Leading Edge Antioxidants Research

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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

ix

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

Preface

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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,


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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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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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

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[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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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.

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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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.

Are Teas the Universal Antioxidants

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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.



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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Mustafa Vecdi Ertekin and Orhan Sezen Table 4. Continued.

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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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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Jayraz Luchoomun, Dominic Sinibaldi and Xi-Lin Chen

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-

Antioxidant Therapy for Chronic Inflammatory Diseases

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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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[199] Venkatraman JT, Chu WC. Effects of dietary omega-3 and omega-6 lipids and vitamin E on serum cytokines, lipid mediators and anti-DNA antibodies in a mouse model for rheumatoid arthritis. J Am Coll Nutr. 1999;18:602-613. [200] Sakai A, Hirano T, Okazaki R, et al. Large-dose ascorbic acid administration suppresses the development of arthritis in adjuvant-infected rats. Arch Orthop Trauma Surg. 1999;119(3-4):121-126. [201] Pattison DJ, Symmons DP, Lunt M, et al. Dietary beta-cryptoxanthin and inflammatory polyarthritis: results from a population-based prospective study. Am J Clin Nutr. Aug 2005;82(2):451-455. [202] Cerhan JR, Saag KG, Merlino LA, et al. Antioxidant micronutrients and risk of rheumatoid arthritis in a cohort of older women. Am J Epidemiol. Feb 15 2003;157(4):345-354. [203] Paredes S, Girona J, Hurt-Camejo E, et al. Antioxidant vitamins and lipid peroxidation in patients with rheumatoid arthritis: association with inflammatory markers. J Rheumatol. Nov 2002;29(11):2271-2277. [204] Helmy M, Shohayeb M, Helmy MH, et al. Antioxidants as adjuvant therapy in rheumatoid disease. A preliminary study. Arzneimittelforschung. 2001;51(4):293-298. [205] Kunsch C. Oxidative stress and the use of antioxidants for the treatment of rheumatoid arthritis. Curr Med Chem - Endoc. Mtabl Agents. 2005;5:249-258.



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.

Impact of Oxidative Stress on Diabetes Mellitus and Inflammatory Bowel Diseases 187

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 5ánd 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 – 5ńuclease) 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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Jana Varvařovská, Rudolf Štětina, Josef Sýkora et al. acetylglucosamine, DHAP –dihydroxyacetone phosphate, DAG-diacylglycerol (activator of protein kinase C)

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)


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].


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)


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


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 DÍnca 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 [DÓdorico]. 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)


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

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