cumulative incidence of hepatocellular carcinoma in patients suffering from chronic HCV infection and liver cirrhosis... Administration of Zn supplement has shown to reduce potential oxidative stress and stabilize erythrocyte membrane. and mitochondrial vitamin C concentrations and mitochondria themselves can produce vitamin C (May et al. 2001). Vitamin C supplementation is observed to significantly enhance NK cells activity. inflammation and oxidative stress). and Chronic Hepatitis C 247 In clinical observation. the daily dose of polaprezinc includes 34 mg elemental Zn for six months that markedly decreases both ALT and aspartate aminotransferase (AST) levels and enhances the response to IFN-a therapy (Takagi et al. On the other hand. However. 1997. Effects of vitamin E were also observed which involving in heme biosynthesis. and GSH (Sagun et al.2. and thereby decrease inflammation and liver enzyme levels. the rate of reduction of ALT levels was observed to positively correlate with that of ferritin (i. T. balancing the immune function (Heuser and Vojdani. 2009). vitamin E (a-tocopherol) is a fat-soluble antioxidant that prevents lipid peroxidation and scavenges lipid peroxyl radicals. Se-containing proteins formation.. Polaprezinc was administrated at 51 mg elemental Zn per day for six months to hepatitis C patients. Nutrient Substances. Vitamin C and E supplementation Vitamin C is an essential and water-soluble antioxidant molecule efficiently protects biological materials against damaging free radicals such as OH* and O2*-.. markedly decreases the carbonyl group content in mitochondrial proteins and enhances SOD and citrate synthase activity (Rosa et al. These observations suggest that Zn supplementation in HCV-infected patients may improve nutritional status.. 2007). 2009). and the integrity of mitochondrial membranes (Mabalirajan et al. and the mitochondrial reduction of vitamin E. 2011).. impaired membrane integrity.Mitochondrial Free Radicals. Antioxidants. and increase the Th1/Th2 ratio.. ascorbic .... suggesting a higher daily Zn dose may be needed to increase the response to IFN-a treatment (Matsuoka et al. Zn administration did not affect virologic response (Takagi et al.. whereas Zn administration did not affect virologic response (Himoto et al. 2006). clinical observation has been shown to have significantly lower plasma concentrations of a-tocopherol... Vitamin C severs as a cofactor for enzymes involved in synthesis of collagen or carnitine (essential for the transport of fatty acids into mitochondria). lipoic acid. a clinical marker of iron storage protein. On the other hand. but not to inhibit virus. and altered membrane fluidity (Maggini et al. ferricytochrome c.and B-lymphocytes. immune system modulation. 2009). SVR. and adverse side effects except for gastrointestinal disturbance (Suzuki et al. Nagamine et al. Studies in an animal model demonstrates that combined administration of vitamin E and C..e.. 2009). 2007). Levine et al. Deficiency in vitamin C can cause oxidative stress and lead to decreased immune response. On the other hand. 2005. For those Zn non-responders. In vivo study has shown that administration of vitamin C supplementation markedly increases plasma.

Patients undergoing IFN-a and ribavirin treatment have markedly higher AA and decreased EPA levels in PBMC. 2006).248 Antioxidant Enzyme acid. which can offers greater benefit in raising GSH concentrations. a combination of vitamin C and other antioxidants may further increase the efficiency of antiviral therapy. Polaprezinc supplementation (equivalent to 34 mg elemental Zn) daily for 12 months . and serum levels of ALT decreased significantly after two weeks of treatment with Vitamin E (500 mg a-tocopherol/day) and C (750 mg ascorbic acid/day) supplementation (Murakami et al. Eight weeks of such treatment led to increases in hemoglobin levels and significantly elevated erythrocyte EPA concentrations in these patients (Hino et al. These patients who take greater amount of vitamin C. 2009). and GSH in HCV. The combined administration of vitamin E (1342 mg a-tocopherol/day) with vitamin C (100 mg ascorbic acid/day) for 48 weeks has been shown to decreases in ribavirin-induced anemia but not SVR in patients with HCV infection (Kawaguchi et al. Plasma a-tocopherol or ascorbic acid levels were negatively correlated with F2-isoprostane and ALT levels (our unpublished results). The combined administration of vitamin E with vitamin C for four weeks prevents the decrease in PBMC EPA and the increase in the ratio of AA to EPA in these patients (Murakami et al. 2012). the dosages of vitamin C can range from 1000 mg to 6000 mg. increase oxidative membrane damage. Thus. Vitamin C... 2011).. further studies will be needed to clarify the effect of a combination of vitamin C and vitamin E on chronic hepatitis C patients treated with IFN-a and ribavirin. 2006). and accelerate hemolytic anemia in the combined therapy of IFN-a and ribavirin (Assem and Yousri..3. A combination antioxidant treatment improves the antioxidant capacity than vitamin E alone in HCV-infected patients.. 500 mg ascorbic acid/day and 40 mg Zn/day for six months) showed significant improvement in antioxidant enzyme activity and ALT reduction (Farias et al. It seems reasonable that co-administration of Zn and antioxidants may be more effective in antiviral therapy. and fibrosis process in HCV-infected patients (Houglum et al. thereby protecting erythrocyte EPA depletion. 2007). and Zn supplementation There is a need for effective antiviral treatments that decrease the inflammation and increase antiviral response. 1997). 2006).. E. virologic response. 7. HCV-infected patients receiving antioxidant supplementation (combination of 800 mg atocopherol/day. Administration of vitamin E (804 mg a-tocopherol/day) for eight weeks has been shown to decrease protein carbonyl group levels. Additionally.infected patients (Lin and Yin. whereas did not significantly affect ALT levels. Based on our previous experience with clinical trials. Further studies indicate that plasma and erythrocyte a-tocopherol and plasma ascorbic acid levels increased. Ribavirin-induced ROS would increase EPA peroxidation and result in alteration in fatty acid compositions of erythrocyte membranes. Studies have demonstrated that ribavirin’s toxicity decrease intracellular energy metabolism. The availability of such treatments would maintain erythrocyte integrity and resistance to hemolysis.

IR... Se deficiency was observed to have reduced T-lymphocytes. E.. and prevent the decrease in polyunsaturated fatty acids of erythrocyte membrane phospholipids in patients during IFN-a plus ribavirin therapy with vitamin E (300 mg a-tocopherol acetate/day) and vitamin C (600 mg ascorbic acid/day) supplementation (Murakami et al. Khan et al. 7. 2005b. and HCV-RNA levels. 2012). and altered innate immunity (NK cells. the supplementation had no effects on ALT. HCV-RNA load. Associations have been observed between plasma MDA. and 200 mg Se/day for six months) had significantly higher plasma levels of ascorbic acid and a-tocopherol and higher erythrocyte GPx activity. protein carbonyl group. However. and Se supplementation Se also plays a vital role in the redox regulation and antioxidant function and immunomodulatory effects.. 2005b. On the other hand.Mitochondrial Free Radicals. and P-C Chen. 2011. Guo et al. these results might be attributed to viral genotypes or a much high viral load. 2012). Hoffmann and Berry. Also. 2005b. increased IR is associated with higher HCV-RNA levels (Ko et al. Thus. Significantly higher viral loads correlate with decreased blood Se and GPx activity in HCV-infected patients (Ko et al. 500 mg ascorbic acid/day. 2007). . which is defined as undetectable HCV-RNA or a less than two log drop in HCV-RNA at week 12.. and neutrophils)(Maggini et al. Vitamin C. HCV. 2006). and Chronic Hepatitis C 249 has been observed to significantly decrease plasma MDA. dendritic cells. W-S Ko... erythrocyte GPx activity and Zn concentrations.4. this dosage may not be enough to be therapeutic for Se therapy. have significantly higher plasma Se concentrations and GPx activity compared to those with nonEVR patients. Se-dependent GPx modules encoded in RNA viruses have been found (Zhang et al. HCV-infected patients who received antioxidant supplementation (633 mg atocopherol/day. Serum and plasma Se levels significantly decrease in proportion to the severity of hepatic fibrosis. and ALT levels with plasma Se concentrations (our unpublished results).. Based on our previous experience with clinical trials. Se status might be a sensitive indicator for the sustained response to therapy in chronic hepatitis C patients. These effects are potentiated by the presence of vitamin E. Antioxidants. and correlate positively with plasma.. decreased Se levels and Sedependent GPx activity either in plasma or in erythrocytes suggests that the anti-oxidative capability is limited in patients with chronic HCV infection (Ko et al. viral load or oxidative markers (Groenbaek et al. 2011). On the basis of the finding. 2008). A similar difference between SVR and non-SVR patients has been observed (C-H Guo.. 2007. impaired lymphocyte proliferation and function. Results from clinical studies suggest that Zn supplementation is more effective against HCV when given along with antioxidants (combinations of vitamin C and vitamin E).infected patients with early virological response (EVR). unpublished results). Nutrient Substances. Himoto et al. Himoto et al. even though the recommended dietary allowances of Se in the USA are 55-70 mg/day for adults.. This finding is difficult to interpret because the potential synergy between vitamin E and Se is well documented. 1999).

2011). Thus. 7. IFN-a and ribavirin treatment can further lead to EPA depletion (Murakami et al.. 2003). which was due to immuno-modulatory effects via EPA and DHA (Mori et al. Treatment with EPA and DHA was observed to reduce in plasma and urinary F2-isoprostanes.. Mitochondrial phospholipid composition. Both EPA and DHA have been shown to exert antioxidant. Both EPA and DHA may induce b-oxidation of fatty acid and upregulation of mitochondrial biogenesis (Ruzickova et al. Vitamin C. 2009). Cardiolipin is composed of four linoleic acid side chains.. 2005). AlGayyar et al. It is proposed that administration of either EPA alone or both DHA and EPA may compensate the loss of EPA by ribavirin induction in erythrocyte membrane.. 2009.. Flachs et al. 2009). Calder. which is essential for normal mitochondrial respiration.. 1999). 2006) and subsequently incorporate into the mitochondrial membranes and maintain the membrane fluidity (Chapkin et al. 2010). both EPA and DHA are essential for mitochondrial function.. HCVinfected patients have markedly higher AA and decreased EPA levels in PBMC compared to the healthy controls. 2004. 2002. there is variability in the absorption and therapeutic mechanism of Se that is related to the forms of Se. anti-inflammatory activities (efficiently suppressed NF-kB activation). 2001) and hepatic lipid metabolism (Araya et al. In addition. After oral treatment with EPA (1. These changes were accompanied by an increase in DHA and EPA level in mitochondrial phospholipids and decreased AA level (Khairallah et al.inflammatory lipid mediators (Merzouk et al. In rat model. Recent study has shown that increased saturated fatty acids and cholesterol associated with alteration in mitochondrial membrane and cardiolipin oxidation. 2004. inhibition of HCV-RNA replication (Liu et al.. 2010).. eicosapentaeoic acid (EPA) and docosahexaenoic acid (DHA).. 2011)..8 g/day) for 12 weeks. are referred to as omega-3 or n-3 fatty acids.. however. EPA treatment lowered plasma triglyceride and increased b-oxidation of fatty acid in hepatic mitochondria and carnitine palmitoyltransferase-1 activity (Madsen et al.. Khairallah et al. 2006). E. These patients had clearly lower plasma .. and reduction of pro. Above observations suggest that EPA and DHA supplementation have potential beneficial effects in HCVinfected patients with and without NAFLD.250 Antioxidant Enzyme Additionally. Further large-scale studies are needed to elucidate the effects of Se alone or in combination in chronic hepatitis C patients treated with IFN-a and ribavirin. particularly in releases of AA and cardiolipin contents are major contributors to trigger MPTP opening. patients were observed to have significantly decreased ALT levels and higher Th1/Th2 ratio. 2003. 2010). substitute such as long chain saturated and monounsaturated fatty acids weaken mitochondrial function (O'Shea et al.. Supplementation with DHA alone or both DHA and EPA significantly delays Ca2+-induced MPTP opening in normal and hypertrophied myocardium (O'Shea et al. which are required for HCV replication (Roe et al.. increases in insulin sensitivity (Ye et al. and eicosapentaeoic acid supplementation Two components of fish oil.5.

2008). and hypertriglyceridemia.. although ethyl ester (EE). Reductions in the rate of non-responders were also observed (manuscript from Dr.Mitochondrial Free Radicals. 2011). glycyrrhiza.. 2007). Neri et al..6. and decreased HCV-RNA loads. increase mitochondrial ATP production. and antioxidants (300 mg a-tocopherol/day and 600 mg ascorbic acid/day). EPA supplementation also decreased the ratio of AA to EPA and increased leukocyte levels (Tomioka et al. Combination of antioxidants and nutrient substances Beside the use of those antioxidants. and to arrest the progression of clinical symptoms. Beneficial therapeutic responses to CoQ (Gane et al. significant increases in oxidative stress and alterations in mitochondrial function have been observed in patients infected with HCV. standard treatment with multiple nutrient supplements (including 2000 mg/day of ascorbic acid. ribavirin. 2005. Such supplements also produce mild beneficial effect in the inflammatory response of patients who are non-responders to IFN-a (Melhem et al. the metabolites of EE-form (catalyzed by carboxy ester hydrolase) that may contribute to the adverse events include gastrointestinal disorder. suggesting treatment with EPA prevents AA accumulation.. Both ethanol and methanol. Thus. P-J. Se. and vitamin B complex) for six months demonstrated significant improvements in immune function.. these observations suggest that the combination of EPA and antioxidants (vitamin C and vitamin E) may ameliorate inflammation and oxidative stress and thereby increase the response of antiviral therapy in HCV-infected patients. EE-form fish oil has shown some unpredictable side effects in clinical application (Data sources from Dr. Patients with chronic hepatitis C who received a combination of natural supplements (CoQ. as reviewed in Tarnopolsky (2008) and Orsucci et al (2009). The bioavailability and efficacy of fish oils are frequently controversial. 800 IU/day of d-a-tocopherol as well as silymarin.. or N-acetyl-cysteine (Cimino et al. Antioxidants. Furthermore. Liu). EPA/DHA. Further. and Chronic Hepatitis C 251 and serum 8-oxo-dG levels after six-months of treatment with IFN-a. 2001.. Thus. Malaguarnera et al. These observations suggest that the synergistic effects of antioxidants and mitochondria-related nutrient substances may be effective in antiviral therapy. 2000) have been observed in patients with hepatitis C. Simon Hsia). some nutrient substances treatments in mitochondrial damage have been reported to produce a positive effect. 7.or triglyceride (TG)-form. vomiting. carnitine (Romano et al. 150 mg/day of GSH. improvements in liver histological status. particularly in chronic HCV-infected patients with NAFLD.. the choice of fish oils for clinical application will have to be considered. 1998.. Nutrient Substances. and decreases in HCV-RNA loads. 150 mg/day of LA. 2008. as well as in animal and cell . Gabbay et al. choline (Niederau et al. reduced adverse side effects. 8. and schizandrae) for six months leads to significant declines in ALT levels. the combined treatment with antioxidants and other nutrients has been show to efficiently decrease mitochondrial oxidative injury.. Kawashima et al. Summary In conclusion. 1998). has recently been introduced into clinical practices. 2010).

Additionally.10:189-199. References Al-Gayyar MM. Starkov AA. Cederbaum AI. . Biol Signals Recept 2001. Oxidative stress in ALS: a mechanism of neurodegeneration and a therapeutic target. Yousri M. Asti A. Pettinelli P. Grisorio B. Barber SC. Mitochondrial metabolism of reactive oxygen species.induced haemolytic anemia in chronic hepatitis C patients: an Egyptian survey. Zaobornyj T. Ribersani M. mitochondrial dysfunction inducted by HCV reduces the b-oxidation of fatty acid and accelerates ROS formation. Mead RJ. Biochem Biophys Res Commun 2003. Boveris A. causing fat accumulation and hepatic lipid peroxidation.31:2129-2239. Holley AK.106:635–643. Biochemistry (Mosc) 2005. [Epub ahead of print] Alvarez S. Di Lorenzo G. et al. Increase in long-chain polyunsaturated fatty acid n-6/n-3 ratio in relation to hepatic steatosis in patients with non. Manganese superoxide dismutase is a mitochondrial fidelity protein that protects Polγ against UVinduced inactivation. et al. Am J Gastroenterol 1999. Institute of Biomedical Nutrition. Belloni G. Biochim Biophys Acta 2006.252 Antioxidant Enzyme models of HCV. Shams ME. Int J Hepatol 2011. Kushnareva YE. Assem M. Dey S. Barbaro G.94:2198-2205. Pharm Biol 2011 Nov 21. Valdez LB.1762:1051-1067. Noel T. Taiwan.305:771-775. Hung-Kuang University. Author details Chih-Hung Guo and Pei-Chung Chen Micro-Nutrition Laboratory. Oxygen dependence of mitochondrial nitric oxide synthase activity. Rodrigo R. Clinical observations indicate that therapeutic approaches targeting mitochondrial biogenesis that decrease oxidative damage and increase the response to antiviral therapy are clinically beneficial for chronic HCV-infected patients undergoing IFN-a and ribavirin treatment. College of Medicine & Nursing. Bakthavatchalu V. Xu Y. Hepatocellular mitochondrial alterations in patients with chronic hepatitis C: ultrastructural and biochemical findings. Reduced mitochondrial biogenesis also contributes to development of IR. et al.70:200-214. Shaw PJ. Andreyev AY. Bai J. Thielemann L. Jungsuwadee P. Araya J. Orellana M. Mitochondrial catalase and oxidative injury.alcoholic fatty liver disease. Videla LA. Impact of pentoxifylline and vitamin E on ribavirin. Republic of China 9. Fish oil improves lipid metabolism and ameliorates inflammation in patients with metabolic syndrome: Impact of nonalcoholic fatty liver disease. Oncogene 2012. Clin Sci (Lond) 2004. HCV-induced mitochondrial oxidative damage and increased ROS production facilitate HCV replication and contribute to the progression of hepatitis C.2011:530949. Barakat EA.

Chen CS. Kim W.34:587-592. Antioxidants. Cheng Y. Mallolas J. Mauss S. Prostaglandins Leukot Essent Fatty Acids 2009. Dharancy S. Bhagavan HN.6:135-139. Gentile I. Junker E. and Chronic Hepatitis C 253 Bäuerle J. Lupton JR. Polymorphic variations in manganese superoxide dismutase (MnSOD). Cardin R. Li M. Nutrient Substances. Brigelius-Flohé R. metabolism and pharmaco. Davidson LA. but decreases eosinophilic infiltration in bronchoalveolar lavage fluid of ovalbumin. Farinati F.kinetics. Chopra RK. Lauterburg BH.81:187-191. Chen H. J Hepatol 2001.46:145-149. and catalase (CAT) contribute to elevated plasma triglyceride levels in Chinese patients with type 2 diabetes or diabetic cardiovascular disease. Am J Clin Nutr 2006.52:189195. inammation. Lipids 2002. McMurray DN. Beckman JS. and cell death. Pathological implications of nitric oxide. Nutr Rev 1988. de Caterina M.28:751-755. Fortunato G. Borutaite V. Zhou W. 57:10471-10476. High dose vitamin C supplementation increases the Th1/Th2 cytokine secretion ratio. n−3 polyunsaturated fatty acids.37:193-199. Homocysteine levels and sustained virological response to pegylated-interferon alpha2b plus ribavirin therapy for chronic hepatitis C: a prospective study. Zhao R.20:205-220. mitochondria. Lin JY. et al. Liver Int 2009. Borgia G. 387:1329-1335. Tiribelli C. Desreumaux P. et al. Dietary docosahexaenoic and eicosapentaenoic acid: emerging mediators of inflammation. Borelli S.363:85-91. and inammatory diseases. Malapel M. DNA oxidative damage in leukocytes correlates with the severity of HCV-related liver disease: validation in an open population study. Hettinger A. Nitric oxide. J Hepatol 1998. IUBMB Life 2001. et al.29:248-452. J Agric Food Chem 2009. Chapkin RS. . Glutathione peroxidases and redox-regulated transcription factors. Yu M. Clément MV. Brown GC. Hepatitis C virus infection downregulates the expression of peroxisome proliferator-activated receptor alpha and carnitine palmitoyl acyl-CoA transferase 1A. et al. Saccoccio G. Role of membrane lipids in metabolic regulation. Biochem Soc Trans 1993. Henderson CE. Dietary n3 PUFA alter colonocyte mitochondrial membrane composition and function. Chapkin RS.11:7591-7596. Laguno M. Bernhard MC. Mol Cell Biochem 2012.21:330-334. Biol Chem 2006. superoxide and peroxynitrite formation. Brenner C. glutathione and homocysteine in plasma of patients with chronic hepatitis C treated with interferon-α with and without supplementation with N-acetylcysteine.40:445-453. Pervaiz S. Free Radic Res 2006.Mitochondrial Free Radicals. Time course of total cysteine. Hong MY. Miquel R. Mitochondrial DNA depletion in liver tissue of patients infected with hepatitis C virus: contributing effect of HIV infection? HIV Med 2005. Calder PC. glutathione peroxidase-1 (GPX1). Borrelli F. Reactive oxygen species and the mitochondrial signaling pathway of cell death. Bras ML.83 (6 Suppl):1505S-1519S. Chang HH.sensitized and challenged mice. Bellentani S. Zhu Q. Fan YY. Masutti F. Berdanier CD. Sanders LM. tissue uptake. Histol Histopathol 2005. World J Gastroenterol 2005. Crow JP. Coenzyme Q10: Absorption. Murillas J.

Am J Physiol Gastrointest Liver Physiol 2006. Mechanisms of liver injury. Farias MS. Mehta R. Floyd RA. Parisotto EB. Free Radic Biol Med 1999. Staubach-Topczewska E. Fargion S. Fracanzani AL. Cimino L. . Sacchetti L. Degan P. Van Thiel DH. Ribeiro CM. et al. Hige S. Mattioli M. Colantoni A. J Gastroenterol Hepatol 2008. O’Halloran TV. Diabetes Metab 2004. et al.30:121-138. De Maria N. Selenium. [Epub ahead of print] Culotta VC. Robin MA.22: 449-456.23:14311436. Halota W. O'Brien PJ. Gastroenterol Hepatol 2012 May 17. [Epub ahead of print] Farinati F. Zachara BA. Molteni V. Franssen-van Hal N. Oxidative stress in the pathogenesis of hepatitis C virus. glutathione and glutathione peroxidase in blood of patients with chronic liver diseases. Yang M. Polyunsaturated fatty acids of marine origin upregulate mitochondrial biogenesis and induce beta-oxidation in white fat. Budni P. Lecis E. Bruce WR.290:G847-G851. Sampietro M. 9:497-503. Salvatore F. Mansouri A. 50:1147-1154. Natsuizaka M. Chuma M. Association between reactive oxygen species and disease activity in chronic hepatitis C.48:2365-2375. Cardin R. Chem Biol Interact 2006. Czuczejko J. Rossmeisl M. Fagiuoli S. J Hepatol 1995. Farinati F. Boldorini R. Belisario MA. Acta Biochim Polon 2003. De Maria N. Cardin R. Brauner P. Ital J Gastroenterol Hepatol 1998. Shangari N. Free Radic Biol Med 1996.30:189-193. Swan D. Kedziora J. Diabetologia 2005. Horakova O.163:113-132. 27:1284-1291. Flachs P. Activation of superoxide dismutase: putting the metal to pedal. Pessayre D. Santos CE. Dias JF. Shanley D.1763:747-758. et al. Hesketh J. Mitochondrial function and toxicity: Role of B vitamins on the one-carbon transfer pathways. Marafin C. et al. Free Radic Biol Med 2012 May 24. Eur J Gastroenterol Hepatol 1997. Antioxidant supplementation attenuates oxidative stress in chronic hepatitis C patients. Farinati F. Oxidative DNA damage in circulating leukocytes occurs as an early event in chronic HCV infection. Liu GJ. Biochim Biophys Acta 2006. Effect of Nacetyl-cysteine on lymphomonocyte glutathione and response to interferon treatment in C-virus chronic hepatitis. Yamamoto Y. Liver iron influences the response to interferon alpha therapy in chronic hepatitis C. lipid peroxidation and glutathione turnover in chronic anti-HCV positive hepatitis.254 Antioxidant Enzyme Choi J. et al. D'Ascoli B. et al. De Maria N. Cole-Ezea P. et al. Fromenty B. Rogers BK. et al. 8-Hydroxy-2'deoxy-guanosine is a risk factor for development of hepatocellular carcinoma in patients with chronic hepatitis C virus infection. Glutathione Peroxidase 4 has a major role in protecting mitochondria from oxidative damage and maintaining oxidative phosphorylation complexes in gut epithelial cells. Ou JHJ. Igoudjil A.21:291-295. Della Libera G. Intrieri M. Nakanishi M. Iron storage. III. Depeint F. Ogawa K. The ins and outs of mitochondrial dysfunction in NASH. Pecina P.

Seeff LB. Keogh GF. Hino K. Gabbay E. Domenech E. An Update on Treatment of Genotype 1 Chronic Hepatitis C Virus Infection: 2011 Practice Guideline by the American Association for the Study of Liver Diseases Hepatology 2011. Mitochondrial membrane biogenesis: phospholipids and proteins go hand in hand. Vallyathan V. Okuda M. Chen PC.41:257-268. Castranova V. Hepatitis C virus core protein inhibits deoxycholic acid-mediated apoptosis despite generating mitochondrial reactive oxygen species. J Gastroenterol 2006. Antioxidant therapy for chronic hepatitis C after failure of interferon: results of phase II randomized. Intrahepatic accumulation of nitrotyrosine in chronic viral hepatitis is associated with histological severity of liver disease. Trace metal imbalance associated with oxidative stress and inflammatory status in anti-hepatitis C virus antibody positive subjects. . Gohil VM.130: 2087-2098. Arduini A.18:985-989. et al. Cu/Zn ratios are Associated with nutritional status. The mitochondriatargeted anti-oxidant mitoquinone decreases liver damage in a phase II study of hepatitis C patients.54:1433-1444. Krarup HB. Shih MY. Am J Respir Crit Care Med 2005. Moreno-Otero R. Clin Biochem 2011. Chen PC. The effect of antioxidant supplementation on hepatitis C viral load. J Hepatol 2000.172:1541-1548. Hansen M. Thomas DL. Pappo O. Yamaguchi Y. Furutani T. Nutrient Substances. Reinhold D. et al. Ghany MG. Groenbaek K. et al. Zinc: A complementary factor in the treatment of chronic hepatitis C? Mol Med Report 2010.44:275-280. Am J Clin Nutr 2008. Hepatology 2009. Rowe M. Hidaka I. Romagnoli M. Guo CH.184:469-472. Kashon ML. Gibson M. Apolinario A. Ko WS. Hara Y. Weilert F. Ring-Larsen H. Thomas DL. Correlates of oxidative stress and free-radical activity in serum from asymptomatic shipyard welders.Mitochondrial Free Radicals. Han SG. Nishina S. Lin KP. Zubia I.33:288-296. Strader DB. Gastroenterology 2006. J Cell Biol 2009. Gane EJ. inflammation and immune abnormalities in patients on peritoneal dialysis. doubleblind placebo controlled clinical trial. Gondo T. Antioxidants. Majano PL. Nelson DR. Yeh MS. Seeff LB.3:371-375.30:1019-1026. and Chronic Hepatitis C 255 Furutani T. Sanz P. management. Eur J Gastroenterol Hepatol 2006.49:1335-1374. Greenberg ML. Diagnosis.13:5317-5323. García-Monzón C. Okuda M. Wang CL. Hepatic iron overload induces hepatocellular carcinoma in transgenic mice expressing the hepatitis C virus polyprotein. Kim Y. et al. Kitase A. oxidative stress. Zabrecky G. Hsiung DY. Oral administration of vitamin C decreases muscle mitochondrial biogenesis and hampers training. Gomez-Cabrera MC. transaminases and oxidative status: a randomized trial among chronic hepatitis C virus-infected patients.87:142-149. Borras C. Strader DB. Guo CH. Hino K. Grüngreiff K.induced adaptations in endurance performance. and treatment of hepatitis C: an update. Ghany MG. et al. World J Gastroenterol 2007. Hemed N. Friis H. Pack DL. Lockhart MM. Pallardo FV. Zigmond E. Orr DW.32:331-338. Liver Int 2010. Environ Toxicol Pharmacol 2012.

High-dose vitamins E and C supplementation prevents ribavirin-induced hemolytic anemia in patients with chronic hepatitis C.and AP-1-signaling induced by hepatitis B virus X. Nakai S.19:291-312. Iwane S. Khanna N. Kakibuchi N. Hino K. Inukai M. Stanley WC. Selenium deficiency is associated with insulin resistance in patients with hepatitis C virus-related chronic liver disease. Cicioğlu Aridoğan B. Manganese Superoxide Dismutase: Guardian of the Powerhouse.12:640-645. Immunopharmacol Immunotoxicol 1997. Bakthavatchalu V. Zinc and prostatic cancer. Successful interferon therapy reverses enhanced hepatic iron accumulation and lipid peroxidation in chronic hepatitis C. Holley AK. Takahashi K. Cancer Lett 2006. Chojkier M. Alpha-tocopherol [corrected] and ascorbic acid attenuates the ribavirin [corrected] induced decrease of eicosapentaenoic acid in erythrocyte membrane in chronic hepatitis C patients. Tsukamoto I.465. Sütüçü R. Nakamura H. Histological assessment of non-alcoholic fatty liver disease. Furutani T. Berry MJ. Matsuki M. Ho E. J Pharmacol Exp Ther 2010. Scand J Gastroenterol 2007. Lyche K. Treatment with docosa. Goda F. A pilot study of the effects of d-alphatocopherol on hepatic stellate cell activation in chronic hepatitis C. Nutr Res 2011. 21:1269-1275.43:325333. Yoneyama H.12:7114-7162. Kanda T. Murakami Y.42:1078-1087.49:450. Am J Gastroenterol 2000. Hosomi N. Mikrobiyol Bul 2006. Houglum K. delays Ca2+-induced mitochondria permeability transition in normal and hypertrophied myocardium. Hübscher SG. Efficacy of zinc administration in patients with hepatitis C virus-related chronic liver disease.31:829-835. Deguchi A.113:1069-1073. Curr Opin Clin Nutr Metab Care 2009. Sesli Cetin E. St Clair DK.256 Antioxidant Enzyme Heuser G. Kinekawa F. Kawasoe H.Eicosapentaenoic acid supplementation for chronic hepatitis C patients during combination therapy of pegylated interferon alpha-2b and ribavirin. Koyabu T.234:143-148. Mol Nutr Food Res 2008. Hara Y. Song Y. et al. but not eicosapentaenoic acid.335:155-162. Kawashima A. Int J Mol Sci 2011. The influence of selenium on immune responses. Himoto T. Yokosuka O. Kobayashi Y. Kurokohchi K. Vojdani A. Enhancement of natural killer cell activity and T and B cell function by buffered vitamin C in patients exposed to toxic chemicals: the role of protein kinase-C. Hepatol Res 2007.40:55-61. et al. Hoffmann PR.hexaenoic acid. Histopathology 2006. et al. Delibaş N. Lipids 2008. Ario K. et al. Kageyama F.37:317-324. Kawasaki T. Nagai A. . Saisho H. Kawaguchi Y. Aktütrk O.95:1041-1050. O'Shea KM. Velez-Roman JM. The relationship between viral load and malondialdehyde and antioxidant enzymes in patients with hepatitis C virus infection. J Gastroenterol Hepatol 2006. Kitase A. Brown BH. Gastroenterology 1997. Khairallah RJ. Toyokuni S. Uchida K. Des Rosiers C. State of hepatitis C viral replication enhances activation of NF-kB.52: 1273-1280. Masugata H. Kawakami T. Venkataramani A. Mizuta T. Nagao K. Himoto T. Kaya S. Murakami Y. et al.

60:634-643. Blood micronutrient. Diabetes 2011. Koya D. Perez-Pinzon MA. and Chronic Hepatitis C 257 Khan MS.11:4697-4702. Ko WS. Arias LJ. Genes Nutr 2012 Feb 22. Vitamin C: a concentration-function approach yields pharmacology and therapeutic discoveries. et al. Guo CH. Increased lipid peroxidation in patients with non-alcoholic fatty liver disease and chronic hepatitis C as measured by the plasma level of 8-isoprostane. Antioxidants. Nutrient Substances. Imaizumi N. Sato EF. Anticancer Res 2009. Oxidative stress and antioxidant defense in patients with chronic hepatitis C patients before and after pegylated interferon alfa-2b plus ribavirin therapy. ROS and innate immunity.142:45-52.25:164-172. a-Tocopherol incorporation in mitochondria and microsomes upon supra. oxidative stress. Ko WS. Adv Nutr 2011. Soma G. Mitochondrial dysfunction in hepatitis C. Cytochrome c association with the inner mitochondrial membrane is impaired in the CNS of G93A-SOD1 mice. Lenaz G. Sumida Y. Effects of zinc supplementation on the treatment of chronic hepatitis C patients with interferon and ribavirin. Kume S. Association of Cu. Li Y. Lee SM. J Clin Gastroenterol 2005b. Korenaga M. J Gastroenterol Hepatol 2006. . Li C. Wang T. Inoue M. Genova ML. Protective effects of combined ischemic preconditioning and ascorbic acid on mitochondrial injury in hepatic ischemia/reperfusion. Saudi J Gastroenterol 2012. Hernandez D. Sun J. Zhou HM. and viral load in patients with chronic hepatitis C. Lee JS. Ayşe E. Rauf N. Levent G. Polat EC. The possible role of selenium concentration in hepatitis B and C patients. Enzyme Res 2011. Iwasa M.nutritional vitamin E supplementation. [Epub ahead of print] Lee WY. Yeh MS.4:25. Formiggini G. Hepatitis C virus core protein inhibits mitochondrial electron transport and increases reactive oxygen species (ROS) production.independent pathway. Chiou YL. Espey MG. Wang T. Ali A. Kirkinezos IG.821. Oca-Cossio J. Jensen SK. Ahmet S. Chen PC. Kira Y. et al. Hsu GSW. Konishi M. Lauridsen C. Fato R.399:96-102. The role of manganese superoxide dismutase in inflammation defense. Okuda M. Li Y. Nishizawa T. Bacman SR.29:817.2011:387176.Mitochondrial Free Radicals. Levine M. Weinman SA. Padayatty SJ.39(4 Suppl 2): S162-S166.38: 614-620. Lin LY. Weinman SA. Otani K.2:78-88. The role of Coenzyme Q in mitochondrial electron transport. Guo CH. et al. Ali I. Mitochodrion 2007. Inagawa H. Clin Biochem 2005a.Zn-type superoxide dismutase with mitochondria and peroxisomes. Yaun SR. 18:106-110.. Araki J. Ahmet A. World J Gastroenterol 2005b. Chan T.280:37481-37488. Kohchi C. Yeh MS. J Biol Chem 2005a. Katsuki A. J Neurosci 2005. J Surg Res 2007. Dilawar S.7S:S8-S33. Hsu GS. Kitada M. Resveratrol improves oxidative stress and protects against diabetic nephropathy through normalization of Mn-SOD dysfunction in AMPK/SIRT1. Aytaç C.21:18211825 Korenaga M. Showalter LA. Arch Biochem Biophys 2002. Kobayashi Y. J Transl Med 2006.

Eicosapentaenoic and docosahexaenoic acid affect mitochondrial and peroxisomal fatty acid oxidation in relation to substrate preference.8:67-89. J Signal Transduct 2012. Hornig DH. Madsen L. BMC Gastroenterol 2010. Holstein D. Ghosh B. Arakawa Y. Yin MC. Qu S. Yang M. Niiyama G. Van Remmen H. Richardson A. Marchi S. and Parkinson's disease. Oshiro S. Agnoletto C. Mitochondria-ros crosstalk in the control of cell death and aging. Antioxid Redox Signal 2004. Immunohistochemical evaluation of oxidative stress markers in chronic hepatitis C.484:87-93. Vacante M. Shen W. Matsumura H. Mitochondrial superoxide production and respiratory activity: biphasic response to ischemic duration. Matsuoka S. J Clin Biochem Nutr 2009. Jang YC. Nutr Neurosci 2005. Bertino G. Wang Y. Arch Biochem Biophys 2009. Frohlich V. Nutrigenomics therapy of hepatisis C virus inducedhepatosteatosis. L-carnitine supplementation improves hematological pattern in patients affected by HCV treated with Peg interferon-α 2b plus ribavirin. Liang H.45:292. Lechleiter J. Ran Q. Jensen LT. Zinc supplementation improves the outcome of chronic hepatitis C and liver cirrhosis. lower iron status and decreased antioxidative defense in patients with chronic hepatitis C treated by pegylated interferon alfa and ribavirin. Weber P. Culotta VC. Adv Drug Deliv Rev 2009. Wintergerst ES. . et al.258 Antioxidant Enzyme Liang H.356:893-898. Brit J Nutr 2007. Ames BN. Bonora M. Kamei A. J Biol Chem 2005.2012:329635.17:4414-4420. Dinda AK. Beveridge S. Mahmood S. Lechleiter J. Dyrøy E. Pennisi M.47: 312-320. Vaagenes H. Aich J. Nakamura H. Bononi A. Matsuzaki S. Selected vitamins and trace elements support immune function by strengthening epithelial barriers and cellular and humoral immune responses. Malaguarnera M. Hayashi J. Clin Nutr 2009. Gpx4 protects mitochondrial ATP generation against oxidative damage. et al.61:1343-1352. Lipids 1999. Suski JM.34:951-963. Sharma SK. Motta M. Liu J. Reducing mitochondrial decay with mitochondrial nutrients to delay and treat cognitive dysfunction. Lin CC. World J Gastroenterol 2011. Mabalirajan U.98 (Suppl 1):S29–S35. Szweda LI. Humphries KM. McDonald-Marsh T. Giordano M. Leishangthem GD. Zhao B. Luk E.107:1285-1292.280:22715-22720. et al. Effects of vitamin E on mitochondrial dysfunction and asthma features in an experimental allergic murine model. Berge RK. Bourbonnais Y. Izumi A. Manganese activation of superoxide dismutase 2 in the mitochondria of Saccharomyces cerevisiae. J Appl Physiol 2009.28:34-38. Alzheimer's disease. Free Radic Biol Med 2009. Wertz K. Nakata K. Maggini S. Giorgi C.303. Biohem Biophys Res Commun 2007. Berge K.6:19-24.10:49. et al. et al. Vitamins B depletion. Glutathione peroxidase 4 differentially regulates the release of apoptogenic proteins from mitochondria. Rustan AC. Targeting mitochondrial biogenesis for preventing and treating insulin resistance in diabetes and obesity: Hope from natural mitochondrial nutrients. Ran Q. Liu Q. Liu J. Kawanaka M. Bengmark S. Zhang P.

Nagamine T. Kawashima A. and Chronic Hepatitis C 259 May JM. Woodman RJ. Moriya Y. Schnabl B. Murakami Y. Murthy S. Liu Z.53:213-218.48: 1420-1429. Croft KD.742.induced oxidative stress suppresses hepcidin expression through increased histone deacetylase activity. Mathur S. Avellini L. Ackerman Z. Chionne F. Mol Chem Neuropathol 1997. Polidori MC. Zinc supplementation prevents the increase of transaminase in chronic hepatitis C patients during combination therapy with pegylated interferon alpha-2b and ribavirin. Mitochondria and human cancer. 13-hydroxy octadecadienoic acid (13-HODE) inhibits triacylglycerol-rich lipoprotein secretion by CaCo-2 cells. Kawakami T. Allergol Immunopathol (Madr) 2008.Mitochondrial Free Radicals. J Clin Gastroenterol 2005. Kodama Y. J Lipid Res 1998. Mitochondrial recycling of ascorbic acid as a mechanism for regenerating cellular ascorbate. Effect of eicosapentaenoic acid and docosahexaenoic acid on oxidative stress and inflammatory markers in treated-hypertensive type 2 diabetic subjects. Khan NA. Cobb CE. Nutrition 2006. Kakizaki S. J Nutr Sci Vitaminol (Tokyo) 2007. Cherubini A. Cao W. et al. Miura K. Nutrient Substances. Vitamin E and C supplementation prevents decrease of eicosapentaenoic acid in mononuclear cells in chronic hepatitis C patients during combination therapy of interferon alpha-2b and ribavirin.105:519–529. Montero Vega MT. Kojima A. Curr Mol Med 2007. Modica-Napolitano JS. Kotera M.38 (Web Server issue):W138-43. Stern M. Prolonged exposure to insulin induces mitochondrion.22:114-122. Mei S.153:2120-2129. Singh KK.39:1254-1262. Hattori M. Tokimatsu T. Israeli E. Mitochondrial membrane fluidity and oxidative damage to mitochondrial DNA in aged and AD human brain. Hino K. Pappo O. Senin U. de Andrés Martín A. Takayma H. Biol Trace Elem Res 2000. Preliminary study of combination therapy with interferon-α and zinc in chronic hepatitis C patients with genotype 1b. Kitase A.derived oxidative stress through increasing mitochondrial cholesterol content in hepatocytes. Antioxid Redox Signal 2009. Field FJ. Beilin LJ. Born E. Mori TA. Muller G. Burke V. Kakibuchi N. Endocrinology 2012.75:53-63 . Toll-like receptors: a family of innate sensors of danger that alert and drive immunity. Kawakami T.31:53-64. Qu ZC. Morawietz H. et al. Beal MF. Mori M. PathPred: an enzyme-catalyzed metabolic pathway prediction server. et al.36:347-357. Hara Y. Implication of lipids in macrosomia of diabetic pregnancy: can n-3 polyunsaturated fatty acids exert beneficial effects? Clin Sci 2003.30:35-48. Kulawiec M. Merzouk H. et al. Gu H. Taura K.11:1711-1731. Shibolet O. Hepatitis C virus. Puddey IB. Nitric oxide. Shigemizu D. Romano G. Kanehisa M. Biofactors 2007. Li L. Takaguchi K. Hepatology 2008. Koyabu T. Free Radic Biol Med 2003. Mecocci P. Murakami Y. Nagai A. Yang X.39:737. Guo H. Goto S.7: 121-131. Nucleic Acids Res 2010. and atherosclerosis.35:772-781. Takagi H. NAD(P)H oxidase. Melhem A. Cecchetti R. Treatment of chronic hepatitis C virus infection via antioxidants: results of a phase I clinical trial. Brenner DA. Antioxidants.

Niederau C.45:797-804. Peter K. Gogvadze V. Clin Biochem 2010. Gogvadze V. Pawlak K.36:288-293. Orsucci D. Kudo M. Heintges T. Zeniya M. Showalter LA. Compadre CM. Filosto M. Siciliano G. Onoda H. Neri S. D'Amico RA. Ierna D. Sampathkumar R. Zhivotovsky B. Traub N. Göpfert E.42:187-192. Paradies G. Bakajsova D. Federici A. et al.260 Antioxidant Enzyme Nagashima M.blind. et al. Lemon SM. Sparagna GC. and antioxidant gene expression are induced by hepatitis C virus core protein. Nihon Rinsho Meneki Gakkai Kaishi 2004. Hayes C. Naoi M. Mancuso M. Strohmeyer G. randomized. Farooq S. Narasimhan S. Xu W. Toda G. . Saito A. oxidative stress. Di Venosa N.16:2799-2817. Chung H. Cu/Zn superoxide dismutase plasma levels as a new useful clinical biomarker of oxidative stress in patients with end-stage renal disease. placebo-controlled trial. Li K. Apoptosis 2007. Osada M. Regulatory failure of serum prohepcidin levels in patients with hepatitis C. Ravikumar R. Takahashi H. Campanile E. Mysliwiec M. Mitochondrial injury. Nutr Rev 2009.38: 700-705. Pearlman BL.12:913-922. Zhivotovsky B. Annu Rev Pharmacol Toxicol 2007. Oxidative stress is independently associated with non-alcoholic fatty liver disease (NAFLD) in subjects with and without type 2 diabetes. The relationship between the intracellular redox status of immune cells and progression of hepatitis C virus related chronic liver disease.47:819-827. Maruyama W. Hauer-Jensen M. Gastroenterology 2002.47:143-183. Monoamine oxidase inhibitors as neuroprotective agents in agedependent neurodegenerative disorders. Khairallah RJ. Okuda M. Sustained virologic response to antiviral therapy for chronic hepatitis C virus infection: A cure and so much more. et al. Nowak G.94:53-59. Nakatani T. Scholle F. γ-Tocotrienol protects against mitochondrial dysfunction and renal cell death.67:427-438 O'Shea KM. Curr Pharm Des 2010. Mitochondrial oxidative stress: implications for cell death. Pistolese M. J Mol Cell Cardiol 2009. Petrosillo G. Gokulakrishnan K.12:366-375. Ott M.52:889-900. Hepatol Res 2006. Antoci S. Ruggiero FM. Orrenius S. Mitochondria. Decrease in mitochondrial complex I activity in ischemic/reperfused rat heart: involvement of reactive oxygen species and cardiolipin. Clin Biochem 2005. double. Hepatogastroenterology 1998.27:315-21. Beard MR. Ishikawa E. oxidative stress and cell death. Hagiwara S. Electron transfer mediators and other metabolites and cofactors in the treatment of mitochondrial dysfunction. Circ Res 2004. J Pharmacol Exp Ther 2012.340:330-338. Noto R. Hecker PA. Dietary omega-3 fatty acids alter cardiac mitochondrial phospholipid composition and delay Ca2+-induced permeability transition. Polyunsaturated phosphatidylcholine and interferon alpha for treatment of chronic hepatitis B and C: a multi-center.43:815-21. Orrenius S. Mohan V. Panminerva Med 2000. Clin Infect Dis 2011. Robillard-Frayne I. Pawlak D. et al. Association of alphainterferon and acetyl cysteine in patients with chronic C hepatitis.

Haase H. Lee VS. Roca B.315:479-482. Feng CG. PLoS One 2011. Role of oxidative stress in the pathogenesis of nonalcoholic steatohepatitis. Colonna V. Prasad AS. Zinc: role in immunity. Prasad AS. Kensicki E. Resino E.53:1114-1121. Ortega RM. Skulachev VP. Inhibition of T-cell inflammatory cytokines. and HCV infection by standardized Silymarin. Nutrient Substances. Freymüller E. Quarato G. Arch Biochem Biophys 1994. Hall WW. Vuagniaux G. Nouailhetas VL. Free Radic Biol Med 2012. Serum B12 levels predict response to treatment with interferon and ribavirin in patients with chronic HCV infection.18:129-134. Ruggiero FM. The cyclophilin inhibitor alisporivir prevents hepatitis C virus-mediated mitochondrial dysfunction.74:1471-1479. Hepatology 2012. Bennasar M. Konstantinov AA.26:965-970. López-Sobaler AM. Phung CD. Ezieme JA. Vitamin C and E supplementation prevents mitochondrial damage of ileum myocytes caused by intense and exhaustive exercise training. Hepatitis C virus co-infection and sexual risk behaviour are associated with a high homocysteine serum level in HIVinfected patients. An inadequate intake of manganese may favour insulin resistance in girls. Romano M. Hydrogen peroxide metabolism in skeletal muscle mitochondria. Hepatology 2007. Aboulafia J. Gavillet B. Shuhart MC. oxidative stress and chronic inflammation. and Chronic Hepatitis C 261 Pereverzev MO.132:1925-1936. Hagen K.55:1333-1343. Palmeira CM. Ribeiro RF. D'Aprile A. Infect Immun 2006. Metabolomic profile of hepatitis C virus-infected hepatocytes. Turrens JF. Transplant Proc 2010. Rinella ME. Piccoli C. Teodoro JS. Wang CC. Bermejo LM.17:2202-2208. FASEB J 2003. Liu Y. Levitsky J. del Monte MC.107:1532-1538. Trends Immuol 2007. Factors associated with sustained virological response in liver transplant recipients with recurrent hepatitis C. Rodríguez-Rodríguez E. Zinc homeostasis and immunity. Reiman RM. Hepatitis C virus protein expression causes calcium-mediated mitochondrial bioenergetic dysfunction and nitrooxidative stress. Ferrero JA. Interleukin-5 (IL-5) augments the progression of liver fibrosis by regulating IL-13 activity. Gargante MP. Cheever AW.52:59-69. Hari D. Pereira FM. Polyak SJ. Nutr Hosp 2011.Mitochondrial Free Radicals. Dig Dis Sci 2008.28:1-4. Vygodina TV. Cytochrome c. hepatocyte NF-kappaB signaling. Curr Opin Clin Nutr Metab Care 2009. an ideal antioxidant.46:58-65. Antioxidants. Vacante M. J Appl Physiol 2009. Roe B. Lecce L. Mol Med 2008. Rosa EF. Mohney R. et al. Moradpour D.42:3647.12:646-652. Pillai AA. Knight R. Zinc in human health: effect of zinc on immune cells. et al.141:w13323. et al. Cammalleri L.14:353-357. Cristaldi E.6:e23641. Gastroenterology 2007. Rolo AP. Quarato G. Paradies G. D'Aprile A. Rink L. Wang E.3651. Biochem Soc Trans 2003. J Viral Hepat 2011. Capitanio N. . Lcarnitine treatment reduces steatosis in patients with chronic hepatitis C treated with alpha-interferon and ribavirin. Swiss Med Wkly 2012. Thompson RW. Lee DY. Scrima R. Role of reactive oxygen species and cardiolipin in the release of cytochrome c from mitochondria. Ripoli M.31(Pt 6):1312-1315. Morishima C. Rosenberg P. Petrosillo G.

Prazak T. Senerb O. 54:47-51.1052:202-211. oxidative stress. Kruzich LA. Redox regulation of hepatitis C in non-alcoholic and alcoholic liver. Zinc: An essential micronutrient. Stojanovi S. Glutathione. Russo AJ. antioxidant defenses. Takagi H. Sheeran F. Am J Clin Nutr 2007. Brain Res 2005. Abe T. Saper RB. J Nutr 2009. Karadurmusa N. The mitochondrial permeability transition pore opening under oxidative stress in ischemia/reperfusion in the tissue of the lower extremities Fiziol Zh 2008. Khurana NC. Wowk M. Suzuki H. Golde DW. Russo AJ.39:1177.129:25-32. Nagamine T.4:782-793.2:9-14. Proteomics Clin Appl 2010.39:200-205. Oxidative stress status and plasma trace elements in patients with asthma orallergicrhinitis. J Viral Hepat 2001. Vitamin C enters mitochondria via facilitative glucose transporter 1 (Glut1) and confers mitochondrial protection against oxidative injury. FASEB J 2005. Sato K. Decreased serum Cu/Zn SOD in children with Autism. Mitochondrially targeted vitamin E and vitamin E mitigate ethanol-mediated effects on cerebellar granule cell antioxidant defense systems. glutathione peroxidase. Smiljana R. Bailey M. Triple therapy of interferon and ribavirin with zinc supplementation for patients with chronic hepatitis C: a randomized controlled clinical trial. Lipids 2004. . Rash R. Stephensen CB.85:173-181. Özelc HE. Kanda D. Dragani S. J Thorac Cardiovasc Surg 2005. Zinc deficiency affects DNA damage.43:869-882. Otsuka T. Manganese superoxide dismutase (MnSOD) catalyzes NO-dependent tyrosine residue nitration. Zinc supplementation enhances the response to interferon therapy in patients with chronic hepatitis C. Siler-Marsiglio KI. 79:768-772. Sahach VF. Sato K. Kakizaki S. Ruzickova J. Leonard SW. Coenzyme Q10 therapy before cardiac surgery improves mitochondrial function and in vitro contractility of myocardial tissue. Rossmeisl M.1185. Ho E. Free Radic Biol Med 2007.139:1626-1631. Horbovets' VS.2:27-35. Mihajlo S. et al. Lyon W. Madorsky I. and selenium status in HIV-positive and HIV-negative adolescents and young adults. Song Y. Traber MG. Pan Q. Am Fam Physician 2009. Bulucua F. Marasco S. Seronello S. Decreased Serum Cu/Zn SOD Associated with high copper in children with attention deficit hyperactivity disorder (ADHD).12:1265-1269. Paiva M. De Re V. Sheikh MY. World J Gastroenterol 2006. Veck M. Omega-3 PUFA of marine origin limit diet-induced obesity in mice by reducing cellularity of adipose tissue. et al.70:601-608. J Central Nerv Sys Dis 2010.262 Antioxidant Enzyme Rosenfeldt F. Milan N. Sagun KC. J Serb Chem Soc 2005.8:367-371. Nutr Metabol Insights 2009. and Vesna N. Wilson CM. Takagi H. Takayama H. Sagdic A. et al. Simula MP. and DNA repair in rats.19:1657-1667. Marquis GS. Choi J. Flachs P. Douglas SD. Sohara N. Cárcamo JM. Heaton MB. Allergol Immunopathol (Madr) 2011. Hepatitis C virus-induced oxidative stress and mitochondrial dysfunction: a focus on recent advances in proteomics. Kakhanovs'kyŭ EF. Sponarova J. Yamaneld L et al.

Analysis of monohydroxyeicosatetraenoic acids and F2-isoprostanes as markers of lipid peroxidation in rat brain mitochondria. Nutrient Substances. Kawakami T. Nat Rev Immunol 2011. et al. Cytochrome C is a hydrogen peroxide scavenger in mitochondria. Li M. García-Arnés J. Free Radic Biol Med 2006. Mitochondria in innate immune responses. Hirsch D.28:9-16. Garg NJ. Biochem J 1996. Increased oxidative stress is correlated with mitochondrial dysfunction in chagasic patients. Cooney GJ. Lück-Lambrecht A. Fanburg BL. nonstructural protein (NS3) triggers dysfunction and apoptosis in lymphocytes: role of NADPH oxidase-derived oxygen radicals. Watson DG. Van Houten B. Myint T. DNA Repair (Amst) 2006. Endo T. Damage to DNA by reactive oxygen and nitrogen species: role in inflammatory disease and progression to cancer. Role of mitochondrial DNA in toxic responses to oxidative stress. García-Serrano S. Garrido-Sánchez L. Vidali M. Stewart SF. Sun H. Interplay between oxidative stress and immunity in the progression of alcohol-mediated liver injury. Tellinghuisen TL. Kumada H. et al.313 (Pt 1):17-29. Woshner V. Clin Chem 2005. Zn-superoxide dismutases related to familial amyotrophic lateral sclerosis. Shadel GS. Kraegen EW. Glycation proceeds faster in mutated Cu. Iino S. Tinahones FJ. The NS5A protein of hepatitis C virus is a zinc metalloprotein.36:1-11. Trends Mol Med 2008. Hellstrand K.14:63-71. Peroxisome proliferator-activated receptor (PPAR)-alpha activation lowers muscle lipids and . Adv Drug Rev 2008.279:48576-48587. Dahlgren C. Marcotrigiano J. A hepatitis C virus-encoded. Wang ZB. Kakibuchi N. Yamada G. Takaguchi K. Yu R.17:938-940. The mitochondrial cocktail: rationale for combined nutraceutical therapy in mitochondrial cytopathies.60:1561-1567. Sembaj A. Murakami Y. Tomioka K. Clin Drug Investig 2008. Romero A. and Chronic Hepatitis C 263 Takamiya R. Reactive oxygen species in cell signaling. Oxidative stress in severely obese persons is greater in those with insulin resistance. Protein Pept Lett 2003. Park YS.11: 389-402.Mitochondrial Free Radicals. Gorbalenya AE.pentaenoic acid supplementation in the treatment of chronic hepatitis C patients. Obesity 2009. Takahashi M. Miyazawa N. Virological response in patients with hepatitis C virus genotype 1b and a high viral load: impact of peginterferon-alpha-2a plus ribavirin dose reductions and host-related factors. Plasma 8-isoprostane concentrations in patients with age-related cataracts. Tarnopolsky MA. Santos JH. Halliwell B.76:1180-1186. Nourooz-Zadeh J. J Leukoc Biol 2004. J Biol Chem 2004. Ye JM. Zhao Y.17:240-246. Am J Physiol Lung Cell Mol Physiol 2000. Yuan Z. et al.51:419-425. Augustin W. Wiseman H. Wang B. Omata M. Wiswedel I. Rice CM. West AP. Murri-Pierri M. Thannickal VJ. Zhu H.5:145-152. et al. J Nutr Sci Vitaminol (Tokyo) 2005. Ghosh S. Iglesias MA. Free Radic Res 2002. Effects of eicosa. Xu JX. Wen JJ. Kita K. Manzur RE. Antioxidants. Yachelini PC. Thorén F. Flechsig A. Pan J.41:270-276. García-Almeida JM.279:L1005-L1028. Lindh M. FASEB J 2003. Doyle PJ. Okuno T. Kiyosawa K.10:247-253.51:1541-1544. Albano E.

Biol Trace Elem Res 1999. Nadimpalli RG. Sollott SJ. Zang Q. Younossi ZM.38:705-709. Barnes DS. J Appl Physiol 2007. Zorov DB. Ong JP. Zhang W. Maass DL. Tavill A. Cardiac mitochondrial damage and loss of ROS defense after burn injury: the beneficial effects of antioxidant therapy. Horton JW. Post A. Seleniumdependent glutathione peroxidase modules encoded by RNA viruses. Ramanathan CS. Taylor EW. White J. Mitochondrial ROS-induced ROS release: an update and review. Bhat AA. et al.264 Antioxidant Enzyme improves insulin sensitivity in high fat-fed rats: comparison with PPAR-gamma activation. Obesity and nonalcoholic fatty liver disease in chronic hepatitis C. Diabetes 2001. J Clin Gastroenterol 2004.102:103-112. .50:411-417.70:97-116. Juhaszova M.1757:509-517. McCullough AJ. Cox AG. Biochim Biophys Acta 2006.

or in follow-up to watch for cancer recurrence. Although there are a multitude of tumor markers. They are usually substances that are released into the circulation and then measured in the blood sample. which permits unrestricted use. Introduction Tumor markers are measurable biochemicals that are associated with a malignancy. Not all tumor receptor marker tests are widely available nor are they widely accepted. they do have a number of clinical applications. to indicate a prognosis. Despite the fact that tumor markers are hardly ever specific enough to be used alone to diagnose cancer. Tumor marker discovering is focuses currently much research and attention. licensee InTech. and reproduction in any medium. such as tissuebound receptors that must be measured in a biopsy from the solid tumor or proteins that are secreted into the urine. Agnieszka Chwilkowska. therapeutic approaches take advantages from determination of oxidative stress markers. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons. Changes in some tumor markers have been sensitive enough to be used as targets in clinical trials. They can be used to stage cancer. Jolanta Saczko. provided the original work is properly cited.Chapter 10 Apoptosis. Free Radicals and Antioxidant Defense in Antitumor Therapy Julita Kulbacka. In this chapter we try to       © 2012 Kulbacka et al. Tumor markers for diagnosis are used in combination with other clinical parameters such as biopsy and radiological findings. They are either produced by tumor cells (tumor-derived) or by the body in response to tumor cells (tumor-associated). However. There are a few exceptions to this.   . to monitor treatment. some of these non-specific markers have found a place in monitoring cancer treatment rather than in Anna Choromańska and Nina Skołucka Additional information is available at the end of the chapter http://dx. In recent times. very few of them have found their way into clinical practice because of their lack of specificity.doi. These markers have gained importance in the evaluation of cancer treatment and prognosis. In the current review we attempt to propose and bring closer some new “cancer markers” connected to oxidative stress and cell death.5772/50357 1. Their final clinical usage is directed by approval from the Food and Drug Administration (FDA) and guidelines established by organizations such as the American Society of Clinical Oncology and the American Cancer Society.0).

In case of cancer process of programmed cell death is inhibited and tumor cells are allowed to tolerate apoptotic signals. Many genes and their proteins products play a dual role in the cell division and apoptosis including p53. In this way organism destroys cells that endanger the homeostasis. During this process the cells are tending to genetic lesions. Thus there is the extrinsic pathway that depends on triggering of death receptors expressed on the cell surface. and cannot communicate with neighboring cells. There are a few control spots of cell cycle. These pathways lead to activation of the specific proteinase caspases and result in DNA fragmentation. are allowed to progress through the cell cycle.5]. senescence (permanent arrest) or apoptosis [1013].266 Antioxidant Enzyme explain the beneficial application of oxidative stress and apoptotic markers for medical requirements [1]. cross-linking of proteins. The restrictive points lead to repair damage in cells or eliminated cells in different ways: necrosis. Problem of selective direct selected cells on apoptotic pathway is still unclear. pRb. Large varieties of different stimuli are able to initiate programmed cell death by apoptosis signaling pathways. overexpressed or modified their . accumulating mutations.1. Apoptosis Apoptosis is the process of programmed cell death which is very important when cells harmful for organism appear. In many types of cancer the mutation of gene responsible for check points are observed [14]. are malign. Bcl-2 family. The cells proliferation is regulated by cell cycle. expression of ligands for phagocytic cell receptors and nally uptake by phagocytic cell [6]. Restore of appropriately induce apoptosis may establish antitumor therapy based on o triggering selective death of cancer cell [2. . formation of apoptotic bodies. mutated. the intrinsic pathway mediated by molecules released from the mitochondria and the third pathway activated by granzmes [4. However. well-organized control mechanisms are shown to exist. and apoptosis [7]. Regulation and inhibition of apoptosis in cancer treatment The cells homeostasis is regulated by different mechanism included proliferation. lose the ability to undergo apoptosis. which detects damage. 2. degradation of cytoskeletal and nuclear proteins. Subsequent stimulation these molecules may induce cell proliferation. The disorder in the balance between cell growth and death often leads to the carcinogenesis. It is now accepted that cancer is more accurately described as being the product of malfunctions within the regulation of the cell cycle. The different result is dependent on different factors. growth arrest.3]. 2. Apoptosis play a central role in the pathogenesis of human disease especially in malignance while the factors controlling the apoptotic progression are suppressed. which respectively inhibit or support apoptotic cell death. cell cycle arrest or cell elimination [8]. such that injured or mutated cells which are normally killed. It may lead to malignancy or the early stages of carcinogenesis [9]. which is an involved sequence of grow and cells replication [8]. Defective Apoptosis has been recognized as a fundamental factor in the development and progression of cancer. cells that ignore the signals of cell cycle regulation.

Apart from granzyme A pathway caspases are essential during apoptosis. 8. acetylation) [15. kinase inhibitors initiate this pathway. Smac/DIABLO. phosphorylation. Consequently caspase-8 recruitment and activation occurs. supports granzyme (A or B) release to the target cell cytosol and.2. TNFα. Their function is essential for the process of programmed cell death. and thus allows the caspases to cleave their substrates. however.Apoptosis. In case of the intrinsic pathway several stimuli. Sutton et al. The group of initiator caspases (2.16]. Granzymes are a different family of serine proteases and the granule protein. 2. by activation of initiator caspase-10 or by direct activation of caspase-3 [23]. activated the pro-apoptotic proteins permeabilize the outer mitochondrial membrane to trigger the release of Smac/DIABLO and cytochrome c to cytosol [17]. Signaling pathways of apoptosis Mitochondria play a crucial role in apoptosis. As a result. This caspase cleaves and activates Bid. Defects in its pathways be able to promote cancer cell survival and also confer resistance to antineoplastic drugs. The study into apoptosis is going at a fast pace and this has led to the possibility of new therapeutic approaches to some human diseases [11]. Effector caspases (3. The extrinsic pathway of programmed cell death involves interaction between ligand and plasma membrane receptor (Fas/CD95. including reactive oxygen species and other cytotoxic elements. They are aspartate-specific cysteine proteases that are present in healthy cells as zymogens. 6 and 7) in turn cleave other protein substrates within . and apoptosis events.19]. promote formation of the apoptosome. During organization of these proteins complex cytochrome c. perforin. Cytochrome c binds apoptotic protease activating factor-1(Apaf-1) using the energy provided by ATP and procaspase 9 is activated into its active form [18. lack of growth factors. show that granzyme B triggers the mitochondrial apoptotic pathway in mouse myeloid cells through direct cleavage of Bid. cleavage of procaspases was stalled when mitochondrial disruption was blocked by Bcl-2 [24]. TRAIL) [20]. 9 and 10) triggers cleaves inactive proenzymes of effector caspases. on entry [21. Perforin/granzyme-induced apoptosis is the main pathway activated by cytotoxic T lymphocytes to eliminate virus-infected or transformed cells. This event results in the activation of caspases 3 and 7 as the downstream effector caspases. Some authors indicated that the release of cytochrome c is tightly regulated by the pro. Caspase-8 may bypass the mitochondria and induce apoptosis by directly activating caspase-3. released into the cytosol. which are usually activated by proteolytic cleave to form a fully functional active site [19]. Granzyme B can operate by specific cleavage of Bid and induction of cytochrome c release. The former mentioned. which releases cytochrome c from the mitochondria to activate the apoptosome. directly binds to cytosolic IAP (inhibitor of apoptosis protein) and removes it from active caspases. Free Radicals and Antioxidant Defense in Antitumor Therapy 267 function (mutation. In case of granzyme A caspase-independent cell death can occur via initiation of DNA cleavage.22].and anti-apoptotic members of the Bcl-2 family [5].

-9 or -10 directly or by with the participation of specific protein trigger effector caspases -3. The colon cancer response to immune attack and chemoradioresistance induced . Important factors involved in the regulation of apoptosis are inhibitors of apoptosis proteins (IAPs) that can block caspase cascade. Interactions and the diversity of apoptotosis signaling pathways. Figure 1. death receptors orgranzyme B.268 Antioxidant Enzyme the cell. In case of colon cancer it was shown abnormal caspase expression. Palmerini et al. Downregulation of caspase 1 expression was observed in colon cancer samples [26]. and 9 [27]. The normal epithelial cells of gastrointestinal tract colonic epithelium strongly express caspases 1 [26]. -6 or -7 which results in cell death. defects in caspase activation often results in chemoresistance. [27] found the downregulation of caspase 7 in most colonic carcinoma tissues. The ability of chemotherapeutic agents to initiate caspase activation appears to be a crucial element of drug efficacy. 3. to initiate irreversible events of the apoptotic process [18]. Caspase cascade can be activated by the apoptosome. Initiator caspases -8. but only some of them directly interact with caspases [25]. 8. Consequently. 7.

and apoptosis. cisplatin and ionizing radiation then SW480 and that Fas receptor and Apaf-1 was decreased in comparison to SW480 cells [28]. Suppression of release of SMAC/DIABLO from mitochondria was reported in melanoma cells [31]. . The monoclonal antibody against the protein CD20 selectively down-regulated the expression of antiapoptotic Bcl-xL and up-regulated the expression of proapoptotic Apaf-1 in Ramos cells [36].Apoptosis. which could be used to the therapy of given kind of cancer. The presence of the specific antigen on the surface of the cell makes it the ideal aim for the antibodies.and caspase-9-mediated apoptosis [34]. whereas U251 cells were markedly sensitive to p53-mediated apoptosis. A-172 cells showed higher endogenous expression of Bcl-X(L) than U251. The relevant levels of Apaf-1 are crucial in the inhibition of tumor progression and for preserving the sensitivity of cancer cells to apoptosis [30]. differentiation. both are downstream components of p53-mediated apoptosis and found that A-172 cells were highly sensitive to Apaf-1. Shan et al. (2000) noticed that caspase-3 may mediate apoptosis induced by Cisplatin derivative in human epidermoid carcinoma cell line A431 [29] . Melanoma cells avoid apoptosis by inhibiting the expression of the gene encoding Apaf-1. Mese et al. Free Radicals and Antioxidant Defense in Antitumor Therapy 269 apoptosis. and leads to cell death by apoptosis [35]. resistant to p53-mediated apoptosis. Jazirehi et al. Shinoura et al. They also observed upregulation of Raf-1 kinase inhibitor protein (RKIP) expression in non-ARL cell line. transduced Apaf-1 and caspase-9 into U-373MG glioma cells and observed increases in chemosensitivity of study cells [33]. CD20 is a B-cell surface antigen that is an effective target for immunotherapy of B-cell malignancies using unmodified or radiolabeled murine monoclonal anti-CD20 antibodies. The protease activating factor Apaf-1. suggesting that high endogenous expression of Bcl-X(L) renders A-172 cells. and transduction of BclX(L) repressed p53-mediated apoptosis in U251 cells. which was identified as the molecular core of the apoptosome executes mitochondria dependent apoptosis. This cell surface phosphoprotein is involved in cellular signaling events including proliferation. show that murine antiCD20 monoclonal antibodies inhibit B-cell proliferation. In the next step researchers transduced A-172 cells and U251 cells with the Apaf-1 or caspase-9 genes. showed that anti-CD20 antibody in ARL cell line diminishes the activity of the p38MAPK signaling pathway resulting in inhibition of the interleukin (IL)-10 leading to the inhibition of constitutive STAT-3 activity and subsequent downregulation of Bcl-2 expression leading to chemosensitization [37]. Treating melanoma cells with the methylation inhibitor 5-aza-29-deoxyctyidine increased Apaf-1 expression and chemosensitized melanoma cells [32]. activation. at least in part. Death receptor (extrinsic) pathway of apoptosis and mitochondrial (intrinsic) pathway of apoptosis were investigated in SW480 and SW620 colon cancer cells. Allelic loss and subsequent absence of Apaf-1 expression in melanoma cells is associated with chemoresistance [32]. In the other study Shinoura et al showed A-172 cells did not undergo apoptosis after p53 transduction. induce nuclear DNA fragmentation. The investigation study revealed that SW620 cell lines appeared more resistant to apoptosis induced by CH-11.

g. and controls cell proliferation and DNA repair. The p53 gene has been called “guardian of the genome”. Mutations of p53 have been observed in over 50% of human cancers (e. In physiological conditions p53 occurs inactive form on the low level in the cells. EC 2. PARP-1. oncogene activation and hypoxia. p53 protein interacts with other proteins. Jin et al. One such protein is a polymerase poly-ADP-ribose (PARP. The decisive function of p53 regulating the verdict of a cell to live or die makes it an attractive target for anticancer therapeutics [51].2. Caspase-dependent and independent mediated apoptosis.50].270 Antioxidant Enzyme Immunotherapy with specific antibody could be applied alone or in combination with chemotherapy. require further explanation of the physiological role of these ligands in the potential application for cancer therapy and prevention [41]. was facilitated by the mitochondria and inhibited by knockdown of Bad or overexpression of Bcl-xL [39]. The role of p53 in cell’s reply to chemotherapy remains unclear. CD95 stimulation as well as anti-cancer agents’ etoposide induces apoptosis. whose job is to protect and preserve DNA stability of the genes.producing group proved a better prognosis than those of the nonproducing group [41]. p53 gene encoded the p53 proteins. Etoposide was also found to induce caspase-8 processing and apoptosis in a CD95-independent fashion because blocking of CD95 receptor function with a specific antibody does not inhibit etoposide-induced apoptosis [38].30) [47]. which positively correlates with the severity of the reaction of proapoptotic [48]. Patients in the TNF. Moreover. The apoptosis-signaling pathways stimulated by TNFs. There was also observed that TNF can induce apoptosis in a limited number of tumor cell lines [40]. An apoptosis promotion involves signaling through members of the tumor necrosis factors (TNF). there are many conflicting studies and . induced by CMTM8 overexpression. The role of p53 and Bcl-2 family in apoptosis Oxidative stress oncogene activation and arrest of cell growth lead to activation of tumor suppressor gene p53.or chemotherapy treatment [49. due to crucial role in protecting the genome against the proliferation of mutated cells [45]. ovarian. 1996). some members of the TNF family can initiate caspase activation. Ito et al. showed that endogenous TNF production peaked after stimulation with OK-432 (Ito et al. which activates apoptosis or senescence [42-44].4. resulting in apoptosis. The effect of TNF induction with anticancer agent OK-432 on the survival rate of colorectal cancer patient was investigated. In the Jurkat clone J16. the mutations are connecting with resistance to radio. The gene p53 is a 53-kDa nuclear phospho-protein that binds to DNA to act as a transcription factor. Under the influence of DNA damaging agents the level of p53 protein increased and stopped the cell cycle in order to repair or cell death [46]. However tumor suppressor protein p53 is induced in response to stress such as DNA damage. DNA damage in the course of therapy cancer increases the expression of PARP and increases the amount of poly-ADP-ribose (PAR) in tumor cells. colon carcinoma). 2007 showed cancer cell lines transfected with chemokine-like factor CMTM8 submit to apoptosis. On binding to their proper receptors. This fact supports that p53 plays an important role in the prevention of tumor development.3. 2.

Many cases showed that p53 is needed if cells are to submit to chemotherapy. the mutations are connecting with resistance to radio. Free Radicals and Antioxidant Defense in Antitumor Therapy 271 approaches which would be the main therapeutic strategy to cancer therapeutics. Loss of p53 and Bcl-2 family take part in a decisive role in apoptosis. The authors showed that p53 is required for radiation-induced apoptosis in mouse thymocytes. The cells isolated from this mouse were totally resistant to -irradiation and died [55]. which demonstrated that p53-defective are or not sensitive to chemotherapy. Moreover. Previous study was based on the idea that activation of p53 can induce apoptosis in the tumor. Some reports suggesting that p53 wildtype are more sensitive to many of anticancer drugs. Similar the fibroblast isolated from the same mice were also resistant to radiotherapy and chemotherapy with adriamycin [56]. colon carcinoma). It is common known that malignant cells undergoing apoptosis p-53 dependent or p-53 independent. The crucial investigation supporting the significance of p53 in mediating DNA damage and induced apoptosis in cells derived from p53 knockout mice. The p53 protein can mediate apoptosis in response to DNA damage caused by chemotherapy but the inducing cell cycle arrest and favoring DNA repair might increase resistance by allowing cells to live after DNA has been damaged by chemotherapeutic treatment [53]. suggesting that can play a decisive role in PDT. The decisive function of p53 regulating the verdict of a cell to live or die makes it an attractive target for anticancer therapeutics [50]. This date create the question: is defective p53 the Achilles heel of the tumor? Mutations of p53 have been observed in over 50% of human cancers (e.g. Additionally. Other are based on the observation that cells with defective p53 are more sensitive to combinations of chemotherapeutic drugs [52]. but there are many investigation.Apoptosis. there are many conflicting studies and approaches which would be the main therapeutic strategy to cancer therapeutics. Other is based on the observation that cells with defective p53 are more sensitive to combinations of chemotherapeutic drugs [52]. Other scientific reports indicated that radiation of T cell lymphoma derived from the same mice with knockout p53 was able to its killing. Similar results obtained after photodynamic treatment in different cancer cells.or chemotherapy treatment [48. The role of p53 in cell’s reply to chemotherapy remains unclear. ovarian. This fact supports that p53 plays an important role in the prevention of tumor development. There are numerous investigations where the cells with defective p53 undergo apoptosis.There are numerous investigations where the cells with defective p53 undergo apoptosis. There are many studies in which the effect of p53 on the cellular response to chemotherapy and radiotherapy is controversial.defective tumors. Previous study was based on the idea that activation of p53 can induce apoptosis in the tumor. Some examination designate that p53 is necessary for executor caspase 3 activation. The p53 protein can mediate apoptosis in response to DNA damage caused by chemotherapy but the inducing cell cycle arrest and favoring DNA repair might increase resistance by allowing cells to live after DNA has been damaged by chemotherapeutic treatment [53].49]. Other study examined the outcome of photodynamic reaction with Photofrin (Ph-PDT) on clear human ovarian carcinoma OvBH-1 with “silent mutation” in the p53 gene. However many examination conducted in recent years have resulted in the discovery of drugs which have been used successfully to treat patients with p53. They suggest that this . radiation therapy is usually applied patients independent of their p53 status [54].induced early apoptosis in malignant tissue [57].

The cells with phosphorylated p53 protein most often were observed in serine 392 and Nterminal and in serine 20 et C-terminal end observed in cells [66]. Some of amino acid in p53 proteins is phosphorylated. that prevent p53 deacethylation along with its promoters allow the development of new anticancer strategy based on the maximization of action of this protein [71. The big influence on anticancer therapy effectiveness is also individual dependent on type of cancer and their environment [61. and Bim. The products of Bcl-2 gene family are divisible into two main groups: antiapoptotic Bcl-2. One of approach. which respectively inhibit or support the effecting of apoptotic cell death [76. significant improvement in patients undergoing therapy combined.62]. however. However. cyclins). which leads to regulation of p53 function. They cause p53 deacethylation which inhibits its activity. It is known that phosphorylation area of p53 protein binding MDM2 inhibits degradation of p53. additional studies demonstrate that PDT can induce apoptosis in cancer cells by pathway independent of p53 [60]. About twenty five members of the Bcl-2 family of proteins have been identified [75]. checkpoint proteins and gene controlling and oncogenic changes [73]. These proteins blocks apoptosis induced by “guardian of the genome” in response to stress. 72]. Recent studies have shown that important role in the effectiveness on anticancer therapy plays a modification by its phosphorylation [63]. The other researcher divided this proteins into three subfamilies based on structural and functional features: pro-survival.70].77]. Differential cells sensitivity on chemo. Bak. The p53 protein is also change in lung cancer. proapototic . protein kinase C. which control cancer cells responding (c-Myc.68]. but there was no strong correlation between changes in expression of this protein (both mutant and native) and course of disease [67. Bcl-2 family is the other important proteins which near and with p53 take part in a decisive role in apoptosis [74]. There was. One of the most important issues is the phosphorylation of p53 at position Ser 15. in which the course of exacerbation p53 protein expression [67]. depends on its posttranslational modifications.and other anticancer therapy is probably dependent not only on p53 status but other genes and their products. In addition the new examination have shown that stimulation or inhibition of tumor growth might be due to changes in proteins modify p53. Ideally anticancer strategy will be therapy adapted to patients based on the p53 status. Bcl-Xl. The modification of this method by chemotherapeutic drug 2-methoxyestradiol leads to apoptotic pathway induction in these cells [59]. which may promote tumor growth The use of Sir2 inhibitors. Bad. It is obvious that only p53-tageted therapeutic strategy is not enough for the treatment of all type of malignant tissue. whose members are most structurally similar to Bcl-2. The in vitro and in vivo studies demonstrate that Sir2 protein is involved in this procedure. Moreover p53 work together with different tumor suppressor family. This process enhanced the stability form of p53 protein. protein kinase A. a C-terminal phosphorylation of Ser392 alters the cell cycle [69. It is common known that chemotherapy resulting in an increased stabilization of this protein [64. and proapoptotic Bax.272 Antioxidant Enzyme mutation may inhibit apoptosis in these cells [58]. Recent investigation have shown that p53 phosphorylation et serine 15 and 20 was necessary to induce apoptosis in ovarian cancer cells after chemotherapy with cisplatin. and Bclw.65].

The fact that key Bcl-2 family genes are p53 targets including pro.82].89]. The pro-apoptotic member of Bcl-2 protein Bid acts crucial function in connecting between the extrinsic and intrinsic apoptotic pathway.77]. These results suggest that p53 can regulate the intrinsic and extrinsic pathway through Bid regulation [93].87].79]. Bid gene succumb p53 regulation in response to chemo. p53 can also independently activate Bax present in the cytoplasm and this protein forms a homodimer and releases cytochrome c from the mitochondria [85]. Bax induced the mitochondrial pathway by outflow of apoptogenic proteins. It has unique ability to connect two different types of apoptosis. Bak and especially Bax were the groups induced by p53 mainly in response to stress [81. nuclear envelope and the external membranes of the mitochondria [76]. The Bcl-2 gene product is located in the membranes of the endoplasmic reticulum. Bax protein takes part in apoptotic response of the developing nervous system to  irradiation and leads to sensitivity fibroblast cells with E1A-expressing to chemotherapy [86.84]. Free Radicals and Antioxidant Defense in Antitumor Therapy 273 Bax and Bak and antagonize their prosurvival functions BH3-only proteins [78. Cellular sensitivity to chemotherapy with adriamycin or 5-fluoroacyl is dependent on wild-type p53 and Bid. Activation of Bid involves cleavage of cytoplasmic Bid by activator caspase 8 and induces post-translational changes. in different studies the involvement of Bax and p53 in different anticancer therapy mediated apoptosis was observed [83. This date was obtained from colorectal cancer cells where the growth arrest through of p21 is the normal rescue response to p53 expression in these cells.91].Apoptosis. This process leads to Bid translocation into the mitochondrial membrane and activates pro-apoptotic Bax and Bak proteins which initiate apoptosome formation. PUMA gene is as well as Bax activating by p53 especially in response to DNA damage.and antiapoptotic [80].and radiotherapy in many type of cancer. Probably PUMA expression promotes mitochondrial translocation and multimineralization of Bax and in consequence inducing apoptosis. Bcl-2 (Bcl-2 its self) residue in the outer mitochondrial membrane . The explanation of enigmatic function of Bax in apoptosis has recently been examined in the context of PUMA. Bax did not appear to be main inductor of cell death [88. It is common that inhibition of apoptosis can lead to cancer. P53 plays a crucial role in regulation of proapoptotic Bcl-2 proteins. In epithelial colon carcinoma undergoing apoptosis in response to radiotherapy. This gene encodes to BH3-domain-conteinig protein: PUMA  and PUMA β [90. However. Moreover. The requirement of Bax and Bak in p53 –activated apoptosis occurs to be cancer celltype dependent. such as cytochrome c. The defect of p21 induced in these cells apoptosis pathway. Bax takes part in the apoptotic death response indirectly target of p53 through PUMA [92]. These results suggest that Bax is absolutely necessary for PUMA – induced apoptosis [92]. Bax gene encoding the protein contains on the promotor sequence the binding location for p53. In the large Bcl-2 family we can find also inhibitors of cell death or growth arrest. A fundamental balance between PUMA and p21 which controlled cell cycle determine growth arrest by senescence or death by apoptosis in cooperation with p53. Thus families of proteins control mitochondrial stability by maintaining the balance between proapoptotic proteins that translocate to the mitochondria and antiapoptotic ones that exist in the mitochondrial membrane [76. whereas PUMA is damage the apoptosis is prevented. Additional studies showed that the level of Bax protein acts not crucial role in inducing apoptosis or growth arrest in other cancer cells.

Acc. The p53 and Bcl-2 pro-apoptotic protein are one of the many proteins that induce the intrinsic signaling pathway. Wild-type p53 can establish complex with Bcl-2 and Bcl-XL and suppress there anti-apoptotic function. high Bcl-2 level is con were connected with high Gleason scores and an increase rate of cancer recurrence after radical prostatectomy. cell growth and apoptosis. Bcl-XL. Hence research is continuing on the use of synthetic inhibitors in preclinical and clinical study (Table 1. to Kang and all [97]. but also are designed to search new possibilities of tumor destruction [52]. National Cancer Institute. Among them are proteins that inhibit apoptosis. However. Agents Apogosypol HA14 Antimycin A Oblimersen sodium Gossypol (AT-101) ABT-737(ABT 263) BH31s GX15-070 Target proteins Bcl-2 Bcl-2 Bcl-2. Previous and present studies yield new information about various factors which regulate apoptosis. Maybridge Chem. The Bcl-2 anti-apoptotic protein inhibits apoptosis in cancer cells and promotes cell survival.) [97]. As an example in primary prostate cancer. Bcl-w. Agents targeting anti-apoptotic Bcl-2 family proteins.96]. Ltd. University. There are many investigations determining the levels of expression of cell death inhibitors in various types of cancer. Bcl-2. Bcl-XL Bcl-2 Bcl-2. This fact decides about therapeutic targets through inhibition of this protein and arrest malignance process [98].274 Antioxidant Enzyme and mainly plays an anti-apoptotic function [94]. cell cycle. Recent studies showed that increasing levels of Bcl-2 and Bcl-XL have associated with a more aggressive malignant phenotype often connected with drug resistance to various type of chemotherapy not only in hematologic but also solid tumors [95. Protein plays the crucial role in regulatory cell development. Bcl-w Sponsor Mcl-1 Brnham (NCI) Maybrige Chem U of Washington Genta Mcl-1(NCI) Abbott Harvard U Mcl-1 Gemin X Stage Preclinical Preclinical Preclinical Phase III Phase I/II Phase I Preclinical Phase I Abbreviations: BH3Is. Bcl-w Bcl-XL Bcl-2. Apoptosis is perturbed in many cancers.. Bcl-XL. These studies afford correlative evidence. U. Also the high expression of BCL-XL in the NCI 60 cell line is strongly correlated with resistance to most chemotherapy agents. NCI. Maybridge Chemical Co. In many malignancies especially in hematologic the overexpression of Bcl-2 family was found. It is the major barrier leads to destruction of cancers. Bcl-XL. Table 1. in 50% of cancers the p53 gene is disrupted and losses its ability to bind to these proteins. The most of them determined the direction of future clinical development and are promising. This procedure leads to correcting the regulation of many metabolic . BH3 inhibitors. The intracellular proteins are selective stabilized or eliminated by ubiquitindepenedent pathways. Many in vitro experiments confirm the preventing role of Bcl-2 in apoptosis activating in different type of cancer [97].

This results in dissociation of HSF (heat shock factor).116]. Apoptosis regulatory molecules have been recognized as substrates and degraded in proteosome. The resistance to apoptosis is one of the major problems in the anticancer therapy. The ubiquitin –proteosome protein damage can inhibit apoptosis by degradation proapoptotic controller. The basis for the classification on these proteins was their chaperone activity and molecular weight. HSP90 inhibits formation of an active apoptosome whereas HSP70 prevents the recruitment of procaspase-9 to the apoptosome complex [60]. then migrates to the nucleus. HSPs can be divided into three subfamilies: large (HSP100. The overexpression HSP27 is often finds in malignant cells for example in ovarian and breast cancer. a coniugating enzyme (E2) and protein ligase (E3) [100-103].Apoptosis. In addition to many function the HSPs protein plays a crucial role in inhibition of apoptotic process. It is often connected with poor prognosis of cancer [112-114]. 3. HSPs are a family which limit the consequences of damage and facilitate cellular recovery [108. There are several reports according to which the use of antisense oligonucleotides directed against HSP27 can be basis of anti-cancer therapy. sHSP in the receptor pathway of apoptosis blocks DAXX protein and through AKT kinase inhibits activation and translocation of Bax to mitochondria [111]. Ubiquitin is a protein complex composed of the activating enzyme (E1). HSP-mediated regulation of apoptosis through inhibition of major pro-apoptotic proteins is involved in this process [115. Anti-apoptotic role of sHSP proteins makes it encourages the development of tumor progression and metastasis. The first line of cellular defense .109]. 90). Bid is the protein which associated two apoptotic pathways intrinsic and extrinsic. Previous investigation involved both HSP70 and HSP27 of Bid dependent apoptosis. Ubiquitin targets the protein substrate for damage via the 26S proteosome. intermediate HSP 70. 60 and 40) and small (sHSP less than 40 kDa). HSP binds injured molecules. The degradation leads to apoptosis resistance in cancer cells. The free ubiquitin is recycled. The heat shock proteins (HSPs) are a highly conserved class of proteins whose expression in increased in cells exposed to different kinds of stress. This process plays a crucial role of many significant signaling pathways and important role in many cellular pathways including apoptosis. Enzymatic antioxidants Defense against oxidative stress is provided by a system of antioxidants enzymes and nonenzymatic antioxidant substances capable of neutralizing free radicals and preventing an excess production of reactive oxidative species (ROS) [117]. Many proteins which can regulate apoptotic pathways have been recognized as target substrates for ubiquitination [103]. When there are damaged proteins. Blocking apoptosis by the sHSP lead to interfere with some cytostatics and decrease the effectiveness of chemotherapy. The heat shock proteins can play an important role in recognition and degradation of damaged proteins by ubiquitination [107]. Free Radicals and Antioxidant Defense in Antitumor Therapy 275 processes in cells. Elements of the cell apoptosis mechanism are often altered in cancer. where it binds with HSE (heat shock rudiments) leading to HSP overexpression [110]. From this studies appear that proteosome inhibitors can apply as antitumor therapies through enhancing apoptosis [103]. To these we can include inter alia members of Bcl-2 family and IAP [104-106].

+2H+ 2H2O2 H2O2 + 2GSH SOD CAT GP H2O2 + O2 2H2O + O2 2H2O + GSSG Figure 2. Catalase is an antioxidant enzyme which catalyzes the conversion of H2O2 to water and oxygen. and red blood cells. SOD2 (MnSOD) is a tetrameric protein that functions in the mitochondria. GPX4. Glutathione sulfide reductase is responsible for converting GSSG to GSH. There are three known human isoforms of SOD. localized mainly in the liver and gastrointestinal tract. Reactive oxidative species (ROS) may cause irreparable damage. The chemical reactions involving ROS and antioxidant enzymes. There has been identified four distinct isoforms of GPX in humans. and catalase (CAT) enzymes. It is concentrated mainly in peroxisomes [130]. catalyzes the reduction of H2O2 and some organic peroxides [127]. SOD is an essential antioxidant enzyme that defends cells against potentially damaging superoxide radicals. DNA . which defends cells against potentially damaging superoxide radicals:   SOD1 (CuZnSOD) is found in the cytoplasm and nucleus in the form of a dimer. Also glutathione transferase (GST) plays an important role in the protective mechanisms. 126]. which catalyze the reduction of hydroperoxides to water and the respective alcohols. Antioxidant enzymes drive chemical reactions to convert ROS into non-toxic molecules: 2O2·. protects against lipid hydroperoxides. which determinant the total cellular antioxidant capacity [125. which is localized in the cytoplasm and mitochondria in the liver. GPX1. extracellular form of the enzyme while each enzyme performs a critical function. kidney. is capable of reducing phospholipid hydroperoxide.129]. SOD3 (CuZnSOD) is a tetrameric. while oxidizing GSH to GSSG [122]. but it is highly detected in plasma.  Enyzmes of the GPX family are selenoproteins. glutathione peroxidases (GPXs).276 Antioxidant Enzyme against oxidative stress enzymes are: the family of superoxide dismutases (SODs). These two compounds serve as the major redox couple within the cell. expressed in the testis. GPX2. including lipid peroxides derived from cholesterol [128. more than 95% of cellular oxygen is metabolized in the mitochondria during oxidative phosphorylation. therein: base modification. lung. SOD2 is particularly important due to its location within the mitochondria [122-124]. It plays an important role in catalyzing the conjugation of reactive electrophilic agent to glutathione (GSH) [118]. GPX3 is has the same function.121. This enzymatic system is complex and highly integrated [119-121]. Persistent oxidative stress is a major initiator to progress cancer [119.131]. They are the main free radical-scavenging enzymes which decomposing superoxide radicals and H2O2.

In initial period its level is increased.139. noted that the absence of a response these enzymes indicate a weakening of antioxidant enzymes system [118]. based on the photochemical reaction produces singlet oxygen and other forms of reactive oxygen. They found no change in the malondialdehyde (MDA) level. cellular signaling. It was observed that SOD. oral cancer and chronic obstructive pulmonary disease [118.145. Its activity increased significantly in cancer patients [141]. while catalase activity is generally lower in tumor cells than in healthy tissue [133-135]. which is consistent with the previous work in patients with oral cancer [117. Apocynin (4-hydroxy-3-methoxyacetophenone) is now used indiscriminately as a NOX4 [154] and as a NOX5 inhibitor [155].142.153] and it might contribute to development of colon cancer through at least two mechanisms: ROS-dependent DNA damage and ROS-dependent enhancement of cell proliferation [150].151]. The changes in enzymatic antioxidants status [118].144].Apoptosis.147]. such as superoxide ion. In some situations ROS are used in anticancer therapies. Vitamin C and GST level.140]. as well as Vitamin E. A variety of studies involving antioxidant enzyme levels and cancer development have been performed. NOX4 homolog of the NADPH oxidase is suggested to promote cell growth in melanoma cells [153]. Carcinogenesis process is accompanied by weakening of the antioxidant enzyme system. and regulation of gene expression. The physiological functions of Nox enzymes include: cell differentiation. NOX1 homolog of the NADPH oxidase is highly expressed in the colon [152. GPx and GSH levels in the erythrocyte and plasma was significantly lower in cervical cancer patients. Oxidative DNA damage in blood and other tissues were detected in various types human carcinogenesis [118. Drugs directly inhibiting the NADPH oxidases activation could successfully inhibit oxidative stress and inflammation caused by this enzymes [149]. but also by high expression ROS-generated enzymes [149]. DNA-protein cross-links [132].146. Following cellular damage initiated the deregulation of cell signaling pathways. the level of lipid oxidation [136] and an increase of DNA breaks number in tumor cells and leukocytes of blood indicate the process of malignancy [137]. hydroxyl radical [156-160]. Free Radicals and Antioxidant Defense in Antitumor Therapy 277 strand breaks. Singlet oxygen and superoxide anion have been demonstrated to play . host defense. The GST is involved in detoxification of carcinogens. It is also observed that the GPx and SOD activities decrease in the group of cancer patients during cancer development [118. hydrogen peroxide. Tumor cells can respond to photodynamic damage by apoptosis or necrosis [161-163].138. The NADPH oxidases (Nox enzymes) share the capacity to transport electrons across the plasma membrane and to generate superoxide and other reactive oxygen species (ROS) [150. In smokers the role of GST is crucial in modulating susceptibility to smoking-related lung cancer. including the process of carcinogenesis [150]. These results suggest possible use of antioxidant supplementation as prophylactic agents for prevention and treatment of this cancer [148]. a promising therapy for solid tumors. Burlakova et al. Those enzymes could also induce a wide range of pathological processes. posttranlational processing of proteins. tumor suppressors and an inhibition of apoptosis [121].143]. Glutathione peroxidase activities have been found to be changeable. Tumor cells nearly always show a decrease in SOD1 and SOD2 expression. but later it decreased [118]. Photodynamic therapy (PDT).

Zinc (II) propoporphyrin IX. Their function is to stabilize the lipid peroxide radicals as well as provide protection against damage from sunlight by absorbing energy or redirecting it to other processes in the cell. estrogen. giving the plants the color yellow. PDT is also antagonized by other cellular antioxidant defense mechanisms: catalase. 165]. Carotenoids are natural antioxidants present in the chloroplasts and chromatopfores. both endogenous (glutathione. 4. Administration of HO-1 inhibitors might be an effective way to potentiate antitumor effectiveness of PDT. 167]. which must be delivered to the body with food because the body is not able to produce them himself. heme oxygenase-1 (HO-1) [171-173]. Biliverdin and bilirubin are potent antioxidants capable of scavenging peroxy radicals and inhibiting lipid peroxidation [174-176]. negatively correlates with the sensitivity of tumor cells to radiation therapy and anticancer drugs [168.278 Antioxidant Enzyme a main role in the cytotoxic effects induced by PDT [164. Blocking these signaling pathways by p38MAPK inhibitors or small interfering RNA (siRNA) for p38MAPK suppress HO-1 increases. Non-enzymatic antioxidants Among the non-enzymatic antioxidants can be distinguished based compounds. This HO-1 stimulation is governed by the p38MAPK (p38 mitogen-activated protein kinase) and PI3K (phosphatidylinositol 3-kinase pathways). 2-methoxyestradiol (2-MeOE2) was shown to selectively inhibit the activity of superoxide dismutases [170]. which significantly increases the effectiveness of therapy [156]. visible especially in autumn. Kocanova et al showed that treatment of HeLa (human cervix carcinoma cells) and T24 cells (human transitional cell carcinoma of the urinary bladder) with hypericin-PDT dramatically induced of HO-1 expression. melatonin. raising the propensity of the cells to undergo PDT-induced apoptosis [178]. Carotenoids ingested with food (beta-carotene) are precursors of retinoids (vitamin A). 166. Induction of HO-1 protects against the cytotoxicity of oxidative stress. lipoamide dehydrogenase. markedly augmented PDT-mediated cytotoxicity towards colon adenocarcinoma C-26 and human ovarian carcinoma MDAH2774 cells [173]. vitamin C. which seems to play a protective role against PDT-induced cell death [173. and HO-1 inhibitor. as well as in laboratory animals exposed to tobacco smoke increases the risk of . red and orange. SOD1 and SOD2 scavenged cells from singlet oxygen and significant extent the antitumor efficacy of PDT [156. Overexpression of SOD2 suppresses apoptosis. 177]. PDT with 2-MeOE2 selectively enhance free radical generation and suppress antioxidant defenses. Vitamin A is fatsoluble antioxidant. HO1 catalyses the rate-limiting step in the oxidative degradation of heme. albumin) and exogenous (carotenoids. vitamin E. Products of the reaction catalyzed by this enzyme are CO and biliverdin which is rapidly converted to bilirubin. the glutathione system. Some studies have shown that supplementation with high doses of β-carotene or carotenoid in smokers. 169]. flavonoids). Combinations of SOD inhibitor with PDT might result in significant increase in the efficiency of anticancer treatment [154].

esophagus.195]. Carotenoids diets have demonstrated some anticarcinogenic activity in animal experiments [187-190]. rectum. protects membranes against oxidation.192]. cervix. Experimental data suggest that these antioxidants such as carotenoids. leukocyte function. Ascorbic acid reduces tocopheryl radical formed by the reaction of vitamin E with lipid radicals. vitamin C and vitamin E can interact synergistically. Ascorbic acid (vitamin C) is antioxidant that works in aqueous environments of the body. Colorectal carcinogenesis may be reflected by greater elevation of MDA and decrease level of vitamin E and vitamin C in the serum [209].Apoptosis. which are both precancerous lesions [201]. bladder. larynx. . it must be provided exogenously in the diet and transported intracellularly. The data reported here suggest that the dose of vitamin C supplement used may induce additional defenses against oxidative damage. through an increase in lymphocyte SOD and CAT activity [196]. at high concentration it exhibits prooxidant effects especially at high oxygen tension [185. and malignant brain tumor. Vitamin C has proven to be beneficial as a factor in preventing cancer of the lungs. Since Vitamin C regenerates Vitamin E. mouth. breast. Free Radicals and Antioxidant Defense in Antitumor Therapy 279 lung cancer [179-181]. and defense against microorganisms. Some authors demonstrated ability to neutralize free radicals produced by exposure to light to compounds of lower toxicity [191. ozone and nitrogen dioxide [193]. The positive effect of Vitamin C has also been found in lung and colorectal cancer [202]. Vitamin C is effective in the defense against oxidative stress-induced damage [203]. stomach. Humans cannot synthesize vitamin C. It has been noticed that in colorectal cancer patients the incidence decrease vitamin E [205207] and the intake of Vitamin E [200 IU] reduced the incidence of colorectal cancer by triggered apoptosis of cancer cells [208]. α-tocopherol Vitamin E is a fat-soluble antioxidant that stops the production of reactive oxygen species formed when fat undergoes oxidation[204]. Vitamin C has important roles in vascular and connective tissue integrity. Prolonged absence of vitamin C in the diet leads to the development of scurvy. pancreas. β-carotene it normally functions as an antioxidant. 211]. Vitamin C plays an important role in the detoxification of substances such as tobacco smoke. Other study reported negative results for Vitamin E in combination with Vitamin C and beta carotene to prevent colorectal cancer adenoma [210. and prevents lipid peroxidation and affect the regeneration of vitamin E [194. The most active form of vitamin E in humans is α-tocopherol and there is considered as a major antioxidant in biomembranes. endometrium. Vitamin C is considered as a most powerful ROS scavenger because of its ability to donate electrons in a number of non-enzymatic and enzymatic reactions. they protect each other from degradation and/or promote their regeneration [197-200]. Low serum levels of Vitamin C in high risk population may contribute to the increased risk of chronic gastritis or gastric metaplasia. A study have shown that administering both vitamin A and vitamin C to the cell culture of human breast cancer cells was three times more effective than the administration of these vitamins separately [184]. 186]. colon. it has been proposed that addition of Vitamin E hinders the protective effect of Vitamin C against oxidative damage.

Apples are commonly consumed and are the major contributors of phytochemicals in human diets. is also responsible for maintaining the appropriate level of glutathione in the cells. glutathione is responsible for maintaining the antioxidant activity of other antioxidants. This tripeptide is produced by the body from three amino acids: cysteine. Quercetin. Some studies have demonstrated that whole apple extracts prevent mammary cancer in rat models in a dose-dependent manner at doses comparable to human consumption of one. Glutathione (GSH) is the most important non-enzymatic cytosolic antioxidant. At high experimental concentrations that would not exist in vivo. and six apples a day. and TF-3. Flavonoids are most commonly known for their antioxidant activity in vitro. Others study demonstrates that in colorectal carcinoma patients. The inhibitory effect of black tea polyphenols on aromatase activities has been investigated. TF-2. soy products). One of the basic functions of glutathione is to maintain the sulfhydryl groups of proteins in the reduced state and inhibition of oxidation by hydrogen peroxide [191. these black tea polyphenols also inhibited the proliferation in MCF-7 cells [217]. 219]. stabilizing its reduced form. Epidemiological studies have shown that regular consumption of fruits and vegetables is associated with reduced risk of chronic diseases such as cancer and cardiovascular disease [213. kaempferol. luteolin. preventing lipid peroxidation. Preliminary results indicate glutathione changes the level of reactive oxygen species in isolated cells grown in a laboratory. the most abundant dietary flavonol. The flavonoids have been reported to have antiviral. Protective effect. glutamic acid and glycine [218].000 flavonoids have been identified. coffee. It is able to regenerate vitamin E and vitamin C back to their active forms. which is reduced by NADPH in a reaction catalyzed by glutathione reductase [220]. Consumption of apples may be an effective strategy for cancer chemoprevention. For the flavonoids and their derivatives with the strongest antioxidant potential include: delfinina. wine and citrus fruits. 222]. Glutathione together with glutathione peroxidase (GSH-Px) reduces hydrogen peroxide H2O2 and lipid peroxides. a very highly significant decrease in total plasma thiols and intracellular glutathione [224]. TF-1. grapes. anti-allergic. vegetables and beverages (tea. the antioxidant abilities of flavonoids in vitro may be stronger than those of vitamin C and E.214]. Over 4. quercetin. which may reduce cancer development [221. which is accompanied by the formation of glutathione disulfide. Black tea polyphenols. and antitumor and antioxidant activities. Equally effective could lead hydroxyl radical HO. Glutathione supplementation increases mean survival time treated mice [223]. In addition to neutralize free radicals. anti-inflammatory. beer.280 Antioxidant Enzyme Flavonoids are polyphenolic compounds that are ubiquitous in nature. In in vivo models. epicatechin. 193. significantly inhibited rat ovarian and human placental aromatase activities. many of which occur in fruits. . It has been reported that fresh apples have potent antioxidant activity inhibit the growth of colon and liver cancer cells in vitro [215]. the most dangerous of free radicals to form water. Fresh fruits could be more effective than a dietary supplement [216]. depending on concentrations tested [212]. is a potent antioxidant because it has all structural features for free radical scavenging activity. three.

nitric oxide synthases). atherosclerosis or aging. neuronal NOS (nNOS) and inducible NOS (iNOS). The initiation phase is connected to the activation of oxygen and is rate limiting. Peroxidation of cell membranes. 5.Apoptosis. asthma and chronic obstructive pulmonary diseases [231-235]. upregulation of enzymes (NADPH oxidase.1. radiation. Selenium deficiency is associated with an increased risk of cancer and cancer death [229. Polyunsaturated fatty acids (the main component of membrane lipids) are receptive to peroxidation. The identification of valid biomarkers of stress is involved with previous characterizing the event of stress and for early identification of the disease development which might follow [236] 5. measurement of NO might be a reliable biomarker to predict earlier oxidative stress mediated cellular response including injury and specific differentiation of stem cells. short-lived. NO has been used for various diseases as a screening marker. Lipid peroxidation Lipid peroxidation is a normal metabolic process extending under regular conditions. propagation and termination. and it is a multifunctional signaling molecule that regulates complex cellular processes. Non-enzymatic antioxidants are relatively ineffective in comparison with the action of antioxidant enzymes. such as cerebral strokes. It is also involved with the increase of lipid peroxidation products and resulting membrane degeneration [237]. xanthine oxidase. The results showed that Selenium could significantly inhibit tumor growth as well as extend the median survival time of tumor-bearing mice [227]. Many studies confirm that selenium reduces the risk of all cancers especially cancer of the liver. ROS and RNS Nitric oxide (NO) is a diffusible. Consequently. hemoxygenase-1. or from the environment (smoking. The antioxidant properties of selenoproteins help prevent cellular damage from free radicals. It can proceed into three steps: initiation. which contain a high concentration of polyunsaturated fatty acids. Selenium significantly inhibits the proliferation cancer cells in vitro [228]. It has been detailed that iNOS gene transcription and promoter activity are increased by oxidative stress and it regulates chromatin modification leading to cellular injury. prostate. for example mitochondria in response to inflammatory conditions. 226]. colorectal and lung cancer [225. industrial pollution) may damage macromolecules such as lipids. The excess of ROS/RNS (reactive oxygen species/reactive nitric species) generated from endogenous sources. The process of lipid peroxidation is one of the most investigated consequences of reactive oxygen species (ROS) actions on membrane structure and function. Production of oxygen radicals increases with clinical progression of disease. 230]. diatomic free radical ubiquitously produced by mammalian cells. Free Radicals and Antioxidant Defense in Antitumor Therapy 281 Selenium is a trace element that is essential in the human diet. Only together with enzymes is effective line of defense against oxidative stress [187]. proteins and DNA and induce neurological disorders. L-arginine derived NO production is mediated by activation of nitric oxide synthase (NOS). There are three isoforms: endothelial NOS (eNOS). is a critical mechanism .

however lipid peroxidation can induce also apoptosis. virus-infected and otherwise damaged cells that threaten our health. 243]. 239]. Saintot et al. and apoptosis. Authors proved that the level of lipid peroxidation was significantly higher for cells after PDT. demonstrated that MDA marker and concentration of –SH groups can be a validate marker for efficiency in PhII mediated photodynamic therapy (PDT) in lung carcinoma cells (A549). women at high risk for breast cancer. depending on the presence of ROS. Decreased concentration of malondialdehyde (MDA) in plasma. The cell death can occur by necrosis. through non-invasive methods such as nipple fluid aspirate sampling. lipid peroxidation markers could also be applied in prognosis. There was also observed increased concentration of MDA (malonodialdehyde) in colorectal carcinoma patients. This type of cell death eliminates precancerous and cancerous. in comparison with respective values in control cells [244]. The consequential damage of proteins may take the form of nitration or oxidation of various residues. comparing to control cells. activating the intrinsic suicide pathway present within all cells [238]. which are accumulated in mucus of patients with bowel disease [237. Authors suggest ROS production in gut due to phagocytes. It has been shown that lipid peroxidation and ROS are triggers and essential mediators of apoptosis [238. both of which are stable markers of oxidative stress. Other authors hypothesize that lipid peroxidation can be a principal mechanism in rodent renal carcinogenesis. maturation. a better understanding of the relationship between breast cancer risk factors and oxidative stress/lipid peroxidation-related biomarkers and genes may prove useful in identifying the dietary or non-dietary exposure. and the induction of differentiation. another lipid peroxidation product. for example. Increased AOPP. Lipid peroxidation levels in breast ductal cells may become a promising cancer biomarker to detect. has been found to be significantly related with severity of prognosis factors for breast cancer.282 Antioxidant Enzyme leading to growth inhibition and cell death. malondialdehyde levels. genotype combinations that put women at the lowest risk. and decreased thiol and nitric oxide concentrations. In addition. may imply that patients are under oxidative stress. also demonstrated thet colorectal carcinogenesis may be associated with greater MDA concentration and decreased level of vitamin C in the patients’ serum [243]. suggested that lipid peroxidation could be verified to be a prediagnostic marker for breast cancer. Saczko et al.2. Some authors also demonstrated that lipid hydroperoxides and oxygenated products of lipid peroxidation degradation as well as lipid peroxidation initiators (ROS) can be involved in the cascade of signal transduction the control of cell proliferation. MDA concentration was significantly lower in the plasma of patients with large tumors or in whom nodes and/or metastasis was observed [239-241]. Protein damage Proteins contained by cells undergo oxidative stress in the presence of various reactive oxygen species (ROS).242. ROS can also induce the formation of advanced oxidation protein products (AOPP) or advanced glycation end products (AGE). They observed much lower concentrations of -SH groups in A549 cells after PDT treatment. In addition. Bahat et al. Proteins damage can provoke reduced cell-specific functional ability and may then allow other mutations to produce signaling components . 5.

[247] proved that lack of filamin-A expression sensitizes cells to chemotherapy reagents. 5. In consequence ROS can play a very important physiological role as secondary messengers [121]. and a wide range of DNA repair activities require filamin-A. others. There was reported that two mitochondrial proteins: aconitase and adenine nucleotide – translocase can be significant targets of longterm oxidative destruction. Yue et al. filamin-A may be used as an effective therapeutic target for these cancers with high or normal level of filaminA expression. Low concentrations of superoxide radical and hydrogen peroxide may stimulate proliferation and enhance survival in a different cell types. However there are many not widely applied proteins that may help in cancer treatment and diagnosis. by the oxidation of sulphydryl groups of cysteine residues to form –S–S– crosslinks. Filamin-A despite of being a cytoskeleton protein. ROS and RNS induce modification in protein structure and function.2. plays a role in the repair of . It has been presented that the hydroxyl radical represents the major species responsible for the oxidation of proteins [121. However this protein marker could also be used as a target to sensitize filamin-A positive cells to therapeutic DNA damage [247]. They presented that the level of filamin-A in melanoma cells correlates with their sensitivity to bleomycin and cisplatin. Filamin-A Recent studies indicated the possibility that filamin-A (cytoskeleton protein) may play a role in cancer response to DNA damage based chemotherapy reagents. Thus. These indicators are associated with many types of cancer. with as few as one. Protein damage is repairable and is a known non-lethal event for a cell. such as bleomycin and cisplatin. These changes observed in protein concentration and structure modification and may be monitored and regarded as biomarkers. This protein can be served as a biomarker to predict cancer prognosis for chemotherapy.246]. These results suggest that filamin-A status may be used as a biomarker for prognosis after treatments.1. including those formed by addition of lysine amino groups to the carbonyl group of an oxidized protein. by interaction of two carbon-centered radicals obtained by the hydroxyl radical-driven abstraction of hydrogens from the polypeptide backbone. only several of them are described below.Apoptosis. filamin-A status in cancer would be a novel marker for prognosis assessment and optimization of individualized treatment planning. or as an inhibition target to sensitize filamin-A positive cancer to therapeutic DNA damage. even an incomplete inhibition of filamin-A expression in C8161 cells can confer a sensitivity to bleomycin and cisplatin treatment in mouse xenograft model. There are some widely used protein tumor markers listed in Table 2. Free Radicals and Antioxidant Defense in Antitumor Therapy 283 which will then go unconstrained aiding tumourigenesis. Thus. Many studies use oxidative protein damage markers for determination of stages in cancer patients’ and disease progression [245]. Protein oxidation by ROS is related with the formation of many different kinds of protein cross-linkages. as shown in. and the oxidation of tyrosine residues to form –tyr–tyr– cross-links. Authors also presented that inhibition of filamin-A sensitizes xenograft tumors to bleomycin and cisplatin treatment. Second.

and when many preventive treatments would probably help prevent long-term health effects. The increased levels of troponin I (TNI) protein in the blood helps identify possible heart damage after cancer treatment [232].284 Antioxidant Enzyme multiple forms of DNA damage. Table 2.pancreatic cancer 9) CA 27-29 (Breast carcinomaassociated antigen) CEA (Carcinoembryonic antygen) breast cancer many cancers. 248]. fascin. long before impairment in heart function and symptoms develop. sPIgR4 and 14-3-3 eta Troponin I B-type natriuretic peptide Beta-HCG (Beta-human chorionic gonadotropin) Application liver. Tumor marker AFP (Alpha-fetoprotein) Her-2/neu Bladder Tumor Antigen Thyro-globulin PSA Leptin. intestinal.2. Furthermore. The TNI concentration measured in blood is a well-established marker of heart muscle injury that’s widely used to diagnose and treat heart attacks and other acute coronary syndromes. and ovarian cancer stage IV breast cancer urothelial carcinoma Thyroid cancer metastasis Prostate cancer Ovarian cancer Lung cancer Myocardial infraction Congestive heart failure testicular cancer and tumors. such as choriocarcinoma and molar pregnancies. testicular. filamin-A can be used as a biomarker to predict cancer sensitivity to therapeutic DNA damage. and as an inhibition target to improve therapy efficacy for filamin-A positive cancers [247]. peritoneal cancer dissemination especially liver.2. indicate TNI as a protein marker for prediction of possible heart damage after chemotherapy. Troponin I TNI is a protein present exclusively in heart cells. that begin in placental cells called trophoblasts CA 125 (Cancer antigen 125) ovarian cancer. prolactin. 5. malignant pleural effusion. However Cardinale at al. non-small cell lung cancer CA 15-3 (Cancer antigen 15-3 breast cancer CA 19-9 (Cancer antigen 19. Commonly applied FDA (Food and Drug Administration) tumor markers [1. However TNI can be assessed and monitored for the safety and effectiveness of different treatments [232] . osteoponin and IGF-II CD98. and pancreatic. TNI categorizes heart disease risk early. Authors also suggest that tracking TNI levels can help form a heart disease prevention plan for some chemotherapy patients.

Molecular biomarker iNOS. antioxidant enzymes and oxidants at different steps of the malignant transformation and in cancer therapeutic applications is evident. The association of free radicals. Conclusions According the current review we tried to assume oxidative stress related markers in Table 3. Mercier et al. Cav-1 is involved in metastatic processes. It has also been shown that Cav-1 expression is induced under oxidative stress conditions. Caveolin. Many details regarding the detailed role of apoptosis. eNOS. Moreover. superoxide dismutase Caveolin-1 Table 3.-dG) Isoprostanes Bityrosine cross-links Filamin A Oxidative scissions Amino acid radicals (i. catalase. nNOS (inducible/endothelial/neuronal nitric oxide synthase) NO Singlet oxygen Malondialdehyde (MDA) 4-hydroxynonenal (HNE) Hydroxypropanodeoxyguanosines (HO-PdGs) Exocyclic etheno DNA adducts (etheno-dA. Process involved in oxidative stress ROS and NO Lipid peroxidation Protein oxidation .2. Molecular biomarkers of lipid and protein oxidation [249. Free Radicals and Antioxidant Defense in Antitumor Therapy 285 5. These results signify that Cav-1 may be an interesting biomarker for the prediction of disease burden [249].Apoptosis. 250]. It was demonstrated that Cav-1 can be a prognostic markers of aggressive (high-grade) forms of prostate cancer [249. lysine. cysteine) paraoxonase-1 Carbonyl and thiol groups GSTpi. histidine. They described Cav-1multiple functions as a controller of estrogen signaling and kinase activity and its lately found role as an important factor monitoring the dynamic relationship between cancer epithelia and stroma position [251]. free radicals and antioxidant markers in multifactor diseases such as cancer are still discovered. indicated Cav-1 as a new therapeutic target for the treatment of breast cancer.3. Caspases. 6.-dC.e. arginine. Authors found that in patients with high serum Cav-1 the antioxidant capacity of the body was reduced.1 Caveolin-1 (Cav-1) plays an important role in cell transformation and the process of tumorigenesis. 252-254]. proline.

Ruefli AA and Lowe SW (2002) Apoptosis: a link between cancer genetics and chemotherapy. [6] Gulbins E. Kodym R (1998) Signal transduction during apoptosis. Wroclaw Medical University. Berneman ZN. Soriano BJ. Shula Y (2010) Role of senescence and mitotic catastrophe in cancer therapy. rev. Cell 108: 153–164. 23: 365-375. cell cycle and apoptosis in cancer. Eur j. Am. pathol. Author details Julita Kulbacka*.com. [5] Elmore S (2007) Apoptosis: A Review of Programmed Cell Death. [4] Igney FH and Krammer PH (2002) Death and anti-death: tumour resistance to apoptosis. Jekle A. Nature 411: 342–348. The ann. Front. Toxicol. j. Figg WD (1997) Apoptosis its role in the development of malignacies and its potential as a novel therapeutic target. George J. Corresponding Author * . van Bockstaele DR (2003) Cell cycle and apoptosis. References [1] Nordenson NJ. [10] Guimareas CA. Grassme H. Bomer MM. Cell prolif. 35: 495-516. implications for cancer therapy. 5: 1-12. [7] Story M. of pharmaco. physiol. 31: 76-81. [3] Johnstone RW.encyclopedia. Lang F (2000) Physiology of apoptosis. [12] Singh R. needs measuring the DNA of healthy patients during a few decades to map the individuals who can develop cancer [121]. Gale Encyclopedia of Cancer.286 Antioxidant Enzyme To determine with confidence which type and what level of oxidative damage can be really a applicable biomarker for cancer. 366: 1638-1650. Lush RM. Radiography 14: 144-149. [11] Vermuelan K. biosci. Agnieszka Chwilkowska. physiol renal. Nat. [8] Foster I (2008) Cancer: A cell cycle defect. Linden R (2004) Apoptosis and alternative death styles. 279: 605–615. 36: 165-175. Poland Acknowledgement The study was supported 2011/01/D/NZ4/01255. biochem. Wroclaw. [9] Dixon S. [2] Evan GI and Vousden KH (2001) Proliferation. Jones CLA (2002) Tumor Markers. Accessed 2012 Apr 3. Jolanta Saczko. by National Science Center research grant UMO- 7. Anna Choromańska and Nina Skołucka Department of Medical Biochemistry. Cell div. cancer 2: 277–288. Ferlinz K. Available: http://www.

Ruefli AA. -3. NFkappaB. Galmiche J-P. Le Neel J-C. J surg. Pelicci PG (2000) PML regulates p53 acetylation and premature senescence induced by oncogenic Ras. Wolf BB. Davis JE. 63: 2705-2715. Ashiya M. [15] Thatte U. Hudig D. Cell 104: 487–501. Carbone R. Laboisse CL (1999) Interleukin 1 and interleukin 1 beta converting enzyme (caspase 1) expression in the human colonic epithelial barrier: caspase 1 downregulation in colon cancer. Weiler S. Alnemri ES. Riha M. cell biol. J. Sedelies K. [28] Huerta S. [20] Locksley R M. -7. [18] Slee EA. Birg F. biochem. Higashimoto Y. McManus BM. Mannella CA and Korsmeyer SJ (2002) A distinct pathway remodels mitochondrial cristae and mobilizes cytochrome c during apoptosis.Apoptosis. Chen D. and AIF. Martin SJ (1999) Ordering the cytochrome cinitiated caspase cascade: hierarchical activation of caspases-2. Buttle K. j. Bleackley RC (2002) Cytotoxic T lymphocytes: all roads lead to death. Dev. Granville DJ (2005) Granzyme B induces endothelial cell apoptosis and contributes to the development of transplant vascular disease. -6. Nat. Appella E. Apaf-1. Sawchuk T. Letessier E. 2: 401-409. Szeto WY. [23] Barry M. [16] Pearson M. immunol. Wang HG. [24] Sutton VR. 32: 461-467. exp. J. Browne KA. Gut 45: 246-251. [19] Twiddy D. Saito S. Sebastini C. Oakes SA. Anguiano-Hernandez YM. cell 2: 55–67. Nature 406: 207-210. [17] Scorrano L. Cancilla M. Devilard E. Green DR. res. Fraser SA. Cioce M. [26] Jarry A. but not direct granzyme B-mediated caspase activation. Rosengard BR (2002) Mechanism of T cell-mediated endothelial apoptosis. Turka LA. IAPs. Minucci S. Cain K (2007) Caspase-9 cleavage. Killeen N and Lenardo M J (2001) The TNF and TNF receptor superfamilies: integrating mammalian biology. Pandolfi PP. 405: e1–e2. Newmeyer DD. Bleackley RC. Wells AD. Drugs 54: 511-532. Cell cycle 3: 1121–1123. immunity and malignancy. -8. . [14] DeVita JVT. Bonavida B. Heinzerling JH. 192:1403-1414. Balsara KR. 5: 494-499. Geisbrecht J. [22] Choy JC. Xerri L (2001) Caspase 7 downregulation as an immunohistochemical marker of colonic carcinoma. Lemarre P. do you need it? J. Cassagnau E. Hellman S. Nicholson DW. Rosenberg SA (1997) Cancer: principles and practice of oncology. Jirik FR. Am. Fagioli M. Krasinskas AM. Trapani JA (2000) Initiation of apoptosis by granzyme B requires direct cleavage of bid. Moreau A. Vallette G. Kluck RM. 142: 184-194. Kerjner A. Casiano CA. Simon HU (2004) An IAP in action: the multiple roles of survivin in differentiation. Harte MT. Dahanukar S (1997) Apoptosis ± clinical relevance and pharmacological manipulation. Livingston EH (2007) Modification of gene products involved in resistance to apoptosis in metastatic colon cancer cells: roles of Fas. Jarry A. Transplantation 74: 871–876. Bou-Hanna C. Cancer res. Johnstone RW. and -10 in a caspase-9-dependent manner. Human pat. 144: 281-292. [21] Krupnick AS. Lin J. Popma SH. Reed JC. [25] Zangemeister-Wittke U. rev. transplant. [27] Palmerini F. Kreisel D. Free Radicals and Antioxidant Defense in Antitumor Therapy 287 [13] Roninson IB (2003) Tumor cell senescence in cancer treatment. Smac/DIABLO. Philadelphia: Lippincott-Raven. Huerta-Yepez S. med. Cruz RP.

de Vries E.. Opitz-Araya X. Cordon-Cardo C. McCombie R. Zhang XY. Han W. De Vos S. Emmanouilides C. Yagita H. cell physiol. [31] Zhang XD. j. Borst J (1998) The anti-cancer drug etoposide can induce caspase-8 processing and apoptosis in the absence of CD95 receptor-ligand interaction. Cell 88: 323-31. [33] Shinoura N. Nguyen T. cancer 2: 1183-1193. the cellular gatekeeper for growth and division. Oncogene 24: 21212143. Jpn. cancer res. He Q. 211: 112–120. Erdlenbruch B (2005) Downregulation of Apaf-1 and caspase-3 by RNA interference in human glioma cells: consequences for erucylphosphocholine-induced apoptosis. Oncogene 22: 2869–2881. Cancer chemother. 87:1160-1164. Hersey P (2003) Activation of ERK1/2 protects melanoma cells from TRAIL induced apoptosis by inhibiting Smac/DIABLO release from mitochondria. [43] Levine AJ (1997) p53. Lakomek M. [37] Jazirehi AR. . Mingava Y. [30] Kugler W. Wang Y. Biochem. Alcalde RE. Sayers TJ (2006) TNF-related apoptosisinducing ligand as a therapeutic agent in autoimmunity and cancer. Köhler F. Polsky D. Cancer 86: 1307-1313. cancer res. Shanker A. Kigawa J. Yin C. Kirino T. Kirino T and Hamada H (2001) Cotransduction of Apaf1 and caspase-9 augments etoposide-induced apoptosis in U-373MG glioma cells. Mol. Song Q and Ma D (2007) CMTM8 Induces Caspase Dependent and –Independent Apoptosis Through a Mitochondria-Mediated Pathway. Hamada H (2000) Transduction of Apaf-1 or caspase-9 induces apoptosis in A-172 cells that are resistant to p53-mediated apoptosis. Lowe SW (2001) Inactivation of the Apoptosis Fffector Apaf-1 in Malignant Melanoma.. Ledbetter JA. 272: 667-673. 84: 87–98. Apoptosis 10: 1163-1174. Asai A. Gerald WL. [34] Shinoura N. res. Chemosensivity and p53-depenedent apoptosis in epithelial ovarian carcinoma. Sakurai S. caspase-3 on cisplatin-induced apoptosis in cisplatin-resistant A431 cell line. [39] Jin C. Herman JG. anti-CD20 mAb) in non-Hodgkin's lymphoma: implications in chemosensitization and therapeutic intervention. [40] Cretney E. [36] Jazirehi AR. Apoptosis 3: 17-25. Bonavida B (2005) Cellular and molecular signal transduction pathways modulated by rituximab (rituxan. Tian L. Borrow JM. 46: 241-245. biophys. Tatekawa I (1996) Tumor Necrosis Factor Production and Colon Cancer. Sasaki A. Immunol. Capodieci P. Matsumura T (2000) The role of caspase family protease. J. [38] Boesen-de Cock JG. Asai A. Esteller M. Williams GT. [35] Shan D. [41] Ito H. Sakurai S. Bonavida B (2003) Rituximab (antiCD20) selectively modifies Bcl-xL and apoptosis protease activating factor-1 (Apaf-1) expression and sensitizes human non-Hodgkin's lymphoma B cell lines to paclitaxelinduced apoptosis. pharmacol. Mora J. Fujitsuka M. Li D. Nature 409: 207–211. Atomi Y. Liu D. Nakayama S. Jpn. Gan XH. Yagita A. Blood 1: 1644-52. j. Smyth MJ. Lazebnik YA. commun. 92: 467-474. [42] Sato S.288 Antioxidant Enzyme [29] Mese H. Zhang Y. Eibl H. and cell biol. Buchholz F. [32] Soengas MS. Press OW (1998) Apoptosis of malignant human B cells by ligation of CD20 with monoclonal antibodies.

Biochim. [56] Love SW. McClatchey A. Bradley A (1992) Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Monden M (2000) CDC25B and p53 are independently implicated in radiation sensitivity for human esophageal cancers. Habilitation. Tolis C and Giaconne G (1999) P53 and chemosensitivity. Ziółkowski P. Cancer res. Sontag E. J clin. Nariai K. [48] Simbulan-Rosenthal CM. commu. Duarte CB (2004) Intracellular signalling mechanism in photodynamic therapy. Bodis S. Yoshikawa T (2007) Early apoptosis and cell death induced by ATX-S10Na(II)-mediated photodynamic therapy are Bax. [49] Donehower LA. 9: 402-412. Rosenthal DS. Chen P & Levine A (2008) Transcriptional control of human p53regulated genes. mol. Ann. Cortes U. [60] Almeida RD. Acta biochim. Remington L. Wysocka T. [45] Kirsh DG. McArthur MJ. [52] Gerl R and Vaux DL (2005) Apoptosis in the development and treatment of cancer. Ruley HE. 10: 1011-1021. Yano M. Housman DE and Jacks T (1993) p53 is required for irradiation-induced apoptosis in mouse thymocytes. [51] Stokłosa T and Gołąb J (2005) Prospects for based cancer therapy. World j. Sumi M. Pharm. Oncogene 22: 7486-7495. Science 266: 807-810. [58] Bar KB. gastroentero. Manadas BJ. Yamamoto H. Kishi K. [47] Tong WM. Chwiłkowska A. biophys. Nishioka K. Nature rev. Słomska I. Remington L. 59: 2190–2194. cancer res.and radiotherapy. Zhang L (2005) The transcriptional targets of p53 in apoptosis control. 16: 3158-3168. pol. biophys. 6: 4859-4865. Luo R. Saczko J. Doki Y. . [57] Mitsunga M. cell biol. Nature 362: 847-849. Fisher DE. Fisher DE.and p53 dependent in human colon cancer cells. acta 1704: 59-86. oncol. Shiozoki H. res. [59] Saczko J (2011) Determination of photodynamic therapy efficiency in clear ovarian cancer resistant to chemo. Butel JS. [53] Ferreira CG. Montgomery CAJ. Carvalho AP. acta 1552: 27– 37. Drąg-Zalesińska M. Kastan MB (1998) Tumor-supressor p53: implications for tumor development and prognosis. rep. oncol. McClatchey A. Ruley HE. Carcinogenesis 26: 263-270. Clin. [50] El-Deiry WS (2003) The role of p53 in chemosensitivity and radiosenssitivity. [55] Lowe SW. 52: 321-328. Nature 356: 215-221.Apoptosis. Wroclaw: Publishing House Wroclaw Medical University. Fujiwara Y. Free Radicals and Antioxidant Defense in Antitumor Therapy 289 [44] Riley T. 331: 851-858. Inoue M. Housman DE and Jacks T (1994) p53 status and the efficacy of cancer therapy. Smulson ME (1999) Poly(ADPribosyl)ation of p53 during apoptosis in human osteosarcoma cells. Biochem. 59: 1734-1140. Bodis S. Duś D (2007) Photofrin II based photosensitization of human ovarian clearcell carcinoma cell Line (OvBH-1). Slagle BL. [46] Yu J. Wang ZQ (2001) Poly(ADP-ribose) polymerase:a guardian angel protecting the genome and suppressing tumorigenesis. [54] Miyata H.13: 692-698. Harvey M. Biochim. Tsubota A.

Satoh K. 26: 28-38. [64] Lee S. Rosenquist R. Jondal M. cancer 33: 125–131. p21 and p27 in cultured human breast tissues. 157: 105–112. Huber LJ (2006) Inhibition of SIRT1 Catalytic Activity Increases p53 Acetylation but Does Not Alter Cell Survival following DNA Damage. 7: 71-80. Braszko JJ (2007) Potranslacyjna fosforylacja białka p53 w komórkach niedrobno komórkowego raka płuca po radio. cancer 97: 769-780. Li Y. 8: 324-330. 276: 36425–36430. 281: 5734-5740. Cancer 2: 389-404. McDonagh T. Okvist A. [63] Yoshida K. Liu H and Miki Y (2006) Proteinkinase C  regulates Ser 46 phosforylation of p53 tumor suppressor in the apoptotic response to DNA damage. 9: 49-89. mol. Elenbaas B. chem. redox sign. and invasive epithelial ovarian tumors. [71] Luo J.. 75: 241-250. Narumi K. Park JW. Via med. j. Magliocco AM (2003) A review of p53 expression and mutation in human benign. Anaesthesia 55: 1081-1093. Wang X & Choi AMK (2007) Mechanisms of cell death in oxidative stress. Griffith J (1995) p53 and its 14 kDa C-terminal domain recognize primary DNA damage in the form of insertion/deletion mismatches. J. Osorio LM (2007) Upregulation of bif-1 is a potential mechanism of chemoresistance in B-cell chronic lymphocytic leukaemia. Xu L. Zheleva D. Chen D. Nutr. Hutchins JR. Mol cell biol. Curtis R. Bhuiyan M. [67] Inoue A. [65] Lian F. Chyczewski L. Sarkar FH (1999) p53-independent apoptosis induced by genistein in lung cancer cells. J. Cell cycle 2008. [68] Hołownia A. Pellecchia M (2005) Apoptosis-based therapies for hematologic malignancies. Fersh NI (2000) Apoptosis: mechanisms and clinical applications. Su F. Hoetzel A. Blood 106: 408-418. Tobin G. [69] Luciani MG. chem. J. Antioxid. Distefano PS. [75] Reed JC. Sugawara S. [73] Friedman JS and Lowe SW (2003) Control of apoptosis by p53. Hupp TR (2000) The C-terminal regulatory domain of p53 contains a functional docking site for cyclin A. [74] Kam PC. [62] Olsson A. Br. [76] Ryter SW.i chemioterapii. Nukiwa T (2000) Administration of wild type p53 adenoviral vector synergistically enhances the cytotoxicity of anti-cancer drugs in human lung cancer cells irrespective of the status of p53 gene. Risto E. Norberg M. Pirkko H. Pasupuleti R. [66] Kmet LM. low malignant potential. Trends cell biol. Nature 408: 377-381. Cook LS. Osterborg FA. 300: 503–518. biol. [70] Mendoza-Alvarez H. Celsing F. Saijo Y. Gu W (2000) Deacetylation of p53 modulates its effect on cell growth and apoptosis. Cell 81: 1013–1020. biol. Nakahira K. Laudański J. [77] Kelekar A & Thompson CB (1998) Bcl-2 family proteins: the role of the BH3 domain in apoptosis. Levine A. Mróz M. Matsubara N. Effects of estradiol and medroxyprogesterone acetate on expressionof the cell cycle proteins cyclin D1. biol. Alvarez-Gonzalez R (2001) Regulation of p53 sequence-specific DNA-binding by Covalent Poly(ADP--ribosyl)ation. Dercov K.. Kozłowski M. Chyczewska E. Choudhury A. Shiloh A. . Oncogene 22: 9030-9040. Kim HP. [72] Solomon JM. Cancer lett.290 Antioxidant Enzyme [61] Natalija F.

natl. [85] Skulachev. Oncogene 19: 649-660. Srinivasan A. Ma LP. Science 288: 1053-1058. [90] Yu J. 100: 1931-1936. Free Radicals and Antioxidant Defense in Antitumor Therapy 291 [78] Bouillet P. cell biol. 115: 1567-1574. 114: 441-449. Immunol. is induced by p53. [79] Packham G. Demicco EG. Zhang ZY (1999) Wild-type p53 protein potentiates phototoxicity of 2-BA-2-DMHA in HT29 cells expressing endogenous mutant p53. acad. [93] Sax JK. Mol. Nat. [88] Attardi LD. a novel proapoptotic gene. Kapsetaki M. Yamashita T. Murphy ME. J. J of Cell Science 116: 4077-4085. Stevenson FK (2005) Bodyguards and assassins: Bcl-2 family proteins and apoptosis control in chronic lymphocytic leukaemia. 94: 2345-2349. Korsmeyer SJ and El-Deiry WS (2002) BID regulation by p53 contributes to chemosensitivity. Patel S.Apoptosis. Shibue T. [86] Chong MJ. PERP. a BH3-only member of Bcl-2 family and candidate mediator of p53 induced apoptosis.138: 189-195. McCurrach ME. Wang Z. Kinzler KW. [84] Bouvard V. [91] Nakano K and Vousden KH (2001) PUMA. Zhang L. before and following ionising irradiation in mice. Vacher M. [87] McCurrach ME. Cancer lett. [80] Haupt S. 423. Proc. Russell HR. Yang HY. cell sci. FEBS Lett. Korsmeyer SJ and McKinnon PJ (2000) ATM and Bax cooperate in ionizing radiation-induced apoptosis in the central nervous system. Hwang P. Cytochrome c in the apoptotic and antioxidant cascades. Mol. 97: 889-894. Choisy-Rossi C. Genes dev. (1998). Reczek EE. [82] Thornborrow EC. Potten CS. Taniguchi T and Tanaka N (2000) Noxa. Lowe SW and Jacks T (2000). Fei P. . Cosmas C. [89] Pritchard DM. an apoptosis-associated target of p53. [81] Oda E. Oncogene 21: 990-999. Straser A (2002) BH3-only proteins-evolutionarily conserved pro-apoptotic Bcl-2 family members essential for initiating programmed cell death. Roberts S and Hickman JA (1999) Damageinduced apoptosis in intestinal epithelia from bcl-2. acad. [92] Yu J. Korsmeyer SJ. mdm2 and waf1/p21.null and bax-null mice: investigations of the mechanistic determinants of epithelial apoptosis in vivo. 275-280. Schwartzfarb EM and Manfredi JJA (2002) Conserved intronic response element mediates direct p53-dependent transcriptional activation of both the human and murine bax genes. Korsmeyer SJ and Lowe SW (1997) Baxdeficiency promotes drug resistance and oncogenic transformation by attenuating p53dependent apoptosis. sci. 4: 842-849. Berger M. Proc. is a novel member of the PMP22/gas3 family. Murray MR. sci. cell 7: 673-682. Tokino T. Knudson CM. [83] Zhang WG. Canivet M. Bernhard E. Murasawa H. Goldberg Z and Haupt Y (2003) Apoptosis-the p53 network. V. natl. P. Oncogene 18: 7287-7293. Kinzler KW and Vogelstein B (2001) PUMA induces the rapid apoptosis of colorectal cancer cells. Ohki R. Nieruchalski M and May E (2000) Tissue and cell-specific expression of the p53-target genes: bax. natl. cell 7: 683-694. Connor TM. Vogelstein B and Zhang L (2003) PUMA mediates the apoptotic response to p53 in colorectal cancer cells. Li XW. Mastropietro AE. Gosink EC. Duthu A. Nemoto J. Wang SW. sci. fas. 14: 704-718. acad. Zaitchouk T. Proc.

292 Antioxidant Enzyme

[94] Minn AJ, Rudin CM, Boise LH, Thompson CB (1995) Expression of bcl-xL can confer a multidrug resistance phenotype. Blood 86: 1903-1910. [95] Reed JC (2008) Bcl-2-family proteins and hematologic malignancies: history and future prospects. Blood 111: 3322-3330. [96] Sellers WR and Fisher DE (1999) Apoptosis and cancer drug targeting. J. clin. invest. 104: 1655-1661. [97] Kang SJ, Kim BM, Lee YJ, Hong SH, Chung HW (2009) Titanium dioxide nanoparticles induce apoptosis through the JNK/p38-caspase-8-Bid pathway in phytohemagglutininstimulated human lymphocytes. Biochem. biophys. res. commun. 386: 682-687. [98] Reed JC (1999) Fenretinide: the death of a tumor cell. J. natl. cancer inst. 91: 1099-1100. [99] Lauria F, Raspadori D, Rondelli D, Ventura MA, Fiacchini M, Visani G, Forconi F, Tura S (1997) High bcl-2 expression in acute myeloid leukemia cells correlates with CD34 positivity and complete remission rate. Leukemia 12: 2075-2078. [100] Orian A, Whiteside S, Isssssrael A, Stancovski I, Scwartz AL and Ciechanover A (1995) Ubiquitin-Mediated Processing of NF-kB Transcriptional Activator Precursor p105. J. biol. chem. 270: 21707–21714. [101] Sudakin V, Ganoth D, Dahan A, Heller H, Hershko J, Luca FC, Ruderman JV and Hershko A (1995) The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis. Mol. biol. cell 6:185197. [102] Haas AL and Siepman TJ (1997) Pathways of ubiquitin conjugation. FASEB j. 14: 12571268. [103] Zhang HG, Wang J, Yang X, Hsu HCh and Mountz JD (2004) Regulation of apoptosis proteins in cancer cells by ubiquitin. Oncogene. 23: 2009-2015.[ [104] Breitschopf K, Haendeler J, Malchov P, Zeiher AM and Dimmeler S (2000) Posttranslational Modyfication of Bcl-2 Facilitates its Proteosome Dependent Degradation: Molecular Characterization of the Involved Signaling Pathway. Mol. and cell biol. 10: 1886-1896. [105] Suzuki Y, Nakabayashi Y, Nakata K, Reed JC, Takahashi R (2001) X-linked Inhibitor of Apoptosis Protein (XIAP) Inhibits Caspase-3 and -7 in Distinct Dodes. J. biol. chem. 276: 27058–27063. [106] Kovalenko A, Chable-Bessia C, Cantarella G, Israel A, Wallach D Courtois G (2003) The tumor suppressorCYLD negativly regulates NF-kappaB signaling by deubiquitination. Nature 6950: 801-805. [107] Aklyama T, Bouillet P, Miyazaki T, Kadano Y, Chikuda H, Chung U, Fukuda A, Khiikita A, Seto H, Okada T (2003) Regulation of Apoptosis by Ubiquitlation of Proapoptotic BH3 only Bcl-2 Family Member Bim. EMBO j. 22: 6653-6664. [108] Beere HM (2004) The Stress of Dying: The Role of Heat Shock Proteins in The Regulation of Apoptosis. J. cell sci. 117: 2641-2651. [109] Kaźmierczuk A, Kiliańska ZM (2009) The Pleiotropic Activity of Heat-Shock Proteins. Postepy hig. med. dosw. 63: 502-521. [110] Morimoto RI (1993) Cells in Stress: Transcriptional Activation of Heat Shock Genes. Science 259: 1409–1410.

Apoptosis, Free Radicals and Antioxidant Defense in Antitumor Therapy 293

[111] Charette SJ, Lavole JN, Lambert H and Landry J (2000) Inhibition of Daxx-Mediated Apoptosis by Heat Shock Protein 27. Mol. cell biol. 20: 7602-7612. [112] Thanner F, Sutterlin M, Kapp L, Rieger AK, Morr P, Kristen P, Dietl J, Gassel AM and Muller T (2005) Heat Shock Protein 27 is Associated with Decreased Survival NodeNegative Breast cancer patients. Anticancer res. 25: 1649-1654. [113] Artsi HJG, Hollema H, Lemstra W, Wilemse PHB, DeVries EGE, Kampinga HH and Van der Zeee AGJ (1999) Heat Shock Protein 27(HSP27) Expression in Ovarian Carcinoma Relation in Response to Chemotherapy and Prognosis. Int. j. cancer 84: 234238. [114] Langdon SP, Rabiasz GJ, Hirst GL (1995) Expression of the Heat Shock Protein HSP27 in Human Ovarian Cancer. Clin. cancer res. 1: 1603-1609. [115] Gabai VL, Mabuchi K, Mosser DD and Sherman NY (2002) Hsp 72 and Stress c-jun Nterminal Kinase Regulate the Bid-Dependent Pathway in Tumor Necrosis FactorInduced Apoptosis. Mol. cell biol. 22: 3415-3424. [116] Paul C, Monero F, GoninS, Kretz –Remy C, Virot S and Arrigo AP (2002) Hsp 27as a Negative Regulator of Cytochrome c Release. Mol. cell biol. 22: 816-834. [117] Lyakhovich VV, Vavilin VA, Zenkov NK, Menshchikova EB (2006) Active defense under oxidative stress. The antioxidant responsive element. Biochemistry–Moscow 71: 962-974. [118] Burlakova EB, Zhizhina GP, Gurevich SM, Fatkullina LD, Kozachenko AI, Nagler LG, Zavarykina TM, Kashcheev VV (2010) Biomarkers of oxidative stress and smoking in cancer patients. J. cancer res. ther. 6: 47-53. [119] Halliwell B (2007) Oxidative stress and cancer: have we moved forward? Biochem. j. 401: 1-11. [120] Yuzhalin AE, Kutikhin AG (2012) Inherited variations in the SOD and GPX gene families and cancer risk. Free radic. res. Epub ahead of print. [121] Valko M, Rhodes CJ, Moncol J, Izakovic M, Mazur M (2006) Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem. biol. interact. 160:1-40. [122] Fridovich I (1986) Superoxide dismutases. Adv. Enzymol. relat. areas mol. biol. 58: 6197. [123] Ho YS, Crapo JD (1988) Isolation and characterization of complementary DNAs encoding human manganese-containing superoxide dismutase. FEBS lett. 229: 256-260. [124] Guidot DM, McCord JM, Wright RM, Repine JE (1993) Absence of electron transport (Rho 0 state) restores growth of a manganese-superoxide dismutase-deficient Saccharomyces cerevisiae in hyperoxia. Evidence for electron transport as a major source of superoxide generation in vivo. J. biol. chem. 268: 26699-26703. [125] Gromer S, Eubel JK, Lee BL, Jacob J (2005) Human selenoproteins at a glance. Cell mol. life sci. 62: 2414-2437. [126] Wu G, Fang YZ, Yang S, Lupton JR, Turner ND (2004) Glutathione metabolism and its implications for health. J. nutr. 134: 489-492. [127] Brigelius-Flohe R (1999) Tissue-specific functions of individual glutathione peroxidases. Free radic. biol. med. 27: 951-965.

294 Antioxidant Enzyme

[128] Forsberg L, de Faire U, Morgenstern R (1999) Low yield of polymorphisms from EST blast searching: analysis of genes related to oxidative stress and verification of the P197L polymorphism in GPX1. Hum. mutat. 13: 294-300. [129] Maiorino M, Thomas JP, Girotti AW, Ursini F(1991) Reactivity of phospholipid hydroperoxide glutathione peroxidase with membrane and lipoprotein lipid hydroperoxides. Free radic. res. commun. 13: 131-135. [130] Mates JM, Perez-Gomez C, Nunez de Castro I (1999) Antioxidant enzymes and human diseases. Clin. biochem. 32: 595-603. [131] Pelicano H, Carney D, Huang P (2004) ROS stress in cancer cells and therapeutic implications. Drug resist. updat. 7: 97-110. [132] Xie J, Fan R, Meng Z (2007) Protein oxidation and DNA-protein crosslink induced by sulfur dioxide in lungs, livers, and hearts from mice. Inhal. toxicol. 19: 759-765. [133] Oberley TD, Oberley LW (1997) Antioxidant enzyme levels in cancer. Histol. histopathol. 12: 525-535. [134] Sato K, Ito K, Kohara H, Yamaguchi Y, Adachi K, Endo H (1992) Negative regulation of catalase gene expression in hepatoma cells. Mol. cell biol. 12: 2525-2533. [135] Li Y, Reuter NP, Li X, Liu Q, Zhang J, Martin RC (2010) Colocalization of MnSOD expression in response to oxidative stress. Mol. carcinog. 49: 44-53. [136] Yanbaeva DG, Dentener MA, Creutzberg EC, Wesseling G, Wouters EF (2007) Systemic effects of smoking. Chest 131: 1557- 66. [137] Stepovaya EA, Novitskii W, Ryazantseva NV, Goldberg VE, Tkachenko SB, Kolosova MV (2003) Structure and properties of lipid bilayer of erythrocyte membranes in patients with malignant tumors. Bull. exp. biol. med. 136: 490-3. [138] Yano T, Shoji F, Baba H, Koga T, Shiraishi T, Orita H, Kohno H (2009) Significance of the urinary 8-OHdG level as an oxidative stress marker in lung cancer patients. Lung cancer 63: 111-114. [139] Nowsheen S, Wukovich RL, Aziz K, Kalogerinis PT, Richardson CC, Panayiotidis MI, Bonner WM, Sedelnikova OA, Georgakilas AG (2009) Accumulation of oxidatively induced clustered DNA lesions in human tumor tissues. Mutat. res. 674: 131-136. [140] Paz-Elizur T, Sevilya Z, Leitner-Dagan Y, Elinger D, Roisman LC, Livneh Z (2008) DNA repair of oxidative DNA damage in human carcinogenesis: Potential application for cancer risk assessment and prevention. Cancer lett. 266: 60-72. [141] Beevi SS, Rasheed MH, Geetha A (2007) Evidence of oxidative and nitrosative stress in patients with squamous cell carcinoma. Clin. chim. acta. 375: 119-123. [142] Patel BP, Rawal UM, Rawal RM, Shukla SN, Patel PS (2008) Tobacco, antioxidant enzymes, oxidative stress, and genetic susceptibility in oral cancer. Am. j. clin. oncol. 31: 454-459. [143] Yanbaeva DG, Wouters EF, Dentener MA, Spruit MA, Reynaert NL(2009) Association of glutathione -S-transferase omega haplotypes with susceptibility to chronic obstructive pulmonary disease. Free radic. res. 43: 738-743. [144] Fiaschi AI, Cozzolino A, Ruggiero G, Giorgi G (2005) Glutathione, ascorbic acid and antioxidant enzymes in the tumor tissue and blood of patients with oral squamous cell carcinoma. Eur rev. med. pharmacol. sci. 9: 361-7.

Apoptosis, Free Radicals and Antioxidant Defense in Antitumor Therapy 295

[145] Patel BP, Rawal UM, Dave TK, Rawal RM, Shukla SN, Shah PM, Patel PS (2007) Lipid peroxidation, total antioxidant status, and total thiol levels predict overall survival in patients with oral squamous cell carcinoma. Integr. cancer ther. 6: 365-372. [146] Gokul S, Patil V, Jailkhani R, Hallikeri R, Kattappagari K (2010) Oxidant- antioxidant status in blood and tumor tissue of oral squamous cell carcinoma patients. Oral dis. 16: 29-33. [147] Gargouri B, Lassoued S, Ayadi W, Karray H, Masmoudi H, Mokni N, Attia H, El Feki Ael F (2009) Lipid peroxidation and antioxidant system in the tumor and in the blood of patients with nasopharyngeal carcinoma. Biol. trace elem. res. 132: 27-34. [148] Grace Nirmala J, Narendhirakannan RT (2011) Detection and Genotyping of HighRisk HPV and Evaluation of Anti-Oxidant Status in Cervical Carcinoma Patients in Tamil Nadu State, India - a Case Control Study. Asian pac. j. cancer prev. 12: 2689-2695. [149] Spychalowicz A, Wilk G, Sliwa T, Ludew D, Guzik TJ (2012) Novel therapeutic approaches in limiting oxidative stress and inflammation. Curr. pharm. biotechnol. Epub ahead of print. [150] Bedard K, Krause KH (2007) The NOX Family of ROS-Generating NADPH Oxidases: Physiology and Pathophysiology. Physiol. rev. 87: 245-313. [151] Jaquet V, Scapozza L, Clark RA, Krause KH, Lambeth JD (2009) Small-molecule NOX inhibitors: ROS-generating NADPH oxidases as therapeutic targets. Antioxid. redox. signal. 11: 2535-2552. [152] Kikuchi H, Hikage M, Miyashita H, Fukumoto M (2000) NADPH oxidase subunit, gp91(phox) homologue, preferentially expressed in human colon epithelial cells. Gene 254: 237–243. [153] Banfi B, Maturana A, Jaconi S, Arnaudeau S, Laforge T, Sinha B, Ligeti E, Demaurex N, Krause KH (2000) A mammalian H+ channel generated through alternative splicing of the NADPH oxidase homolog NOH-1. Science 287: 138–142. [154] Ellmark SH, Dusting GJ, Fui MN, Guzzo-Pernell N, Drummond GR (2005) The contribution of Nox4 to NADPH oxidase activity in mouse vascular smooth muscle. Cardiovasc. res. 65: 495–504. [155] Fu X, Beer DG, Behar J, Wands J, Lambeth D, Cao W (2006) cAMP response element binding protein (CREB) mediates acid-induced NADPH oxidase NOX5-S expression in Barrett's esophageal adenocarcinoma cells. J. biol. chem. 281: 20368-20382. [156] Gołąb J, Nowis D, Skrzycki M, Czeczot H, Barańczyk-Kuźma A, Wilczyński GM, Makowski M, Mróz P, Kozar K, Kamiński R, Jalili A, Kopeć M, Grzela T and Jakóbisiak M (2003) Antitumor Effects of Photodynamic Therapy Are Potentiated by 2Methoxyestradiol. J. biol. chem. 278: 407–414. [157] Hamblin MR, Mróz P (2008) History of PDT: the first hundred years. In: Hamblin M.R., Mróz P, editors. Advances in Photodynamic Therapy: Basic, Translational and Clinical. Boston-London: Artech House. pp. 1-12. [158] Dougherty TJ, Gomer CJ, Henderson BW, Jori G, Kessel D, Korbelik M, Moan J, Peng Q (1998) Photodynamic therapy. J. natl. cancer inst. 90: 889-905.

296 Antioxidant Enzyme

[159] Dougherty TJ, Kaufman JE, Goldfarb A, Weishaupt KR, Boyle D and Mittleman A (1978) Photoradiation therapy for the treatment of malignant tumors. Cancer res. 38: 2628-2635. [160] Saczko J, Kulbacka J, Chwiłkowska A, Drąg-Zalesińska M, Wysocka T, Ługowski M & Banaś T (2005) The influence of photodynamic therapy on apoptosis in human melanoma cell line. Folia histochem. cyto. 43: 129-132. [161] MacCormack MA (2006) Photodynamic Therapy. Adv. dermatol. 22: 219-258. [162] Triesscheijn M, Baas P, Schellens JHM, Stewart FA (2006) Photodynamic Therapy in Oncology. Oncologist 11: 1034-1044. [163] Castano AP, Demidova T N, Hamblin MR (2005) Mechanisms in photodynamic therapy: part two - cellular signaling, cell metabolism and modes of cell death. Photodiag. photodyn. ther. 2: 1-23. [164] Ochsner M (1997) Photophysical and photobiological processes in the photodynamic therapy of tumours J. photochem. photobiol. b 39: 1-18. [165] Pospíšil P. (2012) Molecular mechanisms of production and scavenging of reactive oxygen species by photosystem II. Biochim biophys acta. 1817(1):218-231 [166] Castano AP, Demidova TN, Hamblin MR (2004) Mechanisms in photodynamic therapy: part one - photosensitizers, photochemistry and cellular localization. Photodiagn. photodyn. ther. 4: 279-293. [167] Korbelik M, Parkins CS, Shibuya H, Cecic I, Stratford MRL and Chaplin DJ (2000) Nitric oxide production by tumour tissue: impact on the response to photodynamic therapy. Br j. cancer 82: 1835-1843. [168] Kuroda M, Himei K, St Clair DK, Urano M, Yoshino T, Akagi T, Asaumi J, Akaki S, Takeda Y, Kanazawa S, Hiraki Y (2000) Overexpression of manganese superoxide dismutase gene suppresses spontaneous apoptosis without a resultant alteration in in vivo growth of the mouse fibrosarcoma, FSa-II. Anticancer res. 20: 7-10. [169] Zhao Y, Kiningham KK, Lin SM, St Clair DK (2001) Overexpression of MnSOD protects murine fibrosarcoma cells (FSa-II) from apoptosis and promotes a differentiation program upon treatment with 5-azacytidine: involvement of MAPK and NFkappaB pathways. Antioxid. redox signal. 3: 375-386. [170] Huang P, Feng L, Oldham EA, Keating MJ, Plunkett W (2000) Superoxide dismutase as a target for the selective killing of cancer cells. Nature 6802: 390-395. [171] Kliukiene R, Maroziene A, Nivinskas H, Cenas N, Kirveliene V, Juodka B (1997) The protective effects of dihydrolipoamide and glutathione against photodynamic damage by Al-phtalocyanine tetrasulfonate. Biochem. mol. biol. int. 41: 707-713. [172] Oberdanner CB, Plaetzer K, Kiesslich T, Krammer B (2005) Photodynamic treatment with fractionated light decreases production of reactive oxygen species and cytotoxicity in vitro via regeneration of glutathione. Photochem. photobiol. 81: 609-613. [173] Nowis D, Legat M, Grzela T, Niderla J, Wilczek E, Wilczyński GM, Głodkowska E, Mrówka P, Issat T, Dulak J, Józkowicz A, Waś H, Adamek M, Wrzosek A, Nazarewski S, Makowski M, Stokłosa T, Jakóbisiak M and Gołąb J (2006) Heme oxygenase-1 protects tumor cells against photodynamic therapy-mediated cytotoxicity. Oncogene 25: 3365–3374.

Apoptosis, Free Radicals and Antioxidant Defense in Antitumor Therapy 297

[174] Wagener FA, Volk HD, Willis D, Abraham NG, Soares MP, Adema GJ, Figdor CG (2003) Different faces of the heme-heme oxygenase system in inflammation. Pharmacol rev. 55: 551-571. [175] Stocker R, Yamamoto Y, McDonagh AF, Glazer AN, Ames BN (1987) Bilirubin is an antioxidant of possible physiological importance. Science 235:1043–1046. [176] Kapitulnik J (2004) Bilirubin: an endogenous product of heme degradation with both cytotoxic and cytoprotective properties. Mol. pharmacol. 66: 773–779. [177] Paine A, Eiz-Vesper B, Blasczyk R, Immenschuh S (2010) Signaling to heme oxygenase-1 and its anti-inflammatory therapeutic potential. Biochem. pharmacol. 80: 1895-1903. [178] Kocanova S, Buytaert E, Matroule JY, Piette J, Gołąb J, Witte P, Agostinis P (2007) Induction of heme-oxygenase 1 requires the p38MAPK and PI3K pathways and suppresses apoptotic cell death following hypericin-mediated photodynamic therapy. Apoptosis 12: 731–741. [179] Hennekens CH, Buring JE, Manson JE, Stampfer M, Rosner B, Cook NR, Belanger C, LaMotte F, Gaziano JM, Ridker PM, Willett W, Peto R (1996) Lack of effect of long-term supplementation with beta carotene on the incidence of malignant neoplasms and cardiovascular disease. N. engl. j. med. 334: 1145-1149. [180] Omenn GS, Goodman GE, Thornquist MD, Balmes J, Cullen MR, Glass A, Keogh JP, Meyskens FL, Valanis B, Williams JH, Barnhart S, Hammar S (1996) Effects of a combination of beta carotene and vitamin A on lung cancer and cardiovascular disease. N. engl. j. med. 334: 1150-1155. [181] The Alpha-Tocopherol, Beta Carotene Cancer Prevention Study Group, (1994) The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers N. engl. j. med. 330: 1029. [182] Crabtree DV, Adler AJ (1997) Is β-carotene an antioxidant? Med. hypoth. 48: 183. [183] Zhang P, Omaye ST (2001) Antioxidant and prooxidant roles for β-carotene, αtocopherol and ascorbic acid in human lung cells. Toxicol. in vitro, 15: 13. [184] Kim KN, Pie JE, Park JH, Park YH, Kim HW, Kim MK (2006) Retinoic acid and ascorbic acid act synergistically in inhibiting human breast cancer cell proliferation. J Nutr Biochem. Jul;17(7): 454-62. Epub 2005 Nov 15. [185] Burton GW, Ingold KU (1984) β-carotene: an unusual type of lipid antioxidant. Science. 224: 569. [186] Zhang P Omaye ST (2000) β-carotene and protein oxidation: effects of ascorbic acid and α-tocopherol. Toxicology. 146: 37. [187] Astorg P (1997) Food carotenoids and cancer prevention: an overview of current research. Trends food sci. Technol. 8: 406. [188] Nishino H (1998) Cancer prevention by carotenoids. Mutat. res. 402: 159. [189] Nishino H, Murakosh M, Ii T, Takemura M, Kuchide M, Kanazawa M, Mou XY, Wada S, Masuda M, Ohsaka Y, Yogosawa S, Satomi Y, Jinno K (2002) Carotenoids in cancer chemoprevention. Cancer Mmetastasis Rev. 21: 257.

298 Antioxidant Enzyme

[190] Landrum JT, Bone RA, Herrero C (2002) Astaxanthin, β-cryptoxanthin, lutein, and zeaxanthin, in Phytochemicals in Nutrition and Health. Meskin, M.S. et al., Eds., CRC Press, Boca raton, Florida, chap. 12. [191] Polaczek-Krupa B, Czechowicz-Janicka K (2004) Rola antyoksydantów w profilaktyce i leczeniu chorób oczu. (The role of antioxidants in the prevention and treatment of eye diseases) Ordynator leków. 4. [192] Head K (2001) Natural therapies for ocular disorders, part 2: cataract and glaucoma. Altern. med. rev. 6: 141. [193] Ball S (2001) Antyoksydanty w medycynie i zdrowiu człowieka (Antioxidants in medicine and human’s health). Medyk, Warszawa. [194] Chan PH, Kinouchi H, Epstein CJ, Carlson E, Chen SF, Imaizumi S, Yang GY (1993) Role of superoxide dismutase in ischemic brain injury: reduction of edema and infarction in transgenic mice following focal cerebral ischemia. Prog. brain res. 96: 97104. [195] Kleszczewska E (2002) Witamina C jako naturalny antyoksydant.(Vitamin C as a natural antioxidant) Farm. pol. 58: 913. [196] Khassaf M, McArdle A, Esanu C, Vasilaki A, McArdle F, Griffiths RD, Brodie DA, Jackson MJ (2003) Effect of vitamin C supplements on antioxidant defence and stress proteins in human lymphocytes and skeletal muscle. J physiol, 549.2, pp. 645–652. [197] Chan AC. (1993) Partners in defense, vitamin E and vitamin C. Can j physiol pharmacol 71(9): 725-731. [198] Liu C, Russell RM, Wang XD (2004) a-Tocopherol and ascorbic acid decrease the production of h-apo-carotenals and increase the formation of retinoids from h-carotene in the lung tissues of cigarette smoke-exposed ferrets in vitro. J nutr. 134: 426– 30. [199] Chorvatovicova D, Ginter E, Kosinova A, Zloch Z (1991) Effect of vitamins C and E on toxicity and mutagenicity of hexavalent chromium in rat and guinea pig. Mutat. res. 262:41– 6. [200] Kim KN, Pie JE, Park JH, Park YH, Kim HW, Kim MK (2006) Retinoic acid and ascorbic acid act synergistically in inhibiting human breast cancer cell proliferation. J nutr biochem. 17(7): 454-62. [201] You WC, Zhang L, Gail MH, Chang YS, Liu WD, Ma JL, Li JY, Jin ML, Hu YR, Yang CS, Blaser MJ, Correa P, Blot WJ Fraumeni JF, Xu GW (2000) Gastric cancer: Helicobacter pylori, serum Vitamin C, and other risk factors. J. natl. cancer inst. 92: 1607–1612. [202] Knekt P, Jarvinen R, Seppanen R, Rissanen A, Aromaa A, Heinonen OP, Albanes D, Heinonen M, Pukkala E, Teppo L (1991) Dietary antioxidants and the risk of lungcancer. Am. j. epidemiol. 134: 471–479. [203] Van Poppel G, van den Berg H (1997) Vitamins and cancer. Cancer lett. 114(1-2): 195202. [204] Halliwell B (2001) Role of free radicals in the neurodegenerative diseases: therapeutic implications for antioxidant treatment. Drugs aging. 18: 685-716.

Apoptosis, Free Radicals and Antioxidant Defense in Antitumor Therapy 299

[205] Nea M, Jarmo V, MiKKo V, Demetrius A (1999) The effect of α-tocopherol and β carotene supplementation on colorectal adenomas in middle aged male smokers. Cancer epidemiol. 8: 489–493. [206] Gail ME, Catherine H, Vartouhi J, Elizabeth BS, Peter D, Robert WB (1988) A randomized trial of vitamins C and E in the prevention of recurrence of colorectal polyps. Cancer res. 48: 4701– 4705. [207] Robert S, Zygmunt G, Yousif S, Godwin BE, Maciej S (2005) Cysteine peptidase and its inhibitor activity levels and vitamin E concentration in normal human serum and colorectal carcinomas. World j gastroentnol. 11(6): 850–853. [208] White E, Shannon JS, Patterson RE (1997) Relationship between vitamin and calcium supplement use and colon cancer. Cancer epidemiol. biomark. prev. 6: 769–774. [209] Bhagat Sonali S, Ghone Rahul A, Suryakar Adinath N, Hundekar Prakash S (2011) Lipid peroxidation and antioxidant vitamin status in colorectal cancer patients. Indian j physiol pharmacol. 55 (1): 72–76. [210] Bjelakovic G, Nikolova D, Gluud LL, Simonetti RG, Gluud C (2007) Mortality in randomized trials of antioxidant supplements for primary and secondary prevention: systematic review and meta-analysis, JAMA, 297 (8), 842-857. [211] Greenberg ER, Baron JA, Tosteson TD, Freeman DH, Beck GJ, Bond JH (1994) Clinicaltrial of antioxidant vitamins to prevent colorectal adenoma. N. engl. j. med. 331: 141– 147. [212] Manashi B, Milnes M, Williams C, Balmoori J, Ye X, Stohs S, Bagchi D (1999) Acute and chronic stress-induced oxidative gastrointestinal injury in rats, and the protective ability of a novel grape seed proanthocyanidin extract. Nutrition research. 19 (8): 1189– 1199. [213] Block G, Patterson B, Subar A (1992) Fruit, vegetables and cancer prevention: a review of the epidemiological evidence. Nitr cancer, 18: 1-29. [214] Willett WC (2002) Balancing life-style and genomics research for disease prevention. Science. 296: 695-698. [215] Eberhardt MV, Lee CY, Liu RH (2000) Antioxidant activity of fresh apples. Nature. 405: 903-904. [216] Liu RH, Liu J, Chen B (2005) Apples prevent mammary tumors in rats. J agric food chem. 53: 2341-2343. [217] Way TD, Lee HH, Kao MC, Lin JK (2004) Black tea polyphenol theaflavins inhibit aromatase activity and attenuate tamoxifen resistance in HER2/neu-transfected human breast cancer cells through tyrosine kinase suppression. Eur j cancer. 40: 2165-2174. [218] Czeczot H (2003) Antyoksydacyjne działanie glutationu (Antioxidant activity of glutathione). Farm. pol. 59: 4. [219] Kałużny J, Jurgowiak M (1996) Udział reaktywnych form tlenu w patogenezie wybranych chorób oczu. (Participation of reactive oxygen species in the pathogenesis of eye diseases) Klin. ocz. 98: 145. [220] Dringen R (2000) Metabolism and functions of glutathione in brain. Prog. Neurobiol. 62: 649-671.

(2004) Prognostic value of troponin I in cardiac risk stratification of cancer patients undergoing high-dose chemotherapy. Negrotto S. [224] Avinash SS. Circulation. [230] Batist G (1988) Selenium. 28(1):377-384 . Fiorentini C. Ma N.4-triazole on antimycin A-treated Calu-6 lung cells in relation to cell growth. Lamantia G. 64: 527-542 [227] Yan Yin. Cancer epidem biomark & prev.16(4):417-429. Sudha K. Jing Xing. Yan-Rong Fan. Vinodchandran. Colombo N. Civelli M. Crowell JA. Heliövaara M. [223] Verma AS. 61(2): 288-95. Free radic biol med. Gayathri M. 49(6):997-1007 [234] Huang YJ. Clin colorectal cancer. [229] Fakih M. Dwivedi PD. Oncology reports. Durrani FA. Cun-Shuan X (2009) Antitumor effects of a selenium heteropoly complex in K562 cells. (2011) Nitrative and oxidativeDNA damage as potential survival biomarkers for nasopharyngeal carcinoma. [225] Knekt P. Toxicol lett.Jian-Jun Wang. 385–391. Marniemi J. 16 (8): 1662–1666. Pharmacol rep. [222] Chow HH. Med. Martinelli G. Rustum YM (2005) Selenium protects against toxicity induced by anticancer drugs and augments antitumor activity: a highly selective. Cordova CA. Tang AZ. oncol. Aromaa A (1998) Is low selenium status a risk factor for lung cancer? Am j epidemiol. Schattner M. Rao. 148 (10): 975-982. Ranger-Moore J. [231] D'Atri LP. Mikhael DM. diethyldithiocarbamate or 3-amino-1. and novel approach for the treatment of solid tumors. Pozner RG. Tome ME. Nitric oxide: news from stem cells to platelets. Mishra A. Vining DR.2. Curr med chem. [232] Cardinale D. Beena V. 53 (4): 370–374. Cipolla CM. Latini FR. Zhi-Wei Wu. Indian j physiol pharmacol. reactive oxygen species and glutathione. Colombo A. [228] Jun-Ying Y. Anitha M. Ray PK. Murata M. Cao S. Cerutti JM. Proc nutr soc. Afr j microbiol res. buthionine sulfoximine. Preclinical studies of anticancer therapeutic potential. new. Boeri M. 109(22):2749-2754 [233] Sousa MS. Briehl MM. Zhang BB. Shetty (2009) Advanced oxidation protein products and total antioxidant activity in colorectal carcinoma. Peccatori F. Hakim IA. Biol trace elem res. Alberts DS (2007) Modulation of Human Glutathione STransferases by Polyphenon E Intervention. Huang GW. Romaniuk MA. (1999) Glutathione reduces the toxicity associated with antitumor therapy of ascites fluid adsorbed over Staphylococcus aureus Cowan I in tumor bearing mice. Sandri MT. Qing-Xiao Wang. Monteiro HP.106(2-3):119-127. 2009. [226] Rayman MP (2005) Selenium in cancer prevention:review of the evidence and mechanism of action. Malaver E. 5(2): 132135. Gen-Xing Xu (2011) Antitumor efficacy of Bifidobacterium longum carrying endostatin gene enriched with selenium and the distribution of selenium. (2010) Arginase 2 and nitric oxide synthase: Pathways associated with the pathogenesis of thyroid tumors. Teppo L. Xu Chen. 15:223-229.300 Antioxidant Enzyme [221] Park (2009) The effects of N-acetyl cysteine. 5(31): 5615-5621.

(2007) A list of candidate cancer biomarkers for targeted proteomics. Biochemistry (Mosc). [252] Mercier I. Kizek R. [250] Gumulec J. Nutritional and metabolic aspects. World j gastroenterol. Lugowski M. vitamin C. Adam V. (2003) Levels of plasma vitamin E. 11(3):403-406 [239] Gago-Dominguez M. Pujol H. Babula P. [240] Gerber M. TBARS.49 Suppl 1:82-84. (2012) Caveolin-1 and prostate cancer progression. [242] Gerber M. [247] Ozben T. Nagengast FM. (2011) Age-associated neurodegeneration and oxidative damage to lipids. Hlavna M. (2007) Lipid peroxidation. (2011) Oxidative stress-induced biomarkers for stem cell-based chemical screening. DNA repair (Amst). Sztalmachova M. 96(9):2181-2196.Apoptosis. Sulkowski S. Carcinogenesis. (2012) Filamin-A as a marker and target for DNA damage based cancer therapy. Kanczuga-Koda L. and cholesterol in male patients with colorectal tumors. (2012) Caveolin-1 as a potential high-risk prostate cancer biomarker. 114:211-214. Zhao Z. Hrabec R. (2007) Oxidative stress and apoptosis: impact on cancer therapy. Prev med. Kang KS. Papila C. (2012) Caveolin-1 and breast cancer: a new clinical perspective. Biochimie. Oncol rep. Jiang X. Mol aspects med. Chwiłkowska A. Dec. 27(3):831-841. Cancer lett. Free radic biol med. Astre C. Ghone RA. [248] Yue J. 39(2):182-187. Richardson S. Koltai E. [251] Freeman MR. (2005) High oxygen radical production in patients with sporadic colorectal cancer. J pharm sci. Astre C. Segala C. Berwick M. Goto S. Indian j physiol pharmacol. Rahman I. . Lu H. (2012) Exogenous markers for the characterization of human diseases associated with oxidative stress. Rovny A. Shen Z. Adv exp med biol. Roelofs HM. Masarik M. Scali J. Lisanti MP.1:1-48. [245] Saczko J. (2011) Lipid peroxidation and antioxidant vitamin status in colorectal cancer patients.. Biomark insights. 8 [Epub ahead of print] [236] Vaya J. 32(4-6):305-315. Rocz akad med bialymst. Grenier J. proteins and DNA. Castelao JE. 55(1):72-77 [238] Skrzydlewska E. Free Radicals and Antioxidant Defense in Antitumor Therapy 301 [235] Yang SR. Krizkova S. Di Vizio D. Eckschlager T.9(1):201. (2004) Levels of lipid peroxidation in A549 cells after PDT in vitro. Crastes de Paulet P. Trosko JE. Konukoglu D. (2005) Lipid peroxidation and antioxidant status in colorectal cancer. Adv exp med biol. 17:1267-1271. Crastes de Paulet A: (1989) Relationship between vitamin E and polyunsaturated fatty acids in breast cancer. Suryakar AN. Sulkowska M. Wobbes T. Anderson NL. Yang W. Gerber M: (1996) Tumor progression and oxidantantioxidant status.68(3):325-328. [241] Saintot M.729:83-94. Kulbacka J. Cancer. [246] Radak Z. [Epub ahead of print] [237] Bhagat SS. Breast cancer res. Liu J. Sochor J. Mar 10. 64:2347-2353 [243] van der Logt EM. Saintot M. [244] Saygili EI. Banaś T. Peters WH.729:95-110. [249] Polanski M. Hundekar PS. Pujol H. Akcay T.11(2):192-200. Pujol H: (1997) Tumor progression and oxidant-antioxidant status. Simony-Lafontaine J. oxidative stress genes and dietary factors in breast cancer protection: a hypothesis. Koda M. Zalewski B.

Pol merkur lekarski. Pervaiz S. Panayiotidis MI. (2011) Determination of oxygen derived free radicals producer (xanthine oxidase) and scavenger (paraoxonase1) enzymes and lipid parameters in different cancer patients. Clin lab. Kulbacka J. Pappa A.57(9-10):741-747 . Georgakilas AG. Chem Biol Interact. Dar N. Georgakila S.302 Antioxidant Enzyme [253] Ługowski M. Shaheen S. Banaś T. (2011) [Reactive oxygen and nitrogen species]. (2010) The role of reactive oxygen species and oxidative stress in environmental carcinogenesis and biomarker development. Schoneveld O. Saczko J. Franco R. [254] Ziech D. Athar MA. Malamou-Mitsi V. [255] Samra ZQ. 31(185):313-317.188(2):334-339.

Beta-carotene is found in many foods that are orange in color [6]. which permits unrestricted Introduction Oxygen radicals are continuously formed in all living organisms. Vitamin E compounds (tocopherols and tocotrienols) are well recognized for their effective inhibition of lipid oxidation in food and biological systems [5. Stress plays a significant role in the development of atherosclerotic heart disease (AHD) [7].Chapter 11 Effect of Different Concentrations of Red Palm Olein and Different Vegetable Oils on Antioxidant Enzymes in Normal and Stressed Rat Eqbal M. including free radicals [1. Inherent       © 2012 Dauqan et al. and reproduction in any medium. A stressful condition leads to the excessive production of free radicals which results in oxidative stress an imbalance in the oxidant per antioxidant system [8]. 2]. Production of oxidative species occurs under physiological conditions at a controlled rate. A.5772/48272 1. catalase (CAT) and glutathione peroxidase (GSH-Px) which performs a vital role for detoxification of free radicals. Dauqan. 4]. In foods. Antioxidant enzyme such as superoxide dismutase (SOD) is an important radical superoxide scavenger and it plays an important role in cell protection [10. 2]. The use of antioxidant rich food or antioxidant food supplements became immensely popular since many diseases have been associated with oxidative stress [9].   . licensee InTech. oxygen-containing molecules. carotenoids usually act as a secondary antioxidant. Reactive oxygen species (ROS) is a term which encompasses all highly reactive. with deleterious effects that lead to cell injury and distribution. Aminah Abdullah and Halimah Abdullah Sani Additional information is available at the end of the chapter http://dx.doi.. Carotenoids can act as primary antioxidants by trapping free radicals or as secondary antioxidants by quenching singlet oxygen. there is a natural defense system provided by several enzymes such as superoxide dismutase (SOD). 2]. Under normal conditions. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons. provided the original work is properly cited.0). this CAT or SOD enzyme are very good biochemical markers of stress and their increased activity may attest to a potential for remediation [11]. Therefore. capable of engaging in rapid change reaction that destabilize other molecules and generate many more free radicals [3. Free radicals are an atom or molecule that bears an unpaired electron and is extremely reactive. but it is dramatically increased in conditions of oxidative stress.

It derives its red colour from the high content of alpha. which acts as a super-antioxidant and the carotenoids in red palm oil also act as antioxidants [17]. which can make up 0. and mixed manually and the diets were .08% (w/w) of the crude oil [13]. For the first group the test diet was prepared by mixing RPO with normal commercial rat pellet to contain 5%. corn oil (CO) and coconut oil (COC) were obtained commercially. Corn oil presents a relatively high concentration of polyunsaturated fatty acids (PUFA). The 5% diet was prepared by adding 5g RPO to 95g rat pellet. brownish yellow oil [20]. Problem statement Due to the importance of the role of antioxidants in protection against the oxidative stress which lead to many dangerous diseases such as heart diseases and cancer thus this study was done to investigate the effect of natural antioxidants particularly vitamin E and beta carotene in red palm olein on antioxidant enzymes and compared the results with four different vegetable oils in normal and stress conditions of rats.and beta-carotenes. 3. 14]. Red palm oil is the oil obtained before refining and the characteristic colour of RPO is due to the abundance of carotenoids (500 – 700 mg /L) in the crude oil [15. and superoxide dismutase (SOD). giving rise to the formation of lipid peroxides.045%) provided by Carotino SDN BHD company and palm olein (PO) (Seri Murni). Most people are not aware of the fact that many different kinds of vitamin E occur in nature and that some forms of vitamin E are more beneficial than others. It is the major sources of saturated fat apart from palm kernel.Due to the high levels of unsaturation these lipids are highly susceptible to free radical oxidative reactions. 19]. Many investigations suggest that a large number of polyunsaturated fatty acids produces more lipid peroxides and may have mutagenic activity [18. Effect of different vegetable oils on antioxidant enzymes in normal and stressed rats The evaluated red palm olein (RPO) samples consisted of carotenes (576 ppm). Red palm oil (RPO) is extracted from the oil palm (Elaeis guineenis) fruit [13.304 Antioxidant Enzyme antioxidant defense systems consisting of enzymes. a decrease in the activities or expression of these enzymes may predispose tissues to free radical damage [12]. such as catalase (CAT). As antioxidant enzymes have an important role in the protection against free radical damage. Therefore the objective of this study is to investigate the effect of different concentration of red palm olein and different vegetable oils on antioxidant enzymes in normal and stressed rats. an antioxidant nutrients may participate in coping with oxidative stress. Coconut oil is the principal cholesterol-raising fat because it contains large amounts of lauric (C: 12: O) and myristic (C: 14: 0) acids [21]. Coconut oil is a colorless to pale. They are the only natural sources of lauric oil available to the world market. 2. 16]. vitamin E (>800 ppm) and free fatty acids (0. 10% and 15% of the red palm olein (RPO). Red palm oil contains vitamin E tocotrienols.

Similar process was conducted with 10%. . The 15% diet was prepared by adding 15g RPO. Figure 1. Figure 2. and mixed manually and the diets were then left to absorb the vegetable oils at room temperature overnight and stored at 20o C before the feeding trial was conducted. and 15% RPO. Normal (N) group: Rats were maintained under standard laboratory conditions and fed with respective diet till the completion of the experiment. They were divided into three groups. CO or COC to 85g rat pellet. Universiti Kebangsaan Malaysia. Normal rats Stress (S) group: Rats were restrained by placing them in individual nylon plastic bag for 3 hr/day for one week before killing. For second group the test diet was prepared by mixing vegetable oils with normal commercial rat pellet to contain 15% of the vegetable oils. till the completion of the experiment. Stressed rats One hundred and eighty Sprague Dawley male rats each weighing between 170-250g and approximately 80 days old were obtained from the animal house of the Faculty of Science and Technology. Under these conditions rat were fed with respective diet.Effect of Different Concentrations of Red Palm Olein and Different Vegetable Oils on Antioxidant Enzymes on Normal and Stressed Rat 305 then left to absorb the RPO at room temperature overnight and stored at 20o C before the feeding trial was conducted. PO.

Afterwards.81 g KH2PO4. and was homogenized using a mixer at top speed for 3 min.0 mL of hydrogen peroxide solution (30 mM).0 mL of the liver homogenate suspended in phosphate buffer (50 mM.11. and (b) 8. At the end of the experiment. Tissue was suspended in 2 ml of 50 mM phosphate buffer (pH 7. The reaction mixture consisted of 2. The liver was removed immediately and was washed it with NaCl solution. 4 and 8 weeks. Figure 3.2H2O in distilled water and make up to 100 ml each mix solution (a) and (b) in proporation 1:1. after 2. The second group contains 66 Sprague Dawley male rats which were randomly divided into 11 groups of 6 rats per group and were treated with 15% of RPO.6) was determined based on Aebi’s method [22].4).5 (v/v).0). Phosphate buffer was prepared based on Aebi’s method [22].2 g sample of liver was cut to small pieces. Phosphate buffer 50 mM. They were anesthetized using chloroform. The third group contains 36 Sprague Dawley male rats which were randomly divided into six groups of 6 rats per group (3 normal groups and 3 stressed groups) and were treated with 15% of RPO and PO for 4 weeks. The absorbance was recorded for 2 minutes at 240 nm immediately after adding hydrogen . coconut oil (COC) and control groups for 4 and 8 weeks.90 g Na2HPO4.0: dissolve (a) 6. PH 7. corn oil (CO). the homogenate was centrifuged at 20000 g for 25 min. Catalase activity was measured at 22°C by monitoring the decomposition of hydrogen peroxide. 4 or 8 weeks of treatment the feeding of rats was stopped and the rats were fasted for 18 hours. The rats were fed ad libitum with commercial rat’s pellet containing different concentrations of red palm olein (RPO) for 2.1. In this process the temperature was maintained at 4C0 during the homogenization process. palm olein (PO). Procedure for collecting liver from rat A 0. and 1.306 Antioxidant Enzyme The first group contains 78 rats were divided into 13 groups of 6 rats per group.1. Enzyme activity of catalase (EC. pH 7. It was stored at -80oC until analyzed.

in particular. constitute a major part of this defense [25]. The absorbance was recorded at 750 nm. The absorbance was recorded for 3 minutes at 420 nm immediately after adding the pyrogallol solution. After that. containing 1mM EDTA. 300 μl of 0. After 2 weeks there was no significance difference (p≥0. Figure 4 showed the standard curve of absorbance which was plotted as a function of initial protein concentration and used it to determine the unknown protein concentrations. It is evident from earlier work that different concentrations RPO have differential effects on the activities of antioxidant enzymes [10]. 10% and 15%) for different times (2.6. and let the mixture stand at room temperature for 30–60 min. Antioxidant enzymes.1. Activity superoxide dismutase (EC.4.1 mL of sample or standard was added 0. 4 and 8 weeks) of treatment.1 mL of 2 N NaOH and hydrolyze at 100°C for 10 min in boiling water bath. 0. The reaction mixture consisted of 50 mM of cacodylic acid buffer pH 8. Protein concentrations were determined based on the Lowry method [24]. The hydrolysate was cooled to room temperature and added 1 mL of freshly mixed complex-forming reagent.2 mM pyrogallol. 6 and 7 showed the results of catalase activity at different concentrations of RPO (5%. 300 μl of liver homogenate.2) was assayed based on the method of Marklund and Marklund [23].1. Figure 4. Catalase activity was expressed as moles of hydrogen peroxide reduced/min/mg protein.Effect of Different Concentrations of Red Palm Olein and Different Vegetable Oils on Antioxidant Enzymes on Normal and Stressed Rat 307 peroxide solution. To 0. Effect of different concentrations of red palm olein on antioxidant enzyme of normal rat liver Antioxidant is an important part of a cells defense against free radical damage. Superoxide dismutase activity was determined at 22°C by using the pyrogallol. Figures 5. Let the solution stand at room temperature for 10 min. Superoxide dismutase activity was expressed as units of SOD/minute/mg protein. Protein standard curve 3.05) between the control .1 mL of Folin reagent was added using a vortex mixer.2.

308 Antioxidant Enzyme group and 10% and 15% concentrations of RPO while at 5% there was an increased in the catalase activity. Bars are mean ± SEM (n=6). The catalase activity (u/mg) in liver of rats fed with different type of red palm oil (0%. The catalase activity (u/mg) in liver of rats fed with different type of red palm oil (0%. no significantly different (p>0. 10%. 5%. At 4 weeks there was no significance difference (p≥0.05). no significantly different (p>0. At 8 weeks there was no significance difference between the control group and 5% group while at 10% and 15% there was decreasing of the catalase activity but there was no significance difference (p≥0. 5%. 10% and 15%) for 2 weeks.05).05).05) between the control group and different concentrations groups (5%. Figure 6. . Bars are mean ± SEM (n=6). 10% and 15%) for 4 weeks. and 15%) of RPO. Figure 5.

These results thus suggest that a combination of carotenoids and vitamin E (tocopherol and tocotrinol) in the RPO has an important role in the protection against free radical damage. On the other hand. Although a few of studies explicitly show the effects of vitamin E on the activities of antioxidant enzymes. Bars are mean ± SEM (n=6). 9 and 10 showed the results of SOD activity at different concentration of RPO (5%. There was no significant (p≥0. however. It is therefore not surprising that there are relatively very few studies on their antioxidative effects in oils and fats [2. After 2 weeks there was an increased in SOD activity at 5% while there was a decreased in SOD activity at 10% and 15% groups. On the contrary at 4 weeks the SOD activity increased with increasing duration of treatment in all concentrations compared to the control group. Figures 8. At 8 weeks there was no significant difference (p≥0. there was a significantly decreased (p<0. Tocotrienols are free radical scavenging antioxidants.05) increased in SOD activity at 15% concentration of RPO. 5%. The results of this study showed that 15% treatment of RPO which contain β-carotene and vitamin E for 4 weeks may enhance the antioxidant enzyme (SOD) defence system. The catalase activity (u/mg) in liver of rats fed with different type of red palm oil (0%. there were no significance differences (p≥0. after 4 weeks the activity of SOD was significantly higher (p≤0.05) at 15% of RPO dietary group compared to the control group but the increase in the 10% of RPO dietary group was not statistically significant. 26]. However. Therefore.05) in 15% of RPO dietary group after 2 and 8 weeks. there is no consensus on what might be the responses of antioxidant .05) among these groups. 10% and 15%) for different times (2w.05).Effect of Different Concentrations of Red Palm Olein and Different Vegetable Oils on Antioxidant Enzymes on Normal and Stressed Rat 309 Figure 7.05) between the control group and all treatment groups of RPO except there was decreased in SOD activity at 15% of RPO. 10% and 15%) for 8 weeks. only the αisomer has considerable biological antioxidant activity. Red palm oil contains the highest concentration of tocotrienols compared to other vegetables or plants and the tocotrienols can be 40-60 times more potent as anti-oxidant than tocopherols [26]. 4w and 8w) of treatment. no significantly different (p>0.

310 Antioxidant Enzyme enzymes to vitamin E. Figure 9. The superoxide dismutase (SOD) activity (u/mg) in rat liver fed with different type of red palm oil (0%. Figure 8. Bars are mean ± SEM (n=6). 5%. partly because of different feeding behavior and other ecological conditions [27]. no significantly different (p>0. 10% and 15%) for 4 weeks. Bars are mean ± SEM (n=6).05) at all treated groups with RPO. 10% and 15%) for 2 weeks.05) at 5% and 10%. The superoxide dismutase (SOD) activity (u/mg) in rat liver fed with different type of red palm oil (0%. significantly different (p<0. no significantly different (p>0.05) at 15% . 5%.

β-. The superoxide dismutase (SOD) activity (u/mg) in rat liver fed with different type of red palm oil (0%. Palm oil is a rich source of vitamin E.05) between control group and RPO group.CO and COC groups compared to control group but the CAT liver sample was no significant different (P≥0. After 4 weeks there was no significance different (p≥0.05) between control group and different vegetable oils treated groups while at 8 weeks there was significance decreased (p≤0. 31]. CO and COC) for different times (4 and 8 weeks) of treatment are summarized in Figures 11 and 12. Bars are mean ± SEM (n=6). δ.and β-carotene. .05) in PO. no significantly different (p≥0. it has good potential for routine diets with enrichment carotenoids [28]. PO. CO and COC) on antioxidant enzyme activity of normal rat liver The results of CAT activity at different vegetable oils (RPO. Thus. vitamin E (in the form of α-.140 retinol equivalents per 100 g. Effect of four different vegetable oils (RPO. and 28. 5%.500 mg of β-carotene per 100 g. having both tocotrienols and tocopherols [30. PO.Effect of Different Concentrations of Red Palm Olein and Different Vegetable Oils on Antioxidant Enzymes on Normal and Stressed Rat 311 Figure 10.tocotrienols and tocopherol) [27].05). lycopenes). 3. 10% and 15%) for 8 weeks.000 mg of α-carotene per 100 g for a total of 6. Several studies have illustrated that RPO is a rich cocktail of lipid-soluble antioxidants such as carotenoids (α. and 44 times that of leafy vegetables [29]. Red palm oil has 17.2. Red palm fruit oil (RPO) contains about 15 times more carotenes than that present in the same weight of carrots.

Figure 12.05). different alphabet an each bar indicate significant different (P≤0. Bars are mean ± SEM (n=6). The catalase activity (CAT) in rat liver fed with different vegetable oils for 8 weeks. The catalase activity (CAT) in rat liver fed with different vegetable oils for 4 weeks. Bars are mean ± SEM (n=6). no significantly different (p≥0.05). .312 Antioxidant Enzyme Figure 11.

Effect of Different Concentrations of Red Palm Olein and Different Vegetable Oils on Antioxidant Enzymes on Normal and Stressed Rat 313

The results of SOD activity at different vegetable oils (RPO, PO, CO and COC) for different times (4 and 8 weeks) of treatment are summarized in Figures 13 and 14. After 4 and 8 weeks there was no significance different (p≥0.05) between control group and different vegetable oils treated groups.

Figure 13. The superoxide dismutase (SOD) activity in rat liver fed with different vegetable oils for 4 weeks. Bars are mean ± SEM (n=6), no significantly different (p≥0.05).

Figure 14. Mean superoxide dismutase (SOD) activity in rat liver fed with different vegetable oils for 8 weeks. Bars are mean ± SEM (n=6), no significantly different (p≥0.05).

314 Antioxidant Enzyme

The results from the present study, after different times, showed that under sedentary conditions, ad libitum feeding of RPO. There was no significant difference in level of the catalase in the control group and different concentration groups of RPO treatment. Mazlan et al. [32] reported that the catalase is the slowest of the antioxidant enzymes to respond to an increased level of free radicals. On the other hand, the CAT activity in rat liver treated with PO, CO and COC groups was decreased compared to control group. This study finding was similar to that of Rathnagiri et al. [33] who reported that there were no statistically significant differences between control fed rats with respect to SOD activity in the corn oil. The effect of COC on antioxidant enzyme in this study was not in agreement with Anitha and Lokesh [34] who found that COC increased significantly of SOD activity in the liver but Anitha and Lokesh [34] used coconut oil with groundnut oil or olive oil instead of COC. Vitamins directly scavenge ROS and regulate the activities of antioxidant enzymes. Among them, vitamin E has been recognized as one of the most important antioxidants [27]. This probably involves their actions as antioxidants, reducing the level of free radicals and hence free radical damage. Antioxidant enzymes, such as superoxide dismutase (SOD) play a major role in removing the Reactive Oxygen Species (ROS) [11]. At this time point, it is suggested that different experimental period might lead to different result about the effect of dietary vitamin E on the activities of antioxidant enzymes [27]. In the present study, the 2 weeks period in which this experiment was carried out may be insufficient to witness any change in the activity of this enzyme. In addition to this, Yazar and Tras [35] reported that prior induction of ROS could cause an increase intracellular SOD activity. Hence first induction of ROS may cause changes in SOD activity and then SOD activity may return to the normal level. SOD enzyme, together with CAT, protects cells against damage caused by free radicals and hydrorlipoperoxides [36]. According to Catherine et al. [37] vitamin E work synergistically to decrease the multiplication of free radicals. Vitamin E inhibits the production of lipid hydroperoxide. However, reduced SOD activities may also indicate increased lipid peroxidation endproducts like acid thiobarbituric [38]. Intricately linked to lipid peroxidation are antioxidant enzymes such as SOD and catalase. As a defense against reactive free radicals, the body produces antioxidant enzymes which help to mop them up [39]. As red palm olein was shown to reduce MDA production and increase SOD in 15% group for 4 weeks, it would spare the retina from damage. This effect of palm oil may be related to the ability of β-carotene to quench free radicals and prevent tissue damage [40]. The relatively lower cholesterol level in treated rats and higher antioxidant enzyme activity could be viewed as potentially beneficial for the health of the user population in humans [41]. Presence of high amount of unsaturated fatty acids may be the reason for the low antioxidant enzyme activities of some vegetable oils fed rat since polyunsaturated fatty acids (PUFA) deteriorates the antioxidant status due to their liability to become highly oxidized. Feeding oils high in polyunsaturated fatty acids (PUFA) results and increase the oxidative stress since PUFA are highly susceptible to peroxidation than monounsaturated and saturated fatty acid [42].

Effect of Different Concentrations of Red Palm Olein and Different Vegetable Oils on Antioxidant Enzymes on Normal and Stressed Rat 315

Figure 15. (A) Stressed control group, (B) Stressed red palm olein group, (C) Stressed palm olein group

3.3. Effect of red palm olein and palm olein on antioxidant enzymes in stressed rat liver
Figure 16 shows the results of CAT activity in liver samples of normal and stressed rats that were treated with 15% of RPO and PO for 4 weeks of treatment. After 4 weeks, there was no significant difference (P≥0.05) between control group and 15% RPO and PO normal groups whereas there was significant decreased (P≤0.05) between control group and 15% RPO stressed group and there was significantly higher (P≤0.05) in 15% PO stressed group than

316 Antioxidant Enzyme

the control group. This study finding were similar to that of Benson and Kshama [7] who reported that the CAT activity in RPO group has shown significant decrease compared to PO and RPO groups under stress conditions. Many recent studies emphasize the important role of reactive oxygen species (ROS) in the pathogenesis of various liver diseases. Stress known to increase oxidative stress in the major organs including the liver [42].

Figure 16. The catalase (CAT) activity in normal and stressed rats fed with red palm olein and palm olein for 4 weeks. Bars are mean ±SEM (n=6), different alphabet an each bar indicate significant different (P≤0.05).

Figure 17 shows the results of SOD activity in liver samples of normal and stressed rats that were treated with 15% of RPO and PO for 4 weeks of treatment. After 4 weeks, there was significantly lower (P≤0.05) in 15% RPO and PO normal and stressed groups than the control group. Vitamin E is a major antioxidant vitamins found in the cell and can prevent cell damage through its activity as a free radical chain breaker [43]. Free radicals have been implicated in the etiology of large number of major diseases. They can adversely alter many crucial biological molecules leading to loss of form and function. Such undesirable changes in the body can lead to diseased conditions. Antioxidants can protect against the damage induced by free radicals acting at various levels [43]. β-Carotene has received considerable attention in recent times as a putative chain-breaking biological antioxidant and its ability to interact with free radicals such as peroxyl radicals and to scavenge and quench singlet oxygen is well documented [44]. Defense mechanisms against free radical-induced oxidative damage include the catalytic removal of free radicals and reactive species by factors such as catalase (CAT), superoxide dismutase (SOD) and reduction of free radicals by electron donors, Such as vitamine E (tocopherol and tocotrienol) [45].

Effect of Different Concentrations of Red Palm Olein and Different Vegetable Oils on Antioxidant Enzymes on Normal and Stressed Rat 317

Figure 17. The superoxide dismutase (SOD) in normal and stressed rats fed with red palm olein and palm olein for 4 weeks. Bars are mean ±SEM (n=6), different alphabet an each bar indicate significant different (P≤0.05).

4. Conclusion
In conclusion, palm oil may offer some protection to liver of the treated rats by reducing free radicals damage, as well as increasing SOD. The present study shows no significant difference in level of catalase in control group and different concentration groups of RPO treatment but after 4 weeks 15% of RPO was enhanced the SOD activity level in rat liver. It can be concluded that the effect of different concentrations of RPO appear to depend on the different period of treatment. The current study shows no significant difference in level of catalase in control group and RPO group but the treated rat liver with PO, CO and COC groups were the lowest and it were significantly lower than control group. After 4 weeks of treatment, 15% of RPO enhances the SOD activity level in rat liver.These results could be due to the high content of vitamin E (tocopherols and tocotrienols) and β-carotene in red palm olein. Treatment with 15% RPO and PO diets did not affect the CAT activity after 4 weeks of treatment under normal condition while there was decreased in CAT activity with RPO and increased with PO under stress conditions. Additionally, the results in RPO group showed that higher SOD activity compared to PO and control groups under normal conditions while there were no significant difference (P≤0.05) in SOD between the control group and treated groups under stress conditions.

Author details
Eqbal M. A. Dauqan and Halimah Abdullah Sani School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi Selangor, Malaysia

318 Antioxidant Enzyme

Aminah Abdullah School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi Selangor, Malaysia

This work was supported by the Organization for Women in Science for the Developing World (OWSDW) and the research was funded by UKM-GUP-NBT-27-103, UKM-HEJIMIndustri-16-2010 and UKM OUP-NBT-29-139/2011 We grateful thank to caroteno sdn bhd Malaysia for providing red palm olein sample

5. References
[1] Sapakal, V. D., Shikalgar, T. S., Ghadge, R. V., Adnaik, R. S., Naikwade, N. S. & Magdum, C. S. 2008. In vivo screening of antioxidant profile: A review. Journal of Herbal Medicine and Toxicology 2(2): 1-8 [2] Eqbal, D., Halimah, A. S., Aminah, A.& Zalifah M. K. 2011. Effect of different concentrations of red palm olein on antioxidant enzymes activity of rat liver. African Journal of Biotechnology 10(22): 4651-4655 [3] Sulekha, M., Satish, Y., Sunita, Y., Rajesh, K. N. 2009. Antioxidants: A Review. Journal of Chemical and Pharmaceutical Research 1(1):102-104 [4] Shiv, K. 2011. Free radicals and antioxidants: Human and food system. Advances in Applied Science Research 2(1): 129-135 [5] Kang, K. R., Cherian, G. & Sim, J. S. 2001. Dietary palm oil alters the lipid stability of polyunsaturated fatty acid-modified poultry products. Poultry Science 80:228–234 [6] Hamid, A. A. , Aiyelaagbe, O. O., Usman, L. A., Ameen, O. M. & Lawal, A. 2010. Antioxidants: Its medicinal and pharmacological. African Journal of Pure and Applied Chemistry 4(8): 142-151 [7] Benson, M. K. & Kshama D. 2009. Influence of ω-6/ω-3rich dietary oils on lipid profile and antioxidant enzymes in normal and stressed rat. Indian journal of experimental biology 47: 98-103 [8] Ramasundaram, S. Narayanaperumal, J. P., Aundaramaglingam, M., Govindarajulu S. N. & Rathinasamy S. 2007. Effect of Triphala on oxidative stress and on cell-mediated immune response against noise stress in rats. Molecular and Cellular Biochemistry 283(1-2): 67-74 [9] Akpinar, D., Yargicoglu, P., Derin, N., Aliciguzel, Y. & Agar, A. 2008. The effect of lipoic acid on antioxidant status and lipid peroxidation in rats exposed to chronic restraint stress. Physiol. Res. 57: 893-901 [10] Zullyt, B. Z., Ricardo, G. A., Dailen, G., NelsonMerino, F. H. R., Silvia, M. C., Yaima, A. G. & Siegfried, S. 2007. Antioxidant mechanism is involved in the gastroprotective effects of ozonized sunflower oil in ethanol-induced ulcers in rats. Mediat. Inflamm. 10: 1-6. [11] Malgorzata, K. & Edward, A. G. D. 2003. Antioxidant enzymes in paraquat and cadmium resistant cell lines of horseradish. Biol. Lett. 40(1): 61-69 [12] Jaya, T. V., Poomchai, A., Neera, S. & Gabriel, F. 1998. Effects of dietary n-6 and n-3 lipids on antioxidant defense system in livers of exercised rats. J. Am. Coll. Nutr. 17: 586-594

Effect of Different Concentrations of Red Palm Olein and Different Vegetable Oils on Antioxidant Enzymes on Normal and Stressed Rat 319

[13] Monica, O., Ingrid, V. & Noel, W. S. 2006. Household usage of and recipe creation with condiment sauces based On red palm oil: Exploring the potential for targeted micronutrient delivery to different family members. Journal of Oil Palm Research 18: 181188 [14] Edem, D. O. & Akpanabiatu, M. I. 2006. Effects of palm oil-containing diets on enzyme activities of rats. Pak. J. Nutr. 5(4): 301-305 [15] Edem, D. O. 2009. Haematological and histological alterations induced in Rats by Palm Oil-Containing Diets. Eur. J. Sci. Res. 32: 405-418 [16] Wanna, A. & Yaakob, C. 2010. A rapid method for determination of commercial βcarotene in RBD palm olein by Fourier transform infrared spectroscopy. Asian Journal of Food and Agro-Industry 3(04): 443-452 [17] Azizan, B. A. 2006. Development of HPLC analysis for detection of lycopene on tomato and palm oil, University College of Engineering & Technology Malaysia [18] Michael, J. P. & David, F. W. 1975. Monosomic analysis of fatty acid compostion in embryo lipids of Zea Maysl. Genetics 81: 277-286 [19] Valls, M. G. V., Mun, P., Saez, G. T. & Cabo, J. R. Effect of corn oil and vitamin E on the oxidative status of adipose tissues and liver in rat. Food Chemistry 81(2): 281-286 [20] Cecille, P., Cecilia, J., Rody, S., Felix, E., Punzalan, & Pepito, P. 2010. The effect of virgin coconut oil on lipid profile and fasting blood sugar: A phase i clinical trial. Philippine Journal of Internal Medicine 48(2):1-6 [21] Akpanabiatu, M. I., Umoh, I. B., Eyong1, E. U. & Udoh F. V. 2005. Influence of nauclea latifolia leaf extracts on some hepatic enzymes of rats fed on coconut oil and noncoconut oil meals. Pharmaceutical Biology 43(2):153–157 [22] Aebi, H. 1984. Catalase in vitro. Meth Enzymol. 105: 121-126 [23] Marklund, S. & Marklund, G. 1974. Involvement of the superoxide anione radical in the autoxidation of pyrogallol and convenient assay for superoxide dismutase. Eur. J. Biochem. 47: 469-474 [24] Jakob, H. W. 2009. The Lowry Method for Protein Quantitation. The Protein Protocols Handbook, Third Edition [25] Arunabh, B., Richard, A. L., Aparna, K., Khaliquz, Z., Dongxu, S. & Gabriel, F. 2003 Effect of dietary n-3 and n-6 oils with and without food restriction on activity of antioxidant enzymes and lipid peroxidation in livers of cyclophosphamide treated Autoimmune-Prone NZB/W female Mice. J. Am. Coll. Nutr. 22(5): 388-399 [26] Jacques, V. R. 2005. Can red palm oil protect the heart?. Science in Africa(11/2/2011) // [27] Jinghua, F. U., Wenbingzhang, K. M., Xiuni, F., Wei, X., Zhiguo, L., Hongming, M. A. & Qinghui, A. I. 2007. Effects of vitamin E on antioxidant enzyme activitys and fatty acid compositions in juvenile abalone haliotisdiscus hanna ino. Journal of Shellfish Research 26(3):809–814 [28] Radhika, M. S., Bhaskaram, P., Balakrishna, N., Ramalakshmi, B. A. 2003. Red palm oil supplementation: A feasible diet-based approach to improve the vitamin A status of pregnant women and their infants. Food Nutr. Bull. 24: 208-217 [29] Spinnler, B. A. J. 2003. A place for palm fruit oil to eliminate vitamin A Deficiency. Asia Pac. J. Clin. Nutr. 12(3): 369-372

320 Antioxidant Enzyme

[30] Kamat, P. J. & Devasagayam, T. P. A. 1995. Tocotrinols from palm oil as potent inhibitors of lipid peroxidation and protein oxidation in rat brain mitochondria. Neurosci. Lett. 195: 179-182 [31] Kevin, C., Rahamw, B., Keith, I. & Trevofr, S. 1984. Lipid peroxidation and lipid antioxidants in normal and tumor cells. Toxicol. Pathol. 12: 235-239 [32] Mazlan, M., Abd Halim, F., Mat Top, G. & Wan, Z. 2002. Effect of vitamin E on plasma malondialdehyde, antioxidant enzyme levels and the rates of wound closures during wound healing in normal and diabetic rats. Asia Pac. J. Clin. Nutr. 11: 448-451 [33] Rathnagiri, P., Douglasr, S., Julia, E. S., Mark H. F., Larry, W. O., Amir, R. & Amin, A. N. 1998. Increased Lipid Peroxidation & Impaired Antioxidant Enzyme Function Is Associated With Pathological Liver Injury in Experimental Alcoholic Liver Disease in Rats Fed Diets High in Corn Oil and Fish Oil. Hepatology 27(5): 1317-1323 [34] Anitha, N. & Lokesh, R. B. 2008. Rats fed blended oils containing coconut oil with groundnut oil or olive oil showed an enhanced activity of hepatic antioxidant enzymes and a reduction in LDL oxidation. Food Chemistry 108: 950–957 [35] Yazar, E. & Tras, B. 2001. Effects of fluoroquinolone antibiotics on hepatic superoxide dismutase and glutathione peroxidase activities in healthy and experimentally induced peritonitis mice. Revue de Medecine Veterinaire 152: 235-238 [36] Suleyman, K., Handan, G., Yeter, D., Nihatmert, M., Hakkiyoruk, I. & Tevhide, S. 2007. Studies on serum _-tocopherol, seleniumlevels and catalase activities in lambs with white muscle disease. Bull. Vet. Inst. Pulawy. 51: 281-284 [37] Catherine, J. Field, I. R. Johnson, & Patricia, D. Schley 2002. Nutrients and their role in host resistance to infection. Journal of Leukocyte Biology71: 16-32 [38] Looi, M. L., Noor Aini, A. H. & Yasmin, A. M. Y. 2005. Effects of Palmvitee on Status of Superoxide Dismutase and Glutathione Peroxidase in Rat Liver during Aging. Malaysian Journal of Biochemistry and Molecular Biology 12: 21-24 [39] Eriyamremu, G. E., Ojimogho, S. E., Asagba, S. O. & Osagie, V. E. 2008. Palm oil induced changes in ocular tissue lipid peroxidation, antioxidant enzymes and ATPases of rabbits in cadmium toxicity. Food and Chemical Toxicology 46 3155–3158 [40] Lyn, P. 2000. Beta-carotene: The controversy continues. Alternative Medicine Review 5(6): 530-545 [41] Nandakumaran, M., AL-Sarraf, R. AL-Fadhli, H., AL-Shammari, M., AL-Harmmi, J. & AL-Saleh, E. 2009. Effect of oral administration of coconut oil on hematological and metabolic parameters in female adult rats. Nutritional Therapy & Metabolism 27 (4):183-188 [42] Nevin, K. G. & Rajamohan, T. 2006. Virgin coconut oil supplemented diet increases the antioxidant status in rats. Food Chemistry 99: 260–266 [43] Vaibhav, D. A., Arunkumar, W., Abhijit, M. P. & Arvind, S. 2011. Antioxidants as immunomodulator: an expanding research avenue. International Journal of Current Pharmaceutical Research 3(1):8-10 [44] Olatunde, F. E. & George, B. 1999. Antioxidant activity of palm oil carotenes in organic solution: effects of structure and chemical reactivity. Food Chemistry 64: 315-321 [45] Jorge L. & Maria E. G. 2009. The role of antioxidants and antioxidant-related enzymes in protective responses to environmentally induced oxidative stress. Mutation Research 674: 137–147

Section 3 Antioxidants from Plants             .


Divya Shrivastava and Raosaheb Kale Additional information is available at the end of the chapter http://dx. Dhalla et al. the free radicals namely. Nowadays. This further leads to various diseases like This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.5772/52983 1. The deleterious effect of ROS and RNS is ameliorated majorly through antioxidants. 2000). However. glutathione peroxidase and catalase. 2001). thioredoxin & lipoic acid) and flavonoids. 2001). minerals and photochemicals in these whole foods may act synergistically (World Cancer Research Fund. Antioxidants have therefore been considered as a means to modify and minimize the toxic effect of free radicals. they are guardians of human health. The nonenzymatic antioxidant includes ascorbic acid (vitamin C). Brown et al. carotenoid. They can be categorized into enzymatic and non-enzymatic in nature. α-tocopherol (vitamin E). reactive oxygen species (ROS) and reactive nitrogen species (RNS) are released during metabolic processes.0). 1997. Scientists harbor the popular belief that antioxidants are ‘wonder’ substances and are working round the clock to discover sources of antioxidants. proteins and DNA. 1999). distribution.Chapter 12 Antioxidant Potential of Asparagus adscendens Manju Singh. Introduction Oxidation reactions give rise to free radicals which are molecules with one or more unpaired electrons in their atomic or molecular orbit (Halliwel and Gutteridge..   . licensee InTech. 1997). Recent studies have indicated that naturally occurring plants compounds possess       © 2012 Singh et al. The benefits of eating fruits and vegetables may be much greater as compared to the effects imparted by any of the individual antioxidants they contain because the various vitamins. AICR. if present in higher concentration it gives rise to oxidative or nitrosative stress (Kovacic and Jacintho.doi. cardiovascular diseases and cancer (Dalle & Donne. mankind is overtly conscious of its health and many have included antioxidants as part of their regular dietary regime. At lower concentration ROS and RNS play vital roles during mutagenic activity and response to pathogen attack. Therefore. Due to the presence of unpaired electrons. free radicals are highly reactive in nature. The accumulation of excess of ROS results in the oxidative degradation of vital biomolecules like natural as well as synthetic. thiol antioxidant (glutathione. which permits unrestricted use. AICR. In biological system.. and reproduction in any medium. Overall there is no convincing evidence that antioxidants in the amounts obtained from fruits and vegetables in the diet have deleterious effect on human health (World Cancer Research Fund. 2006. provided the original work is properly cited. The enzymatic antioxidant includes superoxide dismutase (SOD).

Sources and formation of free radicals Human body is constantly exposed to the hazards of free radicals.. 2. Hanu Kogru 1993). This reaction also occurs in the mitochondria (Thannikal and Fanburg 2000). It has traditionally been used in various ailments including diarrhea. they are also triggered by exogenous factors. and many metabolic functions that involve consumption of O 2 occur in peroxisomes. To start with. dysentery. The enzyme xanthine oxidase. their formation.324 Antioxidant Enzyme antioxidant properties. have been used in Ayurveda and Unani tradition of Indian medicine since long... 1995. energy is released.. However. During the passing of electron through the various molecules in the ETC. A wide variety of plants have been associated with antioxidant effects (Deans et al.. The process of oxidation of fatty acid is also a source of H2O2 (Fahl et al. 1993). This process of detoxification is also responsible for the leakage of molecular oxygen to eventually form superoxide radical (Butler 1993). Under stress conditions. 2002). adcendens in particular. These leaked electrons are responsible for the release of superoxide radical in the mitochondria (Benzi et al. Other cellular organelles like endoplasmic reticulum consist of cytochrome P 450 in mammalian cells (Butler 1993). In the following part of the chapter we throw some light on the different free radicals. proteins and DNA. Microsomes release H2O2 which contributes to more than 80% of H2O2 produced in the cell (Stohs and Baghchi 1995). The production of H2O2 also takes place in peroxisomes (Valko et al. It has also been identified as one of the drugs to control the symptoms of AIDS (Trivedi et al. 1984). 1992. 1993.. One of the major sources of molecular oxygen is the electron transport chain (ETC) that occurs in the mitochondria and endoplasmic reticulum of the cell. However. leucorrhea and general debility (Kapoor. Other cellular organelles like microsomes and peroxisomes too release free radical. This is carried out via . This molecular oxygen is then activated to superoxide radical. There are many biochemical reactions occurring in the human body that prompt the production of ROS. Their reactivity particularly incurs damage to the vital molecules of human body like lipids.. 2006). Asparagus adcendens of family Liliaceae. the superoxide radical is produced in excess which inturn accumulates free iron released from iron containing molecules.. The H2O2 breakdown enzyme catalase. Brookes et al. Masaki et al. Yanishlieva et al. 2001.. Since free radicals have an unpaired electron in their outer orbit. 2004). Cytochrome P 450 is responsible for the detoxification of toxic compounds carried out by oxidation of foreign/toxic compounds with the aid of enzyme monooxygenase. effects and how are they neutralized by plant antioxidant in general and A. commonly known as safed musli. some electrons leak from the ETC (Salvador et al. This reaction of conversion of hypoxanthine to xanthine releases superoxide radical as a by product and the conversion of xanthine to uric acid releases hydrogen peroxide (H2O2) as by product (Harrison 2002). molecular oxygen is released during various metabolic processes which is the preliminary molecule for the production of oxygen free radical. Several reports describe that the anticancer activity of medicinal plants is due to the presence antioxidants present in them. catalyzes the hydroxylation of purine. they are highly reactive and unstable. 2001).

Apel and Hirt 2004. NO• can bring alteration in the structure and function of proteins. The RNS includes nitric oxide (NO•) which is released in the tissues of organism by enzyme nitric oxide synthase. Fenton reaction occurs. Apart from the natural endogenous reaction taking place in the body. the simplest form of which exists as hydroperoxy radical (HOO•)and is formed by donating a proton to the superoxide radical (O2•-). These radicals are also released during the ionized decomposition of water molecule and also photolytic decomposition of alkylhydroperoxidase. humans are constantly exposed to the risk of oxidative stress. stress and hazards related to work. 2004). Another free radical found in living organism is the peroxyl radical (ROO•). When superoxide radical and NO• are both produced in large amount. they react together and give rise to a much more reactive free radical called the peroxynitrite anion (ONOO-) which cause DNA fragmentation and lipid peroxidation (Carr 2000). Bergamini et al. The various types of ROS. ROS: Damages and diseases As discussed in the earlier part. when produced in higher amount. ONOOOther sources of ROS include macrophages and neutrophils when activated during pathogen attack. artherosclerosis. excessive production of RNS leads to nitrosative stress (Klatt and Lamas 2000). This enzyme catalyses the conversion of arginine to citrulline and NO• is released as a by product (Ghafourifar and Cadenas 2005). Like ROS. their sources and byproducts are summarized in the table 1.. 3. releasing hydroxyl radical (•OH).. Due to accumulation of excess iron. NO  O2•. chronic inflammation and ischemia-repurfusion (Behrend et al. This free radical is responsible for the peroxidation of fatty acids. Fe 2+ +H 2 O 2  Fe 3   OH+OH The hydroxyl radical is highly reactive and dangerous.The macrophages take up oxygen and release free radicals like NO•. it is apparent that due to reasons like environmental pollution. heavy metal ions. O2•and H2O2 (Conner and Grisham 1996). It has also been well documented that oxidative stress leads to the development of various diseases in humans like diabetes. Inspite of being a stable molecule. carcinogens. When the ROS species incur damage to vital biomolecules like DNA. changes in lifestyles.Antioxidant Potential of Asparagus adscendens 325 [4Fe-4S] cluster-containing enzyme of dehydratase-lyase family (Liochev and Frodovich 1994). 2003. tobacco smoke and certain pesticides. barbiturates. . the generation of ROS/RNS is also elicited by external/environmental factors like exposure to UV radiations or gamma radiations.

DNA is the most vital biomolecule and any irreversible change in the DNA base pairing can lead to mutation. Type of free radical Superoxide radical (O2•-) Enzymatic/nonProducts enzymatic Antioxidant Electron transport chain. rendering the enzyme inactive. high glycolytic activity and lactate production. If the generation of ROS is triggered by metal ions. Bohr and Dianov 1999). Thus any enzyme containing these amino acid can undergo inactivation due to presence of ROS. H2O2 phagocytes. methionine and histidine are more susceptible to ROS damage. irreversible changes occur which mark the beginning of carcinogenesis and ageing. This includes damage in the tumor suppressor gene and increased expression of oncogenes resulting in cancer (Wei 1992. NADPH oxidase. activated Superoxide dismutase H2O + O2. patients have impaired glucose clearance.326 Antioxidant Enzyme lipids and proteins. Lipids that consist of phosphate group are phospho lipids and they are indispensable part of the membranes that surround the cells as well as other cellular structures. such as the nucleus and mitochondria. which in turn blocks the vital biological processes. Cancer and diabetes mellitus show a redox imbalance and generation of ROS increases in mitochondria. H2O + GSSG H2O + O2 H2O Source Hydrogen Product of dismutation of peroxide (H2O2) superoxide radical. Cerutti 1994. then not only DNA but also phospholipids are susceptible to their attack (Siems 1995). This leads to loss of cellular viability and ageing. tumor formation and its spread. Lipid peroxidation is one of the major damage encountered by Phospholipds due to ROS. The damage to DNA caused by ROS involves mispairing mutation . ROS is a potential carcinogen and play role in causing mutagenesis. Xanthine oxidase (SOD) Glutathione peroxidase. In such cases. GSNO Glutathione reductase Source: Nordberg and Arner 2001 Table 1. The major ROS molecules and their metabolism Proteins in the form of enzymes catalyse the vital biochemical reactions in the body. Xanthine oxidase peroxidins Hydroxy radical Product of interaction of transition (OH•) metals like Fe and Cu with O2•and H2O2 Nitric oxide (NO) Nitric oxide synthase Glutathione. cysteine. Catalase. Out of 20 amino acids comprising an enzyme.

. 2003). The activation of AP-1 leads to higher expression of two regulators (c-fos and c-jun) in cell proliferation and can lead to uncontrolled cell division... 1991. after its formation undergoes cyclisation to form endoperoxides which act as precursors to malondialdehyde (MDA. Due to activation of NF-ҚB. 1996). 2001..Antioxidant Potential of Asparagus adscendens 327 or transversions (G-T) (Higinbotham et al. These compounds when formed harm the vital molecules to a great extent. Wang et al. This activation leads to liver carcinoma (Waris and Siddiqui 2003). Santos et al. It leads to the formation of disulphide thiol group.. 2000). 1996.. asbestos and cadmium can lead to cancer (Valko et al. The activation of oncogenes is also a consequence of transversion (Ames 1993). Harris and Hollestein 1993). SOD and A20 which can lead to tumor formation in colon. the damage can be assessed by the amount of production of carbonyl group which is released (Dalle and Donne 2003). The disruption in signaling cascade due to ROS may cause activation of transcription factors like MAP kinase. Ichiba et al. Redox metals are also responsible for carcinogenesis and ageing as they generate free radicals and also bind to thiols (Leonard et al 2004. Arsenic inhibits the activity of enzymes glutathione reductase by binding to its –SH group. Valko 2005).. 1991) and also protects the DNA from damage. 1996). 2001. 1992. This promotes the DNA damage by UV radiations and also cigarette smoke leading to cancer (Waalkes et al. Hepatitis B and C viruses are activated by oxidative stress caused due to consumption of aflatoxins (Kountouras and Lygidakis 2000. TRAF2).. Accumulation of 8-oxo-dG is also observed during liver carcinoma (Shwarz et al. Denissenko et al. Smela et al. Oxidative stress can also enhance the accumulation of compound 8–oxo-dG which is more in the lungs of smokers. 2004). then it gives rise to formation of tumors (Brash et al. MDA is a potential mutagen in bacterial and mammalian cells.. pancreas and other carcinomas (Klaunig and Kamendulis 2004). bcl-xl). If mutation occurs in p53 tumor suppressor gene... 2005). activator protein (AP-1) and nuclear factor-κB (NF-ҚB) which is related to cell proliferation and apoptosis (Valko 2006). Stayner et al. DNA damage caused by oxidative stress due to •OH radical can lead to breast cancer (Jaiyesimi 1992).. The peroxyl (ROO•) free radical. The presence of increased amount of 8-oxodG in the urine of smokers is a reliable biomarker of cancer (Wu et al. Proteins are also attacked by ROS and in this process mainly residues like cysteine and methionine are oxidized (Stadtman 2004). Cadmium can cause activation of protein kinase which through series of phosphorylation and dephosphorylation increases the expression of downstream genes (Valko 2005). breast. Du et al. This compound is a potential mutagen and can lead to fibrosis and tumor development (Zienolddiny et al. During the oxidation process of protein.. The p53 gene has an important role in combating cancer and prevention of tumor generation (Hollestein et al. tumor necrosis factor-receptor associated factor (TRAF1. .. Chromium causes lung cancer due to high levels of free radicals produced in the mitochondria (Pourahamad and Obrien 2001). Santos et al 2005. Exposure to heavy metals like iron. 2000). the genes like B-cell lymphoma (bcl2.. 1994. Under stress G:C base pair is more susceptible to mutation than A:T base pair. 2004). Another byproduct of lipid peroxidation is 4-hydroxy 2-nonenal (HNE) which is also a mild mutagen. the final product of lipid peroxidation) (Fedtke 1990. Mao 1999. It also plays role in causing pancreatic cancer and renal carcinoma in humans.

4..2. Amongst all the various categories of cancer and the accumulation of carcinogen. 1999) (Figure 1).to O2 and H2O2 (which is less reactive) (McCord and Fridovich 1969). Today. it has been well established that by making few changes in the diet by including antioxidants. Enzymes involved in detoxification of O2•. animals and in aerobic organism and is localized in peroxisomes. There are two categories of antioxidants. Of these. thiol antioxidants. Figure 1. Table 2 summarizes the dietary sources of various antioxidants. the occurrence of cancer can be reduced (Khan et al. These include superoxide dismutase (SOD). It carries out the conversion of H2O2 to water and oxygen molecule by the following reaction: . carotenoid. vitamin E and β-carotene are not synthesized in the body and are to be supplied through dietary intake. In humans. catalase (CAT) and glutathione peroxidase (GPX) (Mates et al. flavonoids and metallonin (McCall and Frei 1999). The SOD exists in different isoforms depending on the type of active metal. mitochondria (mitochondrial SOD which includes Mn-SOD) and extra cellular SOD (Landis and Tower 2005).radical. The enzymatic antioxidants include the enzymes that are present in the body to scavenge the ROS/RNS.1. 2008). The non-enzymatic antioxidants include.ascorbic acid (vitamin C). SOD is found in cytosol (cytosolic-SOD which includes Cu and Zn SOD). Increased expression of growth factor receptor (EGF-epidermal growth factors) has been documented during lung and urinary cancer (Drevs 2003). vitamin C. an array of molecules called “antioxidants” come to its rescue.328 Antioxidant Enzyme ROS and presence of metal ions can also lead to anomalies in the growth factor receptor which causes various cancers (Drevs 2003).enzymatic and non-enzymatic antioxidants. Superoxide dismutase (SOD) as antioxidant SOD catalyses the dismutation of O2•. Antioxidants: Role in prevention and cure Before the human body succumbs to the deadly biochemistry of free radicals. 4. Catalase (CAT) Catalase is present in plants. Antioxidants are the chemicals or enzymes that react with the free radicals and protect the vital biomolecule from the damage by terminating the oxidative chain reaction which is the way to cancer and ageing.. the ROS are observed to play a key role. α-tocopherol (vitamin E). 4.

black tea and red wine. green tea. spinach. cheese and yogurt Lipoic Acid Broccoli. strawberries. artichokes. pears. lettuce. cantaloupe. okra. brussels sprouts. broccoli. green beans. and winter squash. plums. Types of antioxidants and their dietary sources . pumpkins. bell peppers. cashews. red grapes grapefruit. pumpkin seeds. eggplants. Flavinoids Blueberries. barley. chard. black grapes. oats. tomatoes. yams and potatoes. okra. potatoes. asparagus. squash. blackberries. pineapple. limes. fish. eggs. broccoli. kale. brown rice. dandelion greens. lemon. sunflower seeds. sunflower seeds. peas. kidney beans. soybean. raspberries. cabbage. spinach. mustard greens and sweet potatoes. cantaloupe. collard greens. grapefruit. papaya. cranberries. pistachios. turnip greens. snap beans. broccoli florets. kale. parsley. Almonds. lima beans. Antioxidants Vitamin C Dietary Sources Broccoli. green beans. bananas. lemons. onions. celery. artichoke. peanuts. beet greens. wheat germ. wheat germ oil. apples. black. cabbage. hazelnuts. Vitamin E Glutathione Avocados. broccoli. cherries. mustard greens. raspberries. beets. meat. tomatoes. Brussels sprouts. walnuts. oranges. peaches. mushrooms. Peanuts. blueberries. milk. cauliflower. fresh peaches. strawberries. peas. currants and raspberries Selenium Table 2. Carotenoids Carrots. pecans. spinach. apricots. Brazil nuts. leeks. breakfast cereals. Brewer’s yeast. cranberries. dates and pomegranates. cauliflower. eggs. romaine lettuce. collard. oranges. turnip greens. asparagus. almonds. okra. turnip. kiwi fruit. kiwi fruit and red bell peppers. spinach. oranges. brazil nuts. blackberries. watermelon. fish dairy. raw tomatoes Grains. garlic. cashews.Antioxidant Potential of Asparagus adscendens 329 H2 O 2  H 2 O+O 2 It minimizes the concentration of H2O2 at lower level and hence plays role in the prevention of cancer. fennel. peanut butter. leafy salad vegetables. tomatoes. summer squash. carrots.

proteins and DNA.. 2005. 2004).330 Antioxidant Enzyme 4. Cuzzorcrea et al. It also regulates the AP-1 signaling pathways. 4. The enzymes reduce peroxides to form selenoles (Se-OH).. 1999). Recently the role of vitamin C has been seen in gene expression. Ascorbic acid (Vitamin C) Vitamin C is a very powerful and effective antioxidant which is functional in aqueous environment.. apoptosis and other vital functions (You et al. One is selenium independent glutathione-S-transferase and other is selenium dependent glutathione peroxidase (Mates et al. It also provides protection against lungs and colorectal cancer (Knekt 1991).4. 4.The important substrates of GPX are H2O2 and organic peroxide (ROOH). respectively. Because of its property of being fat soluble this vitamin can easily get bound to lipid membrane and plays role against lipid peroxidation of the membrane (Pryor 2000). α-tocopherol is regenerated (Kojo 2004). α-tocopherol is the most active and readily absorbed form. The supplemental intakes of this powerful antioxidant have been documented to be useful against cancer..under normal physiological conditions and in lesser amount it is present as AscH2 and Asc2-. Glutathione Peroxidases (GPX) These enzymes exist in two forms that differ in number of sub-unit. It is fat soluble vitamin and a potential antioxidant. Vitamin C also acts a defense against membrane oxidation (Retsky et al. α-Tocopherol (Vitamin E) Amongst the eight related tocopherols and tocotrienols of vitamin E. It is therefore present as diacid (AscH2) and majorly present as AscH.combines with free radical to form a tricarbonyl ascorbate free radical (AscH•). Supplemental . The AscH. 2GSH  H2 O 2  GSSH+2H 2 O 2GSH  ROOH  GSSH+ROH+H2 O Glutathione metabolism carried out by GPX is one of the major antioxidant defence mechanism present in the body. which is responsible for cell proliferation and thus reduces the expression of cancer causing genes. 1999). It plays an important role in inhibiting the reaction between nitrites and amine groups which form N-nitroso compound and provide protection against stomach cancer.. hence it should be taken in the diet.5. It has two ionisable -OH groups. Thus the production of AscH• marks the end of reaction and protects the organism from the oxidative stress (Kasparov et al. which is resonance stabilized and is relatively inert. There have been many instances which show the reduction of oxidative stress mediated damage to lipids. During the redox reaction α-tocopherol is converted to α-tocopherol radical and with the aid of vitamin C. 2000).3. The synthesis of vitamin C does not occur in the human. Thus vitamin C and E act in great accordance in water and fat environment.

GSH acts as a cofactor to many detoxifying enzymes. 2001) . 1997). Glutathione also facilitates the regeneration of vitamin C and E in their active forms and also carries out reduction of tocopherol radical (Valko et al.6. Vitamin E acts in the prevention of free radical formation also. Pathway depicting the interaction between important: Vitamin E. lipoic acid) Amongst the thiol based antioxidants. 4. GSH maintains a reducing environment in the cell. Vitamin C and thiol antioxidants. Glutathione (GSH) is the reduced form and glutathione disulphide (GSSH) is the oxidized form. (Source: Packer et al.Antioxidant Potential of Asparagus adscendens 331 doses of vitamin E have been found to incur reduction in the cases of colorectal cancer (White et al. hence helps in carrying on the DNA repair and normal expression by protein sulphydryls.. 2006) (Figure 2).. thioredoxin. GSH plays role in the redox signaling by both the level of GST and the ratio of GSH/GSSH (Jones et al. Figure 2. 2000). glutathione (GSH) is the most abundantly found intracellular thiol antioxidant. There are many instances when occurrence of cancer has been correlated with disorders of GSH related enzymes and decreased GSH/GSSH ratio (Pastore 2003). GSH also controls many transcription factors like AP-1 and NF-ҚB.. Thiol antioxidants (Glutathione..

It is readily soluble in both aqueous and fat medium due to which it also referred to as “universal antioxidant.8.. When thioredoxin (TRX) is reduced. It controls many transcription factors which in turn control cell proliferation..9. 4. Thioredoxin also inhibits NF-ҚB and AP-1 transcription factors.. .332 Antioxidant Enzyme 4. colon and also in leukemia (Karas et al. The ameliorative effect of lipoic acid has been associated with dreadful diseases like HIV-infection. prostate. it is converted to active state (TR-S) which enters the nucleus. Carotenoids Carotenoids are the tetraterpenoid pigment present in chloroplast and chromoplast of plants. Both ALA and DHLA are powerful antioxidant and protect the body by scavenging ROS.. The beneficial effect of this antioxidant has been documented during various cancers of breast. Lipoic acid or α-lipoic acid (ALA) Lipoic acid or α-lipoic acid (ALA) or thioctic acid is a disulphide derivative of octanoic acid. Bustamante et al. Sharoni et al. 1997).. 2008). This active and reduced state regulates the activity of transcription factors involved in replication. the occurrence of oxidative stress in breast cancer survivor with high dietary and plasma carotenoids was much lower than those with low dietary and plasma carotenoid (Thomson et al. 2004).” It is absorbed from the dietary intake and is stored as dihydro lipoic acid (DHLA) (Smith et al. which activates p53 transcription factor. The TRX also regulated the hypoxia inducible factor (HIF-1) and also cytochrome P-450.. 2000. 4.. 2004). lung. In a recent study. regeneration other antioxidants like Vitamin C and E and also chelate metal ions (Cu2+ and Fe2+) and prevent the promotion of oxidative chain reaction. 2001). There have been many studies in which oral intake of lipoic acid is found to prevent and treat many diseases (Fuchs et al. diabetes and also in the diminishing the effect of radiations (Ramakrishnan et al. Thioredoxin (TRX) Thioredoxin (TRX) contains 2-SH groups in reduced form which gets converted to disulphide unit in oxidized form. It also regulates the protein by directly binding to them. 1992.. Carotenoids and retinoic acid (a metabolite of vitamin A) regulate many transcription factors and prevent the occurrence of cancer (Niles et al. cardiovascular diseases. Due to this β-carotene can efficiently scavenge ROS and also protects the lipid from peroxidative damage. This study indicates important role of carotenoids in oxidative stress mitigation.7. Carotenoids have the ability to delocalize the unpaired electron through conjugated double bond structure (Mortensen et al. TRX regulates the expression of Ref1 (redox effector factor). neurological disorders. This reaction is catalyzed by thioredoxin reductase. There are over 600 types of carotenoids divided to xanthophylls (those which contain oxygen) and carotenes (those are made up of only hydrocarbon and do not have oxygen). They are not synthesized in humans and hence have to be obtained through dietary intake. 2004).. 1998). In the nucleus.

There are many other seleno-proteins with unknown functions. which has a role in cancer prevention. It is present in the proteins as seleno-proteins and is an important component of many antioxidant enzymes. soybean. selenium promotes formation of antibodies. selenium occurs as selenocysteine (Alexander 2007). proteins that act as the body's defense system. which also contain significant levels of bioactive natural compounds. inflammation. as drink or as herbal medicine.Antioxidant Potential of Asparagus adscendens 333 4. the propagation of the reaction does not occur. as spices and condiments.10. breast and lungs due to their antioxidant properties (Damianaki et al. DNA methylation and DNA repair. cereal grains. 2000). 5. it has been found to act as prooxidant and induces apoptosis and is successfully used in anticancer therapy along with other anticancer drugs (Brozmanova 2011). angiogenesis and immune function (Taylor 2004). Flavinoids Flavinoids are the class of plant secondary metabolites. Daily intake of selenium greatly reduce occurrence of some cancers (Harrison et al. 2002). The following reaction occurs: Ph-OH+ROO•  ROOH+PhO• Since the PhO• radical so formed is a stable molecule.11. It is an important class of polyohenolic antioxidants as they play a significant role in curing human diseases (Schroeter et al. These include 4000 types of flavinoids divided into 13 classes. Selenium Selenium in a vital micronutrient required by the body (Thomas 2004). It also activates p53 gene. helps in carrying out normal biochemical processes and protects the body from risk of cancer. carcinogen metabolism. In selenoproteins. iodothyronine deiodinases. apoptosis. Recently. Selenium fights the harmful effects of oxidative stress through involving in processes like. pancreas. The plants as a source of medicines for different diseases and disorders have been attracting the attention of human mind since ages practically in all societies.. may . Selenium plays vital role in protecting the body against the harmful effects of free radicals.. as masticators items. cell proliferation. Flavinoids provide a significant result in lessening the rate of various cancers like that of stomach.. Plants are natural chemical factories. Human societies around the world consume a variety of plants and plant products as a food. The main enzymes are glutathione peroxidases. fish. Selenium is present in legumes. spices. 4. Dietary plants. Plants as source of antioxidants The plant kingdom plays a profound role in the life of humans and animals. meat and Brazil nuts (Whanger 2002). Along with vitamin E. vegetables. 1997). cereals and edible tubers/roots. The phenolic antioxidant (Ph-OH) is capable of terminating the oxidation reaction.such as fruits. hormone production. The diverse kind of plants flourishing on this planet manufactures a vast variety of natural chemical substances. and thioredoxin reductases.

epidemiology and experimental. diet. arthritis. xanthones. Steinmetz and Potter 1991. quercetin. flavonoids (e. infectious agents. the incidence of cancer will increase three-fold.. According to the World Health Organization (WHO). cancer is the leading cause of death all over the world. enzymes (e. These substances possess high antioxidant potential. gallic acid and tannins). ROS generation has been known to cause cellular damage which appears to be a major contributor of many diseases such as aging. There have been considerable scientific evidence.6 million cancer deaths occurred and of these. etc (Somkuwar 2003.g. 2010). 2003). alcohol. 2006). 1989.g. Root is used as appetizing.. flavones. polysaccharides. diabetes. throat complaints and leprosy (Manandhar. cardiovascular diseases and cancer. It is believed that consumption of plant products. diarrhea. flavonones and isoflavones).334 Antioxidant Enzyme provide human health benefits beyond basic nutrition to reduce the risk of many chronic diseases including cancer (Lie.. minerals (e. In light of this chemopreventive potential of A. Projections are that by 2020. diet. Se and Zn). hormones and immune conditions) and environmental/acquired factors (such as tobacco.. Although the hereditary factors cannot be modified. about three quarters of the world's population currently use herbs and other forms of traditional medicines to treat diseases. Mn.7 million new cancer cases and 7.. environmental pollutants and radiation. polyphenols (e. diuretic. In the developed countries about 25% of all medicinal drugs are said to be based on plants and plant products whereas in the developing as well as underdeveloped countries about 75% of all the medicinal drugs are based on the plants or plant products. superoxide dismutase. radiation and infectious organisms). Jain et al. astringent useful in dysentery. catechins. C. The dietary habits play an important role in prevention of cancer. accumulated in these years indicating that a large number of plants. 1990). Root bask is taken with milk for vitality and strength. anthocyanins. despite the enormous amount of research and rapid developments seen during the past decade. Cancer is caused by both internal factors (such as inherited mutations. . it is believed that most of the cancers are not of hereditary origin but because of lifestyle factor such as tobacco. 1980). widely distributed in fruits. Kathiresan et al.. Recent research have shown that the antioxidants of plant origin with free radical scavenging properties could have great importance as therapeutic agents in several diseases caused due to oxidative stress (Ramchoun et al. adcendens root have been discussed which is mainly because of the antioxidant potential of the plant. Among these. and that there will be a disproportionate rise in cancer cases and deaths from the developing countries that have limited resources to tackle the problem (Are et al. ellagic acid.g.. vegetables and other dietary substances possess efficacy to act as cancer preventive agents (Hickman. but a lot improvement can be brought about as far as the lifestyle and environmental factors are concerned. On the basis of current knowledge. approximately 12. 2006. 2009). vegetables and medicinal plants will be conducive in reducing the incidence of cancer. carotene.. E). Rao et al. For many decades. obesity. 56% of all new cancer cases and 63% of cancer deaths were in the less developed regions of the world (Ferlay et al. Cu. Recent reports from the International Agency for Cancer Research indicate that in 2008. Several other reports describe that the anticancer activity of the plants is due to antioxidants such as vitamins (A. aphrodisiac.g. catalase and glutathione peroxidase)... 2010). lignins. fruits. laxative.

The phase II enzyme system can make it inactive to facilitate their excretion outside the body. natural products play a major role in cancer prevention and treatment. 2011). Effect of A. adscendens modulates both phase I and phase II enzymes including cytochrome p450 reductase.. the lowered level of oxidative damage was expected in the group of animals treated with A.. glutathione transferase (GST) and DT-diaphorase (DTD) (Singh et al.1. Chemopreventive potential of A. adscendens on carcinogen/Drug metabolism The parameters that we assessed at the end of the feeding were the inducibility of hepatic enzymes involved in xenobioitc/carcinogen metabolism and maintenance of the cellular antioxidant status. It is well established that free radicals are involved in the initiation and development of cancer. Effect of A. resulting in mutation during cell proliferation. Thus. It has been also reported that more than 50% of all modern drugs in clinical use are of natural products.. it consists of phase I and phase II metabolizing enzyme systems in which. the differences observed in this tissue are considered significant. 6. due to the activity of former. adscendens on antioxidant status The plants having chemopreventive potential are known to contain various antioxidants. showed a significant reduction in tumor incidence and tumor multiplicity in skin and forestomach papillomagenesis at all the three doses (2. To confirm this .Antioxidant Potential of Asparagus adscendens 335 6.2. adscendens (Singh et al. adscendens on peroxidative Damage In the case of increased antioxidant status. These antioxidants actively interact with the reactive oxygen species and try to neutralize them. 2011). 2004). As mentioned earlier. the enzymes involved in the antioxidant function such as catalase. Many biological and molecular events have been identified which are modulated by different natural agents to inhibit the multiple stages of carcinogenesis. the balance between antioxidants and oxidants is believed to be a critical concept for maintaining a healthy biological system. Therefore the test diet containing the roots of A. 4. and 6%) by standard protocol adoption (Singh et al. 6. adscendens. Xenobiotic metabolism plays a crucial role in detoxifying the active carcinogenic dose of a potential carcinogen. Liver being the major site of xenobiotic/carcinogen metabolism and transformation. In this regard. cytochrome b5 reductase. In fact. adscendens Carcinogenesis is a multistep process induced by a variety of carcinogens which ultimately leads to the development of cancer.. superoxide dismutase were found to be enhanced by test diet containing A. it has been shown that the test diet containing A. the epoxide can be formed that is an active form of carcinogen known to bind with the DNA. A. 2011). 6. adscendens which has ability to scavenge free radicals or interfere with the development process of free radical is expected to inhibit the carcinogenesis. adscendens diet. Expectedly. Generally. reactive oxygen species are intimately linked with the process of carcinogenesis. Therefore. Effect of A. many of which have been recognized to have the ability to include apoptosis in various cancer cells of human origin (Rosangkima et al.3.

As expected. which have been used in the Indian traditional medicine system since long for the treatment of various ailments. adscendens. Gujarat. In the future. inducible DNA repair. the identification of all biologically active components could provide mechanistic insight into their preventive and therapeutic potential against various ailments including cancer. Environ Health Perspect 101 (Suppl 5):35-44. Together these studies suggest that the cancer chemopreventive potential of A. This harmful effect is counteracted by the antioxidant action of both enzymatic as well as non-enzymatic antioxidants. The lipid peroxidative damage in the hepatic tissue was measured in terms of MDA content. there was a significant reduction in the activity of lactate dehydrogenase. The decreased level of peroxidative damage is correlated well in accordance with the induction of antioxidant enzymes above the basal level. Shigenaga MK. Gandhi Nagar. New Delhi. 212-8. adscendens in diet was able to inhibit skin and forestomach papillomagenesis induced by DMBA and B(a)P. adscendens which could be mediated through drug metabolizing phase I and phase II enzymes. antioxidants are important way for body protection against the stress. . India Central University of Gujarat. India Raosaheb Kale Jawaharlal Nehru University. India 8.282:143-9. Summary Overproduction of free radicals leads to oxidative stress which is an important contributor to many diseases including cancer. Gold LS (1993) DNA lesions. References [1] Alexander J (2007) Selenium. in mice. as well as free radical scavenging antioxidant enzymes. Oxidative stress induces a cellular redox imbalance which may be related to the oncogene stimulation. Further the test diet containing roots of A. [2] Ames BN. 149-53. respectively. adscendens inhibited phases I and activated II enzymes and antioxidant enzymes. Author details Manju Singh and Divya Shrivastava Jawaharlal Nehru University. adscendens. we evaluated the cancer chemopreventive efficacy of the roots of A.336 Antioxidant Enzyme possibility.. Thus. Novartis Found Symp. There are countless medicinal plants available in nature. and cell division: three key factors in mutagenesis and carcinogenesis. 7. a significant decrease in the level of peroxidative damage was observed also supported this possibility. which have anticancerous properties and majority of them are still to be explored. A. New Delhi. Since the origin of human civilization medicinal plants have been considered to be imperative source of curing various dreaded diseases and cancer is one among those diseases. Therefore. with 4% and 6% test diets of A.

Baden HP. Rossi R. Med. Vijayakumar M (2010). Rihn BH (1998) Alphalipoic acid in liver metabolism and disease. Lin A. Packer L. 20:1716–1723. Mitochondria (2002) Regulators of signal transduction by reactive oxygen and nitrogen species. [16] Conner EM. Simon JA. reactive oxygen species and tissue damage. Med. Shiva S. Dianov GL (1999) Oxidative DNA damage processing in nuclear and mitochondrial DNA. [4] Are C. Klin Onkol. Kouroumalis E. McCall MR. Kampa M. Henderson G. Annu Rev Plant Biol 55:373-399. Biochem. Martin PM. [13] Butler J. McKenna GJ. Dondi A. 31: 1441–1444. Rajaram S. J Surg Oncol 102:100–105. Panagiotou S. Med. [18] Dalle-Donne I. 78:429–441. Marcocci L. 24:1023–1039. Castanas E (2000) Potent inhibitory action of red wine polyphenols on human breast cancer cells. Penzens E (1993) Akademiai Kiado. [15] Cerutti PA (1994) Oxy-radicals and cancer. Curti D (1992) The mitochondrial electron-transfer alteration as a factor involved in the brain aging. Darley-Usmar VM. [19] Damianaki A. Tritschler HJ. [20] Deans SG. Soc. [7] Bergamini CM. [11] Brozmanová J (2011) Selenium and cancer: from prevention to treatment. Nutrition 12:274–277. Thiemermann C. Cell. [9] Brash DE. [12] Bustamante J. Villa RF. Colburn L. Grisham MB (1996) Inflammation. J. Cervellati C (2004) Oxygen. Gemetzi C. free radicals. Noble RC. Lodge JK. Trends Mol. Milzani A (2003) Protein carbonylation in human diseases. Thromb. Frei B (2000) Oxidation of LDL by myeloperoxidase and reactive nitrogen species-reaction pathways and antioxidant protection. Notas G. Lancet 344:862-3. Budapest (1993) p 159. Gambetti S. 9. Oxidative Stress. Neurobiol Aging 13: 361–368. 24(3):171-9. Biol. Hirt H (2004) Reactive oxygen species: Metabolism. Hatzoglou A. Proc Natl Acad Sci U S A 88:10124-8. Ponten J (1991) A role for sunlight in skin cancer : UV-induced p53 mutations in squamous cell carcinoma. [10] Brookes PS. and antioxidants. 169–176. Colombo R. Free Rad Biol Med 33: 755–764. [6] Benzi G. Sarti P. Disparities in cancer care between the United States of America and India and opportunities for surgeons to lead. [14] Carr AC. Dagani F. Curr Pharm Des 10:1611-26. Halperin AJ. . Chem. Biochem. [8] Bohr VA. Salvemini D (2004) Potential therapeutic effect of antioxidant therapy in shock and inflammation.Antioxidant Potential of Asparagus adscendens 337 [3] Apel K. Hoey BM (1993) The one-electron reduction potential of several substrates can be related to their reduction rates by cytochrome-P-450 reductase.. [5] Behrend L. 11:1147–1162.Vasc. Arterioscl. Biochem Biophys Acta 1161: 73–78. Pastoris O. Rudolph JA. [17] Cuzzorcrea S. Biochimie 81:155-60. Marzatico F. Zwacka RM (2003) Reactive oxygen species in oncogenic transformation. and Signal Transduction. Free Rad. Trans. Curr. Giustarini D. Bakogeorgou E. Biol. Levonen AL.

26:190–195. Unger C (2003) Receptor tyrosine kinases: The main targets for new anticancer therapy. 10:235-245. Reed CD. Sci. Sci. Hirooka Y. Hortobagyi G (1992) Inflammatory breast cancer: a review. [30] Harris CC. Liver Int 23:38-45. Tanabe Y. Lanfear J. Harris CC: p53 mutations in human cancers. New York: Marcel Dekker Inc. [36] Ichiba M. Mukoyama T. [37] Jain JB. [35] Hollstein M. Carmichael PL. Drug Targ. Yamada S. Yasui S. Maeta Y. McGarry L. Biol. [25] Fedtke N. Basel. [34] Higinbotham KG. Buzdar AU.3ethenoguanine in rat-tissue DNA. Kurimasa A. Bhattacharya S (2006) Indian J Traditional Knowledge. [33] Hickman G (1989) Prevention of cancer: Vegetables and Plants: Areview. Mathers C. [31] Harrison PR. Ma YH.338 Antioxidant Enzyme [21] Denissenko MF. Medinger M. Phillips DH (1994) Induction of activating mutations in the human c-Ha-ras-1 proto-oncogene by oxygen free radicals. [28] Ghafourifar P. Boucheron JA. Reddy JK (1984) DNA damage related to increased hydrogen-peroxide generation by hypolipidemic drug-induced liver peroxisomes. Hollstein M (1993) Clinical implications of the p53 tumorsuppressor gene. hiota G (2003) Expression of 8hydroxy-2'-deoxyguanosine in chronic liver disease and hepatocellular carcinoma. Swenberg JA (1990) Vinyl chloride-induced DNA adducts. Comp Biochem Physiol. Bray F. Kanbe T. Packer L. Mol Carcinogen 11:170-5. Wu L. Part 2. Sklan D (1993) Electron leakage from the mitochondrial NADPH-adrenodoxin reductase-adrenodoxin p450scc (cholesterol sidechain cleavage) system. Venkatachalam S. Cancer Res 52:4747-51. [29] Hanukoglu I.. [22] Drevs J. Zimmer G (1997) Lipoic acid in health and disease. Biomed Environ Sci. Wani AA (1996) Site-specific induction and repair of benzo[a]pyrene diol epoxide DNA damage in human H-ras protooncogene as revealed by restriction cleavage inhibition. Schmidt-Gersbach C. Perantoni AO (1992) GGT to GTT transversions in codon 12 of the K-ras oncogene in rat renal sarcomas induced with nickel subsulfide or nickel subsulfide/iron are consistent with oxidative damage to DNA. Rice JM. Forman D. Fleming J. Mutat Res 363:27-42. Goel SK. 113–121. Watanabe T. J Clin Oncol 10:1014-24. Proc Natl Acad Sci USA. 93: 201-212. Walker VE. [38] Jaiyesimi IA. 81: 7827–7830. Lalwani ND. Shin HR. Curr. Formation and persistence of 7-(2_-oxoethyl)guanine and n2. Kasprzak KS. Estimates of worldwide burden of cancer in 2008: GLOBOCAN Int J Cancer [Epub ahead of print]. Diwan BA. Blower L (1997) Chemopreventive and growt inhibitory effects of selenium. Okano J. . Kumane SC. Murawaki Y. Trends Pharmacol. Cadenas E (2005) Mitochondrial nitric oxide synthase. Parkin DM (2010). Hong Kong: 205-226. Arch Biochem Biophys 305: 489–498. 4. [24] Fahl WE. [26] Ferlay J. Weber R. Science 253:49-53. N Engl J Med 329:1318-27. Saeki T. [32] Harrison R (2002) Structure and function of xanthine oxidoreductase: Where are we now? Free Rad Biol Med 33: 774–797. Carcinogenesis 11:1287–1292. New York. 5(2): 237-242. Weiner L. [27] Fuchs J. [23] Du MQ. Rapoport R. Sidransky D (1991) Vogelstein B.

Fishman D. Dobrota D (2005) Study of the oxidative stress in a rat model of chronic brain hypoperfusion. 1921–1942. Biol. NP (1980) Medicinal plants of Nepal Himalaya. Liptaj T. Heinonen OP. [45] Klatt P. Acad. Atsumi T. Biochem. 10(3):475-510. Ratna Pustak Bhandar. Kamendulis LM (2004) The role of oxidative stress in carcinogenesis. Med. Sci. Mukhtar H (2008) Cancer chemoprevention through dietary antioxidants: progress and promise. [52] Leonard SS. [44] Khan N.. Int.. LCC. Antioxid Redox Signal. Boopathy NS. Mech. Perez-Gomez C. Nahum A. Hepatogastroenterology 47:855-61. Horecky J. 52: 0115-119. Med. 595–603. Amir H. Kavitha S (2006) Nat. CRC Press. [42] Kasparova S. Biol. Am. Afaq F. Rissanen A. Fridovich I (1994) The role of O2 in the production of OH: In-vitro and invivo. Annu Rev Pharm Toxicol 44:239-267. 601–611. Seppanen R. Jarvinen R. Tower J (2005) Superoxide dismutase evolution and life span regulation. 46. New York Washington D.Antioxidant Potential of Asparagus adscendens 339 [39] Jones DP. Ageing Dev. [56] Mao H. 37. Koifmann A. Biochem. 11:1041–1064. De Castro IN (1999) Antioxidant enzymes and human diseases. Curr. Biol. [46] Klaunig JE. Carlson JL. Nepal. Levy J. Neurochem. Epidemiol. Sakaki S. Lygidakis NJ (2000) New epidemiological data on liver oncogenesis. Marnett LJ. [49] Kountouras J. Vancova O. 517S-520S. Mlynarik V. J. Lamas S (2000) Regulation of protein function by Sglutathiolation in response to oxidative and nitrosative stress. Lynn MJ. Med. [47] Knekt P. Clin. Pukkala E. Ulicna O. Harris GK. [43] Kathiresan K. 55. Harris GK. [58] Mates JM. Schnetz-Boutaud NC. [53] Liochev SI. [41] Karas M. Giat Y. Weisenseel JP. Aromaa A. [55] Manandhar.. Heinonen M. 267: 4928–4944. Mody VC. Eur. Segal S. Shi X (2004) Metal-induced oxidative stress and signal transduction. 365–379. Nutr. Cancer Int. Sharoni Y (2000) Lycopene interferes with cell cycle progression and insulin-like growth factor I signaling in mammary cancer cells. Brezova V. 37:1921–1942. Handbook of Ayurvedic medicinal plants. Chem. Sakurai H. pp. Cai JY. [40] Kapoor LD (2001). U. Free Rad Biol Med 16: 29–33. Prod. Free Radic.36:101–111. 126. Proc. Valko M. Free Rad. Shi XL (2004) Metal-induced oxidative stress and signal transduction. [54] Liu KH (2003) Health benefits of fruits and vegetables are from additive and synergistic combination of phytochemicals. J.C. [50] Landis GN. [51] Leonard SS. [57] Masaki H. Rad . 32. 28:625–635. Biol Pharma Bull (1995) 18-162. Free Rad. [48] Kojo S (2004) Vitamin C: basic metabolism and its function as an index of oxidative stress.S. Danilenko M. 134: 471–479. . Med. Kathmandu. Albanes D.A. 96:6615–6620. Sternberg P (2000) Redox state of glutathione in human plasma. Am J Clin Nutr 78. Natl. J. Teppo L (1991) Dietary antioxidants and the risk of lungcancer. Stone MP (1999) Duplex DNA catalyzes the chemical rearrangement of a malondialdehyde deoxyguanosine adduct.

[66] Ramakrishnan N. Pinto RE. [74] Schwarz KB. retinyl acetate. O’Brien PJ (2001) Biological reactive intermediates that mediate chromium VI toxicity. [73] Schroeter H. 555:81–96. Dig Dis Sci 46:2173-8. Alem C. Biochem. VI:Adv. Dubi N. et al. Boyd C. 26: 90–98. Rocha JBT. [76] Siems WG. Rad. [64] Pourahmad J. Arch. Amrani S. Benlys M. ergocryptine and selenium on DMBA-induced intitiation of mammary carcinogenesis in rats.. superoxide and pH gradients in the mitochondrial matrix: A theoretical assessment. 385:13–19. 4-Hydroxynonenal formation during ischemia and reperfusion of rat small-intestine. 42: 981-988. Wolfe WW. Williams RJ. Biol. Danilenko M. Rimbach G (2001) Molecular Aspects of α-Tocotrienol Antioxidant Action and Cell Signalling. [68] Rao AR. Biophys. 430:89–96. Frei B (1999) Inhibition of copper induced LDL oxidation by Vitamin C is associated with decreased copper-binding to LDL and 2-oxo-histidine formation. (2001) Hydroperoxyl.-Mol.. tocopherol. Weis SN. Hussain SP. Di Bisceglie AM. Chim. Mutagen. Free Rad. Spencer JPE. 500. 159–165. Res.340 Antioxidant Enzyme [59] McCord JM. Neurobiol. Diphenyl diselenide reverses cadmium-induced oxidative damage on mice tissues. J. Biol. 244. React. Groopman J (2001) Increased hepatic oxidative DNA damage in patients with hepatocellular carcinoma. Abrams RA. aminoglutethimide. Indian Journal of Expt. Raosaheb RK (2011) Eur J Cancer Prev 20 (3):240-247. Jannu LN. Biol. 57. Biol. Zeind J. Weber SU. Harnafi H. Skibsted LH. Exp. Fachinetto JM. Zeni G. Kumari MVR and Aradhana (1990) Modulatory influence of tamoxifen. [69] Retsky KL. Med. Rice-Evans C (2002) MAPK signaling in neurodegeneration: influences of flavonoids and of nitric oxide. Klein A. Free Rad Biol Med 31: 1208–1215. [63] Pastore A. [75] Sharoni Y. Britton RS. (2005). Bio. Biol. 203–207. 131(2):369S-373S. Mech. Levy J (2004) Carotenoids and transcription. 785–789. Chen K. Truscott TG (2001) The interaction of dietary carotenoids with radical species. [61] Niles RM (2004) Signaling pathways in retinoid chemoprevention and treatment of cancer. Biophys. Med. Piemonte F (2003) Analysis of glutathione: implication in redox and detoxification. Bertini E. Biol. Aging 23: 861–880. Biochem. Clin. Free Rad. [70] Rosangkima G and Prasad SB (2004) Indian J. Chem.. Catravas GN (1992) Radio-protection of hematopoietic tissues in mice by lipoic acid. [77] Singh M. 151. Chem. Arch. Grune T. Sousa J. Elrhaffari L. Fund. Ben-Dor A. Jones L. 28: 409-16. Nutr. [72] Santos FW. J. Mut. Life Sci. Kew M. . Biol. Med. [65] Pryor WA (2000) Vitamin E and heart disease: basic science to clinical intervention trials. (2009)Pharmacognosy Research 1:106-112. Singh S. Sharma S. Fridovich I (1969) Superoxide dismutase an enzymic function for erythrocuprein Hemocuprein. 130: 360–365. [62] Packer L. Favero AM. Sitzmann J. Interact. Exp. [60] Mortensen A. Res. Esterbauer H (1995). Intermed. 6049–6055. Federici G. [71] Salvador A. 28:141–164. Acta 333:19–39. [67] Ramchoun M. Cadenas E.

Dankovic DA. 160. Occupational exposure to chrysotile asbestos and cancer risk: A review of the amphibole hypothesis.P. deAndrade M. 1105–1112. Cancer Epidemiol Biomarkers Prev 16. 2008. Mazur M. [94] Waalkes MP. [91] Valko M. [87] Thomson CD (2004) Assessment of requirements for selenium and adequacy of selenium status: a review. Prev. Morris H. Hamm ML. Upadhyay KK (1993). Chem. Rhodes CJ. 1–40. Liehr JG. Biol. Pierce JP (2007) Plasma and Dietary Carotenoids Are Associated with Reduced Oxidative Stress in Women Previously Treated for Breast Cancer. Acta. Am. 11. Med Hypotheses 39:267-7. fruits andcancer. [97] Wei H (1992) Activation of oncogenes and/or inactivation of antioncogenes by reactive oxygen species. [82] Stayner LT. Mazur M. Med. Siddiqui A (2003) Regulatory mechanisms of viral hepatitis B and. Hittelman WN. Am J Physiol-Lung cell Mol Physiol 279: L1005–L1028. Rhodes CJ. 31–38. Sayre LM.Chem. Jabalpur. 161–166. 45:821-824. 1161–1208. Morris H. Stendell-Hollis NR. 86. Public Health.D. Med. Liu J. 179–186. [84] Stohs SJ. Mazur M (2006) Free radicals.Sachitra Ayurved.N. . [93] Valko M. [85] Taylor PR (2004) Science Peels the Onion of Selenium Effects on Prostate Carcinogenesis. M. [92] Valko M.Antioxidant Potential of Asparagus adscendens 341 [78] Smela ME. 198. Curr.. 12. Henderson PT. Cronin MTD (2005) Metals. Biophys. A. Rapta P. Essigmann JM (2002) The aflatoxin B(1) formamidopyrimidine adduct plays a major role in causing the types of mutations observed in human hepatocellular carcinoma. Telser J (2004) Role of oxygen radicals in DNA damage and cancer incidence. Biomark.K. A review of vegetables. [89] Trivedi HK. Diwan LA (2004) Mechanisms underlying arsenic carcinogenesis: Hypersensitivity of mice exposed to inorganic arsenic during gestation.V.. [90] Valko M.V. J. (1991). Free Rad Biol Med 18: 321–336. J. Chem. Oxygen free radical generating mechanisms in the colon: Do the semiquinones of Vitamin K play a role in the etiology of colon cancer? Biochim. Rock CL. Mol Cell Biochem 266:37–56. 1527. J. 17:2653–2657. Harris TM. Interact. 5:705–710. K. D. Harris CM. JNCI J Natl Cancer Inst 96 (9): 645-647. Role of oxidant species in aging. [88] Thomson CA. Cussler EC. thesis J. Beckman JS. Fanburg BL (2000) Reactive oxygen species in cell signaling. [79] Smith MA. Proc Natl Acad Sci 99:6655-60.. toxicity and oxidative stress. Izakovic M. Dhingra K. Toxicology. Bilton RF (2001). Ward JM. Cancer Epidemiol. [80] Somkuwar AP. J Biosci 28:311-21. Harris PLR. Curr. [86] Thannickal VJ. Flatt SW. Perry G (1997) Widespread peroxynitrite-mediated damage in Alzheimer’s disease.. Bagchi D (1995) Oxidative mechanisms in the toxicity of metalions. Journal of Epidemiology Cancer Control. (2003) Ph. metals and antioxidants in oxidative stress-induced cancer. [81] Stadtman ER (2004). Lemen RA (1996). Eur J Clin Nutr 58:391-402. Moncol J. [83] Steinmetz. 2: 325–327. Neurosci.. Li DH (1996) Lipid peroxidation-induced putative malondialdehyde–DNA adducts in human breast tissues. [95] Wang MY. [96] Waris G. & Porter. Izakovic M.

Marinova E. Gail MH. Ryberg D. 1997. Food Nutritionand The Prevention of Cancer: A Global Perspective. American Institute for Cancer research. Hu YR. 21(3):223-232. Patterson RE (1997) Relationship between vitamin and calcium supplement use and colon cancer. [99] White E. [103] You WC. [102] Yanishlieva NV. Chiou CC.342 Antioxidant Enzyme [98] Whanger PD (2002) Selenocompounds in Plants and Animals and their Biological Significance. Pokorny J (2006) Eur J Lipid Sci Technol 108-776. Blot WJ. Shannon JS. [100] Wu LL. Natl. Jin ML. 6:769–774. [101] World Cancer Research Fund. Blaser MJ. Biomark. Washington. Ma JL. DC. J. Prev. 92:1607– 1612. Zhang L. and other risk factors. atherosclerosis and diabetics. Wu JT (2004) Urinary 8-OHdG: a marker of oxidative stress to DNA and a risk factor for cancer. Correa P. . Clin Chim Acta 339:1-9. Chang PY. Haugen A (2000) Induction of microsatellite mutations by oxidative agents in human lung cancer cell lines. Yang CS. [104] Zienolddiny S. Carcinogenesis 21:1521-6. serum Vitamin C. Liu WD. Fraumeni JF. Li JY. Chang YS. Xu GW (2000) Gastric cancer: Helicobacterpylori. American Institute for Cancer research. Cancer Inst. J of Am College of Nutr. Cancer Epidemiol.

Balanced content of selenium in human food helps in the case of complications connected with diabetes and affects also the prevention of asthma. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons. as well as biological systems.   . Food. depending on its type and origin. which can contain various amounts and chemical forms of selenium. Through free radicals inhibition selenium moderates harmful effects of radiation [3]. provided the original work is properly cited. Se-proteins) [5]. immune system. M. it can reduce the risk of spontaneous abortions as well [2].SeO42-) and organic forms of selenium (Se-amino acids.. cardiovascular diseases and masculine sterility [1].Chapter 13 Selenium – An Important Antioxidant in Crops Biofortification P. Selenium in human body For human body selenium is essential element playing important role in body antioxidation system. licensee InTech. such as C and E vitamins and in processes protecting the cells from free radicals. Furthermore. J. 1. selenate .0). Ježek. Elzner Additional information is available at the end of the chapter http://dx. slows down the development of AIDS through reducing the speed of HIV development.1. methylated forms. T. Jůzl and Selenium affects in a positive manner human mind and mental wellness [4]. Lošák. which permits unrestricted use. P. Introduction 1.doi. Dominant form of selenium in a food affects acceptability of selenium for body and its availability in proteosynthesis and production of methylated selenium compounds [6]. and reproduction in any medium. Selenium is important for proper function of cerebral neurotransmitters and reduces epileptic waves at children. it is considered individual antioxidant that can cooperate with other antioxidants. distribution.5772/50356 1. In comparison with inorganic forms of       © 2012 Škarpa et al.SeO32-. inhibits virulence. Selenium participates in thyroid hormone metabolism. Hlušek.2. Škarpa. can contain inorganic (selenite .org/licenses/by/3. Sources and forms of selenium in human nutrition Basic source of selenium in human nutrition is food. In such manner selenium protects a body from development of cancer. Selenium deficiency is connected with acceleration of senility and development of Alzheimer´s disease.

27 0.008 0.) [9].004 0.31 0.53 0. Se-cys and from most of plant food.31 0. as they can be absorbed more easily. they are metabolised into structures of Se-proteins and other non-specific proteins and are excreted by renal system in less extent [7].0.97 . During simulated experiment the same forms of selenium were detected in gastric liquids.09 0.76 0.38 0.0.344 Antioxidant Enzyme selenium. containing Se-methylselenocysteine as main Secompound.0.02 0. replacing essential Met or being part of metabolism of selenium [8]. enters directly to metabolism of proteins.) [10] and vegetable treated with SeO32-.03 0.0. if organic selenium is used for nutrition.07 0. On the other hand. Availability of selenium from animal products is intermediate and in some cases even low [8].0.09 0.22 2. In comparison with nutrition using SeO32-. selenium content in urine is lower [3].0. These results indicate that the mentioned plants can be good source of Secompounds for body [9].0.11 .02 .95 % depending on present forms [8].21 1.0.02 .51 0.0.2 .0.1.14 0.50 3. chive plants (Allium schoenoprasum L.02 .03 .78 0.1.07 .43 1.01 .24 *data before Se biofortification Table 1.10 Canada 0.12 .80 0.08 .02 . inorganic selenium is absorbed in mineral form and deposited in tissues in small extent.37 0.01 .0. Content of Se mg.0. Finally.34 .11 0.0.0. located in plant products. For example.03 0.06 .06 0.2.Se-met [11].09 0.48 0.07 0.36 0.54 .20 Czech and Slovakia 0. considerable part of inorganic selenium is eliminated in urine.003 0.84 0.59 0. a body can well use selenium from Se-met.06 0.45 .09 .004 .78 0.19 0.70 0.62 .64 0.1. Meat and fish are rich source of selenium. the organic ones are more available for a Food Pork Beef Chicken Pork liver Beef liver Pig kidneys Beef kidneys Freshwather fish Sea fish Milk Curd Cheese Youghurt Eggs USA 0.01 .0.05 .001-0. occurring mostly in more acceptable form . mostly in Se-cys form. 0.13 1.003 0.0.90 .17 0.10 . vegetable and fruit are lower-grade source of selenium.14 0.39 Finland* 0. Content of selenium in meat and animal products in various countries of the world [12] .04 .16 Germany 0. Content of selenium in meat and animal products is listed in table 1.> SeO32-.24 0.95 0. Surai [3] presents the following acceptability of Se forms: Semet > SeO42.12 .1.05 0.38 0. On the other hand.0.0.02 . Se-met.34 .0.12 0.0.18 .0. Absorption of organic sources of selenium from food is 70 .15 0.75-1.34 0.02 0. Generally.41 0. intestinal liquids and in fresh garlic plants (Allium sativum L.

Asia and part of Africa the intake of selenium from food doesn´t reach recommended daily intake [13]. anyway. Activity of GSH-Px in a body is considered to be an indicator of selenium supply [18]. where it can participate in selenium transport and carries out the function of antioxidant. The most important and known Se-proteins in human body include glutathion-peroxidase (GSH-Px). sportsmen and hardworking people [4]. Next Se-protein is enzyme thioredoxin reductase (TR). Selenium is also the part of proteins. Se-amino acids. 15]. Recommended intake of selenium. chemical. which participates in metabolism in thyroid gland hormone . its deficiency and toxicity Recommended daily intake of selenium for adult men and women is 55 μg per day. Daily intakes of selenium in other parts of the world are listed in table 3. which can be found in plants of Allium and Brassica genus. food and agricultural sciences will be necessary [13]. have anticarcinogenic effect.4. As for retention through tissues and activity of glutathion-peroxidase (GSH-Px = protein with antioxidation effects). 16]. which creates functionally most important group of Se-proteins participating in protection of cells from oxidation caused by free radicals. To understand effect of selenium coming from common food and in particular from a food fortified with selenium to human health in full extent. including pregnant and lactating women. Se-methylselenocysteine and derivates of γ-glutamile. At present there is no universal method for evaluation of selenium availability from food and it is recommended to use combination of various methods [3]. Recommended intakes for other age categories and for pregnant and lactating women are listed in table 2. In Se-proteins selenium is incorporated in form of synthesized Se-cys [16]. the deficiency of selenium in Europe is commonly known [19]. which maintain the integrity of sperm flagellums and W Se-protein. which is 21st biogenic amino acid [17].Selenium – An Important Antioxidant in Crops Biofortification 345 1. In many countries of Europe. Another important group of Se-proteins is iodothironin deiodinase. in comparison with anorganic sources of selenium it is more effective in reduction of cancer presence. based on their specific metabolism [13]. growing and developing children. Se-protein P creates 60 % of total selenium in plasma. SeO32-). This knowledge indicates that bioavailability and effectiveness of selenium in a body is evaluated according to selenium form in the food or in food supplement and function of such Se-compound in a body. Methylated Se-compounds can be important in cancer prevention and are not available for proteosynthesis. selenium from fortified plants of Allium and Brassica genus is as available for body as selenium from inorganic sources (SeO42-. being necessary for muscle metabolism [5. Low intake of selenium endangers mostly the people with reduced intake of food (seniors) and people with higher need of this essential element. the multidisciplinary attitude of a team consisting of research workers in the fields of medical. regulating redox processes in a cell. 1.thyroxine. Function and metabolism of selenium in human body Metabolism of selenium depends on chemical forms of selenium in food and various forms of selenium carry out various functions. . as well as Se-met are transformed to structures of proteins [14.3. 15 20 20 30 40 55 60 70 Children Adults Pregnants Lactation Table 20 55-80 30 – 40 ** 37 48 15 38 30 30 35 31 .day-1) 57 .47 29 29 .199 98 .38 70 30 .350 - *based on concentration of Se in urine of adults. The risk of occurrence of degenerative diseases (in particular cancer.87 28 .) .39 200 . etc.37 10 .43 27 27 .346 Antioxidant Enzyme Life stage Infants Age 0-6 months 7-12 months 1-3years 4-8 years 9-13 years 14 < years --- μg Se.36 106 29 . cirrhosis.4990 38 .48 50 43 104 . **calculated Table 3. Recommended daily intake of selenium by individual age categories [20] Country Australia Belgium Brazil Czech Repubic China Denmark Egypt France Croatia India Ireland Italy Japan Canada Germany Nepal Netherlands Intake of Se (μg Se. Daily intake of selenium from food in various states of the world [6] Deficiency of selenium in human nutrition affects biological functions being in relationship with function of Se-proteins and metabolism of selenium. cardiovascular diseases.25 * 7 .61 28 .54 Country New Guinea New Zealand Poland Portugal Austria Saudia Arabia Slovak Republic Slovenia Serbia Spain Sweden Switzerland Turkey USA UK Venezuela - Intake of Se (μg Se.224 35 23 39 .

Monitored persons showed only moderate symptoms of toxic influence of selenium. no symptoms of overdose with selenium were observed at local inhabitants with daily intakes of selenium higher than 724 μg per day. In China.600 and 3. Maximum safe daily intake of selenium for men and women (pregnant. At immediate overdose the symptoms of toxicity are more intensive at inorganic forms of selenium [20]. The richest mediator of toxic doses of selenium was cereals [22]. Chronically over limit daily intake of selenium can have negative and (depending on doses) even toxic effects. Figure 1 demonstrates the damage of nails at people living in north-western India. This knowledge shows that toxic doses of selenium are more than 10 times higher than physiological need of a body [3]. where the food sources are naturally rich in selenium. The most common symptoms of selenosis are fragility and loss of hair and nails. In case of chronic intake the toxic effects of organic and inorganic forms are similar. as well as lactating) older than 14 years is 400 μg per day. 3]. Content of selenium in hair and nails of intoxicated people was 8-9 times higher in comparison with healthy people [22]. Maximum safe daily intake of selenium for particular age categories is presented in table 4. mottled teeth.Selenium – An Important Antioxidant in Crops Biofortification 347 increases [21]. that means a disease characterized with damage of cartilages causing the deformation of bone structures [ 45 60 90 150 280 400 Table 4. In China the symptoms of selenosis were described at sensitive people with daily intake of selenium higher than 910 μg per day. During an experiment performed also in United States selenium has been administered to cancer patients in doses 1. In United States.12 months 1 . with naturally high content of selenium in soil.200 μg per day. nausea. Life stage Infants Children Adults* *including pregnants and lactation Age 0 . in states South Dakota and Wyoming.3 years 4 . in areas with critical deficiency of selenium two killer diseases are epidemiologically spread: “Keshan disease”. abnormalities of nervous system [3]. . exhaustion garlic odour on the breath. characterized with failures of myocardium function – the so-called “cardiomyopathy” and “Kashin-Beck disease” – the osteoartropathy.6 months 7 .13 years > 14 years μg Se. As for intake of selenium in the frame of improvement of nutritive value of food.8 years 9 . decreased level of haemoglobin. it is important to know its safe upper limit of daily intake separating positive and potential negative effects on human body. diarrhea. Maximum safe daily intake of selenium for particular age categories [20] Selenosis as result of over limit intake of selenium from food at human body was described for the first time in 60-ties in China (province Hubei). hives.

kg-1 of soil. over which these diseases don´t occur is 0. Content of selenium in soils and biological material varies in wide extent. According to Oldfield [25].kg-1 is excessive.348 Antioxidant Enzyme Figure 1. the content of selenium lower than 0. Both these diseases occur in China.33 mg. Feed produced on soils with content of selenium lower that mentioned value causes deficiency of selenium at animals. Kabata-Pendias and Pendias [27] present average content of selenium in soils of the world is already very in bleached sands (podsol) to 0.175 mg.25 mg.123 to 0. . the symptoms connected with over limit content of selenium. Deformation of nails at people intoxicated with selenium in north-western part of India [22] 2. in areas with total content of selenium in arable land lower than 0. Selenium in soil Basic source of selenium in nutrition of man and animals is soil. In China the criterion for evaluation of content of selenium in soil was occurrence of endemic diseases caused by deficiency of selenium – “Keshan disease” and “Kashin-Beck disease”.kg-1 in histosols. Limiting content of selenium in a soil.3 mg Se.6 mg. the so-called selenosis occur [26].37 and average contents of selenium vary depending on soil type and locality from and the content of selenium in soil higher than 3 mg. in areas with contents of selenium in soil higher than 3 mg. On the contrary. Problems resulting from low content of selenium in human nutrition led to mapping selenium content in soils [23].125 of soil in relation to nutrition of animals is limiting and the content lower than Content of selenium in soils is evaluated based on relationship to human physiological needs and physiological needs of livestock and based on average content of selenium in soils of particular monitored regions. Gupta [24] consider that deficient total content of selenium in soil is lower than 0.5 mg.

Availability of selenium applied in such manner is usually 5 to 30 %.3 mg. Natural source of emissions are selenium volatilization in form of dimethyl-selenide ((CH3)2Se) from the soil. This fact results from different physical characteristics of soils containing SeO32.3 mg. Selenates are highly soluble in water and don´t create stable complexes that means. This form of selenium dominates in aerated soils with neutral and higher pH. Residual part is retained in firmly bound with a positive charge [ of soil). With next development of soil acidity and reducing conditions selenium in soil occurs in form of selenides (Se2-). The climate affects the contents of selenium in soil. its mobility and availability for plants are affected with soil of fertilizer [29].14 – 0. Oxidation-reduction relations among individual selenium forms are demonstrated in figure 3. Based on development of these factors. 27] and in acid soils creates stable complexes with iron hydroxides. leached. Another anthropogenic source of selenium includes mineral fertilizers enriched with selenium. individual forms of selenium in a soil undergo transformations regulated with oxidation-reduction processes. This mechanism ensures the release of selenium from a soil to the atmosphere [33]. Selenides in a soil can form the chains of diselenides (RSeSeR) volatilizing from a soil. Dominating form of selenium in a soil. In highly reducing conditions selenium can be present in a soil in its elementary form being available only for some bacteria [34].kg-1 of soil) and. for example as Se-met. With decreasing pH and redox potential in soil SeO32. in Finland now the mineral fertilizers are enriched with selenium in the form of sodium selenate (Na2SeO4) in amount of 10 mg Se.dominate. Dependence of selenium form on pH and redox potential is demonstrated in figure 2. Selenium in soil exists in several inorganic forms: as elementary selenium (Se0). fresh and sea water and volcanic activity [28]. Selenite. create stable complexes with iron hydroxides [27]. For example.[30]. Anthropogenic source of selenium is combustion of coal and metallurgic facilities. similarly to SeO32-. being less available for plants than SeO42.dominate. these compounds represent total selenium soluble in a soil [27]. aeration. as well as with atmosphere.and SeO42. hydrological regime and soil redox potential [31.Selenium – An Important Antioxidant in Crops Biofortification 349 Content of selenium in soil is affected with structure and intensity of parent material (native rock) erosion and its structure [24]. selenite (SeO32-). they represent the form of selenium that can be easily leached and is available for plants. eventually released to atmosphere through volatilization [30]. plants. being the source of selenium air pollutants of natural and anthropogenic origin. which form KHSe. Current researches in China showed that the soils developing in humid and moderately humid tropical and subtropical conditions contain higher amount of selenium soluble in water (> 0. too. 3]. selenate (SeO42-) and in organic forms. the soils developing in humid and moderately humid conditions of mild climate contain altogether small amount of selenium [26]. In soils rich in organic matter and water and without air entry selenates are transformed and reduced to less mobile forms [32].or SeO42and from different mechanism of absorption of both anions. In soils with high content of Ca and Mg CaSeO4 and MgSeO4 are created. being used in areas with low reserves of available selenium in soil. soils developing in steppe and desert conditions contain medium amount of selenium soluble in water (0. finally. . selenide (Se2). NH4HSe and MnSe and also. In cultivated soils SeO32.

SeO4) Figure 3. SeO. SeO3.350 Antioxidant Enzyme Figure 2. reduction (CH3)2Se SeO32 HSeO3Se2- volatilization Me(Se. Distribution of selenium in a soil depending on pH and redox potential [31] Se0 HSe - SeO42oxidation. Diagram demonstrating the transformation of selenium in a soil [27] .

[36] showed that the plants contained the highest amount of selenium after treatment with SeO42-. Selenium is not considered essential element for plants. Selen