Leading Edge Antioxidants Research

LEADING EDGE ANTIOXIDANTS RESEARCH No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

LEADING EDGE ANTIOXIDANTS RESEARCH

HAROLD V. PANGLOSSI EDITOR

Nova Science Publishers, Inc. New York

Copyright © 2007 by Nova Science Publishers, Inc.

All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter cover herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal, medical or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Library of Congress Cataloging-in-Publication Data Leading edge antioxidants research / Harold V. Panglossi (editor). p. ; cm. Includes bibliographical references and index. ISBN: 978-1-61324-512-5 (eBook) 1. Antioxidants--Research. 2. Antioxidants--Therapeutic use. I. Panglossi, Harold V. [DNLM: 1. Antioxidants--therapeutic use. QV 325 L434 2006] RB170.L43 2006 613.2'8072--dc22 2006017578

Published by Nova Science Publishers, Inc. New York

CONTENTS Preface

vii

Chapter 1

Anti- and Prooxidative Effects of Flavonoids Wim Wätjen, Yvonni Chovolou, Andreas Kampkötter and Regine Kahl

1

Chapter 2

Comparison of Antioxidative Properties of Green and Black Tea Agnieszka Augustyniak, Justyna Ostrowska, Wojciech Luczaj and Elzbieta Skrzydlewska

17

Chapter 3

Antioxidants in Foods: A New Challenge for Food Processors G. Oboh and J. B. T. Rocha

35

Chapter 4

Are Teas the Universal Antioxidants Justyna Ostrowska, Wojciech Luczaj and Elzbieta Skrzydlewska

65

Chapter 5

Radioprotective Effects of Antioxidants Mustafa Vecdi Ertekin and Orhan Sezen

89

Chapter 6

Antioxidant Therapy for Chronic Inflammatory Diseases Jayraz Luchoomun, Dominic Sinibaldi and Xi-Lin Chen

Chapter 7

Impact of Oxidative Stress on Diabetes Mellitus and Inflammatory Bowel Diseases Jana Varvařovská, Rudolf Štětina, Josef Sýkora Zdeněk Rušavý, Jaroslav Racek, Silva Lacigová and Konrad Siala

Index

145

179

247

PREFACE In biological systems, the normal processes of oxidation (plus a minor contribution from ionizing radiation) produce highly reactive free radicals. These can readily react with and damage other molecules. In some cases the body uses free radicals to destroy foreign or unwanted objects, such as in an infection. However, in the wrong place, the body's own cells may become damaged. Should the damage occur to DNA, the result could be cancer. Antioxidants decrease the damage done to cells by reducing oxidants before they can damage the cell. Virtually all studies of mammals have concluded that a restricted calorie diet extends the lifespan of mammals by as much as 100%. This remarkable finding suggests that food is actually more damaging than smoking. As food produces free radicals (oxidants) when metabolized, antioxidant-rich diets are thought to stave off the effects of aging significantly better than diets lacking in antioxidants. The reduced levels of free radicals, resulting from a reduction in their production by metabolism, is thought to be a major cause of the success of caloric restriction in increasing life span. Antioxidants consist of a group of vitamins including vitamin C, vitamin E, selenium and carotenoids (such as beta-carotene, lycopene, and lutein). This new book brings together the latest research in this dynamic field. Chapter 1 - Flavonoids are polyphenolic compounds that occur ubiquitously in foods of plant origin. This class of compounds has become increasingly popular in terms of health protection because they possess a remarkable spectrum of biochemical and pharmacological activities. Flavonoids affect basic cell functions such as growth, differentiation and apoptosis. Epidemiological studies have suggested that flavonoids may protect against various stages of the cancer process and are associated with a reduced incidence of coronary heart disease. Flavonoids have been shown to be potent antioxidants because of their radical scavenging activity. Furthermore, flavonoids are able to complex heavy metal ions, e.g. iron and copper which are involved in Fenton-like reactions. The biological actions of flavonoids have long been thought to be due to their antioxidant potential but at present it is by no means clear whether other mechanisms of action contribute to their overall effect or are even more important than their radical scavenging properties. Although some flavonoids act as powerful antioxidants it was also shown that in high concentrations they can generate reactive oxygen species and induce apoptosis. Hydroxyl radicals generated by autoxidation and redox-cycling of the polyphenolic flavonoids may initiate peroxidation of the lipids in cellular membranes. Furthermore, flavonoids can cause an impairment of antioxidative defense systems consisting e.g. of glutathione and glutathione-S-transferase, which in this way indirectly induces oxidative stress in the cell.

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For a number of flavonoids cytoprotective, antioxidative and antiapoptotic as well as cytotoxic, prooxidative and proapoptotic effects have been shown in various cell culture models. Given this wide spectrum of biological actions, it is quite understandable that numerous health claims which are in part mutually exclusive have been linked with flavonoids. Notably in cancer, but also in infections or autoimmune disease, a deficiency in apoptosis is one of the key events. On the other hand, overefficient apoptosis, as observed in fulminant liver failure, or the long term accumulation of apoptotic events in neurodegenerative disorders may be equally harmful for the organism. Given that both beneficial and adverse effects can in principle be caused by flavonoids it must be assumed that – in addition to the cell type or tissue involved and to the presence or absence of a stressor - it depends on dose which action prevails. Chapter 2 - Green tea has been proved to possess antioxidative properties whereas black tea antioxidant properties in vivo have been questioned, considering its components. The aim of this study has been to compare the antioxidative properties of black and green tea manifested by their protective action on the liver, brain and serum antioxidants of two months old rats chronically (4 weeks) intoxicated with ethanol. In order to estimate the intensity of black and green tea action the activity of antioxidant enzymes – superoxide dismutase, catalase, glutathione peroxidase and reductase as well as GSH level were measured by spectrophotometric methods while the levels of vitamin C and E were measured by HPLC methods. Teas antioxidant efficiency was evaluated by lipid peroxidation process intensity estimated as TBARS level. It has been shown that ethanol caused decrease in the activity/level of the examined antioxidants in the liver, brain and serum except glutathione reductase whose activity increased in the liver and serum. Disturbances in antioxidant abilities led to oxidative stress formation manifested by the rice of the level of lipid peroxidation products. Green tea as well as black tea partially prevented changes in the activity/level of antioxidants of all examined tissues caused by ethanol intoxication and significantly protected phospholipids against oxidative modifications. Green tea as well as black tea given to rats receiving alcohol caused significant increase in the activity of all examined enzymes in the brain. Moreover green tea enhanced the activity of superoxide dismutase, glutathione peroxidase and catalase, while black tea increased only glutathione peroxidase and catalase activities in the liver. Serum of alcohol intoxicated and drinking tea rats was characterized by increase in the activity of superoxide dismutase after green tea and in superoxide dismutase and glutathione peroxidase after black tea. Both teas caused significant increased in the liver, brain and serum levels of GSH, vitamin C and E in comparison to alcohol group. However green tea action was a little more effective than that of black tea. In consequence both teas significantly protected phospholipids against oxidative modifications caused by ethanol. The above results clearly indicate beneficial effect of green as well as black tea on antioxidant system and prevention against oxidative stress formation after ethanol intoxication. Chapter 3 - In recent years, human health has assumed an unprecedented important status. A new diet-health paradigm is everlasting which places more emphasis on the positive aspects of diet. Foods have now assumed the status of functional foods, which should be capable of providing additional physiological benefit, such as prevent or delaying onset of chronic diseases, as well as meeting basic nutritional requirements. Recently phytochemicals in fruits and vegetables have attracted a great deal of attention mainly concentrated on their role in preventing diseases caused as a result of oxidative stress. The regular consumption of

Preface

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foods that are naturally high in antioxidants (fruits, vegetables, whole grains, nuts and seeds, legumes and herbal seasonings) is associated with substantial health benefits. Although some of this food are eaten raw without processing, but many of them are subjected to one or more forms of post-harvest food processing techniques such as blanching, cooking, sun drying, frying, soaking, irridation, fermentation, coagulation and baking. Apart from Vitamin C and E that are already established food nutrient with antioxidant properties, other phytochemicals in foods with antioxidant properties includes: Ally sulphide, carotenoids, curcumins, flavonoids, gingerols, indoles and isothiocyanates, isoflavones, lignans, liminoids, phenolic acids, phthalides polyacetylenes, phytates, saponins and terpenes. This phytochemicals varies in their quantities and physico-chemical properties, and their activities in food are either additive or synergistic. Since most of the methods employed in foods processing both at household levels or during the unit operations in food processing industry will bring about a change in quantities and physico-chemicals properties of the food phytochemicals, and this could effect individual / or gross antioxidant properties of the food. In this article, the effect of the various post-harvest food processing techniques popularly employed both at the household or industrial levels on the antioxidant phytochemicals and activity of some commonly consumed plant foods, and the possible remedy is highlighted. Chapter 4 - Cellular metabolism is accompanied by generation of free radicals, which also play pivotal role in the action of numerous xenobiotics. Their increased production is a primary event in many human diseases progression or a secondary consequence of tissue injury. Detoxification of radicals in the cell is provided by the antioxidant defense system. Many synthetic and natural antioxidants are useful when antioxidant systems are no able to cope with radicals action. Among natural antioxidants, the most popular beverage consumed worldwide is tea. Tea is manufactured in three basic forms: green, oolong and black, which are non-, partially- and full-fermented/oxidized, respectively. Therefore, green tea composition is very similar to that of the fresh leaves and contains considerable amount of monomeric polyphenols, particularly catechins. The major components of black tea are multimeric polyphenols - theaflavins and thearubinins – the oxidation products and condensates of catechins. Considerable amount of the original polyphenolic compounds are contained in oolong tea. Monomeric polyphenols possess proved antioxidant properties wich are manifested in the ability to prevent oxygen radical formation, by inhibiting the activity of enzymes participating in their generation as well as in the ability to scavenge free radicals and to chelate transition metal ions. Recently, antioxidant properties of multimeric polyphenols of black tea have also been proved. The antioxidant properties of teas polyphenolic compounds have led to considerable interest in the potential health benefits resulting from teas consumption. Evidence has been collected that teas polyphenols are metabolized extensively and are distributed to all tissues in animal organisms. Metabolites of catechins and their condensates have been also revealed to possess antioxidant properties comparable to their parent compounds. Significant enhancement in tissues antioxidant capacity has been demonstrated following the consumption of teas by animals and humans. In consequence the protective effect of teas polyphenols on DNA, lipids and proteins appears to be very promising in reducing the incidence of many diseases. It was indicated that tea consumption leads among others to decrease in cancer risk but its mode of action is still unclear. Teas polyphenols can block the formation of mutagens and carcinogens from precursor and increase their detoxification. Moreover they influence molecular events at the gene level. However, despite of proved antioxidant and cancer chemoprotective properties of tea

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catechins, attention has been also drawn to their prooxidant action appearing in oxidative damage of cellular and isolated DNA. Chapter 5 - Radiation therapy (RT) is known to be one of most important tools to cure cancer. In recent years, more patients with cancer have been treated with radiation therapy. Because most of them are surviving, there is an increasing need to patients care for RTinduced toxicities. Since X-ray was discovered by Wilhelm Roentgen in 1895, radiationinduced toxicities, such as dermatitis, mucositis, myelosuppression, and tissue’s fibrosis, have been well-known. These toxicities may lead to the delays in administration or to the dosage limitations in radiation therapy, to the increased hospitalisation stays and the costs, and it may have adverse effect on radio-curability of cancer and patient survival. The destructive action of ionizing radiation is mainly due to reactive oxygen species (ROS), including superoxide anion radical (O˙2¯), hydroxyl radical (OH˙), and hydrogen peroxide (H2O2), generated by the decomposition of water, which constitutes around 80% of the cell. These ROS formed in cells contribute to radiation injury in cells, such as damage to cellular DNA and membrane structures, and alterations in the immune system. Although all respiring cells are equipped with protective enzymes such as SOD and CAT or GSH-Px, increased oxidative stress in cells stemming from ionizing radiation may overwhelm the protective systems, leading to cell injury. In this point, there is a question whether oral or parentheral antioxidants might administer concurrent with RT, or not, because some of scientists consider that concurrent administration of oral antioxidants is contraindicated during RT, since antioxidants might reduce oxidizing free radicals created by radiation therapy, and thereby decrease the effectiveness of this treatment. However, previous studies have demonstrated that the use of antioxidants, such as vitamin E and gingko biloba, during radiation therapy might improve the efficacy of radiation therapy by enhancing tumour response and decreasing some of the toxicity towards normal cells. Antioxidant defence mechanisms involve strategies both enzymatic, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) and non-enzymatic, such as vitamins C and E, selenium, zinc, gingko biloba, and melatonin. They work in synergy with each other and against different types of free radicals, and they can offer protection against ionizing radiation-induced oxidants. Therefore, antioxidants might use as a radioprotectant agents. An ideal radioprotectant is one that protects normal tissue while maintaining antitumor effectiveness, and is itself without moderate or severe toxicity. This article reviews the current status of antioxidants as a radioprotective agent in radiation therapy. Chapter 6 - Oxidative signals play important roles in the pathogenesis of inflammatory diseases such as atherosclerosis, arthritis, asthma, and neurodegenerative diseases. Oxidative stress occurs when there is an increased production of reactive oxygen species, reactive nitrogen species, or decreased antioxidant defense mechanisms. Oxidative stress contributes to the initiation and progression of chronic inflammation by promoting cell proliferation, adhesion molecule expression, cytokine and chemokine production, and matrix metalloproteinase generation. A number of animal studies and clinical trials have demonstrated increased levels of biomarkers for oxidative stress in various inflammatory diseases including atherosclerosis, asthma and rheumatoid arthritis. Related studies have also demonstrated that decreased antioxidant capacity enhanced susceptibility to immune and inflammatory diseases; while the use of antioxidants diminished or prevented inflammatory diseases. Therefore, increased cellular antioxidant activity or scavenger ROS may represent a

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novel approach for the treatment of inflammatory diseases. However, several clinical trials using antioxidant vitamins have failed to demonstrate beneficial effects for some inflammatory diseases. The purpose of this review is to summarize our recent understanding of the oxidative signaling events involved in inflammatory processes in the context of experimental and clinical studies that utilize antioxidants for the treatment of inflammatory diseases. Chapter 7 - Formation of reactive oxygen species (ROS) is a natural process during oxidative metabolism. ROS play an important role not only in pathological processes of human organism as usually presented but less attention is paid to their proper important role in cell signaling, biosynthesis or non-specific antiinfectious defence. Overproduction of ROS during numerous pathological situations in presence of insufficient antioxidant protection leads to substantial oxidative changes of lipids, proteins, sugars, and also DNA. Protection against ROS is assured by different extracellular or intracellular antioxidant mechanisms as studied during last decades. Antioxidant enzymes rectifying the oxidative damage are studied with regard to their different activities and usefulness in body protection. Their genetic polymorphisms are certainly involved in different response to oxidative stress. Special attention should be devoted to the topic of oxidative nuclear and mitochondrial DNA damage and its restoring via DNA repair process, especially base excision repair (BER). A large scale of antioxidant enzymes is involved in correction of DNA oxidative damage. Natural trend of worsened DNA repair is usually associated with aging. Other pathologies related with deficient DNA repair are susceptibility to carcinogenesis (lack of apoptosis control) or degenerative diseases. Oxidative stress is involved in the pathophysiology of diabetes mellitus (DM – oxidative stress of mainly metabolic origin) and inflammatory bowel diseases (IBD – oxidative stress of mainly inflammatory origin). In spite of confirmed OS in DM or IBD, the substantial information about the intensity of DNA repair and its possible relationship to the disease course and development of chronic complications is missing. Our pilot studies completed both in adult and pediatric patients with DM or IBD confirmed an increased oxidative stress as well as oxidative DNA damage examined with comet assay. The surprising findings were ascertained in intensity of DNA repair (analysed with modified comet assay). DNA repair process was stimulated in Type 1 diabetes adults without diabetic microvascular complications and still more in diabetic children with short disease duration. On the other hand adults with Type 2 diabetes had substantially increased oxidative DNA damage and extremely low DNA repair. This finding could be the link to increased susceptibility of Type 2 DM patients to cancerogenesis. Patients with IBD (Crohn´s disease - both children and adults) had similar tendencies in OS intensity and oxidative DNA damage and repair but less intensive. Large population studies in DM or IBD studying intensity of OS and expression of DNA repair enzymes are needed in order to get the correlation between individual repair enzymes expression and the long-term course and occurence of complications in DM or IBD.

In: Leading Edge Antioxidants Research Editor: Harold V. Panglossi, pp. 1-16

ISBN 1-60021-274-3 © 2007 Nova Science Publishers, Inc.

Chapter 1

ANTI- AND PROOXIDATIVE EFFECTS OF FLAVONOIDS Wim Wätjen, Yvonni Chovolou, Andreas Kampkötter and Regine Kahl Institute of Toxicology, Heinrich-Heine-University, Düsseldorf, Germany

ABSTRACT Flavonoids are polyphenolic compounds that occur ubiquitously in foods of plant origin. This class of compounds has become increasingly popular in terms of health protection because they possess a remarkable spectrum of biochemical and pharmacological activities. Flavonoids affect basic cell functions such as growth, differentiation and apoptosis. Epidemiological studies have suggested that flavonoids may protect against various stages of the cancer process and are associated with a reduced incidence of coronary heart disease. Flavonoids have been shown to be potent antioxidants because of their radical scavenging activity. Furthermore, flavonoids are able to complex heavy metal ions, e.g. iron and copper which are involved in Fenton-like reactions. The biological actions of flavonoids have long been thought to be due to their antioxidant potential but at present it is by no means clear whether other mechanisms of action contribute to their overall effect or are even more important than their radical scavenging properties. Although some flavonoids act as powerful antioxidants it was also shown that in high concentrations they can generate reactive oxygen species and induce apoptosis. Hydroxyl radicals generated by autoxidation and redox-cycling of the polyphenolic flavonoids may initiate peroxidation of the lipids in cellular membranes. Furthermore, flavonoids can cause an impairment of antioxidative defense systems consisting e.g. of glutathione and glutathione-S-transferase, which in this way indirectly induces oxidative stress in the cell. For a number of flavonoids cytoprotective, antioxidative and antiapoptotic as well as cytotoxic, prooxidative and proapoptotic effects have been shown in various cell culture models. Given this wide spectrum of biological actions, it is quite understandable that numerous health claims which are in part mutually exclusive have been linked with flavonoids. Notably in cancer, but also in infections or autoimmune disease, a deficiency in apoptosis is one of the key events. On the other hand, overefficient apoptosis, as observed in fulminant liver failure, or the long term accumulation of apoptotic events in neurodegenerative disorders may be equally harmful for the organism. Given that both

2

Wim Wätjen, Yvonni Chovolou, Andreas Kampkötter et al. beneficial and adverse effects can in principle be caused by flavonoids it must be assumed that – in addition to the cell type or tissue involved and to the presence or absence of a stressor - it depends on dose which action prevails.

INTRODUCTION Flavonoids are polyphenolic compounds that occur ubiquitously in fruits, vegetables, grains, nuts, tea and wine (di Carlo et al. 1999, Beecher 2003). They are low molecular weight phenylbenzopyrones, the basic structure of a O-heterocyclic ring (B) fused to an aromatic ring (C) with a third ring system (A) attached at C2 of the heterocyclic ring. Up to now, over 6000 flavonoids, low molecular weight phenylbenzopyrones, have been identified.

3´ 4´

2´

8 7

A 6

B

O

5´

C

6´

3 5 4 flavonoid basic structure

OH

OH

OH

OH

HO

HO

O

O

OH OH

OH

O

quercetin

O

luteolin

OH

HO

O

HO

O

OH OH

OH

O

OH

kaempferol

O

galangin

OH

OH OH

HO

O

OH HO

O

OH

OH

OH O

OH catechin

OH

EGCG O

OH OH

Figure 1. Chemical structures of selected flavonoids.

Anti- and Prooxidative Effects of Flavonoids

3

In different epidemiological studies a reduced incidence of coronary artery disease and risk of stroke was associated with a diet rich in flavonoid-containig vegetables and fruits (reviewed by Arts and Hollman 2005). In some of these prospective studies reductions of mortality risk caused by flavonols, flavones or catechins were found to be up to 65%. Associations between the dietary intake of flavonoids and the incidences of a variety of cancers have been studied in several prospective cohort studies and case-control studies. Significant associations were observed only for lung cancer and colorectal cancer, but not for the incidence of cancer of the stomach, urinary tract, prostate, or breast (reviewed by Arts and Hollman 2005). From many in vitro studies, flavonoids are claimed to provide protective effects against several other diseases like neurodegenerative diseases (Youdim et al. 2002), obesity and diabetes (Bhathena and Velasquez 2002), rheumatoid arthritis (Ostrakhovitch and Afanas'ev 2001), chronic obstructive pulmonary disease (Ko et al. 1999), allergic symptoms (Kimata et al. 2000, Muthian and Bright 2004) as well as inflammatory diseases (Pelzer et al. 1998). Because of these diverse potential beneficial effects, flavonoids have become increasingly popular in terms of health protection and are used in food supplements at relatively high doses. However, in epidemiological studies, no associations between the dietary intake of flavonoids and the incidences of rheumatoid arthritis, type 2 diabetes mellitus and cataracts was observed. In the Finnish Mobile Clinic Health Examination Survey a significant inverse association against asthma was observed only for asthma (Knekt et al. 2002), but other studies showed no protective effect (Garcia et al. 2005). Flavonoids have been shown to be potent antioxidants because of their radical scavenging activity. It was also shown that flavonoids are able to complex heavy metal ions, e.g. iron and copper which are involved in Fenton-like reactions (Mira et al. 2002). The biological actions of flavonoids have long been thought to be due mostly to their antioxidant potential but at present it is by no means clear whether other mechanisms of action contribute to their overall effect or are even more important than their radical scavenging properties. Flavonoids possess a remarkable spectrum of biochemical and pharmacological activities (reviewed by Middelton et al. 2000), affecting basic cell functions such as growth, differentiation and apoptosis. An induction of apoptosis by flavonoids was reported e.g. by Wei et al. (1994) and Richter et al. (1999). Several molecular targets are discussed to explain the cytotoxicity of flavonoids e.g. inhibition of enzymes like protein kinase C (Nagasaka and Nakamura 1998), tyrosine protein kinase (Srivastava 1982), 3':5'-cyclic-AMP phosphodiesterase (Picq et al. 1982), DNA topoisomerases (Boege et al. 1996, Chowdhury et al. 2002), glutathione S-transferase (van Zanden et al. 2003) leading to e.g. disturbations in cell cycle followed by apoptotic or necrotic events. Flavonoids are reported to be antagonists or weak agonists of the intracellularly located Ah-receptor (Ciolino et al. 1999, Zhang et al. 2003). Specific interactions of flavonoids with cellular receptors were shown for epigallocatechingallate (EGCG): Tachibana et al. (2004) reported that EGCG binds to the 67-kDa laminin receptor with a Kd-value of 40 nM which is claimed to be a physiologically relevant concentration. This receptor plays a role in retinal angiogenesis and a potent upregulation of this receptor was found in malignant mesothelioma cells. In A549 human lung cancer cells transfected with the gene encoding the laminin receptor, EGCG exhibited a potent growth inhibitory action. In cells transfected with the empty vector, EGCG caused no cytotoxic effect. This effect seems to be specific for EGCG since the structurally related flavonoids catechin, epicatechin, epigallocatechin and quercetin showed no effect. This receptor-mediated effect

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Wim Wätjen, Yvonni Chovolou, Andreas Kampkötter et al.

of EGCG may be an explanation for the antiangiogenic activities of this molecule (Tachibana et al. 2004).

OXIDATIVE STRESS The generation of reactive oxygene species (ROS) is a physiological process due to the oxidative metabolism of the cell. Redox-active heavy metal ions like Fe2+ and Cu2+ also lead to the generation of of ROS through Fenton-like reactions (Stohs and Bagchi 1995). Usually ROS (hydroxyl radicals, superoxide anions, hydrogen peroxide) are inactivated by different antioxidative mechanisms e.g. antioxidative enzymes like superoxide dismutases, glutathione peroxidases and catalase, or low molecular antioxidants like glutathione. If the generation of ROS exceeds the antioxidative potency, this leads to an accumulation of these molecules in the cell a phenomenon called oxidative stress (Sies 1993, 1997). An excess of these highly reactive substances can cause severe damage to lipids and cellular macromolecules like DNA and proteins. On the cellular level, oxidative stress may cause the induction of apoptotic cell death (Buttke and Sandstrom 1994) or a disturbation of intracellular signal transduction pathways (Krejsa and Schieven 1998). Oxidative stress is an important factor in many human diseases e.g. rheumatoid arthritis, neurological disturbances such as morbus Alzheimer, and chronic obstructive pulmonary disease. Oxidative stress may also contribute to the pathology of ageing, inflammation and cancer development. Antioxidants or radical scavengers play an important role in maintaining cellular redox homeostasis, therefore they are used in high concentrations e.g. in food supplements.

ANTIOXIDATIVE EFFECTS OF FLAVONOIDS Many of the biological properties of flavonoids may be related, partially at least, to their antioxidant and free-radical scavenging ability (Noroozi et al. 1998). The antioxidative and radical scavenging activity of the flavonoid molecule depends on the effectiveness of electron donation. Critical structural prerequisites for optimal antioxidant potential of flavonoids are i) presence of a catechol group in the B-ring, ii) a 2,3-double bond, and iii) 3- and 5-hydroxy groups adjacent to the 4-keto structure (Bors et al. 1990). In different other studies (e.g. determination of the trolox equivalent antioxidative capacity) correlations between distinct molecular structure elements and the antioxidative capacity of the flavonoid have been reported. In cellular systems, kaempferol inhibits the generation of superoxide anion radicals from xanthine/xanthine oxidase (Selloum et al. 2001). Luteolin contributes to the antioxidant activity of artichoke leaf extract preventing Cu2+-mediated LDL oxidation (Brown and RiceEvans, 1998). Areias et al. (2001) also showed an antioxidative effect of luteolin in cultured retinal cells measuring a decrease in MDA and also in reactive oxygen species (fluorescent probe DCF). Chemical stress-induced ROS production detected with DCF was decreased by kaempferol (Samhan-Arias et al. 2004). In the comet assay, a protection against oxidative-mediated DNA strand break formation was demonstrated e.g. for quercetin (Musonda and Chipman 1998, Duthie and Dobson 1999,

Anti- and Prooxidative Effects of Flavonoids

5

Johnson and Loo 2000, Wätjen et al. 2005), kaempferol (Noroozi et al. 1998), myricetin (Duthie and Dobson 1999, O'Brien et al. 2000), EGCG (Johnson and Loo 2000, Roy et al. 2003), luteolin (Noroozi et al. 1998, Michels et al. 2005) and rutin (O'Brien et al. 2000). Sasaki et al. (2003) demonstrated that luteolin decreased LOOH cytotoxicity in rat pheochromocytoma PC12 cells. Quercetin and fisetin showed protective effects against H2O2induced cytotoxicity in H4IIE cells (Wätjen et al. 2005). A protection against induction of apoptotic cell death by oxidative stimuli was found e.g. with quercetin (Oyama et al. 1999, Wätjen et al. 2005), epicatechin (Schroeter et al . 2001), kaempferol (Niering et al. 2005), fisetin (Wätjen et al. 2005) and rutin (Chen et al. 2001).

PROOXIDATIVE EFFECTS OF FLAVONOIDS Although some flavonoids act as powerful antioxidants it was also shown that in high concentrations they can generate reactive oxygen species and induce apoptosis (Ochiai et al. 1984, Hodnick et al. 1986, Gaspar et al. 1994, Metodiewa et al. 1999, Yoshino et al. 1999). Due to the electron-donating antioxidative action of flavonoids, the flavonoid molecule itself is oxidized forming a phenoxyradical or semiquinone radical. This flavonoid radical can be further oxidized by donation of an electron to oxygen yielding an superoxide anion radical and a flavonoid quinone. The formation of reactive species during the oxidation of the flavonoid molecule may initiate peroxidation of the lipids in cellular membranes. Furthermore, flavonoids may indirectly induce oxidative stress in the cell by causing an impairment of antioxidative defense systems, e.g. of glutathione and glutathione-S-transferase (Sahu and Gray 1996). Consequently, many reports have described adverse actions of flavonoids on a cellular level. The induction of apoptosis was described e.g. for quercetin (Richter et al. 1999, Choi et al. 2001, Wätjen et al. 2005), fisetin (Chen et al. 2002, Wätjen et al. 2005), morin (Romero et al 2002), myricetin (Kuntz et al. 1999) and rutin (Romero et al 2002). Cytotoxicity, induction of apoptosis and inhibition of cell proliferation in different cell types were caused by kaempferol (Richter et al. 1999, Wang et al 1999, Knowles et al. 2000, Nguyen et al. 2003, Wang et al. 2003, Leung et al. 2004). As further toxic effects of flavonoids in higher concentrations, the induction of DNA strand breaks was reported for quercetin (Musonda and Chipman 1998, Duthie et al. 1997), fisetin (Wätjen et al. 2005), luteolin (Michels et al. 2005), myricetin (Duthie et al. 1997) and kaempferol (Niering et al. 2005). Many flavonoids are positive in the Ames test under aerobic conditions (Nagao et al. 1981). In mice, kaempferol was positive in the micronucleus test (Sahu et al. 1981). The question whether the cytotoxic, pro-apoptotic and DNA-damaging effects of flavonoids involve the formation of reactive oxygen species is controversially discussed. Incubation of human lymphocytes with high concentrations of quercetin increased DNA strand breakage but did not induce oxidative damage to DNA bases (Duthie et al. 1997). Yamashita and Kawanishi (2000) reported that quercetin-induced 8-oxodG formation was dependent on the presence of copper(II)-ions because it was inhibited by bathocuproine, a copper-specific chelator. They suggested that quercetin induces apoptosis via oxidative stress. Luteolin-induced apoptosis in H4IIE cells is accompanied by induction of oxidative stress

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measured as increase in MDA concentration (Michels et al. 2005). Kaempferol has also been shown to exert prooxidative actions (Sahu and Gray 1994, Sahu and Gray 1996). Compared to the cytoprotective antioxidative effects of flavonoids described before, these actions are, in part antagonistic, and some health claims are mutually exclusive. Antiapoptotic actions are claimed to protect against neurodegenerative diseases while proapoptotic actions could be used for cancer chemotherapy. Given that both the antioxidant as well as the DNA-damaging and cytotoxic effects can in principle be caused by the same flavonoid it must be assumed that - in addition to the cell type or tissue involved and to the presence or absence of a stressor - it depends on dose which action prevails. It was reported that luteolin, apigenin, taxifolin and chrysin were antioxidative at low concentrations of Fe-ions, but were pro-oxidative at high concentrations of Fe-ions. Epigallocatechin was antioxidative with all metal ions (Sugihara et al. 1999). It has been demonstrated that myricetin also possesses both antioxidative properties and prooxidative properties. It is a potent anticarcinogen and antimutagen, although it has been shown to promote mutagenesis in the Ames Test (Ong and Khoo 1997). The catechol moiety present in numerous flavonoids is oxidized during the antioxidative reaction yielding a quinone. The 3,5,7-trihydroxy-4H-chromen-4-one group is another antioxidant pharmacophor in certain flavonoids. During the oxidation of quercetin, a quinone is formed from the two phenolic hydroxyl groups in ring B which occurs in four isomeric forms, the ortho quinone and three para quinone methides (Awad et al. 2000). Quinones are known to arylate cellular proteins, but beside this reaction also adducts with SH-groups may be formed. It was reported that the one-electron oxidation of quercetin by horseradish peroxidase and H2O2 in the presence of glutathione (GSH) yields two quercetin-GSH adducts, 6- glutathionyl quercetin and 8-glutathionyl quercetin (Awad et al. 2000, Boersma et al. 2000, Galati et al. 2001). The formation of GH adducts was also reported for taxifolin, luteolin, fisetin and 3,3',4'-trihydroxyflavone (Awad et al. 2002), but not for apigenin and naringenin. As a pharmacological consequence of this adduct formation, essential SH-groups of enzymes may be inactivated. Glutathione transferase P1-1 can be inhibited by quercetin, and it has been shown that this is due to the reaction of quercetin quinones or quinone methides with the cysteine 47 of the human enzyme (van Zanden et al., 2003). The formation of GSH adducts of flavonoids and the inhibition of the GSTP1-1 by flavonoids are models for potential reactions with SH-containing substances of this class of food ingredients in the cell. In general, ortho and para diols are easily oxidized to quinones, as seen with flavonoids with a catechol structure in the B-ring, e.g. quercetin, luteolin, naringenin and apigenin (Galati et al. 1999, Galati et al. 2001). However, similar to the catechol containing flavonoids, the 3,5,7-trihydroxy-4H-chromen-4-one containing flavonoids shift the damage provoked by oxidative stress from lipid peroxidation to thiol arylation. Galangin is a flavonoid without hydroxyl groups in the B-ring, but posesses a 3,5,7-trihydroxy-4H-chromen-4-one group. During the antioxidative reaction of galangin this group is also oxidized and reacts with SHgroups (Michels et al. 2004). For this adduct formation a functionality of the B-ring is not a structural requirement.

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Figure 2. Oxidative metabolism of quercetin: formation of glutathionyl adducts (modified from Galati et al. (2001)).

Metodiewa et al. (1999) demonstrated with an ESR spin-stabilization technique coupled to conventional spectrophotometry that o-semiquinone and o-quinone are the products of enzymatically catalyzed oxidative degradation of quercetin. The former radical might serve to facilitate the formation of superoxide and depletion of GSH, which could confer a specificity of its prooxidative action in situ. The observed one-electron reduction of o-quinone may enrich the semiquinone pool, thereby magnifying its effect. The intracellular oxidative degradation of quercetin was also confirmed under the controlled conditions of model monolayer cell cultures. The results are indicative of the intracellular metabolic activation of

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quercetin to o-quinone, a process which can be partially associated with the observed concentration-dependent cytotoxic effect of quercetin (Metodiewa et al. 1999). Further evidence for the pro-oxidative metabolism of quercetin in a cellular in vitro model was reported by Awad et al. (2002). They reported the formation and subsequently excretetion of glutathionyl quercetin adducts in tyrosinase-containing B16F-10 melanoma cells, expected to be representative also for peroxidase-containing mammalian cells and tissues. Yang et al. (2004) reported that it depends on the experimental conditions, whether EGCG and other polyphenolic compounds can function as either antioxidants or prooxidants. Under most cell-culture conditions, EGCG is oxidized rapidly probably due to the high oxygen tension. The autooxidation leads to the formation of radical species which may lead to glutathione oxidation and quinone species, which may dimerize or form thiol adducts. Some of the apoptotic effects caused by this polyphenol may be mediated by a formation of H2O2, because they are prevented by coincubation with catalase. The formation of H2O2 as well as hydroxyl radicals was also reported by Hayakawa et al. (2004): They showed in vitro that epigallocatechin had a higher prooxidant activity than EGCG measured as generation of H2O2, and that this activity was enhanced in the presence of Cu2+. In the presence of Cu2+ the formation of hydroxyl radicals by epigallocatechin and epicatechin but not EGCG was detected. Therefore the stability of the epicatechin molecule in the presence of Cu2+ in the incubation buffer is very low. In lymphocytes an increased formation of hydrogen peroxide, superoxide anions and TBARS by quercetin was detected (Yen et al. 2003).

METABOLITES Flavonoids are present in food predominantly in a glycosidic form, although a number of free flavonoids such as myricetin occur e.g. in red wine. The glycosides are resorbed by specific transporters or metabolized by intestinal bacteria to the deglycosylated form or further degraded to a variety of phenolic acids such as homovanillic acid (Olthoff et al. 2003). After enteral resorption, flavonoids are extensively metabolized in the liver. Yodogawa et al. (2003) demonstrated that kaempferol was glucurono- and sulfo-conjugated in cultured rat hepatocytes. In humans the predominant form of kaempferol in plasma was kaempferol-3glucuronide (DuPont et al. 2004), in the case of quercetin, quercetin-3-glucuronide, quercetin3´-O-sulfate and isorhamnetin-3-O-glucuronide are detected (Day et al. 2001). The biological effects of flavonoids may be greatly influenced in vivo by metabolism, but only few investigations have studied the influence of metabolism on the bioactivity of flavonoids (Spencer et al. 2004). The metabolic methylation of polyphenols not always leads to an inactivation of the molecule, an increase of pharmacological activity is also reported in the literature. For example Guerrero et al. (2002) examined the vasorelaxant profile of Omethylated quercetin derivates in aortic rings isolated from Wistar rats. The flavonoids inhibited the phenylephrine-induced precontraction in rat isolated aorta with the potency order quercetin 3,7-dimethylether > quercetin > quercetin 3,4',7-trimethylether > quercetin 3,3',4',7-tetramethylether. The dimethylated quercetin derivate had a greater potency for the inhibition than the basic structure quercetin. De Pascual-Teresa et al. (2004) reported that expression of cyclooxygenase-2 in human lymphocytes is inhibited by 100 nM

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3´methylquercetin-3-glucuronide. Day et al. (2000) reported an inhibition of xanthine oxidase by 250 nM 3´methylquercetin. In spite of the potent pharmacological activity of some flavonoid metabolites, the antioxidative capacity is decreased in most of the metabolic reactions by masking OH-groups (O-methylation, O-sulfatation, O-glucuronidation). Luteolin 5,3´ dimethylether as well as the tetramethylated derivate showed no antioxidative capacity in the TEAC assay while the nonmethylated flavonoid luteolin showed a strong antioxidative capacity (Michels et al. 2005b). Lemanska et al. (2004) showed that the antioxidative capacity of luteolin 3´monomethylether and luteolin 4´-monomethylether is lower than that of the nonmethylated flavonoid luteolin. But even without antioxidative capacity, luteolin 5,3´-dimethylether still exerts the same cytotoxicity than the nonmethylated parent compound (Michels et al. 2005b). Because of the extensive metabolism of flavonoids in the organism (Walle 2004) which in most cases decreases the antioxidative capacity of the non-substituated flavonoid, it has to be assumed that most of the pharmacological effects of the substances may not be mediated by simple antioxidative effects. Even if at present it is unclear which flavonoid metabolites possess pharmacological activity, interactions of these flavonoid metabolites with distinct molecular targets (signal transduction network) are discussed. If pharmacological actions of flavonoids are investigated in e.g. cell culture systems, the metabolite which reaches the tissue after metabolisation should be investigated rather than the unmetabolized flavonoid (Williamson et al. 2005). On the other hand, flavonoid metabolites e.g. flavonoid glucuronides may be deconjugated at the site of action, e.g. in endothelial cells, thus regenerating the pharmacologically active form (Wittig et al. 2000, De Santi et al. 2002).

PHARMACOKINETIC OF FLAVONOIDS The pharmacokinetics of quercetin have been studied in rats as well as in humans. Manach et al. (1997) reported that rats fed with quercetin (single meal, 0.2%) exhibited constant plasma concentrations of quercetin metabolites of approximately 50 µM for at least 16 h. Rats adapted to quercetin (0.2%) maintained plasma concentrations of approximately 100 µM (quercetin + metabolites). The authors suggested that the elimination of quercetin metabolites is low and that high plasma concentrations are easily maintained with a regular supply of quercetin in the diet. Data on pharmacokinetics in humans suggest that plasma concentrations in the low micromolar range can be detected: One study with 4 × 250 mg quercetin administered as capsules resulted in plasma concentrations of 1.5 µM (Conquer et al. 1998), however, later studies using quercetin glucosides yielded relatively higher plasma concentrations: 7 µM after quercetin-4´-O-glucoside equivalent to 100 mg quercetin (Graefe et al. 2001), 4.5 µM after 150 mg quercetin 4´-glucoside and 5 µM after 150 mg quercetin 3glucoside (Olthof et al. 2000). When administered in onions (a plant source known to contain quercetin as glucosides) the following plasma concentrations of total quercetin have been reached: 7.65 µM after ingestion of 100 mg quercetin equivalent (Graefe et al. 2001) and 4 µM after ingestion of 300 mg quercetin equivalent (de Pascual-Teresa et al. 2004). Halflifes between 10 and 30 h have been reported (Graefe et al. 2001, Olthof et al. 2000) suggesting that a steady state concentration will be built up on continuous daily intake.

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It has to be stated that all kinetic parameters measured in humans were measured in plasma, while not much information on flavonoid concentrations in different tissues is available. In a recent study by de Boer et al. (2005) the tissue concentration of quercetin and metabolites in different organs of rat were investigated (0,1%, 1% diet for 11 weeks). Blood plasma concentrations (quercetin and metabolites) of approximately 100 µM were found, the highest tissue concentrations were detected in the lung (15.3 nmol/g), testis (14.4 nmol/g) and kidney (11.6 nmol/g). In experiments with pigs (diet 500 mg/kg /day) they found that the flavonoid concentrations in liver (5.87 nmol/g) and kidney (2.51 nmol/g) were higher than the plasma concentration (1.25 µM). This result shows that the plasma concentration may not always be the appropriate parameter to estimate flavonoid concentrations due to accumulation processes in distinct organs. This has to be reminded when flavonoids are used in food supplements. These supplements are available in high dosed preparations and there is a great variety in recommended daily intake values. The daily dietary supplementation with e.g. quercetin as recommended in various internet sources is 1 – 2 g. Since flavonoids are not marketed as drugs they have up to now seldom been subjected to the stringent pharmacological and toxicological testing protocols of drug authorization. Instead, flavonoids are marketed as components of functional food and as flavonoid-containing food supplements, thus avoiding toxicological testing. The pro-oxidative actions of flavonoids in high concentrations are at the moment still far from being understood. Further work is needed to investigate the toxic potential of flavonoids in combination with one-electron-transfer reaction.

CONCLUSION Flavonoids have been shown to be potent antioxidants because of their radical scavenging activity. On the other hand, in high concentrations they can generate reactive oxygen species by autoxidation and redox-cycling. Therefore, for a number of flavonoids cytoprotective as well as cytotoxic and pro-apoptotic effects have been shown in various cell culture models. Given this wide spectrum of biological actions, it is quite understandable that numerous health claims which are in part mutually exclusive have been linked with flavonoids. Notably in cancer, but also in infections or autoimmune disease, a deficiency in apoptosis is one of the key events. On the other hand, overefficient apoptosis, as observed in fulminant liver failure, or the long term accumulation of apoptotic events in neurodegenerative disorders may be equally harmful for the organism. Given that both beneficial and adverse effects can in principle be caused by flavonoids it must be assumed that – in addition to the cell type or tissue involved and to the presence or absence of a stressor - it depends on dose which action prevails.

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Hayakawa, F; Ishizu, Y; Hoshino, N; Yamaji, A; Ando, T; Kimura, T. Prooxidative activities of tea catechins in the presence of Cu2+. Biosci Biotechnol Biochem, 2004, 68(9), 182530. Hodnick, WF; Kung, FS; Roettger, WJ; Bohmont, CW; Pardini, RS. Inhibition of mitochondrial respiration and production of toxic oxygen radicals by flavonoids. A structure-activity study. Biochem Pharmacol, 1986, 35(14), 2345-2357. Johnson, MK; Loo, G. Effects of epigallocatechin gallate and quercetin on oxidative damage to cellular DNA. Mutat Res, 2000, 459(3), 211-8. Kimata, M; Inagaki, N; Nagai, H. Effects of luteolin and other flavonoids on IgE-mediated allergic reactions. Planta Med, 2000, 66(1), 25-9. Knekt, P; Kumpulainen, J; Jarvinen, R; Rissanen, H; Heliovaara, M; Reunanen, A; Hakulinen, T; Aromaa, A. Flavonoid intake and risk of chronic diseases. Am J Clin Nutr, 2002, 76, 560-568 Knowles, LM; Zigrossi, DA; Tauber, RA; Hightower, C; Milner, JA. Flavonoids suppress androgen-independent human prostate tumor proliferation. Nutr Cancer, 2000, 38(1), 116-22. Ko, WC; Kuo, SW; Sheu, JR; Lin, CH; Tzeng, SH; Chen, CM. Relaxant effects of quercetin methyl ether derivatives in isolated guinea pig trachea and their structure-activity relationships. Planta Med, 1999, 65(3), 273-5. Krejsa, CM; Schieven, GL. Impact of oxidative stress on signal transduction control by phosphotyrosine phosphatases. Environ Health Perspect, 1998, 106(Suppl 5), 1179-84. Kuntz, S; Wenzel, U; Daniel, H. Comparative analysis of the effects of flavonoids on proliferation, cytotoxicity, and apoptosis in human colon cancer cell lines. Eur J Nutr, 1999, 38(3), 133-42. Lemanska, K; van der Woude, H; Szymusiak, H; Boersma, MG; Gliszczynska-Swiglo, A; Rietjens, IM; Tyrakowska, B. The effect of catechol O-methylation on radical scavenging characteristics of quercetin and luteolin--a mechanistic insight. Free Radic Res, 2004, 38(6), 639-47. Leung, LK; Po, LS; Lau, TY; Yuen, YM. Effect of dietary flavonols on oestrogen receptor transactivation and cell death induction. Br J Nutr, 2004, 91(6), 831-9. Manach, C; Morand, C; Demigne, C; Texier, O; Regerat, F; Remesy, C. Bioavailability of rutin and quercetin in rats. FEBS Lett, 1997, 409(1), 12-16. Metodiewa, D; Jaiswal, AK; Cenas, N; Dickancaite, E; Segura-Aguilar, J. Quercetin may act as a cytotoxic prooxidant after its metabolic activation to semiquinone and quinoidal product. Free Radic Biol Med, 1999, 26(1-2), 107-116. Michels, G; Haenen, GR; Wätjen, W; Rietjens, S; Bast, A. The thiol reactivity of the oxidation product of 3,5,7-trihydroxy-4H-chromen-4-one containing flavonoids. Toxicology Letters, 2004, 151, 105-11. Michels, G; Wätjen, W; Niering, P; Steffan, B; Tran-Thi, Q-H; Chovolou, Y; Kampkötter, A; Bast, A; Proksch, P; Kahl, R. Pro-apoptotic effects of the flavonoid luteolin in rat H4IIE cells. Toxicology, 2005a, 206, 337-348. Michels, G; Mohamed, G; Weber, N; Chovolou, Y; Kampkötter, A; Wätjen, W; Proksch, P. Effects of methylated derivates of luteolin isolated from Cyperus alopecuroides in rat H4IIE hepatoma cells. Basic and Clinical Pharmacology and Toxicology, 2005b, in press

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Richter, M; Ebermann, R; Marian, B. Quercetin-induced apoptosis in colorectal tumor cells: possible role of EGF receptor signaling. Nutr Cancer, 1999, 34(1), 88-99. Romero, I; Paez, A; Ferruelo, A; Lujan, M; Berenguer, A. Polyphenols in red wine inhibit the proliferation and induce apoptosis of LNCaP cells. BJU Int, 2002, 89(9), 950-954. Roy, M; Chakrabarty, S; Sinha, D; Bhattacharya, RK; Siddiqi, M. Anticlastogenic, antigenotoxic and apoptotic activity of epigallocatechin gallate: a green tea polyphenol. Mutat Res, 2003, 523-524, 33-41. Sahu, RK; Basu, R; Sharma, A. Genetic toxicological of some plant flavonoids by the micronucleus test. Mutat Res, 1981, 89(1), 69-74. Sahu, SC; Gray, GC. Kaempferol-induced nuclear DNA damage and lipid peroxidation. Cancer Lett, 1994, 85(2), 159-64. Sahu, SC; Gray, GC. Pro-oxidant activity of flavonoids: effects on glutathione and glutathione S-transferase in isolated rat liver nuclei. Cancer Lett, 1996, 104(2), 193-6. Samhan-Arias, AK; Martin-Romero, FJ; Gutierrez-Merino, C. Kaempferol blocks oxidative stress in cerebellar granule cells and reveals a key role for reactive oxygen species production at the plasma membrane in the commitment to apoptosis. Free Radic Biol Med, 2004, 37(1), 48-61. Sasaki, N; Toda, T; Kaneko, T; Baba, N; Matsuo, M. Protective effects of flavonoids on the cytotoxicity of linoleic acid hydroperoxide toward rat pheochromocytoma PC12 cells. Chem Biol Interact, 2003, 145(1), 101-16. Schroeter, H; Spencer, JP; Rice-Evans, C; Williams, RJ. Flavonoids protect neurons from oxidized low-density-lipoprotein-induced apoptosis involving c-Jun N-terminal kinase (JNK), c-Jun and caspase-3. Biochem J, 2001 358(Pt 3), 547-57. Selloum, L; Reichl, S; Muller, M; Sebihi, L; Arnhold, J. Effects of flavonols on the generation of superoxide anion radicals by xanthine oxidase and stimulated neutrophils. Arch Biochem Biophys, 2001, 395(1), 49-56. Sies, H. Strategies of antioxidant defense. Eur J Biochem, 1993, 215(2), 213-9. Sies, H. Oxidative stress: oxidants and antioxidants. Exp Physiol, 1997, 82(2), 291-5. Spencer, JP; Abd-el-Mohsen, MM; Rice-Evans, C. Cellular uptake and metabolism of flavonoids and their metabolites: implications for their bioactivity. Arch Biochem Biophys, 2004, 423(1), 148-61. Srivastava, AK. Inhibition of phosphorylase kinase, and tyrosine protein kinase activities by quercetin. Biochem Biophys Res Commun, 1985, 131(1), 1-5. Stohs, SJ; Bagchi, D. Oxidative mechanisms in the toxicity of metal ions. Free Radic Biol Med, 1995, 18(2), 321-36. Sugihara, N; Arakawa, T; Ohnishi, M; Furuno, K. Anti- and pro-oxidative effects of flavonoids on metal-induced lipid hydroperoxide-dependent lipid peroxidation in cultured hepatocytes loaded with alpha-linolenic acid. Free Radic Biol Med, 1999, 27(11-12), 1313-23. Tachibana, H; Koga, K; Fujimura, Y; Yamada K. A receptor for green tea polyphenol EGCG. Nat Struct Mol Biol, 2004, 11(4), 380-1. van Zanden, JJ; Ben Hamman, O; van Iersel, ML; Boeren, S; Cnubben, NH; Lo, Bello M; Vervoort, J; van Bladeren, PJ; Rietjens, IM. Inhibition of human glutathione Stransferase P1-1 by the flavonoid quercetin. Chem Biol Interact, 2003, 145(2), 139-48. Wätjen, W; Michels, G; Steffan, B; Niering, P; Chovolou, Y; Kampkötter, A; Tran-Thi, Q-H; Proksch, P; Kahl, R. Low concentrations of flavonoids are protective in rat H4IIE cells

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In: Leading Edge Antioxidants Research Editor: Harold V. Panglossi, pp. 17-34

ISBN 1-60021-274-3 © 2007 Nova Science Publishers, Inc.

Chapter 2

COMPARISON OF ANTIOXIDATIVE PROPERTIES OF GREEN AND BLACK TEA Agnieszka Augustyniak, Justyna Ostrowska, Wojciech Luczaj and Elzbieta Skrzydlewska* Department of Analytical Chemistry, Medical University of Białystok, 15-230 Białystok, P.O. Box. 14, Poland

ABSTRACT Green tea has been proved to possess antioxidative properties whereas black tea antioxidant properties in vivo have been questioned, considering its components. The aim of this study has been to compare the antioxidative properties of black and green tea manifested by their protective action on the liver, brain and serum antioxidants of two months old rats chronically (4 weeks) intoxicated with ethanol. In order to estimate the intensity of black and green tea action the activity of antioxidant enzymes – superoxide dismutase, catalase, glutathione peroxidase and reductase as well as GSH level were measured by spectrophotometric methods while the levels of vitamin C and E were measured by HPLC methods. Teas antioxidant efficiency was evaluated by lipid peroxidation process intensity estimated as TBARS level. It has been shown that ethanol caused decrease in the activity/level of the examined antioxidants in the liver, brain and serum except glutathione reductase whose activity increased in the liver and serum. Disturbances in antioxidant abilities led to oxidative stress formation manifested by the rice of the level of lipid peroxidation products. Green tea as well as black tea partially prevented changes in the activity/level of antioxidants of all examined tissues caused by ethanol intoxication and significantly protected phospholipids against oxidative modifications. Green tea as well as black tea given to rats receiving alcohol caused significant increase in the activity of all examined enzymes in the brain. Moreover green tea enhanced the activity of superoxide dismutase, glutathione peroxidase and catalase, while black tea increased only glutathione peroxidase and catalase activities in the liver. Serum of alcohol intoxicated and drinking tea rats was characterized by increase in the *

Corresponding author: Elzbieta Skrzydlewska, Department of Analytical Chemistry, Medical University, 15-230 Białystok, P.O. Box. 14, Poland, /fax 48 85 7485707, e-mail: [email protected]

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Agnieszka Augustyniak, Justyna Ostrowska, Wojciech Luczaj et al. activity of superoxide dismutase after green tea and in superoxide dismutase and glutathione peroxidase after black tea. Both teas caused significant increased in the liver, brain and serum levels of GSH, vitamin C and E in comparison to alcohol group. However green tea action was a little more effective than that of black tea. In consequence both teas significantly protected phospholipids against oxidative modifications caused by ethanol. The above results clearly indicate beneficial effect of green as well as black tea on antioxidant system and prevention against oxidative stress formation after ethanol intoxication.

Keywords: green tea, black tea, polyphenols, antioxidants, ethanol, oxidative stress

INTRODUCTION Cellular metabolism of all aerobic organisms is characterized by continuous generation of free radicals. Intracellular free radicals generation results from enzymatic reactions and oneelectron oxidation of reduced form of endo- and exogenous compounds. Because free radicals are very reactive, all cells possess antioxidant system preventing free radicals generation and their reactions with cellular components. It is known that there is a balance between oxidative and antioxidative abilities in the physiological conditions. However even small changes in oxidant or/and antioxidant conditions may disturb this balance. It has been shown that chronic ethanol intoxication enhanced reactive oxygen species generation mainly by superoxide radical and hydrogen peroxide production [1,2]. Enhanced superoxide radical generation is caused by increase in the level of NADH formed during ethanol as well as its metabolite – acetaldehyde oxidation [3]. Decrease in NAD/NADH ratio activates conversion of xanthine dehydrogenase into xanthine oxidase – enzyme responsible for superoxide radical generation [4]. Additionally chronic alcohol intoxication is accompanied by decrease in the activity of ethanol metabolizing enzymes – alcohol and aldehyde dehydrogenase which results in an increase in acetaldehyde accumulation [5]. In such a situation xanthine oxidase may catalyze superoxide radical generation employing acetaldehyde as a substrate [6]. Increase in NADH concentration is also responsible for enhanced release of iron ions (II) from ferritin [7]. Increase in free iron (II) ions that catalyze free radical reactions leads to rise in the levels of reactive oxygen species observed in ethanol intoxication. In addition increased generation of oxygen- and ethanol-derived free radicals has been observed at the microsomal level especially through the intervention of the ethanol-inducible cytochrome P450 isoform [8]. Decrease in antioxidant status found during ethanol intoxication additionally indicates presence of oxidative stress [9]. The oxidative stress may be counteracted by improving antioxidative abilities of the organism, among others by application of antioxidative exogenous substances. More attention has been recently paid to taking advantage of natural antioxidants found in the fruit, vegetables or beverages (supplied to the organism with food) [10,11]. Tea, together with water being popular drink in the world, is one of the beverages characterized by these properties. Tea leaves as well as its brew are known to possess high amounts of polyphenols, mainly catechins [12]. Many properties of tea catechins including antioxidative, anticarcinogenic and hypolipidemic in vitro and in vivo have been revealed [13-16]. Antioxidant effect of green tea has also been confirmed in different oxidative conditions

Comparison of Antioxidative Properties of Green and Black Tea

19

[17,18]. In addition, green tea consumption is associated with beneficial effects on human health [19]. There is a great difference in the phenolic composition of green and black tea due to the fermentation process. Green tea is rich in catechins, whereas black tea is characterized by amount of condensation and oxidation products such as the thearubigins and the theaflavins [20]. These two phenolic fractions make up the majority of phenolic fractions found in black tea [21]. Although questioned in the past, the antioxidant properties of these black tea constituents have been recently proved [22-24]. For this reason, as well as because black tea is consumed in 4 times larger quantities than green tea, an attempt has been made to compare antioxidative properties of black and green tea in oxidative stress conditions caused by chronic ethanol intoxication.

MATERIALS AND METHODS Green tea and black tea - Camellia sinensis (Linnaeus) O. Kuntze (standard research blends - lyophilised extract) were provided by TJ Lipton (Englewood Cliffs, NJ) and were dissolved in the drinking water at a concentration of 3 g/l. The two kinds of tea were prepared freshly three times aweek and stored at 40C until used. The content of drinking vessels was renewed every day. Green tea extract contained epigallocatechin gallate (554 mg/g dried extract), epigallocatechin (82mg/g dried extract), epicatechin (90 mg/g dried extract), epicatechin gallate (86 mg/g dried extract) determined by the HPLC method [25]. Black tea extract contained epigallocatechin gallate (4,84 mg/g dried extract), epigallocatechin (0,74 mg/g dried extract), epicatechin (0,94 mg/g dried extract), theaflavin (TF1) (28,32mg/g dried extract), theaflavin 3-gallate (TF2A) (50,88 mg/g dried extract), theaflavin 3’-gallate (TF2B) (26,72 mg/g dried extract) and theaflavin 3,3’-gallate (TF3) (50,24 mg/g dried extract) determined by the modified HPLC method of Mattila [26].

Animals 2 months old male Wistar rats (n=48) were used for the experiment. They were housed in groups with free access to a granular standard diet and water and maintained under a normal light-dark cycle. All experiments were approved by the Local Ethic Committee in Białystok (Poland) respecting the Polish Act Protecting Animals of 1997. The animals were divided into the following groups: Normal control group (n=12) was treated intragastrically with 1.8 ml of physiological saline every day for 4 weeks. Alcohol control group (n=12) was treated intragastrically with 1.8 ml of ethanol in doses from 2.0 to 6.0 g/kg b.w. every day for 4 weeks. The dose of ethanol was gradually increased by 0.5 g/kg b.w. every three days. Tea group (n=12) including green tea group (n=6) and black tea group (n=6) has been given tea solution ad libitum instead of water for one week. Next it was treated intragastrically with 1.8 ml of physiological saline and received green or black tea solutions ad libitum instead of water every day for 4 weeks.

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Agnieszka Augustyniak, Justyna Ostrowska, Wojciech Luczaj et al.

Alcohol and tea group (n=12) has been given green tea (n=6) or black tea (n=6) solution ad libitum instead of water for one week. Next it was treated intragastrically with 1.8 ml of ethanol in doses from 2.0 to 6.0 g/kg b.w. and received tea solutions ad libitum instead of water every day for 4 weeks.

Tissues Preparation At the next stage of the experiment the rats were sacrified under ether anaesthesia (six animals in each group). Blood was collected by cardiac puncture. Livers and brains were removed quickly and placed in iced 0.15 M NaCl solution, perfused with the same solution to remove blood cells, blotted on filter paper, weighed and homogenized in 9 ml ice-cold 0.25 M sucrose and 0.15 M NaCl with the addition of 6 μl 250 mM BHT (butylated hydroxytoluene) in ethanol to prevent the formation of new peroxides during the assay. Homogenization procedure was performed under standardized conditions; 10% homogenates were centrifuged at 10.000 x g for 15 min at 4°C, and the supernatant was kept on ice until assayed. In the homogenates of liver and brain and in blood serum the levels of biologically active black and green tea components were measured by HPLC method with amperometric detection [27,28].

Biochemical Assays Cu,Zn-SOD (EC.1.15.1.1) activity was determined by measuring the activity of cell cytosolic SOD [29,30]. Mn-SOD of the liver mitochondria is known to be destroyed during this procedure. A standard curve for SOD activity was obtained using SOD from bovine erythrocytes (Sigma Biochemicals St. Louis MO). One unit of SOD was defined as the amount of the enzyme, which inhibits oxidation of epinephrine to adrenochrome by 50%. The enzyme activity was expressed in units per mg of protein. Catalase (EC.1.11.1.9) activity was determined after 30 min preincubation of the postmitochondrial fraction of the liver homogenate with 1% Triton X-100 by measuring the decrease in absorbance of hydrogen peroxide at 240 nm [31]. The rate was determined at 25oC using 10 mM hydrogen peroxide and the activity was expressed in units per mg of protein. Glutathione peroxidase (EC.1.11.1.6) activity was measured in the liver spectrophotometrically [32]. This technique allowed to assay GSSG formation through measuring the conversion of nicotinamide adenine dinucleotide phosphate (NADPH) to NADP. One unit of activity was defined as the amount of enzyme, which catalyzes the conversion of 1 μmol of NADPH/min, at 25oC and pH 7,4. The enzyme activity was expressed in units per mg of protein. Glutathione reductase (EC.1.6.4.2) activity was measured by monitoring the oxidation of nicotinamide adenine dinucleotide phosphate (NADPH) at 340 nm [33]. One unit of activity was defined as the amount of enzyme, which catalyzes the conversion of 1 μmol of NADPH/min, at 25oC and pH 7,4. The enzyme activity was expressed in units per mg of protein.

Comparison of Antioxidative Properties of Green and Black Tea

21

Glutathione (GSH) concentration was measured by means of Bioxytech GSH-400 test. The method is applied in two steps. The first step leads to the formation of substitution products between a patented reagent and all mercaptans (RSH) which are present in the sample. In the second step, the substitution products obtained with GSH are specifically transformed into a chromophoric thione whose maximal absorbance wavelength is 400nm. The HPLC methods were used to determine the level of vitamin C [34] and vitamin E [35]. To measure ascorbic acid level 300μl of liver homogenate was mixed with an equal volume of metaphosphoric acid (100g/l) to precipitate of protein. Before HPLC analysis, samples were centrifuged (3500xg, 4 min) to remove precipitated protein and next immediately assayed. Then the vitamin E was extracted from the liver homogenate with hexane containing 0,025% butylated hydroxytoluene. The hexane phase was removed and dried with sodium sulfate, and 50μl of the hexane extract was injected on the column. The extent of lipid peroxidation in the liver, brain and serum was assayed with thiobarbituric acid (TBA). Chromogenous condensation product of TBA with malondialdehyde (thiobarbituric acid-reactive substances - TBA-rs) was extracted from aqueos phase into butanol and then an absorption at 500, 530 and 560 nm was monitored [36].

Statistical Analysis The data obtained in this study are expressed as mean ± SD. They were analysed by means of standard statistical analyses, one way ANOVA with Scheffe’s F test for multiple comparisons to determine significance between different groups. The values for p<0.05 were considered significant.

RESULTS The levels of biologically active components of black and green tea were detectable and were determined in the examined tissues of rats receiving teas alone or with alcohol (Fig. 1). These compounds were not detectable in tissues of control rats. EGC was found in the liver of rats drinking black tea, in the highest amount, while the level of EGCG was only slightly lower. However the level of EGCG was in the highest concentration in the liver of rats drinking green tea, while the level of EGC was only slightly lower. The EC level was significantly lower than that of the above compounds after drinking of black as well as green tea. The examination of liver homogenates from rats drinking black tea shown that the level of teaflavins was significantly lower than the level of catechins. Among teaflavins TF1 occurs in the highest amounts, while about twice lower were the concentrations of TF2A, TF2B and TF3 and their levels were similar. Alcohol intoxication caused the decrease in the levels of catechins and teaflavins in the liver of rats drinking black or green tea. The concentrations of EC and EGC in the liver of rats drinking alcohol and black tea were only shortly decreased (by about 5% and 10 % respectively), while the level of EGCG was similar to that in the liver of rats receiving only black tea.

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Agnieszka Augustyniak, Justyna Ostrowska, Wojciech Luczaj et al.

Liver

Brain

Blood serum Figure 1. The level of catechins and teaflavins in the liver, brain, blood serum of 2-months old rats drinking black/green tea and rats chronically intoxicated with ethanol and drinking black/green tea Significantly different (p<0,05): a – from normal control group.

Comparison of Antioxidative Properties of Green and Black Tea

23

The levels of teaflavins (TF1, TF2A, TF2B and TF3) in the liver of animals drinking alcohol and black tea were decreased by about 10%, 13%, 13% and 12% respectively. However the levels of EC, EGC and EGCG in rats drinking alcohol and drinking green tea were decreased by about 9%, 7% and 5%, respectively in comparison with the level in the liver of animals drinking green tea alone. The levels of EGC and EGCG in the brain of rats drinking black tea are similar while the level of EC is about ten times lower. The levels of teaflavins were not measured. Their levels were below the limit of detection (<10 ng/g tissue). The highest level was found for EGCG, a little lower for EGC and significantly lower for EC in the brain of green tea group rats. The levels of EC, EGC and EGCG in the brain of rats drinking black tea were slightly decreased after alcohol intoxication - by about 5%, 7% and 3% respectively. The level of catechins in the brain of animals drinking alcohol and green tea was decreased by about 5%, 8% and 10% for EC, EGC and EGCG respectively. EGCG is catechin that occurs in the highest amount in blood serum of black tea group of rats, while the level of EGC is twice lower and the concentration of EC is the lowest. TF2A occurs in the highest amounts mong teaflavins. The concentrations of TF1, TF2B and TF3 are about twice lower and they are at the similar level. The level of EGCG in the blood serum of rat drinking green tea is also the highest, while the amount of EGC is about twice lower and the level of EC is the lowest. The amount of catechins was significantly decreased in the blood serum of rats receiving alcohol and black or green tea in comparison with their levels in the blood serum of animals that received tea alone. The levels of EC, EGC and EGCG are significantly decreased - by about 21%, 15% and 19%, respectively in comparison to rats drinking black tea and by about 20%, 15% and 19%, respectively in comparison to rats drinking green tea. The levels of teaflavins in the blood serum of studies animals drinking alcohol and black tea were below detection limit (<10 ng/g tissue). Black and green tea consumption caused changes in the activities and the levels of antioxidants in the liver (Table 1). Administration of black tea was the reason of increase in the activities of the following antioxidant enzymes: GSH-Px (by 35%), GSSG-R (by 19%), and CAT (by 23%) and in the level of GSH (by 13%) in comparison with the normal control. However the decrease in the activity of Cu,Zn-SOD (by 9%) and in the level of vitamin C (by 4%) and E (by 8%) was observed after black tea administration. Green tea caused higher than black tea increase in the activities of all examined antioxidant enzymes except catalase, whose activity was decreased (by 5%) in comparison with normal control group. Moreover the levels of vitamin C and E were significantly higher after consumption of green than black tea. The level of GSH was also enhanced more by green (by 17%) than black tea (by 13%). Ethanol intoxication caused decrease in antioxidant enzymes activities and in the levels of nonenzymatic antioxidants of rats liver (Table 1). The activities of Cu,Zn-SOD and CAT were decreased by about 10%, while that of GSH-Px by about 34% in comparison with normal control group. However GSSG-R activity was increased by about 68%. The level of liver GSH was diminished only by about 11%, while that of vitamin C and E by about 30 and 40%, respectively. Administration of alcohol to rats receiving black tea caused increase in CAT (9%) and GSH-Px (87%) activities and decrease in the activities of Cu,Zn-SOD (10%) and GSSG-R (32%) in comparison with alcohol control group. However alcohol administration in case of green tea rats was the reason of increase in the activities of CAT, GSH-Px and Cu,Zn-SOD by about 6%, 21% and 20% respectively and led to decrease in GSSG-R activity (by 44%) in comparison with alcohol control group. After alcohol intoxication of black and green tea rats the levels of GSH, vitamin C and E in black and green

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Agnieszka Augustyniak, Justyna Ostrowska, Wojciech Luczaj et al.

tea rats were higher than in alcohol control group. Green tea causes significantly higher changes than black tea. Table 1. Antioxidative parameters in the liver of 2-months old rats chronically intoxicated with ethanol and rats drinking black or green tea and chronically intoxicated with ethanol Analyzed parameter

Group

Normal Control

Alcohol Control

Tea

Alcohol + tea

102 ± 6 91% 121 ± 6a,x 108% 20.5 ± 1.4 a 135% 22.5 ± 1.7a 148% 6.86 ± 0.23a 119% 7.70 ± 0.50a,x 134% 6.27 ± 0.33 a 123% 4.87 ± 0.30a,x 95% 0.98 ± 0.05 a 113% 1.02 ± 0.05a 117% 1.54 ± 0.07a 96% 1.66 ± 0.07x 103% 24.2 ± 1.5 a 92% ± 1.5x 101% a

Cu,Zn–SOD Black tea (U/mg protein) Green tea GSH-Px (U/mg protein)

Black tea

GSSG-R (U/mg protein)

Black tea

CAT (U/mg protein)

Black tea

GSH (μmol/g tissue)

Black tea

Vitamin C (μmol/g tissue)

Black tea

Vitamin E (nmol/g tissue)

Black tea

Green tea

Green tea

Green tea

Green tea

Green tea

Green tea

a

112±5 100%

99±4 88%

15,2±1.1 100%

10,0±1a 64%

5.76±0.27 100%

9,65±0,54a 168%

5.12±0.28 100%

4,62±0,33a 90%

0.87±0.04 100%

0,77±0,04a 89%

1.61±0.07 100%

1,15±0,06 a 71%

26.2±1.3 100%

15,4±1,3 a 59%

89 ± 6a,b,c 79% 119 ± 7a,c,x 106% 18.7 ± 0.7 a, b, c 123% 12.1 ± 0.7a,b,c,x 80% 6.58 ± 0.35a,c 114% 5.40 ± 0.40ab,c,x 94% 5.04 ± 0.37 b,c 98% 4.90 ± 0.30 96% 0.89 ± 0.05 a, c 102% 0.97 ± 0.05a,c,x 112% 1.40 ± 0.08 c 87% 1.53 ± 0.08a,b,c,x 95% 23.4 ± 1.3 a ,c 89% 19.5 ± 1.3a,b,c,x 74%

Data points represent mean ± SD; n=12 for normal control and alcohol control groups and n=6 for teas and alcohol + tea groups; (ap<0.05 in comparison with normal control group; b p<0.05 in comparison with black or green tea group; cp<0,05 in comparison with alcohol control group; x p<0.05 in comparison with black tea group).

Tea consumption influenced also the activity/level of brain antioxidants. Black tea administration caused increase in antioxidant enzymes activities in comparison with normal control group as follows: slight in GSH-Px (by 3%), and GSSG-R (by 7%) and significant (by 35%) in CAT (Table 2). At the same time Cu, Zn-SOD activity was decreased (by 7%). However green tea caused more significant changes than black tea in the activities of the above enzymes. Drinking green tea led to increase in the activities of GSSG-R (by 14%) and CAT (4%) and to significant decrease in the activities of Cu,Zn-SOD (25%) and GSH –Px

Comparison of Antioxidative Properties of Green and Black Tea

25

(20%). Nevertheless black tea as well as green tea did not significantly change the level of nonenzymatic antioxidants. Their levels were similar to normal control group. Table 2. Antioxidative parameters in the brain of 2-months old rats chronically intoxicated with ethanol and rats drinking black or green tea and chronically intoxicated with ethanol Analyzed parameter

Group

Black tea Cu,Zn– SOD (U/mg protein) Green tea GSH-Px (U/mg protein) GSSG-R (U/mg protein)

CAT (U/mg protein)

GSH (mmol/g tissue)

Vitamin C (μmol/g tissue)

Vitamin E (nmol/g tissue)

Black tea Green tea Black tea

Normal Control

Alcohol Control

Tea

69.8±3.1 100%

24,1±2,4 a 35%

31.5±1.6 100%

a

17,4±1,3 55%

64.8 ± 3.2 93%

12.65±0.64 10,33±0,93 a 82% 100%

Green tea Black tea

Green tea

54.2 ± 4.1 a,c,x 78%

32.5 ± 1.2a 103% 25.2 ± 1.9 a,x 80% 13.54 ± 0.86a 107% 14.37 ± 1.01 a,x 114%

26.4 ± 1.4 a, b, c 84% 27.0 ± 1.8 a,c 86% 12.23 ± 0.98 b,c 97% 12.34 ± 0.71 b,c 98%

0.35 ± 0.03 a 135% 0.27 ± 0.02x 104%

0.25 ± 0.02 a,b, c 96% 0.23 ± 0.02 a,b,c 89%

0.41±0.02 100%

0,31±0,03 a 76%

0.43 ± 0.02 105% 0.41 ± 0.03 100%

0.37 ± 0.03 a, b, c 90% 0.37 ± 0.03 a,b,c 90%

1.92 ± 0.09 104% 1.75 ± 0.11 95%

1.67 ± 0.3 c 91% 1.75 ± 0.12 c 95%

27.2 ± 1.4 10% 26.9 ± 1.9 100%

24.1 ± 1.7 a,b c 91% 25.9 ± 1.8 c 97%

1.84±0.1 100%

1,37±0,11 74%

Green tea Black tea

52.3 ± 3.0 a,x 75%

0,20±0,03 a 77%

Green tea Black tea

52.6 ± 3.7 a, b, c 75%

0.26±0.02 100%

Green tea Black tea

Alcohol + tea a

26.6±1.66 100%

21,2±1,7 80%

a

a

Data points represent mean ± SD; n=12 for normal control and alcohol control groups and n=6 for teas and alcohol + tea groups; (ap<0.05 in comparison with normal control group; b p<0.05 in comparison with black or green tea group; cp<0,05 in comparison with alcohol control group; x p<0.05 in comparison with black tea group).

Ethanol administration caused decrease in the activities all examined enzymes – Cu,ZnSOD by about 65%, GSH-Px by about 45%, GSSG-R by about 20% and CAT by about 30% in comparison to normal control group. The levels of nonenzymatic parameters were also

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decreased – GSH and vitamin C – by about 25% and vitamin E by 20%. Administration of ethanol together with black or green tea partially prevented changes caused by alcohol, but green tea acted in most cases more intensively than black tea. In rats drinking black tea activities of all examined enzymes were significantly increased in comparison with alcohol control group (Cu,Zn-SOD – by 118%, GSH-Px by 52%, GSSG-R – by 18% and CAT – by 25%). The activities of examined enzymes in the brain of rats receiving alcohol and green tea were significantly higher in comparison to alcohol control group – the activity of Cu,Zn-SOD was higher by 125%, GSH Px – by 55%, GSSG-R - by 20% and CAT - by 15%. The levels of the examined nonenzymtaic antioxidants were also increased in comparison to alcohol control group after tea administration. Black tea enhanced level of GSH by 19%, vitamin C by 22% and vitamin E by 14%, while green tea causde increase in the levels of these parameters as follows: GSH - by 19%, vitamin C – by 28% and vitamin E – by 22%. Black as well as green tea change antioxidant characteristics of the blood serum. Black tea, given to healthy rats, caused increase in the activities of Cu,Zn- SOD (by 34%) and GSHPx (by 11%) and decrease in the activity of GSSG-R (by 11%) (Table 3). Similarly administration of green tea caused increase in Cu,Zn-SOD activity (by 34%) and decrease in GSSG-R activities (by 11%). Green tea unlike to black tea, caused also significant decrease in GSH-Px activity (by 43%). However the levels of non-enzymatic antioxidants were only slightly changed in both cases - after black as well as green tea administration. Black tea caused increase only in the level of GSH (by 6%), while the levels of vitamins were decreased (vitamin E – by 6% and vitamin C by 1%). After green tea administration the levels of GSH and vitamin E were rised by 3% and 13% respectively, while the level of vitamin C was decreased by 2%. The influence of ethanol intoxication on blood serum antioxidant system was smaller than on the other examined tissues (Table 3). Cu,Zn-SOD and GSSG-R activities were enhanced by 2% and 44%, respectively, while GSH-Px activity was decreased (by 9%) in comparison to normal control group. The levels of nonenzymatic antioxidants were diminished after ethanol administration as follows: vitamins by 10-20% and GSH by more than 30%. Administration of alcohol and black tea together caused slight increase in Cu,Zn-SOD (by 5%) and GSH-Px (by 3%) activities and significant decrease in the activity of GSSG-R (by 25%) in comparison with alcohol control group. However administration of alcohol to rats receiving green tea caused increase in the activity of Cu,Zn-SOD (by 14%) and led to decrease in GSH-Px (by 3%) and GSSG-R (by 35%) activities in comparison with alcohol control group. After alcohol administration to black tea group, the levels of GSH, vitamin C and E were increased – by 48%, 15% and less than 1%, respectively, while to green tea rats, increase of these parameters was as follows: GSH, vitamin C and E by 8%, 27% and 24%, respectively. In the alcohol and tea groups of rats, the 2 kinds of tea significantly protected antioxidant parameters against changes caused by alcohol. Generally the intensity of the protective action of black and green tea was similar.

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Table 3. Antioxidative parameters in the blood serum of 2-months old rats chronically intoxicated with ethanol and rats drinking black or green tea and chronically intoxicated with ethanol Analyzed parameter

Group

Normal Control

Alcohol Control

Tea

Alcohol + tea

0.83 ± 0.03 134% 0.81 ± 0.03 134% 11.09 ± 0.25 a 111% 5.70 ± 0.25a,x 57% 0.40 ± 0.02a 89% 0.40 ± 0.02a 89% 15.6 ± 0.7a 106% 14.9 ± 0.5 103% 55.8 ± 2.9 99% 53.6 ± 2.9 98% 13.4 ± 0.6a 94% 16.0 ± 0.6a,x 113% a

Cu,Zn–SOD (U/ml)

GSH-Px (U/ml)

GSSG-R (U/ml)

GSH (nmol/ml)

Vitamin C (nmol/ml)

Vitamin E (nmol/ml)

Black tea Green tea Black tea Green tea Black tea Green tea Black tea Green tea Black tea Green tea Black tea Green tea

0.62±0.2 100%

0,63±0,04 102%

10.2±0.35 100%

9,38±0,56 a 91%

0.36±0.02 100%

0,52±0,03 a 144%

14.7±0.6 100%

9,87±0,8 a 67%

55±2.1 100%

43,7±2,9 a 79%

14.2±0.5 100%

12,4±0,7 a 87%

0.66 ± 0.03 a,b, c 106% 0.72 ± 0.03a,b,c,x 116% 9.66 ± 0.59 b 96% 9.08 ± 0.59a,b 91% 0.39 ± 0.03 a,c 92% 0.34 ± 0.02b,c,x 94% 14.6 ± 0.8 b, c 99% 10.62 ± 0.7a,b,c,x 72% 50.2 ± 3.0 a, b, c 91% 55.3 ± 3.0c 100% 12.6 ± 0.7 ac 89% 15.4 ± 0.7 a,c,x 108%

Data points represent mean ± SD; n=12 for normal control and alcohol control groups and n=6 for teas and alcohol + tea groups; (ap<0.05 in comparison with normal control group; b p<0.05 in comparison with black or green tea group; cp<0,05 in comparison with alcohol control group; x p<0.05 in comparison with black tea group).

Black and green tea given to healthy rats caused significant inhibition of the lipid peroxidation processes in all examined tissues (Fig. 2). Decrease in the levels of lipid peroxidation products measured as thiobarbituric reactive substances (TBA-rs) was observed after black tea administration – in the liver by about 35%, in the brain by about 20% and in the blood serum by about 25%. Green tea given to rats caused also decrease in the level of TBA-rs in all examined tissues – by 42% in the liver, by 25% in the brain and by 6% in the blood serum. Changes in the activities and level of antioxidants during ethanol intoxication lead to oxidative stress formation and in consequence to lipid peroxidation. The level of TBA-rs was significantly raised in all examined tissues – in the liver by about 54%, in the brain by about 48% and in the blood serum by about 20% in comparison to normal control group. Administration of alcohol to rats drinking black tea significantly reduced lipid peroxidation caused by ethanol. The level of TBA-rs was decreased by 37% in the liver, by 26% in the brain and by 8% in the blood serum in comparison to alcohol control group. However green tea caused decrease in the level of TBA-rs in the liver, brain and blood serum – by 63%, 45%

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Agnieszka Augustyniak, Justyna Ostrowska, Wojciech Luczaj et al.

and 15% respectively in comparison to alcohol control group. Protective action of green tea against lipid peroxidation was significantly more effective in comparison with black tea.

Figure 2. The level of TBA-rs in the liver, brain, blood serum of 2-months old rats drinking black/green tea, chronically intoxicated with ethanol and rats chronically intoxicated with ethanol and drinking black/green tea Significantly different (p<0,05): a – from normal control group value; b – from black/green tea group value c- from alcohol control group value.

Comparison of Antioxidative Properties of Green and Black Tea

29

DISCUSSION Unlike well examined and described properties of green tea, antioxidative abilities of black tea have not been proved yet. Black tea has been regarded, due to its small content of monomeric polyphenols; to have significantly weaker antioxidative properties is comparison to green tea. Taking into consideration the popularity of black tea, researches on its biological properties of it are carried out more often. In order to compare the antioxidative abilities of black and green tea in “in vivo” conditions, these two different kinds of tea have been administered to rats chronically intoxicated with ethanol. It has been revealed that during ethanol metabolism free radicals are generated [37,38]. This plays a major role in ethanol-induced oxidative stress, which may be additionally enhanced by depletion in antioxidant defense system and in consequence by an imbalance between oxidants and antioxidants. Results of this paper have confirmed that chronic ethanol intoxication causes significant changes in antioxidant parameters in rat tissues such as liver, brain and blood serum. The decrease in the activities of superoxide dismutase, catalase and glutathione peroxidase is especially important because it may lead to break down of antioxidant barrier. The decrease in superoxide dismutase and catalase after chronic alcohol consumption was also observed by other authors [9]. Moreover, the data about increase in liver catalase and glutathione reductase activity were also presented [39,40]. The decrease in the antioxidant enzymes activity during chronic ethanol intoxication may be caused by many factors, e.g. by inhibition of protein molecules biosynthesis, which was earlier observed in ethanol intoxication [41]. Moreover ethanol metabolism is accompanied by generation of very reactive metabolites i.e. acetaldehyde and free radicals that can readily react with amino and sulphydryl groups of protein molecules [42-44]. These reactions lead to changes in the structure and function of protein molecules, including enzymes [45]. Similar reactions have been shown between final lipid peroxidation products – malondialdehyde and 4hydroxynonenal and proteins, especially with glutathione peroxidase [46], which can be confirmed by significant increase in membrane lipid peroxidation products after ethanol exposure also observed in the present study. Increase in free radicals generation and changes in antioxidant abilities lead to oxidative stress, which appeared in enhanced level of products of lipid oxidative modifications measured in this paper as thiobarbituric reactive substances. In order to prevent oxidative stress formation black tea or green tea was given to ethanol intoxicated rats. Both kinds of tea contain components possessing antioxidant properties. The main compounds of green tea responsible for its antioxidant properties are monomeric polyphenols – catechins [17]. Catechins as well as green tea antioxidant properties have been extensively examined [18,47]. However, antioxidant abilities of black tea are less connected with catechins, but more with products of their oxidation and di- or polymerization named teaflavins and tearubugins. Recent examinations have proved that multimeric polyphenols– theaflavins and thearubigins possess even stronger antioxidant abilities than their precursors – catechins [20]. After drinking tea, catechins as well as teaflavins are absorbed from gastrointestinal tract and distributed into different tissues [20]. Their presence in the liver, brain and blood serum has also been proved in this study. The present study has revealed that black tea as well as green tea enhanced antioxidant properties of the liver and brain. Similar results were observed in 12 months rats in our

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Agnieszka Augustyniak, Justyna Ostrowska, Wojciech Luczaj et al.

previous experiment [48]. Activities of glutathione peroxidase, reductase and catalase as well as levels of non-enzymatic antioxidants were significantly higher after black tea administration to intoxicated rats in comparison to ethanol intoxicated rats. Moreover, black and green tea protected these tissues against consequences of oxidative stress to similar degree. It has been proved that teaflavins, especially – TF3 possess similarly to catechins ability to prevent oxygen radicals formation, via inhibiting activity of xanthine oxidase enzyme participating in superoxide anion generation [49]. Moreover, teaflavins have been shown to inhibit activities of synthase nitric oxide, cyclooxygenase and lipoxygenases – enzymes participating in free radicals process propagation [50,51]. Moreover, teaflavins as well as catechins possess lower reduction potential than oxygen free radicals and they can scavenge these radicals. It has been proved that teaflavins – TF3 and to a smaller degree TF2 and TF1 are able to scavenge the superoxide anion, singlet oxygen, hydroxyl radical and they react with radicals 10 times faster than the strongest antioxidant among catechins - EGCG [49,52,53]. Long-term ethanol exposure leads to increase in the concentration of free iron ions that catalyze the Haber-Weiss reaction and hydroxyl radical generation and as a consequence to increase in lipid peroxidation [54]. Catechins as well as teaflavins may diminish prooxidative action of transition metal ions by formation of chelate compounds [24,55]. Moreover teaflavins cause decrease in iron absorption from digestive tract [56]. In consequence low level of iron ions causes the diminution of free radicals generation and reactions with integral compounds of cells. Such a situation is crucial especially for proper function of the brain tissue whose high ratio of membrane surface area of brain to cytoplasmic volume and high content of fatty acids, contribute to the accumulation of lipid peroxidation products during oxidative stress, induced by xenobiotics [57]. Moreover, certain brain areas, the striatum and hippocampus, which are rich in iron, being initial factor in lipid peroxidation processes, seem to be particularly vulnerable to membrane lipid peroxidation [58]. These regional differences in lipoperoxidative capacity of the brain areas in vitro are dependent from local endogenous iron contents in the brain regions and their ability to produce lipid peroxides. Lipid peroxidation destroys the integrity of membranes leading to functionality loss. Apart from chelating effect of black tea components, membrane prevention may be connected with the fact that catechins and to a higher degree teaflavins may show partially lipophylic character and can be mostly localized near the surface of membranes penetrating their interior [21]. It is likely that they easily trap the main aqueous lipid oxidation initiator - hydroxyl radical and are accessible to chain-initiating peroxyl radicals more readily than α-tocopherol. It has been also revealed that black tea components are stronger antioxidants that compounds known as typical antioxidants e.g. glutathione, ascorbic acid, α-tocopherol [21]. Our data are in agreement with the studies of Lin et. al. (1998) [59] who proved antioxidative properties of green tea against iron-induced oxidative stress in rat brain homogenates in vitro. However, these authors have not observed any changes during oral administration of green tea for two weeks in the iron induced oxidative injury in nigrostriatial dopaminergic system. These studies have shown that changes in the brain are likely appear a long period of drinking green tea. The influence of black tea on the brain has not been examined yet. In conclusion, long-term drinking of black as well as green tea prevents changes in antioxidant system caused by chronic ethanol intoxication. These results indicate beneficial effect of black tea in particular on the liver. Evidence for the bioactivity of the phenolic

Comparison of Antioxidative Properties of Green and Black Tea

31

compounds of black tea to in vivo supports the notion that their antioxidant properties contribute to important role in the health protection and disease prevention.

ACKNOWLEDGEMENTS The authors wish to thank professor Joe Vinson (University of Scranton) for bestowing the teaflavins standards and for his advice in preparing this manuscript. This investigation was partially supported by a grant from Polish Committee of Scientific Research No. 6P05F01720

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In: Leading Edge Antioxidants Research Editor: Harold V. Panglossi, pp. 35-64

ISBN 1-60021-274-3 © 2007 Nova Science Publishers, Inc.

Chapter 3

ANTIOXIDANTS IN FOODS: A NEW CHALLENGE FOR FOOD PROCESSORS G. Oboh1, and J. B. T. Rocha2 1

Biochemistry Department, Federal University of Technology, P.M.B. 704 Akure, Omdo State, Nigeria 2 Departamento de Química (Bioquímica Toxicológica) Universidade de Federal Santa Maria (UFSM) Campus Universitário – Camobi 97105-900 Santa Maria RS, Brasil

ABSTRACT In recent years, human health has assumed an unprecedented important status. A new diet-health paradigm is everlasting which places more emphasis on the positive aspects of diet. Foods have now assumed the status of functional foods, which should be capable of providing additional physiological benefit, such as prevent or delaying onset of chronic diseases, as well as meeting basic nutritional requirements. Recently phytochemicals in fruits and vegetables have attracted a great deal of attention mainly concentrated on their role in preventing diseases caused as a result of oxidative stress. The regular consumption of foods that are naturally high in antioxidants (fruits, vegetables, whole grains, nuts and seeds, legumes and herbal seasonings) is associated with substantial health benefits. Although some of this food are eaten raw without processing, but many of them are subjected to one or more forms of post-harvest food processing techniques such as blanching, cooking, sun drying, frying, soaking, irridation, fermentation, coagulation and baking. Apart from Vitamin C and E that are already established food nutrient with antioxidant properties, other phytochemicals in foods with antioxidant properties includes: Ally sulphide, carotenoids, curcumins, flavonoids, gingerols, indoles and isothiocyanates, isoflavones, lignans, liminoids, phenolic acids, phthalides polyacetylenes, phytates, saponins and terpenes. This phytochemicals varies in their quantities and physico-chemical properties, and their activities in food are either additive or synergistic. Since most of the methods employed in foods processing both at household levels or during the unit operations in food processing industry will bring about a change in quantities and physico-chemicals properties of the food phytochemicals, and this could effect individual / or gross antioxidant properties of the food. In this article, the effect of the various post-harvest food processing techniques

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G. Oboh and J. B. T. Rocha popularly employed both at the household or industrial levels on the antioxidant phytochemicals and activity of some commonly consumed plant foods, and the possible remedy is highlighted.

INTRODUCTION Antioxidants are powerful free radical scavengers in the body, while free radicals are highly reactive chemical substances such as superoxide, hydroxyl radical, singlet oxygen e.t.c (Alia et. al. 2003) that travel around in the body and cause damage to the body cells. Free radical damage is one of the most prominent causes of devastating diseases that are responsible for killing many people in the world, such as cardiovascular disease, which can manifest as heart attacks, and cancer (Amic et.al. 2003). The aging process has been linked by some researchers, to be due to free radical damage in the body. Free radicals naturally occur in the body as a result of chemical reactions during normal cellular processes such as during the conversion of food into energy by the body. They can also be formed in response to environmental factors such as excess pollution, too much UV rays, exposure to cigarette smoke, automobile exhaust and pesticides. Not getting enough rest or sleep, not managing our stress responses and not eating healthfully can also cause free radical damage (Chu et. al. 2002; Oboh, 2004; Oboh, 2005a; Oboh, 2005b). The human body is equipped with an antioxidant defense system that deactivates these highly reactive free radicals. Antioxidants enzymes (made in the body) and antioxidant nutrients (found in foods) soak up all the excess energy that these free radicals have, turning them harmless particles or waste products that we can then get rid of. So these antioxidant nutrients are functional components of food that have extra health benefits for us (Oboh, 2005a). Current thinking among the “natural health” community indicates that supplements of vitamins and other plant-based chemicals (phytochemicals) are important to ensure that we do not suffer from heart disease, cancer or other chronic diseases. Too much of any antioxidant (which happens when you take a concentrated dose in a supplement form) can actually make the antioxidants work as a prooxidant (Oboh, 2004). Also, some free radicals offer protection to us by attacking harmful bacteria and cancer cells in the body. Antioxidants are powerful substances that can neutralize free radicals before they damage the body's cells. This is the major reasons why the benefits of antioxidants are talked about often in the media, and why people are advised to eat antioxidant foods i.e., foods high in antioxidants (Oboh, 2004; Oboh, 2005a; Oboh, 2006a; Oboh and Akindahunsi, 2004; Sun et. al. 2002; Chu et. al. 2002). Plant foods, including fruits, vegetables and grains, are good for us. Research confirms that some of these foods do, as part of an overall healthful diet, have the potential to delay the onset of many age-related diseases. This appears to be due to high levels of antioxidants and other phytonutrients. Antioxidants comprise many components— some vitamins, minerals, carotenoids, polyphenols—all present in a variety of foods (Jayasinghe et. al. 2003). Some are natural colorants characterized by their distinctive colors—the deep red color of cherries, the red color in tomatoes, the orange color in carrots, and the yellow color of corn, mangos and saffron. The most well-known antioxidants are vitamins A, C and E, beta carotene, and selenium. Citrus fruits and their juices, berries, dark green vegetables (spinach, asparagus, green peppers, brussel sprouts, broccoli, watercress, other greens), red and yellow peppers,

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tomatoes and tomato juice, pineapple, cantaloupe, mangos, papaya and guava are good source of vitamin C (Yang et. al. 2004; Adom et. al. 2005; Liu et. al. 2005), while Vegetable oils such as olive, soybean, corn, cottonseed and safflower, nuts and nut butters, seeds, whole grains, wheat, wheat germ, brown rice, oatmeal, soybeans, sweet potatoes, legumes (beans, lentils, split peas) and dark leafy green vegetables are good sources of vitamin E. Selenium can be gotten by eating Brazil nuts, brewer’s yeast, oatmeal, brown rice, chicken, eggs, dairy products, garlic, molasses, onions, salmon, seafood, tuna, wheat germ, whole grains, most vegetables. Variety of dark orange, red, yellow and green vegetables and fruits such as broccoli, kale, spinach, sweet potatoes, apple, carrots, red and yellow peppers, apricots, cantaloupe and mangos are good source of ß-carotene (Sun et.al. 2002; Chu et.al. 2002; Jiratanan and Liu 2004; Boyer and Liu, 2004; Yang et. al. 2004; Adom et. al. 2005; Liu et. al. 2005). There are about 4000 known plant pigments in our food, including thousands of flavonoids, and hundreds of carotenoids and anthocyanins. These pigments do more than provide color to our foods. They also protect us from disease. Anthocyanins are the watersoluble, reddish pigments found in many fruits, such as strawberries, cherries, cranberries, raspberries, blueberries, grapes and black currants. Since anthocyanins inhibit cholesterol synthesis these fruits provide protection against heart disease. Carotenoids are the pigments found in yellow-orange vegetables (carrots, pumpkins, and sweet potatoes) and leafy, green vegetables (kale, broccoli and collard greens) and the red and yellow-orange fruits (mangoes, pineapple, peaches, oranges, pink grapefruit, tomatoes, strawberries, watermelon, cantaloupe) known to possess significant anti-tumor activity. The carotenoids are also known to enhance immune function. Persons with high blood levels of carotenoids have a reduced risk of heart disease and cancer. Phenols, including flavonoids, protect body cells against ('anti-') the damage caused by oxygen ('oxidation') that's released as a by-product of energy metabolism. A small amount of released oxygen becomes part of highly reactive free radicals, which attack and damage body cells to get the missing electron they need. Antioxidants protect by contributing an electron of their own. In so doing, they neutralize free radicals and help prevent cumulative damage to body cells and tissues. Much of the total antioxidant activity of fruits and vegetables is related to their phenolic content, not only to their vitamin C content. Research suggests that many flavonoids are more potent antioxidants than vitamins C and E (Oboh and Akindahunsi, 2004; Oboh, 2005a, 2005c). Natural polyphenols exert their beneficial health effects by their antioxidant activity, these compounds are capable of removing free radicals, chelate metal catalysts, activate antioxidant enzymes, reduce α-tocopherol radicals, and inhibit oxidases (Amic et.al. 2003; Alia et.al. 2003; Oboh, 2006a). Processed fruits and vegetables have long been considered to have lower nutritional value than their fresh produce based on the loss of vitamin C during processing. Research has shown that the vitamin C in apples contributed less than 0.4% of the total antioxidant activity indicating that most of the activity comes from the natural combination of phytochemicals. This suggests that some processed fruits and vegetables may retain their antioxidant activity in spite of the loss of vitamin C (Oboh and Akindahunsi, 2004; Oboh, 2005a). Furthermore, it has been demonstrated that thermal processing of tomatoes significantly increased the extractable lycopene content and the total antioxidant activity, while thermal processing of sweet corn and green leafy vegetables will significantly increased total antioxidant activity and phytochemical content despite the decline in vitamin C content. In view of this, one

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cannot be equivocal that processed foods have lower nutritional value than fresh produce (Dewanto et.al. 2002a; Dewanto et.al. 2002b). The effectiveness of bioactive compounds in food products can be affected by processing steps as well as by the bioavailability of these compounds from the final products. It is therefore important to know what the effects of processing steps are on the level, and on the activity of bioactive components. Information on the effect of various conventional processing techniques at household and unit operations in industrial levels may have a significant impact on consumers’ food selection and food processing industry. However, the actions of the antioxidant nutrients alone do not explain the observed health benefits of diets rich in fruits and vegetables for chronic diseases because taken alone, the individual antioxidants studied in clinical trials do not appear to have consistent preventive effects. However, the additive and synergistic effects of phytochemicals in fruits and vegetables are responsible for these potent antioxidant and anticancer activities, and that the benefit of a diet rich in fruits and vegetables is attributed to the complex mixture of phytochemicals present in whole foods (Liu, 2003). In this write up, the effect of some commonly employed food processing techniques at household levels and unit operation in the Food processing industry namely: fermentation, cooking, drying, irridation and frying e.t.c. on the antioxidants phytochemicals and activity of some commonly consumed foods will be highlighted.

FERMENTATION Fermentation is one of the oldest applied biotechnologies, having been used in food processing and preservation as well as beverages production for over 6,000 years (Motarjemi, 2002). The fermentation process of staples serves as a means of providing a major source of nourishment for large rural populations, and contributes significantly to food security by increasing the range of raw materials, which can be used in the production of edible products (Adewusi et. al. 1999; Oboh et. al. 2001; Oboh and Akindahunsi, 2003; Oboh, 2006b; Oboh and Akindahunsi, 2005). Fermentation enhances the nutrient content of foods through the biosynthesis of vitamins, essential amino acids and proteins, by improving protein quality and fibre digestibility. It also enhances micronutrient bioavailability and aids in degrading antinutritional factors (Raimbault, 1998; Achinewhu et. al. 1998; Akindahunsi and Oboh, 2003; Oboh et. al. 2002; 2003). Fermentation techniques is more practice in African and South East Asian countries such as China, Japan and Korea and is believed to enhance the neutraceutical value of fermented foods in addition to long storability. Breakdown of food proteins by microbial proteases to produce bioactive peptides may be a possible reason for developing such properties during fermentation. Therefore, interests have been developed to identify biological activities in fermented foods including fish and shellfish (Ichimura et. al., 2003; Kim et. al., 2004; Wong and Mine, 2004). Health related functional properties like antioxidative activity and radical scavenging capacity may present as promising biological benefits of these fermented foods. Fermented marine blue mussel (Mytilus edulis) derived hepa-peptides was reported to be highly effective free radical scavenger and was named as mussel derived radical scavenging peptide (MRSP). Under experimental conditions, MRSP could scavenge superoxide (98%), hydroxyl (96%), carbon-centered (91%) and DPPH radicals (72%) at 200µg/ml concentration

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and the respective IC50 values were found to be 21, 34, 52 and 96µM. In addition, MRSP exhibited a strong lipid peroxidation inhibition at 54µM concentration and it was higher than ∝-tocopherol. Furthermore, it exhibited a 95% higher Fe2+ chelating effect than citrate. Moreover, MRSP could enhance the viability of oxidation induced cultured human cells by 76% following 75 µg/ml of treatment. Therefore, MRSP has been identified as a potent natural antioxidant, which performs its activity via different mechanism of actions (Rajakpase et. al. 2005). “Kimchi”, a Korean fermented traditional food containing different vegetables and spices. The principal spices used in kimchi are paprika, garlic, and ginger, which are recognized as antioxidants in different food systems (Aguirreza´bal et al., 2000; Al-Jalay et. al., 1987; Gassmann, 1992; Gerhardt, 1994; Palic et. al., 1993). It has been demonstrated that they contain antioxidant compounds such as phenolic compounds; carotenoids, flavonoids, phenolic acids, tocopherols, ascorbic acid, nitrate and nitrite (Aguirreza´bal et.al., 1998; Daood et. al., 1996; Elmadfa, 1998;Gerhardt, 1994; Hertog, 1994). Among them, paprika was found to have one of the lowest redox potentials and could have high antioxidant content (Palic et al., 1993). Several components of garlic extracts such as alliin, diallyl sulphide, alyl sulphide, and propyl sulphide exhibited antioxidant activity as hydroxyl radical scavengers (Kourounakis and Rekka, 1991; Yang et. al., 1993). On the other hand, the main materials of baechu-kimchi such as Chinese cabbage, leek or green onion contain a broad spectrum of phenolic compounds, mainly glucosinolate, tocopherols, and ascorbic acid. In an experimental report by Lee and Kunz (2005) on the antioxidant properties of baechu-kimchi, by investigating its effect on the changes in total free fatty acid content, peroxide value, and TBARS during ripening at 7 and 20oC temperature and 5%, 10% and 15% concentrations; the addition of 5% and 10% kimchi inhibited lipid oxidation. However, an increase in the amount of kimchi caused a prooxidant effect. The antioxidant effect of kimchi has been found to be dependant on its fermentation temperature as the batches treated with kimchi fermented at 20oC displayed better protection against the formation of oxidative products than those fermented at 7oC. Kimchi-powder did not show any antioxidant properties (Lee and Kunz, 2005). EM-X is an antioxidant drink derived from fermentation of unpolished rice, papaya and seaweeds with effective microorganisms (EM) of lactic acid bacteria, yeast, and photosynthetic bacteria. ‘EMX’ is the only name of this antioxidant drink and also its commercial name. EM-X contains over 40 minerals, α-tocopherol, lycopene, ubiquinone, saponin and flavonoids, such as quercetin, quercetin-3-O-glucopyranoside and quercetin-3-Orhamnopyranoside. EM-X is widely available in South-East Asia as a beverage and is accepted in clinical practice (Aruoma et. al. 2002). EM-X has been suggested as a prophylactic beverage in the treatment of cancer, hypertension, diabetes, rheumatism, various allergies and chemical substance hypersensitivity, HIV/AIDS, tuberculosis and other infectious diseases (Higa and Ke, 2001). Extensive study using high concentrations of EM-X up to 1800 ml/kg body weight of mice suggest that oral dosing is not toxic and this concentration corresponds to 1500 times the daily dose in the clinic (Zhong et al., 1999). The concentration of EM-X up to 1800 ml/kg body weight means that the daily dose of concentrated EM-X (20-fold) used in the toxicity studies was equated to1800 ml/kg of the original liquid. Ke et al. (2001) reported that EM-X has anti-aging properties and was able to increase serum concentrations of superoxide dismutase and to decrease malondialdehyde levels as well as prolonging the life-span of fruit flies.

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Figure 1. Some of the flavonoid structures found in EM-X.

Oral administration of an EM-X to rats for 7 days inhibited the ferric-nitrilotriacetic acid (Fe-NTA)-dependent oxidation of fatty acids with protections directed towards docosahexanoic, arachidonic, docosapentanenoic acids, oleic, linoleic and eicosadieonoic acids in the liver and kidney. But only the protections of oxidation to docosahexanoic, arachidonic acid in the kidney were statistically significant. Treatment of rats with EM-X prior to the intraperitoneal administration of Fe-NTA led to a reduction in the overall levels of conjugated dienes (CD) measured in the kidney by 27% and in the liver by 19% suggesting inhibition of lipid peroxidation in these organs. The levels of glutathione and ∝-tocopherol were largely unaffected suggesting that the protection by the regular strength of EM-X was confined to the inhibition of lipid peroxidation in vivo, a point dependent on the concentrations of bioactive flavonoids (Aruoma et. al. 2002). Cranberry pomace is a byproduct of the cranberry processing industry. Solid-state fermentation of cranberry pomace using food grade fungus Lentinus edodes improved phenolic profile and antioxidant activity. The phenolic phytochemicals mobilized during fermentation of the pomace improves the antimicrobial functionality against Listeria monocytogenes, Vibrio parahaemolyticus and Escherichia coli-O157: H7 are important food borne pathogens (Vattem et. al.2004; Vattem and Shetty, 2002; USDA, 2001, Sobota, 1984). Fermentation of the pomace for 20 days with Lentinus edodes caused an increase in the total water extractable phenol content and antioxidant activity and the antimicrobial activities of the extracts were also enriched. The bioprocess-based antimicrobial activity depended on different phenolic functional properties of the extracts. Inhibition of L. monocytogenes by the extracts correlated well with the increase in soluble phenolics, antioxidant activity and enrichment of ellagic acid during solid-state bioprocessing. Inhibition of V. parahaemolyticus and E. coli O157: H7 by the extracts correlated with highest ellagic acid and/or β-carotene oxidation system (APF). Sensitivity towards soluble phenolics and the antioxidant activity measured by DPPH radical inhibition assay suggests inhibition by the disruption of the membrane by localized hyper-acidification and potentially disruption of H -ATPase. Whereas, APF and ellagic acid dependent antimicrobial activity suggests potential inhibition

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by membrane-transport and/or electron transport disruption. The variation in sensitivity of pathogens has implications for designing new food-grade antimicrobials. This solid-state bioprocessing strategy is an innovative approach to produce broad-spectrum antimicrobials against important food borne pathogens (Vattem et. al. 2003).

α-tocopherol

Ubiquinone Figure 2. Structure of some other antioxidant phytochemicals in EM-X.

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Cranberry Figure 3. Cranberry and Ellagic acid.

Soy has been a staple of the Southeast Asian diet for nearly five millennia and both its medicinal and nutritional values are deeply rooted in Traditional Chinese Medicine and herbalism (Murphy et.al.1997; Poysa and Woodrow, 2002; Oboh and Omotosho, 2005). On the other hand, consumption of soy in the United States and Western Europe has been limited to the 20th century. Asians on the average eat twenty to fifty times more soy than Americans. Typically soy foods are divided into two categories: non-fermented and fermented soy products. Traditional nonfermented soy foods include fresh green soybeans, whole dry soybeans, soy nuts, soy sprouts, whole-fat soy flour, soymilk and soymilk products, tofu, okara and yuba. Traditional fermented soy foods include tempeh, miso, soy sauces, natto and fermented tofu and soymilk products (Cai and Chang, 1997; Cai and Chang, 1998; Oboh and Omotosho, 2005). In Asia, the traditional fermented soy foods are considered to have more health promoting benefits when consumed in moderate amounts than the super-processed soy products that are consumed in the West (Messina and Barne, 1991). It has been suggested that the fermentation process increases availability of isoflavones in soy (Golbitz, 1995). For example, a study (Baggott et. al. 1990) of the culturing method involved in the production of the Japanese traditional food miso came to the conclusion that the culturing process itself led to a “lower number of cancers per animal” and a “lower growth rate of cancer compared to controls.” The researchers also indicated that it was not the presence of any specific nutrient that was cultured along with the soyabean paste but rather the cultured soy medium itself that was responsible for the health benefits associated with miso consumption. Miso, a fermented or probiotic form of soyabean, is particularly rich in the isoflavone aglycones, genistein and daidzein, which are believed to be cancer chemopreventatives. The fermentation process is thought to convert the isoflavone precursor’s genistin and daidzin to their active anti-cancer isoflavone forms, genistein and daidzein. A recent Korean study concluded that [soy] was one of the foods that decreased the rates of certain cancers such as stomach cancer while heavy salt consumption and cooking methods increased the rates (Lee, 1995). The limited epidemiological evidence so far from these cultures with diets that are high in fermented soy products indicates that such diets may reduce the risk of breast, colon, lung and stomach cancers, as well as offer some protection from cardiovascular diseases, osteoporosis and menopausal symptoms (Lee, 1995, Lissin and Cooke, 2000; Gardiner and Ramberg, 2001). One underlying theoretical paradigm is that primary active ingredients in complex fermented soy “foods” act synergistically with secondary compounds. A second paradigm is that secondary compounds mitigate the undesirable side effects caused by the predominant active ingredients. Another possibility is

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that the multiple ingredients act through multiple discrete pathways to therapeutically affect the host, and that lower concentrations of each of the botanicals or soy phytochemicals can therefore be more efficacious when used together than when used individually. Studies are needed to demonstrate the mechanisms of action and to verify the efficacy. Studies can also compare complexes versus single agents. The benefits of using these complex fermented soy “foods” have historically been based on empirical observation. These concepts help determine which combinations are truly likely to be efficacious (Walker, 1994; Stephen, 1996).

Genistein

Daidzin Figure 4. Structure of some antioxidant phytochemicals in Soybeans.

BLANCHING Blanching could be described as the process of heating vegetables to a temperature high enough to destroy enzymes present in the tissue. It stops the enzyme action, sets the colour, and shortens the drying and dehydration time. It is usually carried out in hot water or in steam; this technique is used by indigenous people to reduce or eliminate the bitterness of vegetables and acid components that are common in leaves (Akindahunsi and Oboh, 1999). In Nigeria, green leafy vegetables are not usually consumed in their fresh form unlike fruits; however, they are usually blanched before consumption or preparation of soup (Akindahunsi and Oboh, 1999;Oboh et.al. 2005a; Oboh 2005c). Due to high bacterial counts of raw vegetables after harvest (up to 106–109 cfu/g), ready-to-use sliced salads are usually

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contaminated by microorganisms (Jaques and Morris, 1995). Bacterial pathogens have been detected in prepackaged salads (Lin, Fernando, andWei, 1995) and lettuce (Park and Sanders, 1992). High initial counts are not substantially reduced during conventional cold washing. Even if chlorine is added to the washing water for decontamination, maximum reductions of about one order of magnitude can be achieved (Marcy, 1985; Seo and Frank, 1999; Weissinger, Chantarapanont, and Beuchat, 2000). Low-temperature blanching (ltb) not involving any chemical treatment, can reduce initial mesophilic counts of leaf salads by more than three orders of magnitude and Enterobacteriaceae counts to less than one cfu/g (Gartner et. al., 1997). It combines food safety with extended shelf-life of the produce (Akindahunsi and Oboh, 1999, Oboh et.al., 2005). Low-temperature (lt) blanching of endive combined with cold storage at 4oC and 97% relative humidity for two days reduced polyphenol content and antioxidant capacity of endive to about 50% compared to the salad washed with cold water (Mayer-Miebach et. al. 2003). Blanching of eight popularly consumed green leafy vegetables in Nigeria namely: Struchium sparjanophora, Amarantus cruentus, Telfairia occidentalis, Baselia alba, Solanum macrocarpon, Corchorus olitorus, Vernonia amygdalina, and Ocimum gratissimum for 5 minutes cause a significant (P<0.05) decrease in the vitamin C [fresh (43.5–148.0 mg/100 g), blanched (15.8–27.3 mg/100 g)] content, reducing power [fresh (0.5–1.5 OD700), blanched (0.1–0.6 OD700)] and DPPH free radical scavenging ability [fresh (20.0–51.4%), blanched (16.4–47.1%)] of the blanched green leafy vegetables except in Struchium sparjanophora, where there was no change in the reducing property (0.6 absorbance) and free radical scavenging ability (59.8%) of the blanched vegetable (Oboh, 2005a).

Corchorus olitorius

Basella alba

Telfairia occidentalis

Figure 5. Some tropical green leafy vegetables.

Recently, Amin et. al. (2006) reported on the effect of longer blanching time (10 and 15min) on the total phenol and antioxidant activity of four varieties of Amaranthus sp. locally known as spinach, namely Amaranthus paniculatus (BP), Amaranthus gangeticus (BM), Amaranthus blitum (BI) and Amaranthus viridis (BPG). Total antioxidant activity of watersoluble components in raw spinach was in the order of BI > BM > BPG > BP, whereas free radical-scavenging activity was in the order of BI > BPG > BM > BP. The total phenolic contents of BM and BP were significantly higher (p < 0.05) than other samples. All the studied spinach species possessed different antioxidant activities and phenolic contents. Antioxidant activities and phenolic contents of all the spinach were in the order of raw >

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blanched 10 min > blanched 15 min. Blanching up to 15 min may affect losses of antioxidant activity and phenolic content, depending on the species of spinach (Amin et. al., 2004). In practice, spinach is cooked with water for quite some time before being consumed (Amin et. al., 2006). In a study carried out by Talcott and co-worker (Talcott et. al.2000) on the effect of blanching Fresh peaches for either a long (20 min) or short (2 min) time, with and without periderm material on the nutritive and physico-chemical properties. Purees containing periderm had higher antioxidant activity (AOX) and individual phenolic acid content after processing and storage, with good correlation to AOX observed with chlorogenic acid (r ) 0.82). Long blanch times resulted in increased levels of extraction and retention of both total soluble phenolics and ascorbic acid. Quantitative descriptive analysis demonstrated increased aromatic perception of skin in samples containing periderm material, but the remaining sensory attributes were indistinguishable between treatments. Macerating peaches without periderm removal was demonstrated to increase levels of bioactive phytochemicals and increase the processing yield by 7.6%, without significantly impacting product quality, color, or sensory attributes (Talcoltt et. al. 2000).

COOKING Cooking is usually carried out in order to increase the palatability and to improve the edibility of some food. The popular thinking is that fresh fruits and vegetables are better for us than cooked ones nutrition wise. However, carotenoids, the colourful pigments in a variety of red, yellow and orange vegetables are more available for absorption from cooked than raw foods. Cooked sweet yellow corn had 44% higher total antioxidant activity than the same corn before cooking. Vitamin C is partially destroyed by cooking, but other antioxidant activity is increased by cooking, this possibly due to freeing the antioxidant phytochemicals from the cells of the corn. One of the beneficial antioxidant chemicals is ferulic acid, found in the cell wall of grains such as corn, wheat and oats. Ferulic acid doubled after 10 minutes of cooking and increased by as much as 900 percent after 50 minutes (Dewanto et.al. 2002a). Although corn cooked for 50 minutes may not be very appetizing, it is no less healthy than uncooked corn and provides much higher levels of antioxidant activity (Dewanto et. al. 2002a). Most vegetables are usually cooked by boiling in water or microwaving (Unlike fruits that are usually consumed in their raw forms) before consumption. These cooking processes could bring about a number of changes in physical characteristics and chemical composition of vegetables (Rehman et.al., 2003; Zhang and Hamauzu, 2004). Sahlin et.al. (2004) reported that boiling and baking had a small effect on the ascorbic acid, total phenolic, lycopene and antioxidant activity of the tomatoes while frying significantly reduced the ascorbic acid, total phenolic and lycopene contents of tomatoes. Zhang and Hamauzu (2004) pointed out that cooking affected the antioxidant components and antioxidant activity of broccoli. While Ismail et. al., (2004) found that thermal treatment decreased the total phenolic content in some vegetables such as kale, spinach, cabbage, swamp cabbage and shallots and antioxidant activity in some of them.

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Figure 6. Maize and its antioxidant phytochemical-Ferulic acid.

Green beans, peas, pepper, squash, broccoli, leek and spinach are common vegetables consumed as cooked, the total phenolics content of the fresh vegetables ranged from 183.2 to 1344.7 mg/100 g (as gallic acid equivalent) on dry weight basis and total antioxidant activity ranged from 12.2% to 78.2%. When those vegetables are subjected to microwave and conventional cooking methods, with the exception of spinach, cooking affected total phenolics content significantly (p < 0.05). The effect of various cooking methods on total phenolics was significant (p < 0.05) only for pepper, peas and broccoli. After cooking, total antioxidant activity increased or remained unchanged depending on the type of vegetable but not type of cooking (Turkmen et. al. 2005).

Green beans

Pepper

Figure 7. Some commonly consumed non-leafy vegetables.

Peas

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Cooking and mashing up carrots may increase their antioxidant value by 34% immediately after cooking carrots and three-times higher than levels in raw carrots when cooked and stored them at 40oC for 4 weeks. Keeping the outer skin on the carrots also boosted antioxidant activity slightly. Numerous phenolic compounds are located in the (skin) of fruits and vegetables, many of which are removed by peeling steps prior to processing (Talcott et.al. 2000).

Carrot Figure 8. Carrot and some of its antioxidant phytochemicals.

Cooking of tomatoes like in the case of preparing soup or sauce makes the fruit hearthealthier and boosts its cancer-fighting ability, despite a loss of vitamin C during the cooking process. The reason, being that cooking substantially raises the levels of beneficial phytochemicals. Dewanto et. al. (2002b) reported that heat processing actually enhanced the nutritional value of tomatoes by increasing the lycopene content, a phytochemical that makes tomatoes red and can be absorbed by the body, as well as the total antioxidant activity. Lycopene, a carotenoid responsible for the red color in tomatoes and other fruits, has long been known as a powerful antioxidant that decreases cancer and heart-disease risk. Carotenoids, along with phenolic acids and flavonoids, are all phytochemicals, the nutritionally beneficial active compounds found in every fruit and vegetable. The research dispels the popular notion that processed fruits and vegetables have lower nutritional value than fresh produce." In the research the tomato samples were heated to 88oC for two minutes, a quarter-hour and a half-hour, vitamin C content decreased by 10, 15 and 29% respectively, when compared to raw, uncooked tomatoes. However, the research revealed that the beneficial trans-lycopene content of the cooked tomatoes increased by 54, 171 and 164% respectively. Levels of cis -lycopene (which the body easily absorbs) rose by 6, 17 and 35% respectively; and antioxidant levels in the heated tomatoes increased by 28, 34 and 62%

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respectively. While the antioxidant activity in tomatoes is enhanced during the cooking process, vitamin C loss occurs when the food's ascorbic acid is oxidized to dehydroascorbic acid and other forms of nutritionally inactive components. Lycopene is the most-efficient single oxygen quencher, and devours more than 10 times more oxygenated free radicals than vitamin E (Dewanto et. al. 2002b).

Tomatoes Figure 9. Tomatoes and some its antioxidant phytochemicals.

Broccoli lost 97% of flavonoids, 74% of sinapics and 87% of caffeoyl-quinic derivatives (three different types of antioxidants) when it was microwaved. When boiled the conventional way (i.e., not in a pressure-cooker), this green lost 66 percent of its flavonoids; when tossed in a pressure cooker, broccoli lost 47 percent of its caffeoyl-quinic acid derivatives. Steamed broccoli, on the other hand, lost only 11 percent, 0 percent and 8 percent, respectively, of flavonoids, sinapics, and caffeoyl-quinic derivatives. The advantage of steaming versus conventional boiling is that you're "not using water directly in contact with the vegetable. The nutritional compounds don't go into the water, once the compounds are in the water, the temperature destroys them much easier. A microwave wreaks havoc because it heats the inside of the vegetable. That, combined with the fact that you normally use water when microwaving, causes the destruction of valuable nutrients. Garlic ( Allium sativumL.) is an essential part of Polish, Ukrainian, and Israeli. Polish garlic has high Polyphenols, tocopherols, proteins, and antioxidant potentials (Goristein et. al. 2005). However, cooking almost leads to the disappearance of the 50 kDa protein and cause a slight decrease in the intensity of the 12 kDa lectin bands of the garlic. The bioactive compounds, antioxidant potential, and proteins in garlic decrease significantly after 20 min of cooking at 100 °C ( P < 0.05). And so, fresh garlic should be added to dishes cooked at 100 °C in the last 20 min of the cooking process (Goristein et. al. 2005).

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Broccoli

Caffeoyl-quinic

Flavonoids Figure 10. Showing Broccoli and some of its antioxidant phytochemicals.

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MILLING Milling is the process of cutting away material by feeding a workpiece past a rotating multiple tooth cutters. The cutting action of the many teeth around the milling cutter provides a fast method of machining. The machined surface may be flat, angular, or curved. The surface may also be milled to any combination of shapes. The machine for holding the work piece, rotating the cutter, and feeding it is known as the milling machine (Homestead Harvest, 2005). Countless essential vitamins and oils are lost in today's commercially processed flours, all in the name of convenience. To ensure that today's store bought flour will last on shelves, all traces of the grains bran and germ must be removed. Because once the bran and germ are milled, they can only last up to 72 hours at room temperature before going rancid. Since the germ is packed with vitamins B and E, much is lost by its removal. This removal process also results in a loss of up to 26 essential vitamins necessary for proper nutrition and valuable roughage which our bodies need to absorb and remove unwanted toxins and poisons within our digestive system (Homestead Harvest, 2005). Commercial processors have tried to 'enrich' flours with vitamins B1, B2, Niacin, and Iron, but it still cannot make up for the many other valuable vitamins lost. Health problems such as Obesity, Diabetes, Hypoglycemia, Heart Disease, Bowel Cancer and Tooth Decay are just some of the major diseases on the rise since the introduction of white flour in the 1900's. Many nutritionists agree that white flour and other refined foods are largely responsible (Homestead Harvest, 2005). Wheat contains phenolic compounds concentrated mainly in bran tissues. Debranning (pearling) of wheat before milling is becoming increasingly accepted by the milling industry as a means of improving wheat roller milling performance, making it of interest to determine the concentration of ferulic acid at various degrees of pearling. Recently Beta et. al. (2005) carried out a study on the distribution of phenolics and antioxidant activities in wheat fractions derived from pearling and roller milling. In the study wheat was pearled incrementally to obtain five fractions, each representing an amount of product equivalent to 5% of initial sample weight. Wheat was also roller milled without debranning. Total phenolic content of fractions was determined using the modified Folin-Ciocalteau method for all pearling fractions, and for bran, shorts, bran flour, and first middlings flour from roller milling. Antioxidant activity was determined on phenolic extracts by a method involving the use of the free radical 2,2-diphenyl-l-picrylhydrazyl (DPPH). The total phenolics were concentrated in fractions from the first and second pearlings (>4,000 mg/kg), while wheat fractions from the third and fourth pearlings still contained high phenolic content (>3,000 mg/kg). A similar trend was observed in antioxidant activity of the milled fractions with ≈4,000 mg/kg in bran and shorts, ≈3,000 mg/kg in bran flour, and <1,000 mg/kg in first middlings flour. Total phenolic content and antioxidant activity were highly correlated (R2 = 0.94) (Beta et. al. 2005). Selenium (Se) is an essential micronutrient for animals and humans, and wheat is a major dietary source of this element. It is important that postharvest processing losses of grain Se are minimized. In the dissection study in order to investigate the distribution of Se and other mineral nutrients in wheat grain and the effect of postharvest processing on their retention using grain dissection, milling with a Quadrumat mill, and baking and toasting studies. The study revealed that Se is more concentrated in the embryo, confirming (along with the milling study) previous findings, however Se (which occurs mostly as selenomethionine in wheat

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grain) are more evenly distributed throughout the grain when compared to other mineral nutrients, and, hence, lower proportions are removed in the milling residue. Postmilling processing did not affect Se concentration or content of wheat products (Lyons et. al. 2005).

Figure 11. Biosynthesis of vanillin from ferulic acid.

Wheat Figure 12. Showing Wheat and the structure of Selenomethionine.

However, earlier study reported by Ferretti and Levander (1974), in which they determine the selenium content of grains and of the products and cereals derived from these grains fluorometrically revealed that wheat flour and farina contained 14 and 29% less selenium, respectively than the original raw wheat. Corn meal, flour, and grits contained 10-28% less selenium than did the raw corn. Milling of oats and rice caused little change in selenium concentration of those grain fractions destined for consumer use. Little or no decline in selenium concentration occurred during the manufacture of nonsugared corn or wheat breakfast cereals. There was a significant decrease in the concentration of selenium in sugared corn and sugared wheat breakfast cereals which was apparently due to dilution of selenium by the sugar. The decrease in selenium concentration in consumer products due to milling or processing of grains appeared to be less than that reported with several other nutritionally essential trace elements (Ferretti and Levander, 1974).

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FRYING Recent consumer interest in “healthy eating” has raised awareness to limit the consumption of fat and fatty foods. Fats and oils are heated at high temperatures during baking, grilling, and pan-frying; however, deep fat frying is the most common method of high temperature treatment (Warner, 1999). Deep fat frying is a popular food preparation method because it produces desirable fried food flavor, golden brown color, and crisp texture. Frying has little or no impact on the protein or mineral content of fried food, whereas the dietary fiber content of potatoes is increased after frying due to the formation of resistant starch. Moreover, the high temperature and short transit time of the frying process cause less loss of heat labile vitamins than other types of cooking. For example, vitamin C concentration of French fried potatoes is as high as in raw potatoes, and thiamine is well retained in fried potato products as well as in fried pork meat (Fillion and Henry, 1998). Although some unsaturated fatty acids and antioxidant vitamins are lost due to oxidation, fried foods is generally a good source of vitamin E (Fillion and Henry, 1998). Because of such large consumption of frying oils and fats, the effect of high temperatures on these oils and fats is of major concern both for product quality and nutrition (Fillion and Henry, 1998; Paul and Mittal, 1997; Warner, 1999). On the other hand, it is well known that dietary fat sources deeply influence several biochemical parameters both in plasma and in mitochondrial membranes (Mataix et al., 1998; Quiles et al., 1999). Fat frying is a popular food preparation method but several components like antioxidant vitamins could be lost due to oxidation and some others with toxic effects could appear. Because of such large consumption of frying oils, the effect of high temperatures on the oils is of major concern both for product quality and nutrition, taking into account that dietary fat source deeply influences several biochemical parameters, especially of mitochondrial membranes. Virgin olive oil possesses specific features for modulating the damages occurred by endogenous and exogenous oxidative stress being particularly rich in antioxidant molecules (Gordon et. al., 2001; Montedoro et.al. 1993). However, Gomez-Alonso et al. (2003) evaluated the extent of modifications suffered by virgin olive oil following a shorttime deep fat frying procedure: vitamin E and phenolic compound as well as total antioxidant capacity decreased, while polar compounds increased. The intake of such altered oil mainly affected the hydroperoxide and TBARS contents of mitochondrial membranes which were enhanced after the dietary treatments. Also, several mitochondrial respiratory chain components (Coenzyme Q, cytochrome b, c C c1, and a C a3) are affected (Lamboni and Perkins, 1996). The concentration of hydroxytyrosol (3,4-DHPEA) and its secoiridoid derivatives (3,4DHPEA-EDA and 3,4-DHPEA-EA) in virgin olive oil decreased rapidly when the oil was repeatedly used for preparing french fries in deep-fat frying operations, at the end of the first frying process (10 min at 180 °C), the concentration of the dihydroxyphenol components was reduced to 50-60% of the original value, and after six frying operations only about 10% of the initial components remained. However, tyrosol ( p-HPEA) and its derivatives ( p-HPEA-EDA and p-HPEA-EA) in the oil were much more stable during 12 frying operations. The reduction in their original concentration was much smaller than that for hydroxytyrosol and its derivatives and showed a roughly linear relationship with the number of frying operations. The antioxidant activity of the phenolic extract measured using the DPPH test rapidly

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diminished during the first six frying processes, from a total antioxidant activity higher than 740μmol of Trolox/kg down to less than 250μmol/kg. On the other hand, the concentration of polar compounds, oxidized triacylglycerol monomers (oxTGs), dimeric TGs, and polymerized TGs rapidly increased from the sixth frying operation onward, when the antioxidant activity of the phenolic extract was very low, and as a consequence the oil was much more susceptible to oxidation. The loss of antioxidant activity in the phenolic fraction due to deep-fat frying was confirmed by the storage oil and oil-in water emulsions containing added extracts from olive oil used for 12 frying operations (Go´mez-Alonso et al. 2003). In a similar study carried out by Andrikopoulos et. al. (2002) on virgin olive oil, sunflower oil and a vegetable shortening were used as cooking oils for the deep-frying and pan-frying of potatoes, for eight successive sessions, under the usual domestic practice. Several chemical and physicochemical parameters (acidic value, peroxide value, total polar artefacts, total phenol content and triglyceride fatty acyl moiety composition) were assayed during frying operations in order to evaluate the status of the frying oils, which were found within expected ranges similar to those previously reported. The oil fatty acids were effectively protected from oxidation by the natural antioxidants. The frying oil absorption by the potatoes was quantitated within 6.1–12.8%, depending on the oil type and the frying process. The retention of - and ( + )-tocopherols during the eight fryings ranged from 85– 90% (first frying) to 15–40% (eighth frying), except for the ( + )-tocopherols of sunflower oil, which almost disappeared after the sixth frying. The retention of total phenolics ranged from 70–80% (first frying) to 20–30% (eighth frying). Tannic acid, oleuropein and hydroxytyrosol-elenolic acid dialdeydic form showed remarkable resistance in all frying sessions in both frying methods, while hydroxytyrosol and hydroxytyrosol-elenolic acid were the faster eliminated. The deterioration of the other phenolic species account for 40–50% and 20–30% for deep-frying and pan-frying, respectively, after three to four frying sessions, which are the most usual in the household kitchen. Deep-frying resulted in better recoveries of all the parameters examined. (Andrikopoulos et. al. 2002).

Oleuropein Figure 13. Some antioxidant phytochemicals in Olive oil.

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DRYING Drying is a popularly way of preserving leafy vegetables which are easily perishable, and not available all year round in tropical Africa for future use (Akindahunsi and Oboh, 1998; Oboh and Akindahunsi, 2004). However, many reports have consistently revealed that drying alters the nutritional and antioxidant potentials of food (Akindahunsi and Oboh, 1998; Oboh and Akindahunsi, 2004, Oboh, 2005b; Oboh et.al. 2005). Sun-drying of green leafy vegetables is popularly practiced in many homes in Nigeria, as a way of preserving green leafy vegetables for future use. Oboh and Akindahunsi (2004) reported that that sun-drying of some Nigerian green leafy vegetables [Struchium sparjanophora (Ewuro-odo), Amarantus cruentus (Atetedaye), Telfairia occidentalis (Ugu), Baselia alba (Amunu tutu), Solanum macrocarpon (Igbagba), Corchorus olitorus (Ewedu), Vernonia amygdalina (Ewuro) and Ocimum gratissimum (Efinrin)] will leads to a significant increase (P<0.05) in the total phenol content (6.45-223.08% gain), reducing property (16.00-362.50% gain) and DPPH free radical scavenging ability (126.00-5757.00% gain) of the green leafy vegetables, while the process caused a significant (P < 0.05) decrease in the Vitamin C content (16.67-64.68% loss), a significant decrease (P < 0.05) in Vitamin C content caused by sun- drying will not reduce the antioxidant activity of the green leafy vegetable, moreover, the phenol constituent of the green leafy vegetables appear to contributes more to the antioxidant properties of vegetables than ascorbic acid, as its increase on sun-drying cause a significant (P < 0.05) increases in the antioxidant properties of the green leafy vegetables, irrespective of the decrease in the ascorbic acid content.

Solanum macrocarpon

Vernonia amygdalina

Amaranthus cruentus

Figure 14. Some tropical green leafy vegetables.

Fruits could also be dried under the sun or in a solar and an artificial dryer, as a way of preserving them for future use when they are out of season (Komiyama and Tsujji, 1987; Marder and Schomaker, 1995). A combination of solar and artificial drying has been used to provide high quality products (Kitagawa and Glucina, 1984). Fresh persimmon (Diospyros kaki L.) is not available all year around it is usually preserved by drying for future use, Park et. al. (2005) reported that when fresh persimmons were subjected to two different processes: sun-drying during 1 month and dehydration at 60oC during 12 h, it was found that the contents of dietary fibers, minerals and trace elements in fresh and dried persimmons fruits were comparable. The polyphenols content and the total radical scavenging activities (TRSAs) are higher in fresh persimmons than in dried fruits; however, both variables are also

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high in dried persimmons; when fresh fruits are not available, proper dried persimmons could be used as a valuable substitute. Herbs, regardless of the purpose they serve, are used as fresh or dried. Enzymatic processes during drying fresh plant tissues may lead to significant changes in the composition of phytochemicals (Jambor and Czosnowska, 2002). When the herbs of lemon balm, oregano, and peppermint were analysed immediately after harvest and after drying to determine their antioxidant activity and content of total phenolics, L-ascorbic acid, and carotenoids. The strongest inhibition of linoleic acid (LA) peroxidation was found for fresh and dried oregano (Capecka et. al. 2005). For peppermint and lemon balm it was significantly lower and decreased after drying. The ability to scavenge the free radical DPPH (2,2-diphenyl-1picrylhydrazyl) was very high in almost all tested samples, exceeding 90%. The three species tested had a very high content of total phenolics and drying of oregano and peppermint resulted in their considerable increase. The highest content of ascorbic acid was determined in fresh peppermint and lemon balm and carotenoid content was at a similar level in all the species tested. Drying caused great losses of these compounds (Capecka et. al. 2005).

IRRADIATION One of the problems associated with the use of spices and herbs is their possible contamination by harmful bacterial, particularly spore-forming bacteria. Although spices and herbs are microbiologically stable products owing to their low moisture content, once they come into contact with water-rich food products such as meat or soup, microorganisms quickly develop (Van den Berg et. al. 1999). Several decontamination methods exist, but the most versatile treatment among them is processing with ionizing radiation. In addition to strict hygiene in preparation, an irradiation dose of 8-10 kGy is particularly valuable as an end product decontamination procedure not affecting sensory, nutritional, and technical qualities (Farkas, 1998). There are not many reports of the influence of irradiation procedures on antioxidant activity of herbs and spices. Murcia et. al. (2004) recently reported that 1, 3, 5, and 10 kGy irradiation of dessert spices (anise, cinnamon, ginger, licorice, mint, nutmeg, and vanilla) did not cause a significant change in the water extractable antioxidant activity as typified by their free radical scavenging activity. Likewise, Farag and Khawas (1998) also reported that γ-Irradiation at 10 kGy and microwave treatments of anise, caraway, cumin and fennel did not cause a change in the antioxidant activity of the essential oils. However, essential oils extracted from the γ-irradiated fruits are more effective as an antioxidant in sunflower oil than those produced from microwaved fruits. Conversely, when sun-dried and dehydrated paprika samples were irradiated at doses from 2.5 to 10 kGy, the capsaicin, dihydrocapsaicin and homodihydrocapsaicin content increased significantly by about 10% in samples irradiated at a dose of 10 kGy (Topuz and Ozdemir, 2004). 10 kGy γ-irradiation treatment nine aromatic herbs and spices (basil, bird pepper, black pepper, cinnamon, nutmeg, oregano, parsley, rosemary, and sage) as reported by Calucci et. al.(2003) resulted in a general increase of quinone radical content in all of the investigated samples, and in a significant decrease of total ascorbate and carotenoid content of some spices. Furthermore, Suhaj et. al. (2005) recently reported that exposure of black pepper to γ-irradiation at doses from 5 to 30 kGy caused some significant changes thiobarbituric acid-reactive substances

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(TBARS) production. Difference between non-irradiated and irradiated samples at the legal European limit dose of 10 kGy reached, on average, 23% and, at the Food and Drug Administration (FDA) 30 kGy limit, 33%. Irradiation affected significantly the DPPH radicalscavenging activity and reducing power of ground black pepper extracts.

Capsaicin

Dihydrocapsaicin

Homodihydrocapsaicin Figure 14. Structure of some antioxidant phyttochemicals in spices.

Mushroom has recently become attractive as functional food; it was considered a source for biological and useful substances. Agaricus blazei Murrill (Agariaceae), Brazilian mushroom, is currently popular food in Taiwan, and sold on the market with various product types. This mushroom was reported to possess antitumor and immunomodulating activities (Kawagishi et al., 1989). The isolated mushroom polysaccharides could stimulate lymphocyte T-cells in mice (Mizuno et. al., 1998). Recently, A. blazei is used for the prevention of cancer and/or as an adjuvant with cancer chemotherapy drugs after the removal of a malignant tumor (Ishihara,1999). Many animal studies and clinical experience have demonstrated that A. blazei showed antitumor activity, immunological enhancement, and also the fungus was effective in treating AIDS, diabetes, hypotension, and hepatitis (Mizuno, 2002). During

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postharvest handling, storage and marketing, A. blazei is prone to microbial contamination and insect infection, which result in quality deterioration and economic loss. Recently Huand and Mau (2005) preserved Agaricus blazei Murrill (Agariaceae) by exposing it 2.5,5,10,15 and 20 kGy dose of gamma irradiation and assessed their effect on and the antioxidant properties of its methanolic extracts of the mushroom. At 7.5 and 10.0 mg/ml, antioxidant activities of methanolic extracts from 2.5 to 20 kGy g-irradiated A. blazei were significantly higher than those of methanolic extract from the non-irradiated control. At 0.5– 7.5 mg/ml, reducing powers of methanolic extracts from A. blazei with 2.5,10,15 and 20 kGy of irradiation and without irradiation were comparable. All methanolic extracts showed excellent scavenging abilities of 95.2–100.7% against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals at 0.5 mg/ml. With regard to the scavenging ability against hydroxyl radicals, unirradiated and γ-irradiated A. blazei were comparable. Total phenols were the major naturally occurring antioxidant components found in the range of 18.77–21.48 mg/g. That indicates the unirradiated and irradiated A. blazei were good in antioxidant properties. Summarily,up to 20 kGy of irradiation did not remarkably affect the amounts of total antioxidant components in A. blazei.

CONCLUSION This write up has clearly shown the effect of some commonly food processing techniques both at household level or unit operation in industrial food processes on the antioxidant phytochemicals and activity. Obviously, virtually all food processing techniques will reduce vitamin C content of food, while same argument will not hold for phenol. Furthermore, it will be wrong to think that, since most food processing techniques will bring about a marked decrease in the composition and biological activity of many food nutrients, that food antioxidant will suffer same. Phenolic compounds (flavonoids inclusive) have been reported to be a more potent antioxidant than either vitamin C or E, in some cases during processing it increases to the extent that it will shield the effect of the decrease in vitamin C and this will consequentially results in an increased antioxidant activity most especially during the cooking and frying of some plant foods. Moreover, since the observed biological activity of many antioxidants are either additive or synergistic, it makes it difficult to just generalized the effect of a certain unit operation on antioxidant activity of a food, rather it will be necessary to generate data for each type of food subjected to food processing techniques and the gross effect of changes in the individual antioxidant phytochemicals on the total antioxidant capacity of the food. Furthermore, in some cases high concentration of some antioxidant phytochemicals can turn out to be prooxidant. These gross inconsistent in the behaviour of antioxidant phytochemical, posses a great challenge to food processors both at household and industrial levels to strike compromise between palatability and nutrition, health and body cells protection. Finally, although antioxidant research is very active all over the world, however, there still very limited data on the antioxidant properties of food from tropical Africa and the effect of traditional processing techniques conventionally used in this part of the world on the antioxidant property vis-à-vis their bioavailability.

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ACKNOWLEDGEMENT The Authors wish to acknowledge the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Brazil and Third World Academy of Science (TWAS) for granting Dr G. Oboh Post-Doctoral fellowship tenable at Biochemical Toxicological Unit of the Department of Chemistry, Federal University of Santa Maria, Brazil, during which period this review was put together.

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In: Leading Edge Antioxidants Research Editor: Harold V. Panglossi, pp. 65-87

ISBN 1-60021-274-3 © 2007 Nova Science Publishers, Inc.

Chapter 4

ARE TEAS THE UNIVERSAL ANTIOXIDANTS Justyna Ostrowska, Wojciech Luczaj and Elzbieta Skrzydlewska* Department of Analytical Chemistry, Medical University of Białystok, 15-230 Białystok, P.O. Box. 14, Poland

ABSTRACT Cellular metabolism is accompanied by generation of free radicals, which also play pivotal role in the action of numerous xenobiotics. Their increased production is a primary event in many human diseases progression or a secondary consequence of tissue injury. Detoxification of radicals in the cell is provided by the antioxidant defense system. Many synthetic and natural antioxidants are useful when antioxidant systems are no able to cope with radicals action. Among natural antioxidants, the most popular beverage consumed worldwide is tea. Tea is manufactured in three basic forms: green, oolong and black, which are non-, partially- and full-fermented/oxidized, respectively. Therefore, green tea composition is very similar to that of the fresh leaves and contains considerable amount of monomeric polyphenols, particularly catechins. The major components of black tea are multimeric polyphenols - theaflavins and thearubinins – the oxidation products and condensates of catechins. Considerable amount of the original polyphenolic compounds are contained in oolong tea. Monomeric polyphenols possess proved antioxidant properties wich are manifested in the ability to prevent oxygen radical formation, by inhibiting the activity of enzymes participating in their generation as well as in the ability to scavenge free radicals and to chelate transition metal ions. Recently, antioxidant properties of multimeric polyphenols of black tea have also been proved. The antioxidant properties of teas polyphenolic compounds have led to considerable interest in the potential health benefits resulting from teas consumption. Evidence has been collected that teas polyphenols are metabolized extensively and are distributed to all tissues in animal organisms. Metabolites of catechins and their condensates have been also revealed to possess antioxidant properties comparable to their parent compounds. Significant enhancement in tissues antioxidant capacity has been demonstrated following the consumption of teas by animals and humans. In consequence the protective effect of teas polyphenols on DNA, lipids and proteins appears to be very promising in reducing *

Corresponding author: Elżbieta Skrzydlewska, Department of Analytical Chemistry, Medical University, 15-230 Białystok, P.O. Box. 14, Poland, /fax 48 85 7485707, e-mail: [email protected]

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Justyna Ostrowska, Wojciech Luczaj and Elzbieta Skrzydlewska the incidence of many diseases. It was indicated that tea consumption leads among others to decrease in cancer risk but its mode of action is still unclear. Teas polyphenols can block the formation of mutagens and carcinogens from precursor and increase their detoxification. Moreover they influence molecular events at the gene level. However, despite of proved antioxidant and cancer chemoprotective properties of tea catechins, attention has been also drawn to their prooxidant action appearing in oxidative damage of cellular and isolated DNA.

Keywords: green tea, black tea, oolong tea, composition, antioxidative properties, metabolism, prooxidant action

INTRODUCTION Tea infusions consumed by 2/3 of the world’s population are prepared from tea dried obtained from the leaves of a plants called Camellia sinensis and Camellia assamica. Tea is regarded as a tasteful drink, but the scientific community has recently re-discovered the therapeutic potential of this beverage. Useful consideration of the physiological and pharmacological effects of the tea requires background information concerning production, leaves composition, availability of the various types of tea and most importantly the chemical changes that take place during the manufacture of the commercial products. Consumption patterns of different types of the tea should also be noted. Tea is manufactured in three basic forms: green, black and oolong tea [1]. Green tea is prepared by dehydration of Camellia sinensis or assamica leaves what precludes the oxidation of contained polyphenols, therefore green tea, contains high concentration of monomeric polyphenols from the catechins group. However black tea, obtained by tea leaves fermentation, is oxidized and contains mainly multimeric polyphenols, whose biological activity has not been fully documented yet. The third form of tea is oolong tea, which is a partially oxidized product. Out of approximately the 2.5 milion metric tons of the manufactured dried tea, 76-78% is black, 20-22% green and less than 2% oolong tea [1].

TEA PRODUCTION AND TEA BEVERAGE COMPOSITION Tea leaves composition varies with climate, season, agricultural practice, variety, and the age of the leaves, i.e. the position of the leaves on the harvested shoot. Fresh tea leaves contain: polyphenolic compounds, carbohydrates, proteins, lignin, ash, amino acids, lipids, organic acids, chlorophyll as well as carotenoids and volatile substances (Table 1) [1]. Polyphenols make up the largest and most important from the biological point of view, group of tea leaves components. However catechins constitute the main group of polyphenols content in tea leaves (Table 2). The most important catechins contained in tea leaves are epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), and epicatechin (EC) [1]. Catechin concentration is highly dependent among others on the leaves age. The leaves bud and the first leaves are richest in EGCG. The basic catechins structure consists of two aromatic rings (ring A and ring B) linked through three carbons that form an oxygenated heterocycle – pyran ring (ring C) (Fig. 1). Two configurations are possible but

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most of the catechin mass is in the so-called epi- form. Three hydroxyl groups present on the B ring characterize gallocatechins. Another variation results from esterification of the OH group on the pyran ring with gallic acid. In the last years, as a result of development of more sophisticated separation and identification techniques, other catechins and related products have been identified in fresh tea leaves but little quantitative data have been reported. These include present in smaller amounts: gallocatechin, epigallocatechin digallate, 3methylepicatechin gallate, catechin gallate, and gallocatechin gallate [2]. Moreover a novel group of compounds called chalkan-flavans has also been identified. These are biomolecular combinations of a catechin attached to a chalcone derivative. Another group of flavanols present in tea leaves are bisflavanols (known as theasinensis) [1]. These are dimeric gallocatechins linked by C-C bonds at the B rings. Dimmeric proantocyanidins are also present in fresh tea leaves. These are condensation products of the catechins linked by C-C bonds between an A ring and a pyran ring. Another group of polyphenols found in tea leaves are flavonols [2]. They occur both in the free state and as glycosides. Their structures are analogous to those of the flavanols but represent a different state of oxidation. The next group of tea components is depsides, which are condensation products of two different hydroxyl [1] acids. Theogallin that is unique compound derived from gallic and quinic acids is one of them. Tea leaves contain also free gallic acid which may enter into interesting oxidation reactions during the manufacture and quinic acid forming from its inclusion in tea depsides. Tea leaves contain also amino acids including an unusual amino acid known as theanine, which constitutes about half of the total amino acids content. However protein fraction of green tea leaves includes the enzymes normally associated with plant cell metabolism e.g. enzymes responsible for catechin synthesis have been identified [3]. The greatest interest is tea polyphenol oxidase, which catalyzes the aerobic oxidation of the catechins. In the growing plant this enzyme is physically separated from the polyphenol substrates contained in the leaf cell vacuoles and moreover its activity is greatest in the youngest leaves. Other enzymes contained in tea leaves are: glucosidases which catalyse the hydrolysis of several aroma precursors, lipoxidases which are responsible for the generation of volatile aldehydes and enzymes responsible for methyl xanthine synthesis [1]. The popularity of tea is partly due to the presence of moderate amounts of caffeine (2,5%-4,%). Other methyl xanthines, theobromine and theophyline, are also present but in very small quantities, 0.1 and 0.02%, respectively. Fresh leaves contain also derivates of carbohydrate such as trigalloylglucose. It contains five galloyl groups bound to the glucose molecule with four or five additional galloyl groups attached by depside linkages. Trigalloylglucose has not been observed in manufactured green or black tea, perhaps because of hydrolysis to the free acid and glucose. Tea leaves contain also small amounts of carotenoid which are important precursors of tea aroma. Violaxanthine, β-carotene, neoxanthin and lutein are among those identified. The volatile fraction of fresh green tea is extremely small but important in determining the acceptance of tea beverage. More than 60 volatile components have been identified in fresh tea leaves. They include alcohols, carbonyls, esters, acids and cyclic compounds. These fractions are greatly augmented in variety and quality during tea manufacture. The mineral content of tea is roughly similar to that of most plants but tea tends to accumulate aluminum and manganese. Tea is relatively rich in potasium, calcium, magnesium and fluoride [1,4]. As mentioned, tea is manufactured in three basic forms: green, black and oolong [1]. The term green tea refers to the products manufactured from fresh leaves preventing oxidation of the polyphenolic components. The manufacturing steps include plucking, rapid enzyme

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inactivation by steaming or pan firing, rolling, and high temperature air drying. In such conditions glycosides of aromatic and terpene alcohols found in the growing leaves are rapidly hydrolyzed after plucking to form free volatile alcohols, however most of polyphenols do not change their chemical structure. Therefore dried green tea leaves composition is similar to that of the fresh tea leaves considering its major components. Careful manufacture results in a light yellow-green infusion exhibiting virtually no polyphenols oxidation. Green tea beverage composition varies with the origin of the leaves and with manufacturing conditions (Table 3). The black tea production steps include only plucking, withering, maceration (rolling) and finally drying. The withering process results in disrupting the cell structure of the leaves and initiation of the fermentation process. In the black tea production process, about 75% of catechins contained in the tea leaves undergo enzymatic transformation consisting in oxidizing and partial polymerization [5,6]. Because the main enzyme taking part in these processes is tea-leave polyphenol oxidase it is essential to assure its direct contact with polyphenols and with atmospheric oxygen. Monophenol monooxidase (tyrosinase) and an odiphenol: O2 oxidoreductase take part in the process together with polyphenol oxidase [7]. Quinones, which are the substrates of consecutive transformations, are produced at the first stage of tea leaves enzymatic oxidation [8]. The resulting catechin quinones react with each other in many ways. The quinone derived from a simple catechin or its gallate may react with a quinone derived from a gallocatechin or its gallate to form seven-membered ring compounds known as theaflavins (Fig. 2). The mechanism of theaflavins formation is presented in Fig. 3. The theaflavins are dimeric catechins. A characteristic element of their structure is the seven-member benzotropolone ring, which is formed by the oxidation of the B ring of either (-)-epigallocatechin or (-)-epigallocatechin gallate, loss of CO2 and simulatenous merger with the B ring of a (-)-epicatechin or (-)-epicatechin gallate molecule [9]. Four theaflavines are formed in these transformations: theaflavin (TF1), theaflavin 3gallate (TF2A), theaflavin 3’-gallate (TF2B), and theaflavin 3,3’-gallate (TF3) [10]. Other benzotropolone compounds were also identified in black tea, but in considerably smaller amounts, compared to the theaflavins [10,11,12]. Among them there are theaflavic acids formed in reactions of catechins quinone and gallic acid quinone. Gallic acid is not a substrate for tea polyphenol oxidase but its quinone can be generated by reaction with quinones derived from some of the catechins [1]. The content of theaflavic acids in black tea is relatively small and this fact suggests that they undergo further transformation in the oxidizing process. Other black tea benzotropolone components are compounds called theaflagallins synthesized as the result of mild oxidizing of gallocatechins and gallic acid mixtures [1]. The formation mechanism of these compounds is not well known but they may be formed by the condensation of two quinones both originating from trihydroxy molecules in contrast to the requirements for the formation of the theaflavins and the epitheaflavic acids. The recently discovered compound is theadibenzenotropolone A, in which the two pyrogallol type rings in EGCG are oxidatively coupled to the catechol type ring B of a EC molecule [14]. Gallocatechin quinones, both in the free or gallated forms, may couple to form a number of bisflavanols that are catechin dimers without seven-membered ring which is characteristic for theaflavins [7]. The bisflavanols, also called theasinensins occur in small amounts in black tea as they probably undergo further oxidation. A new component has been reported recently; it is the product of EGCG oxidation and is called dehydrotheasinensin A. It is equivalent to a hydrated o-quinone of the theasinensin A and is easily converted to theasinensin A and

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theasinensin D by oxidation-reduction dismutation [15]. It has been suggested that most of the catechin mass is transformed, in the process of black tea manufacture, to less structurally defined red-brown compounds - thearubigins with a molecular weight distribution of 1000 40000 [7]. The structures and the formation process of these compounds have not been fully explained yet (Fig. 4). The high oxidation potential of some of the catechin quinones generated at the first fermentation stage is probably the reason of theaflavins, bisflavanols and epitheaflavic acids production and the subsequent incorporation of their oxidation products into the thearubigin formation. (Fig. 5). Both flavanols and depsides – the esters of two or more phenol acids – can participate in this process [1]. This hypothesis is supported by the fact that the content of these compounds decreases during prolonged fermentation [16]. In addition to the already mentioned components, black tea contains flavonols [17]. The most important black tea flavonols are myricetin, quercetin, kaempferol, and rutin mainly occurring as glycosides [18]. At least 14 glycosides of myricetin, quercetin and kaempferol [19], proanthocyanidins [20,21], which are not affected by phenol oxidase, have been recently found in black tea [22]. The contents of individual flavonols in black tea leaves are: myricetin 0,24-0,52 g/kg, quercetin 1,04-3,03 g/kg and kaempferol 1,72-2,31 g/kg [23]. Phenolic acids should also be added to the list of the black tea components, mainly caffeic, quinic and gallic acids, which are methylxanthines primarily in the form of caffeine. Black tea also contains about one third of the caffeine contained in coffee, amino acids including theanine, which occurs in the tea leaves only, and many flavour compounds [24]. The resulting black tea composition depends on the technological process of its production. It is difficult to state a definitive composition for black tea beverage as it varies. An approximate percentage of black tea components in its solid extract and its brew has been presented in table 4 [1]. While during black tea production oxidation is promoted, oolong tea is only partially oxidized product. The manufacture of oolong teas allows only for a short period of oxidation. The process is carried out in several different ways and products vary with respect to degree of catechins oxidation. Normal oolong tea is considered to be fermented only in 50% [25]. Oolong tea extracts contain catechins at the level of 8-20% of the total dry matter [1]. Oolong tea composition would be expected to be intermediate between green and black teas (Table 5). However large number of new polyphenols have been isolated and identified from oolong tea. These include: epigallocatechin esters (p-hydroxybenzoyl and cinamyl) [1], compounds called oolong theanin, which are probably formed by oxidation of one of the B rings of a theasinensin [1], dimeric catechins having methylene bridges between the two pyran rings, designated oolonghomobisflavins [14], epigallocatechin gallate esterified on the pyran ring with ascorbic acid [14] and a series of eight dimeric proanthocyanidins composed of the common catechins [14]. These compunds have not been found in the other two main tea types. However not known all black and green tea components have been found in oolong tea. The techniques practiced in oolong tea production, i. e., controlled partial oxidation, should allow for the production of teas with preferred compositions for organoleptic or pharmaceutical purposes. Major components of green, black and oolong tea beverage were shown in Table 6.

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METABOLISM AND BIOAVAILABILITY OF TEAS POLYPHENOLS In order to understand the mechanisms of action of teas polyphenols, it is important to understand the bioavailability and biotransformation of these compounds, especially catechins and theaflavins in the small intestine and the liver. Recently, the bioavailability of polyphenolic compounds and their distribution in the body have been investigated applying sensitive and accurate methods to analyze metabolites in biological fluids and tissues [26]. It is known that monomeric polyphenols are extensively metabolized either in tissues, once they are absorbed through the gut barrier, or, for the non-absorbed fraction and the fraction re-excreted in the bile, by the colonic microflora (Figure 6). Polyphenol metabolism begins already in mouth, where conversion of EGCG to EGC and presumably ECG to EC take place. It is probably caused by microbial catechins esterase [27]. Separation of the other gallolyl groups occurs in the small intestine [28]. Next free hydroxyl groups of the catechins are coniugated with glucurocic acid, sulphate, glycine or O-methylated what occur in jejunal and ileal sections of the small intestine. The arising catechin derivatives are transported into the liver where not modified ones are conjugated (methylated, suphated and glucuronidated). The capacity of the above catechins biotransformation is particularly active in the liver although it is known that the small intestine plays a major role. The formation of anionic derivatives by conjugation with glucuronides and sulphate groups facilitates their urinary and biliary excretion and explains their rapid elimination. The derivatives of catechins are with the blood stream distributed from the liver to all organs and secreted to the duodenum. In the blood catechins occur mainly as conjugates [28]. After green tea consumption substantial amounts of EGC and EC were found in the esophagus, large intestine, kidney, bladder, lung and prostate, while lower amount were observed in liver, spleen, heart and thyroid [26,29]. However EGCG levels were higher in the esophagus and large intestine, but lower in other organs. Unlike rats, mice had higher lung concentrations of EGCG than EGC and comparable liver concentrations of EGCG and EGC, what suggests relatively higher bioavailability of EGCG in mice than rats. The amounts of EGCG in particular in rat organs are presented in Table 7. In human substantial amounts of catechins were detected in plasma, colon mucosa and prostate surgical samples from patients who consumed tea 12 h prior to surgery [26]. The rats experiments revealed that concentration ratio of EGCG and EGC in the serum was similar to the ratio of their concentration in the administered green tea, which confirms similarity in metabolism of these two compounds [30]. Catechins that are not absorbed in the small intensine, as well as conjugated catechins excreted in the bile, reach the large intestine where they may be metabolized by colonic bacteria and next absorbed. In the colon, bacterial enzymes may deconjugate or break down catechins into more simple compounds. Under the influence of intestine bacteria unabsorbed EGCG may be broken down 4-hydroxybenzoic acid, 3,4-dihydroxybenyoic acid, 3-methoxy4-hydroxy-hippuric acid and vanillic acid [30]. These compounds were identified in human urine [30]. The catechins metabolis is the most frequently examined by trite marked EGCG It has been shown recently that (3H)EGCG may be degraded by microorganisms in the intestinal of human and animals leading to the formation of 5-(3’,4’-dihydroxyphenyl-γ-valerolactone and 5-(3’,4’,5’-trihydroxyphenyl)-γ-valerolactone (Figure 7). These metabolites are the ring fusion products of EGC and EC, respectively. Both are next metabolized like catechins. They (mainly in the glucuronide and sulphate form) have been detected in human urine and plasma.

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In some individuals the amounts of this compounds in urine were several fold higher than their respective precursors [30]. Many studies in humans and rats indicated that EGCG is mainly excreted through the bile while EGC and EC are excreted through urine and bile [29], which is consistent with the observation that EGC and EC, but not EGCG are recovered from human urine samples (Fig. 7) [26]. It has been revealed that 47-58% of the initial amount of green tea catechins gets into the urine and only about 0.1-2% from this amount makes not changed catechins [30]. Both the metabolism and bioavailability of black tea should be considered to be able to estimate its influence on metabolic processes occurring in animal and human organisms. Though metabolism and bioavailability of major antioxidants of green tea - catechins in animal and human organisms are relatively well established, little information concerning bioavailability and biotransformation of black tea polyphenols. This is due to difficulties in quantitative determination of specific components of black tea – theaflavins or thearubigins in urine or blood. However many investigations unanimously confirm increase in catechins levels in plasma after black tea ingestion [31,32, 17]. It has been shown that the level of catechins in plasma, urine and feces constituted about 1,68% of the total content of those components found in the black tea solution [33]. The results may suggest that catechins of black tea are not very well absorbed in human organism or are rapidly metabolized. A conclusion may be also drawn that free catechins are available in human organism more readily than their gallates.

ANTIOXIDATIVE PROPERTIES OF TEAS The principal hypothesis associated with the putative health benefits of tea is linked to the antioxidant properties of their constituents – polyphenols [34]. The antioxidant capacity of teas and their polyphenols has been assessed by several methods [35,36,37,38]. The Oxygen Radical Absorbance Capacity (ORAC) assay measures the relative efficiency of antioxidants in protecting the fluorescent protein β-phycoerythrin against peroxidation. This value is related to that of the water-soluble vitamin E analogue - Trolox (6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid). Using this assay, Cao et al. [39] found that both green and black tea have much higher antioxidant activity against peroxyl radicals than vegetables such as garlic, kale, spinach and Brussels sprouts. Using the Ferric Reducing Ability of Plasma (FRAP) assay [40] it was found that the total antioxidant capacity of green tea was more potent than that of black tea. FRAP assay is chemical assay that determines the antioxidant activity present in extracts. This assay determines the ability of extracts to reduce ferric-2,4,6-tri(2-pyridyl)-s-triazine complex (Fe3+-TPTZ complex) into the ferrous form. The ferrous-TPTZ complex has an intense blue colour and has an absorption maximum at 593nm [40]. Using another the Trolox Equivalent Antioxidant Capacity (TEAC) assay, Rice-Evans et al. [37] ranked epicatechin and catechin among the most potent of 24 plant-derived polyphenolic flavonoids, which they evaluated. The TEAC assay is a spectrophotometric assay that determines the relative efficiency of an antioxidant or prepared extract to quench the ATBS (2,2’-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) radical action. This value is related to the quenching efficiency of the water-soluble vitamin E analogue Trolox and the TEAC values are expressed as Trolox equivalents. It should be stressed that the antioxidant

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capacity of polyphenols determined in vitro is dependent upon the type of assay employed and does not reflect factors such as bioavailability and metabolism. There are many findings concerning relationships between antioxidant activity of tea polyphenols and their chemical structure [41]. All catechins have 3,5,7,3’,4’-pentahydroxy polyphenolic structure (Ryc. 1). Many studies have reported on qualitative structure relationships between catechin-mediated antioxidant activities and have stressed the importance of the catechol moiety (O-dihydroxy) in the B-ring, and the additional presence of 3-, 5- and 7-hydroxyl groups in the catechin molecular structure necessary to achieve an efficient antioxidant action [18]. However the 3-OH group on the C ring is the additional site of antioxidant property [42]. The antioxidant activity responds broadly to the tenet that the structures with the most hydroxyl groups exert the greatest antioxidant activity. It was shown that the antioxidant potentials of the tea catechins, against radicals generated in the aqueous phase are, in order of decreasing effectiveness, as follows ECG≅EGCG>EGC>gallic acid>EC>C [42]. It was shown that catechins antioxidant abilities are significantly higher than antioxidants such as vitamin C and vitamin E (Table 8) [44]. In the case of antioxidant potentials in the lipophilic phase, the sequence of effectiveness of the individual components is ECG≅EGCG≅EC≅C>EGC>gallic acid>EC>C [43]. Moreover it was found that the total green tea polyphenols extract (44% of the dry weight of the green tea preparation) shows a total antioxidant activity accounting for 90% of the antioxidant activity of the tea preparation [45]. Independently of catechins, also theaflavins and thearubigins being multimeric polyphenols contained in black tea reveal antioxidative properties. It was found that antioxidant activities of theaflavins and thearubigins are higher than ascorbic acid, glutathione, and α-tocopherol, but considerably lower than the activity of EGCG, ECG and EGC [40]. Because antioxidatve properties of polyphenols depend on the amount of hydroxyl groups within their molecules, theaflavins as dimers of catechins seem to be stronger antioxidants. Therefore TF3 have been proved to show up higher antioxdative activity than EGCG which is the strongest antioxidant among all catechins and a precursor of TF3 [46,47]. The antioxidant activity for theaflavins and catechins changes as follows TF3>ECG>=EGCG>=TF2B >=TF2A>TF1>=EC>EGC [47]. The present comparisons indicate at least that theaflavins are at least as effective as their precursors. More specific investigations concerning theaflavins structure have shown that depending on the amount and position of hydroxyl groups within their molecules, antioxidative properties change in the following way: TF3>TF2> TF1 [48]. The amount of gallate moieties influence the increase in antioxidative properties owing to which, theaflavins gallates demonstrate stronger antioxidative properties compared with free theaflavins [23]. Antioxidative properties of main tea polyphenols - catechins and theaflavins are manifested particularly by their abilities to inhibit free radical generation, chelate transition metal ions and scavenge free radicals [18]. They have been found to reveal stronger antioxidative properties than typical antioxidants such as glutathione, ascorbic acid or αtocopherol [49,50]. Catechins as well as theaflavins possess the ability to prevent free radical generation, via inhibiting activity of enzymes participating in their generation, xanthine oxidase in particular, enzyme which catalyses oxidation of hipoxanthine and xanthine to uric acid accompanied by oxygen reduction to superoxide radical and hydrogen superoxide [51]. Green tea extract as

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well as catechins and TF3 effectively inhibit xanthine oxidase activity, preventing reactive oxygen species generation in this way [52]. Moreover catechins as well as theaflavins inhibit the activity of inducible nitric oxide synthase (iNOS), preventing another free radical – nitric oxide (NO) generation in this way [52]. Studies on RAW 264.7 mice macrofages have shown that theaflavins and epigalocatechin gallate effective inhibit iNOS [26]. The black tea polyphenols reagarding to this effect was in order of decreasing affectiveness: 3,3’-theaflavin digallate (TF3) > epigalocatechin gallate (EGC) > the mixture of 3-theaflavin gallate (TF2A) i 3’- theaflavin gallate (TF2B) > thearubigins > theaflavin (TF1). In addition solution of black tea containing 1,4 μg of theaflavins has been found to diminish in this way the generation of nitric oxide (NO) by 70% [53]. Catechins also reveal ability to inhibit the activity of mieloperoxidase (MPO) an enzyme which catalyzes the reaction between hydrogen peroxide and chloride ions with reactive hypochloric anions generation [54]. Moreover black, green and oolong tea have been also proved to inhibit the activity of cyclooxygenase COX-2 and 5,12- i 15-lipoxygenase, enzymes participating in enzymatic lipid peroxidation [55,56]. Tea polyphenolic compounds can chelate transition metal ions like iron and copper to prevent their participation in Fenton and Haber-Weiss reactions which in turn preclude the generation of hydroxyl radicals and degradation of lipid hydroperoxides causing reactive aldehydes formation [57,58]. The ability to chelate metal ions mainly such as Fe3+ and Cu 2+ changes in the following way EGCG>EGC>ECG≅EC [59,60]. A likely metal binding site for catechins is the o-3’,4’-OH group on the B ring [26]. It has been revealed that three ECG molecules are necessary to bind two Fe3+ ions, whereas one ion Fe3+ can be chelated by one molecule of EGC or EGCG. Theaflavins as well as catechins show metal chelating abilities [60]. Polyphenols due to generation of complexes with iron, cause reduction of its absorption in gastrointestinal tract. Black tea has been demonstrated to inhibit iron absorption more effectively than green tea [61], but bounding non-haem iron occurs only in the presence of ascorbic acid [61]. Many studies have demonstrated that both catechins and teaflavins besides preventing free radical generation have strong free radical-scavenging abilities both in vitro and in vivo [62]. The conversion of catechins to theaflavins during processing tea leaves does not affect their radical-scavenging potency. Moreover, it was shown that the galloyl moiety of catechins and theaflavins was essential for their scavenging ability [42]. This additional group increases the total number of phenyl hydroxyl groups and improves the ability of the gallate-containing catechins and theaflavins more able to donate a proton due to the resonance delocalization. Ability to scavenge free radicals is partially influenced by value of standard one-electron reduction potential, which is characteristic to a particular chemical compound. Lower catechins reduction potential indicates that less energy is required for hydrogen or electron donation, what is an evidence for their strong scavenging properties. Catechins and teaflavins reduction potential values found in those of black tea are similar to the vitamin E, but higher than those of vitamin C which is one of the strongest free radicals scavenger (Table 9) [57]. Considering standard one-electron reduction potential value polyphenols may scavenge generated in the organism free radicals such as hydroxyl, superoxide and lipid radicals [44]. The exact mechanisms for the radical scavenging activity of catechins are not known, but several structures appear to be important in conferring this activity. It was found that catechol (1,2-dihydroxybenzene) is a potent scavenger of free radicals, whereas phenol (hydroxybenzene) is not, indicating that the catechol in ring A is indeed important for the

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scavenging free radicals. All catechins have at least one orthodihydroxyl group (o-3'4'-OH) in the B ring, which participates in electron delocalization and stabilizes the radical form the gallocatechins (EGC and EGCG) have a trihydroxyl group in the B ring (3'4'5'-OH), while the catechin gallates (ECG and EGCG) have a gallate moiety esterified at the 3rd position in the C ring, adding three more hydroxyl groups [63]. Both the presence of the 3'4'5'-OH group and the gallate moiety have been associated with increased antioxidant activity. Recent studies examining oxidation products of gallocatechins with peroxyl radicals indicate that the 3'4'5'OH group in the B ring is the principal site of antioxidant reaction in EGC and EGCG. The superoxide scavenging abilities of EGC and EGCG are stronger that those of (+) C, EC and EGC but EGC and EGCG are more effective scavengers of the hydroxyl radicals than (+) C, EC and EGC [44]. Therefore an ortho-trihydroxyl group in the B ring and the galloyl moiety may be important in scavenging the superoxide anion and the hydroxyl radicals, respectively. The 3-OH group on the C ring generates an extremely active scavenger [58]. The 5-OH and 7-OH groups may also add scavenging potential in certain cases. Additionally, a study of the oxidation products of gallocatechins with H2O2 indicated that the A ring may also be an antioxidant site for EGC and EGCG [42]. Possible scavenging mechanisms of free radicals by catechins is presented in Figure 8. TF3, TF2, TF1 and EGCG have been found, however to be able to scavenge superoxide radical (O2.-), but TF3 acts the most effectively [52]. In addition theaflavins have been proved to react with superoxide radical over 10 times faster than EGCG [64]. Investigations in vitro have shown that solution of black tea is able to scavenge also other reactive oxygen species such as singlet oxygen (1O2) and hydroxyl radical [52]. Even in cases of very large quantities of ingested tea, the concentrations of catechins and other polyphenols in human blood plasma are from 100 to 1000 times lower than the concentrations of other physiological antioxidants such as ascorbate or glutathione (Table 10) [57]. Though the physiological amounts of tea polyphenols are not high in the organism in comparison with the amounts of endogenous antioxidants (Table 10), their participation in trapping free radicals is significant [44]. Abilities of tea catechins to scavenge DPPH radical, superoxide anion and hydroxyl radical are shown in Table 11. In majority of the examined systems, EGCG was more efficient radical scavenger than EGC, EGC, EC and C [44]. Stable radicals such as the DPPH radical and the ATBS.+ radical cation have also been used to evaluate the scavenging abilities of polyphenols in vitro. Using the TEAC assay, catechins and theaflavins were found to be more effective in reducing the ATBS.+ radical cation than vitamin E and vitamin C [44]. Relative free radicals scavenging abilities among the catechins determined by the TEAC assay have been found to be EGCG >ECG > EGC > EC. The influence of pH on scavenging activity of tea catechins was also studied [43]. The results obtained with DPPH radical at different pH suggest that the radical scavenging efficiency is considerably stronger in neutral to alkaline regions (pH 10). The most efficient scavenger of DPPH radical in all pH regions is EGCG (Table 12). Catechins, have been found also to efficiently scavenge .NO in vitro and it was shown that green tea was about five times more potent .NO scavenger than black tea [26]. It should be mentioned that explanation of tea extract antioxidant activity couldn’t be based only on the activities of the present polyphenols. It can base on antioxidant activity tannins too. It was shown that green tea nonpolyphenolic fraction had significant antioxidant activity, depending on concentration, which shows down linoleic acid oxidation. Pheophytins showed higher antioxidant activity than α-tocopherol and green tea catechins.

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Referring to different composition of different kinds of tea hydrogen-donating ability of tea extracts, measured by 1,1-diphenyl-2-picrylhydrazyl radical - DPPH scavenging, decreased in the following order green>black>oolong (Table 13) [65]. Oolong tea is semifermented and gallocatechins i.e. epigallocatechin and epigallocatechin gallate, which are the strongest free radical scavenger [66], are the first to be oxidized by polyphenols oxidases in the leaves because of their high oxidation potential and high concentration [67]. Oolong tea contains smaller values of gallocatechins but at the same time does not yet contain a great amount of theaflavins and thearubigens, which are found in fully fermented black tea. However black tea contains also gallic acid, a potent hydrogen donator to the DPPH radical [23].The water-soluble solids of oolong tea contained significantly less total polyphenols than green and black tea, with the latter kinds of tea having approximately the same total polyphenol contents (Table 8). This can explain the low DPPH radical scavenging ability of oolong compared with green and black tea (Table 8). However it was found that oolong tea scavenged the DPPH radical better than green and black tea [68].

CONSEQUENCES OF TEAS ANTIOXIDATIVE ACTION The past few years have been rich in information coming from laboratories all around the world concerning the positive impact of teas on human health. Much interest has centered on the role of teas antioxidant in regard to the health benefits of tea consumption, as antioxidant properties of teas polyphenolic compounds have been studies in vitro because it has been known in vitro antioxidant properties of teas polyphenolic compounds. Moreover it has been proved that teas polyphenols they significantly prevent from oxidative modifications of biologically important cellular components such as lipids, proteins and nucleic acids due to their multidirectional antioxidant action [26]. Protective action of teas is connected with the ability of catechins to scavenge free radicals including the most active hydroxyl radical, which may initiate lipid peroxidation. Therefore polyphenols decreasing the concentration of hydroxyl radicals and lipid free radicals terminate the initiation and propagation of lipid peroxidation. Polyphenols also chelate metal ions especially iron and copper which in turn preclude the generation of hydroxyl radicals and degradation of lipid hydroperoxides causing reactive aldehydes formation. Free radicals as well as reactive aldehydes generated during lipid peroxidation causes modification of proteins [69]. The decrease in free radicals and reactive aldehydes level induced by polyphenols may explain the resulting restoration of normalization of lipid peroxidation and decrease in protein oxidation process. The suppression of the increase in protein carbonyl group and bistyrosine formation as well as normalization of the amount of sulphydryl groups and tryptophan residues in different situation oxidative stress generated by alcohol intoxication, cigarette smoke or aging were observed after green as well as black tea administration [70]. Moreover topical administration of EGCG and green tea extract on the mouse skin inhibits single and multple UVB irradiation-induced protein carbonyls formation, by about 70%, [71]. Green tea extract in d.w. also resulted in partially inhibits of protein carbonyl formation enhanced after single and multiple UV exposures [71]. Teas polyphenols, especially catechins, which are water-soluble antioxidants, can reduce the mobility of free radicals in the lipid bilayer as well. Consequently the decreased

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membrane fluidity results in inhibition of lipid peroxidation due to a slower pace of free radical reactions. They can penetrate the lipid bilayer influencing antioxidant capability in biomembranes. Catechins preferentially enter the hydrophobic core of the membrane where they exert a membrane stabilizing effect by modifying the lipid packing order. They can also conserve the α-tocopherol content and delete the lipid peroxidation when membrane phospholipids are exposed to oxygen radicals from the aqueous phase. The oxidative attack from the aqueous phase seems to be an important reaction for initiating membrane lipid peroxidation and perhydroxyl radicals are regarded the most feasible radicals for initiating lipid peroxidation in vivo. It has been shown that teas extracts as well as individual tea components, which also cross blood-brain barrier, protect liver erythrocytes and brain lipids phospholipids against oxidative modifications [72,73]. Oxidative damage to these biomolecules has been implicated in the pathology of a number of chronic diseases, including cardiovascular diseases, cancers and neurodegenerative diseases [26]. Numerous epidemiological studies have addressed the relationship between teas consumption and the incidence of cardiovascular diseases in humans. It has been shown that teas may display a protective role against cardiovascular diseases via a number of different mechanisms, which are determined by their antioxidative properties. The main mechanism involved in the causation of coronary heart disease is the oxidation of LDLcholesterol leading to damage of the vascular system and the heart [74]. As a result of LDLcholesterol oxidation, monocytes are recruited to the arterial wall and monocyte-derived macrophages accumulate the excessive amount of oxidised LDL and become lipidladen foam cells [75]. It has been confirmed that theaflavin digallate (TF3) and to a lesser degree theaflavin, epigallocatechin gallate, epigallocatechin or gallic acid prevent LDL oxidation [76,77]. Moreover studies testing the antioxidant effect of teas polyphenols on LDL and VLDL oxidation indicate that e.g. a lipiprotein-bound antioxidant activity of EGCG has greater than that of tocopherol [78]. Black tea extract also increases the resistance of LDL to oxidation in a concentration dependent manner [79], but at low concentrations, tocopherol is more effective [80]. Theaflavin digallate pretreatment of macrophages or of endothelial cells from mice inhibits, in a concentration- and time- dependent manner, the cell mediated oxidation of LDL [64]. Catechins may change (suppress as well as inhibit) the proliferation of smooth muscle cells of bovine aorta which produces connective tissue leading to luminal narrowing and sclerosis of the arteries [60]. Some experiments in vitro, ex vivo as well as in vivo however, did not confirm the inhibiting influence of black tea on LDL oxidation [76,77]. Independently of the influence of teas polyphenols on oxidation of lipid fraction of LDL these compounds affect the protein part of LDL. The in vitro examination revealed that (+)catechin protects from oxidation of histydyl and lizyl residues of apolipoprotein B-100 contained in LDL [80,81]. Extracts of oolong tea, as well as, though to a lesser degree those of green and black, have been demonstrated to reveal antihiperlipidemic effect [30]. It has been also shown that drinking tea reduces the levels of serum lipids, including cholesterol and triglicerides in animals models and in humans affecting lipid metabolism in this way [30]. It is suggested that the green tea reveals the strongest action [30], although some suggestions have been made about stronger activity of oolong tea [30]. However in another study it was shown no effect of green tea consumption on HDL-cholesterol and trigliceryde level [82]. Reduced absorption of food components was absorbed in rats, which consumed green tea independently of direct influence on the metabolism of lipids. It has been revealed that protein absorption is reduced

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by about 6%, absorption of fats is reduced by oolong tea the most effectively (by about 10%), whereas green and black tea do not cause statistically significant changes in absorption of fats [30]. Teas have recently obtained also significant acceptance as a cancer preventive measure. Epidemiological and laboratory studies have revealed that EGCG as well as green or black tea extract given in drinking water inhibited carcinogenesis in various organs in humans and rodents [83]. Tea consumption appears to decrease cancer risk but its mode of action is still unclear. The mechanisms of carcinogenesis show that induction of cancer involves a sequence of steps leading to clinical cancer. The critical action is transformation of normal cells by genotoxic carcinogens (chemicals, radiation or viruses), which affect specific codons in DNA and represent a somatic mutation of oncogenes or tumor suppressor genes. Teas and their polyphenols inhibit the biochemical activation of genotoxic procarcinogens and formation of mutagens and carcinogens [84]. The major enzyme system responsible for the metabolism of procarcinogens into their DNA-binding metabolites is cytochrome P450. Their binding to DNA is considered essential for tumor initiation. The addition of EC, EGC, ECG, EGCG and green tea extract to microsomes prepared from rat liver resulted in a dose-dependent inhibition of cytochrome P450 [26]. It has been also shown, that teas, black tea in particular, inhibiting cytochrome P450 1A1 activity decreases activation of benzo[a]pyrene (BaP), procarcinogen which participates in DNA damage [85]. Teaflavins, catechins as well as black tea extract were found to act strongly in this process. However it has also been found that drinking green or black tea causes significant increase in cytochrome P450 1A1, 1A2 and 2B1 activities in the liver of rats but changes in 2E1 and 3A4 activities have not been observed [84]. Phase II detoxifying enzymes promote the excretion of potentially toxic or carcinogenic chemicals. Glutathione S-transferases are a family of phase II enzymes that catalyze the conjugation of glutathione to electrophiles, generally reducing their ability to react with nucleic acids and proteins. 0.2% green tea polyphenols supplied in the drinking water to mice for up to 30 days significantly increased glutathione S-transferase activity in liver and small intestine [26]. Feeding rats on green tea leaves (2.5%) also significantly increased liver glutathione Stransferase activity [26], while injecting EGCG into the portal vein of rats increased dose dependently total glutathione S-transferase activity [26]. Moreover tea increases carcinogen detoxification through the induction of higher levels of another phase II enzyme - glucuronyl transferase [26]. Genotoxic carcinogens metabolism and also oxidative processes enhanced during carcinogenesis in cells lead to the formation of reactive oxygen species that alter DNA. Accumulation of free radicals in cells and resulting modifications in DNA structure, enzymatic activity and defence mechanisms all influence the development of the cancer pathogenesis [91]. One key indicator of such alterations is the presence of 8hydroxydeoxyguanine in hydrolyzed fractions of DNA. Studies on RL-34 cell line revealed also that polyphenols contained in teas inhibiting oxidative stress caused by the activity of hydrogen peroxide or tert-buthyl hydroperoxide contribute to decrease of 8-OHdG level which is the most important marker of oxidative DNA damage [56,85]. Similar foundlings have been obtained in experiments on rats, in which after 10 days lasting administration of black tea polyphenols by gavage significantly decreased 8-OHdG generation in colon mucosa induced by the colon carcinogen, 1,2-dimethylhydrazine [86]. Remarkable decrease in 8OhdG level was also found in smokers drinking black tea [87]. Green as well as black tea has

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been recently reported to prevent oxidative DNA damage induced by iron in Jurkat T cells [88,89]. Also oolong tea in in vivo conditions protected against formation of 8-OdG in rats which were exposed to factors induced oxidative stress [90]. Free radicals DNA modification can result in the formation of neoplastic cells [26]. Many factors e.g. transcription factors participate in this process. EGCG effects the action of tumor promotors on transcription factors such as AP-1 or NF-κB, leading to control of the activity of transforming growth factors TGF-α and TGF-β [26]. Moreover the signal transduction cascade including the factors NFκB and AP-1 is redox-regulated and is therefore sensitive to the oxidant/antioxidant status of the cell [91]. NF-κB is a complex of proteins that binds to DNA and activates gene transcription. In unstimulated cells, NF-κB is present in the cytoplasm bound to an inhibitory protein called IκB. A wide variety of stimuli for example inflammantory cytokines and endotoxins may result in the phosphorylation of IκB by IκB kinase, which results in the ubiquitination and degradation of IκBby the proteosome complex. Released NF-κB is able to translocate to the nucleus where it binds DNA and activates the transcription of multiple inflammatory and other genes. Many of the stimuli that active NFκB also induce oxidative stress, and there is some evidence that NF-κB activation is stimulated by free radicals and inhibited by antioxidants [92]. Teas polyphenols have been found to inhibit the activation of NF-κB. In activated macrophages and epidermal cells treated with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), EGCG were found to inhibit phosphorylation and prevent IκB from translocation to the nucleus and binding to DNA [93]. In cultured intestinal epithelial cells, EGCG was found to be the most potent inhibitor of I-κB kinase activity among green tea catechins, with an IC50 (concentration required for 50% inhibition) approximately 18 μM [93]. Recently, EGCG treatment (1 to 10 μM) was found to inhibit proteosome activity of cultured tumor cells, resulting in increased cytosolic accumulation of the IκB-α subunit, which is expected to decrease NF-κB activation [26]. Activator protein –1 (AP-1) is another transcription factor that is affected by the intracellular redox environment and can be affected by both free radicals and certain antioxidants [92]. AP-1 activation is of interest to cancer researchers because high AP-1 activity appears to play a role in tumor promotion of breast, skin and lung cancer [93]. In epidermal cell lines, green tea catechins, especially EGCG have been found to inhibit AP-1 activity induced by UV light, the tumor promoter, TPA and a mutant H-ras gene [93,95]. Topical EGCG has also been found to inhibit UV-induced AP-1 activation in vivo in a transgenic mouse model [96]. In skin cells, catechins appear to inhibit AP-1 activity by inhibiting kinases. It has been reported that EGCG and theaflavin-3-3’-digallate block AP-1, a signal transducer initiating the development of skin carcinogenesis [84]. It has been also proved that the result of incubation of 21BES cells with TF3 (10-20μM) solution results is inhibition of c-jun protein phosphorylation which in turn causes inhibition of transcription factor AP-1 activity which plays a crucial role in transformation and proliferation of cells [97]. Subsequent steps in the development of neoplasia involve the growth control of the early neoplastic cells. The active ingradients in tea can decrease effectively these sequences. The affect of promoters involves blockage of cellular growth control messages through gap junctions. Studies with liposomes have shed light on the mode of action of EGCG on the protein kinase activator, an enzyme related to the cell activation process in the promotion of tumors. EGCG inhibits interactions between proteins and ligands by sealing effect and

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prevents their binding [98]. EGCG and ECG stimulate gap junctional intercellular communication and prevent inhibition by the tumor promoters, mechanisms that plays an important role in the promotion of cancer [84]. Recent research findings indicate that tea polyphenols can protect against the multistage of cancer initiation, promotion and progression. A number of mechanisms concerning tea anticancer actions have been presented as well as results suggesting that the gallate structure of catechins is important for inhibition of growth of tumor cell lines by these compounds. Many studies have been conducted to investigate the effects of tea consumption on human cancer incidence [5,82]. Studies with three human lung cancer cell lines (H661, H1299 and H441) and one colon cancer cell line (HT-29), showed that the potency of inhibiting cell growth by green tea polyphenols had the rank order of EGCG = EGC > ECG > EC [82]. The potency of theaflavin-3–3’-digallate was similar to that of EGCG and higher than that of theaflavin-3 (3’)-gallate, which was still higher than theaflavin. The growth inhibitory activity of green tea extracts appeared to be due to the summation of activities of EGCG, EGC, ECG and EC. These catechins, together with the theaflavins, may account for most of the growth inhibitory activity of black tea extracts. The growth inhibitory activity of thearubigens, the major components of black tea, is not known. The inhibition of EGCG against skin, stomach, colon, and lung carcinogenesis as well as the growth of human prostate and breast tumors in athymic mice have been demonstrated. Theaflavins have been shown to inhibit lung and esophageal carcinogenesis [99]. The activity of EGC has not been tested in carcinogenesis models, but short-term studies showed that it has about the same antiproliferative activity as EGCG. In many studies, black tea has comparable or slightly lower inhibitory activities. The results suggest that the remaining catechins in black tea, the theaflavins, and other components in black tea all contribute to the cancer inhibitory activity. During the last years it has been proved that tea shows protective action also when free radicals generation is enhanced. One of such examples is aging, a process referring to all animals including humans, in which increase in free radicals generation and disturbances in antioxidant abilities are observed [100]. These changes lead to oxidative damage of cellular components [26]. However the increase in the amount of protein and lipid modifications in aging leads to changes in their functions or in expression of their function [101,102]. It has been shown that administration of green tea to rats in different age partially prevents changes in antioxidant parameters including antioxidant enzymes as well as non-enzymatic parameters like GSH or vitamins (Fig. 9). Moreover green tea partially protects lipid and protein against disturbances observed during aging so it may prevent development of many diseases of old age. Metabolism of many xenobiotics including ethanol frequently is accompanied by free radicals generation [103]. Moreover ethanol intoxication causes changes in the antioxidant abilities of different tissues manifested by decrease in the activity of superoxide dismutase, glutathione peroxidase and catalase activity and increase in activity of glutathione reductase as well as by decrease in the level of reduced glutathione, vitamin C, A and E and β-caroten is observed [104]. In consequence alcohol caused the increase in lipid peroxidation products, measured as thiobarbituric acid reactive substances. Green as well as black tea protect the liver, brain and blood antioxidant system against changes observed after ethanol intoxication [105-108]. Teas protect also proteins and membrane phospholipids from oxidative modifications [109]. These results indicate beneficial effect of teas in alcohol intoxication.

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Thus teas have been regarded as one of the most promising chemopreventive agents without any harmful effects. However despite of the proved antioxidant properties of the main components of teas studies of the last years on several human groups and case-control have indicated significant positive relationship between green tea consumption and cancers of various organs. Furthermore, recent study demonstrated that green tea extract does not inhibit but rather tend to increase the incidence and multiplicity of colon tumors in the post-initiation period in an azoxymethane or dimethylhydrazine-induced rat carcinogenesis model [110]. It has been found that catechins, those used in high non-physiological concentrations in particular, may induce oxidative damage both isolated and cellular DNA. Moreover it has been shown that as a result of incubation of human bronchial epithelial 21BES cell lines with TF3, increased generation of H2O2 takes place, which as a consequence leads to apoptosis of these cells after 8-12 hours [97]. Later experiments confirmed that tea polyphenols in some circumstances can act as prooxidants and a possible mechanism of oxidative DNA damage by catechins has been recently suggested [111]. It is speculated that catechins undergo autooxidation mediated by copper ion (II) what lead to generation of copper ion (I) and semiquinone radical. Copper ion (I) reacts with oxygen to generate superoxide anion and subsequently hydrogen peroxide (H2O2). Formed copper ion (I) binding to DNA interacts with hydrogen peroxide (H2O2) resulting in the formation of reactive oxygen species such as copper-hydroperoxo complex Cu(I)OOH. This complex binds hydroxyl radical, which can be released and cause DNA damage. The .OH released from a bound hydroxyl radical immediately attacks an adjacent constituent of DNA, e.g. thymin residue, before it can be scavenged by .OH scavengers. The oxidized form of catechin, such as semiquinone radical or benzoquinone, undergoes nonenzymatic reduction by NADH, resulting in the formation of redox cycle to abundantly produce the reactive oxygen species. Recently it has been also demonstrated that 1,2benzoquinone is converted directly into 1,2-benzenediol through a non-enzymatic twoelectron reduction by NADH. NADH, a reductant existing at high concentrations (100-200 μM) in certain tissues could facilitate the catechin-mediated DNA damage observed in physiological conditions [112]. Moreover it is suggested that epicatechin is oxidized more easily than catechin and that the quinone form of epicatechin is reduced by NADH more easily than that of catechin. These differences can be explained by steric effect of OH group at 3-position of their C ring. Moreover it has been stated that copper ions (II) may also catalyze oxidation of the polyphenols (quercetin and myrcetin),which are contained in some kinds of tea, leading to DNA damage and 8-OhdG generation [113]. These data suggest that independently of antioxidative properties of teas they can act also as prooxidants. Thus the question “Are teas an universal antioxidants” has not been answered yet.

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[98] Fujiki, H., Suganuma, M., Okabe, S., Sueoka, K., Suga, K., Imai, K., Nakachi, K. and Kimura, S. (1999). Mechanistic findings of green tea as cancer preventive for humans. Proc. Soc. Exp. Biol. Med., 59, 225-228. [99] Yang, T. T. C. and Koo, M. W. L. (1997). Hypocholesterolemic effects on chinese tea. Pharmacol. Res., 35, 505-512. [100] Luczaj, W., Waszkiewicz, E., Skrzydlewska, E., and Roszkowska-Jakimiec, W. (2004). Green tea protection against age-dependent ethanol induced oxidative stress. J. Toxicol. Environ., 67, 595-606. [101] Augustyniak, A., Waszkiewicz, E. and Skrzydlewska, E. (2005). Preventive action of green tea from changes in the liver antioxidant abilities of different aged rats intoxicated with ethanol. Nutrition, 21, 925-932. [102] Dobrzyńska, I., Sniecinska, A., Skrzydlewska, E. and Figaszewski Z. (2004). Green tea modulation of the biochemical and electric properties of rat liver cells that were affected by ethanol and aging. Cell. Mol. Biol. Lett., 9, 709-721. [103] Ostrowska, J., Luczaj, W., Kasacka, I., Rozanski, A., and Skrzydlewska, E. (2004). Green tea protects against ethanol-induced lipid peroxidation in rat organs. Alcohol, 32, 25-32. [104] Baltaziak, M., Skrzydlewska, E., Sulik, A., Famulski, W. and Koda, M. (2004). Green tea as an antioxidant which protects against alcohol induced injury in rats - a histopathological examination. Folia Morphol. (Warsaw.), 63, 123-126. [105] Skrzydlewska, E., Ostrowska, J., Stankiewicz, A. and Farbiszewski R. (2002). Green tea as a potent antioxidant in alcohol intoxication. Addict. Biol., 7, 307-314. [106] Yen, G. C., Ju, J. W. and Wu, C. H. (2004). Modulation of tea and tea polyphenols on benzo(a)pyrene-induced DNA damage in Chang liver cells. Free Radic. Res., 38, 193200. [107] Oikawa, S., Furukawa, A., Asada, H., Hirakawa, K. and Kawanishi, S. (2003). Catechins induced oxidative damage to cellular and isolated DNA through the generation of reactive oxygen species. Free Radic. Biol. Med., 37, 881-890. [108] Yoshino, M., Haneda, M., Naruse, M., Murakami, K. (1999). Prooxidant activity of flavonoids: copper-dependent strand breaks and the formation of 8-hydroxy-2’deoxyguanosine in DNA. Mol. Genet. Metab., 68, 468-472. [109] Choi, E. J., Chee, K. M. and Lee, B.H. (2003). Anti- and prooxidant effects of chronic quercetin administration in rats. Eur. J. Pharmacol., 482, 281-285.

In: Leading Edge Antioxidants Research Editor: Harold V. Panglossi, pp. 89-144

ISBN 1-60021-274-3 © 2007 Nova Science Publishers, Inc.

Chapter 5

RADIOPROTECTIVE EFFECTS OF ANTIOXIDANTS Mustafa Vecdi Ertekin* and Orhan Sezen Department of Radiation Oncology, Atatürk University, Faculty of Medicine, 25240, Erzurum, Türkiye

ABSTRACT Radiation therapy (RT) is known to be one of most important tools to cure cancer. In recent years, more patients with cancer have been treated with radiation therapy. Because most of them are surviving, there is an increasing need to patients care for RT-induced toxicities. Since X-ray was discovered by Wilhelm Roentgen in 1895, radiation-induced toxicities, such as dermatitis, mucositis, myelosuppression, and tissue’s fibrosis, have been well-known. These toxicities may lead to the delays in administration or to the dosage limitations in radiation therapy, to the increased hospitalisation stays and the costs, and it may have adverse effect on radio-curability of cancer and patient survival. The destructive action of ionizing radiation is mainly due to reactive oxygen species (ROS), including superoxide anion radical (O˙2¯), hydroxyl radical (OH˙), and hydrogen peroxide (H2O2), generated by the decomposition of water, which constitutes around 80% of the cell. These ROS formed in cells contribute to radiation injury in cells, such as damage to cellular DNA and membrane structures, and alterations in the immune system. Although all respiring cells are equipped with protective enzymes such as SOD and CAT or GSH-Px, increased oxidative stress in cells stemming from ionizing radiation may overwhelm the protective systems, leading to cell injury. In this point, there is a question whether oral or parentheral antioxidants might administer concurrent with RT, or not, because some of scientists consider that concurrent administration of oral antioxidants is contraindicated during RT, since antioxidants might reduce oxidizing free radicals created by radiation therapy, and thereby decrease the effectiveness of this treatment. However, previous studies have demonstrated that the use of antioxidants, such as vitamin E and gingko biloba, during radiation therapy might improve the efficacy of radiation therapy by enhancing tumour response and decreasing some of the toxicity towards normal cells. *

Address for correspondence: Dr. Mustafa Vecdi Ertekin, Atatürk Üniversitesi Tıp Fakültesi, Radyasyon Onkolojisi ABD, 25240 Erzurum, Türkiye, E-mail: [email protected], [email protected], Tel: +90 442 2361212/1608, Fax: +90 442 2361301

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Mustafa Vecdi Ertekin and Orhan Sezen Antioxidant defence mechanisms involve strategies both enzymatic, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) and non-enzymatic, such as vitamins C and E, selenium, zinc, gingko biloba, and melatonin. They work in synergy with each other and against different types of free radicals, and they can offer protection against ionizing radiation-induced oxidants. Therefore, antioxidants might use as a radioprotectant agents. An ideal radioprotectant is one that protects normal tissue while maintaining antitumor effectiveness, and is itself without moderate or severe toxicity. This article reviews the current status of antioxidants as a radioprotective agent in radiation therapy.

INTRODUCTION Reactive oxygen species (ROS) are generated by normal cellular metabolism and exogenous agents during aerobic metabolism [1]. Excess ROS are cytotoxic, leading to cell death, mutations, chromosomal aberrations, or carcinogenesis and may also damage cellular components [1,2]. Oxidative stress arises when rates of ROS production outpace rates of removal [3]. Oxidative stress may cause the damage on cells as a result of one of three factors: 1) an increase in oxidant generation, 2) a decrease in antioxidant protection, or 3) a failure to repair oxidative damage [4]. Radiation is a known producer of reactive oxygen species (ROS). When water, which constitutes around 80% of the cell, is exposed to ionizing radiation, decomposition occurs through which a variety of ROS, such as the superoxide radical (O˙2¯), the hydrogen peroxide (H2O2) and the hydroxyl radical (OH¯) are generated. These ROS formed in cells contribute to radiation injury in cells. Although all respiring cells are equipped with protective enzymes such as superoxide dismutases (SOD) and catalase (CAT), glutathione peroxidase (GSH-Px), increased oxidative stress in cells stemming from ionizing radiation may overwhelm the protective systems, leading to cell injury [5]. One of the indices of oxidative damage is the malondialdehyde (MDA) formation as an end product of lipid peroxidation [3]. Because of the serious damaging potential of ROS, cells depend on the elaboration of the antioxidant defence system (AODS), both enzymatic and non-enzymatic oxidant defence mechanisms [6]. Common antioxidants include the vitamins A, C, E [7,8] amifostine [9,10], zinc [5,11,12], selenium [13], melatonin [3,14,15], and the others [16-18] and the enzymes SOD, CAT and GSH-Px [7,19]. They work in synergy with each other and against different types of free radicals [7], and they can offer protection against ionizing radiation-induced oxidants. In this point, there is a question whether oral or parentheral antioxidants might administer concurrent with RT, or not, because antioxidants might reduce oxidizing free radicals created by radiation therapy, and thereby decrease the effectiveness of this treatment. However, previous studies have demonstrated that the use of antioxidants, such as vitamin E, zinc, gingko biloba and the others [20-24] during radiation therapy might improve the efficacy of radiation therapy by enhancing tumor response and decreasing some of the toxicity towards normal cells. Therefore, antioxidants might use as a radioprotectant agents.

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An understanding of these free radical defenses provides a scientific basis for the use of with antioxidants during cancer treatments [25]. In this chapter, we will debate on radiotherapy, its effect mechanisms on the cancer and healthy cells and the current status of antioxidants as a radioprotective agent in radiation therapy.

1. RADIOTHERAY 1.1. The Definition of Radiotherapy Radiotherapy (RT) is a clinical treatment modality, which ionized beams or atom particles are used in treatment of malignant neoplasms or occasionally in treatment of selected benign diseases [26,27]. The goal of RT is to deliver completely measured doses of ionizing radiation to a defined tumor volume with the minimum accepted injurious effects of ionizing radiation to surrounding healthy tissue by eliminating tumor cells, giving a high quality of life and prolongation of survival at reasonable cost to cancer patients [26,28]. For this purpose, RT is used ionizing radiations in the form of electromagnetic radiation (X-rays, gamma rays) or of particulate radiation (beta particles, electrons, protons, neutrons, negative pi-meson, heavy ions with high energy) [26]. RT is used by itself or together with other treatment methods such as chemotherapy (CT) and surgery in the form of primary treatment, combined treatment modality, adjuvant treatment and palliative treatment. Primary RT, especially on radio-curable tumors, is a treatment method where primary treatment is done by using RT. The goal is to provide cure. Early stage Hodgkin’s disease, glottic area of larynx cancers, nasopharynx cancers, CNS germ cell tumors, cervix carcinomas and skin cancers can be treated quite successfully by RT. Primary treatment is administered by RT in many patients with cancers when patient, with early stage disease, does not give permission for the surgery or when the surgery is risky [26,29]. Combined treatment is a treatment combination where the cure can be provided only when two or more treatment modalities are used together. Both treatment modalities in this treatment contribute equally for keeping the patience alive. RT is used as combined treatment modality together with surgery or CT in patients with late stage cancers. If RT is applied as combined treatment modality, it is able to be administered as pre-operative or post-operative. When RT is applied prior to surgery (called neo-adjuvant radiotherapy), surgery is performed 3-4 weeks after radiotherapy. Neo-adjuvant RT is administered to shrink a previously inoperable tumor to a manageable size to enable surgical excision. RT can be used following surgery (called adjuvant radiotherapy) to destroy any cancer cells that were not removed by surgery and it is administered 2-4 weeks after surgery. The goal of adjuvant RT is to increase the controllability of late stage cancers that is able to do resection with radical surgery [26,28]. Palliative RT, in not curable patients, is a RT type used to eliminate many findings and symptoms that develop as a result of local or systemic effects of primary tumor and of metastases. The goal is to provide palliation and to improve the quality of life [26,28,29].

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X-rays produced or radiations emitted from radioactive source are applied as in the form of two applications. These are external (applied from outside and from a distance) and internal (from inside the body or from a very close proximity) radiotherapy applications.

Figure 1. A Simulator device. Simulation is the first of the procedures carried out when preparing the treatment. In simulation for external radiotherapy, the radiotherapist, in collaboration with the radiotherapy technician, must first of all establish the correct positioning of the patient on the treatment table. They will decide if and how to position immobilisation blocks that will accurately reproduce the position to be assumed by the patient at each treatment session. The next step is to determine the exact port of entry of the radiation the so called "fields": these will be directly drawn on the patient's skin using a marker pen. A simulator film takes to plan and calculate the treatment and to determine protection fields in the radiotherapy fields.

In external radiotherapy, X-ray source or radioactive source is outside the body and the source sends beams to the patient form a distance. All X-ray producing treatment devices (Kilovoltage and megavoltage treatment units, such as Linear accelerator machine), gammaray producing teletherapy treatment units (Cobalt 60 machine) and the treatment units radiating by emitting particles (Cyclotrons) are used as external treatment units. In internal radiotherapy (brachytherapy), radioactive source is placed on the skin (mold therapy), between tissues (interstitial implants) or in body cavities (intracavitary therapy). The most frequently used source in brachytherapy applications are C0-60, Cs-137, Ir-192 (radioactive iridium), Au-198 (radioactive gold), Sr-90 (radioactive stronsiyum), I-125 (radioactive iodine), Yt-90 (radioactive yitrium), Ra-226 (radium) and Cf-252 (Californium). These are in the form of disc, needle, hairpin, wire, seed, tube, disc, globe and cylinder. Dose measurement unit of radiation devices that apply X-ray or gamma-rays with teletherapy is Roentgen (R). One Roentgen is the amount of 2,58x10-4 Coulomb ion that is produced by X-rays or gamma-rays in one kilogram dry air. Absorbed dosage is the radiation energy absorbed by the tissue and its unit is Gray (gy). One Gray is one joule energy absorbed by one kilogram tissue. The activity unit of radioactive materials is Becquerel (Bq).

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Becquerel is the nuclear change (disintegration) number in one second. Equivalent dosage unit is Sievert (Sv). Sievert is used with the purpose of protection from radiation and corresponds to the relative biologic effect in human body caused by one gray radiation [26,27,29,32].

Figure 2. A Linear accelerator (LINAC). A LINAC is the device most commonly used for external beam radiation treatments for patients with cancer. The LINAC also can be used in stereotactic radiosurgery similar to that achieved using the gamma knife on targets within the brain. The LINAC uses microwave technology to accelerate electrons in a part of the accelerator called the "wave guide", and then allows these electrons to collide with a heavy metal target. As a result of the collisions, high-energy x-rays are scattered from the target. A portion of these x-rays is collected and then shaped to form a beam that matches the patient's tumor. The LINAC in our department produces 6 or 18 Million Volt (MV) high-energy x-rays and 3 to 21 Million electron Volt (MeV) electron (e-).

Table 1. Common and System Internationale (SI) Units for Radiation Quantities Quantity Dose

Traditional Units Röntgen (R)

Absorbed dose

Rad (rad)

Radioactivity

Curie (Ci) 1 Ci = 3.7x1010 disint / sn Rem (rem)

Dose equivalent

SI Units Coulomb/Kilogram (C / Kg) Gray (Gy) 1 Gy = 1 Joule / 1 Kg Becquerel (Bq) 1 Bq = 1 / s = s-1 Sievert (Sv) 1 Sv = 1 Joule / 1Kg

Relationship 1 C / Kg = 3876 R 1 R = 2.58x10-4 C / Kg 1 Gy = 100 rad 1 rad = 0.01 Gy = 1 cGy 1 Ci = 3.7x1010 Bq 1 Bq = 2.7x10-11 Ci 1 Sv = 100 Rem 1 rem = 0.01 Sv

disint / sn = disintegration per second; Joule / Kilogram = Joules per kilogram;1 / s or s-1 = per second

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Radical irradiation doses change depending on many factors such as histopathologic type, differentiation degree, size of tumor, residual tumor amount following chemotherapy or surgery and volume of the tissue to be irradiated. Conventional radiotherapy regimes (one fraction per day 180-200cGy, 5 treatments per week) are applied in many radiotherapy centers. Radical radiotherapy doses are around 60-70 Gy/30-35fr/6-7weeks for medium degree radioresistant tumors. It is the fraction type where radiotherapy dose of 180-200cGy is applied once in a day and 5 days a week per fraction. Treatment period is generally around 67 weeks and radiotherapy doses are about 60-70 Gy. This is the radiotherapy regime that is most frequently applied and accepted as a Standard due to the fact that it is convenient for work days and discovered as a result of many radiotherapists experience [26]. As a result the best suitable fraction should be chosen and applied based on type and properties of tumor and properties of regular tissues to increase therapeutic ratio and tumor clinical studies continue on fractionation, such as hyperfractionation and accelerated fractionation [26,32].

1.2. The Short History of Radiotherapy X-rays were discovered by German physician Wilhelm Conrad in 1895. Becquerel defined radioactivity in 1896 as a result of his observations and research on uranium element that exists in the nature. Marie and Pierre Curie isolated polonium (named after the hometown of Marie Curie) radium and other radioactive elements from uranium element. Cure that is related to radiotherapy in a patient with cancer was first reported in 1899. In the beginning of the 19th century when Becquerel forgot a container with 200 mg uranium in his pocket and left it for about 6 hours there the first radiobiological observation and experiment were done and skin erythema and ulcer were defined [26,28,30,31]. American scientist Coolidge pioneered the making of orthovoltage X-ray tube treatment units by inventing X-ray tube with heated catot in 1913. With the development of orthovoltage X-ray (200 kV) treatment units in 1922, malign tumors originated from tissues that are located not too deep were treated and cures were reported in radiosensitive and tumors that are located not too deep, such as Hodgkins disease. In 1934, Joliot Curie defined artificial radioactivity by reporting that when the nucleus of any element was bombarded with the particles inside the nucleus, it gains radioactive properties. Many radioactive materials were obtained artificially because of this invention. Because French researcher Coutard reported in 1934 that radiotherapy applied in multiple fractions were tolerated more than the radiotherapy applied in high dosages and at once, fractional (daily dosages) radiotherapy regimes used today were started. British researchers supplied deep tumors with more beam than neighboring healthy tissues by developing multiple beam collision techniques on deep tumors (multiple beam field beaming technique) and rotational beaming (beaming by rotating) techniques. As a result of development of radioactive cobalt (Co-60) treatment unit in Canada in 1951, the term of teletherapy treatment with megavoltage beams had started and beam dosages that could sterilize the tumor in deeper tissues without the limitation of the skin could be applied. Other linear accelerators that could produce megavoltage beams were developed in 1953, the linear accelerator targeted for treatment were first used in England.

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The structures of linear accelerators have had technical advances in the last 30 years and conformal radiotherapy treatment units have been developed. In addition, treatment planning has had important advances by the usage of diagnosis units, such as CT and MRI, in radiotherapy planning. Cancer treatments with neutron, proton, pi-meson and heavy ions have started in recent years and notable successes were obtained [26,28].

Figure-3. Computerize treatment planning system. It is used in the planning of conformal radiotherapy and other planning techniques for radiaotherapy and to calculate radiation dose in the treatment fields where radiotherapy is applied.

1.3. Physical Basis of Radiation Therapy Ionizing radiations excite and ionize atoms and molecules in the environments they pass through. They are in the form of electromagnetic waves and atomic particles. Electromagnetic waves are made up by energy stacks called photons. They either exist in the nature or are obtained artificially. Radiowaves, microwaves, infrared lights, ultraviolet lights, X-rays and gamma-rays are electromagnetic waves. Frequencies and wavelengths of electromagnetic waves are different however their speeds are the same. They travel at the speed of light in the space. When the wavelengths of electromagnetic lights decrease their energies increase and biologic effects appear. X-rays and gamma-rays have the minimum wavelengths. Radiations are absorbed completely or partially when they pass through material or biologic environments. Partial radiations that are not absorbed exit from the material or living habitat. While some do not collide with any atoms of the material they pass through, others collide, their energy decrease and continue to travel as dispersed light by changing direction.

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Absorption of the radiation occurs by excitation or ionization of atoms and molecules of the environment it passes through. • •

Excitation occurs when the radiation excites the motion of the atoms or molecules, or excites an electron from an occupied orbital into an empty, higher-energy orbital. Ionization occurs when the radiation carries enough energy to remove an electron from an atom or molecule.

Each type of radiation is also classified as to its directness (directly or indirectly ionizing) and its ionizing density (linear energy transfer). Charged atomic particles (electrons, alpha particles, beta particles, protons, negative pi-mesons, heavy ions with high energy) ionize atoms and molecules of the environment they pass through directly (atomic particles themselves) and cause chemical or biological damages. As for electromagnetic waves (X-rays and gamma-rays), they ionize by shooting the electrons that are going to do the ionization from the atoms of the environment they pass through. This type of ionization is called indirect ionization [26,29,32,33]. The amount of energy that the radiation transfers per unit of path length is called its linear energy transfer (LET) and is measured in units of MeV/µm. This feature reflects a radiation’s ability to produce biological damage. Radiation is classified as either high linear energy transfer (high LET) or low linear energy transfer (low LET), based on the amount of energy it transfers per unit path length it travels. Alpha, neutron radiation is high LET; beta, gamma radiation and X-rays are low LET. Some particles (alpha, neutrons) deposit more energy along the path they take through tissue than do x-rays or gamma rays, thus causing more damage to the cells they hit. This type of radiation is often referred to as high linear energy transfer (high LET) radiation. X and Gamma rays are indirectly ionizing radiation. Depending on its energy and the atomic number of the absorbing material, X and gamma rays interacts with an absorber atom by one of three primary mechanisms (photoelectric interaction, Compton scattering, and pair production), which results in the production of highly energetic electrons, which dissipate their energy by interacting with other atoms in their path in exactly the same manner as beta particles (which are electrons) and excite and ionize these atoms. Since the ionizations resulting from X and gamma radiation are due to electrons, gamma radiation is a low LET radiation [26,28,29,33-35].

1.4. Biologic Basis of Radiation Therapy The basic mechanism of deadly effect of ionizing radiation on cells is related to this DNA damages. Radiations that pass through biologic environments cause damages on DNA’s directly or indirectly. In direct radiation damages, the electron or charged particles shot forward causes snapping or breaking on DNA situated in the way of ionization. In indirect DNA damages, ionizing beam first creates some chemical structures (free radicals) in nucleus and these structures cause damage on DNA. For instance, beams cause ionization of water molecules by first removing electrons from water molecules of the cell. Since water molecule lost an electron ion converts into radical (H2O+ and a free electron) and reacts with another water molecule in 10-10 seconds to make H3O+ and free hydroxyl radicals (OH-). Free radicals are highly reactive molecules and they last about a few microseconds, hence they cannot

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travel much. However, if there is a DNA in 100 A (10nm) length regions, they cause breaking in DNA. The life expectancies and biologic effects of free radicals can be increased by oxygen molecules or electronaffinity molecules available in the environment or decreased by chemicals containing sulphydryl. Approximately 2/3 of X-ray related DNA damages in cells of mammals are estimated to be caused indirectly by free radicals that appear as a result of ionization of water and 1/3 of it is estimated to occur directly [26,33]. Table 2. Biologically Significant Free Radicals Reactive Oxygen Species O2·⁻

Superoxide radical

·OH ROO· H2O2 1 O2 NO· ONOO⁻

Hydroxyl radical Peroxyl radical Hydrogen peroxide Singlet oxygen Nitric oxide Peroxynitrite

HOCl

Hypochlorous acid

Another damage of radiotherapy in tumors and normal tissues is that it causes obstructive and vascular changes in time such as endarteritis in the arterioles of the tissues within the irradiated areas. As a result of this damage, growth, feeding and several functions of the tissue is prevented by the decrease in blood flow; atrophy and fibrosis develop in the tissues within the irradiated areas [26]. Biological damage by the indirect action of X rays occurs physical, chemical, and radiobiological basis of ionizing radiation as follows; 1. Primary photon interaction (photoelectric effect, Compton effect and pair production) produces a high energy electron (physical principle). 2. The high energy electron in moving through tissue produces free radicals in water (chemical principle). 3. The free radicals may produce changes in DNA from breakage of chemical bonds. The changes in chemical bonds result in biological effects (radiobiological principle). Radiotherapeutic decisions are made with an understanding of the radiation effects to the tumor and adjacent normal tissues. The four R's of clinical radiotherapy - repair, reoxygenation, repopulation (or regeneration) and reassortment (or redistribution) - have been the basis of considerable tumor biology research [33]. In radiobiologic studies, irradiation doses and cell survival were analyzed in tumor cultures that radiotherapy is applied with X and gamma rays. It was observed that survival curve creates a shoulder region first, and then it drops quickly. Shoulder region is the region where irradiated cells repair radiation damages very quickly (DNA breaks are repaired) since this damage does not cause cell death it is known as sublethal damage. When beam dosage is increased DNA repairs cannot be possible and most tumor cells die. This section of survival curve shows cell death. Aside sublethal damage, there is potential lethal and lethal damages

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as well. In potential lethal damages (PLD), cellular damage that is recovered during the interval between treatment and assay, especially under suboptimal growth conditions. Repair of potential lethal damages shows variations in various tumors. Tumors with low radiocurability do potential lethal damage repair much more effectively. When survivals radiocurable and radiosensitive tumor cultures after irradiation are analyzed it is determined the shoulder region is generally below 1.8-2 Gy. Hence, daily radiotherapy dosage (fraction) is always applied in the doses that exceed shoulder region (1.8-2 Gy) [26,29,33]. The more the amount of cells that divide (clonogenic cell ratio) in a tumor the more radiosensitive it is. It is due to the fact that destruction of dividing cells with radiation occurs more often and much early than it does in stromal cells. Biologic damages caused by X and gamma rays increase depending on the oxygen available in the environment. Why radiation damages are more serious in the environments with oxygen is explained as follows; free radicals formed indirectly have unpaired electrons in their outer orbits. If there is oxygen in environment they are paired immediately, complete their electrons and make peroxides. Peroxides are molecules that are more damaging and longer lasting than free radicals. Hence, oxygenic tissues have more radiation damages [26,33]. Cells are most sensitive to radiations in mitosis and most resistant in late synthesis (late S). Although in the early stages of G1 phase, they are partially resistant, they are sensitive in the late stages and in G2 phase. The relationship between resistance and sensitivity is related to the natural sulphydril component levels in the cell. sulphydril are natural radioprotectors and their level is the highest in S phase and is the lowest in mitosis. Thus, cell is resistant to radiation in S phase, sensitive in mitosis [26,32,33]. Sulphydrils affect with the mechanism that prevent coupling of free radicals that break chemical structures formed as a result of ionization, and oxygen. Protective effects of sulphydril components against radiation are the highest in rays with not dense ionization (x and gamma rays) and the lowest in rays with dense ionizations (alpha particles). Sensitivities of tumors and normal tissue cells to radiation vary in certain degrees depending on variety of ionization beams and their characteristics. Some amount of doses of radiations that ionize differently from each other does not cause the same biological effects. For instance, while irradiation doses that surrounding tissues can tolerate is 60-70 Gy fractions when X and gamma rays are applied in conventional fractions, this dose is around 20 Gy in treatment with neutrons. Eradication of tumor (tumor control) is proportional to the irradiation doses applied. Increasing irradiation doses increase local control of tumor. Irradiation dose are applied in daily dosages (fractions) in clinical radiotherapy. Not applying total irradiation dose that can eradicate the tumor and applying it in fractions and with certain time intervals was originated from the idea of catching the tumors in the mitosis phase more often, where they are most sensitive to radiation and of causing less damage to normal tissues by radiation. While normal tissues repair radiation damage more effectively compared to tumor tissues (repair of sublethal damage) within the time between fractions, deed cells are regenerated through cell repopulation, thus normal tissues are partially protected from radiation damage. On contrary, some of the hypoxic cells re-oxygenate and become more sensitive to radiation and changes occur in cell cycle due to synchronization caused by radiation. At the end of all these, while some tumors are eradicated by radiation surrounding normal tissues that received same dosage radiation do not get damaged much [26,33].

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There are two patterns of morphological changes that are associated with cell death in mammalian cells [33]. The most recognizable is necrosis, which is degenerative in nature and results from the more severe forms of cell injury [36,37]. The second recognized element of radiation-induced cell death is apoptosis, often referred to as programmed cell death. This is a natural process and is essential during normal development and tissue homeostasis, being involved in organogenesis and T-lymphocyte elimination [38-41].

1.5. The Side Effects Radiotherapy on Normal Cells Radiation damages and type of damages that occur in tissues depend on kinetic properties of parenchyma cells that constitute the tissue. While radiation damages appear in early stages in rapidly dividing tissues, radiation damages appear after years and as late radiation damages in slow dividing or in barely regenerative tissues. The changes due to radiotherapy in normal tissues depend on many factors such as radiation quality, radiation parameters (dosage, fraction, duration, irradiation techniques etc.) rate of irradiated normal tissue, cell and tissue properties, whether radiosensitizers or chemotherapy are used together with radiotherapy. The side effects due to radiotherapy are analyzed in three categories in normal tissues. These are acute, sub-acute radiation reactions and late radiation complications. Tolerances of several organs and tissues against X and gamma rays re determined based on clinical observations and experiences. Dosage limiting concepts are minimum and maximum tolerance doses. Minimum tolerance dose (TD 5/5) is the dosage amount that causes 5% late radiation damage on the beamed tissue or organ within 5 tears. Maximum tolerance dose (TD 50/5) is the dosage amount that causes 50% complication on beamed tissue or organ within 5 years [26,29,33].

1.5.1. Acute Radiation Reactions Loss of rapidly dividing cells of the irradiated tissues or organs due to radiotherapy is generally characterized such as hyperemia or edema changes. Appearance of radiation toxicity in normal tissues depends on metabolic activities of front cells that constitute parenchyma of the tissue. Radiation damages appear early in rapidly dividing tissues such as, mucous membrane of gastro-intestinal system, hematopoietic cells in bone marrow, spermatagonium in testis, salivary glands, oral cavity, mucous membrane of oropharyngeal and esophagus mucosa, lymph glands, skin (epidermis), mucous membrane of genitourinary system, mucous membrane of upper respiratory track and in arterioles and capillary vessels. The reasons for this is the death of mucous and parenchyma cells that are damaged during radiation, and that the leading cells (stem cells) producing these cells joins this damage and cannot produce enough cells to cover the shortage temporarily. Acute side effects are named as inflammation of the organ or the tissue that is irradiated. Generally acute side effects are not serious and they do not hinder the treatment and reduced by supportive treatment. If they are serious, treatment should be discontinued and related treatment should be started [26,29,33].

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Table 3. Normal organs or tissues tolerance to radiotherapy applied conventional fraction (one fraction per day 1.8-2 Gy, 5 treatments per week). The table is adapted from Karadeniz, AN. Radyoterapinin Temel İlkeleri. In: Topuz E, Aydıner A, Karadeniz AN. Klinik Onkoloji. İstanbul: Tunç Matbaası; 2000; 16-33 Organs or Tissues Brain Spinal cord Lung Heart Kidneys Liver Bone marrow Stomach

Radiation damage

Radiation Fields

TD5/5

- Transient demyelination - Brain necrosis - Transient demyelination - Progressive radiation myelopathy (infarctus, necrosis) - Subacute / chronic pneumonitis - Pulmonary fibrosis - Subacute / chronic pericarditis - Pancarditis - Acute, subacute, chronic nephritis - Acute, subacute, chronic hepatitis - Aplasia, pancytopenia

- Whole brain - Partial brain (100 cm2) - Whole spine - Partial spine (10x10 cm)

45-50 Gy 60 Gy 36 Gy 45 Gy

- Whole lung - Partial ( 100 cm2) - 60 % of heart

15 Gy 30 Gy 45 Gy

25 Gy 35 Gy 55 Gy

- Whole of two kidneys - Whole Liver - Total body irradiation - Partial - 100 cm2 in irradiated field

15 Gy 25 Gy 2,5 Gy 30 Gy 45 Gy

25 Gy 40 Gy 4,5 Gy 40 Gy 55 Gy

- 400 cm2 in irradiated field - 100 cm2 in irradiated field

55 Gy 65 Gy 75 Gy 80 Gy 80 Gy 100 Gy 2 Gy 7 – 12 Gy

- Ulcer, hemorrhagia, perforation

TD50/5

55 Gy

Bowel

- Ulcer, hemorrhagia, perforation

Esophagus Rectum Bladder Ureter Testis Over Adrenal glands Peripheral nerves Uterus Vagina Soft Tissues Lymph nodes

- Esophagitis, ulceration, stenosis - Ulcer, stricture - Kontraktur - Stricture - Sterilization - Sterilization - Hypoadrenalism

45 Gy 50 Gy 60 Gy 60 Gy 60 Gy 75 Gy 1 Gy 2,5 Gy > 60 Gy

- Neuropathy, neuritis

60 Gy

100 Gy

- Necrosis, perforation - Ulcer, fibrosis, fistula - Fibrosis

> 100 Gy 90 Gy 60 Gy

> 200 Gy > 100 Gy 80 Gy

- Atrophy, fibrosis

50 Gy

> 70 Gy

1.5.2. Subacute Radiation Reactions They appear within a few weeks to 3 months following the end of radiotherapy. They are observed after irradiation of the organs that include tissues with slow proliferating or slow regeneration capability. These are organs such as lung, liver, kidney, heart, spinal cord and brain. Subacute radiation reactions are defined as radiation pneumonitis, hepatitis, nephritis and carditis, subacute radiation myelitis (Lhermitte phenomenon) and subacute demyelization encephalopathy [26,33]. 1.5.3. Late Radiation Reactions They are the complications that appear after the 3rd month following the end of radiotherapy or sometimes even after years. These radiation complications that appear late are due to partially the permanent damage occurred on the vessels by ionizing radiation (chronic

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radiation endarteritis) and partially parenchymal cell and supporting tissue losses of irradiated areas. Fibrosis of connective tissue is generally added to these changes. It is thought that ischemia is due to radiation endarteritine and fibrosis is due to the fact that serum proteins escape out of veins as a result necrosis, perforation of vascular endothelia damage. Fallowing late side effects, ischemia, ulcer that is resulted due to insufficient food intake, hemorrhage, and fistula develop on tissues. Chronic radiation damages due to parenchyma cell losses and organ atrophies develop. (i.e. chronic radiation perikarditis ). Stricture and parenchyma fibrosis due to fibrosis are observed. Late radiation damages are the most feared side effect and complications on radiotherapy applications. They restrict total irradiation dosage. They generally have spreading and permanent character and they are life threading especially when they develop on vital organs (such as brain and spine necrosis, lung fibrosis, pancarditis, and nephrosclerosis) [26,29,33]. Acute, subacute, and late radiation reactions are summarized in table-4. Table 4. Acute, subacute, and late radiation reactions in various organs and tissues occurred during or after conventional radiotherapy. The table is adapted from Karadeniz, AN. Radyoterapinin Temel İlkeleri. In: Topuz E, Aydıner A, Karadeniz AN. Klinik Onkoloji. İstanbul: Tunç Matbaası; 2000; 16-33 Organs or Tissues Brain

Acute Radiation Damage - Brain edema

--

- Transient radiation myelopathy (Lhermitte's sign)

---

---

-Acute conjunctivitis Acute keratitis Enophthalmia Panophthalmia

--

Spinal cord

Hypothalamus – pituitary axis Thyroid Gland Adrenal Glands Eye

Subacute Radiation Damage - Transient disturbance of myelination (Somnolence Syndrome)

--

Late Radiation Damage Focal necrosis Cerebral arthopy Leucoencephalopathy Neuropsychologic damage Cerebrovascular damage Spinal cord atrophy - Progressive radiation myelopathy (infarctus, necrosis) Pituitary insuffiency Selective hormone deficiency Hypothyroidsm Autoimmune thyroiditis - Hypoadrenalism - Chronic Conjunctivitis. telangiectasia, atrophy, ectropion, entropion - Chronic keratitis, Keratitiis Sicca, corneal opasity, ulcer, perforation, - scleral atrophy, necrosis - Cataract, lens opacity - Neovascular glaucoma - Radiation retinopathy - Retinal artery - ven occlusion - Nasolacrimal stenosis, epiphora

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Organs or Tissues Optic nerve

Acute Radiation Damage --

Subacute Radiation Damage - Optic neuritis

Late Radiation Damage

Head-neck

Acute mucositis - Ageusia (loss of taste acuity) - Acute laryngeal edema

--

Ear

- Otitis media

--

Salivary Glands

Acute sialitis Hyposaliva

--

--

--

Erythema Moist desquamation Pigmentation. Dry desquamation

--

- Mucosal atrophy, telengiectasia, ulceration Soft tissue necrosis, ulcer Trismus Laryngeal cartilage necrosis Submandibular neck edema - Deafness - Meniere's syndrome. - Xerostomia Salivary gland atrophy -In child tooth agenesis, enamel dysplasia, hypoplasia of the mandible Radiation dermatitis Skin atrophy Skin pigmentation. Telengiectasia Skin necrosis Fibrosis - Radiation fibrosis - Chronic radiation - Corpulmonale and right heart failure - Constrictive pericarditis - Cardiomyopathy - Coroner artery disease - Valve damage and arrhythmias Esophageal stricture Ulcer, perforation, hemorrhagia Esophageal fistula Chronic gastritis Ulcer, perforation, hemorragia Gastric obstruction and stenosis - Ulcer, perforation, hemorragia and fistula Stenosis Bridge ileus - Chronic radiation proctitis - Ulcer, hemorrhagia, fistula, fibrosis -Chronic radiation hepatitis

Tooth and mandible Skin

-Radiation pneumonitis

Lung --

Heart

Esophagus

Stomach

Bowel

Rectum

- Acute pericarditis - Pericardial effusion - Acute esophagitis - Acute gastritis - Acute ulcer - Hemorrhagia - Acute radiation mucositis

- Acute radiation proctitis

Liver --

- Heart failure - Pancarditis

--

--

--

--

- Subacute radiation hepatitis Veno-occlusive hepatic disease

- Optic neuropathy, optic atrophy

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Table 4. Continued. Organs or Tissues Kidney

Acute Radiation Damage

Subacute Radiation Damage - Radiation nephritis

-Acute cystitis Acute urethritis

- Subacute radiation cystitis

Testis

--

--

Female urogental system

Acute vulvitis Acute vaginitis Acute cervicitis Dysmenorhhea Amenorrhea

--

Bladder-UreterUretra

Bone marrow

Soft tissue Peripheral nerve Arteries and Veins Lymph nodes and lymph drainage

- Neutropenia., thrombocytopenia (total body irradiation) --

--

---

- Neurotis --

--

--

- Anemia - Pancytopenia (total body irradiation)

Late Radiation Damage - Chronic radiation nephritis - Benign - malign hypertension -Hiperreninemik hypertension - Chronic radiation cystitis - Bladder contracture - Ureter \ urethral strictures - Testis atrophy, sterilization, endocrine failure Atrophic vulvitis: Atrophic radiation vaginitis Vaginal fibrosis, stenosis and fistula Cervical atrophy, ulcer, stenosis Uterine necrosis, hematometria, pyometria, hemorrhagia and perforation - Over sclerosis, sterilization, early menopause and endocrine failure Hypoplasia, aplasia, myelofibrosis Pancytopenia Necrosis Atrophy, fibrosis - Neuropathy, paralysis Arteriosclerosis Thrombophlebitis Atrophy Fibrosis Lymphedema

2. REACTIVE OXYGEN SPECIES, RADIOTHERAPY, ANTIOXIDANTS AND RADIOPROTECTION Reactive oxygen species (ROS) are oxygen-containing molecules that have higher reactivity than ground state molecular oxygen. These species include not only the oxygen radicals, such as superoxide (O˙2¯), hydroxyl (OH˙), and peroxyl (ROO˙), but also nonradical molecules like singlet oxygen (1O2) and hydrogen peroxide (H2O2). Radicals are molecules with unpaired electrons [42,43]. ROS are generated by normal cellular metabolism and exogenous agents during aerobic metabolism [1]. ROS are known to be involved a number of clinical conditions, such as cardiovascular disease, sepsis, aging, and carcinogenesis. ROS inactivate enzymes, cause

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DNA damage, and attack polyunsaturated fatty acids in cell membranes resulting in the formation of lipid peroxide radicals triggering an aggressive chain reaction. This situation causes autooxidation. This aggressive chain reaction is known to have major role in membrane damage [44]. Excess ROS are cytotoxic, leading to cell death, mutations, chromosomal aberrations, or carcinogenesis and may also damage cellular components [1,2]. Oxidative stress arises when rates of ROS production outpace rates of removal [3]. Oxidative stress may cause the damage on cells as a result of one of three factors: 1) an increase in oxidant generation, 2) a decrease in antioxidant protection, or 3) a failure to repair oxidative damage [4]. Ionizing radiation has been well-known to enhance the production of ROS in a variety of cells [19,45]. When water, which constitutes around 80% of the cell, is exposed to ionizing radiation, decomposition occurs through which a variety of ROS, such as the superoxide radical (O˙2¯), the hydrogen peroxide (H2O2) and the hydroxyl radical (OH¯) are generated [19]. Because of the serious damaging potential of ROS; cells depend on the elaboration of the antioxidant defence system (AODS), both enzymatic and non-enzymatic oxidant defence mechanisms [6]. Common antioxidants include the vitamins A, C, E [7,8] amifostine [9,10], zinc [5,11,12], selenium [13], melatonin [3,14,15], and the others [16-18] and the enzymes SOD, CAT and GSH-Px [7,19]. They work in synergy with each other and against different types of free radicals [7], and they can offer protection against ionizing radiation-induced oxidants. SOD, the first line of defence against oxygen-derived free radicals, catalyzes the dismutation of O˙2¯ into H2O2 [17,19,46,47]. H2O2 can be transformed into H2O and O2 by CAT and GSH-Px [17,46-48]. Although all respiring cells are equipped with protective enzymes such as SOD and CAT, GSH-Px, increased oxidative stress in cells stemming from ionizing radiation may overwhelm the protective systems, leading to cell injury [5]. When this precarious balance is broken, in favour of free radicals, it causes an oxidative stress. This oxidative stress can induce injury by diverse mechanisms including initiation of lipid peroxidation, inactivation of enzymes, damage to DNA, and protein sulfhydril oxidation [49]. In this process of chemical and biological damages in cells of surrounding normal tissues in irradiated fields; mitotic, interphase, apoptotic, and necrotic cell death occur as a results of damaging effects of ionizing radiation, enhancing the production of ROS that cause the side effects of radiotherapy [50]. In recent years, more patients with cancer have been treated with radiation therapy. Because most of them are surviving, there is an increasing need to patients care for RTinduced toxicities [51], and it was well-known that radiotherapy not only increased production of ROS but also reduced significantly natural antioxidants such as vitamin A, C, E, Selenium, and Zinc and the activities of antioxidant enzymes in plasma and tissue [5,52]. In addition, in previous studies, antioxidant system abnormalities have been reported in various cancer patients [46,53-56]. From all these results, one can consider that significantly reduced natural antioxidants and the activities of antioxidant enzymes during radiotherapy will be able to increase radiation-induced normal cells, tissues or organs toxicities. In order to minimize acute and late radiation damages, it is required that radiation quality and treatments units are good, extensive irradiation fields is not used, suitable set-up and multiple field irradiation techniques are used. In addition, in order to protect surrounding

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normal tissues in the radiotherapy fields, lead blocks, wedge filters and tissue compensators should be used and not be applied irradiation doses over tolerable limits to tissues in the radiotherapy fields, as much as possible. Aside radiation quality, in order to minimize acute and late radiation damages some chemical structures are researched, it has been shown by experimental studies that structures including sulphydril such as cystein and cysteamine lessen the effects of biological damages caused by X and Gamma Rays. These agents are also called radioprotector agents. Radioprotector agents are used in clinical radiotherapy in very limited number of studies. Studies for their usage are still in the stage of research. [26,29,33]. In 1949, Patt et al. [57] reported that cysteine, a sulfur-containing amino acid, could protect rats from a lethal dose of X-rays. After discovery of cysteine as a radioprotectant agent, it has also been considered possible that radiation therapy for cancer patients could be improved by the use of radioprotectors to protect normal tissue, and a large number of agents that have different protective mechanisms were used in the prevention of radiation-induced surrounding normal tissues toxicities in the radiotherapy fields. Antioxidants are only one class of radioprotectors. [58]. Dietary and endogenous antioxidants prevent cellular damage by reacting with and eliminating oxidizing free radicals in physiological conditions [59]. However, in cancer treatment, because radiotherapy involves excess amount of the generation of free radicals to cause cellular damage and necrosis of malignant cells, unfortunately, in this time, dietary and endogenous antioxidants do not prevent-radiation induced cellular damage by reacting with and eliminating oxidizing free radicals. In this increased oxidative stress conditions, in order to eliminate excess free radicals and to protect body organs or tissues against radiation-induced damages, exogenous antioxidants may be supplemented with radiotherapy. In this point, from experimental and clinical studies, there is currently substantial clinical interest in antioxidants, such as amifostine [60,61], melatonin [14,62], vitamin A, C, E [52,63,64], selenium [65,66], zinc [51,67,68], and gingko biloba [17,22] as a protective agent against radiation-related normal tissue injury. In order to understand radioprotective properties of these antioxidants, let us go over quickly amifostine, melatonin, vitamin A, C, E, selenium, zinc, and gingko biloba.

2.1. Amifostine Amifostine was originally developed during the height of the cold war by the Walter Reed Army Institute of Research (Washington D.C., USA) as part of a US Army classified research project to identify potential agents to protect military personel in the event of nuclear warfare. Military experiments show that amifostine had the ability to protect mice, dogs and monkeys from lethal doses of whole body radiation [69-71]. Yuhas and Storer [72] subsequently showed that pretreatment with amifostine effectively protects normal tissues from the toxicities of therapeutic radiation without protecting tumors, and the United States Food and Drug Administration (FDA) approved the use as a cytoprotector to decrease the incidence of moderate-to-severe xerostomia in patients undergoing postoperative radiation therapy for the treatment of head-and-neck cancer [73,74]. It has been developed as a selective cytoprotective agent for normal tissues against the toxicities of chemotherapy and radiation, that is, rapid uptake into normal tissues and slow-to-negligible uptake in tumor cells, is a result of the decreased vascularity of tumors, decreased activity of alkaline phosphatase enzymes in tumor cells and pH dependence activity of the active drug [9].

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Amifostine (WR 2721), a phosphorylated aminothiol pro-drug, is an analogue of cysteamine, the radio protective effect of which is based on the presence of a thiol group in its active metabolite (S-2 [3-aminopropylamino]-ethanethiol), the free thiol WR-1065 [70,71,9]. In the plasma, amifostine is rapidly dephosphorylated (by membrane-bound alkaline phosphatase enzymes) and enters the cells as its active form [75]. WR-1065, the major active metabolite of amifostine, provides cytoprotection by at least three different mechanisms. First, it can bind directly to, and thus detoxify, the active derivatives of antineoplastic compounds [73,76,77]. Second, it acts as a potent scavenger of drug- or radiation-induced oxygen free radicals [9,71,73,76-80]. Third, when administered after exposure to radiation or antineoplastic drugs, it can markedly reduce injury-induced apoptosis [73,74,76,81]. Amifostine is currently the only FDA-approved drug for use as a normal tissue radioprotector in the treatment of cancer by radiation therapy [74]. Because amifostine is ineffective if administered orally, its only approved route of administration at this time is via intravenous injection. But, daily intravenous administration of amifostine during radiotherapy is associated with a high rate of serious adverse effects such as hypotension, vomiting, and allergic reactions, leading to discontinuation of amifostine treatment and sometimes delay of radiotherapy [82]. Thus, there is growing interest in the investigation of subcutaneous administration as a practical alternative [75]. Previous experimental studies demonstrated that amifostine protected oral [83-86] and gastrointestinal mucosal tissues [87,88], salivary glands [89-93], kidney [94], bone marrow [95,96], spermatogenetic cells [70,97], skin [98], hair follicles [99], lung [100], heart [101] and hepatocytes [102] against radiation-induced cell and tissue damages. During the past decade, several clinical studies have investigated the cytoprotective efficacy of amifostine [103-108]. Sasse et al. [103], in their meta-analysis, reported that the efficacy of radiotherapy itself was not affected by amifostine, and this meta-analysis not only confirmed the lack of tumor protection with the use of amifostine, but also proved that patients receiving this drug were able to achieve higher levels of complete response. They also reported that the use of amifostine sometimes cause side effects such as hypotension and nausea, but those were less intense with the subcutaneous route of application. In conclusion, in their meta-analysis, Sasse et al. [103] emphasized that amifostine reduced side effects and improved complete response rate during radiotherapy. Antonadou et al. [60] tested the amifostine during chemoradiotherapy for treatment of lung cancer and concluded that amifostine is effective in reducing the incidence of both acute and late toxicities associated with RCT in patients with locally advanced NSCLC without compromising antitumor efficacy. In a similar study, Komaki et al. [61] reported that amifostine reduced the severity and incidence of acute esophageal, pulmonary, and hematological toxicity resulting from concurrent cisplatin-based chemotherapy and RT. The authors reported that amifostine had no apparent effect on survival in these patients with unresectable non-small-cell lung cancer, suggesting that it does not have a tumor-protective effect. From all these investigations, we can conclude that amifostine, the first drug approved by FDA, is used as a cytoprotector to decrease the incidence of moderate-to-severe xerostomia, has a number of beneficial effects on radiation-induced early and late toxicities.

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2.2. Melatonin Melatonin (MLT) as an endogenous hormone (N-acetyl-5 methoxytryptamine) was synthesized in circadian rhythm by the pineal gland in the human brain [14,109,110]. MLT secretion increases soon after the onset of darkness, peaks in the middle of the night (between 2 and 4 a.m.), and gradually falls during the second half of the night [111]. Once synthesized in the pineal gland, MLT is quickly released into the bloodstream [14,112] and participated in the regulation of a number of physiological and pathological processes [14,109-111]. In addition, it has been demonstrated that melatonin was produced in many other tissues [113]. Melatonin was recently identified as a multifaceted direct free radical scavenger as well as an indirect antioxidant when stimulating antioxidant enzymes [14,15,114] Melatonin scavenges OH˙, nitric oxide (NO˙), O˙2¯ and singlet oxygen (1O2), a high energy form of O2 that exhibits high toxicity at the molecular level [115,116]. Melatonin may decrease the quantity of O˙2¯ in two ways, directly by stimulating SOD and indirectly when the melatonyl cation radical scavenges it. Melatonin stimulates the activity of GSH-Px, which transforms H2O2 to O2 [115-118]. The marked protective effects of melatonin against oxidative stress are aided by its ability to cross all biological membranes that lipid peroxidation is believed to be an important cause of destruction and damage to cell membranes [118-120]. In one study, melatonin seemed to be more effective than other known antioxidants, such as vitamin E in protecting against oxidative damage [121]. Melatonin, as a new member of an expanding group of regulatory factors that control cell proliferation and loss, is the only known chronobiotic, hormonal regulator of neoplastic cell growth [23]. There is evidence from experimental studies that melatonin influences the growth of spontaneous and induced tumors in animals. Pinealectomy enhances tumor growth, and the administration of melatonin reverses this effect or inhibits tumorigenesis caused by carcinogens [122]. The association between melatonin levels and cancer progression has suggested to some that melatonin may be a modifier of cancer progression. The mechanisms by which melatonin may act in this way have not been fully elucidated. One of the potential mechanisms is the possibility that the hormone has antimitotic activity as a result of intranuclear downregulation of gene expression or through the inhibition of growth factor release and activity. There is also evidence to support the inhibition of solid cancer growth in vivo by suppressing tumor linoleic acid uptake and metabolism via a MLT receptor-mediated mechanism. Other possible anti-cancer mechanisms include protection from oxidative damage, anti-angiogenic activity, anti-inflammatory activity, anticachectic properties, and immunostimulation [123-126]. Saez et al. [124] reported that MLT could exert an oncostatic action, lengthening the survival time of mammary tumour-bearing animals, and suggest that this effect is due, at least in part, to regulating the neuroendocrine parameters of tumourbearing animals, bringing them closer to their optimal physiological status. Lenoir et al. [127] clearly showed almost identical preventive and curative effects of MLT on the growth of dimethylbenz [a]anthracene-induced mammary adenocarcinoma, markedly reducing the DNA damage provoked by DMBA. In the last decade, free radical scavenging ability and anti-inflammatory activity of melatonin was used as a rationale for testing its radioprotective ability. Currently, there are increasing evidences, from experimental and clinical studies, suggesting that melatonin could be a beneficial agent in the protection against ionizing radiation-related normal tissue injury [3,14,62,109,115,118-121,128,129]. Melatonin is a new class of radioprotectors against total

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body irradiation lethality [109,130] It was recently shown that whole-body irradiation caused multiple organ damage and melatonin, by its free radical scavenging and antioxidative properties, appeared to ameliorate irradiation-induced organ injury such as the liver, lung, colon, ileum [119] and brain [121,131]. In their study, Yavuz et al. [129] aimed to determine the potential benefits of sparing longitudinal bone growth by fractionated radiotherapy alone compared with pretreatment with melatonin that provides differential radioprotection of normal cells. They reported that fractionated radiation resulted in a mean percent overall limb growth loss and a mean percent overall limb discrepancy. The administration of 5 or 15 mg/kg melatonin before each of the three fractions of radiotherapy reduced the mean percent overall limb growth loss, and the mean percent overall limb discrepancy; these values were significantly different compared with irradiation alone. When compared with fractionated radiation group, the growth arrest recovered by 5 or 15 mg/kg melatonin was 19.7 and 24.1% for the tibia, 7 and 18.6% for the femur, and 17.7 and 21.8% for the total limb, respectively. With these results, the authors proposed that melatonin in combination with fractionated radiation may use in growing children requiring radiotherapy to the extremity for malignant tumors. Karslioglu et al. [3] reported that total cranium irradiation of 5 Gy in a single dose enhanced cataract formation, and melatonin supplementation protected the lenses from radiation-induced cataract formation by the up-regulation of antioxidant enzymes and by scavenging free radicals generated by ionizing radiation. The authors suggested that supplementing cancer patients with adjuvant therapy of melatonin may reduce patients suffering from toxic therapeutic regimens such as chemotherapy and/or radiotherapy and may provide an alleviation of the symptoms due to radiation-induced organ injuries. It has been claimed that the addition of melatonin to radiotherapy attenuates the damage to blood cells by protecting the genetic material of hematopoietic cells and thus makes the treatment more tolerable [111,128,132]. Blickenstaff et al. [130] reported that 950 cGy of whole-body radiation caused the death of all mice within 12 days, whereas when melatonin supplementation (1.076 mM/kg) before a lethal dose of ionizing radiation was administered mice, 43% of the irradiated mice survived at least 30 days. In a similar study, Vijayalaxmi et al. [109] found that 815 cGy of ionizing radiation (LD50/30 dose) resulted in only a 45%–50% survival rate after 30 days; melatonin supplementation at a dose of 125 mg/kg body weight before irradiation increased the survival to 60%, whereas MLT at a dose of 250 mg/kg body weight increased survival up to 85%. Several studies have looked at the effects of giving melatonin to treat cancer. Melatonin has been used alone or in combination with chemotherapy [77,133-134], radiation therapy [62], or hormone therapy (such as tamoxifen) [135] in a number of studies involving different types of cancer. Some of these studies have suggested that melatonin may extend survival and improve quality of life for patients with certain types of untreatable cancers such as advanced cancer [62,133-135]. Some studies reported that MLT protected both normal and cancer cells against genotoxic treatment and apoptosis induced by chemotherapy [77]. Because of its antioxidant [3,14,15,115,116,119], antiapoptotic [136,137], and antiinflammatory actions [137,138], MLT has gained growing attention in the past decade as a radioprotector agent. In this point, there is a question whether MLT might administer concurrent with cancer therapy, or not, since antioxidants might reduce oxidizing free radicals created by radiation therapy or chemothrapy, and thereby decrease the effectiveness of these treatments. In this point, in a recent in vitro study carried out Majsterek et al. [77] was reported that idarubicin induced apoptosis in normal and cancer cells and its level was

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correlated with the extent of DNA strand breaks, and MLT protected both normal and cancer cells against genotoxic treatment and apoptosis induced by idarubicin. The authors concluded that despite its recognized potential as an antioxidant, melatonin should be considered with caution when used in combination with cancer chemotherapy agents, especially in the case of leukemias. The fact that MLT protects both normal and cancer cells against genotoxic treatment and apoptosis induced by idarubicin might be a negative effect of MLT on cancer treatment, and thereby decreases the effectiveness of these treatments. In conclusion, although MLT supplementation in cancer patients with adjuvant therapy of may improve patients suffering from toxic therapeutic regimens such as chemotherapy or radiotherapy or in combination with two and provide an alleviation of the symptoms due to radiation-induced organ injuries, in addition to its antioxidant, antiapoptotic, antiinflammatory and radioprotective actions, it would be worthwhile to study whether MLT has a protective effect on cancer cells treated with chemotherapy or radiotherapy, or not.

2.3. Vitamin A Fat-soluble Vitamin A (VA) compounds include retinol, retinal and retinoic acid. This vitamin group is vital to eye and retina function, protects the mucous membranes of the mouth, nose, throat and lungs from damage, and reduces risk of infection (immune enhancer) and cancer. VA does not occur in plants, but many plates contain carotenoids such as betacarotene that can be converted to vitamin A within the intestine and other tissues. [139,140]. Antioxidant properties of VA are well-known. Studies demonstrated that serum levels of CuZnSOD, GSH-Px, and CAT in VA-deficiency was lower than those of the VA-adequate control, and NO production evaluated as nitrite concentration increased in serum in vitamin A-deficiency. Prooxidant environment and inflammation are induced by vitamin A deficiency [141]. Studies also demonstrated that VA supplementation as retinol increased the activities of GSH-Px and SOD and decreased concentration of TBARS [142-144]. It has been reported that VA supplementation resulted in a decrease in oxidative stress by decreasing the production of superoxide anion, and inhibited lipid peroxidation, and protected DNA-single strand breaks against TCDD-induced production of ROS [145]. Ahlemeyer et al. [146] reported that VA as retinoic acid reduced staurosporine-induced oxidative stress and apoptosis by preventing the decrease in the protein levels of SOD-1 and SOD-2, and thus supported the antioxidant defense system. Retinoic acid plays an important role in controlling cell growth, cell differentiation, and apoptosis, as well as carcinogenesis, and is of potential clinical interest in cancer chemoprevention and treatment [147]. Beta-carotene at high doses induce differentiation, proliferation inhibition, and apoptosis depending on dose and type of antioxidant, treatment schedule, and type of tumor cell, without producing similar effects on most normal cells in vitro and in vivo [148]. VA deficiency, alterations in receptor expression or function could interfere with the retinoid signaling pathway and thereby enhance cancer development even in vitamin A sufficient individuals [149]. Basu [150] reported in his review that there was an association between vitamin A and cancers of epithelial origin, and that vitamin A and its analogues delayed tumor development, retarded tumor growth and terminated tumors induced by carcinogenic polycyclic aromatic hydrocarbons. The author suggested that VA and its analogues may have a prophylactic and a therapeutic role in cancers of epithelial origin.

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Low Vitamin A levels are correlated with increased incidence of several cancers, such as lung [149,151-153], esophagus [154], and stomach [155]. Stahelin et al. [156] reported that cancers of the bronchus and stomach were associated with low carotene levels, and stomach cancers were also associated with low Vitamin A levels. Low plasma carotene was associated with a significantly increased risk for bronchus cancer, low plasma levels of carotene and Vitamin A with increased risk for all cancers, and low plasma Vitamin A in older people with increased risk of lung cancer. Therapeutic trials of vitamin A and related compounds have demonstrated activity in several cancerous and precancerous conditions [157]. De Vries and Snow [158] investigated the role of VA and beta-carotene in the development of head and neck cancers with and without second primary tumors. They found that both groups of patients had decreased levels of beta-carotene, and patients with second tumors had statistically lower levels of VA than people with single tumors, suggesting a role for these vitamins in the development of second head and neck tumors. El Attar and Lin [159] demonstrated that several retinol compounds (retinol; all-trans-retinoic acid; N-4-Hydroxyphenyl) retinamide (N-4-HPR); and carotenoid canthaxanthin all inhibited the bioconversion of arachidonic acid to PGE2 by tongue cancer cells, the most potent inhibitor being N-4-HPR. They suggested that these inhibitory effects, together with antioxidant properties might contribute to retinoids' antiinflammatory and anticancer activity. Santamaria and Santamaria [160] reported that cancer development such as skin, breast, gastric, colon were prevented in 60-100% of animals supplemented with carotenoids (beta-carotene, canthaxanthin and retinol-BC) one month prior to tumor induction (via carcinogenic agents or transplantation). In addition, 15 cancer patients, their primary tumors removed by surgery, who took supplements of beta-carotene and retinol were experienced a longer than expected disease-free interval preliminarily. Thung et al. [161] showed that VA and carotene intakes were modestly associated with a reduced risk of ovarian cancer. Mrass et al. [162] reported that the ability of retinoic acid to regulate the expression of proapoptotic genes and to sensitize keratinocytes to apoptosis may have play a role in their prevention of nonmelanoma skin cancer in transplant patients and patients with DNA-repair deficiencies. Gensler and Holladay [163] reported that dietary supplementation with retinyl palmitate (a form of Vitamin A) and canthaxanthin (a carotenoid) together but not alone, prior to UV irradiation, prevented the enhanced growth of tumor implants. Satake et al. [164] examined the anti tumor effect of VA on head and neck squamous cell carcinoma. They found that VA suppressed the cell proliferation, induced apoptosis, and cell cycle arrest, upregulated sensitivity of the chemotherapeutics drugs and downregulated several angiogenesis factors. The authors suggested that vitamin A and its derivatives are useful for preventing and/or treating patients with HNSCC. Because of antioxidant, apoptotic, anticancer and immune stimulatory effects of VA and its derivatives, scientists have been studying the fate of Vitamin A levels in the body of cancer patients undergoing cancer treatments Other than chemoprevention; VA and its derivatives could have a place in combination cancer chemotherapy or as radiation sensitizers and have developed synergistic cancer treatment applications for Vitamin A, in combination with radiotherapy and chemotherapy. Tumor hypoxia is a poor prognostic indicator during radiotherapy because of reduced local control associated with a raised risk of distant metastases and recurrence [165]. Studies demonstrated that VA as retinoic acid can increase the sensitivity of cervical cancer cell lines [166], and in combination with interferon, headand-neck carcinoma and breast cancer cells to radiation treatment [167-169]. DeLaney et al.

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[170] reported that, using a clonogenic cell survival assay, IFN-alpha-2a (1000 units/ml) or RA (1 microM) alone did not significantly enhance radiation cytotoxicity. The combination of the two agents, however, significantly increased the cytotoxicity of radiation against head and neck squamous cell carcinoma cell line. Komiyama et al. [171] found that VA potentiated the RNA inhibitory action of the chemotherapy drug 5-fluorouracil (5-FU). Furthermore, they applied the triple combination of 5-FU, Vitamin A and cobalt-60 radiation (FAR therapy) to patients with head and neck tumors, with highly effective synergistic effects. Akiyama et al. [172] reported that the blood level of retinol, a strong potentiator of 6-mercaptopurine among vitamin A compounds, was increased in BDF1 mice after retinol palmitate injection. Retinol palmitate was found to enhance the antitumor effect of 6-mercaptopurine against murine L1210 leukemia to a significant extent (69% increase in life span). Nakagawa et al. [173] studied on the combination of retinol palmitate (RP) with six different anticancer agents on ascites sarcoma or leukemia in mice. They reported that with ascites sarcoma, RP considerably enhanced the antitumour activity of 5-fluorouracil (5-FU) methotrexate (MTX) and 1-(4-amino-2-mthyl-5-pyrimidinyl) methyl-3- (2-chlorethyl) -3-nitrosourea (ACNU), but not the action of adriamycin (ADM) or 6-mercaptopurine (6-MP). Against leukemia, RP enhanced antitumor activity of 6-MP, MTX, ADM, ACNU and cis-dichlorodiammineplatinum (CDDP), but not of 5-FU. Kumamoto et al. (174) carried out a study for the use of a triple combination treatment approach consisting of 5-fluorouracil (250 mg/day, i.v.), vitamin A (50,000 unit/day, i.m.) and external radiation (2.0 Gy/day), which they have termed "FAR therapy." in patients with T2N0 laryngeal cancer, because radiotherapy alone is associated with a high risk of local recurrence in patients with T2N0 laryngeal cancer. They found that the local control and ultimate local control rates were 91% (85 of 93), and 99% (92 of 93), respectively. The cumulative 5-year voice preservation and complete laryngeal preservation rates were 91% and 87%, respectively. The cumulative 5-year disease-specific survival rate was 97% and concluded that FAR therapy may be promising for the treatment of patients with T2N0 glottic carcinoma. In another the triple combination of 5-fluorouracil (5-FU), vitamin A and radiation (FAR therapy) study, Nakashima et al. [175] found that 5-FU and radiation caused direct cell death, while vitamin A mainly inhibited cell growth. The combination of these treatments as FAR therapy synergistically enhanced cell death and inhibited cell growth. With flow cytometric evaluation, the authors demonstrated that FARtreated cells were arrested in the G1 phase of the cell cycle before undergoing apoptosis. Previous experimental and clinical studies demonstrated that vitamin A increased therapeutic effects of radiotherapy and decreased radiation induced-toxicities [176-181]. VA enhances irradiation effects on tumor cells by inhibiting the repair of potential lethal damage in cancer cells more effectively than that produced in normal fibroblasts [182]. It has been reported that VA significantly reduced anorectal symptoms of radiation proctopathy and anal ulcer. Vitamin A seems to be very effective in the treatment of radiation-induced anorectal damage, with little toxicity and expense perhaps because of wound-healing effects [176,177]. Mills [178] reported that beta-carotene reduced radiation-induced mucositis without interfering with the efficacy of radiation therapy in patients with cancer of the head and neck. In vitro studies have shown retinoic acid (RA) causes radiosensitization in human tumor cell lines at concentrations which do not cause cellular toxicity. This effect was reversible with removal of RA [180]. In a combination therapy determined the effects of cisretinoic acid (cRA), a derivative of VA, with radiotherapy and interferon-alpha 2a on locally advanced cervical cancer, it has been reported that there were a 47-percent tumor response and 33-

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percent complete remission rate, with no grade 3 or 4 toxicity. Historical controls without cRA treatment had a 42-percent tumor response rate and only 17-percent complete remissions in this study [181]. Radioprotection by dietary vitamin A in mice exposed to partial-body irradiation or total-body irradiation has been reported [183]. Beta-carotene inhibits radiationinduced chromosomal damage in mice [184], and in rats was exposed to high-dose thoracic irradiation, it has been reported that VA supplementation significantly reduced lung inflammation and inhibited radiation-induced pneumonitis [185]. In conclusions, the ability of VA to increase tumor response to radiation while reducing toxicity has been theorized to be due to the stimulation of immune response to tumor tissue [186]. The above studies demonstrate that Vitamin A and carotenoids are of considerable importance to the prevention and treatment of many diverse cancers in a variety of ways: attack and destroy cancer cells; prevent the appearance or proliferation of tumors. Antioxidant, apoptotic, anticancer and immune stimulatory properties of Vitamin A and carotene may have a potential role as a gun to enhance their therapeutic effects and to fight against toxic effects of radiotherapy and chemotherapy.

2.4. Vitamin C Vitamin C (VC) is an acidic molecule with strong reducing activity and an essential component of most living tissues. It acts as a cofactor for 8 enzymes involved in collagen hydroxylation, biosynthesis of carnitine and norepinephrine, tyrosine metabolism and amidation of peptide hormones [187]. As a water-soluble antioxidant, vitamin C (VC) is in a unique position to "scavenge" aqueous peroxyl radicals before these destructive substances have a chance to damage the lipids. It works along with vitamin E, a fat-soluble antioxidant, and the enzyme glutathione peroxidase to stop free radical chain reactions [188]. A decrease free radical scavenging enzymes such as superoxide dismutase (SOD), glutathione-S-transferase (GST) and catalase, as well as levels of total glutathione (GSH) and an increase in level of malondialdehyde (MDA) as an end product of lipid peroxidation due to oxidative stress may be effectivelly restorated by VC [143]. VC oral supplements are among the most popular sold, and gram doses are promoted for preventing and treating the common cold, managing stress, and enhancing well-being [189]. As a powerful antioxidant, VC may help to fight cancer by protecting healthy cells from free-radical damage and inhibiting the proliferation of cancerous cells. Specifically, recent studies have shown that the vitamin may help to remove cancers of the stomach and esophagus by blocking cancer-causing compounds [190,191]. VC can affect cell growth by altering cell proliferation and/or inducing cell death in various cell systems [192,193]. In previous studies, ascorbic acid inhibited the growth of various human melanoma cells [194]; and induced apoptosis in human promyelocytic leukemic HL-60 cells [195], and in fibroblasts [196,197]. Uncontrolled studies reported clinical benefit from oral and intravenous VC administered to patients with terminal cancer at a dosage of 10 g daily [198,199]. Placebocontrolled trials in patients with cancer reported no benefit from oral VC at a dosage of 10 g daily [200,201]. However, in vitro evidence showed that VC killed cancer cells at extracellular concentrations higher than 1000 mol/L [202-204], and its clinical use by some practitioners continues. Cancer treatment is one of the more controversial proposed uses of

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VC. An early study tested VC in a number of cancer patients. Patients received 10 g daily of VC, while the other patients (the control group) did not receive vitamin C. Those taking the VC survived more than four times longer on average (210 days) than those in the control group (50 days) [199]. Benefits were also seen in a similarly designed Japanese study [205]. Toxicity, mutagenicity, teratogenicity and carcinogenicity are low after ingestion of large amounts of VC [206]. Vitamin C is hydrophilic and is an important free-radical scavenger in extracellular fluids, trapping radicals in the aqueous phase and protecting biomembranes from peroxidative damage. It appears to play a role in cellular redox processes mediated by glutathione [207]. In addition to its anti-oxidant effects, VC is involved in the regeneration of tocopherol from tocopheroxyl radicals in the membrane. Thus, vitamins C and E can have interactive effects [208]. Studies in cell cultures show that VC, selectively induce apoptosis in cancer cells while sparing normal cells [209]; other findings, in a model of metastatic growth, show that VC is an angiostatic factor and may have potential in aiding host resistance to tumor growth and invasiveness [210]. VC prevents transformation of normal cells to cancer cells [211]. The antioxidant most widely used for treating cancer is perhaps vitamin C. There are considerable in vitro and animal data showing that VC can protect cells against radiation and chemotherapy [59,212-216]. VC enhanced the antitumor activity of doxorubicin, cisplatin, and paclitaxel in human breast cancer cells in culture [215]. VC also increased drug accumulation and reversed vincristine resistance of human non-small-cell lung carcinoma cells [216]. Prasad and colleagues [217] observed the growth inhibitory effects of VC alone, vitamin E alone, and combinations of VC, vitamin E, betacarotene, and 13-cis-retinoic acid on SK-30 melanoma cells in vitro. They also found that VC, alone or in combination with beta-carotene, vitamin E, and 13-cis-retinoic acid, enhanced the growth-inhibitory effect of cisplatin, dacarbazine, tamoxifen, and interferon-alpha 2b. Vitamin C is an antioxidant that can be used to reduce DNA damage and diminish lipid peroxidation and increase tissue radioresistance [218,219]. VC enhanced the effect of irradiation on neuroblastoma cells but not on glioma cells in culture [220]. The radioprotection of healthy tissue and radiosensitizing effect in tumors with use of VC was confirmed in two other mouse tumor models [212,214]. The administration of VC through drinking water before and after x-irradiation decreased the survival of tumor cells in mice without causing a similar effect on normal cells [212]. In another Mouse study, a single intraperitoneal dose of 4.5 g/kg VC was not cytotoxic to normal tissue and did not change the radiation effect on tumor tissue. The lethal dose of radiation increased and skin desquamation reaction was reduced by VC treatment [214]. In mice, VC (1 g/kg), given intraperitoneally with vitamin K3 (10 mg/kg), increased the therapeutic effect of radiation on solid tumors without causing any signs of toxicity due to the vitamins [213]. A randomized trial with 50 human subjects looked at the effect of concurrent VC (five daily doses of 1 g each) and radiotherapy on different tumor types. More complete responses to radiation were noted in the VC group. Side effects were found to be fewer in the VC-treated subjects as well [221]. VC protects against radiation-induced chromosomal damage in mice even when administered after irradiation [222]. In contrast, some studies demonstrated that VC at low doses may have protected cancer cells against free radical damage produced by chemotherapeutic agents or xirradiation, and VC, when given in a single low dose shortly before x-irradiation, reduced the effectiveness of irradiation on cancer cells in in vitro and in vivo models [223-225].

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In conclusion, vitamin C increases the effect of radiotherapy in humans and in mice and cyclophosphamide, vinblastine, doxorubicin, 5-fluorouracil, procarbazine, and asparaginase in vivo. The use of combination with vitamin K may increase both of the above activities of VC. VC has been shown to increase the effect of cisplatin and paclitaxel in vitro. No in vivo evidence suggests that VC decreases the effect of chemotherapy. VC may increase the resistance to doxorubicin in resistant breast cancer cells [59]. But, these beneficial effects seem to appear in high doses of VC. These results suggest that supplementing cancer patients with adjuvant therapy of vitamin C, separately or in combination with other antioxidants, may improve patients suffering from toxic therapeutic regimens such as chemotherapy or radiotherapy or in combination with two and may provide an alleviation of the symptoms due to radiation-induced organ injuries.

2.5. Vitamin E Vitamin E (VE) was discovered in 1922 and the prefixes α, β, γ and δ were used to distinguish the family of substances. VE is transported around the body as a component of plasma lipoproteins. Dietary VE is transported via the portal vein to the liver in chylomicrons. The lipoprotein that carries most of the VE is the LDL fraction although other lipoprotein fractions contain some VE. VE is classed as a fat-soluble vitamin because of its solubility in solvents of low polarity. Within the cell, VE partitions into the hydrophobic core of the various cell membranes. The relative concentration of VE differs from one membrane to another. The content of tocopherols varies from tissue to tissue [226]. VE, an important lipid-soluble antioxidant, prevents the formation of lipid peroxides, which have been shown to induce oxidative damage [227]. VE is a free radical scavenger, i.e., a sacrificial molecule with which the peroxy radicals preferentially react, rather than with biological molecules, thus preventing damage to cell structures. It also scavenges O˙2¯, OH¯, singlet oxygen, lipid peroxyl radicals and other radical species [228,229]. VE physically stabilizes membrane permeability and fluidity. VE is a potent anti inflammatory agent [230]. VE may not only protect intact tissues by decreasing apoptosis induced by injurious conditions [231], but also increases apoptosis with a direct selective action on cancer cells [232,233]. It has been well-known that VE deficiency results in increased oxidative damage to DNA [226,234]. Such damage to DNA may produce mutations that cause permanent genetic alterations when the cell replicates its DNA and thus increases risk of cancer [227,235]. Umegaki et al. [236] showed that total body irradiation with X-rays at 3 Gy decreased both VE level and antioxidants in various tissues, such as bone marrow, liver, and plasma. They suggested that a decrease in antioxidant vitamins was involved in the mechanism of oxidative damage. It may be considered that radiation-induced organ or tissue damage might associate with decreased level of VE. Vitamin E exists in four common forms, including α-tocopherol, β- tocopherol, γtocopherol, and δ- tocopherol. Of these, α-tocopherol is the most effective scavenger of free radicals and the most predominant tocopherol [237]. Kumar et al. [21] reported that the use of alpha-tocopherol succinate during radiation therapy might have improved the efficacy of radiation therapy by enhancing tumour response and decreasing some of the toxicity towards normal cells. These are only a few of other reasons why VE was used as a radioprotector.

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Ferreira et al. [238] reported that VE decreased the incidence of symptomatic oral radioinduced mucositis in patients with cancer of the oropharynx and oral cavity. Mutlu-Turkoglu et al. [13] found that VE was effective in the prevention of radiation induced-intestinal injury in rats by ameliorating disturbances in prooxidant-antioxidant balance. Yoshimura et al. [239] determined that VE prevented the increase in oxidative damage to lipids and DNA in liver of Osteogenic Disorder Shionogi (ODS) rats given total body X-ray irradiation. Chan et al. [240] showed that, for patients with cerebral radionecrosis, alpha-tocopherol had the potential to be a complementary intervention for patients with cognitive dysfunction due to temporal lobe radionecrosis. Shaheen et al. [241] reported that administration of VE preceding gammaradiation exposure gave a significant radioprotection to the hematological parameters. In a previous study, Karslioglu et al. [1] demonstrated that VE had a protective effect on radiationinduced cataract by decreasing oxidative stress. At the same time, VE was used in combination with a number of protective agents, such as selenium [13], cysteine [241], sodium selenate [242], pentoxifylline [243,244] and beta-carotene [245-246]. In all of these combinations, a radioprotection was observed. In contrast, according to a current study, Biarati et al. [247] reported that alpha-tocopherol supplementation after treatment with radiation therapy produced unexpected adverse effects on the occurrence of second primary cancers and on cancer-free survival. In conclusion, considering the points mentioned above, it is seen that VE have also cytoprotective, apoptotic, immune regulator, antiinflammatory, and anticancer features. When this features come together, it is seen that VE have some beneficial effects in the prevention of radiation-induced organs and/or tissues damage without reducing the therapeutic efficiency of radiotherapy on cancer cells.

2.6. Selenium Selenium is an essential trace element involved in several key metabolic activities via selenoproteins, enzymes that are essential to protect against oxidative damage and to regulate immune function [248,229]. In the form of seleniocysteine, Se plays a main role as an active centre of glutathione peroxidase, one of the most important enzymes both for the detoxifying functions of the body and for the defence of cell membranes, sub cellular structures and macromolecules against a build-up of lipoperoxidases and free radicals [250,251]. Various studies have reported that selenium compounds such as selenoproteins protect cells against oxidative stress [252,253]. Selenium is a well-known, strictly concentration-dependent, modulator of cell-growth. In lower concentrations, selenium stimulates cell-growth and induces synthesis of selenoproteins. In this concentration, selenium is mainly an antioxidant. In higher concentrations, selenium compounds turn from antioxidants to pro-oxidants with potent inhibitory effects on cell-growth. Prooxidant activity may also account for cellular apoptosis and may provide a useful pharmaceutical application for selenium compounds as antibacterial, antiviral, antifungal and anticancer agents [254]. Induction of apoptosis and inhibition of cell proliferation are considered important cellular events that can account for the cancer preventive effects of selenium [255]. Many epidemiological observations support the proposition that Se acts to protect against the development of some cancers [256-258]. Selenium alone has been shown to reduce the risk of prostate cancer [257,259-261] and lung cancer [262]. Vadgama et al. [263] reported that Se

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has a significant anti-neoplastic effect on breast, lung, liver, and small intestinal tumor cells, and supplementation of Se enhanced chemotherapeutic effect of Taxol and Doxorubicin in these cells beyond that seen with the chemotherapeutic drugs used alone. Se at a high dose (0.2 mg/mouse/d or 10 mg/kg of body weight/d) enhances the therapeutic efficacy of some chemotherapeutic agents in athymic mice bearing human squamous cell carcinoma of the head and neck [264]. Reddy et al. [265] showed that selenium inhibited colon cancer incidence (tumours/animal) in rats and that this inhibition may be due to changes in prostaglandin synthesis and selenium-dependent glutathione peroxidase activity. Hussain et al. [266] determined that administration of selenium in drinking water reduced cervical cancer incidence from 72% in control mice to 37% in selenium treated mice as well as decreased hyperplasia and dysplasia. Burke et al. [267] reported that mice treated with selenium had reduced skin inflammation, pigmentation, later onset and lesser incidence of skin cancer following UV irradiation. El-Bayoumy et al. [268] demonstrated that selenium inhibited the incidence of breast cancer in rats and that this may be caused by selenium's inhibition of dimethylbenz(a)anthracene (DMBA)-DNA binding in breast tissue. Kise et al. [269] reported that selenium reduced liver and bile duct tumours in hamsters in incidence rate and number per animal, and significantly inhibited bile duct proliferation, possibly via glutathione peroxidase-mediated activity. Se may have potential in helping overcome the development of drug resistance in cancer cells, one of the serious problems in chemotherapy; a recent report shows that treatment of doxorubicin-resistant human small-cell lung carcinoma cells with selenium resulted in massive apoptosis, while treatment of the parental doxorubicin sensitive cells resulted in necrosis [270]. Subcutaneous injection of 2 mcg/g selenium into tumor-bearing mice led to a 75-percent reduction in tumor mass compared to controls [271]. This inhibitory effect of selenium was confirmed in human breast cancer cells in vitro [272]. Se may have potential in enhancing the efficacy of cancer treatment by radiotherapy and chemotherapy [64,273-276] and also protect against side effects to normal tissues that are associated with these treatments [13,65,274,277]. In a Mouse study, selenium decreased nephrotoxicity of cisplatin, while simultaneously increasing its anti-tumor activity [30]. Other animal studies confirmed these findings [274,275]. In a randomized crossover trial in humans, Hu et al. [278] investigated the effects of 4000 mcg/day selenium supplementation (from four days before until four days post-chemotherapy) on the toxicity of cisplatin. They found that Se consumption was associated with a higher WBC count, even with less consumption of granulocyte stimulating factor, and nephrotoxicity was also significantly less in patients taking selenium. A radioprotective effect of selenium on normal tissue and a possible radiosensitization of tumor cells have been previously suggested [13,64,65,273,277]. Cellular studies on mechanisms of protection by Se against radiation-induced oxidative damage indicate that Se is not likely to have a direct effect on preventing DNA damage through induction of glutathione peroxidase [279]. Schueller et al. [273] and Husbeck et al. [64] demonstrated that Se increased the radiation sensitivity of C6 rat glioma cells and prostate cancer cells by increasing the apoptotic potential and sensitizing them to radiation-induced cell killing, respectively. Se, as a Selenomethionine form, administered IP significantly increases the survival of irradiated mice and provides an extended window of protection. Selenomethionine was equally protective when administered at 24 h, 1 h and 15 min with respect to cobalt-60 irradiation at a low-dose rate (0.2 Gy/ min). Se protects against radiation-induced mutagenesis

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in vitro [280] and inhibits radiation-induced cell transformation [281]. Sodium selenite protected cultured normal human skin fibroblasts, but not squamous cell carcinoma cells, from single-dose and multiple fractionated-dose irradiation [282]. It has been reported that Se has a protective effect against ionizing radiation-induced malformations in mice [283,284]. Sun et al. [285] reported that 60Co-radiation decreased immune response capacity greatly, but that administration of Se counteracts this damage, and the antioxidative ability of Se was correlated with its protecting ability. Most radiation studies support other evidence for the synergistic effects of vitamin E and Se in protection from oxidative damage [13,242]. However, in one study a diet deficient in both vitamin E and Se did not affect the survival of irradiated mice [6] although preliminary data from this group indicated mice were more radiosensitive when fed a deficient diet [277,286]. In a pilot study of radiochemotherapy of advanced rectal cancer, oral sodium selenite (2000 micrograms) was administered after every course of fluorouracil and 400 micrograms daily after irradiation. The Se treatment was well tolerated, but the benefits of Se as an adjuvant in radiotherapy remain to be determined [65]. Supplementation with 200 mg/day of sodium selenite, during therapy (surgery and/or radiation) for squamous cell carcinoma of the head and neck, resulted in a significantly enhanced cell-mediated immune responsiveness during and after therapy [66]. Additionally, evidences suggest that Se has a positive effect on secondary-developing lymphedema edema of the arm and the head-and-neck region caused by radiation therapy alone or by irradiation after surgery [287,288]. In conclusion, GH seems to have beneficial effects both in the prevention of cancer development and the use to be adjuvant with cancer treatment and also the protection against cancer therapy-induced normal tissues damage.

2.7. Zinc Over the past 30 years, many researchers have demonstrated the critical role of zinc (Zn), a group IIb metal, in diverse physiological processes, such as growth and development, maintenance and priming of the immune system, and tissue repair [289]. A number of studies show zinc to be the catalytic component of more than 300 enzymes [290-293], the structural constituent of many proteins, and the regulatory ion for the stability of proteins and in preventing free radical formation. Therefore, zinc is a pivotal element in assuring the functioning of various tissues, organs, including immune response [290,293]. Physiologically bioavailable zinc has been identified as a nutrient essential for normal growth, sexual development, wound healing, ability to fight infections, sense of taste, night vision, healthy epithelial tissue, cell-mediated immunity and other vital functions [294]. Abundant evidence has demonstrated the antioxidant role of Zn [5,12,294-296]. Two mechanisms have been elucidated; the protection of sulfhydryl groups against oxidation, and the inhibition of the production of reactive oxygen species by transition metals [12,296]. Zinc ions may induce the synthesis of metallothionein, sulfhydryl-rich proteins being protective against free radicals [296]. Metallothioneins (MTs) play pivotal roles in metalrelated cell homeostasis because of their high affinity for metals, in particular zinc. Twenty cysteine aminoacids are in reduced form and bind seven zinc atoms through mercaptide bonds forming metal thiolate clusters. Moreover, MTs are antioxidant agents because the zincsulphur cluster is sensitive to changes of cellular redox state, and oxidizing sites induce the

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transfer of zinc from its binding sites in MTs to those of lower affinity in other proteins, as it occurs for superoxide dismutase activation. Thereby, the redox properties of MTs are crucial for their protective roles against the cytotoxic effect of reactive oxygen species, ionising radiation, electrophilic anti-cancer drug and mutagens, and metals. Metallothionein is excellent scavenger of hydroxyl radical. Iron and copper ions catalyse the production of hydroxyl radical from H2O2, and zinc is known to compete with both iron and copper from binding to cell membrane, thus decreasing the production of hydroxyl radical [290,291]. Zn is also the co-factor of copper-zinc superoxide dismutase (Cu-Zn SOD) enzyme that is an important scavenger of superoxide radicals [297]. Copper (Cu) and iron (Fe) can catalyze formation of the highly reactive HO˙ radicals from H2O2 via the Fenton reaction and decompose lipid peroxides to peroxyl and alkoxyl radicals, which favor the propagation of lipid oxidation. Zn could exert a direct antioxidant action by occupying iron or copper binding sites in lipids, proteins, and DNA [298]. It has been suggested that Cu-Zn SOD, in addition to its antioxidant properties, played a role in zinc homeostasis [299]. A major biochemical function of zinc includes the maintenance of membrane structure and function, and zinc has a special role in skin and connective tissue metabolism and in wound healing [293-296,300]. Zinc plays a part in the maintenance of epithelial and tissue integrity through promoting cell growth and suppressing apoptosis and through its under appreciated role as an antioxidant protecting against free radical damage during inflammatory responses. Thus, in case of diarrhea, multiple functions of zinc may help to maintain the integrity of the gut mucosa to reduce or prevent fluid loss [300]. Nowadays, there is no doubt that zinc is an essential trace element for the immune system [301]. A slightly decreased zinc status may first influence the immune system, due to increased number of infections [291,301]. Several data support the view that the impact of zinc on immunocompetence is greater in cell-mediated immunity than humoral [293]. In particularly, chemotaxis by neutrophils and monocytes, thymic endocrine activity, antigen presentation by MHC class II molecules, natural killer (NK) activity, cytokine production and TH1/TH2 balance are the immune functions, which are affected the most by zinc as well as TH3 cells [290,291,293,301,302]. Zinc is present in the cell nucleus, nucleolus and chromosomes, and zinc stabilizes the structure of DNA, RNA and ribosome. Numerous enzymes associated with DNA and RNA synthesis are also zinc metalloenzymes, including RNA polymerase, reverse transcriptases and transcription factor IIIA [303]. The chromosome breaks might be due to increased oxidative damage perhaps due to loss of activity of Cu/Zn superoxide dismutase or the DNArepair enzyme containing zinc [292]. Zinc acts as a signalling substance akin to conventional neurotransmitters in normal physiology [304,305]. Zn is well known as a cytoprotectant, protecting and stabilizing cellular molecules (e.g., proteins and DNA), macromolecular complexes (e.g., microtubules) and subcellular organelles (e.g., membranes) [306]. Provinciali et al. [307] reported that Zn exerted a direct selective action on cancer cells, determining their death through apoptosis. At the same time, Zn protects against the apoptosis induced by diverse physical, chemical, or immunologic stimuli [308,309]. It means that Zn may not only protect intact tissues by decreasing apoptosis induced by injurious conditions, but also increases apoptosis with a direct selective action on cancer cells. Here we have detailed the mechanisms by which Zn diversely acts as:

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(1) an anti-oxidant; (2) an organelle stabilizer; (3) an anti-apopototic agent; (4) an important cofactor for DNA synthesis; (5) a vital component for wound healing; (6) an anti-inflammatory agent. Currently, there are increasing evidences, from human and experimental studies, suggesting that Zn could be a beneficial agent in the protection against radiation-related normal tissue injury [20,51,67,68,310-314]. Zinc salts are a new class of radioprotectors against total body irradiation lethality [310]. It has been reported that Zinc Sulphate supplementation in patients with head and neck cancer delayed the starting week and reduced severity of radiation-induced oropharyngeal mucositis [51] and decreased the rate of oropharyngeal infection [28]. Floersheim et al. [20] reported that Zn did not inhibit the radiotherapeutic effect of gamma rays on human tumors grown as xenografts in immunosuppressed mice and significantly reduced the fall in the haematocrit and numbers of thrombocytes, erythrocytes and leucocytes caused by irradiation, indicating a sparing effect on bone marrow precursors of peripheral blood cells. In addition, we also demonstrated that Zinc Sulphate has beneficial effects on postponing the start of radiodermatitis and decreasing the severity of radiation-induced dermatitis in a rat model [68]. In clinical trials involving patients with taste dysfunction resulting from cancer, reduced taste acuity has been reversed in some patients by means of zinc administration [67,314]. Ali et al. [315] reported that metallothionein induction by Zn was a highly effective approach in preventing cardiotoxicity and hepatotoxicity caused by daunorubicin in the rats. According to the results of their experimental study, authors suggested that the use of Zn in the chemotherapy of cancer patients was able to reduce daunorubicin- induced cardiotoxicity and hepatotoxicity. Briefly, Zn as a radioprotector agent may protect intact tissues against injurious effects of cancer treatments, as chemotherapy or radiotherapy, without an inhibitor effect against their therapeutic effects. In conclusion, Zinc seems to have beneficial effects on radiation-induced some toxicities. These results may be pioneer to studies that will be performed with Zinc to protect from radiation toxicity. It would be worthwhile studying the effects of zinc sulphate supplements in radiation-treated cancer patients, in the hope of reducing radiation-induced toxicity.

2.8. Ginkgo Biloba The leaves of the Chinese tree Ginkgo Biloba (GB) have been cultivated for their medicinal properties for several thousand years. The variety of therapeutic uses for GB stems from its numerous constituents, which provide for a broad of pharmacological activities. It has been used in the treatment of various common geriatric complaints including vertigo, short-term memory loss, hearing loss, lack of attention or vigilance [316]. GB has been used for treatment of peripheral vascular occlusive disease and cerebral insufficiency in European countries for over 20 years [317]. The antioxidant properties of GB have been well-known [318]. GB is standardized to contain 24% flavonoids and 6% terpene lactones (ginkgolides and bilobalide) [319,320]. This

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extract has been reported to be a potent scavenger for several ROS, i.e., O˙2¯ [321-323], OH¯ [324,325], H2O2 [326] and peroxyl radicals [327]. Numerous studies which were initiated in the early 1980s have shown that GB, acting as anti-oxidants, can oppose the deleterious effects of oxidative damage caused by free radicals and related ROS [318]. Recent studies of mitochondria from brain, liver or heart cells exposed to hypoxia and/or ischemic stress show the protective effect of GB against oxidative stress and also limiting effects membrane lipoperoxidation [328-332]. Bridi et al. [320] observed an increase SOD activity in the hippocampus (HC) and substantia nigra (SN), and a decrease of the lipid peroxidation in the HC of patients with Parkinson’s and Alzheimer’ disease which frequently associate with oxidative stress after treated with GB. Naidu et al. [333] carried out a study about protective effect of GB against doxorubicin-induced cardiotoxicity in mice. They found that myocardial SOD and GSH-Px activity decreased and lipid peroxidation increased after treated with doxorubicin. They detected that GB significantly protected the mice from myocardial lipid peroxidation and increased antioxidant enzymes. They suggested that GB protected mice from doxorubicin-induced cardiotoxicity. El-Khatib et al. [334] and Daba et al. [335] reported that GB had a protective effect against to bleomycin-induced acute lung injury in rats. It has been reported that GB has a protective effect against carbon tetrachlorideinduced liver damage [336] and serotonin-induced mitogenesis [322] and UVB-induced cytotoxicity in vitro [337]. Ozkur et al. [338] investigated antioxidant effect of GB against UVB irradiated mice skin. They carried out the study on four groups of mice (n = 6 in each group). The first group was a control group (G1). The second group (G2) was only exposed to acute UVB irradiation. The third group (G3) received 100 mg/kg/day of GB orally for 5 days before UVB irradiation and the fourth group (G4) was given only a single dose of GB immediately after UVB irradiation. They found that the SOD activities and MDA levels in G2, G3 and G4 decreased significantly when compared with G1 (P < 0.05). They detected that the SOD activities of G3 and G4 were higher when compared with G2 (P < 0.05). They suggested that GB might have an important effect, both as a protective and therapeutic agent, in sunburn after UVB irradiation. Orhan et al. [339] investigated the effect of several natural and synthetic compounds on selenite-induced cataract in rat. They found that selenite treatment caused a significant decrease in the activity of erythrocyte SOD and this accompanied by a simultaneous increase in the levels of MDA either in lens or in plasma. They also found that GSH-Px activity was significantly higher than the control in the selenitetreated group. They reported that the activity of SOD and GSH-Px enzymes in the GB-treated group were significantly higher than in the selenite-treated group and MDA level in the GBtreated group significantly lower than in the selenite-treated group. But, they also reported that GB did not affect the cataract formation. Thiagarajan et al. [16] reported that GB’s inherent antioxidant, antiapoptotic and cytoprotective action and potential anticataract ability appeared to be some of the factors responsible for its beneficial effects. Anticancer activities of GB are well-documented [318]. Kim et al. [340] reported that GB induced apoptosis of oral cavity cancer cells and caspase-3 was activated in this apoptosis. Therefore, EGb 761 may be considered as a possible chemopreventive agent against oral cavity cancer. Chen et al. [341] carried out a study to observe the clinical efficacy of Ginkgo biloba exocarp polysaccharides (GBEP) capsule preparation in treating upper digestive tract malignant tumors of middle and late stage. In this study, the authors treated eighty-six patients of the upper digestive tract malignant tumors with GBEP capsule preparation taken

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orally, and the clinical symptoms and the qualities of life of the patients with single GBEP and combined with operation, radiotherapy or intervention chemotherapy were observed. They found that GBEP preparation could markedly improve the patients'clinical symptoms. Karnofsky scoring of the patients markedly increased after treatment. There were 2 CR (complete response, 6.3%), 22 PR (partial response, 68.8%) and 5 SD (stable disease, 15.6%) of 32 cases with single GBEP preparation. The survival periods of the 32 cases were markedly prolonged. The preparation could relieve the inhibited hematopietic function and the weight loss due to radiotherapy. The authors suggested that GBEP capsule preparation has some definite therapeutic effects on upper digestive tract malignant tumors of middle and late stage. Chao et al. [342] evaluated that the effect of Ginkgo biloba extract (EGb 761) on cell proliferation and cytotoxicity in human hepatocellular carcinoma (HCC) cells (HepG2 and Hep3B). They reported that Ginkgo biloba extract significantly could suppress proliferation and increase cytotoxicity in HepG2 and Hep3B cells. Additionally, Ginkgo biloba extract could decrease the expression of proliferating cell nuclear antigen and increase p53 expression in HepG2 cells. In a similar study, Xu et al. [343] studied the therapeutic mechanism of Ginkgo biloba exocarp polysaccharides (GBEP) on gastric cancer. They measured the area of tumors by electron gastroscope before and after treatment, then they calculated inhibitory and effective rates. They found that compared with the statement before treatment, GBEP capsules could reduce the area of tumors, and the effective rate was 73.4%. Ultrastructural changes of the cells indicated that GBEP could induce apoptosis and differentiation in tumor cells of patients with gastric cancer. GBEP could inhibit the growth of human gastric cancer SGC-7901 cells following 24-72 h treatment in vitro at 10-320 mg/L, which was dose-and time-dependent. GBEP was able to elevate the apoptosis rate and expression of c-fos gene, but reduce the expression of c-myc and bcl-2 genes also in a dosedependent manner. The authors suggested that the therapeutic mechanism of GBEP on human gastric cancer may have related to its effects on the expression of c-myc, bcl-2 and c-fos genes, which could inhibit proliferation and induce apoptosis and differentiation of tumor cells. Yamamoto et al. [344] reported that GBE had a inhibitory effect on the growth of cancer cells. The antioxidant, anti-aptotic and cytoprotective properties of GB appear to be some of the factors responsible for its beneficial effects, protecting the organs, tissues and cells against radiation-induced toxicities [17]. Moreover, it was reported that GBE enhanced the radiation effect on tumor in C3H mouse fibrosarcoma, probably by increasing tumor blood flow without increasing acute normal tissue radiation damage. Based on this action, GBE has been suggested to be a possible radio sensitizer [22]. Radiation-induced clastogenic factors (CF) which are characterized by oxidative stress are found in the plasma of Chernobyl accident recovery workers and that their chromosome damaging effects are inhibited by antioxidant treatment with a Ginkgo biloba extract (EGb761). Animal studies demostrated that, in rats irradiated with a radiation dose of 4.5 Gy, CF-induced chromosome damage was regularly prevented by GB due to its ability a potent scavenger for superoxide dismutase radical [345,346] Lamproglou et al. [347] reported that a relatively low dose of total body irradiation induced a substantial acute learning dysfunction in the rat, which persisted fourteen days after TBI and this effect was prevented by the administration of EGb 761 (50 or 100 mg/kg) started twenty-four hours after irradiation. In a recent study, we demonstrated that GB as an antioxidant agent had the protective effect on radiation-induced cataract by its preventing effect on oxidative stress [17].

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These are only a few of the reasons why GBE may be used as a radioprotector agent in radiation-induced toxicities. It would be worthwhile studying the effect of GB supplements in patients with cancer, in the hope of reducing cancer treatments-induced toxicity and increasing tumor response to antitumor activities of these treatments.

CONCLUSIONS Most types of radiation therapy kill cancer cells primarily by inducing oxidative stress in cancerous tissue, i.e., the very high level of free radicals that results from radiation therapy is toxic to cancer (and normal) cells. Radiation toxicities on the surrounding normal tissues in irradiated volume are caused by the oxidative stress Antioxidants, by detoxifying ROS, can reduce or prevent these toxicities without interfering with the anticancer effects of radiotherapy. Antioxidants work in concert with one another by a series of oxidationreduction (redox) reactions to quench reactive oxidant species. Redox buffering systems of the common antioxidants are all related in a stepwise, sequential recycling process, which provides ongoing neutralization of free radicals. An understanding of these free radical defenses provides a scientific basis for the use of with antioxidants during cancer treatment. Considering the points mentioned above, in addition to antioxidant effects, amifostine, melatonin, vitamin A, E, and C, selenium, zinc, and gingko biloba have also cytoprotective, apoptotic, immune regulator, antiinflammatory, and anticancer features. When this features come together, it is seen that these antioxidants have some beneficial effects in the prevention of radiation-induced organs and/or tissues damage without reducing the therapeutic efficiency of radiotherapy on cancer cells. In addition to their antioxidant effects, these substances show antiapoptotic effects on normal tissue cells while increasing apoptosis in cancer cells. This situation may assist in the improvement of radiotherapy effects on cancer cells, and in the prevention of surrounding healthy tissues from radiation-induced damage. Immune regulatory and antiinflammatory features of the antioxidants may have influence upon the prevention of radiation-induced damage. In addition to all these features, it is known that some antioxidants serve as radiosensitizer, such as gingko biloba, vitamin A, at the same time. This situation may improve the therapeutic effects of radiotherapy on cancer cells while having beneficial effects for the prevention of organs and/or tissues damage caused by radiation. For today, even though there are many compounds that are used as radioprotectors and are shown to be beneficial in some aspects, most cancer patients still suffer from organs and/or tissues damages caused by radiation. Unfortunately, there is no effective agent (except amifostine that is used for the therapy of radiation-induced xerostomia approved by FDA) that can be used for the prevention or therapy of organs and/or tissues damages due to radiation, for which researchers are interested in for the last few decades. The radioprotectors have not reached the level of providing the field of medicine with an agent that conforms to all criteria of an optimal radioprotectant, including effectiveness, toxicity, availability, specificity and tolerance. Antioxidants form only one class of radioprotective agents. Researchers should be directed towards the appropriate dosage and combined use under the basis of biological

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effectiveness for the compounds that are scientifically proven to have some effective radioprotective features, or towards the introduction of new compounds in this area. At the same time, there is need the development of target oriented therapy forms by detailed research of physical, biological, and chemical basis of radiation damages. Multidisciplinary studies in the field of radioprotection are to be involved all aspects of the subatomic mechanisms of free radical formation, macromolecular and intracellular radiation-induced alterations, biochemical and physiological homeostatic mechanisms and organ level manifestations. In conclusion, antioxidants discussed above have radioprotective effects in the prevention of radiation induced-organs and/or tissues; it would be worthwhile studying the effect of these antioxidants, in combination, in radiation-treated cancer patients in a randomised clinical study with a larger number of patients, in the hope of reducing radiation-induced toxicity.

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mitochondrial aging by protecting against oxidative stress. Free Radic Biol Med, 1998, 24, 298-304. [330] Fitzl, G; Welt, K; Schaffranietz, L. Myocardium-protective effects of Ginkgo biloba extract (EGb 761) in old rats against acute isobaric hypoxia. An electron microscopic morphometric study. I. Protection of cardiomyocytes. Exp Toxicol Pathol, 1996, 48, 3339. [331] Shen, JG; Zhou, DY. Efficiency of Ginkgo biloba extract (EGb 761) in antioxidant protection against myocardial ischemia and reperfusion injury. Biochem Mol Biol Int, 1995, 35, 125-134. [332] Dumont, E; D'Arbigny, P; Nouvelot, A. Protection of polyunsaturated fatty acids against iron-dependent lipid peroxidation by a Ginkgo biloba extract (EGb 761). Methods Find Exp Clin Pharmacol, 1995, 17, 83-88. [333] Naidu, MU; Kumar, KV; Mohan, IK; Sundaram, C; Singh, S. Protective effect of Gingko biloba extract against doxorubicin-induced cardiotoxicity in mice. Indian J Exp Biol, 2002, 40, 894-900. [334] El-Khatib, AS; Moustafa, AM; Abdel-Aziz, AA; Al-Shabanah, OA; El-Kashef, HA. Ginkgo biloba extract (EGb 761) GBE modulated bleomycin-induced acute lung injury in rats. Tumori, 2001, 87, 417-422. [335] Daba, MH; Abdel-Aziz, AA; Moustafa, AM; Al-Majed, AA; Al-Shabanah, OA; ElKashef, HA. Effects of L-carnitine and ginkgo biloba extract (EG b 761) in experimental bleomycin-induced lung fibrosis. Pharmacol Res, 2002, 45, 461-467. [336] Bahcecioglu, IH; Ustundag, B; Ozercan, I; Ercel, E; Baydas, G; Akdere, T; Demir, A. Protective effect of Ginkgo biloba extract on CCI4-induced liver damage. Hepatol Res, 1999, 15, 215-224. [337] Kim, SJ. Effect of biflavones of Ginkgo biloba against UVB-induced cytotoxicity in vitro. J Dermatol, 2001, 28, 193-199. [338] Ozkur, MK; Bozkurt, MS; Balabanli, B; Aricioglu, A; Ilter, N; Gurer, MA; Inaloz, HS. The effects of EGb 761 on lipid peroxide levels and superoxide dismutase activity in sunburn. Photodermatol Photoimmunol Photomed, 2002, 18, 117-120. [339] Orhan, H; Marol, S; Hepsen, IF; Sahin, G. Effects of some probable antioxidants on selenite-induced cataract formation and oxidative stress-related parameters in rats. Toxicology, 1999, 139, 219-232. [340] Kim, KS; Rhee, KH; Yoon, JH; Lee, JG; Lee, JH; Yoo, JB. Ginkgo biloba extract (EGb 761) induces apoptosis by the activation of caspase-3 in oral cavity cancer cells. Oral Oncol, 2005, 41, 383-389. [341] Chen, HS; Zhai, F; Chu, YF; Xu, F; Xu, AH; Jia, LC. Clinical study on treatment of patients with upper digestive tract malignant tumors of middle and late stage with Ginkgo biloba exocarp polysaccharides capsule preparation. Zhong Xi Yi Jie He Xue Bao, 2003, 1, 189-191 (Abstract). [342] Chao, JC; Chu, CC. Effects of Ginkgo biloba extract on cell proliferation and cytotoxicity in human hepatocellular carcinoma cells. World J Gastroenterol, 2004, 10, 37-41. [343] Xu, AH; Chen, HS; Sun, BC; Xiang, XR; Chu, YF; Zhai, F; Jia, LC. Therapeutic mechanism of ginkgo biloba exocarp polysaccharides on gastric cancer. World J Gastroenterol, 2003, 9, 2424-2427.

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[344] Yamamoto, S; Nakano, K; Ishikawa, C; Yamamoto, M; Matsumoto, Y; Iwahara, M; Furusaki, S; Ueoka, R. Enhanced inhibitory effects of extracts from Ginkgo biloba L. leaves encapsulated in hybrid liposomes on the growth of tumor cells in vitro. Biochem Engineering J, 2002, 12, 125-130. [345] Emerit, I; Arutyunyan, R; Oganesian, N; Levy, A; Cernjavsky, L; Sarkisian, T; Pogossian, A; Asrian, K. Radiation-induced clastogenic factors: anticlastogenic effect of Ginkgo biloba extract. Free Radic Biol Med, 1995, 18, 985-991. [346] Alaoui-Youssefi, A; Lamproglou, I; Drieu, K; Emerit, I. Anticlastogenic effects of Ginkgo biloba extract (EGb 761) and some of its constituents in irradiated rats. Mutat Res, 1999, 445, 99-104. [347] Alaoui-Youssefi, A; Lamproglou, I; Drieu, K; Emerit, I. Anticlastogenic effects of Ginkgo biloba extract (EGb 761) and some of its constituents in irradiated rats. Mutat Res, 1999, 445, 99-104.

In: Leading Edge Antioxidants Research Editor: Harold V. Panglossi, pp. 145-177

ISBN 1-60021-274-3 © 2007 Nova Science Publishers, Inc.

Chapter 6

ANTIOXIDANT THERAPY FOR CHRONIC INFLAMMATORY DISEASES Jayraz Luchoomun, Dominic Sinibaldi and Xi-Lin Chen* Discovery Research, AtheroGenics, Inc., 8995 Westside Parkway, Alpharetta, GA, USA

ABSTRACT Oxidative signals play important roles in the pathogenesis of inflammatory diseases such as atherosclerosis, arthritis, asthma, and neurodegenerative diseases. Oxidative stress occurs when there is an increased production of reactive oxygen species, reactive nitrogen species, or decreased antioxidant defense mechanisms. Oxidative stress contributes to the initiation and progression of chronic inflammation by promoting cell proliferation, adhesion molecule expression, cytokine and chemokine production, and matrix metalloproteinase generation. A number of animal studies and clinical trials have demonstrated increased levels of biomarkers for oxidative stress in various inflammatory diseases including atherosclerosis, asthma and rheumatoid arthritis. Related studies have also demonstrated that decreased antioxidant capacity enhanced susceptibility to immune and inflammatory diseases; while the use of antioxidants diminished or prevented inflammatory diseases. Therefore, increased cellular antioxidant activity or scavenger ROS may represent a novel approach for the treatment of inflammatory diseases. However, several clinical trials using antioxidant vitamins have failed to demonstrate beneficial effects for some inflammatory diseases. The purpose of this review is to summarize our recent understanding of the oxidative signaling events involved in inflammatory processes in the context of experimental and clinical studies that utilize antioxidants for the treatment of inflammatory diseases.

Key Words: Reactive oxygen species, antioxidant, asthma, atherosclerosis, rheumatoid arthritis

*

Corresponding Author: Xilin Chen, M.D., Ph.D. Discovery Research, AtheroGenics, Inc. 8995 Westside Parkway, Alpharetta, GA 30004, Phone: 678-336-2711, Fax: 678-393-8616, E-mail: [email protected]

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INTRODUCTION Oxidative stress occurs when there is an increased generation of reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) or a decrease in antioxidant capacity. In the past, much of the focus on the study of ROS has been regarding their role in antimicrobial defense as part of the innate immune system. Phagocytic leukocytes such as neutrophils and macrophages release a large amount of ROS that are toxic to invading microorganisms and cause destructive effects in adjacent tissues. However, recent research has demonstrated that ROS are also generated by non-phagocytic cells in a controlled fashion and as by-products of cellular metabolism, primarily in the mitochondria. Growing evidence indicates that ROS function as important intercellular second messengers that modulate many downstream signaling molecules, such as transcription factors, mitogen-activated protein (MAP) kinases and protein tyrosine kinases. Induction of these signaling cascades leads to cellular proliferation, apoptosis, expression of pro-inflammatory genes, and modification of the extracellular matrix. Oxidative stress has been implicated in the pathogenesis of inflammatory diseases including rheumatoid arthritis, asthma, chronic obstructive pulmonary diseases (COPD), inflammatory bowel disease, and vascular diseases such as atherosclerosis and restenosis. ROS generated in response to pro-inflammatory cytokines, growth factors, chemical exposure and other stresses serve as important signaling molecules in the activation of key transcription factors, protein kinases, and regulation of many genes involved in the inflammatory response. Recent studies have also demonstrated that decreased antioxidant capacity can result in enhanced susceptibility to immune and inflammatory diseases; while the use of antioxidants diminished or prevented inflammatory diseases. Here we review recent advances in understanding the role of oxidant stress in the pathogenesis of chronic inflammatory diseases and the use of antioxidants as therapy for chronic inflammatory diseases.

ROS, OXIDATIVE STRESS, AND INFLAMMATORY DISEASES Reactive Oxygen Species and Antioxidant Defenses ROS are formed as intermediates in oxidation and reduction (redox) processes, in the conversion of oxygen to water. Eukaryotic cells continuously produce ROS, such as superoxide (O2-.), hydrogen peroxide (H2O2), and hydroxyl radicals (OH.); through the univalent reduction of oxygen in the presence of a free electron (e). Superoxide is the primary radical formed by one electron reduction of molecular oxygen. Hydrogen peroxide is produced primarily from dismutation of O2-.. This reaction can be spontaneous or can be catalyzed by superoxide dismutase (SOD). Unlike O2-, H2O2 is not a free radical and is a much more stable molecule. Hydrogen peroxide is lipid soluble, crosses cellular membranes and has a longer half-life than O2-. Hydrogen peroxide, in the presence of metal-containing molecules such as Fe2+, can also be reduced to generate the highly reactive OH· (hydroxyl radical). The hydroxyl radical is extremely reactive and very short lived and induces local damage where it is formed[1, 2]. Peroxynitrite (ONOO-) is the product of the diffusion-

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controlled reaction between nitric oxide and superoxide and is considered is a strong oxidant[3]. ROS are produced by virtually all types of cells and by a variety of sources. In addition to mitochondrial sources of ROS, O2- and/or H2O2 can be derived from a variety of enzymatic reactions including xanthine oxidase, cyclooxygenase, lipoxygenase, NO synthase, cytochrome p450 oxidase, hemoproteins such as heme and hematin, and NAD(P)H oxidases[2]. However, recent studies have implicated controlled generation of ROS by specialized plasma membrane oxidases such as NADPH oxidase in response to growth factors and cytokines. NADPH oxidase contains a membrane-bound cytochrome b558–like molecule that is responsible for electron transfer from NAD(P)H to oxygen, producing O2-. This cytochrome consists of p22phox (also called Nox2) and the flavoprotein gp91phox, or one of its recently identified homologues including Nox1, Nox3 and Nox4 [4]. Two cytoplasmic subunits, p47phox and p67 phox, and the small G protein rac1 also play regulatory roles[5]. The Nox family proteins comprise 7 homologues and are expressed in a variety of different tissues including the gastrointestinal tract, vascular system, kidney, thyroid, lung, and lymph node tissues. Accordingly, the proposed physiological functions of these enzymes are diverse and include epithelial cell host defense, maintenance of vascular tone, regulation of hormone biosynthesis, and modulation of cell proliferation and differentiation. The enzymes are likely to be involved in a variety of disease processes ranging from immune deficiencies and hypothyroidism to atherosclerosis and vestibular dysfunction[6]. ROS are tightly regulated by anti-oxidants such as SOD, catalase, thioredoxin, glutathione, anti-oxidant vitamins, and other small molecules. SOD is responsible for converting O2- into H2O2. There are three isoforms, CuZnSOD, MnSOD and extracellular SOD (EC-SOD)[7]. H2O2 is then scavenged by catalase and glutathione peroxidase[7]. Under normal conditions, the rate of ROS production is balanced by the rate of elimination. A condition of oxidative stress occurs when cellular production of ROS overwhelms the antioxidant capacity or the cellular endogenous antioxidant capacity is compromised.

Redox Signaling and Inflammatory Gene Expression Recent studies have demonstrated that ROS can serve as important signaling molecules and can act on specific downstream effectors to influence cell activity and function[8]. Physiologic generation of ROS has been implicated in a variety of biological responses such as signal transduction, cell proliferation, gene expression, protein kinase activation, inflammation and apoptosis. A variety of cell types generate ROS in response to proinflammatory stimuli, such as cytokines, lipopolysaccharide (LPS) and viral infection. Intracellular ROS can serve as regulatory signals that modulate expression of early inflammatory genes[8]. Oxidative signals are capable of regulating the expression of cytokines and chemokines including TNF-α, IL-1β [9], IL-6 [10], IL-8 [11], and MCP-1 [12, 13], adhesion molecules such as VCAM-1 and ICAM-1 [12, 13]. Matrix metalloproteinases (MMPs) a family of proteolytic enzymes are involved in tissue destruction in inflammatory diseases such rheumatoid arthritis, asthma and atherosclerosis. Similar to other inflammatory genes, the expression of MMPs including MMP-1, MMP-2 and MMP-9 are regulated by redox-sensitive mechanisms [14-16].

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NF-κB is a ubiquitous family of transcription factors that transduce a wide range of noxious or inflammatory stimuli into the coordinated activation of multiple genes, including those coding for cytokines, cytokine receptors, adhesion molecules, and chemokines [17]. NF-κB is a critical element in immune and inflammatory responses. NF-κB is regulated by a redox-sensitive mechanism. For example, ROS can activate NF-κB in response to a variety of signals including TNF-α, IL-1β, LPS, H2O2 and various forms of cellular stress [18]. This activation can be specifically inhibited by diverse thiol antioxidants, suggesting an important role of ROS in mediating the activation of NF-κB and inflammatory gene expression [18]. Similarly, ROS have been shown to play an important role in the regulation of other transcription factors such as AP-1 [19], hypoxia-inducible transcription factor-1α (HIF-1α) [20] and cAMP-responsive element-binding protein (CREB)[21]. Several protein kinases have been implicated in transducing activation signals from ROS to downstream effector molecules [22]. The p38 MAP kinase pathway is a particularly relevant target for redox modulation in chronic inflammatory diseases. p38 MAP kinase regulates expression of many inflammatory genes such as MCP-1 [23], IL-6 [24], VCAM-1 [25] and TNF-α [26]. Both IL-1β and TNF-α promote the phosphorylation/activation of the p38 MAP kinase pathway in a manner that can be antagonized by the thiol antioxidant Nacetylcysteine [27, 28] and by expression of SOD [29]. Apoptosis signal-regulating kinase 1 (ASK1) is an upstream activator of p38 and JNK/MAPK signaling cascades [30]. ASK1 is activated by various oxidants and plays an important role in oxidative stress-mediated apoptosis and inflammatory responses [31, 32]. The activation of ASK1 has been shown to be negatively regulated by several antioxidant proteins. Thioredoxin, in its reduced form, directly binds to the N-terminal region of ASK1 and inhibits oxidant stress- and cytokineinduced ASK1 activation[31]. The 14-3-3 protein also binds to ASK1 through phosphorylated Ser967 and suppresses ASK1-induced apoptosis [33]. ROS is also important in LPS-induced formation of the TRAF6-TLR4 complex. Treatment with the antioxidant N-acetylcysteine abolished formation or TRAF6-TRL4 complex and inhibited ASK1 and JNK activation by LPS [34]. Additional protein kinases that are redox-regulated include the Src kinase family [35], c-Jun NH2- terminal kinases [36], protein kinase B/AKT [37], ERK1,2 [9], protein kinase C [38] and Erk5 [39]. Recent studies have demonstrated that peroxynitrite can act as signaling molecule. Treatment of endothelial cells with exogenous peroxynitrite induced a time- and dosedependent increase in VEGF and activation of STAT3 [40]. Peroxynitrite serves as a signaling molecule in flow-dependent activation of c-Jun NH2-terminal kinase [41]. Peroxynitrite promotes the synthesis of prostaglandin through activation of cyclooxygenase via oxidation of iron in the enzyme [42, 43]. Furthermore, peroxinitrite can activate Nrf2 transcription factor and increase antioxidant response element (ARE)-driven transcriptional activities [44, 45].

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ANTIOXIDANTS AND ANTIOXIDANT DEFENSE MECHANISMS Endogenous Antioxidant Defense Mechanisms In order to prevent or minimize oxidant stress-mediated injury, the human body uses an elaborate system of biochemical defenses. These antioxidant defense mechanisms include three lines of defense. The first line of defense uses a variety of free radical scavenging enzymes, antioxidant proteins, and metal-chelating proteins to suppress the generation of ROS. The second line of defense utilizes small molecule antioxidants including the glutathione system and a group of diverse radical-scavenging antioxidants such as vitamin E and C to prevent the propagation of reactive species. Finally, a battery of repair and de novo enzymes (such as lipases, proteases, DNA repair enzymes, and transferases) are involved in repairing damage and reconstituting membranes (figure 1)[46]. Intracellular enzymatic antioxidants include SOD, catalase, glutathione peroxidases, glutathione reductases, thiol-disulfide oxidoreductases, and peroxiredoxins. In the extracellular fluid where the major antioxidant enzymes are absent, hydroperoxides of lipoprotein, phospholipids and cholesteryl esters are reduced to alcohol by a non-enzymatic reaction involving methionine residues from apolipoproteins such ApoA-I [47]. Transition metals (if left alone) can readily participate in autooxidation or the decomposition of peroxides to peroxyl, alkoxyl, and hydroxyl radicals. Studies have shown that transition metals such as copper or iron can induce oxidative damage to lipoproteins [48]. Inactive chelates such as ferritin, transferrin, and ceruloplasmin that specifically interact with iron and copper may form an antioxidant defense system by capturing and binding these transition metals [49]. The enzyme heme oxygenase also removes potential prooxidants heme and iron in a process that simultaneously produces the antioxidants biliverdin and bilirubin [50]. Another important antioxidant system is the Nrf2/ARE (antioxidant response element) pathway. ARE is a cis-acting transcriptional regulatory element that regulates expression of genes coding a number of phase II detoxifying enzymes and antioxidant enzymes including NADPH:quinone oxidoreductase (NQO1), glutathione peroxidase (GPx), γ-glutamylcysteine synthase (γ-GCS), glucuronosyltransferase, ferritin, and HO-1 [51-55]. The transcription factor Nrf2 is responsible for both constitutive and inducible expression of ARE-mediated genes [54, 56]. Nrf2-deficient mice have decreased antioxidant capacity and enhanced oxidative stress [57, 58]. In addition, Nrf2 is involved in immune and inflammatory processes. The disruption of Nrf2 significantly enhanced pulmonary injury and increased inflammatory cell infiltration when mice were exposed to hyperoxia and bleomycin [59, 60]. Older Nrf2-deficient female mice developed lupus-like nephritis [59]. Nrf2-deficient mice also had prolonged inflammation during cutaneous wound healing [61], enhanced bronchial inflammation, and susceptibility to cigarette smoke-induced emphysema [62]. Therefore, intracellular antioxidant activity is important in determining potential susceptibility to autoimmune and inflammatory diseases.

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Exogenous Chemical Antioxidants Autoxidation of polyunsaturated fatty acids is a process involving free radical reactions. An initial fatty acid radical reacts with oxygen to form a peroxyl radical which extracts a hydrogen from another fatty acid to generate the hydroperoxide product and a new fatty acid radical. This results in the propagation of the chain reaction. Chemical antioxidants act by donating an electron to a free radical and converting it to a nonradical form. Likewise, reducing compounds can terminate radical chain reactions and reduce hydroperoxides and epoxides to less reactive derivatives. However, chemical antioxidant defense can be both beneficial and harmful. When an antioxidant scavenges a free radical, its own free radical is formed; many antioxidants can act as pro-oxidants by reducing nonradical forms of oxygen to their radical derivatives, particularly if redox cycling occurs. An efficient antioxidant interferes with the autoxidation radical chain process which is propagated by the fatty acid. The exact mix of pro- and antioxidant properties of a reducing compound is a complex interaction involving pH, relative reactivity of radical derivatives, and availability of metal catalysts. For example, the anti- or pro-oxidant properties of sulfhydryl compounds depend upon pH; and those of β-carotene depend upon oxygen concentration [63].

Figure 1. Antioxidant defense machinery – categories and actions

ANTIOXIDANTS AND CARDIOVASCULAR DISEASES Cardiovascular disease is the leading cause of death in industrial countries and many major cities with developing nations. Most cardiovascular events are secondary to atherosclerosis, which is characterized by atherosclerotic plaque formation in artery walls [64]. The earliest fatty streak is compiled of foam cell aggregates with oxidized LDL trapped

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in the sub-endothelial space of the vessel wall, which slowly leads to the development of the occlusive plaque in atherosclerosis [65, 66]. Numerous studies suggest that oxidative damage, in particular oxidized LDL, promotes several key steps in atherosclerosis [67] including endothelial cell damage [68], foam cell accumulation [69-71], recruitment of inflammatory cells[72], and cell proliferation [73]. Circulating levels of oxidized LDL composed of oxidized phospholipids are strongly associated with angiographically documented coronary artery disease [74]. In addition, ROS may directly cause damage to the arterial endothelium [75], promote thrombosis [76], and affect normal vasomotor regulation [77]. While the generation of ROS is inevitable, oxidant-mediated disease is proposed to occur only under circumstances that overwhelm the antioxidant defense machinery. Diverse groups of antioxidant compounds with properties and modes of action are involved in preventing the free radical formation, propagation, and formation of oxidative products, and can participate in the repair of oxidant-induced injury. For example, significantly higher levels of 8,12isoprostane F2α-VI (a marker for reactive oxygen species and a potent vasoconstrictor) and lower levels of antioxidants including vitamins A, C, and E; uric acid; carotenoid; and superoxide dismutase were found in chronic heart failure patients in comparison to healthy controls. In addition, levels of 8,12-isoprostane F2α-VI correlated with the severity of chronic heart failure, and inversely correlated with heart contractile function. These findings support the rationale for intervention trials investigating the beneficial effects of antioxidant micronutrients in cardiovascular disease [78].

Effects of Antioxidant Vitamin Supplementation Dietary antioxidants including vitamin E (α -tocopherol), vitamin C (ascorbic acid), and β -carotene (provitamin A) have received the most attention for targeting cardiovascular disease [79]. Vitamin C is regarded as the most important water-soluble antioxidant in human plasma and an important regulator of iron uptake. It reduces ferric Fe3+ to ferrous Fe2+ ions, thus promoting dietary non-heme iron absorption from the gut and stabilizing iron-binding proteins. Low levels of vitamin C are known to occur in several oxidative stress conditions such as diabetes mellitus [80] and are associated with death from cardiovascular diseases [81]. Vitamin E is lipid soluble and exists as at least 8 naturally occurring compounds that include α, β, λ and δ-tocopherol and α, β, λ and δ-tocotrienol, with α-tocopherol being the most active component of vitamin E. β-carotene along with α-carotene, lycopene, lutein, zeaxanthin and β-cryptoxanthin are the principal dietary carotenoids. Vitamin E and βcarotene have been of particular interest because both are shuttled by LDL particles as well as other lipoproteins and are easily exchanged among lipoproteins. High doses of vitamin E leads to prevention of the oxidative modification of LDL in vitro, a reduction in monocyte adhesion to endothelium, and an inhibition of platelet activation [82]. These findings align with the oxidative modification hypothesis have fueled epidemiological and clinical trials on the role of vitamin E in the prevention and treatment of cardiovascular disease. Several lines of evidence have linked the benefits of vitamins to cardiovascular diseases. Vitamins C and E have protective features in many diseases known to exhibit enhanced oxidative stress. Vitamins C and E can decrease vascular injury in a pig coronary balloon injury model and these effects correlated with the ability to inhibit oxidization of LDLs ex-

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vivo and suppress O2- production in injured vessels [83]. It is possible that the beneficial effects of antioxidant vitamins in coronary artery disease could be related, in part, to alterations in vessel redox state [84]. Although, studies on animals suggested that a number antioxidant compounds decreased lesion formation in animal models of atherosclerosis, there has not been any clear evidence linking the antioxidant protection of LDL with a reduction in atherosclerosis [85]. Vitamins C and E seemed to be beneficial in hypercholesterolemiainduced experimental atherosclerosis through prevention of VEGF and VEGFR-2 upregulation. [86]. Male New Zealand White rabbits fed on vitamin E demonstrated enrichment of vascular tissue with vitamin Eα–tocopherol, which protected the vascular endothelium from oxidized LDL-mediated dysfunction, at least in part, through the inhibition of protein kinase C stimulation [87]. In experimental diabetes mellitus, vitamin E reduced blood glucose levels and improved glucose tolerance in diabetic rats as well as induced weight loss in normal and diabetic rats. Thus, vitamin E may play a role in glucose metabolism and be a useful adjuvant therapy in type I diabetes[88]. A solid proof of principle in animal models together with epidemiological observational studies has warranted the wide clinical use of antioxidants.

Observational Studies Epidemiological studies have clearly shown better protection against cardiovascular diseases in individuals with higher serum levels of vitamin E [89-90]. The WHO/MONICA study reported by Gey et al. in 1991 provided some the first evidence for the hypothesis that antioxidants could reduce the risk of cardiovascular disease. A number of observational studies suggested that long term consumption of antioxidants such as vitamins E, C and βcarotene is beneficial in preventing cardiovascular disease (table 1). The Rotterdam Study [91] showed that high dietary β-carotene intake in the elderly population may protect against cardiovascular disease. In this study, there was no association between vitamin C or vitamin E and myocardial infarction. The effect of vitamin E and vitamin C supplementation was also assessed in relation to mortality risk in 11,178 persons aged 67-105 year who participated in the Established Populations for Epidemiologic Studies of the Elderly in 1984-1993 [92]. The findings were consistent with those for younger persons and suggest protective effects of vitamin E supplements in the elderly. The Iowa Women’s Health Study [93] in postmenopausal women suggests that a high intake of vitamin E from food was inversely associated with the risk of death from coronary heart disease. By contrast, the intake of vitamins A and C was not associated with lower risks of dying from coronary disease. Several other studies such as the Finnish study [94], the Health Professional Follow-up Study [90] and the Nurses’s Health Study [89] have shown that increased intake of Vitamin E was associated with a lower risk of coronary artery disease. In the Finnish study, β-carotene and vitamin C appeared to confer coronary artery protection to women only. While the First National Health and Nutrition Examination Survey (NHANES I) [95] indicated a strong inverse association of vitamin C with cardiovascular diseases in men. The Scottish Heart Study [96] showed a protective effect of all three antioxidants (E, C and β-carotene) against coronary heart disease preferentially in men.

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Table I. Examples of observational studies showing beneficial effect of consuming antioxidant vitamin supplements on cardiovascular diseases Study Size

Population characteristics Age and baseline state

Rotterdam study [91]

4802

EPESE study [92]

11,178

The Iowa Women,s Health Study [93]

Study outcome Duration (yr)

People (55-95) with no history of myocardial infarction 67-105 elderly population

4

34,486

Post-menopausal women(55-69) with no history of CVD

7

Finnish Study [94]

5,133

14

Health Professionals Follow-up Study [90]

39,910

Finnish men and women (30-69) free of heart disease Healthy US male health professionals (40 -75)

Nurses’ Health Study [89]

87,245

Healthy females nurses (34-59)

8

NHANES I [95]

11,348

Non-institutionalized U.S. adults (25-74)

10

Scottish Heart Health study [96]

10,359

Scottish men and women (40-59)

10

8 to 9

4

Beta carotene, but not vitamin C or E, protected against myocardial infarction Beneficial effect of vitamin E (and enhanced by vitamin C) against mortality due to CAD A high intake of Vitamin E, but not A or C protect against death from CAD There was an inverse association between dietary vitamin E intake and coronary Vitamin E reduce risk of coronary artery disease Increased intake of Vitamin E was associated with a lower risk of coronary artery disease Strong inverse association of vitamin C with cardiovascular diseases in men A significant effect on the prevalence of CHD, especially amongst men

Randomized Trial The observations of beneficial effects of antioxidant vitamins have led to the widespread use of such supplements in an attempt to prevent several chronic diseases. However, when randomized controlled trials were conducted to assess the protective role of vitamin antioxidants against the risk of cardiovascular disease, some studies have showed a beneficial role (Table II), others showed no effect, and still some trials have shown adverse effects (Table III). The confusing nature of the results has generated significant debate and caution regarding the use of vitamin E and C. These studies have been critically reviewed by several authors [97-100]. The Cambridge Heart Antioxidant study (CHAOS) was conducted for 1.4 years with 2002 patients. Of these patients, 546 were given a high dose of 800-IU/day of vitamin E, and for safety reasons, the remaining majority were given a lower dose of 400-IU/day of vitamin E. The results showed a reduction in non-fatal myocardial infarctions in the group on higher dose of vitamin E, but no effect on the number of cardiovascular deaths [101]. In the Secondary Prevention with Antioxidants of Cardiovascular disease in End stage renal disease (SPACE), supplementation with 800 IU/day vitamin E reduced composite cardiovascular

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disease endpoints and myocardial infarction [102]. The Antioxidant Supplementation in Atherosclerosis Prevention (ASAP) trial which studied the effect of vitamins E and C on 3year and 6-year progression of carotid atherosclerosis in smoking men found that the supplementation with a combination of vitamin E and slow-release vitamin C reduces atherosclerotic progression in hypercholesterolemic persons [103, 104]. In addition, antioxidant vitamins C and E could retard the early progression of transplant-associated coronary arteriosclerosis measured by intravascular ultrasonography (IVUS) [105]. The MIVIT pilot trial was designed to test the effects of antioxidant vitamins C and E on the clinical outcome of patients with AMI. This randomized pilot trial showed that supplementation with antioxidant vitamins was safe and seemed to positively influence the clinical outcome of patients with AMI [106]. Table II. Examples of controlled clinical trials of vitamin antioxidants on CVD events showing beneficial effects Study and Antioxidant used

Dose

Treatment characteristics Duration (yr)

Population size and type

Study outcome

CHAOS [101] Vitamin E SPACE [102] Vitamin E

800 or 400 IU

1.4

2002 (M, F) Coronary disease subjects

Decreased nonfatal acute MI

800 IU

2

Reduction of composite cardiovascular disease endpoints and MI

ASAP [103 104] Vitamins E and C

182 mg 3 d-α-tocopherol 500 mg vitamin C

196 (M,F) Haemodialysis patients with preexisting cardiovascular diseases 520 subjects with elevated cholesterol levels

IVUS [105] Vitamins E and C

500 mg vitamin C 1 + 400 IU vitamin E

40 subjects after cardiac transplantation

MIVIT Vitamins C and E[106]

1000 mg/12 h 30 days infusion of vitamin C and 600 mg/24 hr of oral vitamin E

800 patients with AMI

Retard the progression of transplant-associated coronary arteriosclerosis Primary end point (cardiac mortality, non fatal new MI) were reduced

Reduction of intimamedia thickness progression in men but not in women

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Table III. Examples of controlled clinical Trials of Vitamin Antioxidants on CVD events showing no effects or adverse effects Study and Antioxidant used

Treatment characteristics

Population size and type

Study outcome

Dose

Duration (yr)

HOPE [107] Vitamin E

400 IU

4.5

9541 subjects (M,F) No effect on MI, CVD or with high CVD risk stroke

GISSI [108] vitamin E

300 mg

3.5

11324 (M, F) post MI No effect on MI, CVD or patients stroke

VEAS [109] Vitamin E

400 IU

3

352 subjects (M,F) with elevated LDLc

PHS [110] β -carotene

50 mg on alternate days

12

22071 (M) health subjects

HPS [111] Vitamin E,C, β -carotene ATBC [112] Vitamin E and β-carotene

600 mg vitamin C, 250 mg vitamin C, 20 mg β-carotene 50 mg vitaminE, 20 mg β -carotene

5

20 536 (M,F) high CVD risk subjects

No effect on CVD mortality

6.1

No effect on CHD or MI. β-carotene increased mortality rate by 8%

CARET [113] β –carotene and vitamin A

30 mg β –carotene and 25,000 IU of vitamin A

Early termination

27271(M) smokers with no MI with a total of 29133 (M) smokers 18,314 (M,F) subjects with high risk of lung cancer

12.5 mg ß-carotene, HATS [114 500 mg vitamin C, 115] Vitamin E,C, β 400 IU vitamin E –carotene Simvastatin and niacin

3

No effect on IMT progression in subjects at low risk for CVD No effect on MI or CVD

No effect on CVD mortality. 28% higher incidence of lung cancer and 17% more deaths 160 subjects with low No effect on CAD. HDL and established Favorable responses of CAD simvastatin + niacin were blunted by the antioxidants

In contrast, several other trials have shown no beneficial effects of antioxidant vitamins. Vitamin E had a negative outcome on cardiovascular risk in the Heart Outcomes Prevention Evaluation (HOPE) trial that had 2545 women and 6996 men 55 years of age or older enrolled [107]. The HOPE trial was the largest trial conducted thus far for the use of antioxidants in diabetes. The SECURE trial [116] designed as a sub-study of the HOPE trial to evaluate the effects of long-term treatment with ramipril and vitamin E on atherosclerosis progression in 732 high-risk patients showed no effect of vitamin E, while ramipril slowed down atherosclerotic changes. In another larger scale antioxidant study called GISSIPrevenzione clinical trial, the effect of vitamin E and/or n-3 polyunsaturated fatty acid (PUFA) was investigated for 3-5 years in 11,324 patients with recent myocardial infarction. The results indicated that in patients who had a myocardial infarction, n-3 PUFA

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supplements, but not the moderate dose of synthetic vitamin E, reduced long-term complications of myocardial infarction [108]. In the small Vitamin E Atherosclerosis Prevention Study (VEAPS) of 353 male and female subjects with LDL cholesterol level ≥ 130 mg/dL and no clinical signs or symptoms of CARDIOVASCULAR DISEASES, vitamin E did not reduce the progression of atherosclerosis in carotid artery over a period of 3 years [109]. The most disappointing results came from the use of β–carotene either alone or in a cocktail with vitamin E or C. Supplementation of 50 mg of β-carotene given on alternate days for 12 years in The Physicians’ Health Study showed neither harm nor benefit in 22,071 male physicians age 40 to 80 [110]. A cocktail of vitamin E (600 mg), vitamin C (250 mg) and βcarotene (20 mg) were given daily to 536 adult age 40 to 80 with coronary artery disease, diabetes, or other occlusive arterial disease in the Heart Protection Study (HPS). Patients showed no positive outcome compared to placebo control when followed for 5 years [111]. The most troubling aspect with the use of β-carotene has been the observation of adverse effects in at least two large trials. The preventive effect of vitamin E and β-carotene supplementation on major coronary events for 6.1 years was assessed in the vitamin E, βCarotene Cancer Prevention (ATBC), a study aimed primarily at cancer, in 27,271 Finnish male smokers aged 50 to 69 years with no history of myocardial infarction. A small dose of vitamin E had a marginal effect on the incidence of fatal coronary heart disease, but no influence on nonfatal myocardial infarction. Supplementation with β-carotene displayed no primary preventive effect on major coronary events [112]. However, in the same study with a total enrollment of 29,133 male smokers, the group taking β-carotene showed an 8% higher mortality rate compared to control without β-carotene, primarily due to more death from lung cancer. A combination of 30 mg of β-carotene and 25,000 IU of vitamin A was administered daily to men and women with high risk of lung cancer in The Beta Carotene and Retinal Efficacy Trial (CARET). The 18,314 patient study was terminated early with no effect on CARDIOVASCULAR DISEASES mortality, but a statistically significant increase of 28% in lung cancer and 17% more deaths than the placebo group [113]. The effect of antioxidant cocktail with 800IU of vitamin E, 1,000 mg of vitamin A, 25 mg of β-carotene and 100ug of selenium was investigated in conjunction with simvastatin and niacin on 160 subjects with established CAD in the High Density Lipoprotein (HDL) Atherosclerosis Treatment Study (HATS). While simvastatin and niacin lead to a statistically significant benefit as measured angiographically, the antioxidant cocktail failed to show any benefit, and rather blunted the beneficial effect of simvastatin and niacin [114, 115]. Experimental and clinical research currently under way on the use of non-vitamin antioxidants may provide promising results.

Effect of Non-Vitamin Antioxidant Supplementation In recent years, the potential hypocholesterolemic effects of several dietary components, such as glucan, soy protein, isoflavones, plant sterols and stanols, and garlic and tocotrienols, have attracted much interest. On the other hand, the use of N-acetylcysteine, NO inducers, probucol and derivatives and catalytic antioxidants are under active investigation for clinical applications against oxidative stress related diseases including cardiovascular diseases.

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Polyphenols Flavanoids are prominent polyphenolic compounds found in fruits, vegetables, and beverages. Flavanoids are derived from plants and consumed regularly within the human diet. Foods thought historically by many societies to have healing properties including cocoa, red wine, and tea, are particularly rich in flavonoids. The dietary intake of flavonoids far exceeds that of the monophenolic antioxidant, vitamin E, and β-carotene on a milligram per day basis [117]. Flavanoids have different phenolic structures and are derived from plant metabolism [118]. Flavanoids are usually subdivided according to their substituents into flavanols (a), anthocyanidins (b), flavones, flavanones, and chalcones. They are powerful chain-breaking antioxidants because of their ability to scavenge free radicals and to chelate metal ions [119]. Recent studies indicate flavonoids possess a wide array of biochemical and pharmacological actions and may represent as potential therapeutic agents. Certain members of this group have long been recognized to possess anti-inflammatory, antioxidant, anti-allergic, anti-thrombotic, antiviral, and anti-carcinogenic activities [120]. Caffeic acid phenethyl ester (CAPE), a natural flavonoid, can specifically block activation of NF-κB. In ApoE-deficient mice, oral CAPE supplementation attenuated the atherosclerotic process, and reduced NF-κB activity and expression of inflammatory genes in the aorta. This was attributable to direct inhibition of NF-κB in the lesion and reduction of systemic oxidative stress [121]. CAPE also decreased lipid peroxidation in streptozotocininduced diabetic rats [122]. Curcumin, a yellow polyphenolic compound from the plant Curcuma ionga, is a commonly used spice and coloring agent with beneficial effects including anti-tumor, antiinflammatory, and antioxidant activities. It can effectively reverse the endothelial dysfunction induced by homocysteine in a porcine coronary artery model and block the detrimental effect of homocysteine on the vascular system. Thus, in addition to preventing cardiovascular diseases, curcumin could be used in patients with hyperhomocysteinemia [123] Resveratrol (3,4('),5-trihydroxystilbene), a phenolic compound found in fruits, nuts, flowers, seeds and bark of different plants is also an integral part of human diet. The cardioprotective effect of red wine has been attributed to resveratrol. It exhibits a wide range of biological effects, including anti-coagulation, anti-inflammatory, anti-tumorgenic, antimutagenic, and antifungal properties. It is also a potent antioxidant, ROS scavenger, and metal chelator. Since resveratrol reduces lipid peroxidation, oxidation and nitration of platelet and plasma proteins, it may be useful for preventing or treating cardiovascular diseases [124]. Pre-ischemic infusion of resveratrol protects the spinal cord from ischemia reperfusion injury in rabbits [125]. Quercetin, a flavonoid present in the human diet, and found in high levels in onions, apples, tea and wine, has been shown to inhibit platelet aggregation in vitro. Consequently, it has been proposed that quercetin may have the protective effects against cardiovascular diseases. A pilot human dietary intervention study demonstrated that quercetin was bioavailable and could inhibit platelet aggregation as well as collagen-stimulated tyrosine phosphorylation and potentially block thrombus formation [126]. Thiol Supplementation Supplementation with the antioxidant N-acetylcysteine has been shown to reduce cardiovascular events in hemodialysis patients. N-acetylcysteine has also been shown to

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reduce LP(a) in patients [127]. N-acetylcysteine is capable of reducing atheroma progression in an animal model of uremia-enhanced atherosclerosis in ApoE-deficient mice, probably via a decrease in oxidative stress [128]. However, other studies have shown no reduction in lesion progression after balloon angioplasty, despite an increase in plasma glutathione level in Nacetylcysteine-treated New Zealand white rabbits [129]. Prophylactic oral administration of N-acetylcysteine in 121 patients with chronic renal insufficiency displayed reduced acute renal damage induced by a contrast agent in patients with chronic renal insufficiency undergoing a coronary procedure [130]. In a separate 79 patient study, N-acetylcysteine was not effective for the prevention of contrast nephropathy after cardiac angiography [131].

Sulforaphane (4-methylsulfinylbutyl isothiocyanate), a naturally occurring sulfur-containing isothiocyanate is the most potent phase 2 protein inducer found in our food sources [132]. NAC is capable of reducing atheroma progression in an animal model of uremia-enhanced atherosclerosis in ApoE-deficient mice, probably via a decrease in oxidative stress [133]. However, other studies have shown no reduction in lesion progression after balloon angioplasty, despite an increase in plasma glutathione level in NAC-treated New Zealand white rabbits [134]. In addition, sulforaphane tested in an ischemic organ damage model, revealed a protective effect on preserved pancreas and may have a potential clinical implication to improve hemodynamically unstable pancreas donor condition [130]. In a separate 79 patient study, NAC was not effective for the prevention of contrast nephropathy after cardiac angiography [131].

α-Lipoic Acid Lipoic acid has a distinct ability to function in both lipid and aqueous regions of the body and thus often termed as a “universal antioxidant”. It was first identified in the 1950s as a component of several human enzyme systems involved in the conversion of carbohydrates and fats into energy. In addition, lipoic acid exhibits significant antioxidant activity, leading researchers to consider its potential for disease prevention and treatment. One study demonstrated that Lipoic acid reduced atherogenic lesions in Japanese quail by preventing oxidation of LDL cholesterol and/or by recycling vitamin E [135].

Edaravone Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one), a synthetic potent scavenger of free radicals, originally produced as a new anti-stroke agent in Japan, has the antioxidant ability and inhibit lipid peroxidation. Mitsubishi Pharma developed edaravone (Radicut) in 2001 for the treatment of patients with acute stage cerebral infarction. However, side effects including liver failure and death were observed in elderly patients forcing doctors to prescribe the drug only with great caution, especially among the elderly population. The Edaravone Acute Infaction Group, a randomized, placebo-controlled, double-blinded, multi-center study on acute ischemic stroke, showed that edaravone (MCI-186) is potentially useful for treating acute ischemic stroke [136]. Yashida et al. demonstrated that edaravone increased eNOS expression and inhibited LDL oxidation. In addition, edaravone can reverse oxidized LDLmediated reduction in eNOS expression in endothelial cells. This mechanism could explain the protective effect of edaravone against ischemic disease [137]. In another randomized,

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controlled, open-label, 80 patient study with acute myocardial infarction, administration of edaravone before myocardial reperfusion was associated with smaller infarcts and a better clinical outcome [138].

Probucol Probucol, a cholesterol-lowering agent found in 1964 while screening for phenolic antioxidants, has potent lipid-soluble antioxidant properties [139]. It has been extensively studied in experimental interventional studies of atherosclerosis and restenosis. Probucol inhibits atherogenesis in hypercholesterolemic rabbits[140, 141] and non-human primates[142]. Furthermore, probucol has been shown to inhibit neointimal thickening and restenosis after angioplasty in rabbits [143] and pigs[144]; and reverses established plaques in rabbits [145] and humans [146]. In clinical trials, probucol has reduced post-percutaneous coronary intervention (PCI) restenosis and progression of carotid atherosclerosis. The Multivitamins and Probucol (MVP) Study tested the effects of a combination of vitamin C (1000 mg/d), vitamin E (400 IU/d), α-carotene (100 mg/d); and probucol (1000 mg/d) versus placebo on the rate and severity of restenosis in humans[147]. The Probucol Angioplasty Restenosis Trial (PART) compared probucol (1000 mg/d) with placebo [148]. Both studies included one month pretreatment followed by 6 month dosing after elective angioplasty and resulted in significant reduction in restenosis relative to placebo. The beneficial effects of probucol were attributed to its antioxidant properties. Results from the MVP study demonstrated that dietary antioxidants alone had no effect and appeared to negate the beneficial effects of probucol when given in combination. In the Fukuoka Atherosclerosis Trial (FAST), the effect of probucol and pravastatin on common carotid atherosclerosis was investigated in 246 asymptomatic hypercholesterolemic patients for two years. Probucol reduced total cholesterol levels and stabilized plaques, leading to lower incidence of cardiac events [149]. Metal-Containing Catalytic Antioxidants The goal of cellular antioxidant defenses is to reduce ROS to water; to this aim metalloproteins such as superoxide dismutases (SODs) and catalase are used to detoxify cellular ROS. Until recently, SOD or catalase have not proven useful therapeutics likely due to multiple limitations including size, low cell permeability, short circulating half-life and high manufacturing costs [150]. However, synthetic metal-containing low molecular-weight catalytic antioxidants are emerging as a novel class of potential therapeutics that can scavenge a wide variety of ROS. Several classes of manganese-containing catalytic antioxidants have shown efficacy in oxidative stress models of human disease. Superoxide dismutase mimetic, Mn(III)tetrakis(1-methyl-4-pyridyl) porphyrin pentachloride indicated a positive outcome in ex vivo model of cardiac hypertrophy [151]. Preincubation of carotid arteries of ApoEdeficient mice with cell-permeable superoxide dismutase mimetic Mn(III) tetra(4-benzoic acid) porphyrin chloride almost normalized NO-mediated endothelial-dependent relaxations to acetylcholine [152]. Another synthetic manganese containing superoxide dismutase mimetic, M40403, exerted a protective effect against ischemia-reperfusion-induced myocardial injury, supporting a key role for superoxide anions in reperfusion injuries [153], and hence a rationale for developing catalytic antioxidant therapeutics.

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ANTIOXIDANT THERAPY FOR ASTHMA AND CHRONIC OBSTRUCTIVE PULMONARY DISEASES (COPD) Oxygen levels in the lungs are higher than any other organ system in the human body. Therefore, various cell types within the lungs are at higher risk of oxidant-mediated damage. The trachea-bronchial tree and alveolar space are exposed to ROS from various sources including airborne pollutants such as smog or tobacco smoke. In addition, numerous biologically active proinflammatory mediators can increase endogenous production of ROS and RNS formation by several inflammatory, immune, and structural cells of the airways. These reactive species lead to oxidation and nitration of proteins, which may alter protein function that are biologically relevant to airway injury/inflammation. Asthma, an inflammatory disease of the airways, is increasing in prevalence worldwide currently affecting over 15 million individuals in the United States and resulting in more than 5500 deaths each year (many of which are children) [154-155]. Asthma is characterized by airway eosinophilia, mucin production, IgE production, and airway hyperresponsiveness (AHR). Eosinophilic inflammation has been correlated with the severity of asthma. The airway inflammation is usually accompanied by increased vascular permeability and plasma exudation with a concomitant recruitment and activation of various inflammatory cells including eosinophils and T cells in the airway mucosa. ROS are produced by inflammatory cells in the airways and/or inhaled directly from environmental contaminants. ROS production, a contributing factor in the pathogenesis of asthma, is increased in asthmatic subjects. In addition, exogenous oxidants such as cigarette smoke, viral infections, and ozone can directly cause asthma exacerbation and simultaneously activate the production of endogenous oxidants resulting in increased inflammation. Therefore, oxidative stress plays a critical role in airway inflammation leading to asthma, and markers of oxidative stress such as nitrotyrosine were higher in asthmatic patients [156, 157]. Concomitant with increased generation of oxidative and nitrosative molecules in asthma, loss of protective antioxidant defenses, specifically SOD, seems to contribute to the overall oxidative stress environment of the asthmatic airway [158]. Further studies have demonstrated that a defect in antioxidant responses could exacerbate asthma severity. Disruption of Nrf2, which regulates many antioxidant proteins, has been shown to cause severe allergen-driven airway inflammation and hyperresponsiveness in mice. Nrf2 disruption causes an increased expression of the T helper type 2 cytokines interleukin (IL)-4 and IL-13 in bronchoalveolar lavage fluid and splenocytes following an allergen challenge. As a result of reduced expression of both basal and inducible antioxidant genes, the lungs have a decreased antioxidant capacity. This underlines the significance of Nrf2-directed antioxidant pathways as major determinant of susceptibility to allergen-mediated asthma [159]. Chronic obstructive pulmonary disease (COPD) is another lung disease with an oxidative stress component which has become a major worldwide health problem with increasing prevalence and mortality [160]. COPD is an obstructive airway disorder characterized by a slowly progressive and irreversible decrease in pulmonary function as measured in forced expiratory volume in one second (FEV1). Varying perturbations in both airway and interstitial lung tissue slowly leads to the narrowing of airway lumen diameter which results in a gradual decrease in pulmonary function.

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The oxidative stress mediated inflammation and imbalance between protease and antiprotease plays an important role the pathogenesis of emphysema. In elastase-induced emphysema, it has been demonstrated that Nrf2 plays an important protective role against disease progression. In Nrf2-deficient mice, elastase-provoked emphysema was markedly exacerbated compared with wild-type mice and is closely correlated with the degree of lung inflammation in the initial stage of elastase treatment. Nrf2-deficient mice display impaired expression of antioxidant and antiprotease genes when compared with wild-type alveolar macrophages in the lungs. These deficiencies could be overcome by transplantation of wildtype bone marrow cells into Nrf2-deficient mice with a resulting inhibition of lung inflammation and emphysema [161]. Similarly, treatment with the antioxidant Nacetylcysteine (NAC) also suppresses elastase-induced emphysema in rats [162]. It is possible that increased oxidative stress is key in the pathogenesis of COPD and could lead to clinical trials to validate the efficacy of N- acetylcysteine treatment COPD in patients.

Effect of Antioxidants Vitamins It seems that there may be a link between consumption of antioxidant rich foods and the occurrence of obstructive airway disease. A recent Scottish study has found that consumption of antioxidant rich fruits and vitamin E were associated with a reduced prevalence of phlegm production and improved pulmonary function [163]. In the Third National Health and Nutrition Examination Survey of 16,000 individuals, it was found that participants with current asthma had lower vitamin C and β-cryptoxanthin concentrations and a lower mean vitamin E/triglyceride ratio than participants without asthma [164]. Troisi et al. used semiquantitative food frequency questionnaire over a 10-yr period in 77,866 women. The authors found that dietary sources of vitamin E may have a modest protective effect against asthma, but antioxidant supplementation during adulthood is not an important determinant of asthma [165]. Increased dietary vitamin E intake may be associated with a reduced incidence of asthma, and combinations of antioxidant supplements including vitamin E has been shown to be effective in reducing ozone induced bronchoconstriction. In a double-blind crossover study, the effects of dietary antioxidants (400 IU vitamin E/500 mg vitamin C) were evaluated on ozone-induced bronchial hyperresponsiveness in adult subjects with asthma. The results suggest that dietary supplementation with vitamins E and C benefit asthmatic adults who are exposed to air pollutants [166]. However, in another study, supplementation with vitamin E for 6 weeks showed no benefit on bronchial hyperresponsiveness to current standard treatments in adults with mild to moderate asthma [167]. A protective effect of fruit and possibly vitamin E intake against COPD has been shown in 2917 men aged 50-69, while intake of vitamin C, β-carotene, vegetables and fish was ineffective[168]. Several population studies have shown a relationship between dietary antioxidants, pulmonary function and the development of COPD. The American Nutrition Examination Survey (NHANES) and the Dutch monitoring project for the risk factors for chronic diseases (MORGAN) have linked antioxidant dietary intake to airflow limitation. NHANES (I) study showed that dietary vitamin C intake was positively and significantly associated with improved pulmonary function in 2526 adults indicating a protective effect of vitamin C on pulmonary function [169]. An inverse relationship between both dietary and

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serum vitamin C and chronic respiratory symptoms was observed in NHANES (II) conducted on 9,074 white and African American adults aged 30 years or older [170]. In NHANES (III) there was a positive correlation between lung function and the levels of dietary vitamin C, vitamin E, selenium, and β-carotene [171]. A higher intake of vitamin C and β-carotene was associated with higher levels of FEV1 in the MORGAN Study and, in particular, fruit and whole grain intake showed independent beneficial associations with COPD [172].

Effect of Non-Vitamin Antioxidants Flavonoids The consumption of flavonoids or flavonoid rich foods seems to have some protective role in the occurrence of asthma and COPD. Tabak et al. demonstrated that flavonol and flavone intake were independently associated with chronic cough only, while solid fruit, but not tea, intake was beneficially associated with COPD symptoms in 13,651 adults during a three year period. Therefore, high intake of catechins and solid fruits are beneficial against COPD [173]. A randomized, placebo-controlled, double-blind study involving 60 subjects, aged 6-18 years old showed that pycnogenol (a proprietary mixture of water-soluble bioflavonoids extracted from French maritime pine) significantly improved pulmonary function for mild-to-moderate asthma patients [174]. However, a population-based, casecontrol study of 1,471 adults aged 16–50 yrs showed no protective effect of three major subclasses of dietary flavonoids (catechins, flavonols and flavones) on asthma [175]. N-Acetylcysteine Thiol antioxidant N-acetylcysteine can be beneficial against cigarette smoke related lung injury, but the pro-oxidant side effects can not be discounted [176]. In a one year follow up study of patients hospitalized for COPD, treatment with N-acetylcysteine dose-dependently reduced the risk of re-hospitalization [177]. However, in a randomized placebo-controlled study of 523 patients with COPD, patients were treated with either 600 mg daily Nacetylcysteine or placebo and followed for three years. There is no improvement in pulmonary function as determined by FEV1 between N-acetylcysteine treated patients and those receiving placebo; and the number of exacerbations per year did not differ between groups. However, subgroup analysis suggested that the exacerbation rate might be reduced with N-acetylcysteine in patients not treated with inhaled corticosteroids and secondary analysis suggested a potential effect on hyperinflation. The authors concluded that Nacetylcysteine was ineffective in preventing lung function deterioration and in reducing exacerbations in patients with COPD [178]. Superoxide Dismutase Mimetics Endogenous SOD levels are known to decrease in atopic asthmatics, therefore efforts to develop SOD mimetics are currently ongoing. Masini et al. have demonstrated that removal of superoxide by the SOD mimetic (SODm) M40403 reduced respiratory and histopathological lung abnormalities caused by ovalbumin (OA) aerosol in a model of allergic asthma in sensitized guinea pigs. This finding supports the potential therapeutic use of SOD mimetics in asthma [179].

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ANTIOXIDANTS THERAPY FOR RHEUMATOID ARTHRITIS Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial tissue proliferation, cartilage loss, bone erosion and progressive joint destruction in peripheral joints. Clinical manifestations of RA include joint swelling, morning stiffness, and joint deformities often resulting in painful disability. RA affects nearly 1% of the population worldwide [180]. The etiology of RA remains unknown, but many factors, including autoimmunity, cytokines, and genetic factors participate in its pathogenesis. ROS have been implicated as mediators of tissue damage in patients with rheumatoid arthritis (RA). Studies have indicated that the development of RA is partly related to the excess production of ROS and a lowered ability to remove oxidative stress [181, 182]. ROS is produced by phagocytes in the synovial fluid and pannus and by synovial endothelial cells during hypoxia-reperfusion events [183]. Plasma malondialdehyde, a degradation product of lipid peroxidation, levels were significantly higher in the synovial fluid and serum of RA patients than that of healthy control subjects [184]. RA patients have significantly lower levels of erythrocyte CuZnSOD compared with healthy counterparts [185]. Also, in children with juvenile rheumatoid arthritis, plasma and red blood cell alpha-tocopherol concentrations were lower compared to those of healthy children [186]. In a Finnish cohort study, low alphatocopherol status was suggested as a risk factor for RA [187]. RA patients not only display low levels of antioxidants in the blood, but altered activity of blood antioxidant enzymes including glutathione peroxidase (GPx) [181], CuZn SOD [185, 188], and catalase [189]. Markers of increased nutritive stress such as nitrite/nitrate, nitrotyrosine, and peroxynitrite have been found to be increased in the blood of arthritic animals [190, 191]. To assess whether ROS/RNS is involved in RA, a variety of methods have been tested that were aimed at increasing the antioxidant capacity and/or decreasing ROS/RNS production systematically or in the joint. Dai and co-workers used an ex vivo gene transfer method to express extracellular (EC)-SOD and catalase in rat knee joints. They found that rats treated with cells over-expressing EC-SOD, catalase, or a combination of EC-SOD and catalase showed significant suppression of knee joint swelling, decreased infiltration of inflammatory cells within the synovial membrane, and reduced gelatinase activity in knee joints of antigen-induced arthritis in rats [192]. Treatment of animals with the thiol antioxidant pyrrolidine dithiocarbamate also attenuates the development of acute and chronic inflammation in carrageenan-induced pleurisy and collagen-induced arthritis [193]. Similarly, treatment with N-acetylcysteine resulted a dose-dependent suppression of arthritis associated with decreased blood ROS levels in mice suffering from type II collagen-induced arthritis [194]. Furthermore, anti-arthritis effects have been observed by systemic administration of liposome entrapped SOD, use of a SOD mimetic, intravascular injection of MnSOD, CuZnSOD, catalase, and peroxidase in antigen- and zymozan-induced arthritis [195-197]. Several studies have demonstrated beneficial effects of dietary antioxidants in RA animal models. Long term treatment with vitamin Ε reduced bone and joint destruction and levels of the proinflammatory cytokine IL-1β, but not TNF-α, in the KRN/NOD mouse which develops spontaneous polyarthritis. There was however, no measurable effect on clinical symptoms such as paw swelling and body weight [198]. Furthermore, treatment with vitamin E delayed the appearance of enlarged lymph nodes, and decreased serum levels of IL-6, IL10, IL-12 and TNF-α in autoimmune-prone MRL/lpr mice (a model for rheumatoid arthritis,

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RA). The authors suggested that these effects vitamin E may delay onset of rheumatoid arthritis [199]. In addition, treatment with ascorbic acid dose-dependently reduced paw swelling and arthritis scores and was attributed to a marked reduction of inflammatory cell infiltration into the synovial tissues [200]. Observational clinical studies have shown that the blood levels of antioxidant vitamins or other antioxidants are associated with the risk of developing RA. In a 20 year follow up study of 1,419 Finnish adults, serum vitamin Ε, β-carotene, and selenium were monitored for their associations with the risk of RA. There was an increased risk of RA associated with low levels of vitamin Ε, β-carotene and selenium, but none of the correlations were statistically significant. However, when all these three micronutrients were analyzed as an overall antioxidant index, a significant association was observed between a low antioxidant index and a risk factor for RA. There was however, no measurable effect on clinical symptoms such as paw swelling and body weight [198]. In another survey of 74 young patients with juvenile rheumatoid arthritis (JRA) and 138 healthy children, JRA children had increased oxidative stress as measured by the blood levels of thiobarbituric acid reactive substances (TBARS, an index of oxidative stress) and low levels of antioxidant vitamin E compared to healthy children [186]. Dietary carotenoids are also associated with a risk of developing inflammatory polyarthritis. In The European Prospective Investigation of Cancer Incidence (EPIC)-Norfolk study, a case-control analysis in 25,000 people was performed to compare carotenoid intake between case subjects and age- and sex-matched control subjects. The subjects in the top onethird of intake of β-cryptoxanthin were at a lower risk of developing inflammatory polyarthritis than were subjects in the lowest one-third β-cryptoxanthin. The author concluded that a modest increase in β-cryptoxanthin intake, equivalent to one glass of freshly squeezed orange juice per day, is associated with a reduced risk of developing inflammatory disorders such as rheumatoid arthritis [201]. In an eleven year prospective study of 29,368 elderly women, increased intake of vitamin C, vitamin E or β-cryptoxanthin was associated with significant decreased incidence of rheumatoid arthritis [202]. Paredes found that RA patients had lower plasma levels of vitamin A compared to healthy controls, while no significant differences in plasma vitamin E levels between RA patients and healthy subjects were observed. Interestingly, the group found a significant inverse correlation in plasma vitamin A and vitamin E levels compared with the inflammation marker C-reactive protein in RA patients [203]. In a study of 30 patients to assess the therapeutic value of adding a high dose of vitamin E on the rheumatoid disease, a high dose of vitamin E significantly decreased clinical symptoms such as morning stiffness. High dose of vitamin E increased patients’ plasma levels of vitamin E and the activity of glutathione peroxidase (GPx), but decreased plasma levels of malonedialdehyde. The authors concluded that use of antioxidant vitamin E as adjuvant therapy in rheumatoid disease worth pursuing [204]. However, there is no large controlled clinical trial to test whether supplementation of antioxidant vitamins will decrease the risk of RA or improve RA clinical signs and symptoms. The use of antioxidant for the treatment of rheumatoid arthritis has been reviewed in detail [205].

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FUTURE DIRECTIONS Recent research has provided abundant evidence demonstrating that ROS/RNS play an important role in the pathogenesis of chronic inflammatory diseases. Numerous animal experiments and clinical trials have documented strong association between chronic inflammatory diseases and elevated indices of oxidant stress or decreased antioxidant capacity. Furthermore, treatment with antioxidants diminished or prevented inflammatory diseases in a number of animal studies. However, for decades the focus on an antioxidant therapy for these diseases was primarily restricted to vitamins E and C. Both of these vitamins can act as both pro- and anti-oxidants; and there has been no convincing beneficial effects obtained from human clinical trials using antioxidant vitamins. In the future, there is a great need for the development of novel, effective, small molecule or biological antioxidant-based drugs. There is also an urgent need for more reliable biomarkers and high-throughput analysis to assess the oxidant status of the patient and allowing a more accurate evaluation of the efficacy of antioxidant therapies.

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heterozygous patient with familial hypercholesterolemia. Atherosclerosis. Feb 1996;120(1-2):181-187. [147] Tardif JC, Cote G, Lesperance J, et al. Probucol and multivitamins in the prevention of restenosis after coronary angioplasty. Multivitamins and Probucol Study Group. N Engl J Med. Aug 7 1997;337(6):365-372. [148] Rodes J, Cote G, Lesperance J, et al. Prevention of restenosis after angioplasty in small coronary arteries with probucol. Circulation. Feb 10 1998;97(5):429-436. [149] Sawayama Y, Shimizu C, Maeda N, et al. Effects of probucol and pravastatin on common carotid atherosclerosis in patients with asymptomatic hypercholesterolemia. Fukuoka Atherosclerosis Trial (FAST). J Am Coll Cardiol. Feb 20 2002;39(4):610-616. [150] Day BJ. Catalytic antioxidants: a radical approach to new therapeutics. Drug Discov Today. Jul 1 2004;9(13):557-566. [151] MacCarthy PA, Grieve DJ, Li JM, et al. Impaired endothelial regulation of ventricular relaxation in cardiac hypertrophy: role of reactive oxygen species and NADPH oxidase. Circulation. Dec 11 2001;104(24):2967-2974. [152] d'Uscio LV, Smith LA, Katusic ZS. Hypercholesterolemia impairs endotheliumdependent relaxations in common carotid arteries of apolipoprotein e-deficient mice. Stroke. Nov 2001;32(11):2658-2664. [153] Masini E, Cuzzocrea S, Mazzon E, et al. Protective effects of M40403, a selective superoxide dismutase mimetic, in myocardial ischaemia and reperfusion injury in vivo. Br J Pharmacol. Jul 2002;136(6):905-917. [154] Woolcock AJ, Peat JK. Evidence for the increase in asthma worldwide. Ciba Found Symp. 1997;206:122-134; discussion 134-129, 157-129. [155] Fuhlbrigge AL, Adams RJ, Guilbert TW, et al. The burden of asthma in the United States: level and distribution are dependent on interpretation of the national asthma education and prevention program guidelines. Am J Respir Crit Care Med. Oct 15 2002;166(8):1044-1049. [156] Wood LG, Gibson PG, Garg ML. Biomarkers of lipid peroxidation, airway inflammation and asthma. Eur Respir J. Jan 2003;21(1):177-186. [157] Hanazawa T, Kharitonov SA, Barnes PJ. Increased nitrotyrosine in exhaled breath condensate of patients with asthma. Am J Respir Crit Care Med. Oct 2000;162(4 Pt 1):1273-1276. [158] Andreadis AA, Hazen SL, Comhair SA, et al. Oxidative and nitrosative events in asthma. Free Radic Biol Med. Aug 1 2003;35(3):213-225. [159] Rangasamy T, Guo J, Mitzner WA, et al. Disruption of Nrf2 enhances susceptibility to severe airway inflammation and asthma in mice. J Exp Med. Jul 4 2005;202(1):47-59. [160] Repine JE, Bast A, Lankhorst I. Oxidative stress in chronic obstructive pulmonary disease. Oxidative Stress Study Group. Am J Respir Crit Care Med. Aug 1997;156(2 Pt 1):341-357. [161] Ishii Y, Itoh K, Morishima Y, et al. Transcription factor Nrf2 plays a pivotal role in protection against elastase-induced pulmonary inflammation and emphysema. J Immunol. Nov 15 2005;175(10):6968-6975. [162] Rubio ML, Martin-Mosquero MC, Ortega M, et al. Oral N-acetylcysteine attenuates elastase-induced pulmonary emphysema in rats. Chest. Apr 2004;125(4):1500-1506. [163] Kelly Y, Sacker A, Marmot M. Nutrition and respiratory health in adults: findings from the health survey for Scotland. Eur Respir J. Apr 2003;21(4):664-671.

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In: Leading Edge Antioxidants Research Editor: Harold V. Panglossi, pp. 179-245

ISBN 1-60021-274-3 © 2007 Nova Science Publishers, Inc.

Chapter 7

IMPACT OF OXIDATIVE STRESS ON DIABETES MELLITUS AND INFLAMMATORY BOWEL DISEASES Jana Varvařovská1, Rudolf Štětina2, Josef Sýkora3 Zdeněk Rušavý4, Jaroslav Racek5, Silva Lacigová6 and Konrad Siala7 1

Department of Paediatrics, Faculty of Medicine Plzen, Charles University Prague, and University Hospital Plzen, Alej Svobody 80, 304 60 Plzen, Czech Republic 2 Department of Toxicology, Faculty of Military Health Sciences Hradec Králové, University of Defence Brno, Czech Republic 3 Department of Paediatrics, Faculty of Medicine Plzen, Charles University Prague, and University Hospital Plzen, Czech Republic 4 Diabetology Center, Ist Clinic of Internal Diseases, Faculty of Medicine Plzen, Charles University Prague, and University Hospital Plzen, Czech Republic 5 Institute of Clinical Biochemistry and Haematology, Faculty of Medicine Plzen, Charles University Prague, and University Hospital Plzen, Czech Republic 6 Diabetology Center, Ist Clinic of Internal Diseases, Faculty of Medicine Plzen, Charles University Prague, and University Hospital Plzen, Czech Republic 7 Department of Paediatrics, Faculty of Medicine Plzen, Charles University Prague, and University Hospital Plzen, Czech Republic

1. ABSTRACT Formation of reactive oxygen species (ROS) is a natural process during oxidative metabolism. ROS play an important role not only in pathological processes of human organism as usually presented but less attention is paid to their proper important role in cell signaling, biosynthesis or non-specific antiinfectious defence. Overproduction of ROS during numerous pathological situations in presence of insufficient antioxidant protection leads to substantial oxidative changes of lipids, proteins, sugars, and also DNA. Protection against ROS is assured by different extracellular or intracellular antioxidant mechanisms as studied during last decades. Antioxidant enzymes rectifying the oxidative damage are studied with regard to their different activities and usefulness in

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Jana Varvařovská, Rudolf Štětina, Josef Sýkora et al. body protection. Their genetic polymorphisms are certainly involved in different response to oxidative stress. Special attention should be devoted to the topic of oxidative nuclear and mitochondrial DNA damage and its restoring via DNA repair process, especially base excision repair (BER). A large scale of antioxidant enzymes is involved in correction of DNA oxidative damage. Natural trend of worsened DNA repair is usually associated with aging. Other pathologies related with deficient DNA repair are susceptibility to carcinogenesis (lack of apoptosis control) or degenerative diseases. Oxidative stress is involved in the pathophysiology of diabetes mellitus (DM – oxidative stress of mainly metabolic origin) and inflammatory bowel diseases (IBD – oxidative stress of mainly inflammatory origin). In spite of confirmed OS in DM or IBD, the substantial information about the intensity of DNA repair and its possible relationship to the disease course and development of chronic complications is missing. Our pilot studies completed both in adult and pediatric patients with DM or IBD confirmed an increased oxidative stress as well as oxidative DNA damage examined with comet assay. The surprising findings were ascertained in intensity of DNA repair (analysed with modified comet assay). DNA repair process was stimulated in Type 1 diabetes adults without diabetic microvascular complications and still more in diabetic children with short disease duration. On the other hand adults with Type 2 diabetes had substantially increased oxidative DNA damage and extremely low DNA repair. This finding could be the link to increased susceptibility of Type 2 DM patients to cancerogenesis. Patients with IBD (Crohn´s disease - both children and adults) had similar tendencies in OS intensity and oxidative DNA damage and repair but less intensive. Large population studies in DM or IBD studying intensity of OS and expression of DNA repair enzymes are needed in order to get the correlation between individual repair enzymes expression and the long-term course and occurence of complications in DM or IBD.

Key words: oxidative stress, reactive oxygen species, antioxidant defense, oxidative DNA damage, DNA repair, Type 1 diabetes mellitus, diabetic microvascular complications, Crohn´s disease

2. INTRODUCTION 2.1. ROS under Physiological Conditions Formation of reactive oxygen species (superoxide, hydrogen peroxide, hydroxyl radical, ROS) is a natural process during oxidative metabolism. ROS play an important role not only in pathological processes of human organism as usually presented but less attention is paid to their proper important role in cell signalling, biosynthesis or non-specific antiinfectious defence. Triplet-state molecular oxygen undergoes process of univalent reduction and formation of superoxide anion (O2-.) nonenzymatically by redox-reactive compounds in the mitochondrial electron transport chain and enzymatically via enzymes such as NAD(P)H oxidases and xanthine oxidase. Further conversion of labile superoxide is into hydrogen peroxide (H20 2) by superoxide dismutase. In the presence of reduced transition metals (e.g. ferrous or cuprous ions), hydrogen peroxide can be converted (via Fenton reaction) into the highly reactive

Impact of Oxidative Stress on Diabetes Mellitus and Inflammatory Bowel Diseases 181 hydroxyl radical (.OH). Alternative conversion of hydrogen peroxide into water is mediated by the enzymes catalase or glutathione peroxidase. In the glutathione peroxidase reaction, glutathione is oxidized to glutathione disulfide, which can be converted back to glutathione by glutathione reductase in an NAD(P)H-consuming process [1](Fig. 1). Superoxide and related ROS formed via NAD(P)H oxidase have important physiological functions such as signal transduction from various membrane receptors, control of ventilation, smooth muscle relaxation, and enhancement of immunological functions, control of erythropoietin production and other hypoxia-inducible functions, cell apoptosis. Under physiological conditions, ROS formation and concentration in biological cells and tissues is determined by the balance between their rates of production and their rates of clearance by various antioxidant compounds and enzymes. Main nonenzymatic compounds are such as α-tocopherol, ascorbate, β-carotene, glutathione, amino acids, albumin, and the most important enzymes involved in antioxidant defence are superoxide dismutase SOD, glutathione peroxidase GPx, glutathione reductase, glutathione transferase, and catalase [1]. Oxidant-antioxidant balance is very important for cells and tissues life and metabolism. A short-term increase in ROS concentrations or a decrease in the activity of one or more antioxidant systems stimulate cell signal cascades and lead to stimulated gene expression and higher production of proteins or antioxidant enzymes (oxidative stress response) [2]. The original balance between ROS production and ROS scavenging capacity is restored and a socalled redox homeostasis is re-established. ROS play important role in receptor-mediated signalling pathways. Growth factors, cytokines or other ligands trigger ROS production in non-phagocytic cells through NADPH oxidase stimulation. Such ROS production mediates further signal transduction including stimulation of protein tyrosine kinases, mitogen activated protein kinases (MAPK) cascade, oxidative activation of protein kinase C as well as activation of transcription factor AP-1 important for differentiation processes and also transcription factor NF-κB, implicated in inflammatory reactions, growth control and apoptosis. ROS are involved in the defence against environmental pathogens and immunological response. In phagocytic cells (macrophages, neutrophils), NADPH oxidase produces ROS after stimulation with microbial products, lipoproteins or by cytokines such as interferon-γ, interleukin-1β, or interleukin-8. The stimulated massive production of ROS is called oxidative burst and plays an important role as a first line of defence against environmental pathogens. The following product created by combination activities of NADPH oxidase and myeloperoxidase is hypochlorous acid (HClO), a potent antimicrobial and oxidant agent. ROS production in lymphocytes is mediated by the enzyme 5-lipoxygenase (5-LO) and may influence their response to cytokines. Exposure of T lymphocytes to relevant concentrations of environmental ROS amplify signalling cascades even after mild stimulation and in case of massive ROS production substantially enhances the lymphocyte reactivity and stimulation. ROS produced during oxidative phosphorylation in mitochondria serve as sensor in carotid body for changes in oxygen concentration and also for expression of erythropoietin. ROS also induce the adherence of leukocytes to endothelial cells in post-capillary venules. ROS are crucial for normal functions of cardiac and vascular cells including vascular tone. Though the physiological and favourable effect of ROS on a normally functioning cell, there is a propensity for ROS to evade antioxidant defences and to modify cell components – lipids, proteins, sugars, and finally DNA. If ROS impact is short, there is no serious damage

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to the cell. On the other hand, prolonged ROS effect leads to cell apoptosis, cellular dysfunction, or neoplastic shift.

2.2. ROS under Pathological Conditions If ROS overproduction during numerous pathological situations in human medicine remains stable and long-lasting, it could be taken as basic pathological mechanism for many different diseases and complex processes as aging, neoplasias, infectious diseases, autoimmune disorders, ischaemia-reperfusion syndromes (myocardial infarct, brain stroke, injuries, organ transplantation), atherosclerosis, diabetes mellitus, toxic factors of different origin, irradiation, extreme physical activity and systemic and organ specific diseases as pancreatitis, glomerulonephritis or interstitial nephritis, inflammatory bowel diseases, hepatitis, different dermatological diseases (atopic dermatitis, psoriasis, infections, pulmonary affections (asthma, dust diseases, ARDS), rheumatoid arthritis, demyelinating diseases [3]. When considering the origin of non-regulated ROS production, it is necessary to refer to the most important situations: a) Mitochondrial electron transport chain (ETC), b) inflammatory processes with excessive stimulation of NDAPH oxidases and lipoxygenases, c) ischaemia and reperfusion with xanthine oxidase stimulation, d) endogenous or exogenous toxic factors including radiation.

I. Generation of superoxide, II. Formation of hydrogen peroxide III. Detoxification of hydrogen peroxide by antioxidant enzymes IV. Protein, lipid and DNA damage by ROS (hydroxyl radical) (by G.P. Eckert) Figure 1. Scheme of oxidative stress in human cell.

Impact of Oxidative Stress on Diabetes Mellitus and Inflammatory Bowel Diseases 183 Oxidative stress (OS) is then defined as the imbalance between high ROS production and insufficient antioxidant (AO) defence. Once formed, ROS deplete antioxidant defences, rendering the affected cells and tissues more susceptible to oxidative damage. Lipids, proteins and DNA are the cellular targets for oxidation, leading to changes in cellular structure and function. The order of preference for modification depends upon a number of factors, such as location of ROS production, relative ability for the biomolecule to be oxidised, and availability of metal ions. Oxidative changes of lipids and proteins may be removed via normal turnover of the molecules; damage to DNA is required to be repaired. Prolonged oxidative stress leads to serious injuries of cell structures and may be finished by cell apoptosis, dysfunction or uncontrolled cell division and neoplasia. Diabetes mellitus and inflammatory bowel diseases represent 2 model diseases with important oxidative stress in their pathogenesis and they will be discussed later.

2.3. Antioxidant Defence of Human Organism There are several approaches how to distinguish components of defence against oxidative stress. The schedule underlines the process of ROS formation or degradation and hence the defence against them [3, 4]. a) Substances preventing the formation of ROS are mainly the compounds binding reduced transition metals (e.g. ferrous or cuprous ions) such as acute-phase proteins (transferrin, ferritin, lactoferrin, haptoglobin, hemopexin, ceruloplasmin), or chelating agents, also inhibitors of enzymes catalysing the formation of ROS (allopurinol blocking xanthine oxidase). b) Substances removing previously formed ROS or restoring reducing agents are either intracellular and extracellular enzymes such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and transferase, or non-enzymatic endogenous and exogenous substances such as glutathione, α-tocopherol, ascorbate, β-carotene, amino acids, uric acid, bilirubin, albumin, and polyphenols. c) Repair systems removing molecules damaged by ROS are principally enzymes degrading modified proteins, or oxidized lipids (phospholipases, glutathione peroxidases), and finally the complex of enzymes repairing DNA damaged by ROS.

2.4. Some Remarks to Antioxidant Enzymes and Substances from the Recent Literature Antioxidant Enzymes are Directly Implicated in Human Protection against Oxidative Stress Their properties, their known polymorphisms and their corresponding phenotype are target of many studies. Superoxide dismutase SOD transforms superoxide into hydrogen peroxide. Three isoforms (cytoplasmic CuZn SOD or SOD1, mitochondrial Mn SOD or SOD2, tetrameric CuZn extracellular EC-SOD or SOD3) exist and have been studied for many years in vitro and in vivo. Relationships among different SOD isoforms and human diseases were found. Down regulated or mutant SOD1 has been associated with stroke, ischaemia/reperfusion

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syndrome, neurodegenerative disorders (Parkinson’s disease) and familial amyotrophic lateral sclerosis with 58 known mutations in the CuZn SOD gene (chromosome 21q22.1). Loss or reduction of the SOD2 activity has been associated with neurodegeneration, heart failure and also death during neonatal period. SOD3 has been detected in several compartments such as the plasma, lymph, ascites, cerebrospinal fluid and lung and is related to inflammation, hyperoxic pulmonary disorders, and focal cerebral ischaemia [5]. The present efforts are done to introduce into clinical practice SOD mimetic drugs [6], either selective (macrocyclics) or non-selective such as manganese metalloporphyrins, manganese salen complexes [7, 8]. Another possibility seems to be dietary delivery system using wheat gliadin biopolymers carrying plant SOD extracts [9]. Glutathione peroxidases GPx scavenge peroxides generated in cells. There are four isoforms with different tissue localisation. Cytosolic GPx1 is ubiquitously expressed in human organism, particularly in erythrocytes, kidney, and liver. Cytosolic GPx2 is expressed primarily in gastrointestinal tissues and plays an important role in protection against ingested lipid peroxides. GPx3 is an extracellular enzyme reducing hydroperoxides of more complex lipids. It is mainly present in plasma, but also in lung, heart, kidney and placenta. GPx4 is detected both in the cytosol and associated with membranes. This enzyme reduces peroxidised phospholipids and cholesterol in membranes [5] (Fig.2). Glutathione reductase GSR has two isoenzymes (cytosolic and mitochondrial) and its function is to restore glutathione into reduced form [5].

Figure 2. Glutathione peroxidase and glutathione reductase action in antioxidant defence and glutathione metabolism.

Catalase decomposes hydrogen peroxide into water and oxygen. This enzyme is particularly abundant in erythrocytes, hepatocytes and the kidney. There are several mutations/polymorphisms in the catalase gene (chromosome 11p13) associated with acatalasemia in erythrocytes, but phenotypes associated with acatalasemia are not prominent [5]. Glutathione transferases GST exist in six cytosolic isoforms and one microsomal GST, the latter now designated membrane associated proteins involved in eicosanoid and glutathione metabolism (MAPEG). GSTs play an important role in detoxification of xenobiotics (chemical carcinogens, environmental pollutants, antitumor agents) and in inactivation of endogenous hydroperoxides and unsaturated aldehydes. GSTs catalyse the conjugation of 4-hydroxynonenal, the degradation product of lipid peroxidation, with glutathione [5, 10, 11]. GST expression is regulated by the cellular redox status and represents a sensor able to transmit the redox variation to the apoptosis machinery. Cytosolic

Impact of Oxidative Stress on Diabetes Mellitus and Inflammatory Bowel Diseases 185 GST isoenzymes are broadly cytoprotective, but their genetic polymorphisms can increase susceptibility to carcinogenesis [12, 13] and inflammatory disease such as chronic obstructive pulmonary disease, asthma, pancreatitis, autoimmune hepatitis, rheumatoid arthritis [5, 14, 15, 16, 17, 18]. It is obvious that cells exposed to oxidative stress express stress-induced genes or genes encoding antioxidant defences [19], and their polymorphisms are certainly involved in various responses to oxidative stress [5].

2.5. Non-Enzymatic Substances of Endogenous or Exogenous Origin Non-Enzymatic Substances of Endogenous or Exogenous Origin are of great importance for antioxidant defence. Some of them are newly synthesized and classified as antioxidant enzymes mimetics and were mentioned concurrently with antioxidant enzymes. Further new antioxidants are analogues of naturally existing substances. Glutathione (GSH - γ-glutamylcysteinylglycine) draw attention in context with different diseases. GSH is the major intracellular non-protein antioxidant and is present in all mammalian tissues, where it plays a crucial role in the detoxification of free radicals. Its synthesis serves to maintain a reduced cellular environment. The liver is the main site of inter-organ glutathione synthesis and supplies GSH into circulation and systemic distribution, but the GSH production occurs also in other body cells such as erythrocytes, GIT epithelium, and bronchiolar epithelium. GSH pool is drawn on for three major applications: for direct free radical scavenging and as an antioxidant enzyme cofactor (glutathione peroxidases GPx, glutathione transhydrogenases reconverting dehydroascorbate to ascorbate), and as cofactor for the glutathione transferases GST. Total glutathione has two forms – the free form which is mainly in its reduced form (GSH) and the form bound to proteins (gluthathionylated proteins) [11, 20, 21]. Conversion of GSH reduced form into oxidised glutathione (GSSG) occurs during oxidative stress and can be reverted to the reduced form by the action of glutathione reductase GSR mentioned above (Fig.3). This enzyme uses as its source of electrons the coenzyme NADPH coming mainly from the pentose phosphate shunt. Low GSH, high GSSG, and a higher GSSG/GSH ratio indicates the presence of oxidative stress and consumption of GSH [11]. Glutathione has also profound importance for cellular homeostasis and for diverse cellular functions such as protein synthesis, enzyme catalysis, transmembrane transport, receptor action, intermediary metabolism, cell maturation, and apoptosis [11, 20]. The importance of glutathione and its disturbances has been highlighted in numerous articles [11, 22, 23]. GSH deficiency certainly plays the role in liver diseases such as cirrhosis, hepatitis and non-alcoholic steatohepatitis [20, 21]. Depletion of glutathione was also found in severe acute pancreatitis [16]. Alterations in alveolar and lung GSH metabolism is widely recognized as a central feature of many inflammatory lung diseases such as idiopathic pulmonary fibrosis, acute respiratory distress syndrome, chronic obstructive pulmonary disease (COPD), and asthma [24, 25, 26]. In patients with cystic fibrosis (CF), CFTR channel is no more taken for a primarily „chloride channel“ but recognized as a channel that also controls the efflux of other physiologically important anions, such as glutathione and bicarbonate. CF mutations in fact cause a primary dysfunction in the reduced GSH system, e.g. CF mutations significantly decrease GSH efflux from cells and this leads to

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deficiency of GSH in the epithelial lining fluid of the lung, as well as in other compartments, including immune system cells and the gastrointestinal tract. Following this, other antioxidants are depleted and this may play a role in initiating the over-inflammation characteristic of cystic fibrosis [26, 27]. The role of glutathione is also alleged in neurodegenerative diseases (Parkinson’s disease, Alzheimer’s disease), in atherosclerosis, and in diabetes whose topic will be discussed later [20, 22]. Strategies for cellular glutathione repletion include mainly dietary interventions. Natural dietary glutathione undergoes hydrolysis by intestinal and hepatic gamma-glutamyltransferase [28], and so food sources or supplements that increase glutathione must either provide the precursors of glutathione, or enhance its production by some other means. Natural foods that boost glutathione levels are fresh and frozen fruits and vegetables (asparagus, broccoli, avocado, spinach, walnuts), fish, and meat [29]. N-acetyl cysteine takes on significance as a dietary GSH source. N-acetylcysteine has been used in several indications, such as the treatment of paracetamol (acetaminophen) intoxication [30], prevention of contrast-induced nephropathy after intravascular angiography [31], treatment of chronic obstructive pulmonary disease [32]. Its application in the treatment of severe sepsis and septic shock remains controversial [33, 34, 35]. N-acetylcysteine amide is currently studied as another possible substance for treatment of neurodegeneration and other oxidation-mediated disorders [36].

GR-glutathione reductase (NADPH-dependent), GS-glutathione synthase, γ-ECS- gammaglutamylcysteine synthase, DHA-dehydroascorbate, DHAR-dehydroascorbate reductase, GSSG- oxidized glutathione, GSHreduced glutathione Figure 3. Metabolism of glutathione in the cell.

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Thioredoxin (Trx) Thioredoxin (Trx) is a small multifunctional protein involved in cellular thiol-redox processes. It contains two redox-active cysteine residues in its active sites. The protein is stress-inducible and enhanced by various types of stresses including ROS. The thioredoxin is kept in the reduced state by the flavoenzyme thioredoxin reductase, in a NADPH-dependent reaction. It acts as an electron donor to peroxidases and ribonucleotide reductase and the administration of recombinant Trx protein, induction of endogenous Trx, or gene therapy can be a therapeutic modality for oxidative stress-associated disorders [37,38, 39, 40]. Coenzyme Q10 (CoQ) Coenzyme Q10 (CoQ) known as ubiquinone, CoQ, and vitamin Q10 is a fat-soluble vitamin-like quinone present in every cell of the body. It is naturally present in different amounts in foods (mostly high in beef meat, soy oil, sardines, mackerel, and peanuts), but it is also synthesized from its precursors Q1-9 or de novo synthesized from acetyl coenzyme A in liver. CoQ-10 is found throughout the body in cell membranes, especially in the mitochondrial membranes and is particularly abundant in the heart, lungs, liver, kidneys, spleen, pancreas and adrenal glands.Q10 is a mobile electron carrier from NADH dehydrogenase complex to cytochrome b-c1 complex within the mitochondrial electron transport chain enabling the H+ flow out of the mitochondrial matrix. Thus electrochemical proton gradient across the inner membrane is formed and finally drives the H+ flow through the ATP synthase, that uses the energy of the H+ flow to synthesize ATP as a carrier of free energy [39]. Q10 is also a powerful antioxidant both on its own and in combination with vitamin E. CoQ is present not only in mitochondria, where bound to mitochondrial membranes serves also as an antioxidant. It is also present in cell membranes where in its reduced form ubiquinol can neutralize a lipid peroxyl radical. The CoQ radical ubisemiquinone is stable and its formation is a normal intermediate between ubiquinone (CoQ) and ubiquinol (CoQH2), and is restored to a non-free-radical state by the respiratory chain Q cycle. Ubisemiquinone and ubiquinol can also regenerate the vitamin E tocopheroxyl radical by electron donation and this is very important as vitamin E neutralizes lipid peroxyl radicals far more readily and is less hydrophobic, allowing it to more freely move throughout the mitochondrial membrane. CoQ is more confined to the centre of the phospholipids layer. Its importance as an endogenously produced lipid-phase antioxidant is indicated with its high concentration not only in cell membranes, but also in Golgi vesicles and microsomes [4, 41, 42]. Supplementation of CoQ is recommended in patients with Parkinson’s disease, mitochondrial encephalomyopathies, with chronic heart failure [41], as CoQ is needed for energy production and antioxidant protection. The problem in question is the supplementation of CoQ in patients treated with statins as HMG-CoA reductase is also the first committed rate-limiting step for the synthesis of a range of other compounds including steroid hormones and ubiquinone. Currently, there is insufficient evidence from human studies to link statin therapy unequivocally to pathologically significantly decreased tissue CoQ levels [43]. Cosupplementation with vitamin E and CoQ reduces circulating markers of inflammation as proved on animal models and this example can be used for further studies dealing with vascular diseases [44].

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Vitamin C Vitamin C is essential for human organism. Under physiological conditions, vitamin C predominantly exists in its reduced form, ascorbic acid (AA), it also exists in trace quantities in the oxidized form – dehydroascorbic acid (DHA). AA functions as a cofactor for enzymes involved in the biosynthesis of collagen, carnitine, norepinephrine, and in the amidation of hormones. In plasma and cells, AA is a powerful antioxidant, quenching ROS. During the process of quenching free radicals, ascorbate donates an electron, becoming the unstable intermediated ascorbyl radical that can be reversibly reduced back to ascorbate via glutathione oxidation. Ascorbyl radical can also donate a second electron and be converted to DHA that can efflux from the cells via the glucose transporters and functions as a readily transportable form of vitamin C. As mentioned above, ROS activate transcription factors, such as NF-κB, that are important in host defence, inflammation, and apoptosis. AA directly quenches ROS intermediates involved in the activation of NF-κB, and is oxidized to DHA, which directly inhibits IκBα kinase (IKKβ) and thus NF-κB cannot be activated [45]. Vitamin E Vitamin E is a fat-soluble vitamin present in many foods, especially certain fats and oils. There are α, β, γ, and δ forms, among them α-tocopherol (α-TOH) is the most abundant and active form with the referring term vitamin E. It is a natural, lipophilic radical-scavenging antioxidant and is described as the most important lipophilic antioxidant in vivo, which has been the subject of many extensive studies [46]. Vitamin E localization in the lipophilic domain of the membranes and lipoproteins inhibits lipid peroxidation, i.e. α-TOH reacts with peroxyl radicals much faster than lipids do and thus it may scavenge the majority of peroxyl radicals before they attack lipids. The efficacy of α-TOH is better when a radical scavenging occurs near the membrane surface. If the radical went deeper into the interior of the membranes, scavenging activity of α-TOH decreased significantly [47]. α-TOH is converted to α-TO· radical which may undergo several reactions. Preferentially, it is reduced by ascorbate in order to regenerate α-TOH provided that α-TO· radical is localized in the membranes and LDL. If the radical goes deeper into the interior of cell or is localized in the LDL core, ascorbate is unable to reduce the radicals. The α-TO· radical can be reduced by other reductants, such as ubiquinol and polyphenolic compounds. Vitamin E may exert its effect by acting as a radical-scavenging antioxidant whose activity depends on many factors (localization, mobility, presence of other collaborating antioxidants). These factors influencing vitamin E efficiency and impact on oxidative stress can explain certain controversies in the antioxidant studies with vitamin E. Nevertheless, vitamin E remains a perspective antioxidant in combination with synergistic antioxidant compounds [46, 47]. Phytochemicals Phytochemicals are a wide range of different naturally plant compounds, such as carotenoids, polyphenols including flavonoids. Their antioxidant effects depend on their chemical structure and lipophilic or hydrophilic character. Carotenoids are plant pigments and animals do not synthesize them. They are responsible for the red, yellows, and orange colour of fruits and vegetables, their number in nature exceeds 600 and can be grouped into carotenes, xanthophylls and lycopene. Carotenoids have been intensively studied in the last decade and as proved by several epidemiological studies, they showed certain preventive or

Impact of Oxidative Stress on Diabetes Mellitus and Inflammatory Bowel Diseases 189 inhibitory effect on atherosclerosis, degenerative diseases, cancer and other diseases [48, 49, 50]. The antioxidant activity of carotenoids arises as a result of the ability to delocalise any unpaired electrons. β-carotene has an excellent ability to quench singlet oxygen without degradation and reacts with free radicals such as peroxyl radical, hydroxyl radical. This is the way that assures lipid protection from peroxidative damage. Polyphenols, among them flavonoids existing for example in green tea, black currant, blackberries, form stable phenolic radicals with ROS, regenerate vitamin C and E and bind transition metals [4]. Resveratrol is a naturally occurring polyphenol (phytoalexin) produced by some higher plants in response to injury of fungal infection. Resveratrol is found in grapevines, also in peanuts and mulberries. It has several activities which include inhibition of the oxidation of low-density lipoproteins, blockade of superoxide anion and hydrogen peroxide in macrophages stimulated by lipopolysacharides (LPS), decrease of arachidonic acid release induced by LPS or by exposure to superoxide or hydrogen peroxide, glutathione-sparing activity thanks to hydroxyl-radical scavenging activity, suppression of lipid peroxidation both by chelation of copper and by scavenging of the free radicals. Resveratrol action is explained by its influence on NADPH-dependent oxidases, xanthine oxidase, 5-lipoxygenase and 15-lipoxygenase, myeloperoxidase. It seems to be a good candidate protecting the vascular walls from oxidation, inflammation, platelet aggregation, and thrombus formation, i.e. atherosclerotic process [51]. The French paradox (low incidence of coronary heart in France) is attributed to the complex antiatherosclerotic role of grape polyphenols regularly consumed in red wine. Isorhapontigenin (ISO), a new resveratrol analogue, has been isolated from Belamcanda chinensis (blackberry lily). Its chemical structure is very similar to that of resveratrol. ISO significantly inhibits malondialdehyde formation induced by hydrogen peroxide and the increase in lipid peroxidation, ISO also inhibits oxidative DNA damage [48, 52]. Figure 4. shows the cellular distribution of some enzymatic and non-enzymatic antioxidants.

Available from: www.medscape.com, by RH Demling and L De Santi Figure 4. Cell structure and location of different endogenous and exogenous antioxidants.

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2.6. Catalytic Antioxidants – New Therapeutic Possibilities As mentioned above, superoxide O2-.and hydrogen peroxide H2O2 participate in the formation of several reactive species that have been directly implicated in tissue damage. Cellular antioxidant enzymes as SODs and catalase detoxify ROS. Unfortunately, their large size, short circulating half-life, and antigenicity are not suitable for therapeutic use in free radical diseases. To overcome many of these limitations, an increasing number of low molecular-weight catalytic antioxidants have been developed. They are designed with redoxactive metal centres that catalyse the dismutation reaction of O2-. and/or H2O2 by a mechanism that is similar to the mode of action of the active-site metals of SOD and catalase. An ideal mimetic is stable and non-toxic and its size and charge of a mimetic can be exploited to target crucial cellular sites of oxidative stress. Three classes of metal-containing catalytic antioxidants, i.e. macrocyclic (M40403), salen (EUK series), and metalloporphyrin mimetics (AEOL series) seem to be important for future clinical use as shown on in vitro and in vivo models. Their potential includes lung fibrosis, chronic obstructive lung diseases, asthma, acute respiratory distress syndrome, bronchopulmonary dysplasia, shock, myocardial infarction, liver failure, colitis, diabetes, ischaemia-reperfusion, transplantation, arthritis, amyotrophic lateral sclerosis, neurodegenerative diseases, stroke [7, 8].

3. DNA OXIDATIVE DAMAGE AND DNA REPAIR 3.1. Nuclear DNA Damage and Repair DNA is constantly exposed to damaging agents from both endogenous and exogenous sources. If these lesions are not repaired, they can lead to mutations and result in cellular dysfunction with either further uncontrolled cell proliferation, or apoptosis. In order to maintain the integrity of the genome, a complicated network of DNA repair pathways remove the majority of deleterious lesions. There are six major repair pathways of DNA repair [53, 54, 55]. The number of human DNA repair genes exceeds 140 and keeps growing [56, 57]. Direct reversal (DR) consists of repair proteins acting directly on a damaged base and reestablishing the correct structure in situ without removing the damaged nucleotide. Exogenous agents such as ionising radiation, tobacco smoke, cytostatics generate larger DNA lesions with abnormal structure or abnormal chemistry (24-29 nucleotides) which are repaired via nucleotide excision repair (NER). NER steps involves a) damage recognition, b) binding of a multi-protein complex at the damaged site, c) double incision of the damaged strand several nucleotides away from the damaged site, on both the 5´and 3´sides, d) removal of the damage-containing oligonucleotide from between the two nicks, e) filling in of the resulting gap by a DNA polymerase and f) final ligation. The genes encoding many of the human NER proteins were first identified in genetic complementation studies of the human DNA repair disease – xeroderma pigmentosum (XP) with mutations in any of 7 genes. In addition to XP, Cockayne´s syndrome (CS) and trichothiodystrophy (TTD) are two other human genetic disease with defects in NER [58, 59]. The mismatch repair (MMR) is a post-replicative DNA repair system contributing to the fidelity of replication and the maintenance of the genome. Recombinational repair (RER)

Impact of Oxidative Stress on Diabetes Mellitus and Inflammatory Bowel Diseases 191 restores deleterious double-strand breaks via homologous recombinational repair (HRR) or non-homologous end-joining (NHEJ). Genes and proteins related to HRR are implicated in chromosomal stability and are critical in maintaining cellular resistance to ionising radiation [60]. Ataxia teleangiectasia (AT) and Nijmegen breakage syndrome (NBS) are chromosome instability syndrome with mutated genes, such as ataxia teleangiectasia mutated (ATM chromosome 11q23) or NBS respectively. AT is an autosomal recessive disease and affected patients develop cerebellar ataxia, teleangiectasia, combined cellular and antibody immunodeficiency, sinopulmonary infections, and malignancies. The Nijmegen breakage syndrome (NBS – chromosome 8q21) is also an autosomal recessive disease characterized by microcephaly, growth retardation, immunodeficiency and cancer predisposition [61, 62]. Germline and somatic mutations of certain HRR genes contribute inevitably to carcinogenesis (e.g. BRCA1, BRCA2 proteins)[63].

ROS

DNA Base Modification

Sugar Damage

Lipid Peroxide 4-HNE, MDA

DNA Base 8-oxo-G Adduct FapyG

Glycosylase hoGG1

AP Sales

Base Propenal Glycosylase MPG

DNA Base Adduct

M 1G, edG, eda

ROS-reactive oxygen species, 4-HNE- 4-hydroxy-2-nonenal, MDA-malondialdehyde, M1G pyrimidopurinone, edG ethenodeoxyguanosine, edA etheno-deoxyadenosine, hOgg1 human 8oxoguanine DNA glycosylase 1, MPG N-methylpurine DNA glycosylase, FapyG 2,6-diamino-4hydroxy-5-formamidopyrimidine, 8-oxo-G 8-oxo-7,8-dihydro-2´-guanosine Figure 5. Schematic diagram of types of DNA damage induced by ROS (published by Powell, CL, Swenberg, JA and Rusyn, I. in Cancer Letters, 2005, 229, 1-11).

Finally, base excision repair (BER) is the most important pathway for oxidative DNA damage. Free radicals, most notably ·OH, react with DNA compounds and form radicals of DNA bases and the sugar moiety. Further reactions of these radicals result in a variety of final products. Major products of oxidative damage to the DNA bases are numerous, among them thymine glycol, 5-formyluracil, 5-hydroxyuracil, 5-hydroxycytosine, 8-hydroxyadenine, 8hydroxyguanine, 2,6-diamino-4-hyroxy-5-formamidopyrimidine. In addition to sugars modified by oxidative stress (e.g. 2-deoxyribonolactone), unaltered sugar moieties within

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DNA, called base-free sites (or apurinic/apyrimidinic site – AP site) are formed from the elimination of modified DNA bases due to the weakening of the glycosidic bond. These lesions can result in single strand breaks. As ROS are continuously generated in all organisms, the oxidized bases, single-strand breaks and AP sites in DNA are repaired primarily via the DNA base excision repair pathway (BER) (Fig. 5, 6). NER is implicated as a back-up system for BER, so oxidative DNA lesions containing oligonucleotide are removed via NER and is taken as a back-up system for BER.

Figure 6. Base excision repair pathway (published by Powell, CL, Swenberg, JA and Rusyn, I. in Cancer Letters, 2005, 229, 1-11).

The developing knowledge about DNA repair processes is bringing the detailed information about purine- and pyrimidine-derived lesions and their removal from DNA. 8-OH-guanine glycosylase OGG1 has 2 major forms hOGG1-1a being targeted to the nucleus and hOGG1-2a being targeted to the mitochondrial inner membrane and protecting mitochondrial DNA. OGG1 is a constitutively expressed gene and its expression can vary with tissue source and between individuals. MutY homologue (hMYH) glycosylase repairs mismatches 8-OH-Gua:A and 8-OH-Gua:G. Human MutT homologue (hMTH1) degrades 8OH-dGTP to 8-OH-dGMP and pyrophosphate. N-methylpurine-DNA glycosylase (MPG protein or 3-methyladenine-DNA glycosylase) has a relatively broad range of substrates including 8-OH-Gua and 3-methyladenine by BER mechanism. Endonuclease III homologue (hNTH1) is involved in the repair of oxidized pyrimidines. Another guanine-derived 2,6diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) is removed via hOGG1 or NEIL1 (glycosylase excising FapyGua). Adenine-derived lesions such as 8-hydroxyadenine (8-OHAde) are removed also via hOGG1 when 8-OH-Ade pairs opposite cytosine. 2-OH-adenine is the substrate for hMTH1. 4,6-diamino-5-formamidopyrimidine (FapyAde) is released by the

Impact of Oxidative Stress on Diabetes Mellitus and Inflammatory Bowel Diseases 193 activity of hNTH1 and also NEIL1 glycosylase. The same enzyme is targeted at 5hydroxycytosine (5-OH-Cyt), 5-OH-uracil lesions and thymine glycol. Thymine glycol could be removed also by thymine glycol glycosylases (primarily mitochondrial TGG1 and nuclear TGG2) [2, 64]. Removal of a damaged nucleotide by a glycosylase forms an AP site. Repair of AP sites inhibits both the mutational and cytotoxic effects of nucleic acid loss. AP endonucleases (APEX1, APEX 2) recognize abasic site and cleave the phosphodiester DNA backbone. Subsequent step is up to the DNA polymerase β which removes the deoxyribose phosphate and adds a nucleotide. Finally, DNA ligase III in partnership with the XRCC1 protein (ligase accessory factor) seals the nick. Cells deficient in polymerase β showed hypersensitivity to monofunctional alkylating agents. The described pathway is called short patch-BER (Fig. 6) [54, 59]. The second BER pathway - long patch-BER is a little bit complicated and involves the activities of glycosylase, AP-endonuclease I, replication factor C, proliferating cell nuclear antigen (PCNA), DNA polymerase δ or ε, flap-endonuclease 1 (FEN1 – 5´nuclease) and DNA ligase I. This involves removal and re-synthesis of a 2-6 nucleotide patch (Fig. 6)[54, 59, 64]. Whichever pathway is used, the product secreted from the cell will be the modified base, because they both pathways involve a glycosylase activity. These excreted oxidized bases could be measured as a marker of endogenous oxidative DNA damage i.e. oxidative stress [2]. There is a large scale of methods for measuring oxidative DNA damage: chromatographic assessment of oxidized purines and pyrimidines, comet assay (or single cell gel electrophoresis SCGE) for determination of single-strand breaks [65], when lesion-specific endonucleases are included, allows detection of oxidized purines and pyrimidines. Slot-blot assays enables the measurement of abasic (AP) sites and with addition of lesion-specific endonucleases allows detection of oxidized purines and pyrimidines [66]. For example, an intensively studied 8-OH-guanine was measured in blood and especially in urine as a marker of oxidative stress in different diseases. Parkinson’s and Alzheimer’s disease, multiple sclerosis, amyotrophic lateral sclerosis, breast cancer, colorectal cancer, haematological disorders, chronic hepatitis, haemochromatosis, Wilson’s disease, cystic fibrosis, lung and gastric cancer, rheumatoid arthritis, systemic lupus erythematosus, Down’s syndrome, diabetes mellitus [2]. Repair enzymes are constitutively expressed, their expression and/or activity may be modulated in response to various potential genotoxic stress or in concert with the cell cycle. Levels of oxidative bases in DNA are the consequence of a balance between lesion induction from radical processes and repair. Reduced repair will result in elevated lesion and an increased risk of disease. Genetic polymorphisms in repair proteins could also affect the relative propensity of individuals to develop various pathologies including even human inherited disease. Biallelic mutations in the BER DNA glycosylase MYH were found in an autosomal recessive syndrome of adenomatous colorectal polyposis with very high risk of colorectal cancer [67]. Several studies focusing the tissue expression of BER enzymes, and their relationship to diseases have been accomplished or are in motion using animal models or human samples [66, 68, 69]. Variable population distribution of polymorphisms in DNA repair genes and inter-individual differences in the DNA-repair efficiency are referred to the different occurrence of oxidative stress related diseases in the world [69, 70].

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3.2. Mitochondrial DNA Damage and Repair BER Repair Pathway Comes about not Only in Nucleus, but also in Mitochondria Mitochondria (originated from maternal egg) are present in many copies in mammalian cells, except for mature erythrocytes (Fig. 7). Each mitochondrion has a few identical copies of its own circular DNA, which codes for 13 mitochondrial enzymes involved in cellular respiration (using a slightly different genetic code than does nuclear DNA). Nuclear genes code for several other mitochondrial proteins. In somatic cells, mitochondrial DNA mutates about 10 to 20 times faster than nuclear DNA, because mitochondria are a major source of reactive oxygen species. These mutations accumulate with age in postmitotic tissues such as neurons, and in cardiac and skeletal muscle. As studied in multiple reports [71] and brilliantly summarized in one of the latest reports [72], there is an evidence that multiple DNA repair pathways exist also in mitochondrion (DR, BER, MMR, RER). BER pathway in mitochondrion shares similar steps like in nuclear BER but taking into account that the responsible enzymes have nuclear and mitochondrial isoforms (OGG1, TGG, APE, MYH). The presence of the only strand could also contribute to the observed increased mutation frequencies of mtDNA compared to nDNA. Though this fact, other non-mutated copies of mtDNA are present and thus mitochondrial function does not necessarily be compromised. Repair of mtDNA and nDNA in principle could be very similar with the difference being that nDNA repair pathways involve a larger number of proteins.

available from http://www.mitoresearch.org/images/q64bigmito.jpg Figure 7. Metabolic pathways in mitochondrion.

If oxidative DNA damage is not repaired completely, several situations can come about. Many oxidative base lesions are mutagenic, other can induce conformational changes

Impact of Oxidative Stress on Diabetes Mellitus and Inflammatory Bowel Diseases 195 in DNA. Lesions as thymidine glycol can block replication one nucleotide before and after the lesion. The length of repetitive sequences of DNA (microsatellites) is constant in normal cells, but microsatellite instability (i.e. variable number of repeats) derives from oxidative DNA damage. If the DNA oxidative lesions are present in the transcribed regions of genes they can lead to mutation. But if they are localized in promoter, elements they can affect transcription factor binding [4, 64].

3.3. Poly(ADP-Ribose) Polymerase Poly(ADP-Ribose) Polymerase 1 (PARP) Plays an Important Role in Oxidative DNA Damage Repair Poly(ADP-ribose) polymerase 1 (PARP-1) is the 113-kDa enzyme, mostly found in nucleus, member of the PARP enzyme family consisting of PARP-1 and several recently identified poly(ADP-ribosylating) enzymes. PARP-1 represents the best studied PARP enzyme and is responsible for more than 90% of the cellular poly(ADP-ribosyl) capacity. The process of poly(ADP-ribosyl)ation plays the role in a variety of physiological and pathophysiological phenomena, such as signalling of DNA damage induced by ionising radiation, alkylating agents and oxidants, DNA-base excision repair, regulation of genomic stability in cells under genotoxic stress, transcriptional regulation, stimulation of nuclear proteasomal function, ageing and longevity, further in pathophysiology of acute and chronic inflammatory disorders (e.g. Crohn´s disease), diabetes mellitus, ischaemia-reperfusion damage in tissue, septic and haemorrhagic shock [73]. The primary signal that cause PARP-1 activation is the recognition of single or double strand DNA breaks caused by various genotoxic agents inclusive oxidative stress in the course of different pathological situation as described above. Activated PARP-1 dimerizes at recessed DNA ends and cleaves NAD+ into nicotinamide and ADP-ribose which forms long branching (ADP-ribose)n polymers. The latter product is then attached to glutamate or aspartate residues of nuclear acceptor proteins, including PARP itself (PARP automodification) (Fig.8). As described by Virag [74], by conferring negative charges to acceptors, this covalent protein modification alters the physico-chemical properties of the modified target proteins and thus regulates their function. The set of polymer acceptor proteins includes various DNA repair proteins, transcription factors, histones, other proteins, and PARP-1 itself. This PARP-1 automodification represents a negative feedback mechanism causing the down regulation of PARP-1 enzyme activity, the poly(ADP-ribosyl)ation is reversible and the enzyme poly(ADP-ribose)glycohydrolase (PARG) degrades the polymer (Fig. 8) As described, nuclear ADP-ribosylation induced by the presence of DNA strand breaks stimulates DNA repair, and finally leads to the cell recovery from moderate DNA damage (Fig. 8). If DNA damage is excessive and unrepairable, PARP is overactivated and mediates a futile energy-consuming cycle inactivating the stress response of the cells [73, 74].PARP-1 depletes the cellular stores of its substrate NAD+ , and in turn NAD+ resynthesis also consumes ATP. Hence, NAD+ and ATP depletion as two key energy metabolites leads to cell death, apoptosis or necrosis (Fig. 9). The switching from apoptosis to necrosis by PARP-1 overactivation may aggravate tissue damage, as leakage of cellular content from

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necrotic cells is pro-inflammatory. It is necessary to stress that the intensity of cellular poly(ADP-ribosyl)ation decreases with age either in humans or in animals [73].

Free radicals and other agents as UV light, ionising radiation or alkylating agents may cause DNA strand breakage. PARP-1 binds to recessed DNA ends resulting in the activation of the enzyme. Active PARP-1 cleaves NAD+ into ADP-ribose (ADPR) and nicotinamide (NA). Then PARP-1 polymerises ADPR covalently linked to itself and histones leading to recruitment of various DNA repair adapter and effector proteins. Poly(ADP-ribosyl)ation of histones causes relaxation of chromatin structure rendering the site of DNA damage accessible to the repair machinery. Automodified PARP-1 detaches from DNA and the repair machinery seals broken DNA ends. PARG (poly(ADP-ribose) glycohydrolase) decomposes the polymer and the chromatin regains its original structure. Figure 8. DNA damage-induced activation of PARP-1 (published by Virag L. Current Vascular Pharmacology, 2005, 3, 209-214, with the kind permission of the author).

Except for the role of PARP-1 in regulation of genomic stability and cell survival, it has also an impact on gene expression. Gene interaction with PARP-1 had either positive or negative effects on the specific transactivation process. An important example is the involvement of PARP-1 in the regulation of NFκB, the specific transcription factor involved in the control of numerous genes, including those of many cellular genes involved in immune responses and inflammation. PARP- has also been implicated in the regulation of activator protein (AP-1)-driven transcriptional activity (Fig. 9). The activites of PARP shows certainly the role in many pathological processes, such as acute ischaemia-reperfusion damage, allergen-induced asthma, septic or haemorrhagic shock, and chronic including cancer, cardiac disorders, Parkinson´s diesease, inflammatory bowel disease and diabetes mellitus. It is

Impact of Oxidative Stress on Diabetes Mellitus and Inflammatory Bowel Diseases 197 necessary to emphasize the fact that the function of PARP-1 and poly(ADP-ribosyl)ation as a regulator of inflammatory signal transduction and transactivation shows marked cell-type and stimulus dependency [75]. This finding might certainly play the role in possible usage of envisaged PARP inhibitor therapy as will be mentioned later.

The central role of PARP-1 in oxidative stress-related pathology. Oxidative-stress-induced DNA strand breakage triggers the activation of PARP-1, leading to apoptosis inducing factor (AIF) release from the mitochondria and AIF-mediated, caspase-independent apoptotic cell death NAD/ATP consumption and consequent necrotic cell death. Oxidative stress also stimulates activation of redox-sensitive transcription factors such as NfκB and activator protein 1 AP-1, key regulators of inflammatory cytokines and chemokines. Figure 9. The role of PARP-1 in oxidative stress-related pathology (published by Virag L. Current Vascular Pharmacology, 2005, 3, 209-214, with the kind permission of the author).

A close association was described between PARP-1 and p53 protein in DNA damageinduced apoptosis [74, 76]. p53 protein is a nuclear protein that plays an essential role in the regulation of cell cycle, specifically in the transition from G0 to G1, the cell cycle arrest at the G1/S transition (which prevents the manifestation of unrepaired chomosome alterations), and also on cell cycle arrest at the G2/M transition, which inhibits the distribution of defective genomes. P53 also initiate apoptosis after the introduction of irrepairable DNA damage. At this point, both PARP-1 activation and p53 activation occur rapidly following DNA damage. The activation of these two mediators is likely to be linked to each other, as PARP-1 has been shown to interact with and poly(ADP-ribosyl)ate p53 [74, 76].

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The described activities of PARP-1 in DNA base excision repair, in apoptosis or cell necrosis induction, stimulation of inflammatory mediators via NFκB and AP-1 transcription factors, and protein degradation have been taken into consideration in several studies with PARP inhibitors on animal models or cell cultures [77](Fig.10) and recently also in clinical trials on oncological or diabetic patients [78, 79, 80]. Most PARP inhibitors developed to date have structural similarity to the substrate NAD+, thus competing for the active centre of the enzyme. The compounds are non cytotoxic on their own at concentrations necessary to achieve PARP inhibition. PARP inhibitors are used for cancer treatment with regard to the effect of PARP inhibition and potentiation of cell necrosis [77]. Another indication is to turn down the stimulation of pro-inflammatory mediators like in chronic obstructive lung disease, diabetes mellitus, or congestive heart failure, and Parkinson´s disease [80]. The question remains what side effects might be observed after a long PARP inhibitor application with regard to the blockage of DNA repair in non-cancerous diseases.

Oxidative-stress-induced DNA strand breakage triggers the activation of PARP-1. If the DNA breakage is low, BER is stimulated, cell cycle is prolonged (in cooperation with p53 protein activation) and DNA is finally repaired. If DNA breakage is high, PARP-1 is over activated, severe NAD+ consumption is in progress and apoptosis or even necrosis is started. PARP inhibitors may block the inflammatory pathways but also BER. Figure 10. DNA breakage and the activity of PARP inhibitors (published by Beneke, S; Diefenbach, J; Bűrkle, A. Poly(ADP-ribosyl)ation inhibitors: promising drug candidates for a wide variety of pathophysiologic conditions. Int J Cancer, 2004, 111, 813-818, with the kind permission of the author A. Bűrkle) .

3.4. Diseases Related with Oxidative Stress and DNA Damage Natural trend of worsened DNA repair is usually associated with aging. Aging as a constant and inevitable process has been related to oxidative stress for many years. Telomere shortening related to a finite number of replication (Hayflick´s model) is observed in normal

Impact of Oxidative Stress on Diabetes Mellitus and Inflammatory Bowel Diseases 199 human somatic cells and is considered as one of the mechanisms of the senescence (in this case – replicative senescence) [81]. Aged cells show age-dependent accumulation of 8-oxoguanine in the nucleus and agedependent decrease in the activity of OGG1 imported into the nuclei and into mitochondria [82, 83]. Also AP endonuclease showed age-associated decreased activity, i.e. less intensive BER corresponds with the theory of aging associated with oxidative stress [83, 84]. Another cause of aging is related to mutation of human WRN gene. The role of normal Werner protein (or RECQ3 helicase) is to interact with many proteins involved in BER, and in this way to facilitate BER by unwinding BER intermediates. Mutated WRN gene give rise to a rare autosomal recessive genetic disorder Werner syndrome with premature aging, predisposition to cancer and early onset of aging symptoms such as osteoporosis, ocular cataracts, greying, loss of hair, diabetes mellitus, atherosclerosis [85, 86, 87]. Facts related to aging and polymorphisms of antioxidant enzymes have not yet been fully established. Reports on mutations of SOD causing amyotrophic lateral sclerosis are known [5]. The mitochondrial theory of aging states that senescent cellular changes are related to the balance between inherited healthy mitochondrial DNA and repaired mtDNA and the load of age-related unrepaired mutations in this DNA [84]. Eventually, damage or loss of mitochondrial DNA prevents postmitotic cells from regenerating new mitochondria, thereby reducing the production of ATP and leading to cell death. Oxidative damage of mtDNA, accumulating with age, should affect mitochondrially encoded proteins, but the high number of mitochondrial genomes allows a certain degree of heteroplasmy (different genomes within a mitochondrion) without effects on phenotype. Clonal accumulations of damaged/mutated mtDNA within individual cells up to homoplasmy of mutant mtDNA, which could be either neutral with regard to phenotype or which could cause substantial phenotype alterations. The degree of age-induced mutations in mitochondrial DNA varies in people and may be under genetic control. Some mitochondrial DNA haplotypes are more common in centenarians than in the general population [88, 89, 90]. Mitochondrial oxidative stress should be considered a hallmark of cellular aging. Mitochondrial ROS production mediates signal transduction pathways sensitive to oxidative stress and this would be involved in the onset and development of age-related degenerative diseases and that limit the mean life span. The second pathway is related to oxidative damage to mitochondrial DNA, proteins, and lipids and would determine the maximum life span [91]. Other pathologies related with oxidative stress and DNA damage are susceptibility to carcinogenesis (lack of apoptosis control), and other non-cancerous pathological conditions, such as degenerative diseases (Alzheimer’s disease, Parkinson’s disease), metabolic diseases (haemochromatosis, Wilson disease, diabetes mellitus), inflammatory diseases of infectious and non-infectious origin (atherosclerosis, autoimmune, and allergic diseases), ischaemiareperfusion injury. The intensity and quality of DNA repair are studied particularly in tissue sections, or tissue cultures, human leucocytes, or on animal models in some pathological conditions, such as malignant tumours [2, 64, 92, 93, 94], neurodegenerative diseases [68, 95], atherosclerosis [88, 96, 97], inflammatory bowel diseases [98], diabetes [99, 100], and in workers exposed to genotoxic effects of different chemicals [101]. The foregoing condensed summary dealing with oxidative stress and oxidative DNA damage and particularly BER serves for further elucidation of specific changes in two models of free radical diseases – diabetes mellitus and inflammatory bowel diseases.

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4. OXIDATIVE STRESS AND PATHOPHYSIOLOGY OF DIABETES MELLITUS AND INFLAMMATORY BOWEL DISEASES Among human diseases mentioned above, diabetes mellitus (DM) and inflammatory bowel diseases (IBD) represent 2 chronic lifelong diseases that could be present as 2 model free radical diseases. ROS in DM are produced via different pathways, among them the most important is the ROS overproduction by the mitochondrial ETC and glucose autoxidation, i.e. oxidative stress of predominantly metabolic origin with consequent inflammatory processes. In IBD, mainly Crohn’s disease, excessive stimulation of otherwise tightly regulated NADPH oxidases during inflammatory process contributes to the ROS formation and the presence of oxidative stress of predominantly inflammatory origin.

4.1. Diabetes Mellitus is defined as a group of chronic, metabolic heterogeneous disease marked by high levels of sugar in the blood. It results from insufficient action of insulin in consequence of its absolute or relative deficiency or both. Diabetes as a life-long disease is accompanied with complex disturbances of sugar, lipid and protein metabolism. The clinical features of diabetes are well-known, such as polyuria and polydipsia, nycturia, loss of weight, fatigue, loss of consciousness even coma, acetone in breath, recurrent infection of skin and urogenital system, later occurs the development of microvascular complications (retinopathy even blindness, nephropathy even renal failure, peripheral and autonomic neuropathy) and atherosclerosis with accompanied hypertension, heart attack, brain stroke, claudication [102](Fig. 11).

Stages Types

Normoglycemia

Hyperglycemia

Normal glucose Impaired Glucose Tolerance or Impaired regulation Fasting Glucose

Diabetes Mellitus Not insulin requiring

Insulin requiring for control

Insulin requiring for survival

Type 1* Type 2 Other Specific Types **

Gestational Diabetes **

Figure 11. The Expert Committee on the Diagnosis and Classification of Diabetes Mellitus ADA (Diabetes Care 26: S5-20, 2003).

Type 1 diabetes (T1DM) develops as a result of autoimmune insulitis in genetically predisposed individuals, typically is diagnosed in childhood, adolescence, or early adulthood.

Impact of Oxidative Stress on Diabetes Mellitus and Inflammatory Bowel Diseases 201 Autoimmune destruction of the ß-cells of the pancreas is reflected with markers of the immune destruction of the ß-cell include islet cell autoantibodies (ICAs), autoantibodies to insulin (IAAs), autoantibodies to glutamic acid decarboxylase (GAD65), and autoantibodies to the tyrosine phosphatases IA-2 and IA-2ß. Also, the disease has strong HLA associations, with linkage to the DQA and B genes, and it is influenced by the DRB genes, these genes being either predisposing or protective. These patients are also prone to other autoimmune disorders such as Graves’ disease, Hashimoto’s thyroiditis, Addison’s disease, vitiligo, and pernicious anaemia. Incidence of the disease is variable with regions, and in Europe an increasing tendency from the South to the North is reported with the highest number of 50/100000 children up to 15 years/year in Finland. The age-related incidence has its peak at the age of 13-15 years. T1DM dominates in childhood and adolescence. The disease onset in adulthood is no more rare (LADA - latent autoimmune diabetes of adults) and has been intensively studied. As T1DM is insulin dependent, different insulin intensified therapies are applied. Besides insulin, also diet, and regular physical activity are necessary for T1DM successful treatment. Type 2 diabetes (T2DM) is characterized by peripheral insulin resistance with an insulin-secretory defect that varies in severity. For type 2 diabetes to develop, both defects must exist: all overweight individuals have insulin resistance, but only those with an inability to increase beta-cell production of insulin develop diabetes. 90% of patients who develop type 2 diabetes are obese. Patients with type 2 diabetes often do not need treatment with oral antidiabetic medication or insulin if they lose weight. There are probably many different causes of this form of diabetes, and it is likely that the proportion of patients in this category will decrease in the future as identification of specific pathogenic processes and genetic defects permits better differentiation among them and a more definitive subclassification. Autoimmune destruction of ß-cells does not occur, and patients do not have any of the other specific causes of diabetes. Both genetic and environmental factors (above mentioned obesity) contribute to insulin resistance. Adipose tissue plays a central role in the pathogenesis of insulin resistance that induces a compensatory increase in β cell mass, which results in normal glucose levels. In other people, β cell response is not adequate and leads to impaired glucose tolerance or even type 2 diabetes. The presence of chronic hyperglycaemia i.e. glucotoxicity induces multiple defects in β cells, including early decreases in glucosestimulated insulin secretion, and late irreversible changes in insulin-gene transcription and β cell mass with consequent low insulin production. The world epidemic of obesity and thus the related increased incidence of T2DM threatens developed countries and represents a serious problem to public health. Worldwide prevalence of T2DM is estimated as 150 million cases in 2002 (Report of a WHO Meeting). Other specific types of diabetes includes genetic defects of the ß-cell (monogenetic defects in ß-cell function - MODY), point mutations in mitochondrial DNA, genetic defects in insulin action (mutations of the insulin receptor), diseases of the exocrine pancreas (acquired processes include pancreatitis, trauma, infection, pancreatectomy, cystic fibrosis and haemochromatosis), endocrinopathies (e.g. acromegaly, Cushing’s syndrome, glucagonoma, pheochromocytoma), drug- or chemical-induced diabetes, infections (rubella, coxsackievirus B, cytomegalovirus, adenovirus, and mumps), uncommon forms of immune-mediated diabetes (systemic lupus erythematosus and other autoimmune diseases), other genetic syndromes sometimes associated with diabetes (Down’s syndrome, Klinefelter’s syndrome, and Turner’s syndrome. Wolfram’s syndrome).

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Gestational diabetes mellitus (GDM) is defined as any degree of glucose intolerance with onset or first recognition during pregnancy. The increasing number of diabetic patients all over the world is an important problem of public health. Quality of life of diabetic patients, chronic diabetic complications, treatment of all types of diabetes and its complications at vast expenses represent important challenges not only for medical science, but also for human society.

In endothelial cells, glucose can pass freely through the cell membrane in an insulin-independent manner via GLUT1. Intracellular hyperglycaemia induces overproduction of superoxide at the mitochondrial level. Overproduction of superoxide is the first and key event in the activation of all other pathways involved in the pathogenesis of diabetic complications, such as polyol pathway flux, increased advanced glycosylation end product (AGE) formation, activation of protein kinase C (PKC) and NF- B, and increased hexosamine pathway flux. Figure 12. The role of increased flux of glucose into the cell and mainly to the mitochondrion (published by Ceriello, A. New insights on Oxidative Stress and Diabetic Complications May Lead to a “Causal” Antioxidant Therapy. Diabetes Care, 2003, 26, 1589-1596, with the kind permission of the author).

4.1.1. ROS and Diabetes Hyperglycaemia is common for all types of diabetes. This metabolic finding is the key for all important consequences related to oxidative stress. High constant influx of glucose under hyperglycaemia via GLUT 1 transporters leads to ROS overproduction at the mitochondrial level (Fig. 12). In diabetic cells with high glucose inside, there is more glucose being oxidized in the citrate cycle (TCA cycle), which in effect pushes more electron donors (NADH and FADH2) into the electron transport chain. As a result of this, the voltage gradient across the mitochondrial membrane increases until a critical threshold is reached. At this point, electron transfer inside complex III is blocked, causing the electrons to back up to coenzyme Q, which donates the electrons one at a time to molecular oxygen, thereby generating superoxide (Fig. 13). Overproduction of superoxide is the first and key event in the activation of all other pathways of hyperglycemic damage involved in the

Impact of Oxidative Stress on Diabetes Mellitus and Inflammatory Bowel Diseases 203 pathogenesis of diabetic complications [103, 104, 105, 106, 107], such as formation of advanced glycation end-products AGEs, stimulation of protein kinase C and transcription factor NFκB, polyol pathway and hexosamine pathway (Fig. 12, 14, 15, 16, 17). The cited work of Brownlee presents an integrated approach to the oxidative stress in diabetes.

Figure 13. Hyperglycaemia-induced production of superoxide by the mitochondrial electron transport chain (published by Brownlee, M. The Pathobiology of Diabetic Complications. Diabetes, 2005, 54, 1615-1625, with the kind permission of the author)

GAPDH - glyceraldehyde-3-phosphate dehydrogenase, GFAT – glutamine:fructose-6-phospate amidotransferase, GLN-glucosamine, glu-glutamine, UDP-uridine diphosphate, GlcNAc- N-

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Figure 14. Mitochondrial overproduction of superoxide activates four major pathways of hyperglycaemic damage by inhibiting GAPDH (published by Brownlee, M. The Pathobiology of Diabetic Complications. Diabetes, 2005, 54, 1615-1625, with the kind permission of the author)

ADPR – ADP-ribose, NA- nicotinic acid, GAPDH- glyceraldehyde-3-phosphate dehydrogenase, PARP- poly(ADP-ribose)polymerase Figure 15. ROS-induced DNA damage activates PARP and modifies GAPDH (published by Brownlee, M. The Pathobiology of Diabetic Complications. Diabetes, 2005, 54, 1615-1625, with the kind permission of the author)

AGEs- advanced glycation endproducts, PKC – protein kinase C, NFκB- nuclear factor κB proinflammatory transcription factor, PW - pathway Figure 16. The unifying mechanism of hyperglycaemia-induced cellular damage (published by Brownlee, M. The Pathobiology of Diabetic Complications. Diabetes, 2005, 54, 1615-1625, with the kind permission of the author)

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Figure 17. General features of hyperglycaemia-induced tissue damage. (published by Brownlee, M. The Pathobiology of Diabetic Complications. Diabetes, 2005, 54, 1615-1625, with the kind permission of the author)

Many previous studies analysed the influence of glucose on protein glycation and formation of advanced glycation end-products [108, 109, 110, 111, 112, 113, 114]. As shown for example by Kalousova and Skrha [115], accumulated glycation products (AGEs, but also advanced oxidation protein products – AOPP and advanced lipoperoxidation end products ALEs) exert not only direct effect on the extracellular matrix, but they are also bound to RAGE specific receptors. Beside other AGE-binding receptors, RAGE receptors are the best known and characterized as a 35kDa protein classified as a member of the immunoglobulin superfamily. RAGE can be expressed on the surface of various cell (e.g. monocytes, macrophages, mesangial cells, neurons, endothelial cells, smooth muscle cells and fibroblasts). It is a multiligand receptor and recognizes also β-amyloid, amphoterins and calgranulins involved in inflammatory processes. RAGE has also physiological function for the development of the central nervous system but during maturation, its presence decreases and is minimally expressed in adults. Under pathological conditions, such as diabetes melllitus, immune-inflammatory processes, Alzheimer´s disease, and cancer, RAGE expression is increased and binds AGE. AGE-RAGE interaction results in the activation of intracellular signal transduction pathways, such as MAPK and JNK kinases, activator protein 1 (AP-1) and nuclear factor NFκB. Subsequently, this activation is followed by overexpression of genes for cytokines (TNF-α, IL-1, IL-2, interferon γ, IL-8), growth factors (platelet-derived growth factor PDGF, insulin-like growth factor IGF-1), adhesion molecules (ICAM-1, VCAM-1). Other processes are also stimulated, among them stimulation of cell proliferation, increased vascular, migration of macrophages, formation of endothelin-1, increased synthesis of collagen IV, fibronectin and proteoglycans, increased procoagulant state [116, 117]. Little is known about the formation and accumulation of AGEs in young

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patients with T1DM. Tsukuhara [118] described increased urinary concentrations of pentosidine as AGE, and 8-hydroxy-2´-deoxyguanosine and acrolein-lysine as markers of OS in children with type 1 diabetes when compared with age-matched healthy control subjects. The mean age of 38 diabetic children was 12.8±4.5 years and their disease duration 5.7±4.3 years and HbA1c 8.0±1.6%, 11 subjects had microalbuminuria >15 mg/g creatinine. The study indicated that the accumulation of AGEs is closely linked to oxidative stress and the subsequent endothelial dysfunction may start early in the course of type 1 diabetes. This means that the risk of vascular complications may be present at an early age and that the best possible glycemic control should be emphasized from the diagnosis of diabetes. Regarding these results, it is important to take into consideration also high and low haemoglobin glycation phenotypes in type 1 diabetes as presented by Hempe [119]. Different glycation phenotype are also associated with susceptibility to the glycation of other proteins, increased AGE synthesis, and greater risk of diabetes complications. Though there are analogies in hyperglycemia, oxidative stress and stimulation of consequent metabolic and inflammatory processes in different types of diabetes, T1DM and T2DM as the most frequent types of diabetes differ in their character. T1DM as autoimmune disease develops towards other autoimmunities and microvascular complications. T2DM (besides microvascular complications) inclines to atherosclerotic (macrovascular) complications, and to carcinogenesis [120, 121]. With regard to a variety of AGEs, there is also a large scale of AGE-inhibitors and AGE-breakers. These substances, either natural or synthetic, could display different reactions. They react with free amino groups of protein to modify them in order to prevent sugar attachment to protein, or they inactivate aldose and ketose sugars before reacting with amino groups of proteins. They could also be metal chelators, and antioxidants themselves like vitamin C or E. Another group are inhibitors trapping reacative dicarbonyl intermediates to form substituted triazines. There are Amadori adducts inhibitors, compounds inducing for enzymatic deglycation at the Amadori level. Finally, inhibitors called cross-link breakers are able to selectively cleave and break the established AGEprotein cross-links in vitro and in vivo. AGE-inhibitors and breakers for clinical use should be non-toxic, well absorbed across the gastrointestinal tract and achieve a broad distribution throughout various tissues. Their therapeutic impact can include not only diabetes, but also other diseases with AGE-formation and accumulation [114, 122]. Also the antioxidant defence is the focus of an intensive research in diabetes. The polymorphisms in the Mn-SOD and EC-SOD (CuZn-SOD) genes play certainly the role in the intensity of detoxifying superoxide. Chistyakov [123] found the relationship between Ala(9)Val substitution in the Mn-SOD gene and diabetic neuropathy in a Russian population. The antioxidant gene enzyme expression and protein activity was studied on skin fibroblast cultures in type 1 diabetic patients with and without nephropathy, and nondiabetic control subjects. Skin fibroblasts of T1DM patients with diabetic nephropathy exposed to long-term hyperglycemia displayed unchanged expression and activity of Mn-SOD, catalase and GPx, whereas T1DM patients without complications, non-diabetic nephropathic patients, and control subjects had an intact stimulated antioxidant response to glucose-induced oxidative stress [124]. The present oxidative stress and low antioxidant defence was found not only in diabetic children, but similar tendency was observed also in their non-diabetic siblings, but not in their non-diabetic parents [125].

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4.1.2. ROS, DNA Damage and DM As mentioned at the beginning of this chapter, oxidative stress injures also DNA. It is impossible to enumerate all articles studying the intensity of oxidative stress and DNA damage in diabetic patients. Krapfenbauer [126] examined in 1998 type 1 and type 2 adult patients and compared them with age-matched non-diabetic controls. He confirmed the increased pentosidine and 8-hydroxy-2´-deoxyguanosine in urine in both diabetic groups and made conclusion that increased glycoxidation was approved but 8-OHdG could be taken either as consequence of increased DNA oxidation or reflection of DNA repair. Collins [127] used comet assay with endonuclease III to detect oxidized pyrimidines, and formamidopyrimidine glycosylase to detect damaged purines including 8-oxo-guanine. The increased formamidopyrimidine glycosylase-sensitive (FPG) sites were found in diabetic patients and the strong correlation was displayed between those sites and serum glucose concentrations. Another studies [128] summarized the importance of detection of 8-hydroxy2´-deoxyguanosine (8-OHdG) in urine in different diseases. The methods for 8-OHdG detection and normal reference range for women and men were also discussed. Special attention was paid to 8-OHdG levels in patients with cancer , chronic inflammation, atherogenesis, and diabetes. An outline was made with regard to the levels of urinary 8OHdG as oxidative stress marker and the presence of accelerated vascular dysfunction, and development of microvascular complications. The level of 8-OHdG could be also used as a marker of the effectiveness of dietary supplements with regard to whether they will reduce the oxidative damage. The review published by Samcova [129] described free radical different diseases and levels of 8OhdG. DNA damage and antioxidant defence were examined in peripheral leukocytes of T1DM patients by Dincer [130]. Comet assay was used in this study that confirmed increased DNA strand breakage and FPG-sensitive sites in diabetic patients. SOD and GPx activities in leukocytes were decreased. The presented findings showed increased oxidative DNA damage and impaired antioxidant defence in leukocytes of adult patients with T1DM. Similar findings of increased DNA damage in diabetic patients were confirmed in another study using comet assay [131]. 4.1.3. ROS, DNA Repair and DM The information about DNA repair process in relation to diabetes and oxidative DNA damage is scarce in humans. Attention is mostly paid to PARP and its inhibitors than to DNA repair process itself. PARP activates three major pathways of hyperglycemic damage in endothelial cells via inhibition of GAPDH activity (Fig. 16)[132]. Activity of PARP was found to be increased in whole retina and in endothelial cell and pericytes of diabetic rats. Long-term administration of PARP inhibitor (PJ-34) led to significantly inhibited death of retinal microvascular cell and prohibited from the development of early lesions of diabetic retinopathy in those animal models. In another experiment, PARP interaction directly with both subunits of NFκB (p50 and p65), mainly under hyperglycemic condition. The usage of PJ-34 blocked the PARP mediated activation of NFκB [133]. Japanese study published in 2004 [134] showed in a large group of T1DM patients that human MTH1 gene Val83Met was associated with type 1 diabetes, particularly in females. The product of MTH1 gene human mutT homologue 1 hydrolyzes 8-hydroxydeoxyguanosine triphosphate (8-OHdGTP) to 8-hydroxy-deoxyguanosine monophosphate in order to prevent transversion mutation. MTH1 is localized not only in nucleus but also in the

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mitochondrial matrix assuring here the integrity of mitochondrial DNA. Val83Met polymorphism of MTH1 gene leads to labile type of MTH1 and its decreased activity, both in nuclear and also in mitochondrial DNA. 8-OHdGTP can pair in DNA with cytosine or adenine and induce transversion mutations in mitochondrial DNA (mtDNA) as well as in nuclear DNA (nDNA). Such accumulated mutations would cause mitochondrial dysfunction, consequently more ROS production, leading to DNA strand breaks and the resultant recruitment of poly(ADP-ribose) polymerase for the repair. Free-radical mediated ß-cell damage has been intensively studied in type 1 diabetes, but not in human type 2 diabetes. Oxidative DNA damage and intensity of DNA repair in pancreatic islets was assessed on pancreatic specimens (post mortem or post surgery) from patients with diabetes for 2-23 years and healthy controls. 8-OH-hydroxyguanine glycosylase (OGG1) DNA repair enzyme expression was examined using immunofluorescent staining of OGG1. The intensity and islet area stained for OGG1 displayed a significant increase in islets from T2DM subjects compared to the healthy controls. A correlation between increased OGG1 fluorescent staining intensity and duration of diabetes was also found. Most of the observed staining was cytoplasmic, suggesting that mitochondrial OGG1 accounts primarily for the increased OGG1 expression. The authors [135] concluded that oxidative stress related DNA damage may be a novel important factor in the pathogenesis of human type 2 diabetes and stimulates OGG1 islet expression. Blasiak [99] published in 2004 the results of study aimed at the intensity of DNA repair in patients with T2DM. Comet assay was used for the assessment of the level of endogenous basal DNA damage (DNA strand breaks and alkali labile sites), as well as endogenous oxidative and alkylative DNA damage (using DNA repair enzymes endonuclease III, formamidopyrimidine-DNA glycosylase and 3-methyladenineDNA glycosylase II) in peripheral lymphocytes. Finally, the measurement of the sensitivity to DNA-damaging agents (hydrogen peroxide and doxorubicin) and the efficacy of removing this induced DNA damage in peripheral blood lymphocytes was performed. The levels of basal endogenous and oxidative DNA damage in diabetic patients were higher than in control subjects. There was no difference between the level of alkylative DNA in the patients and the controls. Diabetic patients displayed higher susceptibility to hydrogen peroxide and doxorubicin and decreased efficacy of repairing DNA damage induced by these agents than healthy control. The conclusion was made that patients with T2DM had the elevated level of oxidative DNA damage and the decreased efficacy of DNA repair and this may be the link between diabetes and cancer. At the same time, Varvarovska [136] published the study comparing the degree of DNA damage and the intensity of DNA repair in T1DM adults and children. Also in this case, comet assay was used for the evaluation of DNA damage and repair. Diabetic adults displayed higher DNA strand breaks and lower DNA repair capacity (measured in peripheral blood lymphocytes) than diabetic children. When adult patients were divided into 2 groups according the presence or absence of diabetic microvascular complications (DMC), the differences were more expressed, i.e. adults with DMC had higher DNA strand breaks and lower DNA repair capacity. The ratio between those two parameters was expressed as DNRI index that displayed large variations in adults without DMC and mainly in children.

4.1.4. Some Contributions to Antioxidant Therapy in Diabetes Mellitus Advance in research of oxidative stress and developing knowledge of its impact on human metabolism has brought new therapeutic possibilities into experiments, clinical trials

Impact of Oxidative Stress on Diabetes Mellitus and Inflammatory Bowel Diseases 209 or clinical practice. In a review published by Schultz Johansen [137] there is a instructive summary of current antioxidant interventions. Several experiments on animal models were mentioned, among them usage of α-lipoic as antioxidant or SOD mimetic experiments in diabetic animals. Recently performed clinical trials with different antioxidants were enumerated. The HOPE trial was the largest trial conducted thus far for the use of antioxidants in diabetes, unfortunately no beneficial effect of vitamin E was approved on cardiovascular outcome or diabetic nephropathy. The Steno-2 trial compared the effect of a multifactorial intensive therapy with that of conventional treatment on modifiable risk factors for cardiovascular disease in T2DM patients. In the intensive treatment group, patients received combined pharmacotherapy (vitamin C, E, folic acid, chromium piconlinate and drugs lowering hyperglycemia, dyslipidemia and hypertension) plus behavior modification including low-fat diet, exercise and smoking cessation. Patients in intensive groups had a 50% decrease in the risk of cardiovascular events than a control group. Evidence is that a multifactorial approach is superior to conventional therapy for the prevention of oxidative stress-induced vascular complications in diabetes. Application of α-lipoic acid in the treatment of diabetic neuropathy had promising results and significantly improved patient symptoms were reached. A number of commonly used drugs have shown promising anitoxidant activity in addition to their primary pharmacological activity, such as thiazolidinediones, 3-hydroxy-3-methyl-glutaryl-Coenzyme A reductase (HMG-CoA) inhibitors (statins), and inhibitors of the renin-angiotensin system (inhibitors of angiotensin II activity, such as angiotensin converting enzyme inhibitors ACEIs and angiotensin II receptor blockers ARBs). In vitro and animal studies confirmed antioxidant effect of those drugs. These antioxidant properties may be a contributing factor to the therapeutic efficacy of these agents. Based on the new developments in the understanding of the pathophysiology of oxidative stress, new agents emerge and seems to be beneficial for the treatment of OS associated clinical problems (SOD and catalase mimetics, L-propionyl carnitine, PKC-β inhibitor). Another review published by Ceriello [106] summarized the choice of different antioxidant drugs and emphasized the importance of thiazolidinediones, statins, ACE inhibitors and AT1 inhibitors (Fig. 18). Da Ros summarized similarly causal antioxidant therapy with new promising tools influencing mitochondrial function and reducing DNA damage, blocking PKC, PARP and peroxynitrite [138, 139]. Reusch [140] reviewed the recent knowledge about diabetic complications and mechanism of glucotoxicity. It is emphasized that conventional antioxidants scavenge free radicals in an inefficient stoichiometric manner so that one molecule of the antioxidant would be neede to neutralize each free radical generated. Novel small mollecular weight compounds functioning as superoxide dismutase mimetics have more reliable benefits due to the catalytic properties that could permit enzymatic detoxification. Suggestion of PARP inhibitors is another therapeutic possibility for diabetic complication prevention. Li [141] pointed to the risk of long-term inhibition of PARP, e.g. premature aging, loss of genome stability. In animal experiment, low-dose PARP inhibitor-containing combination therapies (PARP inhibitor+vasodilators) showed beneficial effect on early peripheral diabetic neuropathy. Another approach how to face diabetic microvascular complications could be the application of aldose reductase inhibitors that block sorbitol formation [142].

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Superoxide overproduction reduces eNOS activity, but through NF- B and protein kinase C (PKC) activates NAD(P)H and increases iNOS expression: the final effect is an increased NO generation. This condition favors the formation of the strong oxidant peroxynitrite, which in turn produces, in iNOS and eNOS, an uncoupled state, resulting in the production of superoxide rather than NO, and damages DNA. DNA damage is an obligatory stimulus for the activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP). Poly(ADP-ribose) polymerase activation in turn depletes the intracellular concentration of its substrate NAD+, slowing the rate of glycolysis, electron transport, and ATP formation, and produces an ADP-ribosylation of the GAPDH. This process results in acute endothelial dysfunction in diabetic blood vessels, which contributes to the development of diabetic complications. NF- B activation also induces a proinflammatory conditions and adhesion molecule overexpression. All these alterations produce the final picture of diabetic complications. SOD or catalase mimetics, L-propionyl-carnitine and lipoic acid, reducing mitochondrial overproduction of superoxide, and reducing DNA damage may be good candidates for causal intracellular antioxidants. This causal therapy would also be associated with LY 333531, PJ34, and FP15, which block protein kinase ß isoform, poly(ADP-ribose) polymerase, and peroxynitrite, respectively. Other current options may be thiazolinediones, statins, ACE inhibitors, and ATI inhibitors, which also have, at different levels, intracellular causal antioxidant activity. AGE, advanced glycosylation end product. Figure 18. Location of action of different types of antioxidants in the treatment of diabetes mellitus (published by Ceriello, A. New insights on Oxidative Stress and Diabetic Complications May Lead to a “Causal” Antioxidant Therapy. Diabetes Care, 2003, 26, 1589-1596, with the kind permission of the author)

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4.2. Crohn's Disease Crohn’s disease (CD) belongs to idiopathic inflammatory bowel diseases (IBD) that also include idiopathic proctocolitis (IPC) and indeterminate colitis (IC), impossible to be ranked precisely into CD or IPC. These diseases cause the intestinal tissue to become inflamed, form sores and bleed easily. Symptoms include abdominal pain, cramping, fatigue, fever, diarrhoea sometimes bloody, malabsorption, loss of weight, failure to thrive in children. CD is characterized by an autoimmune granulomatous chronic process affecting any part of the gastrointestinal tract, from the mouth to the anus (Fig. 19). Patches of inflammation occur, with healthy tissue between the diseased areas [143](Fig. 20). The inflammation can extend through every layer of affected bowel tissue [143]. The generalized inflammation can also affect extragastrointestinal organs and their involvements are presented as skin affections (erythema nodosum, erythema multiforme), bone osteoporosis even osteonecrosis, joint inflammation under the picture of reactive arthritis or ankylosing spondylitis, liver affection as autoimmune hepatitis eventually primary sclerosing cholangitis, eye inflammation (episcleritis or anterior uveitis). Crohn’s disease can not be cured by drugs or surgery, although either or both can help relieve symptoms [144]. Ulcerative colitis affects only the inner layer of the colon, or large bowel. It always starts in the rectum and may extend as a continuous inflammation from there into the rest of the colon. Usually ulcerative colitis can be controlled with medication. The disease can be completely eliminated by surgically removing the colon, but afterward, waste material may have to be stored and expelled through an external appliance [3, 144]. There is no known cause or cure for IBD. People are most frequently diagnosed between the ages of 15-25, or 45-55. IBD is particularly difficult for children and young adults since it often affects a person's self-concept, body image and lifestyle at a time when "being like everyone else" is so important. IBD is unpredictable. Most people experience periods of remission and flare-ups of the disease, often requiring long-term medication, hospitalisation or surgery [145]. Although IBD is found throughout the world, it seems to be more common in North America and northern Europe. CD incidence keeps growing, and is high particularly in developed countries. In Europe, there is an incidence 9-10/100000 people per year, CD prevalence is about 20-80 persons per 100000 people. Both genders are affected equally. Etiopathogenesis of CD is complex and includes genetic, immunological, infectious and other environmental factors. The formation of generalized granulomatous inflammation is accompanied by ROS formation [3, 146, 147, 148]. Initial inflammatory changes disrupt the protective intestinal barrier, then inflammatory cytokines are overproduced and inflammation progresses (cytokine phase). The next step includes tissue damage with increased inflammatory infiltration and simultaneous overproduction of ROS (Fig. 21). The further phase of healing is characterized by connective tissue formation in the intestinal wall and results in consequent stenoses and/or fistulae creation. CD treatment is complex and encompasses anti-inflammatory (mesalazine), immunosuppressive (corticosteroids and azathioprine), and biological therapy with the aim to achieve the remission and to prevent disease complications. An appropriate nutritional support is indispensable, first total parenteral nutrition until the moment when the stabilisation of intestinal inflammation is achieved and the patient is able to stand the enteral

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nutrition without diarrhoea recurrence. The term of biological therapy is in CD reserved for the monoclonal anti-TNFα antibody that has success in refractory CD treatment. If conservative treatment in CD fails, the patient is indicated for surgical therapy with the management of Crohn's specific complications like stenosis, fistula, and abscess by bowel preservation. Limited resection and/or strictureplasty do not influence morbidity and rate and also time of recurrence [145].

Figure 19. Distribution of inflammatory gastrointestinal affection in Crohn’s disease

Impact of Oxidative Stress on Diabetes Mellitus and Inflammatory Bowel Diseases 213

Figure 20. Inflammatory changes of gastrointestinal mucosa in Crohn’s disease

Figure 21. Immunological changes in the intestinal mucosa in CD (published by Shanahan, F. Crohn´s disease. The Lancet, 2002, 359, 62-69.

4.2.1. ROS and IBD As published long ago in 1991 by Yamada [149], the chronically inflamed intestine and/or colon is subjected to considerable oxidative stress whose source are the oxidants produced in phagocytic leukocytes (activated monocytes and polymorphonuclear leukocytes). These cells infiltrate in large numbers the intestinal mucosa and produce significant amounts of ROS in response to different inflammatory stimuli. Because the colonic mucosa contains

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relatively small amounts of antioxidant enzymes (SOD, catalase, GPx), the gut mucosa is overwhelmed during the time of active inflammation that results in intestinal injury [3, 149]. The complex review of oxidative stress as a pathogenic factor in inflammatory bowel disease brought Kruidenier in 2002 [146]. Here, the attention is paid to the origin of ROS and its impact on all cellular and extracellular components (Fig.22). Evidence of increased reactive oxygen metabolite levels is mentioned as it was found in biopsy specimens of IPC or CD correlated with clinical and endoscopic indices of disease activity. The consequences of oxidative stress on the gut are analysed, such as increased lipid peroxidation, presence of the protein carbonyl content as well as peroxynitrite-modified proteins in colonic biopsies, also the presence of DNA oxidation expressed as increased presence of 8hydroxy-deoxyguanosine in the inflammatory changed mucosa, increased apoptosis in the epithelium. In addition, the importance of different antioxidant defences is emphasized. Non-enzymatic antioxidants represented by glutathione and metallothionein are discussed in detail, similarly much attention is devoted to antioxidant enzymes (SOD, GPx, catalase), their activities in IBD mucosa and their expression regulation. It has been confirmed that mainly Mn-SOD is highly inducible under inflammatory conditions as IBD are. Much less information is available concerning the expression regulation of catalase and GPx.. The last part of this review describes some possibilities of antioxidant therapy in IBD and its limitations [150). The other works by Kruidenier et al. [151] contributed to the better knowledge of an imbalance between oxidant and antioxidant mechanisms at the tissue level in patients with IBD. These studies focused on thorough systemic examination of intestinal resection specimens of patients with CD or IPC and compared with normal control mucosa. They confirmed different gut mucosal SOD isoform expression, without major differences between CD and IPC. A marked stepwise increase in Mn-SOD (mitochondrial) levels was observed in non-inflamed and inflamed IBD mucosae, but a proportion of this enzyme was found to be enzymatically inactive. The Cu/ZnSOD (cytosolic) content decreased in epithelium with inflammation. Extracellular EC-SOD was found in low amounts in normal control mucosa, moreover with tendency in IBD patients to be decreased with inflammation. In lamina propria, SOD expression was much lower and not effected by inflammation. Mn-SOD and Cu/Zn-SOD were found mainly in neutrophils and macrophages, and EC-SOD was localized in small vessels, stromal cells, and neutrophils. These findings were extended with further study for the assessment of catalase, GPx, GSH, myeloperoxidase, and metallothionein (OH· hydroxyl radical scavenger) in the intestinal resection specimens of patients with CD or IPC versus normal mucosa. Both in CD and IPC patients, intestinal inflammation was associated with increased activities of catalase, GPx, and MPO, whereas the mucosal GSH content was unaffected. Despite this overall increase in mucosal H2O2 metabolising enzyme capacity, there were found the important differences between epithelium and lamina propria. The risk of direct hydrogen peroxide damage in the lamina propria seemed to be attenuated by the increasing numbers of catalase and MPO positive monocytes/macrophages and neutrophils that infiltrated the inflamed areas. But MPO overexpression might increase the lamina propria levels of hypochlorous acid, a stable ROS with multiple pro-inflammatory effects. Epithelial cells expressing catalase and GPx were found unchanged during inflammation, and moreover hydroxyl radical scavenger metallothionein displayed decreased expression, thus epithelial antioxidant defence is low. Another study performed by Kruidenier et al. confirmed increased levels of peroxynitrite in the inflamed mucosa of IBD patients, and also elevated malondialdehyde concentration as a marker of lipoperoxidation [147]. All these results confirm an imbalanced and inefficient

Impact of Oxidative Stress on Diabetes Mellitus and Inflammatory Bowel Diseases 215 endogenous antioxidant response to ROS produced in the inflamed intestinal mucosa of IBD patients and pointed out the importance of oxidative stress in the pathogenesis of IBD [146, 147, 150, 151]. In another study [152], patients with CD had increased markers of lipid peroxidation as measured by plasma F2 isoprostane, breath pentane and ethane output and significantly lower plasma antioxidant vitamins (ascorbic acid, α- and β-carotene, lycopene, and β-cryptoxanthin) when compared with control subjects. High level of GPx were found in a Turkish group of IBD patients indicating in this way stimulated response against oxidative stress. MDA levels in this study were normal in IBD individuals and this finding suggests the clearance of free radicals without leading to important lipid peroxidation [148]. A large group of IBD patients (94 with IPC, 97 with CD) was compared with 72 healthy controls with regard to blood total antioxidant capacity TAC and corrected TAC cTAC (TAC after subtraction of the interactions due to endogenous uric acid, bilirubin, and albumin). In both groups of IBD, TAC and cTAC were significantly reduced when compared with controls irrespective of disease activity [153]. The study on animal mice model with dextran sulphate sodium (DSS)-induced colitis used the preventive (before colitis induction) or treatment (after colitis induction) with GSH or CoQ. Colitis symptoms were reduced in both groups of animals, hence GSH or CoQ had a beneficial effect on acute signs of IBD. Controversy brought into the problem the histological evaluation that revealed increases in colonic dysplasia and ulceration after GSH or CoQ supplementation [154]. The more favourable result had another animal study with DSS induced colitis. Mice treated with three different antioxidants (S-adenosylmethionine, a GSH precursor; green tea polyphenols; a cysteine prodrug - 2(R,S)-n-propylthiazolidine-4(R )-carboxylic acid – PTCA). All the antioxidants significantly improved diarrhoea and colon lesions, in blood reduced GSH levels returned to normal when treated with antioxidants. Serum amyloid as acute phase protein and tumour necrosis factor-alpha were significantly increased in animals with florid colitis, but they improved with antioxidant treatment. These results may support a beneficial role of antioxidant therapy in IBD individuals [155]. Another recent study used the treatment with Nacetylcysteine (NAC) against toxic damage in the rat colitis induced with acetic acid. NAC was applied locally (into colon) and systemically (intraperitoneally). Treatment with higher levels of NAC (100mg/kg for 7 days) significantly decreased the degree of colonic injury and NAC is judged as having a role in the treatment of IBD [156]. Italian authors published an article on IBD patients and their plasma vitamin A, E, and carotenoid concentrations. They found significantly reduced antioxidant levels in IBD patients (both IPC and CD), particularly in those with active disease, when compared with healthy controls. 8-hydroxy-deoxyguanosine (8-OHdG) were also assessed in the patients´leucocytes. A significantly increased levels of 8-OHdG were found without regarding the disease activity and antioxidant levels [157].

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A. - neutrophil NADPHoxidase, B-xanthine oxidase, C-mitochondrial NADPH cytochrome p450 reductase 1-formation of hydrogen peroxide from superoxide, 2-formation of OH· and OH- from hydrogen peroxide (Fenton reaction) 3-formation of oxygen and water from hydrogen peroxide catalysed by catalase or GPx 4-formation of water nad NADP+ from hydrogen peroxide, H+ and NADPH 5-formation of hypochlorous acid from hydrogen peroxide and chlorine (catalysing enzyme myeloperoxidase) 6-formation of O2, OH· and OH- from hydrogen peroxide and superoxide (iron-catalysed Haber-Weiss reaction) 7-formation of oxygen, chlorine and hydroxyl radical from hypochlorous acid and superoxide 8-formation of OH· and OH- and oxidized iron from reduced iron ions and hypochlorous acid 9-formation of nitrogen dioxide, hydroxyl radical and H+ 10-formation of peroxynitrite anion and H+ from nitric oxide and superoxide No known enzyme exists to facilitate the detoxification of hydroxyl radical OH· , OH·-induced tissue damage may be prevented by the binding of transition metal ions by albumin,k ceruloplasmin, ferritin, transferrin and metallothionein. Figure 22. Schematic representation of ROS in intestinal inflammation (published by Kruidenier, L; Verspaget, HW. Oxidative stress as a pathogenic factor in inflammatory bowel disease — radicals or ridiculous? Alimentary PharmacologyandTherapeutics, 2002, 16, 1997-2015, with the kind permission of the author)

4.2.2. ROS, DNA Damage, DNA Repair and IBD An association between IPC and an elevated risk for colorectal cancer is well established and this carcinogenesis is probably driven by chronic inflammation. The available evidence suggests that DNA damage caused by oxidative stress in the characteristic damageregeneration cycle is a major contributor to colorectal cancer development in IPC patients

Impact of Oxidative Stress on Diabetes Mellitus and Inflammatory Bowel Diseases 217 [158]. Oxidative DNA damage (8-OhdG) in mucosa of ulcerative colitis has been examined in the D´Inca study [159]. Patients with IPC and dysplasia had significantly higher mucosal 8OhdG concentrations, in particular in older patients, in patients with long-standing disease, and active disease when compared with sporadic colon cancer and irritable bowel syndrome. Also the findings in patients with CD demonstrated increased levels of 8-OhdG, this time in peripheral leucocytes [D´Odorico]. An older study on a small number of patients with IPC [160] showed deficient repair of radiation-induced DNA damage compared with a group of healthy controls. The development of colorectal cancer is the most serious long-term complication faced by patients with longstanding extensive ulcerative colitis, with an incidence 20-fold higher than colorectal carcinoma (CRC) in the general population. Thus, more attention is paid to the intensity of DNA repair, expression of DNA repair genes (BER or MMR), and their association with IPC [161,162, 163]. The proper course of the cell cycle is controlled by protein p53 that is overexpressed in the G1 stage of the cell cycle when DNA damage is present. In order to allow DNA repair in stressed or injured cells, protein p53 blocks the continuation of the cell cycle and DNA damage can be repaired. When the expression of p53 was measured in inflamed and regenerated mucosa of patients with IBD, the increased number of cells expressing p53 was found in patients with IPC than with CD. This finding supports the idea of importantly changed DNA and the cell cycle arrest mediated through p53 [164]. Oxidative stress induces DNA strand breaks and their accumulation leads to the stimulation of poly(ADP-ribose)polymerase (PARP), that further initiates BER pathway, but also stimulates pro-inflammatory pathways as mentioned previously. The study on IL-10 deficient animal models showed, that the application of PARP inhibitor (3aminobenzamide) led to the normalization of colonic permeability and downregulation of proinflammatory cytokines, such as tumour necrosis factor-α and interferon [73]. The information about the intensity of DNA repair in CD patients is lacking. Oxidative stress undoubtedly contributes to the pathogenesis of both chronic lifelong diseases - diabetes mellitus and Crohn’s disease though the origin of oxidative stress differs. In DM, oxidative stress is mostly of metabolic origin, in CD, oxidative stress is in particular of inflammatory origin. The extensive research of both diseases has brought recently an enormous knowledge of molecular genetics and participating genes, immunological processes and their consequences, metabolic changes with special attention to oxidative stress. What remains unpredictable is the future course of either diabetes or Crohn’s disease in newly diagnosed patients.

5. OUR STUDY EXAMINING OXIDATIVE STRESS, DNA DAMAGE AND DNA REPAIR IN PATIENTS WITH DIABETES MELLITUS AND CROHN DISEASE Many previously published reports established the fact of increased oxidative stress in patients with diabetes mellitus and inflammatory bowel diseases (IBD), in particular Crohn’s disease (CD). Also our previously published article (JDC) reported increased parameters of oxidative stress in children with type 1 diabetes mellitus (T1DM), and similarly their nondiabetic siblings, when compared with age-matched healthy children. Though the finding of increased oxidative DNA damage in diabetic individuals as well as in subjects suffering

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from IBD has been well documented in several studies, the information about the intensity of DNA repair in these patients is completely lacking. The acknowledged existence of interindividual differences in DNA repair [165] and our acquaintance with a well established method comet assay (single cell gel electrophoresis used for the assessment of DNA strand breaks and in a modified version for the measurement of DNA repair capacity) [65] initiated our hypothesis (Fig. 23).

5.1. Hypothesis Summarizing the hypothesis: these patients with free radical diseases, such as diabetes or IBD, that would have low DNA repair capacity could be expected as those having an early development of complicated course of disease and vice versa, those ones having good or excellent DNA repair capacity should not have an early development of disease complications. In order to get the basic information about the intensity of DNA repair capacity in patients with diabetes mellitus and Crohn’s disease, adult and paediatric patients with type 1 diabetes mellitus, adults with type 2 diabetes, adults and children with Crohn’s disease were recruited and examined for the intensity of oxidative stress (parameters of OS) and for oxidative DNA damage (DNA strand breaks) and DNA repair capacity (DNArc).

Figure 23. Hypothesis of influence of DNA repair on the course of DM or IBD; (in DM diabetic microvascular complications and macrovascular complications, in IBD patients the presence of fistulae, stenosis, abscess)

Impact of Oxidative Stress on Diabetes Mellitus and Inflammatory Bowel Diseases 219

5.2. Study Material T1 diabetes mellitus patients: 53 adult patients, 28 women, 25 men, mean age 38.25±11.02 years, mean disease duration 16.49±3.22 years, mean HbA1c concentration 8.36±1.12 % (normal ranges 4.3-5.8% DCCT, performed 2002) were enrolled in the study. Among them 23 persons without any diabetic microvascular complication (DMC), 30 remaining persons having 1 or more diabetic complications - maximum 4 complications (15 subjects): diabetic retinopathy (18 subjects), diabetic nephropathy (22 subjects) diabetic neuropathy (11 subjects) and diabetic foot (3 patients). Diabetic retinopathy was diagnosed by a specialized ophthalmologist, diabetic nephropathy was defined as microalbuminuria higher than 20μg/min and/or proteinuria higher than 0.20g/24h, diabetic neuropathy was examined using a neurological disability score (NDS), electromyography (EMG), biothesiometer and monofilamenta and diabetic foot was evaluated according to International Consensus on the Diabetic Foot. 5 patients with DMC had simultaneous macrovascular complications (coronary heart disease, or peripheral vascular disease, or cerebral ischaemia). These 2 groups of patients (without complications and with complications) did not substantially differ in age, sex, and diabetes control, but they differ in disease duration (14.70±3.11 years x 17.63±2.57 years, p<0.01). All patients were on diet, and treated with intensified insulin therapy or on insulin pump, other medicaments were applied according to the present diabetic complications. Type 2 diabetes adults (T2DM): 14 patients with T2DM, 6 women, 8 men, mean age 58.0±5.6 years, mean disease duration 11±6.06 years, HbA1c 9.9±2.5% DCCT, treated with insulin, and/or PAD, + diet. 5 patients had macrovascular complications (cardiac artery disease or peripheral vascular disease), 5 patients had microvascular complications (retinopathy in 4 cases, once nephropathy, neuropathy in 3 patients), 3 patients had both types of complications. Crohn’s disease patients: 17 adult patients, 11 women, 6 men, mean age 37±11.36 years, mean disease duration 10.53±7.24 years, CDAI index 68.18±41.10 (normal ranges up to 150 as inactive disease, above 150 active disease) were examined. 11 patients underwent one or more operations for CD complications (stenoses, fistulae), all subjects were on intensive treatment including salicylates, corticosteroids and/or immunosuppressive drugs (Imuran). 1 patient was treated with biologicals (Remicade). T1DM children: 20 children, all individuals were without diabetic microvascular complications, 8 girls and 12 boys, mean age 13.3±2.9 years, mean disease duration 2.94±2.72, mean HbA1c 9.50±2.52% (DCCT) (study performed in 2002-2003) were enrolled. All children were on diet and treated with intensified insulin therapy or on insulin pump. 17 children with Crohn’s disease: 7 girls, 10 boys, mean age 14.29±SD 2.52 years, mean disease duration 3±1.43 years, PCDAI index (activity of disease) was 35.69±16.60 (normal up to 30), CDAI index (activity of disease compared with adults) was 112.94±82,7 (normal up to 150). All patients were treated with salicylates, and/or corticosteroids, at the time of blood collection, none, at the time of blood sample collection, was treated with biologicals. 3 patients underwent surgical treatment for stenosis, fistula, and abscess complications. Control group of healthy adults: healthy control subjects were previously recruited by the corresponding laboratories and their results served as normals for appropriate methods (for markers of oxidative stress 46 women and 42 men, total mean age 34.80±10.32 years, for

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DNA strand breaks and DNA repair capacity 20 women and 22 men, mean age 45.20±8.31 years). Control group consisted of 11 healthy children randomly chosen among children examined for accidental heart murmur (heart defect excluded using echocardiography) 5 girls, 6 boys, mean age 13.7±3.8 years. T1DM and CD children were comparable with regard to their age and sex with the control group of children.

5.3. Methods Methods used for the patients´ assessment: blood samples were examined for markers of oxidative stress, such as superoxide dismutase SOD in erythrocytes (U/g Hb), glutathione peroxidase GPx in blood (U/g Hb), antioxidant plasmatic capacity AOC (mmol/l), reduced glutathione GSH in erythrocytes (mmol/l ery), plasmatic malondialdehyde MDA (μmol/l).Kits from Randox (Crumlin, Great Britain), adapted to an automated Hitachi 717 analyser (Roche, Mannheim, Germany) were used for determination of superoxide dismutase in erythrocytes, glutathione peroxidase in whole blood, and finally for plasma antioxidant capacity. Reduced glutathione concentration in erythrocytes was estimated with kits from Oxis Int. (Bonnenie, Marue, France) while plasma malondialdehyde as a product of lipid peroxidation was determined photometrically as TBARS (thiobarbituric acid reactive substances) [125, 166]. DNA damage and DNA repair capacity in patient’s peripheral lymphocytes were examined with comet assay (single cell gel electrophoresis) and its modified version (Fig. 24, 25, 26). DNA strand breaks (DNAsb) measured by X-ray-calibrated comet assay (alkaline singlecell gel electrophoresis) [65], in peripheral blood lymphocytes isolated from heparinized blood samples were used as a parameter of oxidative DNA damage. Results are expressed as strand breaks per 109 Daltons of the DNA. Briefly, cells embedded in a thin layer of agarose on a microscope slide are lysed with detergent and high salt, and the resulting nucleoids electrophoresed at high pH. DNA is pulled out of the nucleoid to form a “comet tail”. Comets are visualized by fluorescence microscopy, after staining with ethidiumbroide. The relative amount of DNA in the tail compared with the head reflects the number of DNA breaks present. The individual DNA repair capacity (DNArc) restoring oxidative DNA damage was measured by modified comet assay using cell extracts from patients´ lymphocytes. In this case, cultured cells (HeLa) treated with a photosensitiser Ro 19-8022 Hoffman-La Roche and visible light are used as a target cells with a defined amount of oxidative damage. These cells embedded in agarose are treated for 45 min. with cell extracts prepared from lymphocytes isolated from tested patients. The number of DNA breaks (expressed as strand breaks per 109 Daltons) formed in the 8-oxoguanine-containing DNA of target cells by cell extracts expresses the capacity of the examined lymphocytes to initiate the repair of oxidised bases (8oxoguanines) [65, 167]. To express the relationship between the intensity of individual DNA repair capacity and the existing level of oxidative DNA damage, a DNA repair index (DNRI) where DNArc represents the number of strand breaks/109 Daltons induced by cell extract in DNA of target

Impact of Oxidative Stress on Diabetes Mellitus and Inflammatory Bowel Diseases 221 (HeLa) cells containing 8-oxoguanine and DNAsb is the number of strand breaks/109 Daltons measured in DNA of tested lymphocytes [136]. Statistical evaluation: data were expressed as means±standard deviation. Wilcoxon unpaired test, Spearman and Kruskal-Wallis test and rank correlation were used in statistical evaluation of the results. Statistical significance was implied by p-value <0.05. The Local Ethics Committee approved the study, and patients´ informed consent was obtained from all participants before entering the study.

Lymphocytes in 1% agarose microscope slide

Lysis

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nucleoid

0.3M NaOH 10mM EDTA

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25V 30 min

Neutralisation DAPl stain Fluorescence microscopy

% DNA released into tail of comet indicates frequency of DNA breaks

Figure 24. Comet assay for the evaluation of DNA strand breaks (by A.R. Collins).

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Cells + specific DNA damage

Cells

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Accumulation of breaks with time indicates repair activity

Figure 25. Modified comet assay for the evaluation of DNA repair (by A.R. Collins).

5.4. Results All the graphs are expressed in % of values. One group, usually healthy adults or healthy children, were indicated as a baseline 100% value and statistical average and standard deviation of all other groups were expressed as the percentage of the baseline value. Markers of oxidative stress includes superoxide dismutase SOD, glutathione peroxidase GPx, antioxidant plasmatic capacity AOC, reduced glutathione GSH, malondialdehyde MDA. Evaluation of DNA damage is indicated as DNAsb. DNA repair capacity as DNArc. Statistical significance *p<0.05 **p<0.01 ***p<0.001 If standard deviation surpasses the size of the graph, its value is mentioned below the graph. Axis X - examined parameters axis Y - % of values

Impact of Oxidative Stress on Diabetes Mellitus and Inflammatory Bowel Diseases 223

Figure 26. Comet assay - different degrees of DNA damage (1. nucleoid without damage, 3. nucleoid with comet and present DNA breaks 5. completely destroyed nucleoid (published by AR Collins, The comet assay for DNA damage and repair, Molecular Biotechnology, 2004, 26, 249-261, with kind permission of the author).

This pilot study brings some new information about the intensity of DNA damage and DNA repair capacity in seven different groups of individuals, among them five groups of patients with chronic lifelong diseases and still among them, two groups of pediatric children. As we would like to get some knowledge about the development of the assessed parameters not only with regard to the patients´ diagnosis, but also with regard to the patients´age (having in mind the theory of aging), we dared to compare the results of diseased children with diseased adults, and also to relate all the results to a baseline value of healthy adults. Concerning children, we accomplished the comparison between both groups of pediatric patients with healthy controls.

5.4.1. Study of Oxidative Stress, DNA Damage and DNA Repair Capacity of Lymphocytes in Healthy Adults and Healthy Children The level of SOD was significantly higher in healthy children than in adults (p<0.001), on the other hand GPx was lower (p<0.001), the remaining parameters of oxidative stress (AOC, GSH, MDA) did not differ substantially. DNA strand breaks were comparable in both groups (a large variance of results in children influences the statistical significance), but DNA repair capacity was significantly higher in children (p<0.001) together with DNRI (p<0.001). The activities of antioxidant enzymes and levels of other antioxidant substances do not change substantially with age. Our group of healthy children had higher SOD and lower GPx when compared with healthy adults. This result corresponds with the finding of SOD and catalase activation in physically active children [168]. Similar activation of catalase and glutathione reductase and reversely decrease glutathione peroxidase in healthy men after an

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extreme cycling performance was found by Aguilo [169]. It could be expected that our healthy children had certainly more physical activity than healthy adults. Oxidative damage of healthy children was lower than in adults, but their DNA repair capacity and DNRI were significantly higher than in healthy adults. This finding certainly correlates with age difference between children and adults, and thus age-related decreased activity of DNA repair enzymes, as published by several authors and implied in the theory aging [83, 84, 170, 171, 172]. Raji [173] confirmed an increased DNA repair in children. Chen [174] found ageassociated decrease of oxidative repair enzymes, namely human 8-oxoguanine DNA glycosylase. 200

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mean values of healthy adults=100%, DNArc SD in healthy children SD±36%, mean DNRI in healthy children 1044% of the value of healthy adults, DNRI SD in healthy children SD 3026% of the value of healthy adults Figure 27. Markers of oxidative stress, DNA damage and repair.

5.4.2. Comparison of T1DM Patients Versus Healthy Population T1DM patients showed lower GPx (both groups p<0.01) and lower antioxidant plasmatic capacity (both groups p<0.01). Their MDA was very high (both T1DM groups p<0.001). SOD was significantly high in T2DM adults (p<0.001), on the other hand GSH as the important intracellular antioxidant was significantly decreased (p<0.01) in the same group of patients. Their MDA was substantially low (p<0.01). Metabolic compensation (evaluated as the value of HbA1c) of T1DM adults was significantly better (HbA1c 8.36±1.12 % DCCT) than this one of T2DM adults (HbA1c 9.9±2.5% DCCT, p<0.05) and T1DM children (HbA1c 9.50±2.52% DCCT) (p<0.05), (it is not shown in the figure). The findings of lower GPx and AOC and high MDA both in adults and children with T1DM correspond with the results of other authors [130, 175, 176]. Results of patients with T2DM showed stimulation of antioxidant enzymes and low GSH. Low MDA could be explained by MDA incorporation into LDL that are usually elevated in T2DM [177]. The results of T1DM patients behaved differently. Significantly increased DNA repair capacity was found in both adult and pediatric groups of T1DM individuals when compared

Impact of Oxidative Stress on Diabetes Mellitus and Inflammatory Bowel Diseases 225 with adult healthy controls (p<0.001 both), and along with it, DNRI was also extremely elevated (both groups p<0.001). 180% 160% 140% 120% 100% 80% 60% 40% 20% 0% SOD

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mean values of healthy adults=100% Figure 28. Markers of oxidative stress. 600%

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mean values of healthy adults=100%, DNRI in healthy children mean 1144% SD 3026%, T1DM children DNRI mean 919% SD 1307% Figure 29. DNA damage and repair.

There is an apparent finding of significantly elevated DNA strand breaks in patients with T2 diabetes (p<0.05). On the other hand, their DNA repair capacity showed an extremely low level of repairing strand breaks (p<0.001) and similarly DNRI is extremely low (p<0.001). Several studies confirmed oxidative DNA damage in diabetic patients using different methods, mainly with increased loss of oxidized bases into urine or an increased number of DNA strand breaks [3, 126, 127, 129]. In our study, the differences in DNA damage between

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healthy controls and T1DM (adults, children) were not statistically significant. T2DM patients had significantly elevated DNA strand breaks. Similar results were described by Sardas [131] who found worse DNA damage in T2DM than in T1DM. Krapfenbauer [126] also confirmed increased DNA oxidation in both types of diabetes. On the other hand, our T2DM patients had a very low DNA repair. It could be explained either by the older age of this group, or the problem could be hypothetically also in the genetically or environmentally influenced bad DNA repair. Similar results were presented by Blasiak et al.[99] in a casecontrol study and he found also low DNA repair in T2DM subjects. This fact could represent a link to susceptibility of T2DM patients to carcinogenesis. Generally, low DNA repair can lead to extreme DNA damage, and thus initiate apoptosis, or inaccurate repair of damaged DNA leads to mutations and genomic instability and finally to carcinogenesis [2, 3, 53, 55, 56, 57].

5.4.3. Comparison of T1DM Adult Group Versus T1DM Children This graph matches results of diabetic children and adults with T1DM. Though a worse diabetes compensation in children measured as glycated hemoglobin HbA1c (significantly higher, p<0.05) and lower levels of AOC (p<0.05), diabetic children had favourable low levels of MDA (p<0.05), i.e. low lipid peroxidation. They showed also significantly lower DNA damage (p<0.05) and stimulated DNA repair (p<0.001), as well as DNRI (also p<0.001). 200

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mean values of T1DM adults=100%, DNArc diabetic children SD±49%, DNRI children mean 538%, SD 755% Figure 30. Markers of oxidative stress, DNA damage and repair.

The presented results cannot be compared with a similar study in T1DM children and adults. Nevertheless, it is obvious that young organisms coped better with the requirements of hyperglycemic state and oxidative load. The influence of a shorter disease

Impact of Oxidative Stress on Diabetes Mellitus and Inflammatory Bowel Diseases 227 duration (2.94±2.72 years in children versus 16.49±3.22 years in adults, p<0.001) could also influence the presented results. Genetically conditioned individual DNA repair of oxidative damage (via BER) might also affect the results. Unfortunately, the matched results of DNAsb, DNArc and DNRI measured in each patient after a 2-year interval are not yet available, hence it is impossible to declare an attitude towards this question.

5.4.4. Comparison of T1DM Adults Divided into 2 Subgroups Based on the Abscence or Presence of Diabetic Microvascular Complications with T1DM Children (Published by Varvarovska, Biomedicineandpharmacotherapy 2004) 200 180 160 140

**

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###

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*

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100 80 60 40 20 0 HbA1c

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(mean values of adult diabetic patients without microvascular complications=100%) , published by Varvarovska (..) T1DM adults without= T1DM patients without diabetic microvascular complications (DMC), T1DM with=adults with DMC, mean values of T1DM adults without complications=100% *p<0.05 **p<0.01 T1DM children versus adult T1DM patients without diabetic microvascular complications ### p<0.001 T1DM children versus adult T1DM with diabetic microvascular complications DNRI children mean 489%, SD693% Figure 31. Markers of oxidative stress, DNA damage and repair.

In order to support our hypothesis, the group of adults T1DM patients was divided into 2 subgroups. The first one was composed of 23 patients without diabetic microvascular complications, the second group consisted of 30 adults having 1-4 complications (nephropathy, retinopathy, neuropathy, diabetic foot). We compared those 2 subgroups with T1DM children. Though the worse diabetes control (HbA1c) and MDA in children (both values higher on p<0.05 versus T1DM adults without DMC), T1DM children displayed similar DNAsb like both adult subgroups. On the contrary, children´s DNArc was significantly higher when compared with T1DM adults without DMC (p<0.01) and the

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comparison with T1DM adults with diabetic complications showed the statistical significance on p<0.001. DNRI behaved similarly like DNArc, i.e. statistical significance children versus T1DM without DMC p<0.01, and versus T1DM with DMC p<0.001. Hence, it could be concluded that T1DM children had better antioxidant defence against oxidative stress including superior DNA repair process. Adult diabetic patients with microvascular complications showed the worst values of DNA assays and this might agree with our hypothesis, i.e. an individually bad DNA repair could contribute to the earlier development of diabetic microvascular complications. However, it is also necessary to acknowledge a fact, that diabetic patients with DMC had longer disease duration (both subgroups p<0.001 versus diabetic children, not shown in the figure), so DNA repair could be exhausted after a long exposition to oxidative stress originated from the long-lasting hyperglycemia.

5.4.5. Study of CD Patients (Adults and Children) Versus Healthy Adult Population The values of SOD, GPx and GSH in CD patients cannot be compared with the findings in GIT mucosa, as markers of oxidative stress were measured in blood or erythrocytes. Nevertheless, it is obvious that CD patients had lower GPx activity, particularly CD children (p<0.01) when compared with healthy adults. Also total plasmatic antioxidant capacity was significantly low both in CD adults and children (both p<0.01). In CD adults, GSH is significantly higher (p<0.01), also their MDA low levels corresponds with the more localized and inflammatory pattern of the disease (p<0.05). In children, these two markers are presented in a similar way, but there are not significantly changed. 140%

120%

100%

80%

60%

40%

20%

0% SOD

GPx

AOC healthy adults

healthy children

GSH CD adults

MDA

CD children

mean values of healthy adults=100% Figure 32. Markers of oxidative stress.

In correlation with the scarce literary data, our patients had significantly decreased antioxidant plasmatic activity and lower GPx (mainly children) [153]. Lower GPx gives evidence of the worse antioxidant defence of CD patients. The levels of GSH was higher in

Impact of Oxidative Stress on Diabetes Mellitus and Inflammatory Bowel Diseases 229 CD adults (versus healthy adults) what might be in relationship with a special diet applied in those group of patients. 600%

500%

400%

300%

200%

100%

0% DNAsb

DNArc healthy adults

healthy children

DNRI CD adults

CD children

mean values of healthy adults=100%, healthy children DNRI mean 1144%, SD 3026%, CD adults DNRI mean 668%, SD 1256%, CD children DNRI mean 862%, SD 1240% Figure 33. DNA damage and repair.

CD adults and children showed decreased levels of DNA damage (CD adults DNA sb p<0.05 versus healthy adults). On the contrary, the significantly increased DNA repair capacity was found in both groups of CD patients (adults p<0.05, children p<0.001). Similar finding was confirmed in DNRI (both groups p<0.001). The studied group of CD adults and children did not display increased DNA damage as cited in the literature (157). Their levels of DNA repair capacity was significantly high mainly in children, it corresponds with the quality of young organism and is stimulated in inflammatory disease. These results are contradictory to the findings in idiopathic proctocolitis where DNA repair is usually low (but there is a susceptibility to carcinogenesis)[160, 161, 162, 163]. Our results of DNA repair cannot be compared with similar study in the literature.

5.4.6. Comparison of Adult CD Patients with CD Children CDAI index was higher in children, but not significantly (large variance). So their disease was more active (we dared to use CDAI index because pediatric patients were at the older school age or adolescents). CD children had shorter disease duration (p<0.001, not shown here). Markers of oxidative stress did not differ significantly from those of CD adults. DNAsb and DNRI in children were elevated, but not significantly. The intensity of DNA repair was significantly higher in children than in CD adults (p<0.05), certainly reflecting their younger age.

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350

300

250

200

150

100

50

0 CDAI

SOD

GPx

AOC

GSH CD children

MDA

DNAsb

DNArc

DNRI

CD adults

mean values of CD adults=100% Figure 34. Markers of oxidative stress, DNA damage and repair.

5.4.7. Comparison of Adult Diabetic Patients and Patients with Crohn’s Disease 200 180 160 140 120 100 80 60 40 20 0 disease duration

SOD

GPx

AOC

GSH T1DM adults

MDA

DNAsb

DNArc

DNRI

CD adults

mean values of CD adults=100%, T1DM adults DNAsb SD 78%, CD adults DNRI SD 188% Figure 35. Markers of oxidative stress, DNA damage and repair.

Patients with T1DM were comparable with CD patients with regard to their age, but they had longer disease duration (p<0.001). Markers of oxidative stress in T1DM adults displayed lower GSH (p<0.05) than CD adults, and also of course very high MDA (p<0.001). DNAsb were significantly higher in T1DM adults (p<0.01), but their DNArc did not differ substantially. So DNRI in diabetic adults was significantly lower than in CD patients (p<0.05).

Impact of Oxidative Stress on Diabetes Mellitus and Inflammatory Bowel Diseases 231 The presented results displayed the more expressed consequencies of oxidative stress in T1DM patients than in CD adults. It is not excluded that besides the generalized influence of hyperglycemic state, the disease duration (longer in T1DM group) might influence the studied parameters.

5.4.8. Children with T1DM and CD Compared with Healthy Children The children with T1DM had the lowest levels of SOD, stimulated GPx (even if not statistically significant), importantly decreased GSH when compared with healthy children. DNA damage in diabetic children did not differ from the other groups, but DNA repair capacity was statistically increased (p<0.05). CD children did not differ from healthy children though similar tendencies in the examined parameters like in diabetic children were observed (SOD, GPX, AOC, GSH, DNArc and DNRI, but not MDA). 200% 180% 160% 140%

% values

120% 100% 80% 60% 40% 20% 0% SOD

GPx

AOC

GSH T1DM

MDA CD

DNAsb

DNArc

DNRI

healthy

mean values of healthy children=100%, DNRI healthy children SD 264% Figure 36. Markers of oxidative stress, DNA damage and repair/

Table 1. Comparison of T1DM and CD children versus healthy children CHILDREN age disease duration HbA1c PCDAI SOD GPx AOC GSH MDA DNAsb DNArc DNRI

years years % (DCCT - <5%) Index (<30) U/g Hb U/g Hb mmol/l plasma mmol/l ery μmol/l plasma breaks per 109daltons breaks per 109daltons index

T1DM 13.26±2.98 2.94±2.72 9.5±2.52 1227±131*** 53.68±13.44 1.36±0.11 2.02±0.34 * 2.49±0.93 0.43±0.32 1.86±0.58 * 13.88±19.74

CD 14.29±2.52 2.99±1.43 35.69±16.6 1312±148 * 49.05±14.40 1.36±0.14 2.21±0.38 1.94±0.54 0.43±0.33 1.63±0.52 13.02±18.72

HEALTHY 13.73±3.8

1447±115 47.52±9.21 1.41±0.09 2.27±0.24 1.93±0.55 0.42±0.27 1.41±0.28 17.28±45.69

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The results of children with T1DM demostrated the more intensive oxidative stress than in CD and healthy children. It is also obvious that DNA repair was apparently stimulated, and thus correcting the present DNA oxidative damage. Oxidative stress due generalized longlasting hyperglycemic state overwhelms T1DM children and obviously leads to the more intensive oxidative stress than a patchy granulomatous inflammation in CD children. The results cannot be compared with similar findings in the literature.

5.4.9. Comparison between T1DM and CD Children 300%

250%

% values

200%

150%

100%

50%

0% SOD

GPx

AOC

GSH

MDA T1DM

DNAsb

DNArc

DNRI

CD

Values of CD children=100%; mean values of CD children=100%; Comparison between T1DM children and CD children did not show any important differences. Both groups were comparable in their age, sex, and disease duration. Figure 37. Markers of oxidative stress, DNA damage and repair.

5.4.10. Comparison of all Groups of Patients Versus Healthy Adults The summarizing graph brings a complex view on differences among healthy adults and children, CD adults and children, as well as T1DM adults and children, and finally T2DM adults. Both CD patients had significantly low total antioxidant plasma capacity (p<0.01), but on the other hand elevated GSH in CD adults (p<0.01). This finding can be influenced by a special diet in the majority of adult CD patients. CD children had SOD elevated only moderately, but significantly lower GPx (p<0.01). Similar finding was displayed in diabetic adults and children. In addition, they had also significantly low AOC. MDA was elevated in all T1DM patients in agreement with the disease character. In adult T2DM patients, extremely high SOD was found, and reversely low GSH (p<0.01) and MDA (p<0.01) as mentioned above.

Impact of Oxidative Stress on Diabetes Mellitus and Inflammatory Bowel Diseases 233 180%

160%

140%

120%

100%

80%

60%

40%

20%

0% SOD

GPx healthy adults

AOC

healthy children

CD adults

GSH

CD children

T1DM adults

MDA

T1DM children

T2DM adults

mean values of healthy adults=100% Figure 38. Markers of oxidative stress. 800%

700%

600%

500%

400%

300%

200%

100%

0% DNAsb healthy adults

DNArc healthy children

CD adults

CD children

DNRI T1DM adults

T1DM children

T2DM adults

mean values of healthy adults=100%, DNRI healthy children mean 1144%, SD 3026%, DNRI CD adults mean 668%, SD 1256%, DNRI CD mean children 862%, SD 1240%, DNRI T1DM children mean 919%, SD 1307% Figure 39. DNA damage and repair.

It is evident that all groups of children differed from healthy adults in DNAsb, DNArc and DNRI. They had lower levels of DNAsb, i.e. less DNA damage. On the contrary, DNArc was significantly higher in healthy children (p<0.001), CD children (p<0.001), and mainly in T1DM children (p<0.001). Analogous assay tendency was found both in CD and T1DM adults versus healthy adult controls, i.e. lower DNAsb (CD adults p<0.05, similar but not significant finding in T1DM adults), DNArc increased in CD adults (p<0.05) and also in

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T1DM adults (p<0.001). DNRI (=Error!) reflected those presented results, i.e. they were elevated both in CD and T1DM adults (p<0.001). The completely different trend was observed in T2DM adults that showed high DNAsb (p<0.05), extremely low DNA repair capacity (p<0.001) and also DNRI (p<0.001). It could be concluded that both children and adults with Crohn´s disease or type 1 diabetes mellitus displayed stimulated DNA repair of oxidative DNA damage. On the other hand, adult patients with T2DM had very low DNA repair capacity and this might be linked with their susceptibility to carcinogenesis.

6. CONCLUSION Oxidative stress as predominance of reactive oxygen species in organism contributes substantially to many pathological processes in human body. An intensive research has brought a developing knowledge of oxidative stress and its impact on human metabolism. The short survey in the first theoretic part of this chapter summarizes current knowledge of oxidative stress, antioxidant defence, new antioxidant therapeutic possibilities. As oxidative stress injures DNA structure, oxidative DNA damage and DNA repair process has been reviewed with regard to different human diseases. In the second part of this chapter, the authors present their study of oxidative stress, oxidative DNA damage and DNA repair in patients with diabetes mellitus type 1, type 2 and with Crohn´s disease. Summarizing the results, adult patients with type 1 diabetes mellitus had the signs of important oxidative stress, but compensated with a stimulated DNA repair. The subgroup of T1DM with diabetic microvascular complications had lower DNA repair, maybe due to exhaustion of repair machinery in a long disease duration or due to a decreased individual quality of DNA repair. When T1DM adults were compared with T1DM children, the young patients showed better results of parameters of oxidative stress and very high DNA repair, certainly in association with short disease duration and young age, though their diabetes control was not good. The adult patients with type 2 diabetes had very high oxidative DNA damage and extremely low DNA repair, these signs are assumed to be linked to the possible future carcinogenesis. Patients with Crohn´s disease (both children and adults) did not display such an intensive oxidative stress as diabetic patients did. It could be related to the fact that Crohn´s disease need not affect the whole organism like hyperglycemia in diabetes does. The results acquired in T1DM adult patients seems to support our hypothesis i.e. patients with bad DNA repair may have the development of diabetic microvascular complications. Nevertheless, large population studies in DM or IBD studying intensity of OS and expression of DNA repair enzymes are needed in order to get the correlation between individual repair enzymes expression and the long-term course and occurence of complications in DM or IBD.

ACKNOWLEDGEMENTS The study was supported by the grant IGA MZ CR NR7919-3/2004 Individual DNA repair capacity induced by oxidative stress and its influence on the development and

Impact of Oxidative Stress on Diabetes Mellitus and Inflammatory Bowel Diseases 235 prognosis of some chronic non-infectious diseases, and the Research Aim MZ CR MSM 0021620814 Prevention, diagnostics, and the therapy of diabetes mellitus, metabolic and endocrine disorders of human organism.

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INDEX A accelerator, 92, 93, 94 acceptance, 67, 77 access, 19 accounting, 72 accumulation, viii, 1, 4, 10, 18, 30, 78, 113, 136, 151, 173, 199, 205, 217 acetaminophen, 186, 236 acetic acid, 215 acetone, 200 acetylcholine, 159 acid, 15, 21, 34, 39, 40, 42, 43, 45, 46, 48, 50, 51, 53, 54, 55, 67, 68, 70, 71, 72, 74, 75, 76, 79, 84, 97, 105, 107, 109, 110, 111, 113, 132, 133, 134, 135, 136, 150, 155, 157, 158, 159, 164, 172, 173, 176, 181, 188, 189, 209, 210, 214, 216, 220 acromegaly, 201 activation, 7, 11, 13, 14, 16, 34, 77, 78, 82, 83, 86, 118, 127, 131, 141, 143, 146, 147, 148, 151, 157, 160, 166, 167, 172, 181, 188, 195, 196, 197, 198, 202, 205, 207, 210, 223, 239 active oxygen, 61 active site, 187 acute lung injury, 120, 143 acute respiratory distress syndrome, 185, 190 adenine, 20, 192, 208 adenovirus, 201 adhesion, x, 145, 147, 148, 151, 165, 166, 167, 205, 210 adolescence, 200 adolescents, 229 ADP, 127, 195, 196, 197, 198, 204, 208, 210, 217, 239, 242, 243 adrenal gland, 187 adrenal glands, 187 adulthood, 161, 200

adults, xi, 12, 153, 161, 162, 164, 172, 174, 175, 180, 201, 205, 208, 218, 219, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234 aerodigestive tract, 132 affect, vii, 1, 11, 43, 45, 51, 57, 73, 76, 77, 78, 98, 112, 117, 120, 151, 193, 195, 199, 211, 227, 234 Africa, 54, 57 age, 31, 36, 66, 79, 85, 87, 142, 155, 164, 194, 196, 199, 201, 206, 207, 217, 219, 220, 223, 226, 229, 230, 231, 232, 234 ageing, 4, 140, 195, 238, 239, 240 agent, x, 90, 91, 105, 107, 108, 114, 119, 120, 121, 122, 124, 134, 157, 158, 159, 181 aggregates, 150 aggregation, 157 aging, vii, xi, 36, 39, 61, 75, 79, 87, 103, 141, 143, 180, 182, 198, 199, 209, 223, 224, 239, 240, 244 aging process, 36, 240 AIDS, 56 air pollutants, 161 airway hyperresponsiveness, 160 airway inflammation, 160, 174, 175 airways, 160 albumin, 181, 183, 215, 216 alcohol, viii, 17, 18, 21, 23, 24, 25, 26, 27, 28, 29, 31, 32, 33, 75, 79, 87, 149, 175 alcohol consumption, 29 alcoholic liver disease, 31 alcoholics, 31 alcohols, 67, 68 aldehydes, 67, 73, 75, 184 allergen challenge, 160 allergic asthma, 162 allergic reaction, 13, 106 alternative, 11, 106 alters, 54, 170, 195 aluminum, 67

248

Index

alveolar macrophage, 161 American Heart Association, 169 amino acids, 38, 66, 69, 181, 183 amyotrophic lateral sclerosis, 184, 190, 193, 199 androgen, 13 angiogenesis, 3, 110 angiography, 158, 173, 186, 236 angioplasty, 159, 174 angiotensin converting enzyme, 209 angiotensin II, 167, 209 animals, ix, 19, 20, 23, 50, 65, 70, 76, 79, 107, 110, 130, 152, 163, 188, 196, 209, 215 ankylosing spondylitis, 211 ANOVA, 21 antibody, 191, 212 anticancer drug, 138, 139 antigen, 118, 121, 163, 175, 176, 193, 244 antigenicity, 190 antioxidant, vii, viii, ix, x, xi, 1, 3, 4, 6, 14, 15, 17, 18, 19, 23, 24, 26, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 71, 72, 74, 75, 76, 78, 79, 80, 82, 83, 84, 85, 87, 90, 104, 107, 108, 109, 110, 112, 113, 114, 115, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 129, 130, 131, 132, 134, 135, 136, 137, 138, 141, 142, 143, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 157, 158, 159, 160, 161, 162, 163, 164, 165, 167, 168, 169, 170, 171, 172, 175, 176, 179, 180, 181, 182, 183, 184, 185, 187, 188, 190, 199, 206, 207, 209, 210, 214, 220, 222, 223, 224, 228, 232, 234, 235, 236, 241, 242, 243, 244, 245 antioxidative activity, 38 antitumor, x, 56, 90, 106, 111, 113, 122, 130, 133, 134, 138, 140, 184 antitumor agent, 184 anus, 211 aorta, 8, 76, 131, 157 aplasia, 103 apoptosis, vii, viii, xi, 1, 3, 5, 10, 11, 13, 14, 15, 16, 80, 86, 99, 106, 108, 109, 110, 111, 112, 113, 114, 115, 118, 120, 122, 123, 125, 127, 131, 132, 133, 135, 136, 137, 139, 141, 142, 143, 146, 147, 148, 167, 180, 181, 182, 183, 184, 185, 188, 190, 195, 197, 198, 199, 214, 226 ARDS, 182 argument, 57 aromatic hydrocarbons, 109 aromatic rings, 66 arrest, 11, 108, 110, 197, 217 arteries, 76 arterioles, 97, 99

arteriosclerosis, 154, 171 artery, 85, 101, 102, 150, 152, 156, 157, 170, 173, 219 arthritis, x, 3, 145, 163, 164, 176, 177, 190 aryl hydrocarbon receptor, 11, 16 ascites, 111, 140, 184 ascorbic acid, 21, 30, 39, 45, 48, 54, 55, 62, 64, 69, 72, 73, 112, 135, 136, 151, 164, 170, 177, 188, 215 ash, 66 Asia, 42 aspartate, 124, 141, 195 assessment, 123, 124, 193, 208, 214, 218, 220 association, 3, 107, 109, 152, 153, 164, 165, 177, 197, 216, 234, 243 asthma, x, 3, 12, 145, 146, 147, 160, 161, 162, 174, 175, 182, 185, 190, 196, 235 astrocytes, 168 asymptomatic, 159, 174 ataxia, 191, 240 atherogenesis, 159, 169, 207 atherosclerosis, x, 85, 145, 146, 147, 150, 152, 154, 155, 158, 159, 169, 171, 172, 173, 174, 182, 186, 189, 199, 200, 242 atherosclerotic plaque, 150 atoms, 95, 96, 117 atopic asthma, 162 atopic dermatitis, 182 ATP, 125, 187, 195, 197, 199, 210 atrophy, 97, 101, 102, 103, 132, 142 attachment, 206 attacks, 80 attention, viii, x, xi, 18, 35, 66, 108, 119, 151, 179, 180, 185, 207, 214, 217 autoantibodies, 201 autoimmune disease, viii, 1, 10, 201, 206 autoimmune hepatitis, 185, 211, 236 autoimmunity, 163 autonomic neuropathy, 200 autooxidation, 8, 32, 80, 104, 149 autosomal recessive, 191, 193, 199 availability, 42, 58, 66, 122, 150, 183, 236 awareness, 52

B bacteria, 8, 36, 39, 55, 70 balloon angioplasty, 158 beams, 91, 92, 94, 96, 98, 125 beef, 187 behavior, 209 behavior modification, 209

Index beneficial effect, viii, xi, 3, 18, 19, 30, 79, 106, 114, 115, 117, 119, 120, 121, 122, 145, 151, 152, 153, 154, 155, 157, 159, 163, 165, 175, 176, 209, 215 benign, 91, 125 benzo(a)pyrene, 87, 132 beta-carotene, vii, 109, 110, 111, 113, 115, 132, 134, 135, 169 beverages, 14, 18, 34, 38, 82, 84, 157 bicarbonate, 185 bile, 70, 71, 116 bile duct, 116 bilirubin, 149, 183, 215 biliverdin, 149 binding, 11, 14, 33, 73, 77, 78, 79, 80, 85, 116, 118, 132, 148, 149, 151, 166, 183, 190, 195, 205, 216, 241 bioavailability, 12, 38, 57, 58, 70, 71, 72, 82 bioconversion, 110 bioflavonoids, 162 biological activity, 12, 57, 66, 85 biological responses, 147 biological systems, vii biomarkers, x, 145, 165, 240 biomonitoring, 238 biopsy, 214 biosynthesis, xi, 29, 38, 112, 147, 167, 179, 180, 188 black tea, viii, ix, 17, 18, 19, 20, 21, 23, 24, 25, 26, 27, 29, 30, 32, 33, 34, 65, 66, 67, 68, 69, 71, 72, 73, 74, 75, 76, 77, 79, 81, 82, 83, 84, 85, 86 bladder, 70 blends, 19 blindness, 200 blocks, 15, 34, 82, 92, 105, 172, 217 blood, 20, 22, 23, 26, 27, 28, 29, 37, 70, 71, 74, 76, 79, 82, 97, 108, 111, 121, 125, 126, 131, 132, 152, 163, 164, 169, 172, 176, 193, 200, 208, 210, 215, 219, 220, 228 blood flow, 97, 121 blood plasma, 74, 82 blood stream, 70 blood vessels, 210 blood-brain barrier, 76 bloodstream, 107 body, vii, xi, 36, 37, 39, 47, 57, 70, 92, 93, 100, 103, 105, 108, 110, 112, 114, 115, 116, 119, 121, 123, 129, 131, 132, 137, 142, 149, 158, 160, 163, 164, 180, 181, 185, 187, 211, 234, 245 body image, 211 body mass index, 245 body weight, 39, 108, 116, 163, 164 bonds, 67, 97, 117 bone growth, 108

249

bone marrow, 99, 106, 114, 119, 124, 128, 131, 136, 137, 161 bowel, 211, 212 boys, 219, 220 brachytherapy, 92 brain, viii, 14, 17, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 34, 76, 79, 93, 100, 101, 108, 120, 126, 130, 131, 138, 141, 166, 173, 182, 200 brain damage, 130 branching, 195 Brazil, 37, 58 breakfast, 51 breast cancer, 11, 110, 113, 114, 116, 126, 133, 139, 193 breast carcinoma, 136 bronchial hyperresponsiveness, 161, 175 bronchial tree, 160 bronchoconstriction, 161 bronchopulmonary dysplasia, 190 bronchus, 110 buffer, 8

C cabbage, 39, 45 caffeine, 67, 69 calcium, 67 caloric restriction, vii calorie, vii Canada, 94 cancer, vii, viii, ix, x, 1, 3, 4, 6, 10, 11, 14, 32, 36, 37, 39, 42, 47, 56, 60, 61, 66, 77, 78, 79, 81, 82, 84, 86, 87, 89, 91, 93, 94, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 122, 123, 124, 125, 126, 127, 129, 130, 131, 132, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 156, 173, 189, 191, 193, 196, 198, 199, 205, 207, 208, 216, 235, 237, 238, 240, 242 cancer cells, 36, 91, 108, 109, 110, 111, 112, 113, 114, 115, 116, 118, 120, 122, 124, 127, 133, 136, 139, 141, 143 cancer progression, 107 candidates, 198, 210, 239 capillary, 99, 181 capsule, 120, 135, 143 carbohydrate, 67 carbon, 38, 120, 141 carbon tetrachloride, 120, 141 carcinogen, 77, 173 carcinogenesis, xi, 32, 77, 78, 79, 80, 90, 103, 109, 123, 129, 130, 132, 134, 139, 180, 185, 191, 199, 206, 216, 226, 229, 234, 238, 240, 244 carcinogenicity, 113

250

Index

carcinoma, 110, 113, 116, 117, 127, 129, 132, 133, 134, 139, 217 cardiovascular disease, 36, 42, 76, 103, 151, 152, 153, 154, 156, 157, 170, 171, 172, 209 cardiovascular risk, 155 cardiovascular system, 142, 173 carotene, 36, 37, 40, 67, 109, 110, 112, 134, 136, 150, 151, 152, 153, 155, 156, 157, 159, 161, 164, 171, 181, 183, 189, 215 carotenoids, vii, ix, 35, 36, 37, 39, 45, 55, 64, 66, 82, 109, 110, 112, 132, 136, 151, 164, 188 carotid arteries, 159, 174 carotid ultrasound, 172 carrier, 187 cartilage, 102, 163 caspases, 133 catalyst, 176 catalytic activity, 33 catalytic properties, 209 cataract, 34, 108, 115, 120, 121, 123, 124, 141, 143 cation, 74, 107, 129 causation, 76, 86 C-C, 67 cell, vii, viii, ix, x, xi, 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 16, 20, 45, 62, 65, 67, 68, 76, 77, 78, 79, 80, 82, 86, 89, 90, 91, 96, 97, 98, 99, 101, 104, 106, 107, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 121, 125, 126, 127, 128, 129, 131, 133, 134, 135, 136, 139, 140, 141, 143, 145, 147, 149, 150, 159, 160, 163, 164, 165, 166, 167, 169, 179, 180, 181, 182, 183, 185, 186, 187, 188, 190, 193, 195, 196, 197, 198, 199, 201, 202, 205, 207, 208, 217, 218, 220, 236, 237, 239, 240, 244 cell culture, viii, 1, 7, 9, 10, 86, 113, 198 cell cycle, 3, 11, 98, 110, 111, 193, 197, 198, 217 cell death, 4, 5, 13, 90, 97, 99, 104, 111, 112, 127, 167, 195, 197, 199, 236, 239 cell killing, 116, 126, 140 cell line, 13, 77, 78, 79, 80, 82, 86, 110, 111, 125, 133, 135, 140 cell membranes, 104, 107, 114, 115, 187 cell metabolism, 67 cell signaling, xi, 179 cellular immunity, 132 central nervous system, 205 cerebrospinal fluid, 184 ceruloplasmin, 149, 183, 216 cervical cancer, 110, 111, 116, 133 cervicitis, 103 cervix, 91, 134, 139 chaos, 244 chelates, 149 chemical bonds, 97

chemical properties, ix, 35, 45, 195 chemical reactions, 36 chemical structures, 96, 98, 105 chemokines, 147, 148, 197 chemoprevention, 81, 86, 109, 110, 132, 138 chemopreventive agents, 80 chemotaxis, 118 chemotherapeutic agent, 113, 116 chemotherapy, 6, 56, 91, 94, 99, 105, 106, 108, 109, 110, 112, 113, 114, 116, 119, 121, 124, 126, 131, 134, 135, 136, 138, 140 Chernobyl accident, 121 chicken, 37 childhood, 175, 200 children, xi, 108, 160, 163, 164, 176, 180, 201, 206, 208, 211, 217, 218, 219, 220, 222, 223, 224, 225, 226, 227, 228, 229, 231, 232, 233, 234, 242, 244 China, 38, 84 chlorine, 44, 63, 216 chlorophyll, 66 cholangiocarcinoma, 139 cholangitis, 211 cholesterol, 37, 76, 85, 154, 156, 158, 159, 169, 173, 184 chromium, 209 chromosome, 118, 121, 184, 191 chronic obstructive pulmonary disease, 3, 4, 146, 174, 175, 185, 186, 236 cigarette smoke, 36, 75, 149, 160, 162, 168 circadian rhythm, 107 circulation, 185 cirrhosis, 185 classes, 159, 190 claudication, 200 cleavage, 31 clinical oncology, 127 clinical trials, x, 32, 38, 119, 145, 151, 154, 159, 161, 165, 198, 208 clusters, 117 CNS, 91 CO2, 68 coagulation, ix, 35, 59, 157 cobalt, 94, 111, 116, 133 coding, 148, 149, 239 coenzyme, 185, 187, 202, 237, 243 coffee, 69 cognitive dysfunction, 115 cognitive function, 137 cohort, 3, 163, 177 cold war, 105 colitis, 176, 190, 211, 215, 217, 243 collaboration, 92 collagen, 112, 127, 157, 163, 172, 176, 188, 205

Index collisions, 93 colon, 13, 34, 42, 70, 77, 79, 80, 84, 86, 108, 110, 116, 139, 211, 213, 217 colon cancer, 13, 79, 116, 217 colorectal cancer, 3, 131, 193, 216 coma, 200 combination therapy, 111, 133 combined effect, 140 commitment, 15 communication, 79 community, 36, 66 compensation, 224, 226 complications, xi, 99, 100, 101, 156, 180, 200, 202, 203, 206, 207, 208, 209, 210, 211, 218, 219, 227, 234, 240, 241, 243 components, viii, ix, 10, 17, 18, 20, 21, 29, 30, 36, 38, 39, 43, 44, 45, 48, 52, 57, 63, 65, 66, 67, 68, 69, 71, 72, 75, 76, 79, 80, 84, 90, 98, 104, 156, 172, 181, 183, 214 composition, ix, 19, 31, 45, 53, 55, 57, 60, 61, 62, 63, 65, 66, 68, 69, 75, 80, 81, 83 compounds, vii, ix, 1, 2, 8, 18, 21, 29, 30, 31, 37, 38, 39, 42, 47, 48, 50, 52, 53, 55, 57, 62, 63, 65, 66, 68, 69, 70, 73, 75, 76, 79, 81, 106, 109, 110, 111, 112, 115, 120, 122, 123, 138, 139, 150, 151, 152, 157, 180, 181, 183, 187, 188, 191, 198, 206, 209 Compton effect, 97 concentration, 3, 6, 8, 9, 10, 12, 18, 19, 21, 23, 30, 38, 39, 50, 51, 52, 57, 66, 70, 74, 75, 76, 78, 109, 114, 115, 150, 181, 187, 210, 214, 219, 220 condensation, 19, 21, 67, 68 congestive heart failure, 169, 198 conjugated dienes, 40 conjugation, 11, 12, 16, 70, 77, 184 conjunctivitis, 101 connective tissue, 76, 101, 118, 211 consumption, viii, ix, 19, 23, 24, 31, 33, 35, 42, 43, 45, 52, 65, 70, 75, 76, 77, 79, 80, 83, 84, 85, 86, 116, 152, 161, 162, 169, 170, 185, 197, 198 contamination, 55, 57 contracture, 103 control, xi, 3, 13, 19, 21, 22, 23, 24, 25, 26, 27, 28, 57, 61, 78, 80, 81, 98, 107, 109, 110, 113, 116, 120, 125, 136, 156, 162, 163, 164, 180, 181, 196, 199, 206, 208, 209, 214, 219, 220, 226, 227, 234, 241 control group, 19, 22, 23, 24, 25, 26, 27, 28, 113, 120, 209, 220 controlled trials, 112, 130, 153 conversion, 18, 20, 36, 70, 73, 146, 158, 180 cooking, ix, 35, 38, 42, 45, 46, 47, 48, 52, 53, 57, 63, 64 COPD, 146, 160, 161, 162, 185

251

copper, vii, 1, 3, 5, 14, 16, 73, 75, 80, 85, 87, 118, 141, 149, 166, 189 corn, 31, 36, 37, 45, 51, 59 coronary angioplasty, 174 coronary arteries, 172, 174 coronary artery disease, 3, 151, 152, 153, 156, 169, 172 coronary heart disease, vii, 1, 60, 76, 152, 156, 169, 170, 171, 172, 219 correlation, xi, 45, 164, 180, 207, 208, 221, 228, 234, 242 corticosteroids, 162, 211, 219 costs, x, 89, 159 cough, 162 coupling, 98, 167 coxsackievirus, 201 cranium, 108, 124 creatinine, 206 Croatia, 58 Crohn’s disease, 200, 211, 212, 213, 217, 218, 219 cultivation, 84 culture, 8, 14, 61, 113, 134, 136, 166 culture conditions, 8 CVD, 153, 154, 155 cycling, vii, 1, 10, 12, 150, 224 cyclooxygenase, 8, 11, 12, 30, 34, 73, 84, 147, 148 cyclophosphamide, 114, 123 cystic fibrosis, 185, 193, 201, 236 cystitis, 103 cytochrome, 16, 18, 31, 52, 77, 86, 131, 147, 187, 216 cytochrome p450, 147, 216 cytokines, 78, 146, 147, 148, 160, 163, 177, 181, 197, 205, 211, 217 cytomegalovirus, 201 cytoplasm, 78 cytoprotectant, 118 cytosine, 192, 208 cytotoxicity, 3, 5, 9, 13, 15, 111, 120, 121, 133, 143, 169 Czech Republic, 179

D damage, vii, x, xi, 4, 6, 33, 36, 37, 76, 78, 80, 89, 90, 96, 97, 98, 99, 100, 101, 102, 104, 105, 107, 108, 109, 111, 112, 113, 114, 115, 117, 118, 121, 122, 123, 124, 127, 128, 129, 130, 131, 134, 136, 146, 149, 151, 158, 160, 163, 172, 180, 181, 183, 189, 190, 193, 195, 196, 197, 199, 202, 204, 205, 207, 208, 210, 211, 214, 216, 220, 222, 223, 224, 225, 234, 242

252

Index

death, 97, 99, 108, 111, 118, 150, 151, 152, 153, 156, 158, 170, 184, 197, 207 decay, 60 decisions, 97 decomposition, x, 14, 89, 90, 104, 149, 176 defects, 190, 201 defense, vii, ix, x, 1, 5, 15, 29, 36, 65, 82, 109, 123, 125, 140, 145, 146, 147, 149, 150, 151, 165, 176, 180, 242 defense mechanisms, x, 145, 149 deficiency, viii, 1, 10, 101, 109, 114, 131, 132, 185, 200, 239 degradation, 7, 33, 73, 75, 78, 82, 163, 169, 173, 183, 184, 189, 198 dehydration, 43, 54, 66 delivery, 184 demyelinating disease, 182 demyelination, 100 density, 11, 15, 33, 82, 85, 96, 169, 173, 245 deoxyribose, 193 deposition, 169 derivatives, 13, 48, 52, 70, 83, 84, 106, 110, 132, 135, 150, 156 dermatitis, x, 89, 102, 119, 127 destruction, 48, 98, 107, 147, 201 detection, 20, 23, 32, 193, 207 developed countries, 201, 211 developing countries, 62 developing nations, 150 deviation, 222 diabetes, xi, 3, 11, 39, 56, 151, 152, 155, 180, 182, 186, 190, 193, 195, 196, 198, 199, 200, 201, 202, 205, 206, 207, 208, 209, 210, 217, 218, 219, 225, 226, 227, 234, 235, 241, 242, 245 diabetic nephropathy, 206, 209, 219 diabetic neuropathy, 206, 209, 219, 242 diabetic patients, 198, 202, 206, 207, 208, 225, 227, 228, 234, 242 diabetic retinopathy, 207, 219 diacylglycerol, 204 diarrhea, 118 diet, vii, viii, 3, 9, 10, 19, 32, 35, 36, 38, 42, 117, 157, 175, 201, 209, 219, 229, 232 dietary fat, 52, 61 dietary fiber, 52, 54, 58 dietary intake, 3, 157, 161 dietary supplementation, 10, 110, 140, 161 differentiation, vii, 1, 3, 94, 109, 121, 134, 141, 147, 181, 201 diffusion, 146 digestibility, 38, 63 disability, 163 disease activity, 214

disease progression, 161 disorder, 160, 199 distribution, 12, 50, 61, 70, 174, 185, 189, 193, 197, 206, 239 division, 183 DNA, vii, ix, x, xi, 3, 4, 5, 6, 11, 12, 13, 14, 15, 16, 31, 65, 77, 78, 80, 83, 86, 87, 89, 96, 97, 104, 107, 109, 110, 113, 114, 115, 116, 118, 119, 127, 131, 132, 137, 139, 140, 141, 149, 177, 179, 180, 181, 182, 183, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 204, 207, 208, 209, 210, 214, 216, 217, 218, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 238, 239, 240, 242, 244, 245 DNA breakage, 198 DNA damage, xi, 12, 14, 15, 16, 77, 80, 83, 86, 87, 96, 104, 107, 113, 116, 131, 132, 137, 141, 180, 182, 183, 189, 191, 193, 194, 195, 196, 197, 199, 204, 207, 208, 209, 210, 216, 217, 218, 220, 222, 223, 224, 225, 226, 227, 229, 230, 231, 232, 233, 234, 235, 238, 240, 242, 244 DNA lesions, 190, 192 DNA ligase, 193 DNA polymerase, 190, 193 DNA repair, xi, 97, 149, 180, 190, 192, 193, 194, 195, 196, 198, 199, 207, 208, 217, 218, 220, 222, 223, 224, 225, 226, 227, 228, 229, 231, 232, 234, 238, 239, 240, 242, 244 doctors, 158 dogs, 105 domain, 188 donors, 202 dosage, x, 89, 92, 97, 98, 99, 101, 112, 122 dosing, 39, 159 down-regulation, 86 drainage, 103 drinking water, 19, 77, 113, 116 drug resistance, 116 drugs, 10, 12, 33, 56, 106, 110, 116, 165, 184, 209, 211, 243 dry matter, 69 drying, ix, 35, 38, 43, 54, 55, 61, 68 duodenum, 70 duration, xi, 99, 180, 206, 208, 219, 227, 228, 229, 230, 231, 232, 234, 244 dyslipidemia, 209 dysplasia, 102, 116, 215, 217, 244

E East Asia, 38, 39 eating, 36, 37, 52 ecology, 81

Index ectropion, 101 edema, 99, 101, 102, 117 effusion, 102 egg, 194 elaboration, 90, 104 elderly, 152, 153, 158, 164, 170 elderly population, 152, 153, 158 electromagnetic, 91, 95, 96 electromagnetic waves, 95, 96 electromyography, 219 electrons, 91, 93, 96, 98, 103, 185, 189, 202 electrophoresis, 193, 218, 220, 244 embryo, 50 embryogenesis, 138 EMG, 219 emphysema, 149, 161, 168, 174 emulsions, 53 encephalomyelitis, 14, 86 encephalopathy, 100 encoding, 3, 185, 190 endocrine, 103, 118, 235 endocrine disorders, 235 endonuclease, 193, 199, 207, 208 endothelial cells, 9, 16, 76, 127, 148, 158, 163, 165, 166, 167, 181, 202, 205, 207, 242 endothelium, 151, 152, 174 endotoxins, 78 energy, 36, 37, 73, 91, 92, 93, 95, 96, 97, 107, 158, 187, 195 energy transfer, 96 England, 94 enrollment, 156 entropion, 101 environment, 78, 96, 97, 98, 109, 131, 160, 185 environmental factors, 36, 201, 211 enzymatic activity, 77 enzymes, viii, ix, x, xi, 3, 4, 6, 17, 18, 23, 24, 25, 29, 30, 33, 36, 37, 43, 58, 65, 67, 70, 72, 77, 79, 85, 89, 90, 103, 104, 105, 106, 107, 108, 112, 115, 117, 118, 120, 123, 124, 125, 147, 149, 163, 165, 168, 172, 173, 175, 176, 179, 180, 181, 182, 183, 185, 188, 190, 193, 194, 195, 199, 208, 214, 223, 224, 234, 236, 239, 240, 244 eosinophilia, 160 eosinophils, 160 epidemic, 201 epidemiology, 138 epidermis, 99 epinephrine, 20, 32 episcleritis, 211 epithelial cells, 78 epithelial lining fluid, 186 epithelium, 140, 185, 214

253

erosion, 163 erythema multiforme, 211 erythema nodosum, 211 erythrocytes, 20, 76, 119, 138, 176, 184, 185, 194, 220, 228 erythropoietin, 181 esophageal cancer, 132 esophagitis, 102 esophagus, 70, 99, 110, 112, 134 ESR, 7 ester, 157, 172 ethanol, viii, 17, 18, 19, 20, 22, 24, 25, 26, 27, 28, 29, 30, 31, 33, 79, 85, 87 ethanol metabolism, 29 etiology, 163 EU, 12 eukaryotic cell, 166 Europe, 201, 211 evidence, 8, 32, 42, 73, 78, 107, 112, 114, 117, 130, 146, 151, 152, 165, 173, 187, 194, 216, 228 examinations, 29 excision, xi, 91, 180, 190, 191, 192, 195, 198, 238, 240, 244 excitation, 96 excretion, 70, 77, 176 exercise, 61, 63, 209, 244 experimental condition, 8, 38 exposure, 29, 30, 36, 55, 106, 115, 128, 146, 189 expression, x, xi, 8, 11, 79, 109, 110, 121, 126, 133, 145, 146, 147, 148, 149, 157, 158, 160, 161, 165, 166, 167, 168, 169, 173, 180, 181, 184, 192, 193, 205, 206, 208, 210, 214, 217, 234, 241, 242, 243 extracellular matrix, 146, 205 extraction, 45

F failure, 90, 102, 103, 104, 151, 211, 237 failure to thrive, 211 familial hypercholesterolemia, 173, 174 family, 77, 114, 147, 148, 165, 195 fat, 32, 42, 52, 63, 112, 114, 132, 187, 188, 209 fatigue, 200, 211 fatty acids, 30, 40, 52, 53, 130 FDA, 56, 105, 106, 122 feces, 71 feedback, 195 females, 153, 207 femur, 108 fermentation, ix, 19, 35, 38, 39, 40, 42, 59, 62, 63, 66, 68 fermentation technology, 62 ferritin, 18, 149, 183, 216

254

Index

fever, 211 fibroblasts, 111, 112, 117, 135, 166, 205, 206 fibrosarcoma, 121, 124, 140 fibrosis, x, 89, 97, 100, 101, 102, 103, 138, 143, 168, 176, 185, 190 fidelity, 190 Finland, 201 fish, 31, 38, 60, 161, 186 fish oil, 31 flavonoids, vii, viii, ix, 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 32, 33, 35, 37, 39, 40, 47, 48, 57, 71, 81, 82, 83, 84, 87, 119, 142, 157, 162, 172, 175, 188 flavor, 52 fluid, 118, 149, 160, 163 fluorescence, 220 focusing, 193 folic acid, 209 food, vii, ix, 3, 4, 6, 8, 10, 18, 31, 35, 36, 37, 38, 39, 40, 42, 44, 45, 52, 54, 55, 56, 57, 59, 60, 62, 63, 64, 76, 81, 82, 101, 152, 158, 161, 172, 186, 236 food additives, 62 food industry, 81 food intake, 101 food processing industry, ix, 35, 38 food products, 38, 55 food safety, 44, 62 Ford, 175 France, 189, 220 free energy, 187 free radicals, vii, ix, x, 18, 29, 30, 36, 37, 48, 65, 72, 73, 74, 75, 77, 78, 79, 89, 90, 96, 97, 98, 104, 105, 106, 108, 114, 115, 117, 120, 122, 157, 158, 185, 188, 189, 209, 215, 235, 241, 244 fructose, 203 fruits, viii, 2, 3, 12, 35, 36, 37, 38, 43, 45, 47, 54, 55, 61, 63, 157, 161, 162, 175, 186, 188 fungal infection, 189 fungus, 40, 56, 64

G gamma radiation, 96, 130, 137 gamma rays, 91, 96, 97, 98, 99, 119 gastritis, 102 gastrointestinal tract, 29, 73, 128, 147, 186, 206, 211 gel, 193, 218, 220, 244 gene, ix, 3, 11, 66, 78, 107, 121, 147, 148, 163, 165, 166, 168, 181, 184, 187, 192, 196, 199, 201, 206, 207, 235, 236, 240, 244 gene expression, 11, 107, 147, 148, 165, 166, 168, 181, 196 gene therapy, 187

gene transfer, 163 generation, ix, x, 4, 8, 12, 15, 18, 29, 30, 31, 33, 65, 67, 72, 73, 75, 77, 79, 80, 87, 90, 104, 105, 138, 145, 146, 147, 149, 151, 160, 210 genes, 77, 78, 110, 121, 127, 146, 147, 148, 149, 157, 160, 161, 168, 176, 185, 190, 191, 193, 194, 195, 196, 201, 205, 206, 217, 238, 239, 242 genetic code, 194 genetic defect, 201 genetic disease, 190 genetic factors, 163 genetic syndromes, 201 genetics, 217, 238, 240 genome, 190, 209 genotype, 236 Germany, 1, 220 girls, 219, 220 gland, 102, 107, 128 glaucoma, 101 glioma, 113, 116, 139 glomerulonephritis, 182 glucose, 67, 152, 188, 200, 201, 202, 205, 206, 207, 243 glucose tolerance, 152, 201 glucosidases, 67 glucoside, 9, 14 GLUT, 202 glutamate, 195 glutamic acid, 201 glutathione, vii, viii, x, 1, 3, 4, 5, 6, 8, 11, 12, 15, 17, 29, 30, 33, 40, 72, 74, 77, 79, 90, 112, 113, 115, 116, 130, 131, 140, 141, 147, 149, 158, 163, 164, 167, 168, 176, 181, 183, 184, 185, 186, 188, 189, 214, 220, 222, 223, 235, 236, 237, 243, 245 glycine, 70 glycol, 191, 193, 195 glycolysis, 210 glycoside, 81 glycosylation, 202, 210, 241 gold, 92, 176 gold compound, 176 grains, ix, 2, 35, 36, 37, 45, 50, 51, 59, 175 graph, 222, 226, 232 Great Britain, 220 groups, 4, 6, 19, 21, 24, 25, 26, 27, 29, 67, 70, 72, 74, 75, 80, 110, 117, 120, 125, 151, 162, 206, 207, 208, 209, 215, 219, 222, 223, 224, 229, 231, 232, 233 growth, vii, 1, 3, 12, 14, 42, 64, 78, 79, 86, 97, 98, 107, 108, 109, 110, 111, 112, 113, 115, 117, 118, 121, 136, 139, 141, 144, 146, 147, 167, 168, 181, 191, 205, 239, 241 growth factor, 107, 146, 147, 167, 168, 205

Index growth rate, 42, 139 guanine, 192, 193, 207 guidelines, 174 Guinea, 85 gut, 70, 118, 151, 214

H habitat, 95 hair follicle, 106 half-life, 146, 159, 190 haplotypes, 199 haptoglobin, 183 harm, 156 harmful effects, 80 harvesting, 61 hazards, 136 HDL, 76, 155, 156, 172 HE, 14, 175 head and neck cancer, 110, 119, 123, 125, 127, 128, 132, 137, 138, 142 healing, 111, 157, 168, 211 health, vii, viii, ix, 1, 3, 6, 10, 19, 30, 31, 32, 35, 36, 37, 38, 42, 57, 59, 60, 61, 65, 71, 75, 81, 82, 84, 138, 153, 155, 160, 174, 235 health effects, 37, 82, 84 heart attack, 36, 200 heart disease, 12, 14, 36, 37, 83, 84, 85, 152, 153, 170 heart failure, 102, 151, 184, 187 heart murmur, 220 heat, 14, 16, 47, 52, 167 heat shock protein, 16 heating, 43 height, 105 heme, 147, 149, 151 heme oxygenase, 149 hemodialysis, 157 hemoglobin, 226, 241 hemorrhage, 101 hepatitis, 56, 100, 102, 182, 185, 193, 237 hepatitis a, 185 hepatocellular carcinoma, 11, 121, 143 hepatocytes, 8, 15, 16, 33, 85, 106, 129, 184 hepatoma, 13 hepatomegaly, 132 hepatotoxicity, 31, 119 heterogeneity, 139 hexane, 21 high density lipoprotein, 169 hippocampus, 30, 120, 131 HIV, 39 HIV/AIDS, 39

255

HLA, 201 HO-1, 149 Hodgkin's disease, 126 homeostasis, 4, 117, 118, 141, 181, 185 homocysteine, 131, 157, 172 homovanillic acid, 8 hormone, 101, 107, 108, 147 hospitalization, 162 host, 43, 113, 147, 165, 188 human brain, 107 human organisms, 71 human subjects, 32, 113, 245 hybrid, 144 hydrogen, x, 4, 8, 14, 18, 20, 72, 73, 75, 77, 80, 89, 90, 103, 104, 135, 146, 150, 180, 182, 183, 184, 189, 190, 208, 214, 216 hydrogen peroxide, x, 4, 8, 14, 18, 20, 73, 77, 80, 89, 90, 103, 104, 135, 146, 180, 182, 183, 184, 189, 190, 208, 214, 216 hydrolysis, 67, 186 hydroperoxides, 73, 75, 149, 150, 184 hydroxyl, x, 4, 6, 8, 30, 31, 36, 38, 39, 57, 67, 70, 72, 73, 74, 75, 80, 89, 90, 96, 103, 104, 118, 142, 146, 149, 169, 180, 181, 182, 189, 214, 216 hydroxyl groups, 6, 67, 70, 72, 73, 74 hygiene, 55 hypercholesterolemia, 152, 173, 174 hyperemia, 99 hyperglycaemia, 201, 202, 204, 205 hyperglycemia, 206, 209, 228, 234 hyperinflation, 162 hyperplasia, 116 hypersensitivity, 39, 193 hypertension, 39, 103, 200, 209 hypertrophy, 159, 174, 237 hypoplasia, 102 hypotension, 56, 106 hypothesis, 69, 71, 151, 152, 218, 227, 234 hypothyroidism, 147 hypoxia, 110, 120, 143, 148, 163, 165, 181 hypoxic cells, 98

I IBD, xi, 180, 200, 211, 213, 214, 216, 217, 218, 234 ICAM, 147, 205 ideas, 125 identification, 12, 67, 82, 84, 201 idiopathic, 185, 211, 229 IFN, 111 IL-13, 160 IL-6, 147, 148, 163, 165 IL-8, 147, 166, 205

256

Index

ileum, 108 images, 194 immune function, 37, 115, 118 immune response, 112, 117, 140, 196 immune system, x, 89, 117, 118, 146, 186 immunity, 117, 118, 138, 140, 141 immunodeficiency, 191 immunoglobulin, 205 immunoglobulin superfamily, 205 immunosuppressive drugs, 219 immunotherapy, 127, 134 in vitro, 3, 8, 11, 12, 16, 18, 30, 32, 34, 72, 73, 74, 75, 76, 83, 84, 85, 86, 108, 109, 112, 113, 114, 116, 117, 120, 121, 127, 133, 134, 136, 138, 141, 143, 144, 151, 157, 183, 190, 206, 239, 244 incidence, vii, ix, 1, 3, 66, 76, 79, 80, 105, 106, 110, 115, 116, 155, 156, 159, 161, 164, 171, 189, 201, 211, 217, 238 inclusion, 67 India, 81 indication, 198 indices, 90, 137, 165, 214, 241 indigenous, 43 individual differences, 193, 239, 244 inducer, 158 induction, 3, 4, 5, 11, 13, 31, 77, 86, 110, 116, 119, 137, 139, 166, 167, 168, 169, 187, 193, 198, 215, 238 industry, 38, 40, 50 infancy, 142 infarction, 154, 155, 158, 173 infection, vii, 57, 109, 119, 142, 200, 201 infectious disease, 39, 182, 235 inflammation, x, 4, 14, 84, 99, 109, 112, 116, 131, 145, 147, 149, 160, 161, 163, 164, 166, 167, 168, 174, 176, 184, 186, 187, 188, 189, 196, 207, 211, 214, 216, 232, 236, 237, 240, 243, 244 inflammatory bowel disease, xi, 146, 180, 182, 183, 196, 199, 200, 211, 214, 216, 217, 243, 244 inflammatory cells, 151, 160, 163 inflammatory disease, x, 3, 138, 145, 146, 147, 148, 149, 160, 163, 165, 185, 199, 229 inflammatory mediators, 198 inflammatory responses, 118, 148 influence, ix, 8, 26, 30, 33, 52, 55, 66, 70, 71, 72, 74, 76, 77, 118, 122, 136, 141, 147, 154, 156, 181, 189, 205, 212, 218, 226, 231, 234, 235 informed consent, 221 ingestion, 9, 71, 82, 113 inhibition, 3, 5, 6, 8, 14, 27, 29, 32, 39, 40, 55, 76, 77, 78, 79, 83, 84, 86, 107, 109, 115, 117, 130, 131, 140, 151, 152, 157, 161, 189, 198, 207, 209, 239

inhibitor, 61, 78, 110, 119, 166, 167, 197, 198, 207, 209, 217 initiation, x, 68, 75, 77, 79, 80, 104, 132, 145 injury, ix, x, 30, 31, 65, 87, 89, 90, 99, 104, 105, 106, 107, 115, 119, 124, 126, 130, 143, 149, 151, 159, 160, 162, 165, 166, 168, 169, 170, 172, 173, 174, 176, 189, 199, 214 innate immunity, 167 insight, 13 instability, 191, 195, 226, 240, 244 insulin, 60, 200, 201, 202, 205, 219, 245 insulin resistance, 201 integrity, 30, 118, 190, 208 intensity, viii, xi, 17, 26, 48, 180, 196, 199, 206, 207, 208, 217, 218, 220, 223, 229, 234 interaction, 96, 97, 150, 196, 205, 207 interactions, 3, 9, 31, 61, 78, 126, 136, 141, 215 interest, ix, 50, 52, 58, 65, 67, 75, 78, 105, 106, 109, 151, 156 interferon, 110, 111, 113, 133, 136, 181, 205, 217 interferon-γ, 181 interleukin-8, 181 internet, 10 interphase, 104 interpretation, 174, 241 interstitial lung disease, 236 interstitial nephritis, 182 interval, 98, 110, 227 intervention, 18, 115, 121, 151, 157, 159, 172 intestine, 70, 109, 128, 213 intima, 154 intoxication, viii, 17, 18, 19, 21, 23, 26, 27, 29, 30, 31, 32, 33, 75, 79, 87, 186 iodine, 92 ionization, 96, 98 ionizing radiation, vii, x, 55, 89, 90, 91, 96, 97, 100, 104, 107, 117, 124, 125, 126, 127, 130, 131, 140 ions, vii, 1, 3, 4, 5, 6, 14, 15, 18, 30, 73, 75, 80, 91, 95, 96, 117, 151, 157, 180, 183, 216 iridium, 92 iron, vii, 1, 3, 14, 18, 30, 31, 34, 73, 75, 78, 118, 141, 143, 148, 149, 151, 216 irradiation, 55, 57, 59, 60, 62, 63, 64, 75, 94, 97, 98, 99, 100, 101, 103, 104, 108, 111, 113, 114, 115, 116, 119, 120, 121, 123, 126, 127, 128, 129, 130, 131, 133, 136, 137, 142, 182 irritable bowel syndrome, 217 ischemia, 33, 101, 157, 159, 172 ischemia reperfusion injury, 157 Islam, 63 Italy, 235 IV collagenase, 166

Index

J Japan, 38, 60, 64, 158 joint destruction, 163, 176 joint swelling, 163 joints, 163 juvenile rheumatoid arthritis, 163, 164, 176

K keratinocyte, 168 kidney, 10, 40, 59, 70, 100, 106, 128, 147, 184 kidneys, 100, 187 kinase activity, 78, 82, 86, 240 kinetic parameters, 10 kinetics, 11, 82 knowledge, 142, 192, 208, 214, 217, 223, 234 Korea, 38, 61

L lactic acid, 39 lactoferrin, 183 large intestine, 70 laryngeal cancer, 111 larynx, 91 LDL, 4, 76, 84, 85, 114, 150, 151, 152, 156, 158, 168, 169, 170, 173, 188, 224 lead, x, 4, 8, 27, 29, 55, 77, 79, 80, 89, 105, 156, 160, 161, 181, 190, 195, 226, 241 leakage, 195 learning, 121 lens, 34, 101, 120, 123 lesions, 127, 158, 169, 190, 192, 194, 207, 215 leucine, 168 leukemia, 111, 136 life span, vii, 111, 199 lifespan, vii lifestyle, 211 ligands, 11, 78, 181 lignin, 66 limitation, 94, 161 linkage, 201 links, 206 lipases, 149 lipid metabolism, 76 lipid oxidation, 30, 39, 118 lipid peroxidation, viii, 6, 15, 17, 21, 27, 29, 30, 31, 34, 39, 40, 61, 73, 75, 76, 79, 84, 87, 90, 104, 107, 109, 112, 113, 120, 123, 125, 127, 130, 131, 137, 143, 157, 158, 163, 168, 172, 174, 176, 177, 184, 188, 189, 214, 220, 226

257

lipid peroxides, 30, 114, 118, 184 lipids, vii, ix, xi, 1, 4, 5, 65, 66, 75, 76, 85, 112, 115, 118, 137, 177, 179, 181, 183, 184, 188, 199 lipoproteins, 114, 149, 151, 181, 188, 245 liposomes, 78, 144 liquid chromatography, 32, 82 Listeria monocytogenes, 40 liver, viii, 1, 8, 10, 12, 14, 15, 16, 17, 20, 21, 22, 23, 24, 27, 28, 29, 30, 31, 32, 33, 40, 58, 59, 70, 76, 77, 79, 83, 87, 100, 108, 114, 115, 116, 120, 123, 129, 131, 137, 143, 158, 172, 184, 185, 187, 190, 211, 237 liver cells, 33, 87 liver damage, 120, 143 liver disease, 32, 33, 185 liver failure, viii, 1, 10, 158, 190 localization, 188, 243 location, 183, 189 longevity, 31, 195 loss of consciousness, 200 low risk, 155 low-density lipoprotein, 168, 171, 189 lumen, 160 lung cancer, 3, 14, 78, 79, 106, 110, 115, 126, 131, 139, 155, 156, 172 lung disease, 160, 185 lung function, 162 lupus, 149 lutein, vii, 67, 151 lycopene, vii, 37, 39, 45, 47, 151, 188, 215 lymph, 99, 103, 147, 163, 184 lymph gland, 99 lymph node, 147, 163 lymphedema, 117, 140 lymphocytes, 5, 8, 12, 14, 16, 166, 181, 208, 220, 221, 245 lysine, 33, 206

M machinery, 150, 151, 184, 196, 234 macrocyclics, 184 macromolecules, 4, 115 macrophages, 11, 34, 76, 78, 86, 131, 146, 169, 181, 189, 205, 214 magnesium, 67 magnetic resonance, 237 magnetic resonance spectroscopy, 237 major cities, 150 malabsorption, 211 malignant melanoma, 135 malignant mesothelioma, 3 malignant tumors, 108, 120, 143

258

Index

mammalian tissues, 185 management, 175, 212 mandible, 102 manganese, 67, 159, 184 manipulation, 240 manufacturing, 67, 159 market, 56 marketing, 57 markets, 63 marrow, 100, 103 masking, 9 mass, 67, 69, 116, 201 matrix, x, 145, 166, 187, 208, 241 matrix metalloproteinase, x, 145, 166, 241 maturation, 185, 205 maxillary sinus, 133 MCP, 147, 148 MCP-1, 147, 148 measurement, 92, 193, 208, 218 measures, 71, 85 meat, 52, 55, 186, 187 media, 36, 62, 102, 154 medication, 201, 211 MEK, 14 melanoma, 8, 112, 113, 136 melatonin, x, 90, 104, 105, 107, 108, 109, 122, 123, 126, 127, 129, 130, 131 membrane permeability, 114 membranes, vii, 1, 5, 14, 30, 52, 63, 107, 118, 136, 146, 149, 184, 187, 188 men, 152, 153, 154, 155, 161, 170, 175, 207, 219, 223 menopause, 103 mesangial cells, 205 mesons, 96 messages, 78 messengers, 146 metabolism, vii, ix, xi, 4, 7, 8, 9, 12, 15, 16, 18, 29, 34, 37, 65, 66, 70, 71, 72, 76, 77, 82, 84, 90, 103, 107, 112, 118, 141, 146, 152, 157, 173, 179, 180, 181, 184, 185, 200, 208, 234, 236 metabolites, 9, 10, 11, 12, 15, 29, 70, 77, 82, 125, 131, 137, 165, 195 metabolizing, 18, 34 metalloproteinase, 241 metals, 117, 140, 149, 190 metastasis, 126, 131 methylation, 8, 9, 13 MHC, 118 MHC class II molecules, 118 mice, 5, 39, 56, 62, 70, 73, 76, 77, 79, 105, 108, 111, 112, 113, 114, 116, 119, 120, 129, 131, 132, 133, 135, 136, 139, 140, 141, 142, 143, 149, 157, 158,

159, 160, 161, 163, 166, 168, 172, 174, 176, 215, 243 microcephaly, 191 micrograms, 117 micronucleus, 5, 15 micronutrients, 136, 151, 164, 177, 243 microsatellites, 195 microscope, 220 microscopy, 63, 125, 220 microsomes, 31, 33, 77, 187 microwaves, 59, 95 migration, 205 military, 105 milk, 82 mitochondria, 20, 31, 120, 146, 181, 187, 194, 197, 199 mitochondrial DNA, xi, 180, 192, 194, 199, 201, 208, 239 mitogen, 14, 146, 166, 167, 181 mitosis, 98 mixing, 59 MLT, 107, 108, 109 MMP, 147, 166 MMP-2, 147 MMP-9, 147 MMPs, 147 mobility, 75, 188 mode, ix, 66, 77, 78, 190 model system, 165 modeling, 128 models, viii, 1, 6, 10, 14, 76, 79, 113, 152, 159, 163, 176, 187, 190, 193, 198, 199, 207, 209, 217, 239, 244 moisture, 55 moisture content, 55 molasses, 37 mold, 92 molecular oxygen, 103, 146, 180, 202 molecular structure, 4, 72 molecular weight, 2, 69 molecular weight distribution, 69 molecules, vii, 4, 29, 52, 68, 72, 73, 95, 96, 98, 103, 114, 118, 146, 147, 148, 160, 166, 183, 205 monitoring, 20, 161 monolayer, 7, 16 monomers, 53 monounsaturated fatty acids, 59 morbidity, 212 morning, 163, 164 morning stiffness, 163, 164 morphology, 128 mortality, 3, 132, 152, 153, 154, 155, 156, 160, 170, 175

Index

259

mortality rate, 155, 156 mortality risk, 3, 152 motion, 96, 193 MRI, 95 mRNA, 166 mtDNA, 194, 199, 208 mucin, 160 mucosa, 34, 70, 77, 84, 86, 99, 118, 160, 213, 217, 228, 244 mucous membrane, 99, 109 mucous membranes, 109 multiple sclerosis, 193 mumps, 201 mutagenesis, 6, 14, 116, 140 mutant, 78, 183, 199 mutation, 77, 194, 195, 199, 235 myelofibrosis, 103 myocardial infarction, 152, 153, 155, 159, 170, 171, 173, 190 myocardial ischemia, 143

Nigeria, 35, 43, 44, 54, 58, 62 nitric oxide, 11, 30, 34, 73, 107, 125, 147, 167, 169, 173, 216 nitric oxide synthase, 11, 34, 73, 167, 173 nitrogen, x, 145, 146, 216 nitrogen dioxide, 216 nodes, 100, 103 non-smokers, 86 norepinephrine, 112, 188 normal development, 99 North America, 211 Nrf2, 148, 149, 160, 161, 168, 174 nuclei, 15, 199 nucleic acid, 75, 77, 193 nucleolus, 118 nucleotides, 190 nucleus, 78, 94, 96, 118, 192, 195, 199, 207 nurses, 153 nutrients, 36, 38, 48, 50, 57, 126, 175 nutrition, 45, 50, 52, 57, 131, 172, 212

N

O

NaCl, 20, 59 NAD, 18, 147, 165, 168, 180, 195, 196, 197, 198, 210 NADH, 18, 80, 187, 202 nasopharynx, 91 nausea, 106 neck cancer, 105 necrosis, 99, 100, 101, 102, 103, 105, 116, 123, 125, 195, 198, 215, 217 nephritis, 100, 103, 149 nephropathy, 158, 173, 186, 200, 206, 219, 227, 236 nerve, 102, 103, 166 nerve growth factor, 166 Netherlands, 172 network, 9, 131, 190 neuritis, 100, 102 neuroblastoma, 113, 136 neurodegeneration, 184, 186 neurodegenerative disorders, viii, 1, 10, 184, 238 neurological disability, 219 neuronal cells, 142 neurons, 15, 194, 205 neuropathy, 102, 209, 219, 227 neuroprotection, 86 neutrons, 91, 96, 98 neutrophils, 15, 118, 146, 181, 214 New Zealand, 61, 152, 158 niacin, 155, 156, 172 nicotinamide, 20, 195, 196 nicotinic acid, 204

obesity, 3, 11, 201 observations, 94, 99, 115, 153 obstruction, 102 obstructive lung disease, 190, 198 occlusion, 101 ODS, 115, 137 OH-groups, 9 oil, 31, 52, 53, 55, 60, 62, 63, 187 oils, 37, 50, 52, 53, 55, 58, 59, 64, 188 old age, 79 older people, 110 omega-3, 177 oncogenes, 77 ophthalmologist, 219 orange juice, 164 organ, 99, 101, 108, 109, 114, 123, 125, 130, 158, 160, 182, 185, 237 organelles, 118 organism, viii, xi, 1, 9, 10, 18, 71, 73, 74, 179, 180, 184, 188, 229, 234, 235 osteoporosis, 42, 199, 211 outline, 207 output, 215 ovarian cancer, 110, 124, 133, 238 overproduction, 182, 200, 202, 204, 210, 211 overweight, 201 oxidation, vii, ix, 4, 5, 6, 8, 11, 12, 13, 18, 19, 20, 29, 31, 39, 40, 52, 53, 65, 66, 67, 68, 69, 72, 74, 75, 76, 80, 81, 83, 84, 85, 104, 117, 122, 146, 148,

260

Index

157, 158, 160, 169, 171, 173, 183, 186, 188, 189, 205, 207, 214, 226, 239, 242, 244 oxidation products, ix, 19, 65, 69, 74 oxidative damage, x, xi, 5, 13, 59, 66, 79, 80, 85, 86, 87, 90, 104, 107, 114, 115, 116, 118, 120, 131, 137, 142, 149, 151, 179, 180, 183, 191, 199, 207, 220, 227, 232, 236, 243 oxidative reaction, 14 oxidative stress, vii, viii, x, xi, 1, 4, 5, 6, 10, 13, 14, 15, 17, 18, 19, 27, 29, 30, 33, 34, 35, 52, 62, 75, 77, 78, 86, 87, 89, 90, 104, 105, 107, 109, 112, 115, 120, 121, 122, 123, 124, 125, 131, 132, 137, 138, 143, 145, 147, 148, 149, 151, 156, 157, 158, 159, 160, 161, 163, 164, 166, 168, 180, 181, 182, 183, 185, 187, 188, 190, 191, 193, 195, 197, 198, 199, 200, 202, 206, 207, 208, 213, 216, 217, 218, 219, 220, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 237, 239, 241, 242, 244, 245 oxygen, ix, 5, 8, 13, 18, 30, 33, 36, 37, 48, 65, 68, 72, 74, 76, 80, 83, 90, 97, 98, 103, 104, 106, 107, 114, 125, 129, 130, 136, 141, 145, 146, 147, 150, 166, 167, 181, 184, 189, 216, 238 ozone, 160, 161, 175

P p53, 121, 133, 197, 198, 217, 239, 244 paclitaxel, 113, 114, 129, 136 pain, 211 Pakistan, 61, 62 palliative, 91 pancreas, 158, 187, 201 pancreatic cancer, 131 pancreatitis, 138, 182, 185, 201, 236 paralysis, 103 parameter, 10, 24, 25, 27, 220 parenchyma, 99, 101 parenchymal cell, 101 parents, 206 Parkinson’s disease, 184, 186, 187, 199 parotid, 128 parotid gland, 128 particles, 36, 91, 92, 94, 95, 96, 98, 151 partnership, 193 pathogenesis, x, 77, 145, 146, 160, 161, 163, 165, 175, 176, 183, 201, 202, 203, 208, 215, 217, 236, 240, 243 pathogens, 40, 44, 64, 181 pathology, 4, 34, 76, 197 pathophysiology, xi, 129, 180, 195, 209, 239

pathways, 4, 43, 160, 167, 181, 190, 193, 194, 198, 199, 200, 202, 204, 205, 207, 217, 237, 238, 239, 242 peptides, 38 perforation, 100, 101, 102, 103 pericarditis, 100, 102 peripheral blood, 119, 208, 220, 245 peripheral vascular disease, 219 permeability, 159, 160, 165, 217 permit, 209 peroxidation, vii, viii, 1, 5, 17, 27, 29, 30, 31, 40, 55, 71, 75, 76, 120, 126, 189, 215, 243, 245 peroxide, 20, 39, 53, 80, 97, 104, 142, 143, 146, 166, 180, 189, 208, 216 peroxide radical, 104 peroxynitrite, 148, 163, 167, 168, 169, 176, 209, 210, 214, 216 perspective, 188 pH, 20, 74, 105, 150, 220 pharmaceuticals, 33 pharmacokinetics, 9, 129, 134 pharmacology, 81 pharmacotherapy, 209 phenol, 40, 44, 53, 54, 57, 62, 69, 73 phenotype, 183, 199, 206 pheochromocytoma, 5, 15, 201 phospholipids, viii, 17, 76, 79, 149, 151, 169, 184, 187 phosphorylation, 78, 85, 86, 148, 157, 166, 167, 181 photons, 95 phycoerythrin, 71 physical activity, 182, 201, 224, 244 physiology, 118, 141 pigs, 10, 12, 159, 162, 175 pilot study, 117, 134, 223, 237 pine, 162 pineal hormone melatonin, 130, 131 placebo, 123, 126, 134, 135, 138, 156, 158, 159, 162, 171, 173, 175 placenta, 184 planning, 95 plant sterols, 156 plants, 59, 60, 66, 67, 109, 129, 157, 173, 189 plaques, 159, 173 plasma, 8, 9, 10, 12, 15, 32, 52, 70, 71, 82, 85, 104, 106, 110, 114, 120, 121, 129, 147, 151, 157, 158, 160, 163, 164, 169, 170, 184, 188, 215, 220, 231, 232, 241, 243, 244 plasma levels, 110, 164, 169 plasma membrane, 15, 147 plasma proteins, 157 platelet aggregation, 157, 172, 189 platelets, 169

Index platinum, 111 pleurisy, 163 PM, 140, 170, 236 pneumonitis, 100, 102, 112, 134 point mutation, 201 Poland, 17, 19, 65 polarity, 114 pollutants, 160, 184 pollution, 36 polonium, 94 polydipsia, 200 polymerase, 118, 193, 195, 204, 208, 210, 217, 239, 242 polymerization, 29, 68 polymers, 195 polymorphism, 208 polyunsaturated fat, 104, 143, 150, 155 polyunsaturated fatty acids, 104, 143, 150 polyuria, 200 poor, 110 population, xi, 60, 66, 136, 161, 162, 163, 170, 177, 180, 193, 199, 206, 217, 234, 235 port of entry, 92 portal vein, 77, 114, 129 positive correlation, 162 positive relation, 80 positive relationship, 80 potato, 52 power, 44, 56, 82 preference, 183 pregnancy, 202 preparation, 43, 52, 55, 72, 120, 143 pressure, 48 prevention, viii, 18, 30, 56, 86, 105, 110, 112, 115, 117, 122, 123, 126, 127, 129, 136, 138, 139, 140, 142, 151, 152, 158, 168, 171, 172, 174, 186, 209, 236, 240, 243 primary tumor, 91, 110, 132 primate, 173 priming, 117 principle, viii, 2, 6, 10, 97, 152, 194 probe, 4 probiotic, 42 proctitis, 102 production, vii, ix, x, 4, 13, 15, 18, 31, 38, 42, 56, 64, 65, 66, 68, 69, 84, 86, 90, 96, 97, 104, 109, 117, 118, 130, 132, 145, 147, 152, 160, 161, 163, 165, 166, 169, 170, 181, 182, 183, 185, 186, 187, 199, 201, 203, 208, 210 prognosis, 235 program, 174

261

proliferation, x, 5, 13, 15, 76, 78, 107, 109, 110, 112, 115, 121, 134, 135, 141, 143, 145, 146, 147, 151, 163, 190, 205 promoter, 78, 86, 195 propagation, 30, 75, 118, 149, 150, 151 prophylactic, 39, 109 proposition, 115 prostate, 3, 13, 70, 79, 115, 116, 126, 137, 139 prostate cancer, 115, 116, 137, 139 prostate carcinoma, 126 protective mechanisms, 105, 136 protective role, 76, 118, 140, 153, 161, 162 protein kinase C, 3, 148, 152, 167, 170, 181, 202, 203, 204, 210 protein kinases, 146, 148, 181 protein oxidation, 75 protein synthesis, 33, 185 proteins, ix, xi, 4, 6, 29, 38, 48, 65, 66, 75, 77, 78, 79, 85, 101, 117, 118, 132, 147, 148, 149, 151, 160, 167, 179, 181, 183, 184, 185, 190, 191, 193, 194, 195, 196, 199, 206, 214, 238 proteinuria, 219 proteoglycans, 205 proteolytic enzyme, 147 protons, 91, 96 psoriasis, 182 public health, 201, 202 pulse, 34 purines, 193, 207 pyrimidine, 192 pyrophosphate, 192

Q quality of life, 91, 108 quinone, 5, 6, 7, 8, 11, 55, 68, 80, 149, 168, 187

R radiation, x, 77, 89, 90, 91, 92, 93, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 108, 109, 110, 111, 112, 113, 114, 115, 116, 118, 119, 121, 122, 123, 124, 125, 126, 127, 128, 129, 133, 134, 136, 137, 138, 139, 140, 142, 182, 190, 191, 195, 196, 217 Radiation, x, 89, 90, 93, 95, 96, 99, 100, 101, 102, 103, 121, 122, 124, 125, 126, 133, 144 radiation cystitis, 103 radiation damage, 96, 97, 98, 99, 101, 104, 121, 123, 128, 140 radiation therapy, x, 89, 90, 91, 104, 105, 106, 108, 111, 114, 115, 117, 122, 126, 128, 129, 138, 142

262

Index

radical formation, ix, 33, 65, 117, 123, 151 radical reactions, 18, 76, 150 radio, x, 89, 91, 106, 115, 121, 134 radiosensitization, 111, 116 radiotherapy, 91, 92, 94, 95, 97, 98, 99, 100, 101, 104, 105, 106, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 119, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 134, 136, 142 radium, 92, 94 rancid, 50 range, 9, 38, 57, 148, 157, 187, 188, 192, 207 raw materials, 38 reactive arthritis, 211 reactive oxygen, vii, x, xi, 1, 4, 5, 10, 12, 15, 18, 33, 73, 74, 77, 80, 83, 87, 89, 90, 117, 118, 123, 132, 137, 140, 145, 146, 151, 165, 166, 167, 174, 176, 179, 180, 191, 194, 214, 234 receptors, 3, 133, 148, 169, 181, 205 recognition, 190, 195, 202, 245 recovery, 121, 134, 140, 195 rectum, 211 recurrence, 110, 212 recycling, 122, 158 red blood cells, 85, 237, 245 redistribution, 97 reduction, vii, 7, 30, 40, 52, 69, 72, 73, 80, 81, 116, 122, 146, 151, 152, 153, 157, 158, 159, 164, 166, 172, 180, 184 reflection, 207 regenerate, 187, 188, 189 regeneration, 97, 100, 113, 216 regression, 138, 170, 173 regression analysis, 170 regulation, 107, 108, 131, 146, 147, 148, 151, 165, 166, 168, 174, 195, 196, 197, 214, 236, 237, 239 regulations, 125 regulators, 197 relationship, xi, 52, 76, 84, 98, 161, 170, 180, 193, 206, 220, 229, 242 relationships, 13, 33, 72, 83 relatives, 242 relaxation, 174, 181, 196 relevance, 138, 236 reliability, 238 remission, 112, 211 renal failure, 200 renin, 209 repair, xi, 90, 97, 98, 104, 110, 111, 117, 118, 149, 151, 180, 190, 191, 192, 193, 194, 195, 196, 198, 207, 208, 217, 218, 220, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 238, 239, 240, 244, 245 replication, 190, 193, 195, 198

resection, 91, 212, 214 residues, 75, 76, 149, 187, 195 resistance, 53, 76, 85, 98, 113, 114, 124, 133, 136, 173, 191, 201 resources, 60 respiration, 13, 194 respiratory, 52, 99, 140, 162, 174, 175, 187 responsiveness, 117 restenosis, 146, 159, 173, 174 retardation, 191 retention, 45, 50, 53, 176 retina, 109, 135, 207 retinol, 33, 109, 110, 111, 132, 134 retinopathy, 101, 200, 219, 227 reverse transcriptase, 118 rheumatoid arthritis, x, 3, 4, 14, 145, 146, 147, 163, 164, 175, 176, 177, 182, 185, 193, 236 rhythm, 129 ribose, 195, 196, 204, 208, 210, 217, 239, 242 ribosome, 118 rice, viii, 17, 37, 39, 51 rings, 8, 67, 68, 69, 223 risk, ix, 3, 11, 12, 13, 37, 42, 47, 60, 64, 66, 77, 109, 110, 114, 115, 133, 136, 152, 153, 155, 160, 161, 162, 163, 164, 169, 170, 171, 172, 175, 176, 177, 193, 206, 209, 214, 216, 235, 242 risk factors, 12, 161, 209, 235 RNA, 111, 118 rodents, 77 room temperature, 50 rubella, 201 rural population, 38

S safety, 153 salicylates, 219 salivary glands, 99, 106, 128 salmon, 37 salts, 119, 141 sample, 21, 50, 170, 219 saponin, 39 scattering, 96 school, 229 sclerosis, 76, 103 scores, 164 seafood, 37 secretion, 60, 107, 128, 201 security, 38 seed, 92 selectivity, 33, 235

Index selenium, vii, x, 36, 51, 59, 61, 90, 104, 105, 115, 116, 122, 123, 124, 127, 137, 138, 139, 140, 156, 162, 164, 176 self, 175, 211 self-concept, 211 senescence, 199, 239, 244 sensitivity, 41, 98, 110, 116, 133, 139, 140, 208 separation, 67 sepsis, 103, 186, 237 septic shock, 131, 186, 237 series, 61, 69, 122, 190 serotonin, 120, 142 serum, viii, 17, 20, 21, 22, 23, 26, 27, 28, 29, 33, 39, 70, 76, 84, 101, 109, 132, 152, 162, 163, 164, 176, 177, 207 severity, 106, 119, 151, 159, 160, 169, 201 sex, 164, 219, 220, 232 sexual development, 117 shares, 194 shear, 167 shellfish, 38 shock, 167, 190, 195, 196 shoot, 66 shortage, 99 short-term memory, 119 shrimp, 64 siblings, 206, 217 side effects, 42, 99, 101, 104, 106, 116, 129, 158, 162, 198 sign, 101 signal transduction, 4, 9, 13, 78, 147, 165, 181, 197, 199, 205 signaling pathway, 109, 167 signalling, 118, 141, 166, 180, 181, 195 signals, x, 145, 147, 148, 166 similarity, 70, 198 simulation, 92 sites, 85, 117, 118, 129, 190, 192, 193, 207, 208 skeletal muscle, 194 skin, 45, 47, 75, 78, 79, 85, 91, 92, 94, 99, 106, 110, 113, 116, 117, 118, 120, 124, 128, 136, 139, 141, 168, 200, 206, 211 skin cancer, 91, 110, 116, 139 Slovakia, 244 small intestine, 70, 77, 83 smog, 160 smokers, 77, 86, 155, 156 smoking, vii, 154, 175, 209 smooth muscle, 76, 167, 169, 181, 205 smooth muscle cells, 76, 167, 169, 205 sodium, 21, 115, 117, 124, 127, 134, 137, 140, 215 solid tumors, 113, 136, 138 solubility, 114

263

solvents, 114 somatic cell, 194, 199 somatic mutations, 191 Southeast Asia, 42 soybean, 37, 63 species, vii, x, xi, 1, 4, 5, 8, 10, 12, 15, 18, 33, 44, 53, 55, 58, 59, 61, 73, 74, 77, 80, 83, 87, 89, 90, 103, 114, 117, 118, 122, 127, 132, 136, 137, 140, 141, 145, 146, 149, 151, 160, 165, 166, 167, 174, 176, 179, 180, 190, 191, 194, 234 specificity, 7, 122 spectrophotometric method, viii, 17 spectrophotometry, 7 spectrum, vii, viii, 1, 3, 10, 39, 41, 128 speed, 95 speed of light, 95 spin, 7 spinal cord, 100, 157, 172 spine, 100, 101 spleen, 70, 187 spore, 55 Sprague-Dawley rats, 132, 139 squamous cell, 110, 111, 116, 117, 129, 133 squamous cell carcinoma, 110, 111, 116, 117, 129, 133 stability, 8, 117, 191, 195, 196, 209 stabilization, 7, 239 stages, vii, 1, 98, 99 standard deviation, 221, 222 standards, 31, 125 starch, 52 statin, 187 stem cells, 99 stenosis, 100, 101, 102, 103, 212, 218, 219 steroids, 61 stimulus, 197, 210 stomach, 3, 42, 61, 79, 110, 112, 132 storage, 44, 45, 53, 57, 61, 64 strategies, x, 90, 175 strength, 40 stress, viii, x, xi, 4, 5, 11, 14, 15, 18, 29, 30, 36, 78, 90, 104, 109, 112, 120, 121, 122, 131, 145, 146, 148, 149, 160, 161, 163, 164, 165, 166, 167, 171, 174, 177, 180, 183, 185, 187, 193, 195, 197, 198, 199, 200, 203, 206, 207, 209, 214, 216, 217, 222, 228, 232, 234, 235, 236, 240, 241, 242, 243, 244 striatum, 30 stroke, 3, 155, 158, 182, 183, 190, 200 stromal cells, 98, 214 styrene, 240 subacute, 100, 101 substitution, 21, 206 substrates, 67, 68, 192

264

Index

subtraction, 215 sucrose, 20 sugar, 51, 61, 191, 200, 206 sulfur, 105, 158 sulphur, 61, 117 Sun, 32, 36, 37, 54, 59, 63, 117, 124, 134, 140, 143 supply, 9 suppression, 14, 33, 75, 83, 163, 189 surface area, 30 survival, x, 89, 91, 97, 106, 107, 108, 111, 113, 115, 116, 121, 126, 128, 129, 131, 135, 140, 166, 196 survival rate, 108, 111 susceptibility, x, xi, 82, 85, 145, 146, 149, 160, 168, 174, 180, 185, 199, 206, 208, 226, 229, 234 swelling, 163, 164 switching, 195 Switzerland, 166 symptoms, 3, 42, 91, 108, 109, 111, 114, 121, 156, 162, 163, 164, 175, 199, 209, 211, 215 synchronization, 98 syndrome, 102, 184, 190, 191, 193, 199, 201, 239, 240 synergistic effect, 38, 111, 117 synovial fluid, 163 synovial membrane, 163 synovial tissue, 163, 164 synthesis, 37, 67, 98, 115, 116, 117, 118, 119, 129, 148, 166, 169, 185, 187, 193, 205 systemic lupus erythematosus, 193, 201 systems, vii, ix, x, 1, 4, 5, 9, 34, 39, 65, 74, 89, 90, 104, 112, 122, 125, 158, 181, 183, 241

T T cell, 78, 86, 125, 160 T lymphocytes, 125, 181 Taiwan, 56 tamoxifen, 108, 113, 131, 136 tannins, 74 targets, 3, 9, 93, 183, 243 TBI, 121 technician, 92 technology, 81, 93 teeth, 50 telangiectasia, 101 temperature, 39, 43, 44, 48, 52, 60, 61, 64, 68 temporal lobe, 115, 137 tension, 8 terpenes, ix, 35, 142 tetrachlorodibenzo-p-dioxin, 132 TGF, 78 theory, 199, 223, 224 therapeutic agents, 157

therapeutics, 159, 174, 235 therapy, x, 33, 86, 89, 90, 92, 108, 109, 111, 114, 117, 122, 123, 124, 126, 127, 133, 134, 135, 146, 152, 164, 165, 171, 172, 177, 187, 197, 209, 210, 211, 214, 219, 235, 236, 241, 243 thermal treatment, 45 thiazolidinediones, 209 thinking, 36, 45 Third World, 58 threshold, 202 thrombocytopenia, 103 thrombosis, 151 thrombus, 157, 189 thymine, 191, 193 thyroid, 70, 147 thyroiditis, 101, 201 tibia, 108 tics, 162 time, 24, 43, 44, 45, 52, 59, 64, 75, 76, 97, 98, 105, 106, 107, 115, 118, 121, 122, 123, 126, 148, 202, 208, 211, 212, 214, 217, 219 tissue, viii, ix, x, 2, 6, 9, 10, 23, 24, 25, 30, 43, 65, 90, 91, 92, 94, 96, 97, 98, 99, 101, 102, 103, 104, 105, 106, 107, 112, 113, 114, 116, 117, 118, 119, 121, 122, 127, 134, 147, 152, 160, 163, 165, 166, 184, 187, 190, 192, 193, 195, 199, 201, 205, 211, 214, 216, 236, 237, 241 tissue homeostasis, 99 TLR4, 148, 167 TNF, 147, 148, 163, 166, 205 TNF-alpha, 166 TNF-α, 205 tobacco, 86, 132, 160, 190 tobacco smoke, 160, 190 tocopherols, 39, 48, 53, 114 total cholesterol, 159 total parenteral nutrition, 211 toxic effect, 5, 52, 112 toxicity, x, 15, 39, 89, 90, 99, 106, 107, 111, 112, 113, 114, 116, 119, 122, 123, 126, 129, 134, 135, 138, 139, 140, 142 TPA, 78, 83, 141 trace elements, 51, 54 trachea, 13, 160 transcription, 11, 12, 78, 118, 146, 148, 149, 165, 166, 168, 181, 188, 195, 196, 197, 198, 201, 203, 204 transcription factors, 78, 146, 148, 166, 188, 195, 197, 198 transducer, 78 transduction, 237 transferrin, 149, 183, 216 transformation, 68, 77, 78, 86, 113, 117

Index transformations, 68 transforming growth factor, 78 transition, ix, 30, 65, 72, 73, 117, 149, 180, 183, 189, 197, 216 transition metal, ix, 30, 65, 72, 73, 117, 149, 180, 183, 189, 216 transition metal ions, ix, 30, 65, 72, 73, 216 translocation, 78, 131 transplantation, 110, 154, 161, 182, 190 transport, 41, 180, 182, 185, 187, 202, 203, 210 transversion mutation, 207 trauma, 166, 201 treatment methods, 91 trend, xi, 50, 180, 198, 234 trial, 113, 116, 126, 127, 129, 131, 134, 135, 137, 138, 154, 155, 164, 171, 173, 175, 209 triggers, 197, 198 tryptophan, 75 tuberculosis, 39 tumor, 13, 15, 34, 37, 56, 77, 78, 79, 84, 86, 90, 91, 93, 94, 97, 98, 105, 106, 107, 109, 110, 111, 112, 113, 116, 121, 122, 129, 133, 136, 139, 140, 144, 157, 166, 167 tumor cells, 15, 78, 91, 97, 105, 111, 113, 116, 121, 144 tumor growth, 107, 109, 113 tumor necrosis factor, 166, 167 tumors, 59, 61, 78, 79, 80, 91, 94, 97, 98, 105, 107, 109, 110, 111, 112, 113, 119, 120, 130, 133, 134, 139 tumours, 32, 116, 199 turnover, 183 type 1 diabetes, 206, 207, 208, 217, 218, 234, 240, 241, 242, 245 type 2 diabetes, 3, 201, 208, 218, 234, 240, 242 tyrosine, 3, 15, 112, 146, 157, 167, 181, 201

U UK, 125 ulcer, 94, 101, 102, 103, 111 ulcerative colitis, 211, 217, 244 ultrasonography, 154 ultraviolet irradiation, 139 United States, 42, 105, 160, 170, 174 uranium, 94 urethral strictures, 103 urethritis, 103 uric acid, 72, 151, 183, 215 urinary tract, 3, 63 urinary tract infection, 63 urine, 70, 71, 82, 193, 207, 225 UV, 36, 75, 78, 110, 116, 196

265

UV irradiation, 110, 116 UV light, 78, 196 uveitis, 211

V vaginitis, 103 validation, 238 values, 10, 21, 39, 42, 71, 73, 75, 108, 222, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233 variable, 195, 201 variables, 54 variance, 223, 229 variation, 41, 67, 140, 184 vascular cell adhesion molecule, 167 vascular diseases, 146, 187 vascular wall, 189 vasculature, 165, 166 vasomotor, 151 VCAM, 147, 148, 165, 166, 205 vector, 3 vegetables, viii, 2, 3, 12, 18, 35, 36, 37, 38, 39, 43, 44, 45, 46, 47, 54, 58, 60, 61, 62, 63, 64, 71, 157, 161, 186, 188 VEGF expression, 167 vein, 16 ventilation, 181 venules, 181 vertebrates, 129 vertigo, 119 vessels, 19, 99, 100, 152, 214 viral infection, 147, 160 viruses, 77 vision, 117, 235 vitamin A, 104, 105, 109, 110, 111, 122, 131, 132, 133, 134, 155, 156, 164, 215 vitamin C, vii, viii, 14, 17, 21, 23, 26, 37, 44, 47, 52, 57, 72, 73, 74, 79, 112, 113, 114, 134, 135, 136, 151, 152, 153, 154, 155, 156, 159, 161, 164, 169, 170, 171, 175, 188, 189, 206, 209 vitamin E, vii, x, 21, 26, 37, 48, 52, 61, 71, 72, 73, 74, 89, 90, 107, 112, 113, 117, 123, 124, 130, 132, 135, 137, 138, 140, 149, 151, 152, 153, 154, 155, 157, 158, 159, 161, 163, 164, 169, 170, 171, 172, 177, 187, 188, 209, 237 vitamin K, 113, 114, 135 vitamin supplementation, 138, 171 vitamins, vii, x, xi, 26, 33, 36, 37, 38, 50, 52, 79, 90, 104, 110, 113, 114, 131, 132, 134, 135, 136, 138, 145, 147, 151, 152, 153, 154, 155, 161, 164, 165, 170, 171, 172, 177, 215 vitiligo, 201 VLDL, 76

266

Index

voice, 111 vomiting, 106 vulvitis, 103

X

W water, x, 18, 19, 20, 33, 37, 40, 43, 44, 45, 48, 53, 55, 64, 71, 75, 89, 90, 96, 97, 104, 112, 146, 151, 159, 162, 181, 184, 216 wavelengths, 95 weight loss, 121, 152 well-being, 112 Western Europe, 42 wheat, 37, 45, 50, 51, 58, 61, 184 wheat germ, 37 white blood cells, 86 women, 126, 152, 153, 154, 155, 161, 164, 170, 177, 207, 219 words, 180 work, x, 10, 36, 50, 90, 94, 104, 122, 203 workers, 121, 163, 199 wound healing, 117, 118, 119, 149

xenografts, 119, 139 xeroderma pigmentosum, 190 xerostomia, 105, 106, 122 X-irradiation, 135, 136 x-rays, 93, 96

Y yeast, 37, 39 yield, 45, 58, 63 young adults, 211

Z zinc, x, 58, 90, 104, 105, 117, 118, 119, 122, 123, 124, 127, 140, 141, 142, 166 zinc sulfate, 127