Nutrigenomics (Oxidative Stress and Disease)

Nutrigenomics

edited by

Gerald Rimbach Jürgen Fuchs Lester Packer

Boca Raton London New York Singapore

A CRC title, part of the Taylor & Francis imprint, a member of the Taylor & Francis Group, the academic division of T&F Informa plc.

Published in 2005 by CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2005 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 0-8247-2663-4 (Hardcover) International Standard Book Number-13: 978-0-8247-2663-8 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe.

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Series Introduction

Oxygen is a dangerous friend. Through evolution, oxygen—itself a free radical— was chosen as the terminal electron acceptor for respiration. The two unpaired electrons of oxygen spin in the same direction; thus, oxygen is a biradical. Other oxygen-derived free radicals, such as superoxide anion or hydroxyl radicals, formed during metabolism or by ionizing radiation are stronger oxidants, i.e., endowed with a higher chemical reactivity. Oxygen-derived free radicals are generated during oxidative metabolism and energy production in the body and are involved in regulation of signal transduction and gene expression, activation of receptors and nuclear transcription factors, oxidative damage to cell components, the antimicrobial and cytotoxic action of immune system cells, neutrophils and macrophages, as well as in aging and age-related degenerative diseases. Overwhelming evidence indicates that oxidative stress can lead to cell and tissue injury. However, the same free radicals that are generated during oxidative stress are produced during normal metabolism and, as a corollary, are involved in both human health and disease. In addition to reactive oxygen species, research on reactive nitrogen species has been gathering momentum to develop an area of enormous importance in biology and medicine. Nitric oxide or nitrogen monoxide (NO) is a free radical generated by nitric oxide synthase (NOS). This enzyme modulates physiological responses in the circulation such as vasodilation (eNOS) or signaling in the brain (nNOS). However, during inflammation, a third isoenzyme is induced, iNOS, resulting in the overproduction of NO and causing damage to targeted infectious organisms and to healthy tissues in the vicinity. More worrisome, however, is the fact that NO can react with superoxide anion to yield a strong oxidant, peroxynitrite. Oxidation of lipids, proteins, and DNA by peroxynitrite increases the likelihood of tissue injury. iii

iv

Series Introduction

Both reactive oxygen and nitrogen species are involved in the redox regulation of cell functions. Oxidative stress is increasingly viewed as a major upstream component in the signaling cascade involved in inflammatory responses and stimulation of adhesion molecule and chemoattractant production. Hydrogen peroxide decomposes in the presence of transition metals to the highly reactive hydroxyl radical, which by two major reactions—hydrogen abstraction and addition—accounts for most of the oxidative damage to proteins, lipids, sugars, and nucleic acids. Hydrogen peroxide is also an important signaling molecule that, among others, can activate NF-kB, an important transcription factor involved in inflammatory responses. At low concentrations, hydrogen peroxide regulates cell signaling and stimulates cell proliferation; at higher concentrations it triggers apoptosis and, at even higher levels, necrosis. Virtually all diseases thus far examined involve free radicals. In most cases, free radicals are secondary to the disease process, but in some instances free radicals are causal. Thus, there is a delicate balance between oxidants and antioxidants in health and disease. Their proper balance is essential for ensuring healthy aging. The term oxidative stress indicates that the antioxidant status of cells and tissues is altered by exposure to oxidants. The redox status is thus dependent on the degree to which a cell’s components are in the oxidized state. In general, the reducing environment inside cells helps to prevent oxidative damage. In this reducing environment, disulfide bonds (S –S) do not spontaneously form because sulfhydryl groups are maintained in the reduced state (SH), thus preventing protein misfolding or aggregation. This reducing environment is maintained by oxidative metabolism and by the action of antioxidant enzymes and substances, such as glutathione, thioredoxin, vitamins E and C, and enzymes such as superoxide dismutases, catalase, and the selenium-dependent glutathione reductase and glutathione and thioredoxin hydroperoxidases, which serve to remove reactive oxygen species (hydroperoxides). Changes in the redox status and depletion of antioxidants occur during oxidative stress. The thiol redox status is a useful index of oxidative stress mainly because metabolism and NADPH-dependent enzymes maintain cell glutathione (GSH) almost completely in its reduced state. Oxidized glutathione (glutathione disulfide, GSSG) accumulates under conditions of oxidant exposure and this changes the ratio GSSG/GSH; an increased ratio is usually taken as indicating oxidative stress. Other oxidative stress indicators are ratios of redox couples such as NADPH/NADP, NADH/NAD, thioredoxinreduced/thioredoxinoxidized , dihydrolipoic acid/a-lipoic acid, and lactate/pyruvate. Changes in these ratios affects the energy status of the cell, largely determined by the ratio ATP/ ADP þ AMP. Many tissues contain large amounts of glutathione, 2 –4 mM in erythrocytes or neural tissues and up to 8 mM in hepatic tissues. Reactive oxygen and nitrogen species can oxidize glutathione, thus lowering the levels of the most abundant nonprotein thiol, sometimes designated as the cell’s primary preventative antioxidant.

Series Introduction

v

Current hypotheses favor the idea that lowering oxidative stress can have a health benefit. Free radicals can be overproduced or the natural antioxidant system defenses weakened, first resulting in oxidative stress, and then leading to oxidative injury and disease. Examples of this process include heart disease, cancer, and neurodegenerative disorders. Oxidation of human low-density lipoproteins is considered an early step in the progression and eventual development of atherosclerosis, thus leading to cardiovascular disease. Oxidative DNA damage may initiate carcinogenesis. Environmental sources of reactive oxygen species are also important in relation to oxidative stress and disease. A few examples: UV radiation, ozone, cigarette smoke, and others are significant sources of oxidative stress. Compelling support for the involvement of free radicals in disease development originates from epidemiological studies showing that an enhanced antioxidant status is associated with reduced risk of several diseases. Vitamins C and E and prevention of cardiovascular disease are a notable example. Elevated antioxidant status is also associated with decreased incidence of cataracts, cancer, and neurodegenerative disorders. Some recent reports have suggested an inverse correlation between antioxidant status and the occurrence of rheumatoid arthritis and diabetes mellitus. Indeed, the number of indications in which antioxidants may be useful in the prevention and/or the treatment of disease is increasing. Oxidative stress, rather than being the primary cause of disease, is more often a secondary complication in many disorders. Oxidative stress diseases include inflammatory bowel diseases, retinal ischemia, cardiovascular disease and restenosis, AIDS, adult respiratory distress syndrome, and neurodegenerative diseases such as stroke, Parkinson’s disease, and Alzheimer’s disease. Such indications may prove amenable to antioxidant treatment (in combination with conventional therapies) because there is a clear involvement of oxidative injury in these disorders. In this series of books, the importance of oxidative stress and disease associated with organ systems of the body is highlighted by exploring the scientific evidence and the medical applications of this knowledge. The series also highlights the major natural antioxidant enzymes and antioxidant substances such as vitamins E, A, and C, flavonoids, polyphenols, carotenoids, lipoic acid, coenzyme Q10 , carnitine, and other micronutrients present in food and beverages. Oxidative stress is an underlying factor in health and disease. More and more evidence indicates that a proper balance between oxidants and antioxidants is involved in maintaining health and longevity and that altering this balance in favor of oxidants may result in pathophysiological responses causing functional disorders and disease. This series is intended for researchers in the basic biomedical sciences and clinicians. The potential of such knowledge for healthy aging and disease prevention warrants further knowledge about how oxidants and antioxidants modulate cell and tissue function. Lester Packer Enrique Cadenas

Preface

Nutrition research commenced more than 200 years ago in the dawn of the chemical revolution. The “golden age of nutrition” began in the early 1910s and continued into the 1940s when nutritional sciences focused primarily on diseases associated with single nutrient deficiencies. This led to the formulation of the Recommended Daily Allowance (RDA) of each nutrient. After almost all of the essential nutrients had been discovered, nutrition research focused on the problem of multifactorial chronic diseases, many of which are caused not by nutritional deficiency but by overnutrition. In the following years, the revolutionary progress in recombinant DNA technology and genomics culminated in 2001 with completion of the Human Genome Project and sequencing of the entire human genome. As a result of all these developments, genomics, transcriptomics, proteomics, and metabolomics are increasingly being used in nutritional research. Nutritional genomics, also called nutrigenomics, is an emerging field in the life sciences and is considered as one of the next frontiers in the postgenomic era. Its fundamental concept is that a healthy phenotype can develop into a chronic disease phenotype via alterations in gene expression or epigenetic phenomena and that the diet contains substances having the potential to modify these processes. Nutrigenomics focuses on the relationship between dietary nutrients and gene expression using state-of-the-art technology. The development of DNA microarrays and protein chips make large-scale genomic and proteomic investigations possible by allowing simultaneously high throughput monitoring of the expression of thousands of genes in response to diet. The emerging knowledge will aid in the understanding of how nutrients modify cancer risk, chronic diseases, and aging. It is generally recognized that most human diseases are largely avoidable by lifestyle changes. This places nutrigenomics at the forefront of preventive medicine. vii

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Preface

The present book was compiled to update the reader on recent advances in nutrigenomics with special emphasis on the gene regulatory activity of oxidants, antioxidants, phytochemicals, and micronutrients in human health and disease. Gerald Rimbach Ju¨rgen Fuchs Lester Packer

About the Editors

GERALD RIMBACH is Professor of Food Science and Director of the Institute of Human Nutrition and Food Science, Christian Albrechts University, Kiel, Germany. Prior his appointment in Kiel, he worked at the University of California, Berkeley, and was Lecturer at the University of Reading, United Kingdom. Dr. Rimbach is a member of the German and British Nutrition Society and the Society of Nutrition Physiology (GfE) and is the vice-president of the German Society for Quality Research of Plant Food (DGQ). He is the author, coauthor or coeditor of over 100 journal articles and book chapters. Dr. Rimbach received the M.Sc. and Ph.D. degrees from the University Giessen, Germany. ¨ RGEN FUCHS is Professor, Department of Dermatology, Johann Wolfgang JU Goethe-Universita¨t, Frankfurt, Germany. The author of approximately 250 published articles and the coeditor of Environmental Stressors in Health and Disease, Lipoic Acid in Health and Disease, Oxidative Stress in Dermatology, Vitamin C in Health and Disease, and Vitamin E in Health and Disease (all titles, Marcel Dekker, Inc.), he received the Ph.D. (1985) and M.D. (1986) degrees from the University of Frankfurt, Germany. LESTER PACKER is Adjunct Professor of Molecular Pharmacology and Toxicology, University of Southern California School of Pharmacy, Los Angeles. He is the author, coauthor, or coeditor of over 800 journal articles and book chapters, and many books including Antioxidants in Diabetes Management; Environmental Stressors in Health and Disease; Flavonoids in Health and Disease; Free Radicals in Brain Pathophysiology; Handbook of Antioxidants, Second Edition; Redox Regulation of Cell Signaling and Its Clinical Application; ix

x

About the Editors

Vitamin C in Health and Disease; and Vitamin E in Health and Disease (all titles, Marcel Dekker, Inc.). Dr. Packer received the B.S. (1951) and M.S. (1952) degrees from Brooklyn College, New York, and the Ph.D. degree (1956) from Yale University, New Haven, Connecticut.

Contributors

R. Ambra Italy

National Institute for Food and Nutrition Research (INRAN), Rome,

Max O. Bingham Food Microbial Sciences Unit, School of Food Biosciences, University of Reading, Reading, UK Raymond K. Blanchard Nutritional Genomics Laboratory, Food Science and Human Nutrition Department, Center for Nutritional Sciences, University of Florida, Gainesville, Florida, USA R. Canali Italy

National Institute for Food and Nutrition Research (INRAN), Rome,

Aedin Cassidy School of Medicine, Health Policy & Practice, University of East Anglia, Norwich, UK Rainer Cermak Institute of Animal Nutrition and Physiology, Christian Albrechts University, Kiel, Germany Kyung-Joo Cho Laboratory of Cell Biology, Korea Advanced Institute of Science and Technology, Daejeon, South Korea An-Sik Chung Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, South Korea Robert J. Cousins Nutritional Genomics Laboratory, Food Science and Human Nutrition Department, Center for Nutritional Sciences, University of Florida, Gainesville, Florida, USA S. G. Cremers School of Animal and Microbial Sciences, University of Reading, Reading, UK xi

xii

Contributors

Sonia De Pascual-Teresa Department of Plant Food Science and Technology, Instituto del Frı´o, Consejo Superior de Investigaciones Cientificas, Madrid, Spain School of Food Biosciences, University of Reading, Reading, UK

B. A. Ewins

Alexandra Fischer Department of Cardiovascular Medicine, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK Glenn R. Gibson Food Microbial Sciences Unit, School of Food Biosciences, University of Reading, Reading, UK R. D. Gill-Garrison K. Grimaldi

Sciona, Ltd., Havant, UK

Sciona, Ltd., Havant, UK

Robert Francis Grimble Institute of Human Nutrition, Biomedical Sciences Building, University of Southampton, Southampton, UK O. Gulati

Horphag Research Ltd., Geneva, Switzerland

Shuji Honda Aging Redox Regulation Research Group, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan Yoko Honda Aging Redox Regulation Research Group, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan John K. Lodge School of Biomedical and Molecular Sciences, University of Surrey, Guildford, Surrey, UK Silvia Mandel Eve Topf and USA National Parkinson Foundation Centers of Excellence for Neurodegenerative Diseases Research and Department of Pharmacology and Rappaport Family Research Institute, Technion-Faculty of Medicine, Haifa, Israel Anne M. Minihane Hugh Sinclair Unit of Human Nutrition, University of Reading, Reading, UK Ken Mills Department of Haematology, Wales College of Medicine, Cardiff University, Cardiff, Wales, UK Hadi Moini Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, Los Angeles, California, USA B. A. Nier School of Animal and Microbial Sciences, University of Reading, Reading, UK Estibaliz Olano-Martin Reading, UK

School of Food Biosciences, University of Reading,

Contributors

xiii

Lester Packer Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, Los Angeles, California, USA Josef Pallauf Institute of Animal Nutrition and Nutrition Physiology, JustusLiebig-University of Giessen, Giessen, Germany Gerald Rimbach Institute of Human Nutrition and Food Science, Christian Albrechts University, Kiel, Germany Cristina Rota Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy Abulkalam M. Shamsuddin Professor of Pathology, The University of Maryland School of Medicine, Baltimore, Maryland, USA Jeremy P. E. Spencer Reading, UK

School of Food Biosciences, University of Reading,

Paul Sharp

School of Health and Life Sciences, King’s College, London, UK

J. L. Slater

Sciona, Ltd., Havant, UK

F. Virgili Italy

National Institute for Food and Nutrition Research (INRAN), Rome,

Johannes von Lintig Department of Animal Physiology and Neurobiology, Institute for Biology I, University of Freiburg, Freiburg, Germany Stefan Weber Klinik und Poliklinik fu¨r Ana¨sthesiologie und Operative Intensivmedizin, Universita¨tsklinikum Bonn, Rheinische Friedrich-WilhelmsUniversita¨t, Bonn, Germany Peter D. Weinberg Physiological Flow Studies Group, Department of Bioengineering, Imperial College, London, UK Orly Weinreb Eve Topf and USA National Parkinson Foundation Centers of Excellence for Neurodegenerative Diseases Research and Department of Pharmacology and Rappaport Family Research Institute, Technion-Faculty of Medicine, Haifa, Israel Siegfried Wolffram Institute of Animal Nutrition and Physiology, Christian Albrechts University, Kiel, Germany Moussa B. H. Youdim Eve Topf and USA National Parkinson Foundation Centers of Excellence for Neurodegenerative Diseases Research and Department of Pharmacology and Rappaport Family Research Institute, Technion-Faculty of Medicine, Haifa, Israel

Contents

Series Introduction

.......................................

iii

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . About the Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii ix

Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xi

1.

Application of Nutrigenomics Tools to Analyze the Role of Oxidants and Antioxidants in Gene Expression . . . . . . . . . . . . . . . Gerald Rimbach and Sonia De Pascual-Teresa

1

2.

Oxidative Stress and Human Genetic Variation . . . . . . . . . . . . . . R. D. Gill-Garrison, J. L. Slater, and K. Grimaldi

13

3.

Analysis of Microarray Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ken Mills

43

4.

Oxidative Stress, Gene Expression, and Lifespan . . . . . . . . . . . . . Yoko Honda and Shuji Honda

67

5.

Anti-Oxidant Modulation in Immune Function . . . . . . . . . . . . . . . Robert Francis Grimble

97

6.

Concentration-Dependent Gene and Protein Expressions of Neuroprotective and Neurotoxic Activities of Antioxidants, Including Nutrients . . . . . . . . . . . . . . . . . . . . . . . . . 123 Orly Weinreb, Silvia Mandel, and Moussa B. H. Youdim xv

xvi

Contents

7.

Effects of Antioxidants on Gene Expression in Endothelial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 B. A. Nier, B. A. Ewins, S. G. Cremers, and Peter D. Weinberg

8.

Fatty Acids, Gene Expression, and Coronary Heart Disease (CHD) . . . . . . . . . . . . . . . . . . . . . . . . . . 181 Anne M. Minihane

9.

Cell Regulatory Activity of Tocopherols and Tocotrienols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 Cristina Rota, Anne M. Minihane, Peter D. Weinberg, Stefan Weber, John K. Lodge, Lester Packer, and Gerald Rimbach

10.

Molecular Analysis of the Vitamin A Biosynthetic Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 Johannes von Lintig

11.

Molecular Mechanisms Underlaying the Health Promoting Activity of Lycopene . . . . . . . . . . . . . . . . . . . . . . . . . 241 Estibaliz Olano-Martin

12.

Cellular Redox Activity and Molecular Functions of Ascorbic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257 John K. Lodge

13.

Cell Signaling Properties of a-Lipoic Acid: Implications in Type 2 Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . 283 Hadi Moini, Lester Packer, Kyung-Joo Cho, and An-Sik Chung

14.

Dietary Isoflavones and Coronary Artery Disease—Proposed Molecular Mechanisms of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301 Aedin Cassidy and Sonia De Pascual-Teresa

15.

Anti-Carcinogenic Properties of Soy Isoflavones . . . . . . . . . . . . . . 327 Max O. Bingham and Glenn R. Gibson

16.

Effect of Ginkgo biloba Extract EGb 761 on Differential Gene Expression in the Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341 Rainer Cermak and Siegfried Wolffram

Contents

xvii

17.

Interactions of Flavonoids and Their Metabolites with Cell Signaling Cascades . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353 Jeremy P. E. Spencer

18.

Antioxidant and Gene Regulatory Properties of Procyanidins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379 R. Canali, R. Ambra, O. Gulati, and F. Virgili

19.

Cell Signaling Properties of Inositol Hexaphosphate (IP6) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397 Abulkalam M. Shamsuddin

20.

Modulation of Gene Expression by Dietary Iron . . . . . . . . . . . . . . 421 Paul Sharp

21.

Dietary Selenium and Gene Expression . . . . . . . . . . . . . . . . . . . . 441 Alexandra Fischer and Josef Pallauf

22.

Modulation of Gene Expression by Dietary Zinc Raymond K. Blanchard and Robert J. Cousins

Index

. . . . . . . . . . . . . 457

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473

1 Application of Nutrigenomics Tools to Analyze the Role of Oxidants and Antioxidants in Gene Expression Gerald Rimbach Christian Albrechts University, Kiel, Germany

Sonia De Pascual-Teresa Instituto del Frı´o, Consejo Superior de Investigaciones Cientificas, Madrid, Spain

What is Nutrigenomics? Do Oxidants and Antioxidants Affect Gene Expression? Methods and Applications in Nutrigenomics References

1 3 4 9

WHAT IS NUTRIGENOMICS? The rapid progress in the understanding of the human genome has opened up new avenues to study interactions between diet, gene expression, genetic variability, health, and disease. Nutrigenomics considers the relationship between specific nutrients or diet and gene expression (1), whereas nutrigenetics determines how genetic variability affects the response to diet (2). Nutrigenomics is a modern discipline at the interface between genetics, molecular nutrition, molecular biology, pharmacogenomics, and molecular medicine (Fig. 1.1). 1

2

Rimbach and De Pascual-Teresa

Figure 1.1 Nutrigenomics as a discipline at the interface between genetics, molecular nutrition, molecular biology, pharmacogenomics, molecular medicine, and bioinformatics.

Nutrigenomics applies high-throughput molecular biology techniques including sequencing and genotyping (genomics), transcriptomics, proteomics, and metabolomics. Transcriptomics determines patterns of gene expression in response to a nutrient, whereas proteomics studies the effect of nutrients on protein synthesis, protein structure, and patterns of protein expression. The profile and function of metabolites are analyzed by metabolomic techniques, whereas the comprehensive data handling is finally accomplished by bioinformatic tools (Fig. 1.2). By combining information from transcriptomics, proteomics, metabolomics, and bioinformatics one can gain a comprehensive understanding of nutrient related homeostasis.

Figure 1.2 Genomics, transcriptomics, proteomics, and metabolomics as analytical tools in molecular nutrition.

Application of Nutrigenomics

3

DO OXIDANTS AND ANTIOXIDANTS AFFECT GENE EXPRESSION? It has been clearly shown that the cellular oxidant/antioxidant equilibrium is a key factor in determining redox-dependent signal transduction pathways both in vitro and in vivo. Antioxidant nutrients interact with cell receptors (e.g., isoflavones bind to estrogen receptor alpha and beta) and modulate key enzymes such as phosphatase and kinases. Changes in transcription factor activity leads to changes in mRNA and protein levels as summarized in Fig. 1.3. Furthermore, antioxidants can directly interact with enzymes (e.g., through protein-binding properties), thereby changing their activity. Molecular and cell biology has changed our understanding of how antioxidants can mediate their biological properties. A good example is vitamin E—the most important lipid-soluble antioxidant. Since its discovery, studies of the constituent tocopherols and tocotrienols have focused mainly on their antioxidant properties. In 1991, Angelo Azzi’s group first described nonantioxidant, cell signaling functions for vitamin E, demonstrating that vitamin E regulates protein kinase C activity in smooth muscle cells (3). At the transcriptional level, vitamin E modulates the expression of the hepatic a-tocopherol transfer protein (TTP) (4) as well as the expression collagenase gene (5) and a-tropomyosin gene (6). Recently, a tocopherol-dependent transcription factor (tocopherol associated protein, TAP) has been discovered (7). In cultured cells, it has been demonstrated that vitamin E inhibits inflammation, cell adhesion, platelet aggregation, and smooth muscle cell proliferation (8). Many of these cellular functions of vitamin E seem to be independent of its antioxidant properties. Thus, antioxidants do not act only as scavengers of reactive oxygen and nitrogen species, thereby

Receptors Phosphatase Kinases

Transcription Factors Oxidants

Antioxidants Gene Expression

Protein levels

Enzyme Activity

Figure 1.3 Cell receptors, cellular key enzymes, and transcription factors as molecular targets of oxidants and antioxidants.

4

Rimbach and De Pascual-Teresa

preventing oxidative damage towards lipids, protein, and DNA, they are also cell signaling molecules. Both the free-radical scavenging as well as the cell signaling activity of antioxidants may contribute to their potential beneficial effects in preventing atherogenesis, carcinogenesis, and neurodegneration (Fig. 1.4). Various transcription factors such as NF-kB, AP-1, Nrf-1, and SP-1 are regulated by the cellular redox status. NF-kB controls the expression of different genes involved in inflammatory and proliferative responses. A spectrum of key genes known to be involved in the development of atherosclerosis have been shown to be regulated by NF-kB, including those encoding for cytokines, chemoattractants, and cell adhesion proteins (8). Several lines of evidence including the inhibition caused by various antioxidants, suggest that NF-kB is subject to redox regulation. Owing to its pivotal role in inflammation and atherogenesis, a significant effort has focused on identifying nutrients that regulate NF-kB activity. In this scenario, flavonoids may play an important role, either by directly affecting key steps in the activation pathway of NF-kB, or by modulating the intracellular redox status, which is, in turn, one of the major determinants of NF-kB activation. Consistent experimental data is accumulating, which suggests that the anti-inflammatory properties of flavonoids are in part due to their ability to down-regulate NF-kB (9). METHODS AND APPLICATIONS IN NUTRIGENOMICS There is an increasing evidence indicating that antioxidants such as vitamin E, carotenoids, ascorbic acid, lipoic acids, bioflavonoids, and ginkgo biloba as well as trace elements (e.g., Zn, Fe, Se) affect differential gene expression in

Figure 1.4 Antioxidants as free-radical scavengers, metal chelators, and redox signaling molecules—both prevention of oxidative damage towards lipids, proteins, and DNA as well as redox signaling contributes to their potential beneficial effects.

Table 1.1

Epigallocatechin-3-gallate, melatonin Fish oil

Antioxidants Ascorbic acid Coenzyme Q10 Copper Epigallocatechin-3-gallate

Ozone UVB radiation

Human Human Mouse Human

Aortic smooth muscle cells Endothelial cells Lung Keratinocytes

Human Human Human Human Human Human Human Human Rat Mouse

Human

Retinal pigment epithelium cells

Keratinocytes Skeletal muscle Macrophages Cervical cancer cells Lung cancer cells Lung cancer cells Prostate carcinoma cells Neuroblastoma cells Brain Liver

Mouse Human

Species

Swiss 3T3 Breast cancer cells

Cell/Tissue

588 12,000 6,800 384 588 588 250 25 3,200 6,521

35,932 588 4,000 6,000

1,176

513 17,000

Number of genes monitored

(continued )

Catani et al. (17) Linnane et al. (18) Svensson et al. (19) Ahn et al. (20) Fujiki et al. (21) Okabe et al. (22) Wang and Mukhtar (23) Weinreb et al. (24) Kitajka et al. (25) Takahashi et al. (26)

Sukhanov et al. (13) Virgili et al. (14) Gohil et al. (15) Sesto et al. (16)

Weigel et al. (12)

Bosio et al. (10) Chuang et al. (11)

Reference

Studies on the Effect of Oxidants and Antioxidants on Differential Gene Expression in Cultured Cells, Laboratory Animals and

Oxidants Cigarette smoke Hydrogen peroxide, menadione t-butyl hydroperoxide Hydrogen peroxide 4-hydroxynonenal t-butyl hydroperoxide Oxidized LDL

in Humans

Application of Nutrigenomics 5

Continued

Vitamin E (Tocotrienol) Vitamin E and Selenium Zinc

Vitamin E

Sulphoraphane Vitamin A Vitamin A and E, selenium Vitamin D3

Genistein Indole-3-carbinol Lycopene, Vitamin E Melatonin Methylseleninic acid Proanthocyanidin extract from grape seed Procyanidins from pine bark Resveratrol Selenium

Folic acid Ginkgo biloba

Table 1.1

Human Rat Mouse Human Human Rat Rat Human Human Human Human Rat Mouse Mouse Human Rat Rat Human Mouse Rat Human Rat Rat Rat Rat

Keratinocytes Prostate cancer cells Mammary epithelial organoids Intestine Small intestine Airway tissues Skeletal muscle Osteosarcoma cells Prostate cancer cells Kidney Liver Aortic smooth muscle cells Fetal brains Liver Mucosa cells of small intestine Liver

Species

Nasopharyngeal carcinoma cells Brain Brain Prostate cancer cells Prostate cancer cells Prostate Retina Premalignant breast cells Endothelial cells

Cell/Tissue

588 2,400 588 6,347 6,000 30,000 800 5,000 20,000 12,422 7,000 10,000 8,000 465 1,185 2,500

2,200 8,000 7,000 557 22,215 7,000 24,000 316 2,400

Number of genes monitored

Rihn et al. (36) Narayanan et al. (37) Dong et al. (38) Rao et al. (39) Thimmulappa et al. (40) Soref et al. (41) Sreekumar et al. (42) Farach-Carson and Xu (43) Krishnan et al. (44) Li et al. (45) Barella et al. (46) Villacorta et al. (47) Roy et al. (48) Fischer et al. (49) Blanchard et al. (50) tom Dieck et al. (51)

Jhaveri et al. (27) Li et al. (28) Watanabe et al. (29) Suzuki et al. (30) Li et al. (31) Siler et al. (32) Wiechmann (33) Dong et al. (34) Bagchi et al. (35)

Reference

6 Rimbach and De Pascual-Teresa

Application of Nutrigenomics

7

cultured cells, in laboratory animals, and in humans. In addition, oxidative stress, induced by oxidants such as ozone, cigarette smoke, UV radiation, and oxidized LDL, is associated with changes in differential gene expression. Recent studies on the effect of oxidants and antioxidants on differential gene expressions are summarized in Table 1.1. The potential applications of transcriptomics in the field of antioxidant and free-radical research are manifold. Several methods have been developed for the quantitative and comprehensive analyses of changes in mRNA expression such as differential display, serial analysis of gene expression, DNA microarrays, and gene chips (Fig. 1.5). Gene-arrays have been used in order to analyze redox-sensitive signal transduction pathways, thereby getting more insights into the molecular functions of oxidants and antioxidants. A novel application of gene-array technology may be the development of new biomarkers of oxidative stress, which is still an open issue. Furthermore, differences in the biopotency and bioavailability among different antioxidants may be detected by gene-array technology. Finally, gene-arrays can be applied in order to study the toxicity of oxidants and antioxidants as well as for screening new antioxidants. Once candidate genes have been identified by array technology, the corresponding mRNA sequence has to be obtained from a data base (e.g., gene bank), and a blast of the sequence against the genome is performed in order to retrieve the DNA sequence, chromosomal loci, as well as the upstream sequence. This procedure is followed by a search for homology in regulatory elements and transcription factors (52) as summarized in Fig. 1.6.

Figure 1.5 Analytical techniques and potential applications of transcriptomics in the field of free-radical research.

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Identify Candidate Genes by Gene Chip Technology

Obtain mRNA Sequences from Data Base (e.g. Gene Bank) and Blast Sequence against Genome

Retrieve DNA Sequences, Chromosomal Loci and 10Kb Upstream Sequence

Search for Homology in Regulatory Elements and Transcription Factors

Describe potential signal transduction pathways

Figure 1.6 pathways.

Major steps in the identification of redox-sensitive signal transduction

In order to obtain a comprehensive understanding of the molecular mechanisms of action of oxidants and antioxidants, rigorous study designs and statistical analysis are necessary. Time-point measurements need to be introduced in order to elucidate whether differences in the gene expression profile are manifested consistently over a prolonged period of time. In previous studies, differential changes in gene expression in response to dietary treatments

Target tissue (e.g. rat liver)

RNA Extraction

Gene chip hybridization

Biotinylated cRN A

cDN A Synth esis

I mage An alysis/ Bioinformatics

Identification of C andidate Genes

Verification by R T -PCR/ Nor thern blotting

Treatment (e.g. antioxidant)

Functional Endpoint Measurements

Verification by Western blotting/ ELISA/Proteomics

Figure 1.7 Schematic representation of the analytical steps involved in a gene chip experiment.

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were often monitored only at one time-point and in pooled samples. Furthermore, differences in gene expression levels observed by gene chips technology should always be confirmed by independent methods such as real-time PCR and northern blotting, and then substantiated by functional parameters (Fig. 1.7). A set of guidelines (Minimum Information About a Micorarray Experiment, MIAME) have been established to outline the minimum information required for microarray experiments. Overall, gene expression profiling using array technology is rapidly becoming an important tool in nutrition and free-radical research. Array technology enables to study nutrient gene interactions on large scale that would be impossible using conventional analysis. Nutrigenomics has the potential to validate and to extend many of the strategies used in human nutrition on a molecular level, thereby improving human health.

REFERENCES 1. Ommen B, Stierum R. Nutrigenomics exploiting systems biology in the nutrition health arena. Curr Opinion Biotechnol 2002; 13:517 – 521. 2. Elliot R, Ong TJ. Nutritional Genomics. Br Med J 2002; 324:1438 – 1442. 3. Boscoboinik D, Szewczyk A, Azzi A. Alpha-tocopherol (vitamin E) regulates vascular smooth muscle cell proliferation and protein kinase C activity. J Biol Chem 1991; 266:6188– 6194. 4. Hosomi A, Arita M, Sato Y, Kiyose C, Ueda T, Igarashi O, Arai H, Inoue K. Affinity for alpha-tocopherol transfer protein as a determinant of the biological activities of vitamin E analogs. FEBS Lett 1997; 409:105 – 108. 5. Ricciarelli R, Maroni P, Ozer N, Zingg JM, Azzi A. Age-dependent increase of collagenase expression can be reduced by alpha-tocopherol via protein kinase C inhibition. Free Radic Biol Med 1999; 27(7 –8):729 – 737. 6. Aratri E, Spycher SE, Breyer I, Azzi A. Modulation of alpha-tropomyosin expression by alpha-tocopherol in rat vascular smooth muscle cells. FEBS Lett 1999; 447(1):91– 94. 7. Stocker A, Zimmer S, Spycher SE, Azzi A. Identification of a novel cytosolic tocopherol-binding protein: structure, specificity, and tissue distribution. IUBMB Life 1999; 48(1):49 –55. 8. Rimbach G, Minihane AM, Majewicz J, Fischer A, Pallauf J, Vigili F, Weinberg PD. Regulation of cell signalling by vitamin E. Proc Nutr Soc 2002; 61:415 – 425. 9. Saliou C, Valacchi G, Rimbach G. Assessing bioflavonoids as regulators of NF-kappa B activity and inflammatory gene expression in mammalian cells. Methods Enzymol 2001; 335:380 –387. 10. Bosio A, Knorr C, Janssen U, Gebel S, Haussmann HJ, Muller T. Kinetics of gene expression profiling in Swiss 3T3 cells exposed to aqueous extracts of cigarette smoke. Carcinogenesis 2002; 23:741 – 748. 11. Chuang YY, Chen Y, Gadisetti C, Chandramouli VR, Cook JA, Coffin D, Tsai MH, DeGraff W, Yan H, Zhao S, Russo A, Liu ET, Mitchell JB. Gene expression after treatment with hydrogen peroxide, menadione, or t-butyl hydroperoxide in breast cancer cells. Cancer Res 2002; 62:6246– 6254.

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12. Weigel AL, Ida H, Boylan SA, Hjelmeland LM. Acute hyperoxia-induced transcriptional response in the mouse RPE/choroid. Free Radic Biol Med 2003; 35:465– 474. 13. Sukhanov S, Hua Song Y, Delafontaine P. Global analysis of differentially expressed genes in oxidized LDL-treated human aortic smooth muscle cells. Biochem Biophys Res Commun 2003; 306:443– 449. 14. Virgili F, Ambra R, Muratori F, Natella F, Majewicz J, Minihane AM, Rimbach G. Effect of oxidized low-density lipoprotein on differential gene expression in primary human endothelial cells. Antioxid Redox Signal 2003; 5:237– 247. 15. Gohil K, Cross CE, Last JA. Ozone-induced disruptions of lung transcriptomes. Biochem Biophys Res Commun 2003; 305:719 –728. 16. Sesto A, Navarro M, Burslem F, Jorcano JL. Analysis of the ultraviolet B response in primary human keratinocytes using oligonucleotide microarrays. Proc Natl Acad Sci USA 2002; 99:2965 – 2970. 17. Catani MV, Costanzo A, Savini I, Levrero M, de Laurenzi V, Wang JY, Melino G, Avigliano L. Ascorbate up-regulates MLH1 (Mut L homologue-1) and p73: implications for the cellular response to DNA damage. Biochem J 2002; 364:441 – 447. 18. Linnane AW, Kopsidas G, Zhang C, Yarovaya N, Kovalenko S, Papakostopoulos P, Eastwood H, Graves S, Richardson M. Cellular redox activity of coenzyme Q10: effect of CoQ10 supplementation on human skeletal muscle. Free Radic Res 2002; 36:445– 453. 19. Svensson PA, Englund MC, Markstrom E, Ohlsson BG, Jernas M, Billig H, Torgerson JS, Wiklund O, Carlsson LM, Carlsson B. Copper induces the expression of cholesterogenic genes in human macrophages. Atherosclerosis 2003; 169:71– 76. 20. Ahn WS, Huh SW, Bae SM, Lee IP, Lee JM, Namkoong SE, Kim CK, Sin JI. A major constituent of green tea, EGCG, inhibits the growth of a human cervical cancer cell line, CaSki cells, through apoptosis, G(1) arrest, and regulation of gene expression. DNA Cell Biol 2003; 22:217 –224. 21. Fujiki H, Suganuma M, Okabe S, Sueoka E, Sueoka N, Fujimoto N, Goto Y, Matsuyama S, Imai K, Nakachi K. Cancer prevention with green tea and monitoring by a new biomarker, hnRNP B1. Mutat Res 2001; 480 – 481:299 – 304. 22. Okabe S, Fujimoto N, Sueoka N, Suganuma M, Fujiki H. Modulation of gene expression by (2)-epigallocatechin gallate in PC-9 cells using a cDNA expression array. Biol Pharm Bull 2001; 24:883– 886. 23. Wang SI, Mukhtar H. Gene expression profile in human prostate LNCaP cancer cells by (2) epigallocatechin-3-gallate. Cancer Lett 2002; 182:43 –51. 24. Weinreb, Mandel S, Youdim MB. cDNA gene expression profile homology of antioxidants and their antiapoptotic and proapoptotic activities in human neuroblastoma cells. FASEB J 2003; 17:935 – 937. 25. Kitajka K, Puskas LG, Zvara A, Hackler L, Barcelo-Coblijn G, Yeo YK, Farkas T. The role of n-3 polyunsaturated fatty acids in brain: modulation of rat brain gene expression by dietary n-3 fatty acids. Proc Natl Acad Sci USA 2002; 99:2619 – 2624. 26. Takahashi M, Tsuboyama-Kasaoka N, Nakatani T, Ishii M, Tsutsumi S, Aburatani H, Ezaki O. Fish oil feeding alters liver gene expressions to defend against PPARalpha activation and ROS production. Am J Physiol Gastrointest Liver Physiol 2002; 282:G338– G348. 27. Jhaveri MS, Wagner C, Trepel JB. Impact of extracellular folate levels on global gene expression. Mol Pharmacol 2001; 60:1288 – 1295.

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28. Li W, Trovero F, Cordier J, Drieu K, Papadopoulos V. Prenatal exposure of rats to Ginkgo biloba extract (EGb 761) increases neuronal survival/growth and alters gene expression in the developing fetal hippocampus. Brain Res Dev Brain Res 2003; 4(2):169– 180. 29. Watanabe CM, Wolffram S, Ader P, Rimbach G, Packer L, Maguire J, Schlutz PG, Gohil K. The in vivo neuromodulatory effects of the herbal medicine ginkgo biloba. Proc Natl Acad Sci USA 2001; 98(12):6577– 6580. 30. Suzuki K, Koike H, Matsui H, Ono Y, Hasumi M, Nakazato H, Okugi H, Sekine Y, Oki K, Ito K, Yamamoto T, Fukabori Y, Kurokawa K, Yamanaka H. Genistein, a soy isoflavone, induces glutathione peroxidase in the human prostate cancer cell lines LNCaP and PC-3. Int J Cancer 2002; 99:846– 852. 31. Li Y, Li X, Sarkar FH. Gene expression profiles of I3C- and DIM-treated PC3 human prostate cancer cells determined by cDNA microarray analysis. J Nutr 2003; 133:1011– 1019. 32. Siler U, Barella L, Spitzer V, Schnorr J, Lein M, Goralczyk R, Wertz K. Lycopene and vitamin E interfere with autocrine/paracrine loops in the Dunning prostate cancer model. FASEB 2004; 18(9):1019– 1021. 33. Wiechmann AF. Regulation of gene expression by melatonin: a microarray survey of the rat retina. J Pineal Res 2002; 33:178 –185. 34. Dong Y, Ganther HE, Stewart C, Ip. C Identification of molecular targets associated with selenium-induced growth inhibition in human breast cells using cDNA microarrays. Cancer Res 2002; 62:708– 714. 35. Bagchi D, Bagchi M, Stohs S, Ray SD, Sen CK, Preuss HG. Cellular protection with proanthocyanidins derived from grape seeds. Ann NY Acad Sci 2002; 957:260– 270. 36. Rihn B, Saliou C, Bottin MC, Keith G, Packer L. From ancient remedies to modern therapeutics: pine bark. Pine bark uses in skin disorders revisited. Phythother Res 2001; 15(1):76– 78. 37. Narayanan BA, Narayanan NK, Re GG, Nixon DW. Differential expression of genes induced by resveratrol in LNCaP cells: P53-mediated molecular targets. Int J Cancer 2003; 104:204 –212. 38. Dong Y, Lisk D, Block E, Ip C. Characterization of the biological activity of gammaglutamyl-Se-methylselenocysteine: a novel, naturally occurring anticancer agent from garlic. Cancer Res 2001; 61:2923 –2928. 39. Rao L, Puschner B, Prolla TA. Gene expression profiling of low selenium status in the mouse intestine: transcriptional activation of genes linked to DNA damage, cell cycle and oxidative stress. J Nutr 131(12):3175 – 3181. 40. Thimmulappa RK, Mai KH, Srisuma S, Kensler TW, Yamamoto M, Biswal S. Identification of Nrf2-regulated genes induced by the chemopreventive agent sulforaphane by oligonucleotide microarray. Cancer Res 2002; 62:5196– 5203. 41. Soref CM, Di YP, Hayden L, Zhao YH, Satre MA, Wu R. Characterization of a novel airway epithelial cell-specific short chain alcohol dehydrogenase/reductase gene whose expression is up-regulated by retinoids and is involved in the metabolism of retinol. J Biol Chem 2001; 276:24194 – 24202. 42. Sreekumar R, Unnikrishnan J, Fu A, Nygren J, Short KR, Schimke J, Barazzoni R, Nair KS. Impact of high-fat diet and antioxidant supplement on mitochondrial functions and gene transcripts in rat muscle. Am J Physiol Endocrinol Metab 2002; 282:E1055 –E1061.

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43. Farach-Carson MC, Xu Y. Microarray detection of gene expression changes induced by 1,25(OH)(2)D(3) and a Ca(2þ) influx-activating analog in osteoblastic ROS 17/2.8 cells. Steroids 2002; 67:467 – 470. 44. Krishnan AV, Peehl DM, Feldman D. Inhibition of prostate cancer growth by vitamin D: Regulation of target gene expression. J Cell Biochem 2003; 88:363– 371. 45. Li X, Zheng W, Li YC. Altered gene expression profile in the kidney of vitamin D receptor knockout mice. J Cell Biochem 2003; 89:709 – 719. 46. Barella L, Mu¨ller PY, Schlachter M, Hunziker W, Sto¨cklin E, Spitzer V, Meier N, De Pascual-Teresa S, Minihane AM, Rimbach G. Identification of hepatic molecular mechanisms of action of alpha-tocopherol using global gene expression profile analysis in rats. Biochim Biophys Acta 2004; 1689(1):66– 74. 47. Villacorta L, Graca-Souza AV, Ricciarelli R, Zingg JM, Azzi A. Alpha-tocopherol induces expression of connective tissue growth factor and antagonizes tumor necrosis factor-alpha-mediated downregulation in human smooth muscle cells. Circ Res 2003; 92:104– 110. 48. Roy S, Lado BH, Khanna S, Sen CK. Vitamin E sensitive genes in the developing rat fetal brain: a high-density oligonucleotide microarray analysis. FEBS Lett 2002; 530:17– 23. 49. Fischer A, Pallauf J, Gohil K, Weber SU, Packer L, Rimbach G. Effect of selenium and vitamin E deficiency on differential gene expression in rat liver. Biochem Biophys Res Commun 2001; 285:470 – 475. 50. Blanchard RK, Moore JB, Green CL, Cousins RJ. Modulation of intestinal gene expression by dietary zinc status: effectiveness of cDNA arrays for expression profiling of a single nutrient deficiency. Proc Natl Acad Sci USA 2001; 98:13507 –13513. 51. Tom Dieck H, Doring F, Roth HP, Daniel H. Changes in rat hepatic gene expression in response to zinc deficiency as assessed by DNA arrays. J Nutr 2003; 133(4):1004– 1010. 52. Gohil K, Chakraborty AA. Application of microarray and bioinformatics tools to dissect molecular responses of the central nervous system to antioxidant nutrients. J Nutr 2004; 50– 55.

2 Oxidative Stress and Human Genetic Variation R. D. Gill-Garrison, J. L. Slater, and K. Grimaldi Sciona, Ltd., Havant, UK

Manganese Superoxide Dismutase MnSOD Background MnSOD Polymorphisms MnSOD and Health Conditions Late-Onset Neurological Disorders Cancer Copper, Zinc Superoxide Dismutase SOD3 Background SOD3 Polymorphisms The Glutathione-S-Transferases The GST Genes GSTM1—Glutathione-S-Transferase M1 GSTM1 Background GSTM1 Biomarkers GSTM1 and Health Conditions Glutathione-S -Transferase P1 GSTP1 Background GSTP1 Biomarkers GSTP1 and Health Conditions Glutathione-S-Transferase T1 GSTT1 Biomarkers 13

15 15 16 17 17 17 18 19 19 19 20 20 20 21 21 22 23 23 23 24 24

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GSTT1 and Health Conditions Cancer Endothelial Nitric Oxide Synthase eNOS Summary Functional Studies of the 894G . T (Glu298Asp) Polymorphism eNOS Glu298Asp (G . T) and Cardiovascular Disease eNOS and Oxidative Stress of Smokers Potential Dietary Interventions Catalase CAT Background CAT Polymorphisms NADPH Oxidase NADPH Oxidase Background NADPH Polymorphisms NADPH Polymorphisms and Health Conditions Glutathione Peroxidase GPX1 Background GPX1 Polymorphisms GPX1 Polymorphisms and Health Myeloperoxidase MPO Background MPO Polymorphisms NADPH Dehydrogenase, Quinone 1 NADPH Background NADPH Polymorphisms Conclusion References

24 24 25 25 25 26 26 26 27 27 27 28 28 28 28 29 29 29 29 30 30 30 31 31 31 31 32

Oxidative stress has been attributed to a host of human disorders ranging from cancer to premature aging. Although the role of oxidative stress in damaging tissues ultimately leading to disease or disrepair is widely accepted, science is progressing rapidly in terms of understanding the impact of interindividual variation in genes responsible for dealing with oxidative stress in cells. As molecular techniques have advanced and the technology has become more accessible, it has become much easier to include analysis of genetic variation in epidemiological studies as well as biochemical studies modeling particular genetic variations. Single nucleotide polymorphisms (snps) are the most common variations that exist in the human genome, occurring at 1500 bp intervals in the human genome. Snps are considered distinct from rare mutations in that a snp by definition must occur in at least 1% of the population. Many of these sequence

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alterations may occur in noncoding regions of the genome, and may have no significance, others may lead to alterations in the amino acid sequence of the gene product, leading to a functional change in the protein, still others may alter the promoter region, thus having an impact on efficiency of transcription of the gene. Often, snps are identified through molecular epidemiological methods that do not have a clear functional effect, and these variations are assumed to be in linkage disequilibrium with some other, yet unidentified functional variation. Other variations that can occur include insertions and deletions, in one common example, the complete sequence of the GSTM1 gene, for example, is deleted in 50% of the Caucasian population. Useful online resources for studying human variation include the Database of Single Nucleotide Polymorphisms http://www.ncbi.nlm.nih.gov/SNP, GeneCards http:// bioinformatics.weizmann.ac.il/cards, the Human Gene Mutation Database http://archive.uwcm.ac.uk/uwcm/mg/hgmd0.html, OMIM (Online Mendelian Inheritance in Man) http://www.ncbi.nlm.nih.gov/OMIM, and the SNP consortium http://snp.cshl.org. This chapter is an overview of several of the genes associated with antioxidant activity and the most common variations found within those genes. Associations of the variations with disorders often attributed to oxidative stress are included, as well as dietary interventions, where possible, which demonstrate an altered response in the population in epidemiological studies. MANGANESE SUPEROXIDE DISMUTASE Gene: MnSOD—SOD2—Manganese superoxide dismutase Polymorphisms: C(228)T—Ala(29)Val T175C—Ile58Thr . . . . . .

Function: Destroys radicals which are normally produced within the cells and which are toxic to biological systems Biochemical activity: 2 superoxide þ 2Hþ ¼ O2 þ H2O2 Cofactor: Manganese Structure: Homotetramer Location: Mitochondria Population frequency: C(228)T 50% Caucasian, 12% Japanese

MnSOD Background MnSOD, also known as SOD2, is an enzyme that acts as a free-radical scavenger. The SOD2 gene is located on chromosome 6 in humans (region 6q25), with 5 exons and 4 introns, spanning 11,091 bp (1,2) and the active enzyme is generally found within the mitochondria of cells. This is the site of many oxidative reactions; thus, free radicals are generated here which can cause damage to the DNA, proteins, cell membranes, and the mitochondrion itself. The MnSOD

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enzyme converts the superoxide free radical into hydrogen peroxide at near diffusion limiting rates at the catalytic manganese site (3). The active MnSOD enzyme is a homotetramer with the four subunits each contributing to the formation of the catalytic site (4). Knock-out mice, in which the mouse equivalent gene was removed, died within 10 days of birth with dilated cardiomyopathy, accumulation of lipid in the liver and skeletal muscle, and metabolic acidosis, leading to the conclusion that the MnSOD is a vital enzyme, necessary for life (5). MnSOD Polymorphisms There have been two polymorphisms described for this gene, the Ala(29)Val polymorphism and the Ile58Thr polymorphism. The Ala(29)Val polymorphism was discovered by cloning the MnSOD gene derived from patients with diseases of premature aging, progeria, Werner syndrome, and Cockayne syndrome; and comparing them with normal samples. The polymorphism consists of a single nucleotide change; a cytosine is replaced by a thymine, leading to the substitution of a valine (Val) amino acid in place of an alanine (Ala) in the region of the gene known as the signal sequence, which directs the enzyme into the mitochondria. The authors hypothesized that these diseases of premature aging may be due to a so-called disease of distribution, in which an essential enzyme is not targeted to the proper location in the cell, leading to deficiencies of critical enzymes in specific locations within the cell. In this case, the movement of the enzyme within the cell, rather than its actual activity is defective (6). Further studies of the signal sequence demonstrated that the Ala allele codes for the ideal helical protein structure that would allow maximal transport into the mitochondria, and this helical structure is disrupted when valine is present (7). Most of the recent research on the MnSOD Ala(29)Val polymorphism has focused on epidemiological data; few studies have measured function or biomarkers associated with this polymorphism. However, one study has measured a reduction of processing into the mitochondria of 11% when the Val residue is present (8). Earlier work determined that the signal sequence is essential for protection of mitochondria from ionizing radiation (9). One study has measured the formation of 8-hydroxy-deoxyguanosine adducts, an indicator of oxidative stress, in term pregnant women; the investigators found higher levels of adducts associated with the Ala(29) allele (10). The population distribution for the Ala(29)Val is random (50%) for the Caucasian population of North America and Western Europe (6), whereas the occurrence in the Japanese population is much lower at 12% (7). This has led to some confusion in the literature, with some investigators referring to the polymorphism as Val(29)Ala; however, the convention used on the major human sequence databases such as EMBL and OMIM is that of Ala(29)Val which reflects the functionality of the polymorphism. The second polymorphism identified, Ile58Thr, occurs much more rarely in the population, and there are no studies to date that have measured the occurrence

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in particular ethnic groups. This polymorphism occurs at a position that causes two packing defects in the assembly of the four subunits into the active tetrameric enzyme. The defect causes a reduction in stability of the enzyme; studies in vitro have shown the polymorphic enzyme to exist in a dimeric form in solution (with two subunits only) due to a lack of thermostability. Predictions are that the enzyme would be even less stable at normal body temperatures (4). Further studies of cells in culture demonstrated that the wild-type (Ile) enzyme had three times the activity of the polymorphic (Thr) enzyme (11). MnSOD and Health Conditions Late-Onset Neurological Disorders The Ala(29)Val polymorphism has been associated with increased risk of occurrence in several disorders, including idiopathic dilated cardiomyopathy (9) and diabetic neuropathy. The Val allele has been implicated in early studies of Parkinson’s disease (7) and progeria-type diseases (6); however, further studies have not found a link with Parkinson’s disease (12,13). The contribution of this polymorphism to these late-onset neurological diseases has been suggested to be due to the accumulation of mitochondrial oxidative damage in the absence of adequate levels of MnSOD enzyme activity (14). A Chinese study has reported a synergistic effect of the Val allele together with the ser9 allele of the dopamine receptor (DR3) ser9gly polymorphism in the development of tardive dyskinesia in a population of patients with schizophrenia, suggesting a link between increased mitochondrial oxididative stress and dopamine receptor function in the onset of tardive dyskinesia (15). The Ala allele has been associated with increased risk of other conditions including age-related macular degeneration with an odds ratio of 10.14 (16), as well as motor neurone disease, with an odds ratio of 2.9 for the homozygous Ala allele, which increased to an odds ratio of 5.0 when females alone were considered (14), and severe alcoholic liver disease, with a 6 –10-fold increase in cirrhosis and alcoholic hepatitis (17). Cancer The Ala allele has been associated with increased risk of developing certain forms of cancer. This association is particularly interesting because it is the Ala allele that has the signal sequence intact, and should have the most efficient transport into the mitochondria. A study by Ambrosone et al. in 1999 demonstrated an increased risk of breast cancer (odds ratio 4.3) in premenopausal women with homozygous Ala alleles. In this study, levels of fruits and vegetables in the diet were also measured, and the odds ratio for premenopausal women with the lowest consumption of fruits and vegetables increased to 6.0, whereas the odds ratio for those with the highest consumption was lower, at 3.2. When vitamin supplementation was considered, the risk for premenopausal women with the homozygous Ala genotype was only observed among women who did not take supplements of vitamin C (odds ratio 4.8) and a-tocopherol (odds ratio 3.8).

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Premenopausal women taking supplements did not show increased risk with the homozygous Ala allele. (18). The link between breast cancer and the MnSOD Ala allele has been repeated in another study, the odds ratio reported was 1.5; however, there was no dietary information included in the study (19). A further study carried out by Egan et al. (20) has given limited support to this association; the odds ratio reported were much lower; however, there was no data reported on antioxidant supplementation to contribute to the analysis, which prevents direct comparison with the previous study. Further evidence has been presented which demonstrated a link between the Ala allele, dietary consumption of antioxidants, and risk of ovarian cancer. In this study, women with low intake of antioxidants and the Ala allele were at increased risk of ovarian cancer, with an odds ratio of 2.6 (21). These studies have provided a potential link between consumption of antioxidants and reduction of risk associated with a specific genotype. A study of colorectal cancer in young individuals (below the age of 40) found the frequency of the Ala allele was greatly increased in patients with the disease, the authors calculated an odds ratio of 7.5 for the homozygous Ala allele in individuals under 40, and an odds ratio of 1.5 for Ala/Val heterozygotes. The authors suggest that this polymorphism may be a useful marker to identify young individuals at a high risk of developing colorectal cancer (22) and have filed a US patent covering the use of a genetic test of the MnSOD gene polymorphism to screen for colon cancer risk in this population (see http://www. usc.edu/academe/otl/3015w.htm). Similar results were reported in a study of colorectal cancer in a Hispanic population (23). The Ala allele has also been associated with elevated prostate cancer risk (24,25). Not all studies have shown positive correlations however, which may indicate differences in tissue specificity, age-related differences, or other mechanistic contributions to the etiology of cancer. Also, the studies rarely include dietary information, which has been shown to have an impact on the incidence of breast cancer in women (18). Negative results have been reported recently in distal colorectal adenomas in men aged 50–74 (26), and in lung cancer risk in Taiwan (27). COPPER, ZINC SUPEROXIDE DISMUTASE Gene: SOD3—CuZn SOD—SOD3—Copper, Zinc superoxide dismutase— CuZn SOD Polymorphisms: C760G Arg213Gly . . . . . .

Function: Destroys free radicals Biochemical activity: 2 superoxide þ 2Hþ ¼ O2 þ H2O2 Cofactors: Copper and zinc Structure: Homotetramer Location: Extracellular Population frequency of polymorphisms: 3 – 4% Caucasian

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SOD3 Background EC-SOD or SOD3, the extracellular form of SOD, is found in plasma, lymph, and synovial fluid as well as in tissues. SOD3 is the major antioxidant enzyme system of the vessel wall of the cardiovascular system. It is a tetrameric glycoprotein with an apparent subunit molecular weight of 30,000 Da. Like the CuZn SOD (SOD1), SOD3 contains one Cu2þ and one Zn2þ ion per subunit; however, the amino acid compositions of SOD1 and SOD3 are quite different and no cross-reactivity is observed in immunologic studies. SOD3 also has a different tissue distribution than SOD1. The SOD3 enzyme is synthesized with a putative 18-amino acid signal peptide preceding the 222 amino acids in the mature enzyme, indicating that the enzyme is a secretory protein. The primary characteristic distinguishing SOD3 from SOD1 and SOD2 is the heparinbinding capacity of SOD3. SOD3 binds on the surface of endothelial cells through the heparan sulfate proteoglycan and eliminates the oxygen radicals from the NADP-dependent oxidative system of neutrophils (28,29). SOD3 Polymorphisms One SOD3 SNP that has been studied extensively, Arg213Gly, results in the accelerated release of the enzyme from the tissue interstitium into the plasma and is accompanied by reduced tissue SOD3 activities. The Arg21Gly SNP was discovered by Adachi et al. (29), who developed an immunoassay system for SOD3 in order to measure SOD3 levels in the serum of healthy subjects. They found that 6% of these persons had an SOD3 level that was 10 –15-fold higher than the mean SOD3 level in all subjects, which was familial in nature (30). Sandstrom et al. (31) reported that 2% of the plasma donors in Sweden had an 8– 10-fold higher SOD3 level and that a single base substitution of C to G at position 760 of the cDNA was responsible for the high level in plasma. The polymorphism is located in the region associated with the heparin affinity of the enzyme. The resulting amino acid substitution may result in a decrease of heparin affinity which favors the presence of SOD3 in the serum; thus, the high plasma activity can be explained by an accelerated release from the tissue interstitium heparan sulfate to the vasculature which is accompanied by significantly reduced tissue SOD3 activities. Marklund (32) observed the frequency of the Arg213Gly variant was 3.8% in the Northern Swedish population. Their data also suggested that plasma levels of the wild-type form of SOD3 may be modulated by lifestyle factors, such as smoking, and show a complex covariation with many of the conventional cardiovascular risk factors. THE GLUTATHIONE-S-TRANSFERASES The enzymes belong to a super-family with broad and overlapping substrate specificities. The active enzymes exist as dimeric structures and catalyze conjugation reactions between glutathione and aromatic radicals and epoxides. The active

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enzymes contain a highly conserved G-site, which is the binding site for glutathione, and a divergent H-site, which provides a site for binding of electrophiles (33). The majority of the conjugation activity is believed to occur in the liver, with a transfer of intermediates into the kidney for excretion. Glutathione-Stransferases provide a major pathway of protection against chemical toxins and carcinogens, as well as reactive oxygen species, and are thought to have evolved as an adaptive response to environmental insult, thus accounting for their wide substrate specificity (34). There are four family members: A, M, T, and P. Polymorphisms have been identified in each family (35). These enzymes catalyze reactions in which the products of Phase I metabolism are conjugated with glutathione, thus making them more water soluble and more easily excreted from the body. When the activity of the enzymes is enhanced, the products of Phase I metabolism should be excreted more rapidly from the body, and therefore less likely to interact with the DNA and proteins of the cells. Individuals with polymorphisms in the GST Phase II detoxification genes have a decreased rate of detoxification, with a corresponding increase in levels of carcinogen—DNA adduct formation and also an increased level of chromosomal aberrations (36,37). Cruciferous vegetables, such as broccoli, and members of the allium family, such as garlic and onion, have been shown to be potent inducers of these enzymes, which would be expected to increase clearance of potential toxins from the body (38,39). THE GST GENES GSTM1—Glutathione-S-Transferase M1 Gene: GSTM1—Glutathione-S-transferase mu, M1 Polymorphisms: Gene deletion . . . . .

Function: Conjugation of glutathione to hydrophobic compounds Biochemical activity: RX þ glutathione ¼ HX þ R-S-glutathione Structure: Homodimer Location: Cytoplasm of the cell, in the liver Population frequency: 50% Caucasian

GSTM1 Background The GSTM1 subtype has the highest activity of the four types of GSTs and is predominately located in the liver (34). Approximately half of the Caucasian population has a complete deletion of this gene (35,40). There have been reports of two alleles in the literature and on public domain websites: GSTM1A and GSTM1B reported to differ in position 172; however, this is an error that has arisen due to homology with GSTM4 (41).

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GSTM1 Biomarkers The effects of diet on activity of the GST enzymes have been demonstrated in several studies. For example, in a feeding study with healthy volunteers, activity of GSTA and GST serum levels were measured in GSTM1(þ) and GSTM1(2/2) subjects. Brassica vegetable diets increased GSTA by 26% and GST serum activity by 7% in the GSTM1-null individuals, particularly in women. Among the GSTM1(þ) women, GSTM activity was increased by both brassica (18%) and the allium (26%) diets (42). Palli et al. (43) have previously shown that that DNA adducts, a reliable indicator of genotoxic damage and, possibly, of cancer risk, are modulated by plasma levels of selected micronutrients. In a follow-up study, stratification by GSTM1 genotype showed strong inverse associations of DNA adduct levels with increasing consumption of vegetables, particularly raw and leafy vegetables, as well as vitamins E and C. In contrast, no associations were found among 295 GSTM1 wild-type individuals. The authors conclude that the results suggest that the role of a diet rich in antioxidants in preventing or reducing DNA adduct formation is restricted to subjects lacking the detoxifying activity of GSTM1 isoenzyme (50% of the general population) (44). Plasma autoantibodies (aAbs) against the oxidized DNA base derivative 5-hydroxymethyl-20 -deoxyuridine (5-HMdU) are potential biomarkers of cancer risk and oxidative stress. Current smokers lacking GSTM1, particularly men, have shown greater aAb titers compared with nonsmokers or persons with intact GSTM1 (45). GSTM1 and Health Conditions Cancer: There have been a host of studies examining the association between GSTM1-null individuals and increased cancer risk, with many studies reporting positive finding (46). The following is a brief synopsis of studies that have investigated cancer incidence, GSTM1 status, and consumption of isothiocyanates, which are known to increase the activity of glutathione-S-transferase enzymes; however, these compounds can also be conjugated and thus removed from the body by GST enzymes. Isothiocyanates are contained in cruciferous vegetables, and have been shown to have chemopreventive effects in laboratory animals. The investigators found that isothiocyanates appeared to reduce the risk of lung cancer in Chinese men, with reduction of risk more pronounced in individuals with homozygous GSTM1-null genotype, odds ratio 0.36, and more so if both GSTM1 and GSTT1 were deleted, odds ratio 0.28 (47). Similar results were described in studies of lung cancer (48), colorectal adenomas (49, 50), and head and neck cancer (51). Lung disease: Ozone is a powerful oxidant associated with impairment of pulmonary function and increased airway inflammation. Romieu et al. studied 158 asthmatic children in Mexico city, an area with high ozone exposure and looked at the effect of GSTM1 genotype and antioxidant supplementation (vitamins C and E). The GSTM1-null genotype was present in 39% of the

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children. In those given placebo, the deleterious effect of ozone on forced expiratory flow was worse in GSTM1-nulls than in those carrying the gene. In all children receiving antioxidant supplementation, there was no decrease suggesting that the supplementation abolished the effect of the null genotype (52). A recent report found that individuals with GSTM1-null or GSTP1 Ile105 wildtype genotypes showed enhanced nasal allergic responses to diesel-exhaust particles. The GSTM1-null individuals had a significantly larger increase in IgE and histamine in nasal lavage fluid after challenge with diesel-exhaust particles or allergen than children with a functional GSTM1 genotype (53). The data was highly reproducible between individuals and provides additional evidence supporting the concept of individual sensitivity to air pollution (54,55). Other disorders: Focusing on the antioxidant activity of GSTM1 and publications of the last year, one study has compared levels of 8-hydroxy guanine adducts, GSTM1 and GSTT1 genotypes in glaucoma patients. The glaucoma patients had higher levels of DNA adducts than healthy controls, and there were more GSTM1-null primary open angle glaucoma patients than healthy controls. The authors conclude that oxidative damage is increased in glaucoma patients, particularly in the trabecular meshwork, and that the GSTM1-null genotype predisposes glaucoma patients to more severe oxidative DNA damage (56). Other reports include increased biomarkers of lung inflammation in GSTM1 individuals exposed to ozone (57), protection against loss of hearing owing to oxidative stress in cochlear tissue of workers exposed to work-place noise for GSTM1(þ) individuals (58), and increased levels of 8-OH guanine adducts in pregnant GSTM1 women, also in MnSOD variant women (11). A study of patients with acute myocardial infarction (AMI) found that, in contrast to the other disorders described in this chapter, the GSTM1 positive allele conferred an increased risk, with significant differences more pronounced in smokers (59). The GSTM1-null allele was also found to have a protective effect in the development of cortical cataracts in an Estonian population. The GSTM1(þ) genotype was shown to confer risk in this study, with an odds ratio of 1.88 (60). Further study is required to determine the mechanism for this paradoxical effect. Glutathione-S-Transferase P1 Gene: GSTP1—Glutathione-S-transferase P1, pi Polymorphisms: A313G—Ile105Val C341T—Ala113Val . . . . .

Function: Conjugation of glutathione to hydrophobic compounds Biochemical activity: RX þ glutathione ¼ HX þ R-S-glutathione Structure: Homodimer Location: Cytoplasm of the cell, located in the lungs Population frequency of polymorphisms: A313G, 50% Caucasian, C341T, 10% Caucasian

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GSTP1 Background The GSTP1 gene is known to metabolize many carcinogenic compounds and is the most abundant subtype in the lungs (34). Two snps have been reported to date. The first is GSTP1
B (Ile/Val), with a single nucleotide change at position 313 in the DNA, leading to the substitution of a valine (Val) amino acid in place of an isoleucine (Ile) at position 105 of the enzyme. The second is GSTP1
C, with the previous change, together with a single nucleotide change at position 341 of the gene leading to the substitution of a valine residue for an alanine at position 113 of the enzyme (Ala/Val). The latter polymorphism may also occur on its own, the current designation is GSTP1
D. The enzymes of these polymorphic genes have decreased activity compared with the wild-type due to changes in the active pocket of the enzyme (61 – 63) and non-Hodgkin’s lymphomas (64). GSTP1 Biomarkers Vegetable juice has been shown to induce the expression of GSTP1 (65). The results of five large intervention studies showed considerable induction of GSTP activity after consumption of Brussels sprouts and red cabbage (66). A further study has identified a component of Japanese horseradish wasabi, 6-methylsulfinylhexyl isothiocyanate (6-HITC), as potent inducer of GSTP enzymes (67). GSTP1 enzyme activity is reduced by the presence of the polymorphisms, reduction in conjugation activity was 82% of wild-type for the heterozygous Ile/Val(105), and 70% for the homozygous Val/Val(105) (68); these results correspond with previous studies carried out in vitro (69). A study of DNA adducts resulting from oxidative damage in breast cancer tissue found lower levels of DNA adducts in patients with the wild-type Ile/Ile allele (70). A study that examined combinations of GSTM1 and GSTP1 found the highest level of DNA adducts in smokers with one or two copies of the Ile/Val polymorphism, together with the GSTM1(2/2) genotype (37). GSTP1 and Health Conditions Val allele associations: The GSTP1 Val polymorphism has been associated with an increased risk of developing prostate cancer (71,72), lung cancer (27,73), and recurrent early pregnancy loss (74). Asthma: The Val/Val genotype was found to be protective against asthma (75). The Ile/Ile was more prevalent in cases of atopy with increasing severity of airflow obstruction and bronchial hyper-responsiveness, implying a protective effect of the Val/Val polymorphism (76). Ile allele associations: The Ile allele was associated with greater risk of severe disability in patients with muscular dystrophy of duration greater than 10 years (77), squamous cell carcinoma of the esophagus (78). A recent publication which examined dietary patterns using cluster, as well as genetic polymorphisms in both GSTP1 and GSTM1 alleles found that smokers that were heterozygous for

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the Ile/Val allele and a “healthy” diet had a lower risk of lung cancer than individuals homozygous for the Ile allele with an “unhealthy” diet, OR ¼ 0.16 (79). The diets were categorized using four major dietary constituents, including carbohydrate, fat, protein, and fiber. Healthy diets were considered low in protein and fat and high in carbohydrate and fiber, whereas unhealthy diets had high levels of protein and fat, with low levels of carbohydrate and fiber. GLUTATHIONE-S-TRANSFERASE T1 Gene: GSTT1— Glutathione-S-transferase theta, T1 Polymorphism: Gene deletion . . . . .

Function: Conjugation of glutathione to hydrophobic compounds Biochemical activity: RX þ glutathione ¼ HX þ R-S-glutathione Structure: Homodimer Location: Cytoplasm of the cell, located in variety of tissues Population frequency: 20% Caucasian, 80% Asian

GSTT1 is considered one of the most ancient forms of glutathione-Stransferases. GSTTs are found in mammals, fish, plants, and bacteria (80). The GSTT1 gene is deleted in 20% of the Caucasian population and 80% of the Asian population (80). The enzyme is found in a variety of tissues, including red blood cells, liver, and lung (81), and the liver and kidney have the highest expression of GSTT1 (80). GSTT1 Biomarkers Dietary isothiocyanates have been shown to induce GSTT1 (82). A study of polycyclic aromatic hydrocarbon-induced chromosomal breaks revealed a higher levels of breaks associated with GSTT1(2/2) genotypes (83). Lymphocytes from GSTT1(2/2) individuals are also more prone to strand breaks induced by butadiene (84). A study of DNA adducts in smokers revealed higher levels of DNA adducts in individuals with both GSTT1(2/2) and NAT2-slow genotype (1.80) compared with GSTT1(þ)/NAT2 fast individuals (0.96) (85). GSTT1 and Health Conditions Cancer The deletion is associated with an increased risk of lung, larynx, prostate, cervical, and bladder cancers (34,80,86 –89), and asthma (90). When broccoli consumption was factored into a study of the incidence of colorectal adenomas, a protective effect was observed in GSTT1(2/2) with individuals consuming highest amounts of broccoli (91).

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ENDOTHELIAL NITRIC OXIDE SYNTHASE Gene: eNOS—Endothelial Nitric Oxide Synthase (NOS3) Polymorphisms: G894T—Glu298Asp . . . . . .

Function: Nitric oxide production which is implicated in relaxation of vascular muscle Biochemical activity: L -Arginine þ N NADPH þ M O2 ¼ citrulline þ nitric oxide þ N NADPþ Cofactors: Heme protein, FAD, FMN, BH4 Structure: Homodimer Location: Platelets Population frequency: 29% Caucasion, 9% Japanese

eNOS Summary Endothelium-derived nitric oxide (NO) plays a key role in the regulation of vascular tone, peripheral resistance and also has vasoprotective effects by suppressing platelet aggregation, leukocyte adhesion, and smooth muscle cell proliferation. As eNOS mediates basal vascular wall nitric oxide production, and altered nitric oxide production has been implicated in the development of coronary atherosclerosis, variants in the eNOS gene have been speculated to promote atherosclerosis via altered eNOS function (92). Four polymorphisms: 2786T . C; Glu298Asp; Intron 4 VNTR, and Intron 13 CA repeat have been included in a large number of genetic epidemiology studies (92).

Functional Studies of the 894G > T (Glu298Asp) Polymorphism A study by Tesauro et al. (93) suggests that the Glu298Asp variant may affect proteolytic cleavage of the enzyme. Because glutamate and aspartate are considered to be conservative replacements, the polymorphism was thought to be a marker for a functional locus elsewhere in the gene. Using transfected cells, primary human endothelial cells, and human hearts, Tesauro et al. showed that eNOS with aspartate, but not glutamate, at position 298 is cleaved, resulting in the generation of 100 and 35 kDa products. Thus, the eNOS gene with polymorphisms at nucleotide 894 generates protein products with differing susceptibility to cleavage, suggesting that, in contrast to prior predictions, this polymorphism has a functional effect on the eNOS protein. A recent report examining the function of wild-type and polymorphic enzymes in a yeast expression system did not find differences in the enzyme activity or cofactor binding, so clearly further work is required to fully understand the impact of the polymorphism (94).

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eNOS Glu298Asp (G > T) and Cardiovascular Disease Associations with the Glu298Asp polymorphism and cardiovascular disease have been reported in many studies (95,96), The Asp298 polymorphism has been shown to have significantly higher (P ¼ 0.001) median plasma NOx than those without this mutation (97). How this reflects NO levels in the vasculature is unclear. Leeson et al. (98) studying vascular function by endothelium-dependent, flow-mediated brachial artery dilatation (FMD) and endothelium-independent dilatation response to glyceryl trinitrate observed that vascular function was lower in Asp298 carriers, particularly in smokers and that n-3 fatty acids had a positive effect in this group, but not in Glu298 homozygotes. Not all studies have shown positive associations: Nassar et al. (99) examined the prevalence of this polymorphism in patients with early-onset coronary artery disease (CAD) compared with those manifesting CAD later in life and found no association of the eNOS Asp298 allele with premature CAD, no association was found in a Taiwanese population (100), or a Finnish population (101). eNOS and Oxidative Stress of Smokers The effects of smoking on white matter lesions, such as lacunar infarction and leukoaraiosis, are still controversial; recently the hypothesis that the eNOS T-786C genotype was a modulating factor for the effect of smoking on cerebral circulation was examined. Smokers were showed greater oxidative stress, as estimated by urinary F(2)-isoprostane excretion. In smokers, 2786CC homozygotes showed a significant decrease of cerebral blood flow and a significant increase of cerebrovascular resistance, whereas the eNOS genotype did not affect these parameters in nonsmokers (102). Further evidence for a role of smoking in modulating the activity of eNOS was demonstrated in vitro using luciferase reporter vectors with the various haplotype combinations of the 2786T . C and intron 4 repeat polymorphisms. Transcription efficiency in the T promoter was lower than in the C promoter. Treatment of the constructs with cigarette smoking extract increased the transcription efficiency significantly in the T promoter (1.7-fold, P , 0.01), it reduced further the already lower C promoter efficiency (by 10– 15%) (103). Potential Dietary Interventions A role for omega-3 fatty acids in modulating enzyme activity is provided by Leeson et al. (98), in which there is a significant positive relationship between the number of Asp alleles and flow-mediated dilation and omega-3 fatty acids levels. Benefit of vitamin E supplementation is suggested by Hingorani et al. (104). The authors state that the altered production of NO mediated by the Glu298Asp polymorphism might explain the unexpectedly large benefit of a-tocopherol in preventing MI in the CHAOS study (105). The authors

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propose that a-tocopherol may act to prevent the accelerated NO destruction caused by free oxygen radicals in the atherosclerotic vessel wall. CATALASE Catalase—CAT Polymorphisms: C(2264)T, BstXI C . T . . . . .

Function: Decompose hydrogen peroxide into water and oxygen Biochemical activity: 2H2O2 ¼ O2 þ 2H2O Structure: Homotetramer Location: Cytoplasm of the cell, located in variety of tissues Population frequency of polymorphisms: 2262C/T 28% Caucasian

CAT Background CAT is a ubiquitous enzyme found in all known organisms, it is most abundant in liver, kidney, and erythrocytes. CAT has a very high turnover number, rapidly decomposing H2O2 into O2 and H2O. Together with SOD and glutathione peroxidases (GPX), CAT constitutes a primary defense against oxidative stress (106). The human CAT gene consists of 13 exons and is located on chromosome 11p13. Several rare mutations/polymorphisms have been reported in the CAT gene, most of them being associated with acatalasemia, an autosomal recessive trait characterized by erythrocyte CAT levels 0.2– 4% of normal. CAT Polymorphisms A common functional polymorphism (28% in a Swedish population) in the promoter region has been identified. This is a C . T substitution at position 2262 and the variant appears to bind different transcription factors. The T variant was reported to be expressed at higher levels in HepG2 and K562 cell lines and it was confirmed that in vivo levels in liver and blood cells were higher for the T-allele. Individuals homozygous for TT and heterozygous CT have significantly higher CAT concentrations compared with individuals homozygous for CC (107). There are also other CAT polymorphisms that have been reported to affect enzyme levels and be associated with disease states. For example, a recent report has described the association of a T/C SNP in exon 9 of the CAT gene (BstX I) with vitiligo susceptibility. Vitiligo susceptibility is a complex genetic trait that may involve genes important for melanin biosynthesis, response to oxidative stress, and/or regulation of autoimmunity, as well as environmental factors. The authors chose the CAT gene as a candidate gene because of the reduction of CAT enzyme activity and concomitant accumulation of excess hydrogen peroxide observed in the entire epidermis of vitiligo patients. They observed

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that T/C heterozygotes are more frequent among vitiligo patients than controls and that the C allele is transmitted more frequently to patients than controls. The authors suggest that the SNP or nearby variations may contribute to a quantitative deficiency of CAT activity in the epidermis and the accumulation of excess hydrogen peroxide (H2O2) (108). NADPH OXIDASE NADPH oxidase Polymorphisms: C242 p22phox subunit, BstXI C . T . .

Function: Membrane bound enzymes that catalyze the one-electron reduction of oxygen Location: Integral membrane protein

NADPH Oxidase Background NAD(P)H oxidases are membrane-associated enzymes that catalyze the oneelectron reduction of oxygen using either NADH or NADPH as the electron donor, and they are the major oxidases in vascular tissue. NAD(P)H oxidase comprises several distinct subunits; gp91phox and p22phox are electron-transfer proteins, and both are expressed in endothelial cells (109). NADPH Polymorphisms The C242T polymorphism results in substitution of Tyr for His at residue 72 of p22phox modifying one of the two heme-binding sites that is thought to be essential for the stability of the protein (110). This polymorphism is associated with significantly reduced superoxide production in patients carrying the 242T allele, suggesting a role for genetic variation in modulating vascular superoxide production (111). NADPH Polymorphisms and Health Conditions The role of this polymorphism and disease association is still controversial, with reports of increased risk of cerebrovascular disease (112), whereas others have reported reduced risk of artherosclerosis associated with reduction in superoxide production (113) and reduced risk of CAD (114). Hayaishi-Okano looked at the role of the variant in atherosclerosis in type 2 diabetic subjects, they also looked at the level of 8-hydroxy-20 deoxyguanosine (8-OHdG) DNA adducts as an index of oxidative DNA damage. The diabetic subjects with TC or TT genotypes displayed less vascular thickening than those with the CC genotype and insulin levels were also affected. They concluded that the C242T polymorphism in the p22 phox gene is associated with progression of asymptomatic atherosclerosis

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in the subjects with type 2 diabetes and is also associated with insulin resistance in nondiabetic subjects (115).

GLUTATHIONE PEROXIDASE Glutathione peroxidase—GPX1 Polymorphisms: GCG repeats (Ala4, 5, 6), Pro198Leu . . . . .

Function: Reduce peroxides by coupled oxidation with GSH Biochemical activity: 2 glutathione þ H2O2 ¼ oxidized glutathione þ 2H2O Cofactor: Selenium Structure: Homotetramer Location: Cytoplasm

GPX1 Background Human cellular glutathione peroxidase 1 (hGPX1) is a selenium-dependent enzyme that participates in the detoxification of hydrogen peroxide and a wide range of organic peroxides by reducing organic peroxides and hydrogen peroxides through the coupled oxidation of reduced glutathione (GSH). GPX1 Polymorphisms An inframe GCG trinucleotide repeat was reported in 1994 by Shen et al. They looked at the GPX1 gene in 55 individuals and the allele frequencies for 4, 5, and 6 GCG repeats were 0.40, 0.35, and 0.25, respectively (116). A proline to leucine polymorphism at position198 has been reported in Caucasians, but has not been found in Asian populations (117). GPX1 Polymorphisms and Health Preliminary examination of the relationship of the GCG repeats with lung cancer and DNA damage [specifically 8-hydroxydeoxyguanosine (8-OHdG) adducts] indicated a trend towards lower 8-OHdG levels in normal lung tissue possessing the ALA6 allele of the GPX1 gene, implying a possible functional change in enzyme activity as a result of sequence characteristics affecting its protection against DNA (118). In contrast, a recent study of association of five, six, or seven alanine (ALA) repeats and CAD found a significant association between individuals with at least one ALA6 allele and an increased risk of CAD (119). The leucine polymorphism at codon 198 was reported to be associated with an increased lung cancer risk (117) and breast cancer (120).

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MYELOPEROXIDASE Myeloperoxidase—MPO Polymorphisms: 2129G/A, 2436G/A . . . . .

Function: Catalyzes formation of hypochlorous acid Biochemical activity: Donor þ H2O2 ¼ oxidized donor þ2H2O Cofactor: Heme, iron(III), and calcium Structure: Tetramer Location: Lysosomes

MPO Background MPO, an iron-containing protein, is found in neutrophils and in the lysosomes of monocytes in humans, it is most abundant in the granules of neutrophils. MPO plays a key role in immune activity within a cell. When neutrophils become activated, they undergo a process referred to as a respiratory burst which produces superoxide, hydrogen peroxide, and other reactive oxygen derivatives. These reactive oxygen species are released from the cell and are toxic to bacteria. MPO catalyzes the conversion of hydrogen peroxide and chloride ions (Cl) into hypochlorous acid, which is a potent agent for killing microbes, more so than hydrogen peroxide. Other targets include fungi, parasites, protozoa, viruses, tumor cells, natural killer cells, red cells, and platelets. The MPO – hydrogen peroxide –Cl system is also believed to be involved in terminating the respiratory burst and may be involved in down-regulating the inflammatory response (121). MPO Polymorphisms Two polymorphisms have been identified in the promoter region of the MPO gene, 463G/A and 129G/A, which reduce MPO enzyme activity in neutrophils, with the 463G/A showing gender- and age-specific effects (122). Examination of women on hormone replacement therapy revealed an association of the loweractivity 463A allele and increased progression of atherosclerotic lesions (123), and examination of men found increased fibrotic lesions associated with the 463A allele in men under the age of 53 years (124). In studies of brain infarction, an association with outcome rather than cause was reported, with patients with G-129 allele showing larger brain infarction, and patients with G-463 allele demonstrating worse short term functional outcome (125). In contrast, other studies have reported a protective effect associated with the A allele. A study of CVD in end-stage renal disease, found reduced incidence associated with the A allele, possibly due to lower production of reactive oxygen species (126). A protective effect against the development of hepatoblastoma was

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also reported recently (127) with the authors suggesting that the lower activity of the A allele has a reduced rate of activation of potentially carcinogenic substances. Reports of reduction of risk of lung cancer have produced conflicting results; however, analysis of tumor histological type may account for some of the conflicting results (128). NADPH DEHYDROGENASE, QUINONE 1 NADPH Dehydrogenase, Quinone 1—NQO1 Polymorphisms: C609T . . . . .

Function: Acts as two-electron quinone reductase in detoxification and biosynthetic pathways Biochemical activity: NAD(P)H þ acceptor ¼ NAD(P)þ þ reduced acceptor Cofactor: FAD Structure: Homodimer Location: Cytoplasm

NADPH Background NAD(P)H:quinone oxidoreductase (NQO1), also known as DT-diaphorase is an enzyme catalyzes 2- or 4-electron reductions of quinones, both endogenous and exogenous. The reduction reactions prevent redox cycling, which leads to the generation of free radicals. Synthetic antioxidants and extracts of cruciferous vegetables, including broccoli, are potent inducers of NQO1 (129). NADPH Polymorphisms A snp (C ! T) at position 609 of the NQO1 gene has been shown to reduce activity of the gene. This polymorphism has been associated with susceptibility to cancer (130 – 132), including lung cancer in smokers (27) and bladder cancer in smokers (133). In a population of GSTM1-null children exposed to high levels of ozone in Mexico, the NQO1 polymorphism was found to have a protective effect (134). CONCLUSION It is clear from the literature that there is significant interindividual variability in genes which play a role in the body’s antioxidant defenses. The impact of this variation on human health is being unraveled now as techniques of molecular epidemiology are more widely applied. We look forward for further improvements in techniques and experimental design, so that variation in not only one or two

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specific genes, but panels of hundreds to thousands of gene variations can be examined during the course of a well-planned study. The burden will turn to the existing data management and statistical analysis tools to accurately interpret the results. Given the growing popularity of alternative health methods, which often claim miraculous antioxidant or detoxification treatments, building a credible body of evidence to understand reponse to dietary antioxidant and detoxification agents becomes an increasingly important goal, both from a consumer protection and a public health standpoint. As future studies continue to examine associations of many and various genetic polymorphisms with health conditions, it is acknowledged that many initial positive associations may have negative results, and the converse may occur, as well. Continuing to develop the body of knowledge of individual variation, particularly in genes with antioxidant activity, should help to advance the understanding of the mechanisms in which oxidative stress contribute to disease and aging; to turn the widely accepted “truism” of the impact of oxidative stress on human health into a well-proven mechanism which explains how oxidative damage sustained by a cell can ultimately lead to aging or disease.

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129. Benson AM, Hunkeler MJ, Talalay P. Increase of NAD(P)H:quinone reductase by dietary antioxidants: possible role in protection against carcinogenesis and toxicity. Proc Nat Acad Sci 1980; 77:5216– 5220. 130. Zhang JH, Li Y, Wang R, Geddert H, Guo W, Wen DG, Chen ZF, Wei LZ, Kuang G, He M, Zhang LW, Wu ML, Wang SJ. NQO1 C609T polymorphism associated with esophageal cancer and gastric cardiac carcinoma in North China.World J Gastroenterol 2003; 9(7):1390– 1393. 131. Zhang J, Schulz WA, Li Y, Wang R, Zotz R, Wen D, Siegel D, Ross D, Gabbert HE, Sarbia M. Association of NAD(P)H:quinine oxidoreductase 1 (NQO1) C609T polymorphism with esophageal squamous cell carcinoma in a German Caucasian and a northern Chinese population. Carcinogenesis 2003; 24(5):905 – 909. 132. Sarbia M, Bitzer M, Siegel D, Ross D, Schulz WA, Zotz RB, Kiel S, Geddert H, Kandemir Y, Walter A, Willers R, Gabbert HE. Association between NAD(P)H: quinone oxidoreductase 1 (NQ01) inactivating C609T polymorphism and adenocarcinoma of the upper gastrointestinal tract. Int J Cancer 2003; 107(3):381 – 386. 133. Park SJ, Zhao H, Spitz MR, Grossman HB, Wu X. An association between NQO1 genetic polymorphism and risk of bladder cancer. Mutat Res 2003; 536(1–2):131–137. 134. David GL, Romieu I, Sienra-Monge JJ, Collins WJ, Ramirez-Aguilar M, del Rio-Navarro BE, Reyes-Ruiz NI, Morris RW, Marzec JM, London SJ. Nicotinamide adenine dinucleotide (phosphate)reduced:quinone oxidoreductase and glutathione S-transferase M1 polymorphisms and childhood asthma. Am J Respir Crit Care Med 2003; 168(10):1199 –1204.

3 Analysis of Microarray Data Ken Mills Cardiff University, Cardiff, Wales, UK

Introduction Normalization Methods Per Array Normalization Normalize to a Median or Percentile Normalize to Positive Control Genes Normalize to a Constant Value Per Gene Normalizations Normalization to Specific Samples Normalize to Median Data Analysis Fold Changes in Expression Levels Class Discovery—Unsupervised Learning Methods k-Means Clustering Self-Organizing Maps Principal Component Analysis Hierarchical Trees (Clustering) Class Prediction—Supervised Learning Methods Class Prediction Cross-Validation Validation of Microarray Analysis In silico Validation Laboratory Based Validation 43

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INTRODUCTION Microarray analysis of gene expression has become one of the most widely used functional genomic tools, since its development in the mid 1990s (1,2). This is reflected in the number of publications containing or arising from some aspect of microarray technology. The development has allowed researchers from all areas of biological research to simultaneously monitor the expression levels of thousands of genes. However, the efficient application of this powerful technique requires robust and reproducible protocols to be developed. These protocols must not only encompass all aspects of the technical processes but also include robust strategies for data normalization and in silico data analysis (3). As in any experiment, conventional or microarray, initial experimental design should be considered, planned, and refined to ensure that the data produced is truly meaningful (4). For example, will comparison of control and test samples produce data that reflects the biological question rather than simply reflect a difference in maturation status of the cells. The production of the raw intensity data is only the first step in a microarray experiment. The post-technical stages can then be divided into two broad sections: normalization steps and data analysis. NORMALIZATION METHODS Data normalization procedures are related to, and dependent, on the design of the experiment. In microarray studies, large number of genes and samples are usually analyzed producing amounts of data unimaginable 5 years ago. Some arrays contain up to 30,000 gene probe sets (e.g., the Affymetrix# U133 arrays) and have been used in experiments of up to 500 samples; this produces approximately 15,000,000 data points. Therefore, experiment normalizations are used to standardize microarray data and to differentiate between real (biological) variations in gene expression levels and variations due to the measurement process. It must also be noted that normalizing procedures also scale your data so that you can compare relative gene expression levels between samples within an experimental set. Data produced from some cDNA arrays undergo some aspect of normalization as a consequence of the technique—ratios of the fluorescent dye Cy3 and Cy5 labeled probes are used to produce raw data as image files that have

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to be transformed into gene expression formats. This process requires raw data manipulation, owing to differences in the chemistry of the dyes, before differences in transcript levels can be identified. It is necessary to normalize the fluorescence ratios in order to compensate for systematic variations (5). Most of the currently used normalization methods are linear; however, nonlinear methods have recently started to appear. One of these methods is that of QQ Quantile Normalization with log centering, which can be used for the normalization of microarray data (6). In this section, we concentrate on the type of linear normalizations methods available (Fig. 3.1). Per Array Normalization Per array normalizations are used to control array-wide variations in intensity. Such variations may be due to inconsistent washing, inconsistent sample preparation, or other microarray production or microfluidics imperfections. Normalize to a Median or Percentile With this option, all of the measurements on each array are divided by a specific value; this is usually the median value of all the intensity values on the array. Other values or percentiles usually can be specified depending on the overall range of data, although these should be carefully considered before use. Signal strength of gene A in sample X Median signal strength of all genes in sample X In any case, the method of global per array normalization is not recommended for any experiment where .50% of the genes on the array are likely to be affected similarly by the experimental conditions. For example, if a focused gene microarray containing only known growth factors were used to study gene expression in malignant and benign tumors, it might be expected that a majority of the genes

Figure 3.1 A schematic example of microarray expression data. Int. 1,1 is the intensity of gene 1 in array 1, Int. 1,2 is the intensity of gene 1 in array 2, whereas Int. 2,1 is the intensity of gene 2 in array 1.

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will be differentially expressed. In this case, applying a median normalization would mask the changes in expression. Normalize to Positive Control Genes Many arrays have positive controls (mRNA from another genome or housekeeping genes) as part of the array. These can be used to normalize intensity differences and are often used to control differences during drug treatment or time course experiments. The formula for this difference is: Signal strength of gene A in sample X Median signal strength of the positive controls in sample X Normalize to a Constant Value Some gene array commercial technologies will calculate their own values for normalization on the basis of a constant value. For example, if the Affymetrix Global ScalingTM algorithm is used, which centers the data on a specific value, usually 100, then the data would be normalized to 100 to center it around 1. Signal strength of gene A in sample X Constant value Per Gene Normalizations Per gene normalizations have the effect of normalizing each gene on the array across all the samples within the experiment. If additional microarray sample data are added to an experiment, per gene normalizations need to be recalculated. Only one type of per gene normalization is required for an experiment, as they address the same issue. Normalization to Specific Samples In normalization to specific samples, each gene is divided by the intensity of that gene in a specific control sample or by the average intensity in several control samples. The formula for this is: Signal strength of gene A in sample X Signal strength of gene A in control sample or Signal strength of gene A in sample X Average signal strength of gene A in control samples This type of normalization might be used, for example, during a time course experiment; and changes in gene expression are related to the untreated or time zero sample array. Different samples within an experiment may be normalized to different control samples. For example, when comparing the effect of a

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drug treatment in two different cell lines: time points for cell line A may be related to untreated cell line A; whereas time points for cell line B may be related to untreated cell line B. It should be noted that, although patterns of gene expression during the time course of each cell line can be identified using this method, it does not allow the direct comparison of expression levels of individual genes between cell lines A and B. Normalize to Median Normalize to median is the most common type of normalization. Per gene normalization is related to the median signal intensity of each gene across all of the samples in an experiment. Signal strength of gene A in sample X Median of every measurement taken for gene A throughout experiment It allows direct comparison of gene expression levels for each gene across all the samples within an experiment. However, care should be taken that all other procedures within the microarray techniques are comparable; different RNA extraction procedure or even location of RNA extraction may result in step changes in expression levels unrelated to biological variations.

DATA ANALYSIS At the moment, analysis of microarray data is perhaps the most exciting area of biology. It has the potential to identify changes in gene expression related to disease classification, disease diagnosis, responses to pharmacological or toxicological reagents, and in a multitude of basic biological science studies. As a consequence, a new and developing field of bioinformatics and biostatistics has been developed. Expression databases that follow standardized methods for reporting array data have been implemented, which allows more uniformity and confidence when comparing expression levels. Standardized approaches such as that implemented by minimal information about a microarray experiment (MIAME) (7,8) need to be applied to allow the comparison of data produced in different laboratories and different array platforms. The microarray gene expression data (MGED) (www.mged.org) organization provides well defined guidelines for the cross-comparison of array data and the need for comprehensive records of sample type, extraction processes, and labeling and hybridization techniques. Other databases are also available (9 – 14). All the questions asked of microarray data can probably be reduced to one overall question: “How can microarray data be mined to identify hidden patterns of gene expression?”. In particular, can we identify changes in genes expression patterns between different samples within an experiment or can we identify groups of genes that have similar expression patterns. These patterns will be

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Figure 3.2 Schematic illustrating the differences between class discovery and class prediction. Class discovery starts with no concept of groupings and the class discovery clustering algorithms place samples into groups based on similarities. Training/Test sets are used to identify genes that will be able to predict which known sub-groups an unknown sample should be allocated.

complex and interpretation will be compounded by the gene array data from a sample being associated with several parameters or factors. These parameters will be related to the sample type, treatment, or origin; however, the quality of sample, extraction procedures, and labeling methods should also be considered. The identification of genes that are differentially expressed among predefined set of samples with known parameters or classes within an experiment is called class comparison. Class prediction is related to identifying a set of genes that are capable of predicting which parameter group a specific sample should belong (Fig. 3.2). An increasing number of statistical methods have been, and continue to be, developed to extract reliable biological information from the microarray data. The analysis methods ranging from the basic to the more complex and some of these will be considered in this section. Whichever method is used to identify differentially expressed genes, further validation or selection is then required (see section “Validation of Microarray Analysis”). Fold Changes in Expression Levels Although this is one of the more basic methods of mining relevant data from microarrays, it can have disadvantages, which are usually associated with the lack of comparison of sample type within the experiment. In general, the

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Figure 3.3 Fold change in a series of 12 samples. (A) The normalized level of expression of one gene selected to show at least 2-fold higher expression in series of 12 samples (A1– C4). (B) The same gene expression level averaged over the four samples in each of three of discrete variables (A– C).

expression data is systematically analyzed for genes that show up or down changes in expression when comparing samples with two or more different parameters (Fig. 3.3). Often changes of
2-fold may be sufficient to identify a list of candidate genes. The accuracy of the candidate genes list will be increased when the issues of replicates are also considered. Several replicates must be done in order to attach statistical significance of the expression data. Ideally, at least three replicates should be done for each condition or parameter; however, in the case of disease analysis the replicates could also be biological replicates rather than technical replicates. A simple type of analysis of microarrays, containing several thousand genes, may result in a large list of genes, particularly, if a small number of samples are also analyzed. This is because the “noise level” from the array data is reduced when a greater number of samples, or replicates, are analyzed. For example, if an array contains 10,000 genes, then in any one sample or replicate, it may be that approximately 5000 genes will have either intensity values above the background threshold or are called “present.” A replicate of this sample may have a similar number of genes, but only 95% of these genes are in common, and so on as more samples or replicates are analyzed. By combining the data from the replicates, the number of genes consistently over the threshold, or called “present,” is rationalized. Furthermore, the intensity levels now have values statistically associated with them (median and standard deviation). Therefore, the identification of genes showing .2-fold change in expression is now statistically relevant, although the list may still be relatively large. The degree of fold change also has to be considered. Owing to the data normalization methods, many genes may be identified that show
2-fold changes in expression level, but when the raw intensity data are examined some of the genes may have very low levels. These genes should probably be considered as absent and removed from further consideration. In general, microarray data cannot be

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used to identify gene expression changes with less than an overall
2-fold with great confidence. This can be a major consideration if a sample does not contain 100% of the same cell type. For example, in a sample in which only 40% of the cells show a response to a drug treatment, candidate gene expression levels would need to change by at least
3.5-fold in all of those 40% of cells to result in an overall
2-fold change in expression level. Another method of identifying genes with significant changes within an experiment is to use a one- or two-way analysis of variance (ANOVA) filter. ANOVA is performed separately for each gene, only those genes that pass the significance level are retained for further analysis and therefore the changes in expression that are identified are probably likely to be due to the defined parameter rather than random fluctuations. If we assume that some genes are significantly identified during these analysis, the gene list may still be number several hundred. These candidate genes can be reduced by further by adding further significance levels. Tests such Bonferroni correction or “significance analysis of microarrays (SAM)” can be used (15 – 17). The former correction can often be restrictive and may result in no significant genes being identified, the latter method is now becoming a standard statistical technique for identifying genes lists of differentially expressed genes. Class Discovery—Unsupervised Learning Methods One of the major strengths of the gene microarrays is the ability to identify and reveal hidden patterns of gene expression. Various methods of data reduction and classification have been formulated that will link together groups of genes that have similar patterns of gene expression throughout the whole experiment. These methods reduce the initial main list of genes into smaller sub-groups or clusters. These sub groups are based on similar patterns of gene expression in the experiment. Basically, genes are clustered together because their expression is similar; each gene will show an increased or decreased expression which follows the same pattern as other gene members of the same sub-group. These clusters are identified without prior knowledge or input into the group, parameter or class that an individual samples is a member (Fig. 3.2). Gene expression signatures have been used to classify disease outcome in several disease types. The main successes have been reported within cancer research (18 – 21) using supervised learning algorithms have been developed to classify samples into distinct specific sub-sets based on gene expression. Classical examples of this are the first example of the feasibility of a cancer classification which was based solely on gene expression in acute leukemia (20,21) and the identification of a novel class of patients with diffuse large B-cell lymphomas (20). k-Means Clustering k-Means clustering divides genes into a (user) defined number of groups on the basis of their expression patterns (22,23). This produces groups of genes with a

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high degree of similarity within each group and a low degree of similarity between k groups. k-Means clustering does not show the relationship between clusters, but instead, k-means clusters are constructed so that the average behavior in each group is distinct from any of the other groups. For example, in a time series experiment, k-means clustering could be used to identify unique classes of genes that are up-regulated or down-regulated in a time dependent manner (Fig. 3.4). A k-means clustering algorithm will typically divide genes into a userdefined number (k) of equal-sized groups on the basis of the order in the selected gene list. It will then create centroids around the average location of each group of genes. This is then done over several iterations and genes are reassigned to the group with the closest centroid. When all the genes have been assigned, the location of the centroids is recalculated and the process is repeated until the number of user-defined iterations has been reached. Self-Organizing Maps Self-organizing maps (SOM) is a technique similar to k-means clustering. SOMs show the relationship between groups by arranging them in a two-dimensional map as result of dividing genes into groups on the basis of expression patterns (Fig. 3.5). SOMs can visualize distinct expression patterns and determine which of these patterns are variants of each other by Refs. (24,25). SOMs can be used to analyze many kinds of data, and some applications to gene expression analysis were described by Tamayo et al. (26). SOM algorithms create a two-dimensional grid of nodes in the space of gene expression. In each iteration, one gene is selected and all of the nodes within a user-defined “neighborhood” are moved closer to it. This process is repeated with each gene in the selected gene list until the maximum number of iterations has been reached. With each iteration, the “neighborhood radius” is incrementally reduced and nodes are moved by smaller amounts to produce convergence. In this way, the grid of nodes is stretched and wrapped to best represent the variability of the data, while still maintaining similarity between adjacent nodes. After the iteration is complete, genes are assigned to the nearest node, and a display grid of gene expression graphs is generated, corresponding to the initial grid of nodes. Principal Component Analysis Principal component analysis (PCA) is a “data reduction” technique used to identity uniquely expressing genes (27). PCA is not a clustering technique, but is a tool that will characterize the most abundant components that re-occur within many genes in an experiment. PCA is a decomposition technique that produces a set of expression patterns that are known as principal components. Diagonal or 3D combinations of these patterns can be assembled to represent the behavior of all of the genes in a given data set (28) (Fig. 3.6). In this procedure, the expression data from the “genes X expression” space are transformed to produce the principal components of a data set that are

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Figure 3.5 Self-organizing maps using the same series of 12 samples (A1– C4) as in Fig. 3.2 using the list of genes showing above background levels of normalized expression.

known as “eigenvectors,” which are obtained from an eigenvector – eigenvalue decomposition of the covariance matrix of the data (29,30). The eigenvalue corresponding to an eigenvector represents the amount of variability explained by that eigenvector. The eigenvector of the largest eigenvalue produces the first principal component. The eigenvector of the second largest eigenvalue is the second principal component and so on. There are never more principal components than there are conditions in the data. PCA identifies the correlations between gene expression profiles and attempts to explain a majority of the variance in the entire data set. Hierarchical clusters will group together gene expression profiles with similar distances.

Figure 3.4 k-Means clustering using the same series of 12 samples as in Fig. 3.2. (A) A 9 cluster k-means separation of the gene as showing above background levels of normalized expression. (B) The same k-means cluster showing the averaged patterns of gene expression in the three main populations (A – C).

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Figure 3.6 Principal component analysis. (A) 2-Dimensional map of the principal components showing distinct separation into three groups each comprising the four related samples. (B) 3-Dimensional map of (A) showing three distinct groupings of related samples. Note, however, that one sample (arrowed) lies in the same horizontal plane as the other related samples, but is slightly removed in the Z-direction; this may indicate that this sample has a slightly different phenotype from the other members of the group, but distinct from the other groups.

Hierarchical Trees (Clustering) The classification of organisms into phylogenetic trees is a central concept of biology: organisms that share properties are clustered together. The tree branches indicate how far close or diverse two sets of organisms are. The distance from two organisms to a common branch can be considered as a measure of how different

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the organisms are. Other concepts can also can be classified in a similar manner (31,32); for example, cluster analysis will place genes whose expression patterns are similar to the adjacent branches in a tree. Such mock-phylogenetic trees are also called dendrograms (Fig. 3.7). Complex trees can be made from multiple experiments or by tightly defining the types of data used. Cluster analysis is a useful method of class discovery. This is because the clustering algorithms do not depend on sample parameters—and as such are classified as unsupervised analyses.

Figure 3.7 A typical dendrogram of 50 genes. Two main branches or clusters can be seen, with various sub-branches. Genes with related expression features are placed closest together, and the point where they join is the node, or junction, of the branches.

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Measurements of similarity, distance, and separation: Hierarchical analysis techniques are based on measures of gene similarity. Similarity or “nearness” between genes is a measure of the degree of correlation between the expression profiles of the two genes. Measures of similarity. Measures of similarity take two expression patterns and produces a number representing how similar the two genes are. Most of the measures of similarity are correlation measures, and their value varies from 21 (exactly opposite) to 1 (the same). For a measure of distance, the result will vary from 0 (the same) to infinity (different). Measures of confidence vary from 0 (no confidence) to 1 (perfect confidence). Both distance and confidence can be considered as measures of dissimilarity: this means that “small” relates to “close”; whereas “large” means “far away.” Similarity definitions. The default correlation is the standard correlation, Pn i¼1 Ai Bi qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Pn   Pn  2 2 i¼1 Ai i¼1 Bi To make a tree, the correlation for each gene with every other gene in the set is calculated, the pair of genes with the highest correlation is then taken, and their expression profiles averaged. This new composite gene is then compared with all of the other unpaired genes. This process is repeated until all of the genes have been paired. Separation ratio. The minimum distance and the separation ratio affect the branching behavior of the tree. The separation ratio determines how large the correlation difference between groups of clustered genes must be for them to be considered discrete groups. This number should be between 0 and 1 and determines how large the correlation difference between groups of clustered genes has to be for the groups to be considered discrete groups and not be joined together. Increasing separation increases the “branchiness” of the tree, whereas a separation ratio of 0 indicates that all gene expression profiles can be regarded as identical. Minimum distance. The minimum distance deals with how far down the tree discrete branches are depicted. A value ,0.001 has very little effect, this is because most genes are not more closely related than that. A higher number tends to add more genes to a group, making the groups less specific. The minimum distance deals with how far down the tree discrete branches are depicted. A higher number tends to combine more genes to a group, making the groups less specific (Fig. 3.8). Experiment and gene clusters: It is becoming increasingly common for clustering algorithms to be done on both the gene lists and the samples. The resultant dendrograms is a graphical matrix of the relationship between genes and the corresponding samples (Fig. 3.9). The extent of gene expression is usually shown by changes in color. Typically, red depicts high expression and green depicts

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Figure 3.8 The effect of different statistical methods for the calculation of hierarchical clusters. (A) The sample dendrogram produced using a standard correlation of similarity measurement. (B) The related dendrogram when distance is used of similarity measurement.

lower or absent expression. The gradient of color change between these two colors is proportional to the signal intensity (Fig. 3.10) of an individual gene. Cluster analysis can be a very powerful tool. Genes with similar expression profiles across a series of samples are clustered together; often these genes do not have functional similarity, which may lead to a re-examination of the biological hypothesis. It should always be noted that similar expression levels or patterns does not always mean that the genes are related in the same biological pathway. This is because any genetic abnormality or external stimulus may initiate several different, and separate, pathways that exhibit concordant gene expression patterns, but these may be coincidental. A related strategy is to identify genes whose expression patterns are similar or opposite to that of a known

Figure 3.9 A two-way cluster analysis, or heat-map, of gene expression. Vertical columns within the heat-map represent expression within each sample. Horizontal rows show the expression level of individual genes across the samples.

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Figure 3.10 The vertical color gradient represents the relative expression levels: white at the bottom of the bar represent low or absent expression, whereas black are high levels of normalized expression. The gradient between these two extremes correlates with the expression levels. The horizontal axis of the color bar indicates a measure of reliability of the data. The darker the region towards the right represents increasing trust or reliability of the data. The trust is a representation of the (the median value of the chip)  (the median value of the gene).

candidate gene. These approaches will allow relationships between, and within, molecular proliferation, differentiation, or apoptotic pathways. Hierarchical clustering of samples within an experiment will usually group together those samples that show distinct phenotypes. However, it should be noted that the concept of “majority voting” is very important in the sample clustering. This means that each biological parameter within an experimental sample population should be equally represented as in majority voting, the results of presenting a pattern to a number of networks are tallied, and the majority classification is taken as correct. QT clustering: QT clustering identifies clusters of genes so that each gene in the cluster is within a specified distance metric of every other gene in the cluster. In QT clustering, the “diameter” of a cluster refers to the largest distance between any two genes in the same cluster (Fig. 3.11). A cluster built by QT clustering starts with a single gene. The diameter at that point is 0. It then adds the

Figure 3.11 Three of the 57 QT clusters, grouped on the basis of the criteria of minimum cluster size of 25 genes with minimum similarity of 0.1 and standard correlation of similarity measurement.

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gene that is closest to the starting gene. The diameter of the cluster is now equal to the distance between the two genes. It continues adding genes one at a time, always choosing a gene that will result in the smallest cluster diameter. Eventually, it reaches a point where no genes can be added without the diameter growing beyond a defined cutoff. The cluster is then complete. Importantly, the first cluster obtained is dependant on the initial gene is chosen. Therefore, it independently builds clusters starting from each gene in the user-selected gene list. The cluster with the most genes is kept, and is part of the final classification. All others are discarded. That stage results in a single cluster. The genes contributing to that cluster are removed from the overall gene list, and the clustering process is begun again. Again, a new cluster is built from every gene in the reduced gene list, the largest one is kept. This process is repeated until the number of genes in the largest cluster is smaller than a user-defined cutoff. Class Prediction—Supervised Learning Methods In the earlier sections, methods are described which can be considered as “class discovery.” These are used with out a preconception of classes or parameters associated with any sample (Fig. 3.2) and k-means, SOMs, and hierarchical cluster analysis are types of this analysis. Class prediction methods, or supervised learning methods, use sample associated parameters or classes to identify gene lists that can associate unknown samples with these parameters or classes (33 – 35) (Fig. 3.2). Class Prediction The class predictor is designed to predict the value, or “class,” of an individual parameter in an uncharacterized sample or set of samples. This is usually performed on a split sample method. The number of samples is divided into two groups: a training set (usually comprising two-third of the total sample size); and a test set (the remaining one-third). In the training set, multiple kinds of parameters can be defined; however, the parameters within the separate test set are not used for the development of the prediction model. In the first of two steps, the class predictor algorithm examines all genes in the training set individually and ranks them on their power to discriminate each class from all the others. In the next step, it uses the most predictive genes to classify the test set (in this set the parameter value is unknown). For example, a disease type could be predicted from the expression data from samples whose disease status is known. Class predictor can also be used to identify genes whose behavior is related to a given parameter by examining the list of predictor genes. Ordering all the measurements for a given gene according to their normalized expression levels assembles the list of predictor genes. For each parameter value, the predictor places a mark in the list where the relative abundance of the class on one side of the mark is the highest in comparison to the other side of the mark. The genes that are most accurately segregated by these markers are

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considered to be the most predictive. A list of the most predictive genes is made for each class and an equal number of genes are taken from each list. Cross-Validation Cross validation, or “jack knifing” is an alternative to the split sample method of class prediction (36 – 38). This employs a “leave one out” cross validation method in which the test set consists of only one sample. The remainder form the training set, which is used to develop the class prediction gene model. This model is then used to predict the class of test sample on the basis of the expression profile. If the model-test sample does not correlate, a new cycle of training-omitted sample is run in which a different sample is omitted from the training set. At the end of n models, where n is the number of samples, a cross-validated error rate is calculated. This is an estimate of error rate that could be used for future samples, if the relationships between class and expression profiles are the same. VALIDATION OF MICROARRAY ANALYSIS As indicated previously, microarrays represent an extremely powerful technique for the analysis of gene expression. The range of experiments is endless and the data produced from even a limited experiment using an array with only a limited number of genes can be produced enough candidates for several new research projects. However, it should be recognized that there are technical limitations that may distort the data analysis. The size (in bp), the type (cDNA or oligonucleotides), and the location in the target gene of microarray probes may all contribute to efficiency of the hybridization reaction and thus the ability of the technique to accurately detect differences in gene expression levels. One of the easiest ways to reduce erroneous expression results is to ensure that array experiments are fully optimized. Validation approaches can be divided into two main areas: in silico or laboratory approaches. In silico Validation This is based on bioinformatics and allows data from microarray experiments to be analyzed against publicly or privately available databases. Publicly available expression data is often available as an appendix to published articles. However, comparison of locally produced data with that within any other database should be examined with several provisos, many of which relate to the technical procedure and arrays analyzed. A wide range of information about candidate genes can also be mined via bioinformatics databases. Gene annotations can be obtained from the National Center for Bioinformation Technology (www.ncbi.nlm.nih.gov/) or The Institute for Genome Research (TIGR) (www.tigr.org). The GeneCards is a database of human genes, their products and their involvement in diseases (http:// bioinformatics.weizmann.ac.il/cards/). Other relevant databases include those

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for literature information such as MEDLINE (http://ncbi.nlm.nih.gov/PubMed/), genetic variations in sequence such as registered by dbSNP (http://ncbi.nlm.nih. gov/SNP) or HGBase (http://hgbase/interactiva/de), or location to specific metabolic pathways using KEGG (http://kegg.genome.ad.jp/kegg/). The Gene Ontology (GO) Consortium (www.geneontology.org) produces a controlled vocabulary that can be applied to all organisms as the knowledge of genes and the role of their proteins accumulates. The ontologies relate genes into three broad categories: molecular function; biological processes; or cellular component. Numerous other databases of promoter function, structural motifs, or phenotype data are becoming available. Laboratory Based Validation Laboratory-based validation of data provides a second level of independent, experimental verification of gene expression levels. Methods that can be used to confirm, and increase confidence, in microarray expression results include northern blotting and quantitative RT-PCR (RQ-PCR), RNA protection assays, in situ hybridization or immunohistochemistry using tissue microarrays (TMAs). RQ-PCR allows the continuous measurement of products produced during the course of PCR reaction. This can be done using the TAQman based fluorescent probe method or by using Sybr Green to detect double stranded DNA products. Both methods rely on the detection of product molecule present throughout the numerous cycles occurring during a complete PCR reaction. The exponential growth in PCR products is related to the cycle threshold point and from this crossing point, the number of targets present in the input sample can be determined. TMA containing numerous microscopic sections of tissue are another approach for the validation of array data in a large set of diverse tissue specimens unrelated to the initial experimental samples. Perhaps the most relevant, but also time-consuming method of validation is to examine protein levels of the relevant candidate genes. Protein analysis can be done on several levels depending on the resources and questions asked. Proteomics can study of the function of all expressed proteins; and although rapid progress has been made in the past few years in generating large-scale data sets of protein profiles, the technique still requires further improvements to achieve its potential. Protein analysis will allow post-translational modifications to be detected—which microarray techniques will fail to identify. Changes in protein levels can be done by western blot analysis, in combination with immunoprecipitation, or by sensitive flow cytometry methods. MICROARRAY ANALYSIS SOFTWARE The statistical methodologies described earlier are complicated and involve very large data sets and calculations. Numerous software packages have been designed to analyze interpret, and analyze the images and intensity data obtained from

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microarrays. Three general categories of software are available. The first group could be called “stand-alone” packages and will accept data from several different array scanners and imagers: ArrayGauge (Fujifilm Medical Systems) and ImageMaster Array 2 (Amersham Pharmacia). A second group are designed to interact with only with data produced from specific microarray scanners; QuantArray (Packard BioChip Technologies) and GenePix Pro 3.0 Array Analysis Software (Axon Instruments). The third group consists of software for the analysis of array-specific systems. The best known, owing to the high use of GeneChips is the Microarray Suite (MAS) Software (Affymetrix Inc.). ArrayToolsTM (Incyte Genomics) allows the investigation of data generated by Life ArrayTM microarrays; whereas Pathways 3 Microarray Analysis Software (Research Genetics) of Huntsville, Ala., analyses GeneFilters. There are also available a number of post-imaging analysis software packages that use data mining tools. VistaLogic software has methods for visualizing gene expression profiles, and has the novel approach of using 3-D plot with peaks and valleys to depict up and down regulated genes. This plot rotates and has a zoom feature designed to extract additional information from the data. GeneSpringTM Expression Analysis software (Silicon Genetics) has a host of visualization and analysis tools. The visualization options include graphics showing the physical position of a gene on a chromosome and expression profile graphs. Individual profiles are hyperlinked to annotations about the underlying gene and can find genes with a similar expression profile based on a variety of similarity measures (e.g., Pearson correlation or Euclidian distance). Spotfire Array Explorer (Spotfire Inc.) contains numerous clustering packages, whilst BioDiscovery’s GeneSight offers some 19 color maps for data visualization. A comprehensive list of commercial microarray analysis software packages has been published (http://www.the-scientist.com/images/yr2001/pdfs/microarray_010430.pdf). However, new packages and databases are constantly being developed by academics (39 –44) and commercial companies. SUMMARY Microarray analysis of gene expression is still in an early phase of development. Statistical algorithms can be used to identify numerous genes that change in expression levels: may only be expressed in certain sub-groups of samples; or can predict which type of group a specific sample should be placed. However, perhaps the overall goal of any microarray experiment is to place changes in gene expression within the concept of molecular networks and interactions. If one gene shows, for example, a 3-fold increase in expression, what effect does that have on other genes within the same pathway, on related pathways, or on the cellular phenotype. The more the microarray data analyzed and validated, the more we will understand of interactions and nodes of interactions. It is only when these complex networks are identified and validated will we truly understand how normal and abnormal cells function differently.

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REFERENCES 1. Schena M, Shalon D, Davis RW, Brown PO. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 1995; 270:467– 470. 2. DeRisi J, Penland L, Brown PO, Bittner ML, Meltzer PS, Ray M et al. Use of a cDNA microarray to analyse gene expression patterns in human cancer. Nat Genet 1996; 14:457– 460. 3. Hegde P, Qi R, Abernathy K, Gay C, Dharap S, Gaspard R et al. A concise guide to cDNA microarray analysis. Biotechniques 2000; 29(3):548 – 556. 4. Dobbin K, Simon R. Comparison of microarray designs for class comparison and class discovery. Bioinformatics 2002; 18(11):1438– 1445. 5. Bilban M, Buehler LK, Head S, Desoye G, Quaranta V. Normalizing DNA microarray data. Curr Issues Mol Biol 2002; 4(2):57 – 64. 6. Bolstad BM, Irizarry RA, Astrand M, Speed TP. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 2003; 19(2):185 –193. 7. Brazma A, Hingamp P, Quackenbush J, Sherlock G, Spellman P, Stoeckert C et al. Minimum information about a microarray experiment (MIAME)-toward standards for microarray data. Nat Genet 2001; 29(4):365– 371. 8. Stoeckert CJ Jr, Causton HC, Ball CA. Microarray databases: standards and ontologies. Nat Genet 2002; 32(suppl):469 –473. 9. Craigon DJ, James N, Okyere J, Higgins J, Jotham J, May S. NASCArrays: a repository for microarray data generated by NASC’s transcriptomics service. Nucl Acids Res 2004; 32(1):D575– D577. 10. Tong W, Cao X, Harris S, Sun H, Fang H, Fuscoe J et al. ArrayTrack—supporting toxicogenomic research at the US Food and Drug Administration National Center for Toxicological Research. Environ Health Perspect 2003; 111(15):1819– 1826. 11. Killion PJ, Sherlock G, Iyer VR. The Longhorn array database (LAD): an opensource, MIAME compliant implementation of the Stanford Microarray Database (SMD). BMC Bioinformatics 2003; 4(1):32. 12. Taylor CF, Paton NW, Garwood KL, Kirby PD, Stead DA, Yin Z et al. A systematic approach to modeling, capturing, and disseminating proteomics experimental data. Nat Biotechnol 2003; 21(3):247– 254. 13. Brazma A, Parkinson H, Sarkans U, Shojatalab M, Vilo J, Abeygunawardena N et al. ArrayExpress—a public repository for microarray gene expression data at the EBI. Nucleic Acids Res 2003; 31(1):68– 71. 14. Spellman PT, Miller M, Stewart J, Troup C, Sarkans U, Chervitz S et al. Design and implementation of microarray gene expression markup language (MAGE-ML). Genome Biol 2002; 3(9):RESEARCH0046. 15. Singhal S, Kyvernitis CG, Johnson SW, Kaiser LR, Liebman MN, Albelda SM. Microarray data simulator for improved selection of differentially expressed genes. Cancer Biol Ther 2003; 2(4):383– 391. 16. Zhao Y, Pan W. Modified nonparametric approaches to detecting differentially expressed genes in replicated microarray experiments. Bioinformatics 2003; 19(9):1046– 1054. 17. Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA 2001; 98(9):5116– 5121.

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18. Cardoso F. Microarray technology and its effect on breast cancer (re)classification and prediction of outcome. Breast Cancer Res 2003; 5(6):303 – 304. 19. Wright G, Tan B, Rosenwald A, Hurt EH, Wiestner A, Staudt LM. A gene expressionbased method to diagnose clinically distinct subgroups of diffuse large B cell lymphoma. Proc Natl Acad Sci USA 2003; 100(17):9991– 9996. 20. Alizadeh AA, Eisen MB, Davis RE, Ma C, Lossos IS, Rosenwald A et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling [see comments]. Nature 2000; 403(6769):503– 511. 21. Golub TR, Slonim DK, Tamayo P, Huard C, Gaasenbeek M, Mesirov JP et al. Molecular classification of cancer: class discovery and class prediction by gene expression monitoring. Science 1999; 286(5439):531– 537. 22. Sarkar M, Leong TY. Fuzzy K-means clustering with missing values. Proc AMIA Symp 2001; 588– 592. 23. Schachter AD, Kohane IS. An unsupervised self-optimizing gene clustering algorithm. Proc AMIA Symp 2002; 682–686. 24. Kohonen T, Somervuo P. How to make large self-organizing maps for nonvectorial data. Neural Netw 2002; 15(8 – 9):945– 952. 25. Kohonen T. Comparison of SOM point densities based on different criteria. Neural Comput 1999; 11(8):2081– 2095. 26. Tamayo P, Slonim D, Mesirov J, Zhu Q, Kitareewan S, Dmitrovsky E et al. Interpreting patterns of gene expression with self-organizing maps: methods and application to hematopoietic differentiation. Proc Natl Acad Sci USA 1999; 96(6):2907– 2912. 27. Yeung KY, Ruzzo WL. Principal component analysis for clustering gene expression data. Bioinformatics 2001; 17(9):763–774. 28. Remy I, Michnick SW. Dynamic visualization of expressed gene networks. J Cell Physiol 2003; 196(3):419– 429. 29. Eisen MB, Spellman PT, Brown PO, Botstein D. Cluster analysis and display of genomewide expression patterns. Proc Natl Acad Sci USA 1998; 95(25):14863–14868. 30. Alter O, Brown PO, Botstein D. Singular value decomposition for genomewide expression data processing and modeling. Proc Natl Acad Sci USA 2000; 97(18):10101– 10106. 31. Shannon W, Culverhouse R, Duncan J. Analyzing microarray data using cluster analysis. Pharmacogenomics 2003; 4(1):41 – 52. 32. Guess MJ, Wilson SB. Introduction to hierarchical clustering. J Clin Neurophysiol 2002; 19(2):144– 151. 33. Soinov LA. Supervised classification for gene network reconstruction. Biochem Soc Trans 2003; 31(Pt 6):1497– 1502. 34. Lagreid A, Hvidsten TR, Midelfart H, Komorowski J, Sandvik AK. Predicting gene ontology biological process from temporal gene expression patterns. Genome Res 2003; 13(5):965– 979. 35. Soinov LA, Krestyaninova MA, Brazma A. Towards reconstruction of gene networks from expression data by supervised learning. Genome Biol 2003; 4(1):R6. 36. Nelander S, Mostad P, Lindahl P. Prediction of cell type-specific gene modules: identification and initial characterization of a core set of smooth muscle-specific genes. Genome Res 2003; 13(8):1838– 1854. 37. Bura E, Pfeiffer RM. Graphical methods for class prediction using dimension reduction techniques on DNA microarray data. Bioinformatics 2003; 19(10):1252–1258.

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38. Tanay A, Sharan R, Shamir R. Discovering statistically significant biclusters in gene expression data. Bioinformatics 2002; 18(suppl 1):S136 –S144. 39. Azuaje F. Clustering-based approaches to discovering and visualising microarray data patterns. Brief Bioinform 2003; 4(1):31 – 42. 40. Dudoit S, Gentleman RC, Quackenbush J. Open source software for the analysis of microarray data. Biotechniques 2003; (suppl):45 – 51. 41. Anderle P, Duval M, Draghici S, Kuklin A, Littlejohn TG, Medrano JF et al. Gene expression databases and data mining. Biotechniques 2003; (suppl):36 – 44. 42. Satagopan JM, Panageas KS. A statistical perspective on gene expression data analysis. Stat Med 2003; 22(3):481– 499. 43. Tamames J, Clark D, Herrero J, Dopazo J, Blaschke C, Fernandez JM et al. Bioinformatics methods for the analysis of expression arrays: data clustering and information extraction. J Biotechnol 2002; 98(2 – 3):269– 283. 44. Weinstein JN, Scherf U, Lee JK, Nishizuka S, Gwadry F, Bussey AK et al. The bioinformatics of microarray gene expression profiling. Cytometry 2002; 47(1):46 – 49.

4 Oxidative Stress, Gene Expression, and Lifespan Yoko Honda and Shuji Honda Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan

Introduction Lifespan Regulation in C. elegans Aging of C. elegans Stress, Hormesis, and Lifespan in C. elegans Insulin/IGF-I Signaling and Lifespan Regulation Insulin/IGF-I Signaling Pathway and Stress Resistance DAF-16 Transcription Target Reproductive System and Lifespan Nervous System and Lifespan Mitochondrial Electron Transport and Lifespan Lifespan Regulation in Drosophila Lifespan Regulation in Mammals Replicative Lifespan and Oxidative Stress Conclusion References 67

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INTRODUCTION The lifespan of metazoans can be extended by environmental conditions: caloric restriction (CR) in a wide range of organisms (1), low temperature in some poikilothermic animals (2), and low oxygen concentrations in the nematode Caenorhabditis elegans (3). The mechanisms by which each condition slows the aging rate have not yet been fully elucidated. Function of CR has been postulated as hormonal changes, altered gene expression, lowered metabolic rate, and a reduced generation rate of mitochondrial reactive oxygen species (ROS). Lifespan could also be lengthened by environmental perturbations. Hormesis is a phenomenon occurring when agents that are harmful at high doses or over long periods, actually produce beneficial effects, such as lifespan extension, when used at low doses or over short periods. C. elegans shows lifespanextension hormesis when exposed to low doses of radiation (4) or short-term heat (5), hyperoxia (6), or hyperbaric oxygen (7). These treatments are associated with adaptive resistance to lethal thermal or oxidative stress, and the gene expression of stress-defense proteins (5,6,8). Recently, lifespan-extension mutants of the nematode C. elegans have been extensively isolated, and the gene network responsible for its longevity has been unraveled [for a review see Ref. (9)]. Two main classes of lifespan-extension mutants have been reported; one class is related to the activity of the mitochondrial electron transport chains, such as clk-1 (10) and isp-1 (11), and the other is related to hormonal mechanisms, especially an insulin/IGF-I signaling pathway, such as daf-2 (12,13) and age-1 (14,15). The insulin/IGF-I signaling pathway regulates the activities of DAF-16, the fork head transcription factor (16,17) and HSF-1, heat shock transcription factor 1 (18) to activate the transcription of genes that have more direct effect on lifespan determination. This pathway confers resistance to a variety of stresses, including heat (5), UV (19), metal (20), and oxidative stress (21 –23) as well as lifespan extension in C. elegans. Recent investigations suggest that an analogous pathway also regulates stress resistance and longevity in Drosophila (24,25) and mammals (26,27). The close association between stress resistance and longevity suggests the possibility that the molecular mechanisms protecting against stress may overlap to retard the aging process. The free-radical theory of aging, first proposed by Harman (28), is attracting considerable attention (29). According to this theory, aging is the result of accumulated ROS-induced oxidative stress. Indeed, a large body of correlative evidence is consistent with this hypothesis (30). Recently, studies into the insulin/IGF-I signaling pathway that promotes longevity and changes gene expression in C. elegans (31,32) have been initiated, and the RNAi inactivation effect on lifespan of these genes has been systematically analyzed. The gene expression profiles of various tissues in calorically restricted mammals or lifespan-extension mutant mammals have also been studied (33,34). These studies will provide a molecular description of organismal mechanisms regulating the aging processes.

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LIFESPAN REGULATION IN C. ELEGANS Aging of C. elegans The wild-type C. elegans lives for 3 weeks. As an animal ages, various senescence symptoms become apparent (35). From studies of ultrastructural observation and the visualization of specific cell types with green fluorescent protein (GFP), the nervous system is shown to be intact even in advanced old age, however, a gradual and progressive deterioration of muscles becomes apparent with increasing age resembling human sarcopenia (36). The stochastic features in the aging processes were clearly shown. Using full-genome microarrays, the gene expression changes during aging were examined (37). One hundred and sixty four genes, ,1% of the genome, show significant changes in expression as aging advances. Dynamic changes in the expression of four insulin/IGF-I-like genes and two sir-2 homologues are observed during aging. The expression of two heat shock proteins, HSP16 and HSP70, also shows age-related changes. Murakami and Johnson (38) showed that the expression of old-1 gene increase with advancing age. The old-1 gene product, OLD-1, is a transmembrane tyrosine kinase protein that is upregulated in daf-2 and age-1 lifespanextension mutants in a daf-16-dependent manner. Various stresses, such as heat, UV, and starvation induce old-1 expression. The overexpression of old-1 induces stress resistance and lifespan extension. In contrast, the genomic knockout (KO) of old-1 in wild-type animals causes lifespan shortening and stress hypersensitivity. Furthermore, the genomic KO or RNAi inactivation of old-1 in lifespan-extension mutants restores the normal lifespan. This indicates that OLD-1 is a positive regulator of stress resistance and longevity.

Stress, Hormesis, and Lifespan in C. elegans We showed that the lifespan of C. elegans decreases with increasing environmental oxygen concentration (39). On the other hand, low oxygen concentrations extend the lifespan (3), indicating that environmental oxygen concentration is one of lifespan determinants. Since ROS are thought to arise in organisms depending on the oxygen concentration (40), these findings suggest that ROS are involved in determination of lifespan. On the other hand, short-term exposure to hyperoxia slightly lengthened lifespan (6), short-term exposure to hyperbaric oxygen also increases lifespan (7). These effects are thought to be a form of hormesis for lifespan. In response to oxidative stress, the level of SOD activity and the expression of CuZnSOD, MnSOD, and catalase genes and oxidative stress resistance (6,8) increase in C. elegans. Pretreatment with hyperbaric oxygen or juglone (an intracellular generator of ROS) significantly increased subsequent resistance to the same or reciprocal stressors (7). The most widespread explanation is that an increased ability to remove ROS induced by oxidative stress could reduce normally occurred oxidative stress that may cause aging.

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High doses of ionizing radiation reduce the lifespan of C. elegans, but low doses induce moderate lifespan extensions (4). We reproduced this result (S. Honda, Y. Honda, and S. Suzuki, unpublished observation) but Cypser and Johnson (7) found no lifespan extension by low-dose ionizing radiation as well as UV. These hormetic effects may be weak and occasional. Heat treatment at sublethal temperatures induces increased resistance to subsequent lethal heat stress and modestly extends the lifespan of C. elegans (5). Heat shock induces a small heat shock protein (SHSP), HSP-16, in wildtype animals (41). Hsu et al. (42) also showed the increased expression of shsp genes, hsp-16.1, hsp-16.49, hsp-12.6, and sip-1 by heat shock. Yokoyana et al. (43) showed the induction of hsp70F and lifespan extension by heat shock. The overexpression of hsp70F, predominantly in the muscles, induces lifespan extension, and overexpression of the hsp16 gene induces thermal resistance and extended lifespan (44). These HSPs may play an important role as the molecular chaperon for preventing improper protein associations accumulated in the aging process. Treatment of C. elegans worms with salen– manganese complexes, EUK-8 or EUK-134, synthetic SOD/catalase mimetics increased lifespan by 44% (45), but other investigators could not reproduce their results (46). The extension of lifespan by SOD/catalase mimetics may only occur under very particular culture conditions. SOD/catalase mimetics was reported to have cytoprotective activities in ischemic rat brain injury (47), and reverse age-related learning impairment and brain protein oxidation in mice (48). Insulin/IGF-I Signaling and Lifespan Regulation One of the two main classes of the lifespan-extension mutations of C. elegans is related to the insulin/IGF-I signaling. Insulin/IGF-I signaling is mediated by the DAF-2 insulin/IGF-I receptor. The daf-2 mutants that reduce the activity of DAF-2 remain youthful and active much longer than the wild-type animals and live more than twice as long [reviewed in Ref. (9)]. The lifespan-extension phenotype of the daf-2 is suppressed by mutations in daf-16, indicating that daf-16 is negatively regulated by DAF-2 signaling and is the major downstream effector. The daf-16 encodes a FOXO transcription factor (16,17). Binding of insulin/IGF-I-like ligands to the DAF-2 insulin/IGF-I receptor controls insulin/IGF-I signaling. There are at least 38 genes (ins) encoding insulin/IGF-I-like peptides in C. elegans (49,50). Many of these genes are divergent insulin superfamily members, and as the specific ligand has not yet been identified, these members may be possible to have complex and redundant roles. The daf-28 gene encodes insulin-like peptide. A dominant-negative allele of the daf-28 mutant lives 10% longer than wild-type animals. A phenotype of the daf-28 mutant is rescued by ins-4 or ins-6 transgene, suggesting a redundant nature (51). Some ins genes are expressed in sensory neurons (50,51). Environmental cues such as food, pheromones and temperature may affect

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insulin/IGF signaling through different expression and the secretion of various INS peptides. The mutation of age-1, which encodes the PI3 (phosphoinositide-3-OH) kinase catalytic subunit, doubles the lifespan in C. elegans (14,15). The current model of insulin/IGF-I signaling is as follows (Fig. 4.1). The DAF-2 insulin/IGF-I receptor transduces signals by activating AGE-1. AGE-1 PI3 kinase phosphorylates PIP2 to generate the second messenger PIP3.

Environmental Cues Food Temperature Pheromone

cGMP Signaling DAF-11 Guanyl Cyclase

Sensory Neurons

DAF-21 HSP90

TGF-β Signaling DAF-7 TGF-β

DAF-1 DAF-4 Receptor

DAF-8 DAF-14

DAF-9 P450

DAF-3 SMAD

DAF-12 Nuclear Hormone Receptor

Serotonergic Signaling Insulin/IGF-I Insulin/IGF-I Signaling

DAF-2 Receptor

AKT-1/AKT-2/SGK-1 AGE-1 PI3K PDK-1 PIP2 PIP3 DAF-18 PTEN

DAF-16 FOXO Transcription Factor

SOD-3 MTL-1 SCL-1

Longevity

SHSP Stress

Germ Line

HSF-1 Heat Shock Transcription Factor

Reproductive System Gonad

Figure 4.1 The signaling pathways regulating lifespan in C. elegans. C. elegans senses environmental cues including food, pheromones, and temperature by sensory organs. The environmental information is transduced into at least four signaling pathways including TGF-b, cGMP, serotonin, and insulin/IGF-I. The serotonergic signaling affects the TGF-b and the insulin/IGF-I signaling. TGF-b is secreted by a pair of specific sensory neurons. Insulin/IGF-I ligands appear also to be secreted by sensory neurons and are rendered in neuroendocrine system to bind to the receptor DAF-2. This signal is finally transduced to regulate the activity of the transcription factor DAF-16. TGF-b, cGMP, and insulin/IGF-I signaling pathways converge in the regulation of the activity of P450 DAF-9 to synthesize lipophilic (steroid?) hormones. Those appear to circulate systemically and activate the nuclear hormone receptor DAF-12. The gonadal tissue and germline cells send the signal that regulates to activate DAF-16 and DAF-12. Various stresses also regulate to activate heat-shock transcription factor HSF-1 as well as DAF-16. These transcription factors in concert regulate the aging rate and lifespan by controlling transcription of the target genes.

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On the other hand, DAF-18 PTEN dephosphorylates PIP3 and thus antagonizes the action of AGE-1. Thus, the PIP3 level is determined by a balance between generation by AGE-1 PI3 kinase and degradation by DAF-18 PTEN. PIP3 activates PDK-1 (3-phosphoinositide-dependent kinase-1), which in turn phosphorylates and activates AKT-1/AKT-2/SGK-1 Ser/Thr kinase. AKT-1/ AKT-2/SGK-1 phosphorylates and inactivates the DAF-16 transcription factor to be sequestered from the nucleus to the cytoplasm. In this state, adults age rapidly. On the contrary, when DAF-2 signaling is reduced, DAF-16 is eventually translocated to the nucleus to promote transcription of target genes. In fact, disrupting AKT-consensus phosphorylation sites in DAF-16 causes nuclear accumulation, although the nuclear accumulation is not sufficient for lifespan extension (52,53). C. elegans worms grow through four larval stages (L1 –L4) before reaching maturity. However, when the food supply is limited and the population density is high at the L1 stage, animals become dauer larvae after the L2 stage. The dauer larva is a developmentally arrested dispersal stage and lives up to several months, greatly exceeding the normal adult lifespan of about 3 weeks under stressful environmental conditions (53). It seems that the dauer stage is nonaging, because the post-dauer life span is not affected by a prolonged dauer stage of up to 2 months (54). The dauer larva is more resistant to a variety of environmental stresses, including hypoxia, heat, desiccation, and oxidative stress and has increased levels of SOD and catalase (21,55). The expression of the MnSOD gene (sod-3) is higher in the dauer larvae than in the adults (23). As dauer larvae live much longer than adults, some genes expressing altered levels in dauer state may be the key to longevity. By using serial analysis of gene expression (SAGE), Jones et al. (56) found that the expression of tts-1 (transcribed telomere-like sequence), a variant histone H1 and a nucleosome assembly protein possibly relating to the structure or stability of chromatin is high in dauer larvae. These results suggest that the chromatin structure may change to be more stable in the dauer state than in the growing state. Holt and Riddle (57) examined gene expression profiles of carbohydrate metabolism in dauer larvae by using SAGE. A high gene expression of pyruvate kinase, alcohol dehydrogenase, a putative cytosolic fumarate reductase, two pyruvate dehydrogenase components, and a succinyl CoA synthetase a subunit implies that anaerobic metabolism is prominent in dauer larvae. By genetic analysis of mutants displaying “dauer larva formation abnormal,” Daf phenotype, a number of genes that regulate dauer formation have been identified (53). These genes have been assembled into four neuroendocrine signaling pathways: TGF-b/SMAD, cGMP, serotonin (58) and insulin/IGF-I. DAF-7, a TGF-b family member expressed in a pair of sensory neurons, signals through transmembrane receptor kinases DAF-4 and DAF-1. These receptors regulate the activities of DAF-8 and DAF-14, dwarfin/MAD/DPC-4, and DAF-3, SMAD transcription factors. The cGMP pathway is composed of DAF-11, transmembrane guanyl cyclase and DAF-21, HSP90. The mutant

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adults in the TGF-b and cGMP pathway do not exhibit a lifespan-extension phenotype. The serotonin pathway affects TGF-b and insulin/IGF-I signaling. The mutant of the serotonin pathway has a longer reproductive period than wild-type animals (58). TGF-b, insulin/IGF-I, and cGMP signaling pathways converge on DAF-9, a member of the cytochrome P450 hydroxylase family (59,60) that is implicated in the synthesis of a lipophilic hormone acting upstream of DAF-12, nuclear hormone receptor (61,62). Sterols may be the DAF-9 substrate and DAF-12 ligand because cholesterol deprivation displays daf-9 mutant phenotype. DAF-12 is expressed within almost all cells, whereas DAF-9 is expressed within two sensory neurons, hypodermal cells, and somatic gonadal cells thought to be endocrine tissues. In these pathways, the mutations in the insulin/IGF-I pathway mainly affect the adult lifespan. The simplest interpretation of these observations is that the dauer larvae have an efficient life-maintenance mechanism for long dauer survival under stressful conditions, and that the insulin/IGF-I pathway is closely related to this mechanism (12,63). Although the mutants in the TGF-ß and cGMP pathways do not display a lifespan-extension phenotype, these pathways interact with the insulin/IGF-I pathway at daf-9 position to affect lifespan (Fig 4.1). The daf-9 mutations extend lifespan at a certain temperature (59,60). The daf-9 and daf-12 mutations enhance the lifespan-extension of certain daf-2 mutants (63,64). Insulin/IGF-I Signaling Pathway and Stress Resistance To investigate the relationship between lifespan-extension and oxidative-stress resistance, we screened the oxidative-stress resistance phenotype in various mutants (6,23). We examined the survival period of each mutant under experimentally induced, acute oxidative stress. We used paraquat, an intracellular superoxide O2 2 generator, under hyperoxia for acute oxidative stress. The daf-2 mutants with an extended lifespan, survived for a longer period of time than wild-type animals in the presence of paraquat under hyperoxic or normoxic conditions. The daf-2 mutant is also more resistance to menadione, another intracellular O2 2 generator, under hyperoxia, than wild-type animals. The mutants in the TGF-b pathway and cGMP pathway do not show oxidative-stress resistance. The oxidative-stress resistance seen in the daf-2 mutants is suppressed by mutations in daf-18 or daf-16 indicating that daf-16 and daf-18 act downstream of daf-2 to confer oxidative-stress resistance, as well as extended lifespan. Vanfleteren (22) and Larsen (21) showed that the lifespan-extension mutant age-1 is more resistant to oxidative stress in old age than wild-type animals at the same age. We showed that the age-1 mutants of young adults also display the oxidative-stress resistance. The oxidative-stress resistance in age-1 mutants is suppressed by daf-16 mutation, indicating that daf-16 is located downstream of age-1 in the pathway for regulating oxidative-stress resistance. †

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On the other hand, oxidative-stress resistance in two alleles of age-1 mutants (m333 and mg44) is not fully suppressed by daf-18 mutation, indicating that daf-18 does not act downstream of age-1. daf-16 and daf-18 act downstream of daf-2 in the insulin/IGF-I signaling pathway for oxidative-stress resistance (23). Taken together, we postulate the following pathway for oxidative-stress resistance: daf -2
! daf -18
! age-1
! daf -16
! Oxidative-stress resistance This pathway is essentially identical to the pathway regulating longevity (63), suggesting a strong association between lifespan-extension and oxidative-stress resistance. However, Dorman and Canyon (65) demonstrated that the daf-18 mutation suppressed the lifespan-extension phenotype of another allele of age-1 (hx546) indicating that daf-18 acts downstream of age-1. Such differences could be attributed to the differences in severity of the age-1 alleles used. Two alleles of age-1 mutants (m333 and mg44) display the Daf phenotype, which is completely suppressed by daf-18 or daf-16 mutations, indicating the following pathway for dauer formation: daf -2
! age-1
! daf -18
! daf -16
! dauer formation Thus, oxidative-stress resistance is closely associated with longevity but not with dauer formation. The PIP3 level is maintained under a balance between generation by AGE-1 PI3 kinase and degradation by DAF-18 PTEN, which could determine the impact of this pathway. The loss or reduction of function mutations in age-1 could reduce PI3 kinase activity to drop this second messenger level. When DAF-18 is reduced, only the preexisting pool of the second messenger may be insufficient to inhibit longevity and oxidative-stress resistance but sufficient to inhibit dauer formation. Dillin et al. (66) found that the inactivation of daf-2 during adulthood by RNAi extends lifespan and increases oxidativestress resistance. Since dauer formation is switched in the early larval stage, the insulin/IGF-I pathway controls the dauer switch and oxidative-stress resistance/longevity independently. There are several genes in C. elegans that encode SOD enzymes: sod-1 encodes cytosolic CuZnSOD (21), sod-2 and sod-3 each encodes mitochondrial MnSOD (67 – 69), and sod-4 encodes extracellular CuZnSOD (70). The level of sod-3 mRNA in daf-2 mutants is higher than that in the wild-type animals (23). The levels of mRNA transcripts of sod-1, sod-2, and catalase in the daf-2 are similar to those in the wild-type animals. The level of sod-3 mRNA in daf-2 mutants increases as it develops from the egg to the L2 larval stage coinciding with increased in oxidative-stress resistance. The elevated level of sod-3 mRNA in the daf-2 mutants is suppressed by daf-16 and daf-18 mutation (23). The level of sod-3 mRNA in the age-1 mutants is higher than that in wild-type animals (6). The elevated level of sod-3 mRNA in age-1 mutants is

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suppressed by daf-16 mutation but is not fully suppressed by daf-18 mutation. These results provide further evidence that the insulin/IGF-I signaling pathway regulates extended lifespan, oxidative-stress resistance and sod-3 expression in a similar way. These results suggest that the extended lifespan is correlated with the efficient withdrawal of ROS generated in mitochondria during normal metabolism. Murakami and Johnson (19) showed that the insulin/IGF-I pathway confers resistance to UV exposure. Lithgow (71) indicated that the insulin/ IGF-I pathway also confers increased Cd- and Cu-ion resistance. Metallothioneins are metal-binding proteins that are induced in response to a wide variety of stresses including metal ions and oxidative stress. In C. elegans, there are two isoforms of metallothioneins. Metallothionein-1 (MTL-1) is induced by Cd and heat in intestinal cells. Levels of mtl-1 mRNA are high in daf-2 mutants compared with wild-type animals under normal conditions. Cd challenge induces mtl-1 and mtl-2 in daf-2 mutants more greatly than in wild-type animals. The daf-2 mutant is resistant to hypoxia. Scott et al. (72) showed that daf-2 is important for preventing hypoxic death in myocyte and neurons. The signaling pathway for hypoxia resistance is somewhat distinct from insulin/IGF-I signaling for longevity. The age-1 and daf-2 mutants survive longer in acute thermal stress than wild-type animals (5,64). The age-1 mutant has elevated levels of HSP-16 at normal temperature, and when challenged by heat shock, accumulates greater levels of HSP16 compared with wild-type animals (41). The hsp-16 transgene induces heat-stress resistance and extended lifespan both in wild-type and age-1 mutant animals. The DAF-16 transcription factor is essential for maximal hsp-16 expression and for lifespan-extension induced by the hsp-16 transgene (44). DAF-16 translocates into the nucleus upon heat and oxidative stress (73). Taken together, these results suppose that molecular chaperons play an important role in the extension of lifespan by preventing the accumulation of conformationally altered protein associated with aging. Mun˜oz and Riddle (74) isolated thermotolerant mutants of C. elegans, and 80% of these mutants exhibit an extended lifespan, suggesting a strong correlation between stress resistance and lifespan extension. From the overall screening of RNAi inactivation of chromosome I genes, Garigan et al. (18) found that the inactivation of HSF-1, a transcription factor regulating the response to heat and oxidative stress, shortens the lifespan and causes premature aging. These findings raise the possibility that the activation of thermal and oxidative stress response mechanisms may slow down the rate of aging. Hsu et al. (42) introduced extra-copies of the hsf-1 gene into animals, resulting in resistance to heat and oxidative stress (paraquat) and lifespan extension. The lifespan extension by hsf-1 extra-copies requires daf-16, suggesting that DAF-16 and HSF-1 may act together to promote longevity. DAF-16 also appears to act independently of HSF-1, because hsf-1 RNAi does not prevent DAF-16 from

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accumulating in the nucleus of daf-2 mutants or activating two known DAF-16downstream genes, mtl-1 and sod-3. The expression of several shsp genes, hsp-16.1, hsp-16.49, hsp-12.6, and sip-1 is increased in daf-2 mutants and decreased in daf-16 mutants. HSF-1 is required for increased shsp gene expression in daf-2 mutants, thus, HSF-1 functions in the insulin/IGF-1 system. DAF-16 as well as HSF-1 is required to activate shsp expression after heat shock. Furthermore, both the DAF-16 binding site (GTAAAc/tA) and HSF-1 binding site (TTCTa/cGAA) are located at the regulatory regions of the shsp genes. The RNAi inactivation of each shsp genes partly shortens the lifespan of daf-2 mutant and HSF-1 overexpressed animals. These results suggest that DAF-16 from insulin/IGF-I signals and stress, and HSF-1 from stress, act together to activate the transcription of a variety of genes inducing lifespan extension (Fig. 4.1). The RNAi inactivation of shsp accelerates the onset of polyglutamine-expansion protein aggregation in a C. elegans model for triplet repeat disease. This result suggests that SHSPs may influence the aging rate and polyglutamine aggregation coordinately by in part, preventing the improper association of oxidized or abnormally folding proteins. DAF-16 Transcription Target Lee et al. (32) identified genes bearing the DAF-16 binding site, within the promoter region in C. elegans and Drosophila. In addition to sod-3 (MnSOD) (23), an FK506-binding protein and a nucleolar protein (75) previously known to be regulated by DAF-16, they found several DAF-16 target genes that are regulated by insulin/IGF-I signaling in a daf-16-dependent manner in C. elegans. The expression of a ser/thr phosphatase (C25E10.12) gene is upregulated in daf-2 mutants in a daf-16-dependent manner. When C25E10.12 is inactivated by RNAi, it shortens the lifespan of the age-1 mutant but does not alter the lifespan of the wild-type animals. Ookuma et al. (76) surveyed genes with the DAF-16 consensus-binding site within the regulatory region in C. elegans. They found a candidate DAF-16 transcriptional target gene, scl-1, that is a putative secretory protein with an SCP domain and is homologous to the mammalian cysteine-rich secretory protein (CRISP) family. The expression of scl-1 is upregulated in daf-2 and age-1 mutants. The inactivation of scl-1 by RNAi reduces both the lifespan and stress resistance of these mutants. Using DNA microarray analysis, Murphy et al. (31) identified genes whose expression increases or decreases when insulin/IGF-I signaling activity is reduced. Many daf-2 (2)-induced genes encoded proteins that may be involved in the synthesis of downstream signaling molecules such as a steroid or lipidsoluble hormone. The reduction of these gene activities with RNAi shortened lifespan. These results suggest that insulin/IGF-I signaling produces a putative secondary endocrine system that promotes longevity. In addition to sod-3 (23) and mtl-1 (17), they found that the expression of the catalase genes

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(ctl-1: peroxisomal and ctl-2: cytosolic), the glutathione-S-transferase gene, and the small heat-shock protein genes (hsp-12.6, hsp-16.1, hsp-16.2, hsp-16.11, hsp-16.49, and sip-1) are all increased when DAF-2 activity is reduced. The inhibition of the activities of these genes with RNAi generally shortens the lifespan of daf-2 mutants. This supports the hypothesis that genes that increase resistance to environmental stress contribute to longevity. They also found that some antimicrobial and metabolic genes are upregulated when DAF-2 activity is reduced. In addition to old-1 (38), they found that reducing DAF-2 activity downregulates specific life-shortening genes including guanyl cyclase and vitellogenin. These results suggest that multiple effector genes, whose expression is regulated by the insulin/IGF-I signaling pathway, act in a cumulative manner to influence longevity. They found that the DAF-16 consensus site, GTAAAc/tA is present not only in the promoter of daf-2 (2)-induced genes, but also in the promoters of daf-2 (2)-downregulated genes, indicating that DAF-16 may both directly repress and activate gene expression. They also found a new site, CTTATCA, in the promoter of many of these genes suggesting that DAF-16 regulates these genes with another transcription factor. Reproductive System and Lifespan The somatic gonad and germ line can be selectively deleted by laser beam irradiation of their precursor cells at the early larval stage in C. elegans. Hsin et al. (77) ablated the germ line and left the somatic gonad intact resulting in a large extension of lifespan in wild-type animals. Ablation of the somatic gonad (entire gonad organ) has no effect on wild-type lifespan. In the case of certain daf-2 mutants, ablation of the somatic gonad as well as the germ line promote an extended lifespan. In daf-16 mutants, ablation of the germ line does not extend lifespan and ablation of the somatic gonad rather shortens lifespan. One model to explain these results is that the germ line produces a signal that inactivates DAF-16 to shorten lifespan and that the somatic gonad emits a signal reducing insulin/IGF-I signaling to extend lifespan (77) (Fig. 4.1). In fact, the ablation of germ line induces DAF-16 translocation into the nucleus in the intestine (52). In daf-12 mutants, ablation of the germ line does not extend the lifespan but ablation of the somatic gonad rather shortens lifespan. Gerisch et al. (59) showed that ablation of the germ line does not extend lifespan in certain daf-9 mutants. Thus, DAF-9 and DAF-12 are required for germ-line ablation to extend the lifespan. As DAF-9 is expressed within the somatic gonad, the lifespan-regulating signal from the gonad may be a DAF-9-related hormone. The extended lifespan in certain daf-2 mutants is promoted by daf-9 or daf-12 mutations (59,63,64). This result suggests the existence of cross-talk between DAF-9/DAF-12 signaling and insulin/IGF-I signaling. Germ-line ablation induces heat and oxidative-stress (paraquat) resistance and increased sod-3 expression as well as lifespan extension. The heat resistance

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as well as lifespan-extension is reproduced by mutations in the mes-1 and glp-1 genes that lack germ line (78). Nervous System and Lifespan Apfeld and Kenyon (79) found, by mosaic analysis, that daf-2 controls lifespan in a cell-nonautonomous manner in that the loss of daf-2 (þ) extrachromosomal duplication from the neuron-generating cell lineage of daf-2 mutant restores the extended lifespan (79). The expression of normal daf-2 (þ) or age-1 (þ) genes in neurons but not muscle, or intestines restores the normal lifespan in daf-2 or age-1 mutants, respectively. These findings suggest that insulin/IGF-I signaling in neurons alone is sufficient to specify a normal lifespan (80). C. elegans senses environmental signals including food, temperature, pheromones, and osmotic pressure through ciliated sensory neurons located in sensory organs in the head and tail. Mutations that disrupt the cilium structure or signal transduction in sensory neurons extend the lifespan. This lifespan extension is suppressed by the daf-16 mutation (81). These results support the notion that environmental cues are transduced into insulin/IGF-I signaling, which exerts its function for regulating lifespan in the nervous system. Mitochondrial Electron Transport and Lifespan The other lifespan-extension mutants are those in a set of clk genes (clk-1, clk-2, clk-3, gro-1, and isp-1), which display altered biological timing, including the defecation cycle, pharyngeal pumping for food intake and cell cycle, and duration of development (10). CLK-1 is involved in the biosynthesis of coenzyme-Q, a substance that regulates energy production in the mitochondria. The clk mutants do not display the Daf phenotype. The maximum life span is extended in a clk-1 mutant is only 40%. However, the mean life span of the double mutant, daf-2; clk-1, is over five times the normal lifespan (10). This synergistic effect indicates that the insulin/IGF-I signaling pathway and the clk mechanism interact in determining life span. Mutants in clk-1 lack coenzyme-Q9 and instead accumulate the biosynthetic intermediate demethoxy-Q9, which does not seem to function in electron transport (82). ATP levels are normal or slightly elevated in clk-1 mutants (83). Withdrawal of coenzyme-Q from the diet extend the lifespan of wild-type and daf-2 mutant animals (84). Mouse and human genes homologous to C. elegans clk-1 restore normal rhythmic movement and lifespan in clk-1 mutants (85). CLK-2 is homologous to Saccharomyces cerevisiae Tel2p, an essential DNA-binding protein that regulates telomere length in yeast. The clk-2 gene is found to be the same with rad-5, that is, DNA damage checkpoint gene (86). Mutants in clk-2/rad-5 are hypersensitive to UV- and X-irradiation. CLK-2/ RAD-5 acts in a pathway that partially overlaps the MRT-2 checkpoint pathway, in which genotoxic stress induces cell cycle arrest and apoptosis of germ-line cells. In the mutant of mrt-2, which is homologous to the S. pombe

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rad1 checkpoint gene, the germ line cannot be passed indefinitely (87). The mrt-2 mutant exhibits progressive telomere shortening, late-onset chromosome fusions and x-ray hypersensitivity of the germ line. The mutant in cep-1, a homologue of the mammalian p53 tumor suppressor gene, also shows resistance to DNA damage-induced apoptosis of germ-line cells independent of the casparse pathway (88). The effect of clk-2/rad-5 mutation on telomere length has not yet been established (86,89,90) and the relationship between altered biological timing and the DNA-damage checkpoint is not yet known. GRO-1 is a tRNA-modifying enzyme in the mitochondria. Mutants in isp-1, which encodes iron sulfur protein of mitochondrial Complex III, inhibit mitochondrial respiration, show slow biological timing, and extend lifespan (11). Mutants in mitochondrial leucyl-tRNA synthetase (lrs-2) that is predicted to compromise mitochondrial electron transport through suppressing the translation of 12 mitochondrial polypeptides encoded by the mitochondrial genome, show slow biological rhythms and lives 200% longer than wild-type animals. The extended lifespan of lrs-2 does not require DAF-16 (91). Mutants in clk-1 do not display oxidative-stress resistance or increased expression of sod-3. However, clk-1 mutation largely promotes oxidative-stress resistance and an increased expression of sod-3 in daf-2 mutants (23) as well as extended lifespan(10). On the other hand, mutation in isp-1 cannot promote an extended lifespan of daf-2 and itself causes oxidative-stress resistance and increased expression of sod-3 (11). Insulin/IGF-I signaling seems to regulate the expression of sod-3 in concert with mitochondrial energy metabolites. Mammalian MnSOD has a dual role: one is as a protective function against the damaging effects of ROS (92 –95), and another is as a regulator of levels of ROS that mediate signal transduction (96 –99), [reviewed in Ref. (100)]. Interestingly, there is an intriguing link between an insulin signaling, and the gene expression of MnSOD in vertebrates: TNF-a, which interferes with insulinreceptor signaling (101), induces the gene expression of MnSOD (102). The FOXO3a transcription factor, a mammalian homologue of DAF-16, activates MnSOD transcription and the subsequent reduction of ROS (103). ROS particularly H2O2, has been found to be involved in insulin or IGF-I signaling (104). The link between insulin signaling and ROS, may have been conserved among diverse species. Further studies are needed to clarify the roles of sod-3 in C. elegans aging. The functions of two MnSOD isoforms, sod-2 and sod-3, in C. elegans are now under investigation. From systematic RNAi screening of 5690 chromosome I and II genes in C. elegans, Lee et al. identified genes that are related to lifespan. Fifteen percentage of the genes influencing lifespan are specific for mitochondrial functions, including mitochondrial carriers, electron-transport chain components, and a mitochondrial ribosomal subunit. The responses to heat and oxidative stress (paraquat and H2O2) of the RNAi inactivation of these genes are different from each other, suggesting that the lifespan-extension by impaired mitochondrial function is not simply due to reduction in ROS generation. Lifespan-extension

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by RNAi inactivation of some genes is suppressed by daf-16 mutation, however, DAF-16 translocation into the nucleus is not observed when any of these genes influencing lifespan are RNAi inactivated (91). Dillin et al. (105) reported the reduction of electron transport chain activity by RNAi of NADH/ubiquinone oxidoreductase (nuo-1, complex I), cytochrome-c reductase (cyc-1, complex III), cytochrome-c oxidase (cco-1, complex IV), and ATP synthase (atp-3, complex V). These RNAi treatments all decreased ATP levels and increased lifespan. Lifespan-extension by these RNAi treatments is not suppressed by the daf-16 mutation, and is promoted by the daf-2 mutation and germ-line ablation, but not by somatic – gonad ablation. In contrast to these RNAi treatments, somatic – gonad ablation shortens the lifespan of clk-1 mutants to the wild-type level. Therefore, the mechanism of lifespan-extension of these RNAi-treated animals is likely to differ from that of clk-1 mutation. In fact, ATP levels are normal or slightly elevated in clk-1 mutants (83). Interestingly, these RNAi treatments in early development are critical for lifespan-extension, in contrast with insulin/IGF-1 signaling, which functions exclusively in adulthood to influence lifespan, suggesting that a regulatory system monitoring mitochondrial activity during development specifies the aging rate. As animals with reduced ATP levels by these RNAi treatments only in adulthood do not live longer than untreated animals, the mechanism of lifespan-extension by CR in adulthood is not likely to reduce the respiratory rate. Together, these findings indicate that mitochondrial electron transport is generally a regulator of lifespan as well as behavioral rates. This regulator seems to mainly function independently on insulin/IGF-I signaling pathway but there seems to be possible cross-talk between them (Fig. 4.2).

Development

Adult Reproduction

Respiratory Chain Activity

Aging

Insulin / IGF-I Signaling

Caloric Restriction

Figure 4.2 Timing of lifespan regulator’s action in C. elegans. A regulatory system monitoring respiratory chain activity during development specifies the aging rate. On the other hand, insulin/IGF-I signaling and CR act in adulthood to influence the aging rate. Conditions of reproductive system influence the aging rate mainly through insulin/ IGF-I signaling. There seems to be possible cross-talk between respiratory chain activity, insulin/IGF-I signaling and CR.

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Rea and Johonson (106) proposed a general model in which the utilization of fermentative malate dismutation as alternative energy generation can induce longevity in a variety of lifespan-extension mutants. Sir-2 has been postulated to play a role in lifespan-extension by CR, and mediates chromatin silencing through histone deacetylase activity that depends on NAD (107). The transgene of sir-2.1, one of four in the C. elegans SIR-2 family extends the lifespan. This extended lifespan is suppressed by daf-16 mutation and is not promoted by daf-2 mutation, indicating that SIR-2.1 functions upstream of daf-16 in the insulin/IGF-I signaling pathway (108). In yeast, CR extends the lifespan by increasing the activity of Sir-2. Resveratrol, a polyphenol found in red wine, stimulates the activity of SIRT1, a human Sir-2 homologue, and increases DNA stability and extends the lifespan of yeast, C. elegans, and Drosophila (109).

LIFESPAN REGULATION IN DROSOPHILA The mutations of Drosophila melanogaster in the chico gene that encodes an insulin receptor substrate in insulin/IGF-I signaling extend lifespan by up to 48% (24). The chico mutations increase resistance to starvation stress but not to heat stress. Slight resistance to oxidative stress (paraquat) is seen in chico heterozygotes chi2/þ but not in homozygotes chi2/chi2. The effects on stress resistance of chico mutations are not so marked as in the daf-2 and age-1 mutations of C. elegans. Total SOD activity is higher in chico homozygotes than chico heterozygotes and wild-type flies (24). On the other hand, when the food supply is ,65% of the control food, the chico mutant has a rather shorter lifespan than wild-type flies, suggesting that CR and the insulin/IGF-I pathway in Drosophila act through overlapping mechanisms (110). A heteroallelic insulin-like receptor (InR) mutation of Drosophila results in dwarf and extends lifespan by up to 85%. The InR mutation impairs juvenile hormone (JH) synthesis. Treatment of the InR mutants with a JH analog methoprene, restores the lifespan to the normal level. Fruit flies overwinter as a reproductive diapause controlled through the downregulation of JH synthesis. Ovaries of InR dwarf females morphologically resemble the ovaries of diapause wild-type flies. Diapause flies do not age and are stress resistant. SOD activity of the InR mutants increases 2-fold. Reduced JH may induce InR mutants to inappropriately express an efficient survival diapause program resulting in extended lifespan (25). A loss-of-function mutation in the gene, Methuselah, extends lifespan by 35% (111). The methuselah mutant is resistant to a number of different stresses, including starvation, heat and oxidative stress (paraquat). The methuselah gene encodes a G-protein-coupled seven-transmembrane-domain receptor suggesting that a signal-transduction pathway regulates lifespan and stress resistance. An insertional mutation in Indy, a homologue of a mammalian sodium dicarboxylate cotransporter, a membrane protein that transports Krebs cycle

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intermediates, extends lifespan. Indy may induce a metabolic state that mimics CR (112). Orr and Sohal (113) indicated that the simultaneous overexpression of CuZnSOD and catalase extends the lifespan of Drosophila (113). Sun and Tower (114) demonstrated that the overexpression of CuZnSOD extends lifespan, and that catalase overexpression has no lifespan-extension effect. Parkes et al. (115) showed that the overexpression of human cytosolic CuZnSOD (SOD1) in the motor neuron extends the lifespan of Drosophila and rescues the lifespan of a short-lived SOD1 null mutant (115). They also showed that overexpression of SOD1 and catalase combination in the motor neuron diminishes the lifespan-extension effect of SOD1 overexpression (116). Mockett et al. (117) showed that the overexpression of MnSOD in the mitochondria of Drosophila increases heat tolerance but rather decreases lifespan. On the other hand, Sun et al. (118) demonstrated that the overexpression of MnSOD extends lifespan in a dose-dependent manner without decreasing the metabolic rate. The mitochondrial overexpression of catalase causes an increase in resistance to oxidative stress (H2O2 and paraquat) and cold stress, but there is no lifespan-extension effect (119). The overexpression of glutathione reductase in Drosophila increases the survival period under hyperoxia but no effect on lifespan under normoxia, suggesting that glutathione reductase is critical for protecting against robust oxidative stress but not against aging-associated damages (120). Orr and Sohal (121) posed the question whether transgenes of these antioxidant enzymes could decrease the aging rate on the basis that the lifespan of the controls was too short. They introduced a combination of antioxidant-enzyme genes into relatively long-lived Drosophila strains and examined their lifespan. The transgenes of various combinations of CuZnSOD, MnSOD, catalase, and thioredoxin reductase, all have no lifespan-extension effect, although activities of some enzymes actually increase above wild-type levels (122). Currently, it may be difficult to assert the establishment of the free-radical theory of aging from antioxidant-enzyme trangene experiments with Drosophila. Methionine sulfoxide reductase A (MSRA) catalyzes the repair of oxidized methionine in proteins by reducing methionine sulfoxide back to methionine. Overexpression of the msrA gene predominantly in the nervous system, increases resistance to oxidative stress (paraquat) and extends the lifespan of Drosophila (123). Using DNA microarray, Zou et al. (124) assessed age-related changes in gene expression levels in Drosophila and compared these changes with those induced by oxidative stress (paraquat). Age-related downregulation genes are involved in reproduction, metabolism, detoxification, chaperone, and protein turnover. Age-related downregulation genes functioning as chaperones or detoxification agents are SHSP, Hsp26, alcohol dehydrogenase, a-tocopherol transfer-related protein, and a homologue of microsomal epoxide hydrolase. One-third of the age-related genes overlap the paraquat-regulated genes. Both

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age-related and paraquat-regulated upregulation genes are homologues of arsenite-translocating ATPase and glutathione S-transferase D1 that are involved in detoxification (124). Treatment of Drosophila with 4-phenylbutyrate (PBA), an inhibitor of histone deacetylase, induces resistance to starvation and oxidative stress (paraquat) and extends lifespan. PBA dramatically changes the gene expression pattern associated with increased histone acetylation, and the SOD expression is increased greatly (125). LIFESPAN REGULATION IN MAMMALS In mice, the constitutive and ubiquitous overexpression of CuZnSOD in various tissues does not extend lifespan (126). However, ROS may not necessarily be unrelated to aging in mammals. In fact, KO mice of the p66Shc gene that show extended lifespan indicate resistance to oxidative stress in various aspects. Cultured p66shc2/2 cells are resistant to H2O2- or UV-induced apoptosis (127). In p66Shc KO mice, systemic oxidative stress including plasma LDL oxidation and arterial oxidative damage is reduced (128). In mammals, the DAF-16 homologues regulate apoptosis and cell-cycle progression in response to stress. Nemoto et al. (129) demonstrated that mutations in p66shc2/2 activate the FOXO transcription factor, a mammalian DAF-16 homologue, through the reduction in cellular ROS levels. FOXO transcription factor regulates both H2O2 scavenging and oxidative stress resistance in response to oxidative stress. The DAF-16 homologue activates MnSOD to protect cells from apoptosis caused by glucose deprivation (103). It has been found that DAF-16 homologues regulate transcription of the DNA-damage response gene, Gadd45 that functions as a G2 – M checkpoint in response to UV-induced damage (130) or oxidative stress (131). DAF-16 homologues downregulate D-type cyclins to promote cell-cycle progression (132). Several single-gene mutants have been reported to have a long lifespan [for review see Ref. (133)]. The Ames dwarf mouse (df/df), a mutant of the Pro-1 gene (134); and the Snell dwarf mouse (dw/dw), a mutant of the Pit-1 gene (135), live more than 40 –60% longer than wild-type mice. Both genes are required for the development of the pituitary cells that secrete growth hormone (GH), thyroid stimulating hormone and prolactin. Furthermore, KO of GH receptor, GHR-KO mice (Ralon model) are found to live longer than heterozygotes and wild-type mice (136). GHR-KO decreases plasma IGF-I and insulin levels (137). Thyroid hormone synthesis is also reduced in these dwarf mice. Hypothyroidism induced by a thyroxin injection into neonatal rats actually extends lifespan (138). Hypothyroidism in rats decreases heart mitochondrial H2O2 production and suppresses oxidatively damaged DNA (8-oxodG) (139). Heterozygous KO mice of Igf1r, an IGF-I receptor gene, live longer than wild-type controls. Lifespan-extension is associated with increased resistance to oxidative stress in vivo (paraquat) and in vitro (H2O2). Mutation in Igf1r

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reduces the phosphorylation of p66Shc that is a substrate of IGF-IR (26). IGF-I injection with an adeno-associated virus into motor neurons prolongs the lifespan of amyotrophic lateral sclerosis (ALS) model mice with mutated CuZnSOD (140). IGF-I may prevent ROS-mediated impairment (140), although how the CuZnSOD mutations induce the neurodegeneration is not yet clearly understood. Disruption of the insulin receptor gene in the fat tissue also extends the lifespan of mice. These animals have reduced fat mass, which is often seen upon CR (27). CR also leads to a decrease in plasma IGF-I levels early in adult life, suggesting the involvement of IGF-I in CR-induced lifespan-extension (141, 142). However, CR extends further the lifespan of Ames dwarf mice, suggesting that the mechanisms regulating lifespan-extension in CR and dwarf mice are not identical (143). Bonafe et al. (144) examined the relationship between human longevity and polymorphic variants of the IGF-I response pathway genes, namely, IGF-I receptor, PI3-kinase, insulin receptor substrate-1, and FOXO1A. Free IGF-I plasma levels and human longevity are co-regulated by a set of IGF-I signaling pathway genes. Insulin/IGF-I signaling may regulate longevity in a wide range of organisms from worms to humans. Genome-wide searches for aging-, CR-, stress-, and lifespan-extension mutation-associated gene expression changes in mammals have been conducted using DNA microarray. The gene expression profile of the skeletal muscle between adults and elderly mice and the effect of CR were examined (33). Fifty-eight genes of 6347 genes, express more markedly in the elderly animals than in the adults. Of the 58 genes upregulated with age, 16% were related to stress responses, including the heat shock factors Hsp71 and Hsp27, protease Do, and the DNA damage-inducible gene GADD45. The largest differential expression between adult and elderly animals is the mitochondrial sarcomeric creatine kinase, a critical target for ROS-induced inactivation. Most alterations are prevented by CR. Transcriptional patterns of calorie-restricted animals suggest that CR retards the aging process by inducing a metabolic shift toward increased protein turnover and decreased macromolecular damage. Lee et al. (145) further examined the gene-expression changes in the neocortex and cerebellum of mice during aging and showed that aging induces a gene-expression profile indicative of an inflammatory and oxidative stress response. CR selectively attenuates the age-associated induction of genes related to inflammatory and stress responses such as JunB, Fos, and DnaJ homologues. Kayo et al. (146) examined the expression of 7070 genes in muscles between young and aged rhesus monkeys and the effect of CR. Aging resulted in the selective up-regulation of transcripts involved in inflammation and oxidative stress, and a downregulation of genes involved in mitochondrial electron transport. These age-related changes are not inhibited by adult-onset CR. The retardation of age-related changes in gene expression may depend on the species. Miller et al. (34) assessed CR-induced changes in the expression of 2352 genes in the liver and compared them with long-lived GHR KO mice. A total

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of 352 genes are increased or decreased by CR. A parallel alteration in the expression of 29 genes can be seen between the calorically restricted and GHR KO mice. The gene expression profile of the intestine of Se(2)-diet fed mice was examined. A low Se state actually reduces the activity of glutathione peroxidase, a Se-containing antioxidant enzyme, and upregulates the expression of genes related to genetic instability and oxidative stress, including XP-E, GADD34, GADD45, metallothionein-I, HSP27, and HSP40. A low Se state appears to induce a stress response at the transcriptional level (147). Edwards et al. (148) searched genes among a 9977-gene array that are differentially expressed in the heart after treatment with paraquat, with variously aged mice. They demonstrated that only young mice display a significant increase in the expression of all three isoforms of GADD45, a DNA damage-responsive gene. Additionally, the number of immediate early response genes found to be induced by paraquat was considerably higher in the younger animals. These results indicate that there is an age-related impairment of specific inducible pathways in response to oxidative stress in the murine heart.

REPLICATIVE LIFESPAN AND OXIDATIVE STRESS Cultured normal cells generally have a finite replicative lifespan as originally shown by Hayflick and Moorhead (149). Human diploid fibroblasts lose the capacity to replicate after vigorous proliferation, and enter a viable but nonproliferative state of senescence. Senescent cells are characterized by the upregulation of cyclin-dependent kinase inhibitors, p21SDI1/WAF1/CIP1 (150), and p16INK4a (151), and hypophosphorylated Rb (152). Human fibroblasts grown in hypoxia have an extended replicative lifespan (153). Mouse embryonic fibroblasts (MEFs) senesce after vigorous proliferation, and then grow into an immortal cell in normoxia (20% oxygen), whereas human normal fibroblasts never achieve immortality. Parrinello et al. (154) found that MEFs do not senesce in hypoxia (3% oxygen) and that MEFs accumulate more DNA damage in normoxia than hypoxia, and more damage than human fibroblasts in normoxia. These results suggest that human cells have a superior ability to prevent or repair oxidative DNA damage compared to murine cells. Stress-induced premature senescence (SIPS) occurs when cells are grown in hyperoxia (155), and treated with hyperbaric oxygen (156) and H2O2 (157), with appearance of several biomakers of replicative senescence. Hyperoxia (40% oxygen) increases the rate of telomere shortening from 90 bp per population doubling (normoxia) to more than 500 bp per population doubling (hyperoxia) (158), suggesting that telomere shortening causes premature senescence. On the other hand, Gorbunova et al. (159) indicated that the overexpression of the catalytic subunit of telomerase cannot prevent SIPS but can protect from stress-induced apoptosis and necrosis. This finding suggests that SIPS may not be due to telomere shortening.

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The addition of N-acetylcysteine, a cellular ROS scavenger, into culture medium extends the replicative lifespan of human embryonic fibroblsts (160). On the other hand, the addition of L -buthionine-(R, S)-sulfoximine, a specific inhibitor of GSH synthetase, shortens the replicative lifespan (160). These results suggest that cellular ROS levels are a determinant of replicative lifespan. The inactivation of cytosolic CuZnSOD (SOD1) by RNAi induces premature senescence in human fibroblast depending on p53 induction (161). In human fibroblasts with inactivated p53, the SOD1 RNAi is without effect suggesting that oxidative DNA damage mediates premature senescence. The overexpression of extracellular CuZnSOD decreases the intracellular peroxide content, slows the telomere-shortening rate, and extends the replicative lifespan under normoxia and hyperoxia (162). The overexpression of an activated V12Ras induces premature senescence in human fibroblasts and induces increased mitochondrial ROS generation (162). In hypoxia, the overexpression of the V12Ras cannot induce premature senescence or increase the level of p21 that is related to senescent phenotype (163). These findings suggest that ROS play a role in the regulation of the replicative lifespan.

CONCLUSION In the last decades, gene-manipulation studies have revealed that gene networks exist for the determination of lifespan in diverse species. There appear to be common and different features among species. ROS appear at various points in the aging processes, and play a variety of roles, including the manifestation of oxidative stress and the mediation of signal transduction. The precise role of ROS in the aging process is not yet clear. DNA microarray technology allows us to show global gene expression profiles governing the lifespan and aging rate, although these studies are just beginning. Using the data from DNA microarray analysis, painstaking studies are needed to clarify the aging mechanism to analyze how various gene products work in concert to regulate the aging rate.

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expression related to insulin-like growth factor-1 in rat muscle. Aging (Milano) 2001; 13:273 –281. Sonntag WE, Lynch CD, Cefalu WT, Ingram RL, Bennett SA, Thornton PL, Khan AS. Pleiotropic effects of growth hormone and insulin-like growth factor (IGF)-1 on biological aging: inferences from moderate caloric-restricted animals. J Gerontol 1999; 54:B521– B538. Bartke A, Wright JC, Mattison JA, Ingram DK, Miller RA, Roth GS. Extending the lifespan of long-lived mice. Nature (London) 2001; 414:412. Bonafe` M, Barbieri M, Marchegiani F, Olivieri F, Ragno E, Giampieri C, Mugianesi E, Centurelli M, Franceschi C, Paolisso G. Polymorphic variants of insulin-like growth factor I (IGF-I) receptor and phosphoinositide 3-kinase genes affect IGF-I plasma levels and human longevity: cues for an evolutionarily conserved mechanism of life span control. J Clin Endocrinol Metab 2003; 88:3299–3304. Lee CK, Weindruch R, Prolla TA. Gene-expression profile of the ageing brain in mice. Nat Genet 2000; 25:294 – 297. Kayo T, Allison DB, Weindruch R, Prolla TA. Influences of aging and caloric restriction on the transcriptional profile of skeletal muscle from rhesus monkeys. Proc Natl Acad Sci USA 2001; 98:5093 – 5098. Rao L, Puschner B, Prolla TA. Gene expression profiling of low selenium status in the mouse intestine: transcriptional activation of genes linked to DNA damage, cell cycle control and oxidative stress. J Nutr 2001; 131:3175 – 3181. Edwards MG, Sarkar D, Klopp R, Morrow JD, Weindruch R, Prolla TA. Age-related impairment of the transcriptional responses to oxidative stress in the mouse heart. Physiol Genomics 2003; 13:119– 127. Hayflick L, Moorhead PS. The limited in vitro lifetime human diploid cell strains. Exp Cell Res 1961; 25:595 –621. Noda A, Ning Y, Venable SF, Pereira-Smith OM, Smith JR. Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen. Exp Cell Res 1994; 211:90 –98. Smith JR, Pereira-Smith OM. Replicative senescence: implications for in vivo aging and tumor suppression. Science 1996; 273:63 – 67. Stein GH, Beeson M, Gordon L. Failure to phosphorylate the retinoblastoma gene product in senescent human fibroblasts. Science 1990; 249:666 – 669. Packer L, Fuehr K. Low oxygen concentration extends the lifespan of cultured human diploid cells. Nature (London) 1977; 267:423 –425. Parrinello S, Samper E, Krtolica A, Goldstein J, Melov S, Campisi J. Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts. Nat Cell Biol 2003; 5:741– 747. Balin AK, Goodman DB, Rasmussen H, Cristofalo VJ. The effect of oxygen and vitamin E on the lifespan of human diploid cells in vitro. J Cell Biol 1977; 74:58– 67. Honda S, Matsuo M. Shortening of the in vitro lifespan of human diploid fibroblasts exposed to hyperbaric oxygen. Exp Gerontol 1983; 18:339 –345. Chen Q, Ames BN. Senescence-like growth arrest induced by hydrogen peroxide in human diploid fibroblast F65 cells. Proc Natl Acad Sci USA 1994; 91:4130 – 4134. von Zglinicki T, Saretzki G, Docke W, Lotze C. Mild hyperoxia shortens telomeres and inhibits proliferation of fibroblasts: a model for senescence? Exp Cell Res 1995; 220:186– 193.

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159. Gorbunova V, Seluanov A, Pereira-Smith OM. Expression of human telomerase (hTERT) does not prevent stress-induced senescence in normal human fibroblasts but protects the cells from stress-induced apoptosis and necrosis. J Biol Chem 2002; 277:38540 – 38549. 160. Honda S, Matsuo M. Relationships between the cellular glutathione level and in vitro life span of human diploid fibroblasts. Exp Gerontol 1988; 23:81 –86. 161. Blander G, Machado De Oliveira R, Conboy CM, Haigis M, Guarente L. SOD1 knock down induces senescence in human fibroblasts. J Biol Chem 2003; 278:38966 – 38969. 162. Serra V, von Zglinicki T, Lorenz M, Saretzki G. Extracellular superoxide dismutase is a major antioxidant in human fibroblasts and slows telomere shortening. J Biol Chem 2003; 278:6824 – 6830. 163. Lee AC, Fenster BE, Ito H, Takeda K, Bae NS, Hirai T, Yu ZX, Ferrans VJ, Howard BH, Finkel T. Ras proteins induce senescence by altering the intracellular levels of reactive oxygen species. J Biol Chem 1999; 274:7936– 7940.

5 Anti-Oxidant Modulation in Immune Function Robert Francis Grimble University of Southampton, Southampton, UK

Introduction The Immune Response to Infection Injury and Inflammatory Agents The Function of Pro-Inflammatory Cytokines During the Normal Response to Infection and Injury Adverse Effects of Pro-Inflammatory Cytokines Anti-Oxidant Defenses are Interlinked and Interdependent A Decline in Anti-Oxidant Defenses and Increased Oxidant Damage Follows Infection and Injury Aging Increases Oxidative and Inflammatory Stress Mechanisms Underlying Low-Grade Inflammation During Aging Mechanisms of the Effects of Oxidants and Anti-Oxidants on Inflammation and Immune Function Effects of Anti-Oxidants on Immune Function Effects of Vitamin E Ascorbic Acid and Immune Function Glutathione and Immune Function Effects of Substances that Act as Precursors for GSH or Cofactors in Enzyme Pathways Associated with GSH on Immune Function Effects of Precursors of GSH on Immune Function Effects of Vitamin B6 on Immune Function 97

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INTRODUCTION The production of oxidant molecules is both an integral part of the immune response and a major modulator of immune function. As a corollary to this concept anti-oxidant defences play a pivotal role in immune function by potentially interacting at both of these levels. In practice, however, anti-oxidants exert only a modulatory influence on the effects of oxidants on immune function. The reduction in anti-oxidant defenses that occurs during the normal process of inflammation may be an attempt by the body to expose pathogens to the full strength of oxidants produced by the immune system. This phenomenon is, as will be seen later, not without risk to the host. In this chapter, the circumstances under which oxidant production occurs will be described, the effect of oxidants on immune function will be examined, the mechanisms whereby oxidants produce these effects will be outlined, and the interaction of dietary and endogenously produced anti-oxidants on immune function will be discussed. THE IMMUNE RESPONSE TO INFECTION INJURY AND INFLAMMATORY AGENTS The immune system has a large capability for immobilizing invading microbes, creating a hostile environment for them, and bringing about their destruction (1). The immune system may also become activated, in a similar way to the response to microbial invasion, by a wide range of stimuli and conditions that do not directly involve pathogens; these include burns, penetrating and blunt injury, the presence of tumor cells, environmental pollutants, radiation, exposure to allergens, and the presence of chronic inflammatory diseases. The response of the immune system to this diverse range of agents and conditions contains many common elements. These, however, vary in intensity according to their impact on the body. The elements of the response include the production of immunomodulatory proteins (cytokines), oxidant molecules (hydrogen peroxide, superoxide, hypochlorous acid, and nitric oxide), antiinflammatory hormones (cortisol), natural antagonists (cytokine receptor antagonists), and anti-oxidants enzymes (superoxide dismutase, catalase, and glutathione peroxidase) (2).

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THE FUNCTION OF PRO-INFLAMMATORY CYTOKINES DURING THE NORMAL RESPONSE TO INFECTION AND INJURY The pro-inflammatory cytokines, interleukin-1b (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-a (TNF-a), have widespread metabolic effects on the body and bring about the process of inflammation. Many of the signs and symptoms experienced after infection and injury, such as fever, loss of appetite, weight loss, negative nitrogen, sulfur and mineral balance, and lethargy are caused directly or indirectly by pro-inflammatory cytokines (Fig. 5.1). Indirect effects of cytokines are mediated by neural actions on the adrenal glands and endocrine pancreas resulting in increased secretion of the catabolic hormones adrenalin, noradrenalin, glucocorticoids, and glucagon. Insulin insensitivity occurs in addition to this “catabolic state.” The biochemistry of an infected individual is thus fundamentally changed in a way which will ensure that the immune system receives nutrients from within the body (Fig. 5.2). Muscle protein is catabolized to provide amino acids for synthesizing new cells, glutathione (GSH), and proteins for executing and controlling the immune response. Furthermore, amino acids are converted to glucose (a preferred fuel, together with glutamine, for the immune system (1). The extent of the rearrangement in protein metabolism is evident from changes in excretion of substances in the urine following infection and injury. An increase in urinary nitrogen and sulfur excretion

Raised blood lipids

Fever

Glucose synthesis

Plasma copper Production of oxidant molecules

Effects of cytokines TNF, IL1 and IL6

Plasma Zn Plasma iron

Acute phase proteins

Loss of lean tissue and fat

Appetite loss and lethargy Increased urinary nitrogen sulphur and mineral losses

Figure 5.1 Alterations in body function, composition, and metabolism that result from production of pro-inflammatory cytokines following infection and injury and during chronic inflammatory disease.

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Trauma/infection/burns Immune system activation

Pro-inflammatory cytokines

Oxidants

Feedback systems IL10, Heat shock proteins

Antioxidant defence

Pathogen killing

Tissue damage

T and B cells

Glucose

Nutrient release from host tissues

Glutamine

Sulphur amino acids

Creation of a hostile environment Appetite loss

Figure 5.2 and injury.

Immunonutrition

Glutathione synthesis

Antioxidant defences strengthened

Main metabolic events and their purpose during the response to infection

occurs. There is an increase in urinary nitrogen excretion from 9 g/day in mild infection to 20 –30 g/day following major burn or severe traumatic injury (3). The loss of nitrogen from the body of an adult during bacterial infection, may be equivalent to 60 g of tissue protein and in a period of persistent malarial infection, equivalent to over 500 g of protein. However, during the response to infection and injury the urinary excretion of sulfur increases to a lesser extent than that of nitrogen (4), suggesting that sulfur amino acids are preferentially retained and so “spared” from catabolism. Large decreases in plasma glycine, serine, and taurine concentrations occur following infection and injury. These compounds are metabolically related to sulfur amino acids. The changes may reflect enhanced utilization of these amino acids. Many substances produced in enhanced amounts in response to pro-inflammatory cytokines, are particularly rich in these amino acids. These substances include GSH, which contains glycine, glutamic acid, and cysteine; metallothionein (the major zinc-transport protein), which contains glycine, serine, cysteine, and methionine to a composite percentage of 56%; and a range of acute phase proteins that contain up to 25% of these amino acids in their structure. Among these compounds, GSH, metallothionein, and the acute phase protein ceruloplasmin are important endogenously derived anti-oxidants. If an increased demand for sulfur and related amino acids is created by the inflammatory response, then provision of additional

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supplies of these amino acids may assist the response (2). Indeed whey protein, which is rich in sulfur amino acids, has been shown to be beneficial in the treatment of children suffering from burn injury (5). Children receiving whey protein, rather than a standard high protein enteral formulation, had higher plasma C3 complement and IgG concentrations and less bacteremic days and better survival. Infection with human immunodeficiency virus (HIV) has been shown to cause substantial excretion of sulfate in the urine during the asymptomatic phase of the disease (6). The losses reported were equivalent to 10 g of cysteine per day, in contrast to losses of 3 g/day for healthy individuals on a “Westernized diet.” As cysteine is the precursor for both sulfate and GSH, this finding may be linked with the decline in tissue glutathione pools that has been observed in HIV infection (7). Clearly, such a depletion of anti-oxidant defenses will have serious effects. ADVERSE EFFECTS OF PRO-INFLAMMATORY CYTOKINES Although pro-inflammatory cytokines are essential for the normal operating of the immune system, they play a major role in tissue damage during many inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, asthma, psoriasis, multiple sclerosis, and cancer (2,8). In these type of disease, combating pro-inflammatory cytokine production with antibodies to TNF-a has shown clinical benefit. Good examples of success with this strategy have been observed in Crohn’s disease and rheumatoid arthritis (9,10). Proinflammatory cytokines are also thought to be important in the development of atheromatous plaques in cardiovascular diseases (11). In conditions such as cerebral malaria, meningitis, and sepsis, pro-inflammatory cytokines are produced in excessive amounts and are an important factor in increased mortality (8). Clearly, in all of the diseases mentioned earlier, cytokines are being produced in the wrong biological context. Cytokines exert an adverse influence on anti-oxidant defenses, because in most of these diseases and conditions, pro-inflammatory cytokines bring about a loss of lean tissue, which is associated with depleted tissue-GSH content. ANTI-OXIDANT DEFENSES ARE INTERLINKED AND INTERDEPENDENT Many of the components of antioxidant defense interact to maintain antioxidant status (1). Glutathione and the enzymes that maintain it in its reduced form are central to effective anti-oxidant status. For example, when oxidants interact with cell membranes, the oxidized form of vitamin E that results is restored to its reduced form by ascorbic acid. Dehydroascorbic acid formed in this process is reconverted to ascorbic acid by interaction with the reduced form of glutathione. Subsequently, oxidized glutathione formed in the reaction, is reconverted

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Methionine Homocysteine Vit B6 Cysteine

Oxidants

Vit E reduced

Dehydro ascorbic acid

Glutathione GSH

Vit E oxidised

Ascorbic acid

Glutathione GSSG

Glutathione reductase Riboflavin

Figure 5.3 Main linkages and relationships between key components of the anti-oxidant defenses system.

to the reduced form of glutathione by glutathione reductase (Fig. 5.3). Vitamins E and C and glutathione are thus intimately linked in anti-oxidant defense. The interdependence of the various nutritional component of anti-oxidant defense is illustrated in a study in which healthy subjects were given 500 mg ascorbic acid per day for 6 weeks (12). A 47% increase in glutathione content of red blood cells occurred. Nutrients that do not have anti-oxidant properties also contribute indirectly to the robustness of anti-oxidant defenses. These include Vitamin B6 and riboflavin. Vitamin B6 is the cofactor in the metabolic pathway for the biosynthesis of cysteine (Fig. 5.3). Cellular cysteine concentration is rate limiting for glutathione synthesis. Riboflavin is a cofactor for glutathione reductase, which maintains the major part of cellular glutathione in the reduced form (Fig. 5.3). A DECLINE IN ANTI-OXIDANT DEFENSES AND INCREASED OXIDANT DAMAGE FOLLOWS INFECTION AND INJURY There is a growing body of evidence that anti-oxidants suppress inflammatory components of the response to infection and trauma and enhance components related to cell-mediated immunity. The reverse situation applies when antioxidant defenses become depleted. Although the body strives to maintain them, observations in experimental animals and patients indicate that anti-oxidant defenses become depleted during infection and after injury. For example, in mice infected with influenza virus, there were 27%, 42%, and 45% decreases in the vitamin C, vitamin E, and glutathione contents of blood, respectively (13). In a further study on mice, given a dose of endotoxin sufficient to cause septic shock, both GSH and ascorbic acid

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were shown to fall precipitately in spleen, lymph nodes, and peritoneal macrophages (14). In asymptomatic HIV infection, substantial decreases in glutathione concentrations in blood and lung epithelial lining fluid have been noted (15). In patients undergoing elective abdominal operations, the glutathione content of blood and skeletal muscle fell by over 10% and 42%, respectively, within 24 h of the operation (16). Blood concentrations returned rapidly to preoperative values; however, concentrations in muscle were still depressed 48 h after the operations. A diverse range of clinical treatments and diseases, all of which involve the inflammatory process, have been shown to lead to a decrease in tissue anti-oxidant concentrations. These include hepatitis C, ulcerative colitis, and cirrhosis. In patients with malignant melanoma, metastatic hypernephroma, and metastatic colon cancer, plasma ascorbic acid concentrations fell from normal to almost undetectable levels within 5 days of commencement of treatment with IL-2 (17). In patients with inflammatory bowel disease, substantial reductions in ascorbic acid concentrations occurred in inflamed gut mucosa (18). As a general consequence of the weakening of anti-oxidant defenses, during disease, oxidative damage is apparent in a wide range of clinical conditions in which cytokines are produced. Lipid peroxides and increased thiobarbituric acid reactive substances are present in blood of patients with septic shock, asymptomatic HIV infection, chronic hepatitis C, breast cancer, cystic fibrosis, diabetes mellitus, and alcoholic liver disease. Peroxides also increase following cancer chemotherapy, open heart surgery, bone marrow transplantation, and hemodialysis (17). There is evidence, from studies on experimental animals and patients, that the decrease in strength of anti-oxidant defenses may exert a deleterious influence. When glutathione status was reduced in rats by injection of diethyl maleate, which binds irreversibly to GSH rendering it inactive, a sublethal dose of TNF became lethal (19), thus illustrating the importance of GSH in protection from the adverse effects of pro-inflammatory cytokines. A parallel phenomenon was noted in patients with sepsis. The onset of sepsis in patients led to a transient decrease in the total anti-oxidant capacity of blood plasma (a functional measure of the total anti-oxidant content) (20). The capacity returned to normal values over the following 5 days. However, this was not the case for patients who subsequently died, in whom values remained well below the normal range. As well as increasing the risk of direct oxidant damage, a reduction in the strength of anti-oxidant defenses also indirectly increases the risk of damage to the host via transcription factor activation leading to upregulation of pro-inflammatory cytokine production (see later). AGING INCREASES OXIDATIVE AND INFLAMMATORY STRESS Although inflammation is an integral part of the response to pathogens, it would appear that the general level of inflammatory stress increases during aging

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irrespective of the presence of infections and inflammatory disease. It is interesting to note that aging is also associated with a decline in immune function. An age-related increase of IL-6 concentration has been found in serum, plasma, and supernatants of mononuclear blood cell cultures from apparently healthy elderly people and centenarians (21 –23). Increases in serum levels of this cytokine have also been found as early as 30 –40 years of age (24), particularly in men (25). Furthermore, population studies have shown that the magnitude of increase in the concentration of IL-6 is a reliable marker for functional disability and a predictor of disability and mortality in the elderly (26,27). An enhanced capacity for the release of pro-inflammatory cytokines by leucocytes may contribute to the pathogenesis of ischaemic stroke. Grau et al. (28) investigated the lipopolysaccharide (LPS)-induced release of IL-1, IL-6, IL-8, and TNF-a in whole blood from 20 patients with a history of ischaemic stroke under the age of 50, and 21 age- and sex-matched healthy control subjects. Release of IL-8 was significantly higher and release of TNF-a and IL-6 tended to be higher in young stroke patients than in control subjects. The authors concluded that high inducible release of IL-8, TNF-a, and IL-6 may contribute to the odds of suffering from ischaemic stroke in young adults (28). The question of whether aging is associated with chronic elevation of cytokine production or whether an increased capacity for cytokine production develops during aging is an interesting matter for consideration. An insight into this issue can be gained from the response to surgery where an inflammatory stimulus is applied at a defined moment in time, making it easy to follow the subsequent response, Ono et al. (29) investigated the age-related changes in the inflammatory response to surgical stress in patients with gastric cancer, undergoing distal gastrectomy. Patients were divided into 2 groups: .75 years of age (elderly group) and
75 years of age (young group). Serum IL-6 levels, TNF-a production, and CD11b/CD18 expression by monocytes, and the postoperative clinical course were compared between the 2 groups. The clinical course, serum IL-6 levels, monocyte production of TNF-a, and monocyte expression of CD11b/CD18 were used as markers of the systemic response: TNF-a production by LPS-stimulated monocytes and CD11b/CD18 expression on monocytes after the inflammatory stimulus of surgery were significantly higher in the elderly than in the young group. Moreover, serum IL-6 levels on the first postoperative day in the elderly group were significantly higher than those in the young group. The incidence and duration of systemic inflammatory response syndrome were significantly greater in the elderly than in the young group. The authors concluded that activation of monocytes and raised blood concentrations of pro-inflammatory cytokines occur more readily in the elderly than in young subjects. Paradoxically, both loss of body weight and lean tissue and obesity are found in elderly populations. Is there a link between this phenomenon and increased levels of inflammation?

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Mechanisms Underlying Low-Grade Inflammation During Aging There are a number of potential mechanisms for the higher level of chronic inflammation and hence oxidant stress, observed in the elderly than in younger subjects. The first of these is that the elderly are experiencing a higher level of asymptomatic bacteriuria. This possibility was studied in 40 consecutive patients (age 70 –91 years) admitted to hospital for functional disability. Patients were examined for the presence or absence of bacteria in urine. Twenty subjects had positive urine culture and 20 sex- and age-matched subjects had negative urine culture. Patients with asymptomatic bacteriuria had significantly increased levels of circulating tumor necrosis factor receptors (sTNFR-I) and a higher number of neutrophils in the blood compared with the group without bacteriuria. Thus, the study provides some support for the hypothesis that asymptomatic urinary infections are associated with low-grade inflammatory activity in frail, elderly subjects (30). A second potential mechanism resides in endocrine changes during aging. In aging, dysregulation of secretion of hormones that come under the regulation of the HPA axis may occur. This may have impact on the regulation of cortisol secretion. Cortisol is important as an anti-inflammatory agent. The effect of aging on glucocorticoid (GC) sensitivity of pro-inflammatory cytokine production was examined in elderly men, testosterone-treated elderly men, and young controls. Stress-induced increases in cortisol did not differ significantly between experimental groups, but GC sensitivity increased significantly in young controls and testosterone-treated elderly men, whereas a decrease was found in untreated elderly men. As the increase in GC sensitivity after stress serves to protect the individual from detrimental increases of pro-inflammatory cytokines, the disturbed mechanism in elderly men may result in enhancement of inflammation. The decrease in sensitivity is linked to decreased testosterone production during aging as impaired sensitivity was partly restored by testosterone treatment (31). Now, there is a large body of evidence suggesting that the decline in ovarian function with menopause is associated with spontaneous increases in pro-inflammatory cytokines. Studies in men and postmenopausal women indicate a remarkable individual constancy in the ability of PBMCs to produce TNF-a ex vivo. Genetic determinants underlie this constancy. However, in premenopausal women, production is highly variable at an individual level indicating how ovarian hormones are able to override the influence of genotype (32). The exact mechanisms by which estrogen interferes with cytokine activity are still incompletely known but may potentially include interactions of the estrogen receptor with other transcription factors, modulation of nitric oxide activity, anti-oxidative effects, plasma membrane actions, and changes in immune cell function. Experimental and clinical studies also strongly support a link between the increased state of pro-inflammatory cytokine activity and postmenopausal bone loss (33). The third potential mechanism is the general increase in the incidence in inflammatory disease that occurs with aging. However, the link between inflammation and aging is of a two-way nature, as chronic inflammation may contribute

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to the pathogenesis of many diseases encountered with greater frequency in the elderly population. A fourth mechanism for the increase in inflammatory and oxidant stress during aging may be the increased association with obesity in the middle-aged and the elderly populations. It is well known that the adipocyte is able to produce a wide range of pro-inflammatory cytokines. Furthermore, a body mass index of 25 is associated with increased signs of inflammation (raised plasma acute phase protein concentrations), which decline if a program of weight loss is initiated [for review see Ref. (34)]. A fifth potential mechanism involving changes in peroxisome proliferatoractivated receptor-a (PPARalpha) activity has been revealed by recent studies on aged mice (see next section). MECHANISMS OF THE EFFECTS OF OXIDANTS AND ANTI-OXIDANTS ON INFLAMMATION AND IMMUNE FUNCTION The oxidant molecules produced by the immune system to kill invading organisms may activate at least two important families of proteins that are sensitive to changes in cellular redox state. The families are nuclear transcription factor kappa B (NFkB) and activator protein 1 (AP1). These transcription factors act as “control switches” for biological processes, not all of which, as illustrated earlier, are of advantage to the individual. NFkB is present in the cytosol in an inactive form, by virtue of being bound to IkB. Phosphorylation and dissociation of IkB renders the remaining NFkB dimer active. Activation of NFkB can be brought about by a wide-range of stimuli including pro-inflammatory cytokines, hydrogen peroxide, mitogens, bacteria and viruses and their related products, and UV and ionizing radiations. The dissociated IkB is degraded and the active NFkB is translocated to the nucleus where it binds to response elements in the enhancer and promoter regions of genes. A similar translocation of AP1, a transcription factor composed of the proto-oncogenes c-fos and c-jun, from cytosol to nucleus, also occurs in the presence of oxidant stress. Binding of the transcription factors is implicated in activation of a wide-range of genes associated with inflammation and the immune response, including those encoding cytokines, cytokine receptors, cell adhesion molecules, acute phase proteins, and growth factors (35) (Fig. 5.4). Unfortunately, NFkB also activates transcription of the genes of some viruses, such as HIV. This sequence of events, in the case of HIV, accounts for the ability of minor infections to speed the progression of individuals who are infected with HIV towards AIDS because, if anti-oxidant defenses are poor, each encounter with general infections results in cytokine and oxidant production, NFkB activation, and an increase in viral replication. It is thus unfortunate that reduced cellular concentrations of GSH are a common feature of asymptomatic HIV infection (15).

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Inflammatory stimuli LPS, oxidants,stress

Transcription factors

Cell proliferation

AP1

NFkB

IL2

Acute phase proteins

Adhesion molecules

IL1, IL6, IL8 TNF

HIV replication

GSH synthesis

Figure 5.4 The influence of activation of AP1 and NFkB on gene transcription following infection and injury.

Oxidant damage to cells will indirectly create a pro-inflammatory effect by the production of lipid peroxides. This situation may also lead to upregulation of NFkB activity, as the transcription factor has been shown to be activated in endothelial cells cultured with linoleic acid, the main dietary n-6 poly-unsaturated fatty acid, an effect inhibited by vitamin E and NAC (36). The interaction between oxidant stress and an impaired ability to synthesize glutathione that results in enhanced inflammation is clearly seen in cirrhosis, a disease that results in high levels of oxidative stress and an impaired ability to synthesize GSH (37). In this study, an inverse relationship between glutathione concentration and the ability of monocytes to produce IL-1, IL-8, and TNF-a was observed. Furthermore, treatment of the patients with the GSH prodrug, oxothiazalidine-4-carboxylate (procysteine), increased monocyte GSH content and reduced IL-1, IL-8, and TNF-a production (Fig. 5.5). Thus, anti-oxidants might act, to prevent NFkB activation, by quenching oxidants. However, NFkB and AP1 may not respond to changes in cell redox state in the same way. When rats were subjected to depletion of effective tissue GSH pools by administration of diethyl maleate, there was a significant reduction in lymphocyte proliferation in spleen and mesenteric lymph nodes (38). An increase in inflammatory stress would be expected in this study. In an in vitro study using HeLa cells and cells from human embryonic kidney, both TNF and hydrogen peroxide resulted in activation of NFkB and AP1 (39). Addition of the anti-oxidant sorbitol

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Methionine Vit B6 Folic acid

NAC

Cysteine

OTZ

Glutamic acid

Glutathione synthesis OTZ (Procysteine)L- 2oxothiazolidine-4-carboxylate

Glutamine

Glycine

NAC n- acetyl cysteine

Figure 5.5 Nutrients and drugs that may be used to improve glutathione synthesis during treatment for infection and injury.

to the medium suppressed NFkB activation (as expected) but (unexpectedly) activated AP1. Thus, the anti-oxidant environment of the cell might exert opposite effects upon transcription factors closely associated with inflammation (e.g., NFkB) and cellular proliferation (e.g., AP1). Evidence for this biphasic effect was seen when glutathione was incubated with immune cells from young adults (40). A rise in cellular glutathione content was accompanied by an increase in IL-2 production, and lymphocyte proliferation, and a decrease in production of the inflammatory mediators, PGE2 and LTB4. Modification of the glutathione content of liver, lung, spleen, and thymus in young rats, by feeding diets containing a range of casein (a protein with a low sulfur amino acid content) concentrations, changed immune cell numbers in lung (41). It was found that in unstressed animals, the number of lung neutrophils decreased as dietary protein intake and tissue glutathione content fell. However, in animals, given an inflammatory challenge (endotoxin) liver and lung GSH concentrations increased directly in relation to dietary protein intake. Lung neutrophils, however, became related inversely with tissue glutathione content. Addition of methionine to the protein deficient diets normalized tissue glutathione content and restored lung neutrophil numbers to those seen in unstressed animals fed a diet of adequate protein content (Fig. 5.6). Thus, it can be hypothesized that anti-oxidants exert an immuno-enhancing effect by activating transcription factors that are strongly associated with cell proliferation (e.g., AP1) and an anti-inflammatory effect by preventing activation of NFkB by oxidants produced during the inflammatory response. The molecular mechanisms that underlie the increased inflammatory and oxidant stress that accompanies aging has been examined in aged mice. The role of changes in PPAR-a activity in the process has been examined by Poynter and Daynes (42). Their findings suggest a role for PPAR-a in the maintenance of redox balance during the aging process.

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Neutrophils/lung area*

25 Influence of GSH and NF B activity?

Rats injected with LPS

20 15

Control rats injected with sterile saline

10

Influence of GSH and AP1 activity?

5 0 0

0.5

1

1.5

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GSH (umol/g lung tissue) Figure 5.6 The relationship between lung neutrophil and glutathione content in animals fed diets with sulfur amino acid contents ranging from 2.2 to 6.5 g/kg and then given either a control saline or lipopolysaccharide injection intraperitoneally. Note: Neutrophils were counted in total lung sections and are expressed as the total number  100 observed per subdivision of the graticule field.

In aged mice, the administration of agents capable of activating the alpha isoform of the PPAR-a receptor was able to restore the cellular redox balance. Evidence for this effect came from the observation that tissue lipid peroxidation was decreased, NF-kB activity decreased, and spontaneous inflammatory cytokine production was reduced. Aged animals bearing a null mutation in PPAR-a failed to elicit these changes following treatment with PPAR-a activators, but remained responsive to vitamin-E supplementation, thereby highlighting independent effects of anti-oxidants and PPAR-a on inflammation. Aged mice were also found to express reduced transcript levels of PPAR-a and the peroxisome-associated genes, acyl-CoA oxidase and catalase. Supplementation of aged mice with PPAR-a activators or with vitamin E caused elevations in these transcripts to levels seen in young animals. A number of natural endogenous molecules have been found that are capable of activating PPARs. For example, 15-deoxy-D12,14 prostaglandin J2 represents a natural PPAR-g ligand (43,44). Many specific fatty acid species and their derivatives, especially polyunsaturated fatty acids (45 –48), the leukotriene B4 (49), and the eicosanoid 8(S)-hydroxyeicosatetraenoic acid (50), have been shown to be ligands for PPAR-a. Activation of PPARs has been demonstrated to antagonize signaling through an array of important pathways, including STATs, AP-1, and NF-kB (45,51 – 55). Poynter and Daynes (42) demonstrated that NF-kB is present in an active state in the macrophages and lymphocytes that reside in the spleens of aged mice (56). This active NF-kB correlated with the expression of the

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NF-kB-regulated genes IL-6, IL-12, macrophage migration inhibitory factor, cyclooxygenase-2, and TNF (56). The administration of specific PPAR-a activators, or vitamin E, to aged rodents effectively reduced the elevated levels of active NF-kB, re-established control over pro-inflammatory cytokine production, and reduced lipid peroxide levels in various tissues (56).

EFFECTS OF ANTI-OXIDANTS ON IMMUNE FUNCTION Effects of Vitamin E A number of studies in which anti-oxidant status has been raised by dietary supplementation indicate that improvement of anti-oxidant status is associated with an increase in cellular aspects of immune function. Vitamin E exerts modulatory effects on both inflammatory and immune components of immune function. In general, vitamin E deficiency and low tissue vitamin E content enhance components of the inflammatory response and suppress components of the immune response. Dietary vitamin E supplementation brings about the opposite effect. Studies in animals have demonstrated that vitamin E deficiency impairs cellular and humoral immunity and is associated with an increased incidence of disease. Supplementation of the diet with vitamin E, at levels that are several fold greater than requirements, increases resistance to a number of pathogens. Resistance of chickens and turkeys to Escherichia coli and of mice to pneumococci, was enhanced by vitamin E supplementation. A similar phenomenon may also occur in humans, since epidemiological evidence shows lower incidence of infectious disease in subjects with high plasma a-tocopherol concentrations. Rats consuming diets that were deficient in vitamin E, and given injections of endotoxin, showed a greater degree of anorexia and greater concentrations of plasma a-1-acid glycoprotein and IL6, than animals consuming adequate amounts of the vitamin. In smokers, a low intake of vitamin E was associated with an increased intensity of the inflammatory response to cigarette smoke. Plasma concentrations of a-1-acid glycoprotein was 50% higher in subjects in the lowest tertile of intake compared with the values for subjects in the highest tertile (57). Large doses of a-tocopherol 50 mg/kg given i.p. significantly decreased the number of neutrophils in airspaces of rats given endotoxin aerosols; however, there appeared to be no change in NFkB or AP1 activation (58). Supplementation of the diet of healthy subjects, and smokers with 600 IU/ day a-tocopherol, for 4 weeks, suppressed the ability of PBMCs to produce TNF-a (59). Intense exercise of healthy young and elderly subjects results in the appearance of a mild inflammatory response characterized by raised blood IL1, IL6, and acute phase protein concentrations. A twice daily supplement of 400 IU of a-tocopherol inhibited the response. A dose of 600 IU/day a-tocopherol given to healthy elderly subjects, for 235 days, increased delayed type hypersensitivity and raised antibody titers to hepatitis B (60). When elderly subjects were supplemented with 800 IU of a-tocopherol for 30 days, there was a 50% increase in the delayed type

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hypersensitivity response, a 65% increase in IL-2 production, and a decrease in oxidative stress as indicated by a major decrease in plasma thiobarbituric acid substances (TBARS). An enteral feed, enriched with vitamin E, vitamin C, and taurine, given to intensive care patients decreased total lymphocyte and neutrophil content in bronchio-alveolar lavage fluid (decreased inflammation) and resulted in a reduction in organ failure rate, reduced requirement for artificial ventilation, and a reduction of 5 days in the requirement for intensive care (61). These results highlight that reduced inflammation and improved immune function are interrelated. Ascorbic Acid and Immune Function The observation that high concentrations of vitamin C are found in phagocytic cells has underpinned the concept that ascorbic acid is an important nutrient for optimal immune function. However, although the role of vitamin C as a key component of antioxidant defense is well established, most studies have shown only minor effects on a range of immune functions, except in cases where the vitamin may be acting by interacting with GSH metabolism. Unlike deficiencies in vitamins B6 , E, and riboflavin, deficiency of vitamin C does not cause atrophy of lymphoid tissue. In a study of ultra marathon runners, dietary supplementation with 600 mg/day of ascorbic acid reduced the incidence of upper respiratory tract infections after a race by 50% (62). It is interesting to note that strenuous exercise has been shown to deplete tissue glutathione content. The interrelationship between glutathione and ascorbic acid may therefore play a role in the effect of exercise on immune function. When immunological parameters and anti-oxidant status were measured in adult males fed 250 mg/day of vitamin C for 4 days followed by 5 mg/day for 32 days, plasma ascorbic acid and glutathione decreased and impairment of antioxidant status became evident from a doubling in semen 8-hydroxydeoxyguanosine concentration (a measure of oxidative damage to nucleic acids) during the second dietary period (63). A fall in vitamin content in peripheral blood mononuclear cells was noted and the delayed type hypersensitivity reaction to seven recall antigens was significantly reduced in intensity. These results again highlight that the strength of inflammation and immune function is interrelated in an inverse manner. A further potential facet of the effect of ascorbic acid on immune function, other than by modulating anti-oxidant defenses, was shown in an in vitro study that examined the effects of physiological concentrations of the vitamin on binding of AP1 to its receptor in macrophages. The vitamin enhanced binding. The effect was shown to be independent of the oxidation state of iron, which is important in binding of AP1 to its receptor (64). Glutathione and Immune Function One of the first indications that glutathione influences aspects of immune function, which are related to T lymphocytes, came from a study in which the GSH content of these cells was measured in a group of healthy volunteers (65). The numbers of helper (CD4þ) and cytotoxic (CD8þ) T cells increased in parallel

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with intracellular GSH concentrations up to 30 nmol/mg of protein. However, the relationship between cellular glutathione concentrations and cell numbers was complex, with numbers of both subsets declining at intracellular glutathione concentrations between 30 and 50 nmol/mg of protein. The study also revealed that cell numbers were responsive to long-term changes in GSH content. When the subjects engaged in a program of intensive physical exercise daily for 4 weeks, a fall in glutathione concentrations occurred. Individuals with glutathione concentrations in the optimal range before exercise, who experienced a fall in concentration after exercise, showed a 30% fall in CD4þ T cell numbers. The decline in T cell number was prevented by administration of N-acetyl cysteine (NAC is metabolized to cysteine, see later section). This study suggests that immune cell function may be sensitive to a range of intracellular sulfhydryl compounds including glutathione and cysteine. In HIVþ individuals and patients with AIDS, a reduction in cellular and plasma glutathione has been noted (15). At present, it is unclear whether the depletion in lymphocyte population that occurs in these subjects is related to this phenomenon. However, in a large randomized, double-blind, placebo-controlled trial, administration of 600 mg/day of NAC for 7 months, resulted in both anti-inflammatory and immunoenhancing effects (6). A decrease in plasma IL-6 concentration occurred, together with an increase in lymphocyte count and in the stimulation index of T lymphocytes in response to tetanus toxin. The precise mechanism underlying the complex effects of changes in cellular glutathione content are not clear, and whether they are related to GSH function as an anti-oxidant or to some other property, such as the effect of GSH on thioredoxin, is not apparent. However, a recent study suggests that glutathione promotes IL-12 production by antigen-presenting cells so driving T helper cells along the Th1 pathway of differentiation (66). Paradoxically, when GSH is processed by glutathione-S-transferases (GST), in pathogens, suppressive effect on immune function are achieved. The enzyme in Faciola hepatica in rat is responsible for inhibition of T cell function and downregulation of nitric oxide production thereby allowing the parasite to evade the animals immune defenses. Likewise, the human parasites Onchocerca volvulus and Trapanozoma cruzi may also, via GST, interfere with immune function in the host. On the basis of these observations, a hypothesis has been proposed that some pathogens via release of GST interfere with the functions of cells of the immune system through their ability to scavenge GSH (67).

EFFECTS OF SUBSTANCES THAT ACT AS PRECURSORS FOR GSH OR COFACTORS IN ENZYME PATHWAYS ASSOCIATED WITH GSH ON IMMUNE FUNCTION Effects of Precursors of GSH on Immune Function A number of strategies have evolved to raise GSH concentrations in depleted individuals. As shown in Fig. 5.4, there are three potential ways of enhancing

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cellular GSH content: administration of the three amino acids (cysteine, glutamic acid, and glycine) that comprise the tri-peptide, either singly or in various combinations; administration of cofactors for the metabolic pathways leading to GSH production, that is, vitamin B6, riboflavin, and folic acid; administration of synthetic compounds, which become converted to precursors of GSH. Although cysteine supplies are the primary determinant of the ability to synthesize GSH, in some circumstances an insufficiency in the other two amino acids, from which it is made, might limit synthesis. Glutamine (a precursor of glutamate), for example, has been shown to maintain hepatic GSH in animals poisoned with acetaminophen, to enhance gut GSH synthesis in rats, when given by gavage, and to enhance hepatic GSH synthesis when given intravenously to rats (68). In human studies, a similar effect on gut GSH concentrations was noted (69). Glycine supplements have been shown to raise hepatic GSH in rats exposed to hemhorragic shock (70). In this condition, however, the metabolic demand for glycine is increased as glycine is the sole nitrogen donor for haem synthesis and, therefore, becomes rate limiting for GSH synthesis. There are many studies that illustrate the ability of sulfur amino acid availability to influence tissue GSH concentrations (71). Studies using animal models of inflammation have shown that a low protein diet will suppress glutathione synthesis, a situation that is reversed by provision of cysteine or methionine (41,72). Because cysteine is unstable in its reduced form, is toxic in high doses, and is mostly degraded in the extracellular compartment, several compounds have been used to deliver cysteine directly to cells. These are L -2-oxothiazalidine-4carboxylate (OTC) and NAC. OTC is an analog of 5-oxoproline in which the 4-methylene moiety has been replaced by sulpfur. It provides an excellent substrate for 5-oxoprolinase (an intracellular enzyme). The enzyme converts OTC to S-carboxy-L -cysteine, which is rapidly hydrolyzed to L -cysteine. NAC rapidly enters the cell and is immediately deacylated to yield L -cysteine. Recent animal and clinical trials with NAC and OTC have demonstrated the ability of the compounds to enhance GSH status (7,73,74). In studies on patients with sepsis, NAC infusion was shown to increase blood GSH, decrease plasma concentrations of IL-8 and soluble TNF receptors (an index of TNF production), improve respiratory function, and shorten the number of days needed in intensive care (74,75). Although not affecting mortality rates, NAC shortened hospital length of stay by .60%. OTZ increased whole blood GSH in peritoneal dialysis patients, normalized tissue GSH in rats fed with sulfur amino acid deficient diet, and decreased the extent of inflammation in a rat peritonitis model (74). In a randomized double blind controlled study on asymptomatic HIV-infected patients, oral OTC treatment increased GSH concentrations in whole blood (6). Other randomized studies on asymptomatic HIV positive patients in the presence and absence of anti-retroviral therapy (ART), have shown that NAC can raise blood GSH, increase natural killer cell activity and enhance stimulation indices of T cells incubated with mitogen or tetanus toxin (6,76). Interestingly, the rise in T-cell function was accompanied by a fall in plasma IL-6 in subjects receiving

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ART as well as the drug. Furthermore, studies have shown that survival time was improved in HIVþ patients who maintained high concentrations of GSH in CD4þ T lymphocytes (77). It could therefore be surmised that improved T-cell function and reduced inflammation are modulated by improvement on anti-oxidant status in these patients. a-Lipoic acid provides a further means of enhancing tissue GSH content (73). The compound is reduced to dihydrolipoic acid, which converts cystine to cysteine. This change has functional significance for glutathione status in lymphocytes, because the xc-transport system, which is needed to take up cystine into the cells, is weakly expressed and is inhibited by glutamate, whereas the neutral amino acid transport system that takes up cysteine is functional. Cysteine, upon gaining entry to the immune cells is rapidly converted to GSH. Flow cytometric analysis of freshly prepared human peripheral blood lymphocytes shows that lipoic acid is able to normalize a subpopulation of cells with severely compromized thiol status rather than increasing the level in all cells above normal values (78). Therefore, lipoic acid may also prove to be a useful clinical agent for restoring cellular GSH concentration in immunocompromised subjects. Effects of Vitamin B6 on Immune Function Vitamin B6 , although having no anti-oxidant properties, plays an important part in anti-oxidant defenses because of its action in the metabolic pathway for the formation of cysteine, which, as indicated earlier, is the rate limiting precursor in glutathione synthesis. Vitamin B6 status has widespread effects on immune function (79). Vitamin B6 deficiency causes thymic atrophy and lymphocyte depletion in lymph nodes and spleen. Antigen processing is unaffected. However, the ability to make antibodies to sheep red blood cells is depressed. In human studies, the ability to make antibodies to tetanus and typhoid antigens is not seriously affected. Various aspects of cell-mediated immunity are also influenced by vitamin B6 deficiency. Skin grafts in rats and mice survive longer during deficiency, and guinea-pigs exhibit decreased delayed hypersensitivity reactions to bacille Calmete-Guerin (BCG) administration. Deficiency of vitamin B6 is rare in humans but can be precipitated with the anti-TB drug isoniazid. However, experimental deficiency in elderly subjects has been shown to reduce total blood lymphocyte numbers and decrease the proliferative response of lymphocytes to mitogens (80). Similarly, IL-2 production is reduced by deficiency of the vitamin. Restoration of vitamin B6 intake to normal by dietary supplements restores immune function. It is unclear, at present, whether a similar situation occurs in younger subjects. One mechanism for the effect of vitamin B6 on immune function may be due to the importance of the vitamin in cysteine synthesis, as outlined earlier. Deficiency of the vitamin may limit the availability of cysteine for glutathione synthesis. In rats, vitamin B6 deficiency resulted in decreases of 12% and 21% in glutathione concentrations in plasma and spleen, respectively (81). In

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healthy young women, large doses of vitamin B6 (27 mg/day for 2 weeks) resulted in a 50% increase in plasma cysteine content (82), presumably by increased flux through the transulfuration pathway. As cysteine is a rate-limiting substrate for glutathione synthesis, these findings may have implications for the response to pathogens because of the importance of glutathione in lymphocyte proliferation and anti-oxidant defense. However, although vitamin B6 has cellular effects on the immune system, evidence is lacking of any effect on the inflammatory response. Effects of Folic Acid on Immune Function Folic acid plays a crucial role in DNA synthesis suggesting that every aspect involving cell proliferation might be affected by deficiency in the vitamin. Indeed, cell-mediated immunity is especially affected by deficiency in humans and animals (83). Folate deficiency also impairs natural killer cell activity in rats (84). As the vitamin is also intimately involved in sulfur amino acid metabolism, it might be expected that the vitamin would modulate anti-oxidant status and immune function. However, there is evidence that only the second of these two effects occurs. Indeed, oxidative stress may impair folate metabolism. In a double-blind, placebo-controlled crossover intervention in healthy subjects, it was found that although a folate rich diet and folate supplements caused a fall in plasma homocysteine concentrations, there was no change in anti-oxidant activity (plasma and red blood cell glutathione peroxidase activity and red cell superoxide dismutase activity) or oxidant damage (plasma malonaldehyde) (85). Infection and inflammation are often associated with hyperhomocysteinemia, an indicator of folate insufficiency (86). It has been hypothesized that the underlying cause of this effect is that the active form of folate (tetrahydrofolate) is susceptible to oxidation during the oxidative stress involved in infection and inflammatory disease (86). Therefore, folate insufficiency is the result rather than the cause of a weakening in anti-oxidant defenses during the immune response. Taurine and Immune Function Taurine, together with sulfate, can be regarded as biochemical end products of cysteine metabolism. However, it is apparent that taurine also plays a role in immune function. It is the most abundant free nitrogenous compound (often incorrectly classified as an amino acid) in cells. It is a membrane stabilizer and regulates calcium flux thereby controlling cell stability. It has been shown to possess anti-oxidant properties and to regulate the release of pro-inflammatory cytokines in hamsters, rats, and humans (87 – 89). The possibility that taurine might have immunomodulatory properties was indicated in studies in obligate carnivores, such as cats, in which taurine is an essential nutrient owing to an inability to synthesize the compound. In cats

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deprived of taurine, substantial impairment of immune function occurs (88). A large decline in lymphocytes, an increase in mononuclear cells, and a decrease in the ability of these cells to produce a “respiratory burst” and to phagocytose bacteria, occurs. There was a rise in g-globulin concentrations in deficient animals. Spleen and lymph nodes showed regression of follicular centers and depletion of mature and immature B lymphocyte numbers. The changes were reversed by inclusion of taurine in the diets. Studies in other species have also reported effects of supplementation on immune system and function. In mice, administration of taurine prevented the decline in T cell number that occurs with aging and enhanced the proliferative responses of T cells in both young and old mice (17). The effect was more marked in cells from old than young animals. Taurine has been shown to ameliorate inflammation in trinitrobenzene sulfonic acid-induced colitis. Taurine interacts with hypochlorous acid, produced during the “oxidant burst” of stimulated macrophages, to produce taurine chloramine (TauCl). This compound may have important immunomodulatory properties and may be responsible for properties that have been ascribed earlier to taurine. TauCl has been shown to inhibit NO, PGE2, TNF-a, and IL-6 production from stimulated macrophages in culture and to inhibit the ability of antigen-presenting cells to process and present ovalbumin (17). In in vitro studies with murine dendritic cells, the compound altered the balance of Th1 to Th2 cytokines suggesting that it might play a role in maintaining the balance between the inflammatory response and the acquired immune response. CONCLUSIONS Oxidant stress is both an integral part of the body’s response to invasion by pathogens and a modulator of immune function. The modulation may take the form of an upregulation of inflammatory components of the response and downregulation of cell-mediated immunity. This apparent paradoxical response is due in part to the action of oxidant stress on the activity of transcription factors such as NFkB and AP1. The former has an impact on inflammation and the latter on cellmediated immunity. In addition to these molecular factors, the age of the host may influence the balance between inflammation and cell-mediated immunity. As aging proceeds, inflammation increases in intensity, even in the absence of pathogenic invasion of the body. Cell-mediated immunity may also weaken. Studies on PPAR-a in mice indicate that the normal restraining influence of this group of transcription factors on inflammation may weaken, thereby, contributing to an inflammatory phenotype in the aged. Paradoxically, the inflammatory response may deplete anti-oxidant defenses, an action that may lead to upregulation of inflammation and impairment of cell-mediated immunity. Many studies in humans and animals have shown that nutrients, which contribute to anti-oxidant defenses within the body, in general have an antiinflammatory, immunostimulatory influence. For some nutrients such as ascorbic

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acid, glutathione, and its precursors, the modulatory effect is via actions on NFkB, for other nutrients, such as a-tocopherol, actions may be via an influence on AP1 activity or by unknown mechanisms.

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67. Ouaissi A, Ouaissi M, Sereno D. Glutathione S-transferases and related proteins from pathogenic human parasites behave as immunomodulatory factors. Immunol Lett 2002; 81:159– 164. 68. Cao Y, Feng Z, Hoos A, Klimberg VS. Glutamine enhances gut glutathione production. J Parenteral Enteral Nutr 1998; 22:224 – 227. 69. O’Riordain MG, De Beaux A, Fearon KC. Effect of glutamine on immune function in the surgical patient. Nutrition 1996; 12(suppl):S82– S84. 70. Spittler A, Reissner CM, Oehler R, Gornikiewicz A, Gruenberger T, Manhart N, Brodowicz T, Mittlboeck M, Boltz-Nitulescu G, Roth E. Immunomodulatory effects of glycine on LPS-treated monocytes: reduced TNF-alpha production and accelerated IL-10 expression. FASEB J 1999; 13:563– 571. 71. Stipanuk MH, Coloso RM, Garcia RAG. Cysteine concentration regulates cysteine metabolism to glutathione, sulfate and taurine in rat hepatocytes. J Nutr 1992; 122:420– 427. 72. Hunter EAL, Grimble RF. Dietary sulphur amino acid adequacy influences glutathione synthesis and glutathione-dependent enzymes during the inflammatory response to endotoxin and tumour necrosis factor-a in rats. Clin Sci 1997; 92:297– 305. 73. Deneke SM. Thiol-based antioxidants. Curr Topics Cell Regululat 2000; 36:151– 180. 74. Bernard GR, Wheeler AP, Arons MM, Morris PE, La Paz H, Russell JA, Wright PE. The Antioxidant in ARDS study Group. A trial of antioxidants N-acetylcysteine and procysteine in ARDS. Chest 1997; 112:164– 172. 75. Spapen H, Zhang H, Demanet C, Vleminckx W, Vincent JL, Huyghens L. Does N-acetyl-L -cysteine influence cytokine response during early human septic shock? Chest 1998; 113:1616 – 1624. 76. Simon G, Moog C, Obert G. Effects of glutathione precursors on human immunodeficiency virus replication. Chem Biol Interact 1994; 91:217 – 224. 77. Herzenberg LA, De Rosa SC, Dubs JG, Roederer M, Anderson MT, Ela SW, Deresinski SC, Herzenberg LA. Glutathione deficiency is associated with impaired survival in HIV disease. Proc Natl Acad Sci USA 1997; 94:1967 – 1972. 78. Sen CK, Roy S, Han D, Packer L. Regulation of cellular thiols in human lymphocytes by alpha-lipoic acid: a flow cytometric analysis. Free Radic Biol Med 1997; 22:1241– 1257. 79. Rall LC, Meydani SN. Vitamin B6 and immune competence. Nutr Revs 1993; 8:217– 225. 80. Meydani SN, Ribaya-Mercado JD, Russell RM, Sahyoun N, Morrow RD, Gershoff SN. Vitamin B6 deficiency impaires IL-2 production and lymphocyte proliferation in elderly adults. Am J Clin Nutr 1991; 53:1275 – 1280. 81. Takeuchi F, Izuta S, Tsubouchi R, Shibata Y. Glutathione levels and related enzyme activities in vitamin B-6 deficient rats fed a high methionine and low cysteine diet. J Nutr 1991; 121:1366– 1373. 82. Kang-Yoon SA, Kirksey A. Relation of short-term pyridoxine hydrochloride supplements to plasma vitamin B6 vitamers and amino acid concentration in young women. Am J Clin Nutr 1992; 55:865 – 872. 83. Dhur A, Galan P, Hercberg S. Folate status and the immune system. Prog Food Nutr Sci 1991; 15:43– 60. 84. Kim YI, Hayek M, Mason JB, Meydani SN. Severe folate deficiency impairs natural killer cell-mediated cytotoxicity in rats. J Nutr 2002; 132:1361– 1367.

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85. Moat SJ, Hill MH, McDowell IF, Pullin CH, Ashfield-Watt PA, Clark ZE, Whiting JM, Newcombe RG, Lewis MJ, Powers HJ. Reduction in plasma total homocysteine through increasing folate intake in healthy individuals is not associated with changes in measures of antioxidant activity or oxidant damage. Eur J Clin Nutr 2003; 57:483 –489. 86. Fuchs D, Jaeger M, Widner B, Wirleitner B, Artner-Dworzak E, Leblhuber F. Is hyperhomocysteinemia due to the oxidative depletion of folate rather than to insufficient dietary intake? Clin Chem Lab Med 2001; 39:691 –694. 87. Kontny E, Szczepanska K, Kowalczewski J, Kurowska M, Janicka I, Marcinkiewicz J, Maslinski W. The mechanism of taurine chloramine inhibition of cytokine (interleukin-6, interleukin-8) production by rheumatoid arthritis fibroblast-like synoviocytes. Arth Rheum 2000; 43:169– 177. 88. Grimble RF. Sulphur amino acids and the metabolic response to cytokines. Adv Exp Med Biol 1994; 359:41 – 49. 89. Huxtable RJ. Taurine past, present, and future. Adv Exp Med Biol 1996; 403:641– 650.

6 Concentration-Dependent Gene and Protein Expressions of Neuroprotective and Neurotoxic Activities of Antioxidants, Including Nutrients Orly Weinreb, Silvia Mandel, and Moussa B. H. Youdim Technion-Faculty of Medicine, Haifa, Israel

Introduction Cellular Viability in Response to Antioxidants Expression of Apoptosis and Cell Survival-Related Genes and Proteins in Response to Antioxidants The Effect of the Antioxidants on Caspase-3 Protein Level and Activity The Molecular Mechanism of Action of Antioxidants Conclusion Acknowledgment References

123 125 125 129 134 137 137 137

INTRODUCTION Parkinson’s disease (PD) is a progressive and age-dependent neurodegenerative disease, characterized at cellular level by a depletion of dopamine (DA) in DA neurons of substantia nigra pars compacta (SNPC). Although the etiology 123

124

Weinreb, Mandel, and Youdim

of the neuronal cell death is still unclear and no casual therapy is available yet, the current view supports oxidative stress (OS) as a key factor of neurodegeneration (1,2). Analysis of Parkinsonian brain samples demonstrated certain apoptotic cell features in DA neurons of SNPC, but the reported results remain highly controversial (3). However, in vitro and in vivo experiments with neurotoxins such as 6-hydroxydopamine (6-OHDA) (4) and N-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) (5) have shown OS-dependent apoptosis in death of DA neurons and many of the neurochemical changes reported in substantia nigra of Parkinsonian brains (6), and thus it may be a contributing pathway to dopaminergic neuronal cell death in PD (7). Previous studies have shown that high concentrations of antioxidants, such as DA, DA D1-D2-receptor agonist, R-apomorphine (R-APO), green tea polyphenol (2)-epigallocatechine-3-gallate (EGCG), and the pineal indoleamine hormone, melatonin, can be cytotoxic to neuronal cells (8 – 12), raising the question whether long-term treatment with these compounds will contribute to the degeneration of the dopaminergic neurons of the substantia nigra in PD (13). Nonetheless, the few clinical studies done with these compounds have not exhibited such a phenomenon. Thus, the in vitro studies with these antioxidant agents are not compatible with in vivo studies. The neuroprotective therapy aimed to interfere with cytotoxic processes or promote neural growth and function have been successfully demonstrated in cell and animal models of PD with several antioxidants, but not in the clinic. The few clinical neuroprotection so far done has been far more difficult to establish. Several catechol derivatives, especially L-dopa, or the DA D1-D2-receptor agonists, such R-APO, bromocriptine, and pramipexole, are employed for the therapy of PD (14). Also, the antioxidant, melatonin, has been suggested to have clinical potential in the treatment of PD (15). Previous studies have observed that in vitro low concentrations of DA receptor agonists, such as R-APO (16,17), tea extracts (18), the major polyphenol component of green tea EGCG (10), and melatonin (19), protected neuronal cells from the toxic effects of 6-OHDA and were able to protect against MPTP-induced neurotoxicity in vivo in mice (20 – 23). Iron chelators and radical scavengers such as R-APO, DA, EGCG, and melatonin possess concentration-dependent biphasic actions in preventing and promoting neuronal cell death in models of PD. Potential candidates possessing a key role in cell survival/death are the conserved group of mitochondrial Bcl-2 family members. The apoptotic proteins, Bax and Bak, and the BH-3-only proteins (e.g., Bad, Bid, Bim, Noxa, and Puma) may trigger the opening of the mitochondrial megachannel, or a specific channel in the outer mitochondrial membrane, both of which promote the fall in mitochondrial membrane potential, leading to cytochrome c release. The anti-apoptotic members, Bcl-2 and Bcl-xL proteins, prevent this, probably by inhibiting Bax or Bad translocation and insertion into mitochondrial membrane, or via a direct interaction with the channels (24,25). Indeed, the anti-Parkinson neuroprotective anti-apoptotic drug,

Neuroprotective/Neurotoxic Activities of Antioxidants

125

rasagiline, was recently shown to prevent the fall in mitochondrial membrane potential and the opening of mitochondrial voltage-dependent anion channel via the increase in bcl-2 and bcl-xl mRNAs and their proteins (26,27). An important aspect of neuroprotection in PD or other neurodegenerative diseases [Alzheimer’s disease (AD), Huntington’s disease, amyotrophic lateral sclerosis, Friedreich’s ataxia] or their animal models is the onset of OS resulting from generation of reactive oxygen species. In vivo and cell culture studies with antioxidant neuroprotective agents have revealed that these compounds have a bell shaped, concentration-dependent pharmacological response and duration of action, which would be translated into a complex set of gene alterations (10,21,28). The significant questions are whether the concentration-dependent pharmacological actions of antioxidant as associated with alteration in different gene expressions and whether antioxidant neuroprotective drugs are engaged in a common genetic program, since they exhibit similar neuroprotective activity, at least in the PD models. In this article, we discuss the molecular mechanisms involved in the cell survival and cell death action of the catechol derivatives, DA and R-APO, and the radical scavengers, EGCG and melatonin, at low and high concentrations, since there are numerous reports that such compounds exhibited concentrationdependent effects, where at low concentrations they are antioxidants and at high concentrations they are prooxidants. We have therefore employed customized cDNA and proteomics to illustrate the dose-dependent actions of these compounds. We have shown a considerable extent of similarity between DA, R-APO, EGCG, and melatonin with respect to cell survival/cell cycle-related gene and protein expression at their neuroprotective and neurotoxic concentrations. Furthermore, their concentration-dependent ability to induce cell survival and death are related to expression of genes that promote such activities. CELLULAR VIABILITY IN RESPONSE TO ANTIOXIDANTS In order to examine whether the four antioxidants, DA, R-APO, EGCG, and melatonin, display a biphasic mode of action, NB SH-SY5Y cell viability studies were conducted with these compounds over a broad range of concentrations. The antioxidant agents demonstrated a dose-dependent effect on the viability of NB SH-SY5Y cells. At low concentrations (1 – 10 mM) they had no effect on cell survival, whereas at higher concentrations (.10 mM) they induced a gradual decrease with rank order being R-APO . EGCG . DA . melatonin (Fig. 6.1). EXPRESSION OF APOPTOSIS AND CELL SURVIVAL-RELATED GENES AND PROTEINS IN RESPONSE TO ANTIOXIDANTS The advent of genomic tools such as cDNA microarrays and proteomics provide an entirely new approach for molecular characterization of neurodegenerative

Cell Viability (% of control)

126

Weinreb, Mandel, and Youdim

110

Dopamine

100

R-apomorphine

90 80

Melatonin EGCG

70 60 50 40 30 20 10 0

1

10

100

1000

Concentration (mM) Figure 6.1 The effect of DA, R-APO, EGCG, and melatonin on cellular viability. NB SH-SY5Y cells were pretreated with increasing concentrations of DA, R-APO, EGCG, and melatonin for 24 h. Cell viability was assessed by MTT test, and expressed as a percent of untreated control without the compounds. The results are expressed as the mean + SD. The experiments were repeated at least three times in duplicates.

diseases and their models. The combined implementation of these methods may shed light on the extent of homology existing between the models of PD, whether the models are relevant to the disease pathology and on the effect of drugs reputed to exert neuroprotective activity in such models. It may contribute to the characterization of novel genes implicated in the pathogenesis of neurodegeneration, or other pathways leading to cell death, for which novel neuroprotective drugs need to be developed. Employing a customized cDNA array containing 25 human cDNA fragments of genes coding for protein related to apoptosis and cell survival pathways, we have observed (28) that low concentrations of DA (10 mM), R-APO (1 mM), and melatonin (1 mM) had no effect on gene expression and only EGCG (1 mM) decreased the expression of bad, bax, and tumor necrosis factor ligand member 10 (TRAIL) mRNAs (Table 6.1). However, similar induction of mRNAs coding for pro-apoptotic proteins was observed with the high toxic concentrations of the antioxidants (Table 6.1). DA (500 mM), R-APO (50 mM), and melatonin (50 mM) increased apoptosisrelated cysteine proteases (e.g., caspase-3, -10), tumor necrosis factor receptor members fas and fas-ligand, nuclear factor kappa B (NF-kB p105 subunit), and tumor suppressor protein p53. DA (500 mM), R-APO (50 mM), and EGCG (50 mM), but not melatonin (50 mM), increased the expression of pro-apoptotic Bcl-2 family members bad, bax, and caspase-6 mRNAs. Only DA and R-APO affected the expression of TRAIL, TRAIL receptor DR5, and DNA-damage inducible transcript gadd45.

0.897+ 0.032 0.899 + 0.050 0.964 + 0.208 0.896 + 0.023 0.911 + 0.098 0.896 + 0.028 0.942 + 0.174 0.95 + 0.192 0.932 + 0.150 0.936 + 0.137 0.897+ 0.033 0.896 + 0.028 0.930 + 0.157 0.900 + 0.055 0.899 + 0.049

DA (10 mM)

R-APO (1 mM) 0.865 + 0.038 0.871 + 0.073 0.946 + 0.225 0.881 + 0.046 0.996 + 0.210 0.863 + 0.009 0.996 + 0.210 0.995 + 0.286 0.928 + 0.190 0.975 + 0.249 0.903 + 0.154 0.871 + 0.065 1.005 + 0.360 0.886 + 0.096 0.922 + 0.190

DA (500 mM) 1.292 + 0.045
" 1.211 + 0.010
" 0.940 + 0.019 1.029 + 0.005 1.385 + 0.054
" 1.303 + 0.007
" 1.370 + 0.087
" 1.475 + 0.047
" 1.309 + 0.09
" 1.354 + 0.017
" 1.266 + 0.006
" 1.033 + 0.007 1.550 + 0.011
" 1.281 + 0.040
" 1.322 + 0.009
"

Apoptosis and Cell Survival Gene Expression Analysis Identified by the cDNA Microarray

bad (U66879) bax (L22474) bcl-2 (M14745) bcl-xL (U59747) caspase-3 (U13737) caspase-6 (U20537) caspase-10 (U60519) DR5 (AF016266) fas (X63717) fas-ligand (U08137) gadd45 (M60974) gadd45b (AF078077) NF-kB (M58603) p53 (M14694) TRAIL (U37518)

Gene name (Unigene)

Table 6.1

(continued )

1.377 + 0.020
" 1.463 + 0.039
" 0.986 + 0.031 1.146 + 0.245 1.483 + 0.139
" 1.593 + 0.135
" 1.739 + 0.037
" 1.589 + 0.119
" 1.406 + 0.052
" 1.499 + 0.039
" 1.383 + 0.030
" 1.346 + 0.012
" 1.744 + 0.137
" 1.344 + 0.056
" 1.535 + 0.032
"

R-APO (50 mM)

Neuroprotective/Neurotoxic Activities of Antioxidants 127

Continued Melatonin (50 mM) 1.088 + 0.019 1.065 + 0.032 1.125 + 0.037 1.090 + 0.098 1.383 + 0.023
" 1.120 + 0.012 1.331 + 0.017
" 1.062 + 0.053 1.444 + 0.009
" 1.219 + 0.012
" 1.164 + 0.098 1.367 + 0.045
" 1.372 + 0.039
" 1.218 + 0.099
" 0.927 + 0.004

Melatonin (1 mM)

1.034 + 0.032 1.135 + 0.076 1.198 + 0.127 0.917 + 0.109 1.045 + 0.093 1.070 + 0.051 0.926 + 0.134 0.998 + 0.053 1.002 + 0.069 0.991 + 0.087 1.009 + 0.122 1.028 + 0.076 1.001 + 0.021 1.011 + 0.121 0.993 + 0.198

EGCG (50 mM) 1.693 + 0.114
" 1.594 + 0.119
" 1.005 + 0.091 0.547 + 0.096
# 1.109 + 0.054 1.663 + 0.072
" 1.187 + 0.078 1.012 + 0.033 1.779 + 0.123
" 1.005 + 0.029 1.105 + 0.032 1.694 + 0.173
" 1.123 + 0.054 1.009 + 0.041 0.921 + 0.028

EGCG (1 mM) 0.395+ 0.058
# 0.795 + 0.008
# 1.001 + 0.097 0.873 + 0.072 1.024 + 0.096 0.877 + 0.035 0.987 + 0.119 0.928 + 0.019 1.009 + 0.012 0.907 + 0.087 1.003 + 0.061 0.528 + 0.200 1.051 + 0.021 1.018 + 0.045 0.412 + 0.123
#

Note: NB SH-SY5Y cells were treated without or with DA (10 and 500 mM), R-APO (1 and 50 mM), melatonin (1 and 50 mM), and EGCG (1 and 50 mM) for 6 h. cDNA probes were hybridized to a microarray, containing 25 genes related to cell survival and apoptotic pathways. The amount of each product was normalized to bactin and expressed as fold stimulation of untreated control cells, set arbitrarily as 1. The results are the mean of three separate experiments, performed in duplicates. t-test:
p , 0.05 vs. control. The arrows indicate alterations of gene expression.

bad (U66879) bax (L22474) bcl-2 (M14745) bcl-xL (U59747) caspase-3 (U13737) caspase-6 (U20537) caspase-10 (U60519) DR5 (AF016266) fas (X63717) fas-ligand (U08137) gadd45 (M60974) gadd45b (AF078077) NF-kB (M58603) p53 (M14694) TRAIL (U37518)

Gene name (Unigene)

Table 6.1

128 Weinreb, Mandel, and Youdim

Neuroprotective/Neurotoxic Activities of Antioxidants

129

Verification of apoptosis-related gene changes (Table 6.2) was assessed using quantitative real-time RT –PCR of RNA samples isolated from NB SH-SY5Y cells treated with the antioxidants. Low neuroprotective concentrations of DA, R-APO, and EGCG (10, 1, and 1 mM, respectively) induced an acute decrease of bax mRNA, evident as early as 1.5 h. A delayed and less marked reduction in bax expression was observed with melatonin (1 mM), being induced at 6 h of incubation. In parallel, an immediate substantial reduction in Bax protein content was induced by the low concentrations of the compounds after 1.5 h of exposure and except for R-APO, subsequently declined to control values (Fig. 6.2). The expression of the cell-cycle inhibitor gadd45 and fas-ligand mRNAs were reduced by EGCG, R-APO, and DA at 1.5 h and both R-APO and DA treatments accompanied by an increase in the expression of anti-apoptotic bcl-2 and bcl-xL mRNAs. This effect was transiently manifested 1.5 h after drug administration, and returned to control levels at 6 h. A strict correlation between bcl-2 gene and protein expression was observed with R-APO and DA, since both of them upregulated Bcl-2 protein already at 1.5 h of treatment, whereas R-APO induced a sustained upregulation of protein levels up to the 6 h incubation period. Low concentration (1 mM) of EGCG decreased the expression of bax and caspase-6 mRNA at 6 h of treatment. The high toxic concentrations of DA, R-APO, and melatonin (500, 50, and 50 mM, respectively) caused a continued increase in the expression of bax, gadd45, and fas-ligand mRNAs, which was accompanied by an increase in Bax protein content. However, the effect of melatonin was comparably lower than the other three compounds DA, R-APO, and EGCG, markedly reduced bcl-2 and bcl-xL mRNA expression, whereas no effect was obtained with melatonin (Table 6.2).

THE EFFECT OF THE ANTIOXIDANTS ON CASPASE-3 PROTEIN LEVEL AND ACTIVITY Caspase-3, -6, and -10 are members of a family of cysteine proteases and important components of the apoptosis machinery (29). DA, R-APO, EGCG, and melatonin affected, either way, the mRNAs coding for these proteases (Tables 6.1 and 6.2). Caspase-3, an apoptotic marker, is activated by multiple proteolytic cleavage of its 32 kDa precursor form to generate an enzymatically active p17/p12 complex. In control-untreated NB SH-SY5Y cells or cells treated with the low concentrations of DA (10 mM), R-APO (1 mM), or melatonin (1 mM), only the 32 kDa precursor was detected. However, exposure of NB SH-SY5Y cells to the high concentrations of DA (200 and 500 mM), R-APO (20 and 50 mM), or melatonin and EGCG (50 mM) for 1.5– 6 h induced the cleavage of caspase-3, as demonstrated by the appearance of the 17 kDa fragment detected with specific antibody for caspase-3 [Fig. 6.3(a)] and stimulation of caspase-3 enzymatic activity by 1.5– 2.5-fold over the control at 3 and 6 h after exposure [Fig. 6.3(b)].

0.251 + 0.001
# 2.296 + 0.089
" 1.465 + 0.007
" 0.935 + 0.031 0.358 + 0.028
# 0.220 + 0.01
#

0.851 + 0.242 1.071 + 0.083 1.030 + 0.003 0.962 + 0.011 0.572 + 0.011
# 0.610 + 0.006
#

For 6 h bax (L22474) bcl-2 (M14745) bcl-xL (U59747) caspase-6 (U20537) fas-ligand (U08137) gadd45 (M60974)

DA (10 mM)

1.360 + 0.005
" 1.074 + 0.010 0.773 + 0.004 1.510 + 0.024
" 1.410 + 0.024
" 1.502 + 0.039
"

1.426 + 0.005
" 0.641 + 0.018
# 0.626 + 0.012
# 0.783 + 0.062 1.480 + 0.070
" 1.853 + 0.011
"

DA (500 mM)

0.971 + 0.045 1.81 + 0.132
" 0.973 + 0.012 1.051 + 0.060 1.390 + 0.160 1.106 + 0.073

0.571 + 0.029
# 2.270 + 0.082
" 1.991 + 0.054
" 1.143 + 0.144 1.084 + 0.161 0.33 + 0.01
#

R-APO (1 mM)

Apoptosis and Cell Survival Gene Expression Analysis Identified by Quantitative Real-Time RT– PCR

For 1.5 h bax (L22474) bcl-2 (M14745) bcl-xL (U59747) caspase-6 (U20537) fas-ligand (U08137) gadd45 (M60974)

Gene name (Unigene)

Table 6.2

1.321 + 0.049
" 1.293 + 0.200 0.645 + 0.002
# 1.620 + 0.012
" 1.593 + 0.050
" 1.420 + 0.121
"

1.657 + 0.087
" 0.352 + 0.012
# 0.199 + 0.008
# 1.395 + 0.145 2.063 + 0.125
" 2.218 + 0.231
"

R-APO (50 mM)

130 Weinreb, Mandel, and Youdim

1.031 + 0.069 0.845 + 0.040 0.863 + 0.002 3.271 + 0.142
" 1.310 + 0.001
" 0.805 + 0.132

1.272 + 0.032
" 1.139 + 0.127 1.164 + 0.022 0.868 + 0.063 1.083 + 0.354 2.330 + 0.143
"

Melatonin (50 mM)

0.603 + 0.13
# 0.819 + 0.002 1.065 + 0.07 0.473 + 0.04
# 1.001 + 0.071 0.936 + 0.53

0.560 + 0.11
# 1.05 + 0.08 0.99 + 0.13 0.61 + 0.09
# 0.630 + 0.09
# 0.470 + 0.03
#

EGCG (1 mM)

1.52 + 0.105
" 0.453 + 0.09
# 0.491 + 0.09
# 1.101 + 0.05 1.010 + 0.01 2.967 + 0.27
"

0.86 + 0.16 0.505 + 0.01
# 0.561 + 0.04
# 0.86 + 0.23 1.02 + 0.34 0.69 + 0.23

EGCG (50 mM)

Note: NB SH-SY5Y cells were treated with DA (10 and 500 mM), R-APO (1 and 50 mM), melatonin (1 and 50 mM), and EGCG (1 and 50 mM) for 1.5 and 6 h. The amount of each product was normalized to the housekeeping gene 18S-rRNA, and expressed as fold stimulation of untreated control, arbitrarily set as 1. The results are the mean of three separate experiments, performed in duplicates. t-test:
p , 0.05 vs. control. The arrows indicate alterations of gene expression.

0.870 + 0.013 0.864 + 0.112 0.901 + 0.021 0.863 + 0.021 1.061 + 0.021 0.863 + 0.053

For 6 h bax (L22474) bcl-2 (M14745) bcl-xL (U59747) caspase-6 (U20537) fas-ligand (U08137) gadd45 (M60974)

Melatonin (1 mM)

1.008 + 0.321 1.055 + 0.118 1.825 + 0.029
" 0.804 + 0.059 1.003 + 0.328 1.081 + 0.110

Continued

For 1.5 h bax (L22474) bcl-2 (M14745) bcl-xL (U59747) caspase-6 (U20537) fas-ligand (U08137) gadd45 (M60974)

Gene name (Unigene)

Table 6.2

Neuroprotective/Neurotoxic Activities of Antioxidants 131

(a)

0.0

0.5

1.0

1.5

2.0

2.5

10

(mM) C

200 DA

200 DA

500

500

1 50 Melatonin

*

1 50 Melatonin

*

*

1

1

*

20 50 R-APO

20 50 R-APO

*

200 DA 500 1 50 1 20 50 Melatonin R-APO

*

*

*

0.0

1.0

1.5

2.0

0.0 10 200 500 1 50 1 20 50 DA Melatonin R-APO

*

0.5

C

*

0.5

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Figure 6.2 The effect of DA, R-APO, and melatonin on protein levels: (a) Bcl-2 and (b) Bax. NB SH-SY5Y cells were treated with increasing concentrations of DA (10, 200, and 500 mM), R-APO (1, 20, and 50 mM), and melatonin (1 and 50 mM), and harvested at different time intervals (1.5, 3, and 6 h). Western blotting were probed with anti-Bax and anti-Bcl-2. An antibody for b-actin was used to normalize the expression level of the proteins. The bands were quantified by densitometry and represented graphically. The results are the mean of three independent experiments, performed in duplicates. t-test:
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Figure 6.3 The effect of DA, R-APO, and melatonin on caspase-3 protein level and activity. NB SH-SY5Y cells were exposed to DA, R-APO, EGCG, and melatonin, and harvested at different time intervals (1.5, 3, and 6 h). Activation of caspase-3 was analyzed in cell lysates and analyzed with: (a) Immunoblotting using antibody against pro-caspase-3 (32 kDa) and the activated cleaved caspase-3 fragments (17 kDa and 12 kDa). An antibody for b-actin was used to normalize the expression level of the proteins. The data are from one representative experiment of three independent experiments that exhibited similar results. (b) Caspase-3 like protease activity was measured with the caspase-3 substrate colorimetric assay in accordance with the protocol supplied by the manufacturer (Calbiochem, CA). The assay was preformed in 96-well microtiter plates. The colorimetric substrate (Ac-DEVD-pNA) was added to 20 mg of cell lysates in 100 ml reaction buffer. After 4 h incubation at 378C, absorbance was determined at 405 nm. The results are expressed as the mean + SD. The experiments were repeated at least three times.
p , 0.01 vs. control.

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THE MOLECULAR MECHANISM OF ACTION OF ANTIOXIDANTS Antioxidants have neuroprotective, antioxidant anti-apoptotic activity at low concentrations, whereas at high concentrations they induce pro-oxidant and neurotoxic pro-apoptotic actions in cell cultures and in vivo models of neurodegenerative diseases. Our recent studies (10,28) provide new insights into the molecular events involved in the dose-dependent anti- and/or pro-apoptotic activities of catechol-derived and indoleamine compounds at low and high concentrations. For this purpose, low neuroprotective and 50-fold higher concentrations were chosen. cDNA microarray has demonstrated a concentrationdependent homology among antioxidants R-APO, DA, melatonin, and EGCG for modulation of cell survival/cell death-related gene pathways. The gene microarray analysis provides the first evidence for a selective, dose-dependent regulation of a number of mRNAs by these drugs. One important aspect of our study is the significant homology between the catechol-derived compounds and differences observed with the indoleamine, melatonin (Fig. 6.4). The extremely

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low pro-apoptotic activity of melatonin, when compared with DA, R-APO, and EGCG, may be partially explained by its mild or lack of effect on the expression of Bcl-2 family genes. No significant gene changes were observed with the low concentrations of R-APO, DA, and melatonin, previously reported (30 – 33) to induce effective neuroprotection in both neuronal primary and cell line cultures. However, when quantitative real-time RT-PCR method was applied, specific gene expression changes as a function of concentration and time were observed. This discrepancy may result from the sensitivity thresholds of both methods. Low DA, R-APO, and melatonin concentrations induced an immediate expression of anti-apoptotic bcl-xL and/or bcl-2 mRNAs, whereas bax mRNA was reduced. The gene changes were correlated with alterations in protein levels of both Bcl-2 and Bax. The increased bcl-2 or bcl-xL to bax ratio as a result of R-APO, DA, and melatonin suggests the involvement of these pathways in their neuroprotective and anti-apoptotic actions. Similarly, the potent antioxidant, EGCG, at low concentration decreased pro-apoptotic genes bax and caspase-6 supporting previous findings (10). However, a pro-apoptotic pattern of gene expression was observed at high concentrations of the antioxidants. A similar expression profile was obtained after exposure to R-APO, DA, and melatonin, including upregulation of pro-apoptotic caspases-3 and -10, tumor necrosis factor receptor fas and fas-ligand, NF-kB p105 subunit, and tumor suppressor protein p53 mRNAs. DA and R-APO displayed a greater homology of gene expression when compared with melatonin, suggesting that these drugs may share a similar mechanism in their cell death action. The wide range between the neuroprotective and the toxic concentrations of melatonin may be of critical importance in its pharmacotherapy, since it may provide a safer dosage window than R-APO and DA. One possible explanation for the high cell viability in the presence of high melatonin concentration may be that caspase-3 activation, per se, or its upstream/downstream effectors may not be indispensable for onset of apoptosis, as has been previously suggested (34). The observation that neither bcl-2 nor bcl-xL mRNA level was altered by high concentration of melatonin emphasizes their pivotal role in cell survival. This is in contrast to what was observed with high concentrations of R-APO, DA, and EGCG. Schematic overview suggesting potential gene targets involved in the pro-apoptotic and anti-apoptotic action of DA, R-APO, melatonin, and EGCG is described in Fig. 6.5. The gene expressions are associated with regulation of their proteins and these effects are concentration- and time-dependent. In vitro cell culture studies have suggested that the neurotransmitter DA serves as an endogenous neurotoxin, thereby participating in neurodegenerative processes in PD (35 –37). This assumption is based on observations that high concentrations (200 –1000 mM) of DA induce apoptosis in neuronal cell culture but not in vivo. They have implicated endogenous DA as a neurotoxic culprit in PD (38). In vivo however, the highly active intraneuronal and extraneuronal (cells such as glia, astrocytes) monoamine oxidase (MAO) never allows the build-up

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Figure 6.5 Schematic overview indicating potential gene targets involved in the antiapoptotic and pro-apoptotic action of low and high concentrations of antioxidants in NB SH-SY5Y cells. Solid arrows and dotted lines indicate induction and inhibition of gene expression, respectively.

of such high, nonphysiological concentration of intraneuronal or extraneuronal DA. Only when the brain MAO is inhibited, neuronal and synaptic DA levels increase. Selective and nonselective MAO inhibitors (tranylcypramine, selegiline, moclobemide, and rasagiline) (39) have been employed in the treatment of depressive illness and PD. There is no evidence for increased incidents of PD or neurodegeneration in depressive illness or that such treatment aggravates the neurodegeneration process in PD subjects. The present study has clearly shown that DA, at low concentrations, which is relatively closer to its physiological concentration, does not affect cell viability, but rather activates cell survival genes and their associated proteins. This supports the most recent view that low DA concentration may indeed have a beneficial effect on DA neuron plasticity (40). This assumption may be also relevant for the dopaminergic receptor agonist R-APO, since it may have a neuroprotective action in addition to its favorable symptomatic action in PD patients. The phenomenon described for DA may also apply to the action of other neurotransmitters and antioxidants. Implementation of cDNA microarrays represents an invaluable tool for the identification of gene alterations at the level of the mRNA, providing the possibility of assessing the simultaneous expression of thousands of genes at a specific time and conditions. As shown in the studies with antioxidant, the activities of these compounds to alter gene expressions are concentration- and time-dependent. Thus, they have a window of activities that need to be considered when being applied in vitro, cell culture, in vivo, and clinical studies. The global picture obtained with microarray and proteomic studies indicates a domino

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cascade of events in drug action and drug response in neurodegenerative diseases (41). This may explain why up to date single neuroprotective drug therapy has failed in clinical neuroprotection, and thus serious consideration needs to be given to their concentration-dependent action and multidrug cocktail, as currently implemented in other diseases such as cancer and cardiovascular diseases. Future in vivo studies with multiple antioxidant compounds as therapeutic agents will be aimed to prevent changes in mitochondrial membrane potential involving gene expression cascades associated with programed cell survival and cell death. It will address the questions whether combined antioxidant agents would have a synergistic action and can be more effective in clinical treatment of neurodegenerative diseases. CONCLUSION Significant evidence has been provided to support the hypothesis that OS and inflammatory processes trigger a cascade of events leading to apoptotic/necrotic cell death in neurodegenerative disorders such as PD, AD and Huntington’s disease, stroke, and amyotrophic lateral sclerosis. The novel therapeutic approaches aimed at neutralization of OS-induced neurotoxicity, support the application of reactive oxygen species scavengers, transition metals (iron and copper) chelators, and nonvitamin natural antioxidants, in monotherapy, or as part of antioxidant cocktail formulation for these diseases. Recent studies indicate that the radical scavenger property of antioxidant agents, such as DA, R-APO, green tea polyphenol EGCG, and melatonin, is unlikely to be the sole explanation to their neuroprotective effects in models of PD and AD, but a wide spectrum of cellular signaling events may also account for their biological actions. This article provides a new insight into the gene and protein mechanisms involved in both the neuroprotective and anti-apoptotic activities of antioxidant drugs, demonstrating a concentration- and time-dependent correlation between R-APO, DA, EGCG, and melatonin in modulation of cell survival/ cell death-related gene pathways. ACKNOWLEDGMENT The authors acknowledge the support of National Parkinson Foundation (Miami), Stein Foundation (Philadelphia) and Rappoport Family Research, TechnionIsrael Institute of Technology, and Friedman Fund for Parkinson’s Disease (Technion). REFERENCES 1. Halliwell B. Reactive oxygen species and the central nervous system. J Neurochem 1992; 59:1609 –1623. 2. Gotz ME, Kunig G, Riederer P, Youdim MBH. Oxidative stress: free radical production in neural degeneration. Pharmacol Ther 1994; 63:37 – 122.

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3. Jellinger KA. Cell death mechanisms in Parkinson’s disease. J Neural Transm 2000; 107:1– 29. 4. Saner A, Thoenen H. Model experiments on the molecular mechanism of action of 6-hydroxydopamine. Mol Pharmacol 1971; 7:147– 154. 5. Burns RS, Chiueh CC, Markey SP, Ebert MH, Jacobowitz DM, Kopin IJ. A primate model of parkinsonism: selective destruction of dopaminergic neurons in the pars compacta of the substantia nigra by N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Proc Natl Acad Sci USA 1983; 80:4546 – 4550. 6. Jellinger KA. The pathology of Parkinson’s disease. Adv Neurol 2001; 86:55– 72. 7. Gotz ME, Freyberger A, Riederer P. Oxidative stress: a role in the pathogenesis of Parkinson’s disease. J Neural Transm Suppl 1990; 29:241 –249. 8. Walkinshaw G, Waters CM. Neurotoxin-induced cell death in neuronal PC12 cells is mediated by induction of apoptosis. Neuroscience 1994; 63:975– 987. 9. Simantov R, Blinder E, Ratovitski T, Tauber M, Gabbay M, Porat S. Dopamineinduced apoptosis in human neuronal cells: inhibition by nucleic acids antisense to the dopamine transporter. Neuroscience 1996; 74:39– 50. 10. Levites Y, Amit T, Youdim MBH, Mandel S. Involvement of protein kinase C activation and cell survival/cell cycle genes in green tea polyphenol (2)-epigallocatechin-3-gallate neuroprotective action. J Biol Chem 2002; 277:30574– 30580. 11. Gassen M, Gross A, Youdim MBH. Antioxidant and Cytoprotective Properties of Apomorphine. New York: Plenum Press, 1998:421 –427. 12. Wolfler A, Caluba HC, Abuja PM, Dohr G, Schauenstein K, Liebmann PM. Prooxidant activity of melatonin promotes fas-induced cell death in human leukemic Jurkat cells. FEBS Lett 2001; 502:127– 131. 13. Walkinshaw G, Waters CM. Induction of apoptosis in catecholaminergic PC12 cells by L-DOPA. Implications for the treatment of Parkinson’s disease. J Clin Invest 1995; 95:2458– 2464. 14. Baas H, Harder S, Burklin F, Demisch L, Fischer PA. Pharmacodynamics of levodopa coadministered with apomorphine in Parkinsonian patients with end-of-dose motor fluctuations. Clin Neuropharmacol 1998; 21:86– 92. 15. Reiter RJ, Cabrera J, Sainz RM, Mayo JC, Manchester LC, Tan DX. Melatonin as a pharmacological agent against neuronal loss in experimental models of Huntington’s disease, Alzheimer’s disease and parkinsonism. Ann NY Acad Sci 1999; 890:471– 485. 16. Gassen M, Pinchasi B, Youdim MBH. Apomorphine is a potent radical scavenger and protects cultured pheochromocytoma cells from 6-OHDA and H2O2-induced cell death. Adv Pharmacol 1998; 42:320– 324. 17. Youdim MBH, Gassen M, Gross A, Mandel S, Grunblatt E. Iron chelating, antioxidant and cytoprotective properties of dopamine receptor agonist; apomorphine. J Neural Transm Suppl 2000; 83– 96. 18. Levites Y, Youdim MBH, Maor G, Mandel S. Attenuation of 6-hydroxydopamine (6-OHDA)-induced nuclear factor-kappaB (NF-kappaB) activation and cell death by tea extracts in neuronal cultures. Biochem Pharmacol 2002; 63:21 – 29. 19. Mayo JC, Sainz RM, Uria H, Antolin I, Esteban MM, Rodriguez C. Melatonin prevents apoptosis induced by 6-hydroxydopamine in neuronal cells: implications for Parkinson’s disease. J Pineal Res 1998; 24:179– 192. 20. Grunblatt E, Mandel S, Berkuzki T, Youdim MBH. Apomorphine protects against MPTP-induced neurotoxicity in mice. Mov Disord 1999; 14:612 –618.

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21. Grunblatt E, Mandel S, Maor G, Youdim MBH. Gene expression analysis in N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mice model of Parkinson’s disease using cDNA microarray: effect of R-apomorphine. J Neurochem 2001; 78:1 – 12. 22. Levites Y, Weinreb O, Maor G, Youdim MBH, Mandel S. Green tea polyphenol (2)-epigallocatechin-3-gallate prevents N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurodegeneration. J Neurochem 2001; 78:1073– 1082. 23. Antolin I, Mayo JC, Sainz RM, del Brio Mde L, Herrera F, Martin V, Rodriguez C. Protective effect of melatonin in a chronic experimental model of Parkinson’s disease. Brain Res 2002; 943:163 – 173. 24. Bernardi P, Petronilli V, Di Lisa F, Forte M. A mitochondrial perspective on cell death. Trends Biochem Sci 2001; 26:112 – 117. 25. Cory S, Adams JM. The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer 2002; 2:647 – 656. 26. Maruyama W, Takahashi T, Youdim MBH, Naoi M. The anti-Parkinson’s drug, rasagiline, prevents apoptotic DNA damage induced by peroxynitrite in human dopaminergic neuroblastoma SH-SY5Y cells. J Neural Transm 2002; 109:467 – 481. 27. Maruyama W, Akao Y, Youdim MBH, Davis BA, Naoi M. Transfection-enforced Bcl-2 overexpression and an anti-Parkinson’s drug, rasagiline, prevent nuclear accumulation of glyceraldehyde-3-phosphate dehydrogenase induced by an endogenous dopaminergic neurotoxin, N-methyl(R)salsolinol. J Neurochem 2001; 78:727 – 735. 28. Weinreb O, Mande S, Youdim MBH. cDNA gene expression profile homology of antioxidants and their anti-apoptotic and pro-apoptotic activities in human neuroblastoma cells. FASEB J 2003; 17:935 – 937. 29. Nicholson DW, Thornberry NA. Caspases: killer proteases. Trends Biochem Sci 1997; 22:299– 306. 30. Riederer P, Sofic E, Rausch WD, Schmidt B, Reynolds GP, Jellinger K, Youdim MBH. Transition metals, ferritin, glutathione, and ascorbic acid in parkinsonian brains. J Neurochem 1989; 52:515 – 520. 31. Youdim MBH, Grunblatt E, Levites-Royak Y, Mandel S. Drugs to prevent cell death in Parkinson’s disease: neuroprotection against oxidative stress and inflammatory gene expressions. Adv Neurol 2001; 86:115 – 125. 32. Olivieri G, Hess C, Savaskan E, Ly C, Meier F, Baysang G, Brockhaus M, Muller-Spahn F. Melatonin protects SH-SY5Y neuroblastoma cells from cobaltinduced oxidative stress, neurotoxicity, and increased beta-amyloid secretion. J Pineal Res 2001; 31:320 – 325. 33. Shen YX, Xu SY, Wei W, Wang XL, Wang H, Sun X. Melatonin blocks rat hippocampal neuronal apoptosis induced by amyloid beta-peptide 25 – 35. J Pineal Res 2002; 32:163– 167. 34. Cheng EH, Wei MC, Weiler S, Flavell RA, Mak TW, Lindsten T, Korsmeyer SJ. BCL-2, BCL-X(L) sequester BH-3 domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis. Mol Cell 2001; 8:705– 711. 35. Linert W, Herlinger E, Jameson RF, Kienzl E, Jellinger K, Youdim MBH. Dopamine, 6-hydroxydopamine, iron, and dioxygen—their mutual interactions and possible implication in the development of Parkinson’s disease. Biochim Biophys Acta 1996; 1316:160 –168. 36. Gerlach M, Double K, Riederer P, Hirsch E, Jellinger K, Jenner P, Trautwein A, Youdim MBH. Iron in the Parkinsonian substantia nigra. Mov Disord 1997; 12:258– 260.

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37. Offen D, ZivI I, Gorodin S, Barzilai A, Malik Z, Melamed E. Dopamine-induced programmed cell death in mouse thymocytes. Biochim Biophys Acta 1995; 1268:171 –177. 38. Barzilai A, Melamed E, Shirvan A. Is there a rationale for neuroprotection against dopamine toxicity in Parkinson’s disease? Cell Mol Neurobiol 2001; 21:215 –235. 39. Konradi C, Riederer P, Jellinger K, Denney R. Cellular action of MAO inhibitors. J Neural Transm Suppl 1987; 25:15 – 25. 40. Todd RD. Neural development is regulated by classical neurotransmitters: dopamine D2 receptor stimulation enhances neurite outgrowth. Biol Psychiatry 1992; 31:794– 807. 41. Mandel S, O Weinreb O, Youdim MBH. Using cDNA microarray to assess Parkinson’s disease models and the effects of neuroprotective drugs. Trends Pharmacol Sci 2003; 24:184 – 191.

7 Effects of Antioxidants on Gene Expression in Endothelial Cells B. A. Nier, B. A. Ewins, and S. G. Cremers University of Reading, Reading, UK

Peter D. Weinberg Imperial College, London, UK

Introduction Oxidants, Antioxidants, and NF-kB Mediated Gene Expression in the Endothelium Oxidants, Antioxidants, and AP-1 Mediated Gene Expression in the Endothelium Vitamin E and Endothelial Gene Expression Regulation of Transcription Through Changes in Chromatin Structure Applicability to Physiological Conditions Discussion Conclusion Acknowledgments References

141 143 148 152 157 160 164 167 167 167

INTRODUCTION Thirty years ago, the main roles of endothelial cells—the cells lining the inner surface of all blood vessels—were thought to be the largely passive ones of providing a nonthrombogenic surface and of keeping macromolecules within 141

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plasma, while allowing the exchange of smaller solutes with underlying tissue. Since then, however, endothelial cells have been shown to play an active and key role in a large number of physiological processes that are critical for vascular homeostasis (1). The explosion of interest in these cells has undoubtedly been fueled by their central involvement in the development of atherosclerosis. This disease underlies most myocardial infarctions and cerebrovascular accidents, and in many countries it constitutes the major cause of death. Several aspects of its pathogenesis remain to be established but there is currently a consensus that key events include the activation or perturbation of endothelial cells by various mechanical, chemical, or biological stimuli, entry of lipoproteins and inflammatory cells into the arterial wall, the migration of smooth muscle cells (SMC) into the arterial intima and their subsequent proliferation, and interactions between the atheromatous plaque and circulating platelets, all of which are strongly influenced by the endothelium (2). In parallel with the increasing interest in endothelial cells, there has been increasing interest in the proatherogenic role of oxidative stress and, consequently, in the possible atheroprotective role of dietary and other antioxidants. Initially, this interest resulted from the probable involvement of oxidized low density lipoprotein (LDL) in the development of the disease. A characteristic of atherosclerotic lesions is the presence of foam cells containing large numbers of cholesterol-rich lipid droplets within their cytoplasm. LDL is the major carrier of cholesterol in the human circulation, but its uptake by cells is tightly controlled: increasing uptake of cholesterol leads to downregulation of LDL receptors in a classical negative feedback loop (3). However, uptake of modified forms of LDL occurs by different routes, and is not regulated. It has been shown that LDL oxidation can occur in vivo, which oxidized LDL is present in lesions but rarely elsewhere, that oxidized LDL is taken up by scavenger receptors rather than LDL receptors, and that incubation of macrophages with oxidized LDL leads to the formation of foam cells (4). More recently, other potentially important roles of oxidative stress have emerged. These stem from a re-emphasis (5) of the role of inflammation in atherogenesis, a concept dating back at least as far as Virchow, and from the realization that oxidative stress, or reactive oxygen species (ROS), are potent pro-inflammatory stimuli in endothelial cells (6,7). Hence, the balance between ROS and dietary and other antioxidants might control the rate of atherogenesis through pro- and anti-inflammatory actions, as well as by modifying the rate of oxidation of LDL. Sources of ROS within cells include mitochondrial respiration, NAD(P)H oxidase, nitric oxide synthases (NOS), cyclo-oxygenases (COX), lipoxygenases, xanthine oxidase, cytochrome P-450 mono-oxygenase, heme oxygenases, peroxidases, and hemoproteins. There is evidence that NAD(P)H oxidases are the primary source in cultured endothelium. The main enzymatic defenses are superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) (6,7). Of course, effects of exogenous ROS and antioxidants must also be considered.

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This review concerns the roles of ROS and antioxidants in modifying expression of genes within endothelial cells. Although relatively novel, this subject is already a large one. The review takes a critical approach to the current orthodoxy, and it concentrates on (i) the control and actions of the two principal redox-sensitive transcription factors, Nuclear Factor kappa B (NF-kB), and Activator Protein-1 (AP-1), (ii) the effects of the most important lipid-soluble antioxidant in the diet, vitamin E, (iii) the possibility that oxidants and antioxidants might affect gene expression other than through influences on transcription factors, (iv) the likelihood that effects of oxidants and antioxidants observed in culture are modified in vivo, particularly by shear stresses, and (v) whether the putatively anti-atherogenic effects of antioxidants observed in vitro are supported by results from dietary intervention trials.

OXIDANTS, ANTIOXIDANTS, AND NF-kB MEDIATED GENE EXPRESSION IN THE ENDOTHELIUM The biology of NF-kB in endothelial cells, and its possible modification by dietary components, has attracted considerable attention because of accumulating evidence that this transcription factor might play a major role in atherogenesis. First, activated NF-kB is present in the atheromatous plaque—in SMC and macrophages as well as endothelial cells—but is absent in cells of the healthy arterial wall (8). Second, NF-kB is activated by an array of stimuli thought to play a key role in the development of atherosclerotic lesions, including tumor necrosis factor alpha (TNF-a), interleukin-1 (IL-1), bacterial lipopolysaccharide (LPS), advanced glycation end products (AGEs), hyperglycemia, platelet activating factor, shear stress, oxidized lipids, oxidant stress, and hypoxia/reperfusion (9). Third, NF-kB regulates genes responsible for key atherogenic proteins, including pro-inflammatory cytokines (TNF-a), chemokines such as monocyte chemoattractant protein-1 (MCP-1), cell adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and E-selectin, and inflammatory enzymes such as inducible nitric oxide synthase (10,11)—a more complete list is presented below. Consequently, regulation of the action of the transcription factor, or of related upstream and downstream signaling events, might inhibit the formation of lesions or aid in their regression. The normal regulation and activation pathways of NF-kB are considered first, with particular reference to studies in endothelial cells and to stimuli and genes relevant to atherosclerosis. NF-kB is a ubiquitously expressed protein (12). It belongs to a family of proteins that share a conserved central region of 300 amino acids called the Rel homology domain. Five members of the NF-kB family have been described: p50, p65 (RelA), c-Rel, RelB, and p52; p105 and p100 are the precursors of p50 and p52, respectively (12). NF-kB usually exists as a dimer. The classical form is a heterodimer composed of the p50 and p65 subunits.

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NF-kB exists in two states: in quiescent cells it is bound to an inhibitory protein, inhibitory kappa B (IkB), but it dissociates from IkB when activated (13,14). Binding to IkB keeps NF-kB in the cytoplasm and therefore incapable of acting upon its target genes. The Rel domain plays an important role in this interaction. Multiple mammalian forms of IkB exist. Those identified so far are IkB-a, -b, -g (p105), d (p100), and 1. [The C-terminal regions of the p50 and p52 precursors, p105 and p100, have IkB-like segments; free C-terminal segments are therefore termed IkB-g (p105) and IkB-d (p100).] Removal of IkB requires activation of IkB-a, -b, and -g kinases (IKK-a, -b, and -g, respectively). Their activation leads to phosphorylation of two specific serine residues near the N-terminus of IkB-a (Ser32 and Ser36), an event that can occur within minutes. IkB-b is phosphorylated on two equivalent serine residues (Ser19 and Ser23) by both IKK-a and IKK-b (15,16). Homologous sites are found at Ser157 and Ser161 in IkB-1 (17). Phosphorylation targets IkB for ubiquitination and thus rapid degradation by the 26S proteasome (18). The IKK belong to a larger, more complicated protein complex called the IKK signalosome. This complex includes IKK complex-associated protein (IKAP) and NF-kB essential modulator (NEMO, or IKK-g), which are essential for NF-kB activation (19,20). In the endothelium, the activation of IKK in the IKK signalosome, and the subsequent phosphorylation and degradation of IkB, is triggered by extracellular stimuli that lead to the activation of intermediate protein kinases such as mitogen-activated protein kinase (MAPK), protein kinase C (PKC), protein tyrosine kinase, NF-kB-inducing kinase (NIK) (21), and MAPK/ERK kinases 1, 2, and 3 (MEKK-1, -2, and -3) (22,23). When the NF-kB heterodimer is released by these processes, its nuclear localization sequence is unmasked and it rapidly enters the nucleus. Once it enters, it binds to recognition elements, called kB sites, in the enhancer and promoter regions of inducible genes, and regulates their expression. Since its discovery in 1986 (24), NF-kB has been shown to play a key role in the regulation of many genes, especially those related to inflammatory and immune responses. A list of NF-kB-regulated proteins that are relevant to the vasculature can be found in Table 7.1 and the activation process is summarized in Fig. 7.1. Interestingly, NF-kB can bind to kB elements in the promoter region of its own inhibitory protein, IkB-a. Hence, activated NF-kB levels are regulated by negative feedback. This autoregulatory system ensures a transient activation of NF-kB, with a subsequent return to a quiescent state. Positive feedback loops also exist, since NF-kB is upregulated by some of the products of the genes that contain NF-kB-binding domains, such as TNF-a. Much recent research has attempted to elucidate further the molecular mechanisms underlying activation of NF-kB. The majority of this research has centred on the p50/p65 heterodimer and its association with IkB-a as this appears to be the predominant form of NF-kB in mammalian cells. Although many compounds are known to activate NF-kB, it is unclear what unified mechanism, if any, explains how such a diverse array of stimuli can have the same

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Interleukins and growth factors Cytokine and cell adhesion receptors

Immunomodulatory Others

IL-1, IL-6, IL-8, TNF-a, G-CSF, M-CSF, GM-CSF, MIP1-k, MCP-1, RANTES E-selectin, ICAM-1, VCAM-1, MAdCAM-1, Lox-1, RAGE, A20, A1, XIAP, c-IAP1, c-IAP2 MHC-I, MHC-II, IRF-1 iNOS, COX-2, tissue factor, PLA2, IkBa, MnSOD, MMP-2, MMP-9

Note: Interleukin, IL; tumor necrosis factor alpha, TNFa; colony stimulating facor, CSF; granulocyte macrophage, GM; macrophage inflammatory protein-1a, MIP-1a; monocyte chemoattractant protein1, MCP-1; regulated upon activation normal T-cell expressed and secreted, RANTES; intercellular adhesion molecule-1, ICAM-1; vascular adhesion molecule-1, VCAM-1; mucosal addressin cellular adhesion molecule-1, MAdCAM-1; lipoxygenase, Lox; receptor for AGE, RAGE; x-linked inhibitor of apoptosis, XIAP; interferon regulatory factor, IRF; inducible nitric oxide synthase, iNOS; cyclooxygenase-2, COX-2; phospholipase A2, PLA2; inhibitory kappa B alpha, IkBa; superoxide dismutase, SOD; matrix metalloproteinase, MMP. Modified from De Martin et al. (25).

effect. It has been postulated that NF-kB is regulated by the redox status of the cell (26,27). This view led to NF-kB being termed “the redox sensor of the cell,” but recently met with controversy. Although pathways leading to the activation of NF-kB have been delineated, there have been few data to link ROS directly to these mechanisms. The first suggestion that ROS were involved in the modulation of NF-kB came in 1990, just 4 years after the discovery of the transcription factor (28). Shortly after, Schreck et al. put forward the hypothesis that diverse agents activated NF-kB through an increase in ROS and oxidative stress within the cell (29,30). This hypothesis was based on four main lines of evidence. First, NF-kB is activated in cells exposed to hydrogen peroxide (H2O2). Second, ROS are found in increased levels in cells treated with agents that activate NF-kB (30,31). Third, compounds with antioxidant properties are able to inhibit the activation of NF-kB (30). Fourth, modulation of molecules known to regulate intracellular levels of ROS, such as glutathione (32) and SOD (30), can affect the activation of NF-kB. These lines of evidence led to the widely accepted view that H2O2 acts as a second messenger in the activation of NF-kB. However, it is becoming clear that the role of H2O2, and perhaps of ROS in general, may have been overstated; its involvement in the activation of NF-kB is restricted to certain cell lines, and its role is well characterized only in lymphocytes (33,34). Thus, H2O2 is a potent activator of NF-kB in Wurzburg subclone of T cells, L6 skeletal muscle myotubes, human breast MCF-7 cells, and 70Z/3 pre-B cells (27,29,35). However, activation in a number of cell types have been shown to be completely insensitive to H2O2 . These include Jurkat cells (36), monocytic cell lines, EL4.NOB-1 T

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Figure 7.1 Activation pathway of NF-kB. Many external stimuli activate signal transduction pathways ultimately leading to the activation of the IKK signalosome. This in turn results in the phosphorylation of IkB, which is in turn polyubiquitinated by a specific E3 ligase. Ubiquitinated IkB is then sent for degradation by the 26S proteasome. (Mitogen activated protein kinase, MAPK; MAPK/ERK kinases 1, 2, and 3, MEKK-1, -2, and -3; protein kinase C, PKC; NF-kB-inducing kinase, NIK; transforming growth factor b-activated protein kinase 1, TAK-1.)

cells, KB epidermal cells, astrocytoma, J. Jhan lymphoblastoid T cells, and human umbilical vein endothelial cells (HUVEC) (37,38). It has already been mentioned that antioxidant compounds are able to inhibit the activation of NF-kB. This is one of the most compelling arguments for the involvement of ROS and oxidative stress. Several natural antioxidants, such as curcumin (39), gallic acid (40), quercetin (41), vitamin C (42), and vitamin E (43), as well as artificial compounds, have been reported to inhibit the cytokine-mediated activation of NF-kB in endothelial and non-endothelial cell lines. However, recent work suggests that they do not consistently do so, that they may act through diverse pathways, and that they may exert influences unrelated to their antioxidant properties. Examples are given as follows. Two nondietary antioxidants widely studied with regard to NF-kB activation are the glutathione precursor N-acetyl cysteine (NAC) and the thiol

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pyrrolidine dithiocarbamate (PDTC). Both have been reported to inhibit activation of NF-kB in many cell types (29,30,36,44). Indeed it was NAC, an antioxidant that can increase intracellular levels of glutathione and therefore directly scavenge ROS, that was used in the initial experiments of Schreck et al. which led to the redox-regulation hypothesis. However, there are now numerous reports of NAC- and PDTC-insensitive pathways of NF-kB regulation; these demonstrate that the effect is not only cell specific, but also stimulus specific within the same cell type (36,38,45). (Possible non-redox mediated effects of thiol antioxidants such as PDTC are considered in the following section.) Different antioxidants often inhibit activation of NF-kB through different mechanisms. The inhibition of cytokine-mediated NF-kB activation by salicylates and by curcumin results from the inhibition of IkB phosphorylation and degradation (39,46). IKK-a and IKK-b are inhibited by these antioxidants at the phosphorylation level. These antioxidants also inhibit the activation of additional kinases, such as MEKK-1, resulting in the inhibition of other downstream transcription factors such as Fos and Jun. It can be therefore be postulated that these antioxidants act on early steps in the signaling cascade, although their specific target has not yet been identified. Conversely, caffeic acid phenethyl ester and gallates reduce NF-kB translocation and binding without influencing IkB-a degradation (40,47). Similarly, Schubert et al. (48) have shown that NAC and pomegranate wine both have antioxidant effects in TNF-a-stimulated bovine aortic endothelial cells (BAEC) and both inhibit the activation and nuclear translocation of NF-kB, but do not do so by reducing Ser32 phosphorylation of IkB-a, or IkB-a degradation (which depends on such phosphorylation). Differences between NAC and pomegranate wine were also observed by Schubert et al. (48). In the TNF-a-stimulated BAEC, NAC reduced p65 Ser536 phosphorylation, a recently postulated step in the cascade of events leading to the activation of NF-kB (49), but the wine did not. Conversely, subsequent de novo synthesis of IkB was inhibited by the wine but not by NAC. Other pathways by which antioxidants inhibit NF-kB activation include S-nitrosylation of cysteine residues on NF-kB subunits, which interferes with their binding to DNA but not with nuclear translocation (50,51). The most important dietary antioxidant that has been studied in detail with respect to NF-kB and the endothelium is vitamin C. Vitamin C inhibits NF-kB activation in the human endothelial cell line ECV304 and in HUVEC. However, the effect may be unrelated to its antioxidant activity (42). HUVEC were activated through multiple pathways using an array of stimuli including IL-1 and TNF-a, whereas H2O2 was used to stimulate the ECV304 cells. The inhibitory effects were independent of both the cell type and the stimulus used, something not seen with either NAC or PDTC. In particular, vitamin C inhibited NF-kB when the latter was activated through pathways previously shown to be insensitive to other antioxidants, such as those stimulated by IL-1 (38).

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Furthermore, the effect of vitamin C was not potentiated by co-incubation with vitamin E, even though vitamins C and E are known to have a cooperative effect (42). Experiments such as these provide evidence against the general hypothesis that ROS are central to the activation of NF-kB in endothelial cells. If NF-kB were universally controlled by ROS, less diversity in the inhibitory effects of antioxidants would be expected. Nevertheless, they do suggest that dietary antioxidants might exert an atheroprotective effect by inhibiting its activation. Possible additional modes of action that require further investigation are effects on the IKK complex, on the ubiquitination and degradation of IkB, on NF-kB nuclear import/export, and on its interactions with DNA. OXIDANTS, ANTIOXIDANTS, AND AP-1 MEDIATED GENE EXPRESSION IN THE ENDOTHELIUM Although NF-kB has been shown to play a pivotal role in EC activation (8,52,53), increasing evidence indicates the importance of the transcription factor AP-1 in endothelial homeostasis and dysfunction. AP-1 can be either a homo- or heterodimer, involving members of the c-Jun (c-Jun, Jun B, and Jun D) and c-Fos (c-Fos, Fra-1, Fra-2, and Fos B) protooncogene families (54). The AP-1 subunits contain the basic-leucine zipper (bZIP) motif, where the leucine zipper mediates dimerization and the basic region is directly involved in DNA binding. Owing to the biochemical properties of Fos and Jun leucine zippers, all of the Jun protein family can form homo- and heterodimers, which are capable of binding to AP-1 DNA binding sites. Fosproteins do not associate with each other, but are capable of associating with any member of the Jun family to form stable heterodimers that have higher DNA-binding activity than Jun –Jun homodimers (55). Jun and Fos can also form heterodimers with other bZIP transcription factors, such as members of the cyclic adenosine monophosphate response element binding protein (CREB) and activating transcription factor (ATF) family to form an AP-1 complex (56,57). Jun – Jun and Jun –Fos forms of AP-1 bind to a specific DNA sequence, referred to as the 12-O-tetradecanoyl-13-phorbol acetate (TPA) response element (TRE). This sequence is present in the promoters of many inducible genes implicated in cell proliferation, differentiation, inflammation, and stress response (58), including genes for ICAM-1, VCAM-1 (59), E-selectin (60) MCP-1 (61), and tissue factor (TF) (62,63). c-Jun and c-Fos proteins also contain several transcriptionally active regions distinct from the leucine zipper and DNA basic region, including several autonomous transactivation domains, a carboxyl-terminal transrepression domain (c-Fos), and a region that interacts with the TATA box-binding protein (64,65). These domains in Fos and Jun function additively and perhaps cooperatively in transcriptional regulation. Furthermore, binding by Jun homodimers leads to DNA binding that is distinct from

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binding by Fos– Jun heterodimers, which may result in highly specific protein – protein interactions between AP-1 factors and other promoter-bound transcription complexes (65). Therefore, the distinct AP-1 proteins can exhibit distinct transcriptional properties, and the composition of the AP-1 complex may be critical to its regulatory function with diverse biological consequences. The activity of AP-1 can be controlled by transcriptional mechanisms, leading to increased amounts of AP-1 protein, and by posttranslational mechanisms, acting on pre-existing AP-1 proteins, in response to a variety of extracellular stimuli, including mitogens, phorbol esters, and differentiation signals. Expression of the various AP-1 dimers is differentially regulated temporally during cell cycle progression and in response to many stimuli (66). The promoter for the c-Jun gene itself contains a TRE region, so it can be activated by AP-1 in a positive autoregulatory way (67,68). The c-Fos promoter lacks the TRE region and thus is not subject to this type of autoregulation. The transcriptional and posttranslational regulators of AP-1 activity are in turn regulated through different signaling cascades, thereby explaining the versatility of AP-1 in responding to a broad spectrum of stimuli (68 –70). In various cell types, three different MAPK cascades are known to be involved in the induction of AP-1 activity. These are extracellular signal-regulated kinases (ERK-1 and -2), c-Jun-N-terminal protein kinases (JNKs)—also known as stressactivated protein kinases (SAPKs)—and p38 kinase cascades (69,70) (Fig. 7.2). In general, the ERK pathway is strongly activated by growth factors and cytokines. Its activation is related to the stimulation of upstream tyrosine kinase receptors, which start a signaling cascade involving Ras activation, recruitment of Raf kinase to the plasma membrane, and sequential activation/ phosphorylation of MAPK/ERK kinase 1, 2 (MEK-1, -2) and ERK-1 and -2. The major regulatory element of the c-Fos promotor, the serum response element (SRE), is recognized by the ternary complex factor (TCF) member Elk-1 and the dimeric serum responsive factor (SRF). When Elk-1 is phosphorylated by ERK-1 and -2, it combines with SRF to form a ternary complex with SRE which leads to the transcriptional induction of c-Fos (71). JNK and p38 cascades are only weakly activated by mitogens but are highly stimulated by exposure to pro-inflammatory cytokines such as TNFalpha and IL-1 and a wide variety of environmental stress inducers such as LPS. JNKs are phosphorylated by SAPK/ERK-1 (SEK1), also known as MAPK kinase 4 (MKK4), which in turn is activated through phosphorylation by MEKK-1. The activation/phosphorylation of MKK3/6 leads to the activation of p38 (69,72). These cascades can also be activated by members of the Ras superfamily of small GTPases, such as Rac and Cdc42, two Rho-like proteins that in the case of the JNK cascade have been reported to act synergistically with Ras. Ras therefore appears to be an alternative upstream component of this pathway (69,73). JNK and p38 cascades are connected with the activation of AP-1 in a similar fashion to the ERK cascade described earlier. Two members of the TCF family, Elk-1 and Sap-1a, are substrates of JNK in activated

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Figure 7.2 Regulation of AP-1 by extracellular stimuli. Extracellular signals received by membrane receptors are transduced into the nucleolus through three different MAPK subgroups ERK 1/2, JNKs, and p38. The transcription factors ELK-1, Sap-1a, c-Jun, and ATF-2 couple the activation of the different MAPKs with the transcriptional activation of c-Fos and c-Jun promoters. (ERK, extracellular signal-regulated kinase; JNK, c-JunN-terminal protein kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK/ ERK kinase; MEKK, MEK kinase; MKK, MAPK kinase; SEK, stress-activated protein kinase (SAPK)/ERK; SRE, serum response element; SRF, serum responsive factor; TCF, ternary complex factor; TRE, TPA (12-O-tetradecanoyl-13-phorbol acetate) responsive element.)

cells, and p38 has been reported to phosphorylate Elk-1 (69,71 – 74). Therefore, the three pathways converge at the TCF level and are potential regulators of the c-Fos gene through the transcriptional activation of the SRE. However, ATF-2 and c-Jun are also substrates of JNK, and p38 has been reported to phosphorylate ATF-2 (69,71,72). Thus, JNK and p38 are also involved in the transcriptional and posttranslational regulation of c-Jun and/or ATF-2. AP-1-binding activity is regulated in vitro by the redox status of a single conserved cysteine residue in the DNA-binding domains of Jun and Fos, which

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has to be in the reduced state for DNA binding to occur (75,76). The nuclear redox factor Ref-1, which was initially identified in HeLa nuclear extracts, was shown to stimulate DNA binding of AP-1 in vivo via reduction of the conserved cysteine residues (77). Ref-1 activity is itself regulated by a redox mechanism involving Thioredoxin (TRX), another pleiotropic cellular factor with thiolmediated redox activity that facilitates protein –nucleic acid interactions. TRX enhances DNA-binding activity of Jun and Fos via direct association with Ref-1, acting as a hydrogen donor (78). Although not formally demonstrated in endothelial cells, the ubiquitous nature of Ref-1 and TRX makes it likely that this redox cascade may modulate AP-1 activity in these cells. In several cell types, AP-1 behaves as a redox-sensitive transcription factor which is activated, to different extents, under pro-oxidant conditions generated by treatment with agents such as H2O2 , UV light, g-radiation, or various cytokines (55,68). In endothelial cells, agents such as H2O2 (79), oxidized LDL (80), and native LDL (81), which has been demonstrated to increase the amount of free radicals in ECs (82), have been reported to activate AP-1 DNA-binding activity. Regulation of the endothelial expression of genes like MCP-1 and ICAM-1 by H2O2 is mediated through AP-1-binding elements in the promoters of these genes (83,84). Overexpression of AP-1 components suggested that AP-1 itself is sufficient to induce ICAM-1 and MCP-1 genes in endothelial cells and that AP-1-induced gene expression is mediated through a mechanism independent of NF-kB (85). Induction of VCAM-1 by LDL through activation of the VCAM-1 promoter is concomitant with increased AP-1-binding activity (81). Recently, Wang et al. (86) demonstrated for the first time that the AP-1 signaling pathway and its cognate binding motif are of major importance for ICAM-1 expression in endothelial cells activated by LDL. Stimulation of cultured HUVEC with leptin, which is concomitant with increased intracellular accumulation of ROS, led to enhanced AP-1 DNAbinding activity and enhanced expression of MCP-1 (87). The pathways of ROS-mediated AP-1 activation and involvement of different MAPKs are less well characterized in endothelial cells than in other cell types. Laminar shear stress on endothelial cells is associated with peroxynitritedependent activation of JNK (88). Chen et al. (89) found that AP-1 activation by H2O2 in porcine aortic endothelial cells was mediated by the activation of the JNK pathway. This JNK activation by H2O2 was in turn mediated by Src-dependent epidermal growth factor (EGF) receptor transactivation without involvement of PKC. Activation of AP-1 by LDL in human endothelial cells was shown to involve the JNK-c-Jun and the p38-ATF-2 pathways through Ras activation (90), but not the ERK/c-Fos pathway (90,91). Bouloumie et al. (87) showed that leptin activated the JNK pathway, as demonstrated by enhanced JNK activity and AP-1 binding. Unlike NF-kB, AP-1 is activated not only by oxidants but also, paradoxically, by a number of antioxidants, including dithiocarbamates, the antioxidant enzyme TRX, and NAC. These have been shown to stimulate the DNA

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binding and transcriptional activity of AP-1 in several cell types (92). In endothelial cells, ICAM-1 expression induced by PDTC correlated with increased AP-1 binding to the PDTC-responsive region of the ICAM-1 promoter (93). PDTC was also shown to augment cytokine-induced AP-1 activation and ICAM-induction (94). On the other hand, antioxidants have also been shown to block AP-1 transcriptional activity in the endothelium. Shau et al. (95) demonstrated that the antioxidant enzyme TRX peroxidase-1 blocked AP-1 activation induced by TNFa. Similarly, NAC and catalase prevented the cyclic strain- or H2O2induced AP-1 binding to an AP-1 element in the MCP-1 promoter and MCP-1 expression (84). Enhanced AP-1-binding activity and expression of MCP-1 in leptin-activated HUVEC was also abolished by NAC (87). The mechanisms by which certain kinds of antioxidants selectively activate AP-1 in endothelial cells are poorly understood. Recently, JNK and ERK pathways were revealed as upstream regulatory mechanisms in PDTC-induced c-Jun and c-Fos activation, with concomitant ICAM-induction, in endothelial cells (94). Whereas JNK and c-Jun activation was sustained, upregulation of ERK and c-Fos was only transient. The thiol-antioxidant PDTC may activate AP-1 through pro-oxidant effects since it is known to act as a copper ionophore (96 –98). Recently Kim et al. (99) demonstrated in bovine cerebral endothelial cells (BCEC) that the activation of AP-1 and suppression of NF-kB by PDCT was mediated by zinc. This idea was based on the observation that PDTC treatment enhances zinc influx into BCEC (99). Activation of AP-1 and inhibition of NF-kB could be blocked by zinc chelation with Ca2þ-EDTA, but not by Zn2þ-EDTA. Zinc sulfate mimicked the action of PDTC in activating AP-1 and inhibiting NF-kB, and depleted the cellular glutathione store in a manner that could be reversed by thiol antioxidants like NAC but not nonthiol antioxidants like Trolox, ascorbic acid, and butylated hydroxyanisole. Finally, thiol antioxidants, but not nonthiol antioxidants, could reverse activation by zinc of AP-1 and inhibition of NF-kB (100). These results suggest that thiol antioxidants prevent the reciprocal actions of PDTC on AP-1 and NF-kB by acting as metal-chelators, rather than by scavenging oxygen free radicals or replenishing the cellular glutathione content. Thus, antioxidant-mediated AP-1 activation and gene expression remain a complex field, and further studies will be necessary to reveal the diverse mechanisms. As only effects of synthetic antioxidants on AP-1 activation have so far received extensive study, investigation of nutritionally relevant antioxidants would be of great interest. VITAMIN E AND ENDOTHELIAL GENE EXPRESSION Vitamin E is the most important lipid-soluble antioxidant in the diet. It consists of a group of tocopherol and tocotrienol compounds that have in common a 6-chromanol ring coupled to a C16 aliphatic side chain. For tocopherols, this

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side chain is saturated, whereas for tocotrienols, it contains three double bonds. The tocopherols (a, b, g, and d) are subdivided according to the number and position of methyl groups—1, 2, or 3—on the phenolic ring. The antioxidant properties are conferred by a hydroxyl group, also on the phenolic ring. The ability of this group to donate an H atom is dependent on the number of methyl groups. a-Tocopherol, with three methyl groups, is thought to be most potent. Antioxidant effectiveness in vivo also depends on factors other than the number of methyl groups. For example, the mobility of the molecule within lipid layers, which depends on the nature of the aliphatic side chain, seems important (101,102). Additionally, bioavailability may differ between homologs. Vitamin E circulates in the blood within lipoprotein particles. It is transferred from the gut to the liver in chylomicrons, but in hepatocytes is transferred into very low density lipoprotein by a process that involves a cytosolic a-tocopherol transfer protein (a-TTP). Circulating and tissue vitamin levels are therefore determined by the affinities of a-TTP for the different tocopherols. For the b-, g-, and d-forms, affinities are 38%, 9%, and 2% that for a-tocopherol, respectively (103). Finally, effectiveness is also determined by the presence of other antioxidants. In particular, ascorbate can regenerate vitamin E from the tocopheroxyl radical that is formed when vitamin E donates an H atom (104). The possibility that vitamin E might reduce the oxidation of LDL and hence slow atherogenesis has motivated a large number of in vitro and in vivo studies. However, in addition to the inhibition of such oxidation, vitamin E has been identified as a favorable modulator of many other processes at the molecular and cellular levels that may be of importance in atherogenesis (Table 7.2). Several of these potentially beneficial effects may be mediated through influences of the vitamin on gene expression. Thus, for example, the expression of CD36, a-TTP, a-tropomyosin, and collagenase are affected by a-tocopherol at

Table 7.2 Potential Mechanisms by Which Vitamin E Inhibits Atherosclerosis # # # # # # # " "

LDL oxidation, # macrophage uptake of oxLDL (105) Endothelial cell injury (106 –110) Adhesion molecule expression (107,109,111) Immune/endothelial cell adhesion (107,112) Inflammatory cytokines and chemokines (109) Smooth muscle cell proliferation (113– 115) Platelet aggregation (116,117) NO production, " arterial dilatation (118,119) PGI2 , # TXA2 (116)

Note: Low density lipoprotein, LDL; oxidized low density lipoprotein, oxLDL; nitric oxide, NO; prostacyclin, PGI2; thromboxane A2 , TXA2 .

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the transcriptional level (120). The effects of vitamin E on expression of inflammatory genes has received particular attention. It inhibits the production of cytokines (IL-1b, IL-6, IL-8) and suppresses the expression of adhesion molecules (ICAM-1, VCAM-1, E-selectin) and chemoattractants (MCP-1) in endothelial cells, and reduces their adhesive interaction with monocytes. Table 7.3 gives further details of these studies of protein (and, where indicated, mRNA) expression. Although there is strong evidence for effects of vitamin E, the mechanisms involved are less clear. The authors of many of these studies speculate that the vitamin is altering gene expression by inhibiting NF-kB activation. However, the evidence for this in endothelial cells is exceptionally limited. Of the studies cited, only that by Islam et al. (123) showed, by gel shift assay, that NF-kB activation actually decreased. Several studies have shown that vitamin E modulates PKC activity. This could explain any influence on NF-kB that does occur, since PKC affects the IKK signalosome (see earlier). There is considerable recent evidence for a direct effect of PKC on NF-kB activity. Ogata et al. (128), for example, have demonstrated that ROS-induced NF-kB activation in human endothelial cells was abolished by PKC inhibitors. The influence of vitamin E on PKC was first demonstrated by Boscoboinik et al. (129) in SMC, where the vitamin inhibits proliferation induced by various stimuli. The effect, which occurs at physiological vitamin E levels, is not limited to this cell type or response, however. For example, vitamin E inhibits platelet aggregation by almost complete abrogation of the phosporylation of a 47 kDa PKC substrate (130). Consistent with this, it causes a delay in intra-arterial thrombus formation (131). In endothelial cells, vitamin E exerts an inhibitory action on thrombin-induced PKC activation and endothelin secretion (132). A putative mechanism for the influence of vitamin E on PKC is the activation of protein phosphatase 2A (PP2A) (115,133,134), which causes the dephosphorylation and hence inactivation of PKCa (115). The expression of PKC is not affected. b-Tocopherol or Trolox, which have antioxidant capacity similar to a-tocopherol, do not exert such an effect, showing that a-tocopherol inhibits PKC activity in a specific manner (135). The phenolic hydroxyl group on the chromanol ring does not appear to be involved. Freedman and Keaney (130) found that blocking this group with an acetate ester does not alter the inhibition of PKC. Furthermore, b-tocopherol (which has the same hydroxyl group) does not appear to be active (129). At least one study suggests that vitamin E modifies gene expression independently of its antioxidant function and of its effect on PKC. Faruqi et al. (121) demonstrated that vitamin E inhibits monocyte adhesion to the endothelium induced with phorbol 12-myristate 13-acetate (PMA, a well-established activator of PKC) and thrombin. However, it did not inhibit phosphorylation of the myristoylated alanine-rich C kinase substrate protein, which is specifically phosphorylated by PKC and is a good indicator of PKC activity. In addition, it did not

HUVEC

Monocytes from healthy humans, HUVEC HUVEC

HAEC HUVEC, U937

Normocholesterolemic rabbits

HAEC, U937

Devaraj et al. (107)

Cominacini et al. (122)

Martin et al. (61) Islam et al. (123)

Fruebis et al. (111)

Wu et al. (109)

Cell type/Animal

Experiment

Vitamin E (0.1%), Probucol (low: 0.04– 0.075%, high: 0.5%) 1. Vitamin E (20, 40, 60 mmol/L) 2. Cells stimulated with IL-1, vitamin E (20, 40, 60 mmol/L)

a-Tocopherol (cells pretreated); stimulaton with IL-1, thrombin, PMA a-Tocopherol for 8 weeks (1200 IU/d) 1. Pretreating cells with vitamin E 2. Pretreating LDL with vitamin E (5 mM before oxidation) and probucol (incorporation of antioxidants into lipoproteins) Incubated with LDL, vitamin E a-Tocopherol (25, 50, 100 mM)

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Faruqi et al. (121)

Authors

Table 7.3

(continued )

1. IL-8 # 2. ICAM-1 #, VCAM-1 #, E-selectin #, MCP-1 #, IL-8 # (at 40 and 60 mmol/L), IL-6 # (at 40 and 60 mmol/L)

sICAM-1 #, Prostacyclin I2 " CD11b #, VLA-4 # , reduced agonistinduced adhesion to HUVEC VCAM-1 (mRNA, Protein) #

IL-1b #, monocyte-endothelial cell adhesion # 1. VCAM-1 #, ICAM-1 #, E-selectin 2. VCAM-1 #, ICAM-1 #, E-selectin

E-selectin # (mRNA and surface protein expression)

Genes/Proteins

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Vitamin E (400 IU/d or 800 IU/d) Treatment with LDL, pretreatment with vitamin E Cells activated with IL-1b, vitamin E Vitamin E (control: 40 mg/kg diet, suppl. group: 800 mg/kg diet)

Experiment

MCP-1 # (mRNA and protein)

MCP-1 #

sVCAM-1 # ICAM-1 #, VCAM-1 #

Genes/Proteins

Note: Endothelial cells, EC; human umbilical vein endothelial cells, HUVEC; human aortic endothelial cells, HAEC; human monocyte cell line, U937; phorbol 12-myristate 13-acetate, PMA; integrin alpha M or Mac-1 alpha (leukocyte) CD11b; very late antigen-4 (expressed by leukocytes), VLA-4; apolipoprotein E, Apo E; interleukin, IL; monocyte chemoattractant protein-1, MCP-1; intracellular adhesion molecule-1, ICAM-1; vascular cell adhesion molecule-1, VCAM-1; E-selectin, cell adhesion molecule; nuclear transcription factor-kappa B, NF-kB; protein kinase C, PKC; low density lipoprotein, LDL; real time polymerase chain reaction, rtPCR.

Apo E knockout mice

Human EC

Zapolska-Downar et al. (126) Peluzio et al. (127)

Cell type/Animal

Hypercholesterolemic patients EC

Continued

Desideri et al. (124) Yoshida et al. (125)

Authors

Table 7.3

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affect the activation of NF-kB at vitamin concentrations that caused marked decreases in E-selectin mRNA and protein expression. Consistent with this result are studies showing an absence of effect of vitamin E on NF-kB in nonendothelial cells. Suzuki and Packer (43) showed that although vitamin E acetate and a-tocopheryl succinate dose-dependently inhibit TNF-a-induced NF-kB activation in Jurkat cells, a-tocopherol does not. Similarly, Nakamura et al. (136) found that pretreatment with 50 mM a-tocopheryl succinate produced a 43% inhibition of NF-kB binding to DNA when cells of the human macrophage cell line THP-1 were activated with LPS, but a-tocopherol itself was without effect. Thus although it seems clear that vitamin E affects gene expression, the transcription factors responsible have not been clearly identified. Whether or not NF-kB is involved, perhaps through the influence of vitamin E on PKC, there is evidence that the vitamin is not acting through an antioxidant effect. REGULATION OF TRANSCRIPTION THROUGH CHANGES IN CHROMATIN STRUCTURE Changes in gene expression are mediated primarily through the action of transcription factors. However, activation or inhibition of the transcription factors themselves, discussed in the preceding sections, are not the only ways in which their action is controlled; modification of chromatin structure affects whether transcription factors are able to influence gene expression. Chromatin is a filamentous complex of DNA, histones (the principal protein), and other proteins, which are predominantly transcription factors. Some is tightly coiled (“heterochromatin”) and transcriptionally inert, whereas the remainder (“euchromatin”) is transcriptionally active in places. DNA is divided between nucleosomes and linker segments. In the nucleosome, a DNA segment 146 bp long is wrapped around an octomer of histones (two each of types H2A, H2B, H3, and H4). Linker regions of DNA having variable length separate the nucleosomes; the nucleosomes and the linker DNA are associated with histone H1. Structural modifications of the histones and of the DNA itself have been implicated in the control of gene expression. Such modifications are enzymatically controlled, and can reflect dynamic changes in cellular environment. However, they can also be heritable (mitosis and meiosis). This leads to the fascinating possibility that environmental effects in one generation may influence gene expression for several generations. Because the structural changes do not modify the genome sequence itself, they have been termed “epigenetic.” Electrostatic interactions between histones and DNA are regulated by acetylation, methylation, and phosphorylation of the histones. Broadly speaking, acetylation of histones allows transcriptional machinery to access DNA, whereas deacetylation and methylation block it, preventing transcription factors from recognizing their response elements. Histone phosphorylation also seems to play a role. Chromatin remodeling complexes aid these processes by continually

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shuffling the positions of individual nucleosomes, which are transcriptionally inactive, to expose random sequences for short periods (137,138). Thus, an interplay exists between chromatin remodeling and histone modification. For example, expression of a gene may require disruption of nucleosomes positioned at the promoter region by a chromatin remodeling complex before an enzyme required for histone acetylation can be recruited (139). Alternatively, expression of genes may require that histone acetylating enzymes and even RNA polymerase bind to the promotor prior to recruitment of the chromatin remodeling complex (140). Specific histone modifications and chromatin remodeling complexes have also been implicated in silencing at some loci (137). DNA methylation is the addition of methyl groups, after replication, to the 50 position of cytosine rings within guanine – cytosine (CpG) dinucleotides. This results in alterations in the structure of DNA—specifically to the conformation of the groove—and hence alters the binding of proteins to it (141). For example, methylated CpG dinucleotide sites near a gene can recruit specific DNAbinding proteins, which in turn recruit histone deacetylases, resulting in loss of histone acetylation and silencing of gene expression (142). Approximately 70% of CpG pairs in the mammalian genome are constitutively methylated (143). There are short DNA sequences, however, with an unusally high guaninephosphate – cytosine content and a high frequency of unmethylated CpG dinucleotides (144). These regions, called CpG islands, are primarily located in 50 regulatory regions. They are present in all housekeeping and most tissue-specific active genes (145) and have also been proposed to function as replication origins (146). Table 7.4 lists genes of importance in atherogenesis which are known to be at least partially regulated by DNA methylation.

Table 7.4 Examples of Genes Implicated in Atherogenesis Which Are At Least Partially Regulated by DNA Methylation Gene

Name

Reference

IFN-g PDGF-A MMP-2 MMP-7 MMP-9 TIMP-3 ICAM-1 p53

Interferon-g Plateled-derived growth factor Matrix metalloproteinase-2 Matrix metalloproteinase-7 Matrix metalloproteinase-9 Tissue inhibitor of metalloproteinase Intracellular adhesion molecule Estrogen receptor

ERa

Extracellular superoxide dismutase

EC-SOD

Tumor suppressor

White et al. (147) Lin et al. (148) Sato et al. (149) Sato et al. (149) Sato et al. (149) Wild et al. (150) Tanaka et al. (151) Schroeder and Mass (152), Guevara et al. (153) Post et al. (154), Ying et al. (155) Laukkanen et al. (156)

Source: From Hiltunen and Yla-Herttuala (157).

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Direct evidence for the methylation of genes in atherogenesis is emerging. To date, the evidence is strongest for the estrogen receptor a (ERa). This receptor is activated by estrogen and regulates a variety of cellular activities of potential importance in the disease. For example, activated ERa increases the expression of eNOS, resulting in enhanced nitric oxide (NO) production that, in turn, is known to inhibit SMC proliferation, platelet aggregation, leukocyte adhesion, etc. Post et al. (154) found increased methylation of CpG islands in the ERa gene in atherosclerotic human tissue compared with normal arterial tissue. Furthermore, evidence for ERa gene methylation was obtained in contractile vascular smooth muscle (VSMC) but not in proliferative VSMC in culture, indicating that methylation had occurred during the phenotypic switch (155). However, human aortic endothelial cells did not show differential methylation of ERa in culture. More limited evidence is available for extracellular superoxide dismutase (EC-SOD), which protects arteries against deleterious effects of superoxide anions and the development of atherosclerosis. Laukkanen et al. (156) cloned and characterized the rabbit EC-SOD gene and showed it had a marked reduction in the amount of methylated CpG dinucleotides in atherosclerotic arteries compared with normal arteries, perhaps indicating some form of compensatory upregulation of gene expression. However, the methylation was in the coding region, and it is not established that such modification alters transcription. There is an intriguing suggestion that DNA methylation may explain why age is a risk factor for atherosclerosis (158). A number of studies have demonstrated an increased methylation of specific genes, and a global drop in methylation, with age. Consistent with this, the study by Post et al. (154) found decreased methylation of the ERa gene with age in cardiac mycocytes. As with aging, experimental atherosclerosis was found to decrease global methylation in rabbit aortas (156). There is also a link with hyperhomocysteinaemia, which causes oxidative stress (159), and is another risk factor for atherosclerosis. Homocysteine can be methylated to methionine, which in turn is metabolized to products involved in the methylation of DNA. One of the methyl donors for the conversion of homocysteine to methionine is produced by the action of the enzyme methylenetetrahydrofolate reductase. Mice deficient in this enzyme have elevated homocysteine, global hypomethylation of DNA, and aortic lipid deposition (160,161). To date, there is no evidence for epigenetic modification by dietary antioxidants in endothelial cells. We speculate that such evidence will be obtained, however, because a recent study has demonstrated that shear stress does lead to modifications of this type; as explained in the following section, there is a strong link between shear stress and oxidative stress. Illi et al. (162) have demonstrated that shear stress induces histone H3 acetylation and phosphorylation and cooperates with the histone deacetylase inhibitor trichostatin to enhance histone H3 phosphoacetylation and histone H4 acetylation. These histone modifications,

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which lead to chromatin remodeling and DNA unwinding, may play a fundamental role in the shear stress-dependent regulation of gene expression.

APPLICABILITY TO PHYSIOLOGICAL CONDITIONS The data discussed in the preceding sections have been obtained almost exclusively by using endothelial cells in static culture. The use of cell culture, and the absence of cyclic strain and shear stress (which are present in vivo as a result, respectively, of pulsatile pressure and blood flow), could have had a large influence on the results. In culture, it is conventional to incubate cells under a gas mixture of 95% air/5% CO2 . This gives an oxygen partial pressure of 150 mm Hg, whereas the endothelium will be exposed to a partial pressure in systemic arteries—the site of atherosclerosis—of 105 mm Hg and in veins to 40 mm Hg or less, depending on tissue activity. The hyperoxic culture environment has definite effects on cell biology. Cells grown under these conditions (20% O2) are preadapted and preselected to survive oxidative stress (and have increased accumulation of oxidant damage compared with cells grown at 3% oxygen) (163). The adaptation appears to involve upregulation of heat shock protein (164) and other genes. Halliwell (165) has recently reviewed the culture artifacts likely to affect investigations of ROS-mediated effects on cells. First, the high oxygen tension will lead to elevated leakage of ROS from the electron transport chain. Then the media themselves can be pro-oxidant, since they may contain iron or copper ions, and can generate further ROS from substances added to them (including ascorbate, polyphenolics, and thiols). Serum added to the media may also release metal ions. Additionally, media are generally deficient in ascorbate and vitamin E, and also in selenium which is a cofactor for antioxidant enzymes such as TRX and GPx. Hence, effects of exogenous antioxidants may be exaggerated in culture. Cyclic strain is also known to influence oxidative stress and redox-sensitive gene expression (84,166). Here, however, it is shear stress (the frictional force acting per unit area on the endothelium, parallel to its surface) that is considered in detail, since it has been more extensively studied. Although it is well known to modify redox-sensitive signaling and gene expression, the effects are complex and not fully understood. A key consideration is that shear varies substantially in magnitude and temporal pattern from site to site within the vasculature. Hence, the phenomena observed in static culture might be modified in different ways at different locations in vivo. This is potentially important because atherosclerosis is a patchy disease—its prevalence varies substantially with location. Paralleling the confusion concerning the influences of shear on redox signaling, however, there has been confusion about the locations and hemodynamic environment predisposing to atherosclerosis. This confusion arises from neglect of effects of age and species on the location of disease, and because of

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the technical difficulty in measuring wall shear stress with adequate spatial resolution under physiological conditions (167,168). It has been known for more than a decade that the application of fluid dynamic shear stress to cultured endothelial cells can affect gene expression (169 – 171). Recent studies have demonstrated that over 100 genes are regulated by the level or type of shear (172). It has subsequently emerged that shear also affects levels of ROS in endothelial cells. Most notably, Laurindo et al. (173) demonstrated this in intact vessels. In rabbit aortas perfused with a spin trap, radical adducts, detected by electron paramagnetic resonance spectroscopy, increased with increase in flow rate. This effect was completely blocked by endothelial denudation and by SOD, but not by NG-nitro-L -arginine methyl ester (L-NAME) or indomethacin (inhibitors of NOS and COX, respectively). Similarly, when flow was increased in iliac arteries in vivo by infusing saline through an extracorporeal circuit or by pharmacological or physiological means, levels of the ascorbyl radical, a stable oxidation product of ascorbate, were increased. Again, this response was completely blocked by SOD, but not by L-NAME, indomethacin, or catalase. Stopping the flow decreased ascorbyl levels. Shear alters many signaling pathways in endothelial cells (174,175), so the increase in ROS need not necessarily explain the altered gene expression. However, there is increasing evidence for a causal relation. Several experiments have shown that shear increases ROS levels and putatively proatherogenic gene expression in endothelial cells, and that antioxidants block the change in expression. Thus, for example, Chiu et al. (176) exposed endothelial cells from human umbilical cords to a shear stress of 20 dynes/cm2 and detected increased superoxide concentrations after 15 min, with a peak at 30 min. Levels then declined but were still elevated by 6 h if flow was maintained. Shear also increased ICAM-1 mRNA at 3 or 6 h, an effect that was reduced by NAC and abrogated by catalase. A reporter gene assay demonstrated altered transcription, and surface ICAM-1 expression was also increased. A similar result was obtained by Yeh et al. (177), who additionally obtained evidence for the involvement of the glycosphingolipid, lactosylceramide. When an inhibitor of the enzymes involved in its synthesis was applied, there was no shear-induced increase in superoxide production or ICAM-1 expression. Hsieh et al. (178) showed an increase in ROS in HUVEC exposed to 15 – 40 dynes/cm2 that was blocked by NAC or catalase, and reduced by deferoxamine mesylate or a hydroxyl radical scavenger. An increase in c-Fos gene expression was also seen; this increase was reduced by NAC and catalase. Although such experiments suggest that shear stress has effects similar to cytokines, other studies have demonstrated greater complexity. As mentioned above, Mohan et al. (179) exposed human aortic endothelial cells to a shear stress of 2 dyne/cm2 for 6 h and detected increased NF-kB activation and mRNA and protein expression for VCAM-1 (which has NF-kB-binding sites in its promoter). The antioxidant PDTC blocked all three, but NAC only slightly

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decreased NF-kB, did not affect VCAM-1 mRNA levels and increased VCAM-1 protein expression. With cytokine stimulation, both PDTC and NAC inhibited all three. In this case, therefore, shear and cytokines acted through different pathways. Other studies have shown that shear increases ROS but that this downregulates putatively pro-atherogenic genes. Thus, Masatsugu et al. (180) found that endothelin converting enzyme-1 (ECE-1) and endothelin-1 (ET-1) mRNA were downregulated in bovine carotid artery endothelial cells and in HUVEC by shear (1.5 – 15 dyne/cm2) (and by H2O2). Shear stress increased intracellular peroxide concentrations, and the downregulation of both mRNAs was almost completely blocked by NAC. An additional complication arises from studies demonstrating that shear can oppose effects of ROS on gene expression. Tsao et al. (181) found, as expected, that incubation with oxidized LDL or with LPS and TNF-a increased superoxide production, NF-kB production, and VCAM-1 expression in human aortic endothelial cells. However, these effects were inhibited by a 4 h preexposure to shear, in complete contrast to the effects of shear alone described earlier. The effects of flow were abolished by nitro-L -arginine (another NOS inhibitor) and mimicked by an NO donor, so NO may have been influencing oxidantmediated transcription. Similarly, Hojo et al. (182) found that shear can block signaling events induced by ROS. JNK activation (which is thought to be pro-atherogenic through phosphorylation of c-Jun, activation of AP-1, and stimulation of pro-inflammatory genes such as ICAM-1) was induced in bovine lung microvascular endothelial cells by H2O2 ; this effect was reduced by a 10 min pre-exposure to a shear stress of 12 dynes/cm2. Shear increased the activity of glutathione reductase and increased the ratio of reduced to oxidized glutathione. When glutathione reductase was inhibited, shear was ineffective. And shear can also block ROS-induced apoptosis. Hermann et al. (183) induced apoptosis in HUVEC by incubation with H2O2 or TNF-a. Concomitant shear stress (15 dynes/cm2) completely inhibited this effect. The effect of shear was, in turn, partly reduced by N G-monomethyl-L -arginine (L -NMMA, a further NOS inhibitor) and by inhibition of the GSH pathway with buthionine sulfoximine (BSO). L -NMMA and BSO together completely blocked the effect of shear. Dimmeler et al. (184) additionally showed that the inhibitory effect of shear on apoptosis in HUVEC could be reduced by an antisense oligonucleotide to Cu/Zn SOD. Indeed, although it has been known for some time that shear upregulates SOD gene and protein expression (185,186), recent studies, including those using microarray technologies, show that shear induces a wide range of antioxidant genes (187 – 190). Chen et al. (191) note that a large number of the antioxidant defense genes upregulated by shear have an antioxidant response element (ARE) or an ARE-like sequence in their promoters. These genes include NAD(P)H:quinone oxidoreductase (NQO1), heme oxygenase-1 (HO-1), ferritin (heavy and light chains), microsomal epoxide hydrolase, glutathione S-transferase, and gamma-glutamylcysteine synthase. Shear appears to activate ARE-mediated transcriptional activity in endothelial cells. For example,

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mutation of ARE, expression of antisense Nrf2 (a transcription factor for ARE), a dominant negative Nrf2, or an Nrf2 inhibitor all prevented shear upregulating NQO1. How can the contradictory effects of shear on redox status and its effects be reconciled? This question cannot be answered at the moment, but a number of possibilities are emerging. One is that both ROS and antioxidant defenses are increased by shear—the latter may be a response to the former. Reference has already been made to the study by Hsieh et al. (178) in which shear increased ROS. The same study also found that shear increased antioxidant capacity: when exogenous H2O2 was added to extracts of the endothelial cells, it was decomposed more quickly in those cells that had been exposed to shear. This effect was blocked by an inhibitor of catalase. Another possibility gaining increasing support is that the type of shear is itself important for the influence on redox status or redox-sensitive transcription factors. (This explanation obviously fits well with the non-uniform localization of atherosclerosis in the vasculature; all cells will be exposed to shear, but its level and temporal pattern will vary from site to site.) Topper et al. (186) exposed HUVEC to laminar shear stress or to turbulent shear stress of comparable time-averaged magnitude (10 dynes/cm2) for 1 –24 h, and then examined effects on Mn SOD. Protein levels and mRNA increased with laminar but not with turbulent shear. Nuclear runoff analysis showed similar changes in transcriptional rates. Comparable data were obtained for eNOS mRNA levels. de Keulenaer et al. (192) compared effects on HUVEC of oscillatory and steady shears, both at 5 dynes/cm2, for 1 – 24 h. The oscillatory shear caused a sustained increase in NADH oxidase activity, superoxide levels, and gene and protein expression of HO-1, whereas the steady shear only transiently increased NADH oxidase activity and HO-1 expression, and gave lower superoxide levels. However, steady shear, unlike oscillatory shear, did increase Cu/Zn SOD. Comparable data concerning superoxide levels were obtained in a similar experiment by McNally et al. (193); use of a series of inhibitors and other interventions demonstrated that the superoxide was generated by xanthine oxidase and that this in turn was maintained by NAD(P)H oxidase. Nagel et al. (194) inserted a bar across their culture dish to obtain regions of disturbed and then uniform flow (12 dynes/cm2). Compared with culture in the absence of flow, the nuclear concentration of the ROS-sensitive transcription factors NF-kB, Egr-1, c-Jun and c-Fos, assessed by quantitative immunofluorescence, were all upregulated more by the disturbed than the uniform shear. All these studies suggest that steady laminar shear stress might have a more atheroprotective, less atherogenic effect than turbulent, oscillatory, or disturbed flows. Finally, we note that recent evidence directly supports the view that different shear stresses have different influences on the response of endothelial cells to antioxidants. de Nigris et al. (195) exposed human coronary endothelial cells to shears of 1 or 15 dyne/cm2. eNOS protein expression increased more at 1 than at 15 dyne/cm2, and treatment with a-tocopherol and ascorbic acid

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(50 and 10 mM, respectively) substantially increased expression at 1 dyne/cm2, but had little or no effect at 15 dyne/cm2. Conversely, expression of the redoxsensitive transcription factors Elk-1 and p-CREB increased at 1 dyne/cm2 and increased more at 15 dyne/cm2. The antioxidants reduced the effect at 15 dyne/cm2 but had no effect at 1 dyne/cm2. Because effects of antioxidants on transcription factors and gene expression are likely to be modulated in complex ways by shear stress, measurement of net effects in vivo—preferably in lesion prone and protected sites—is essential. Such studies are starting to emerge. One such trial was conducted by de Nigris et al. (196). Levels of cMyc (an oxidant-sensitive transcription factor) and its binding partner Max were examined in coronary arteries from pigs fed a cholesterol-enhanced diet. Higher levels were found in lesion prone areas than in protected vessels, and even higher levels where mild lesions were present. The cMyc-regulated genes GAD4 and p53 were down- and up-regulated, respectively, in the same distribution. All these changes were attenuated by dietary antioxidant vitamins. Subsequently, the same group found that pigs fed a cholesterol-enhanced diet had elevated activation of NF-kB, decreased eNOS expression and decreased radical scavenging activity in coronary artery tissue compared with animals on a normal diet. In a third group of animals, administered 100 IU/kg day vitamin E and 1 g/day vitamin C concurrently with the cholesterol, NF-kB activation and NO bioactivity were normalized (197). Along similar lines, de Nigris et al. (195) examined healthy vessels, areas with small lesions and areas with large lesions from LDL-receptor knockout (LDLR2/2 ) mice fed with high-fat diet for 6 months, with or without an additional 1 week of dietary antioxidant supplementation. (Endothelial morphology, which is flow-dependent, suggests that the areas with large lesions experience low time-averaged levels of shear with complex patterns, whereas the areas with small lesions experience high unidirectional shear.) eNOS levels were lower in small lesions than in healthy vessels, and even lower in large lesions, and were increased modestly by the antioxidants. Conversely, Elk-1 and p-CREB levels were increased, not reduced, in small lesions and more so in large lesions, and were decreased by the antioxidants. No substantial differences were seen in p-JUN expression. The complex and contradictory nature of the data emerging from studies of cultured endothelial cells means that further studies of this type are urgently required.

DISCUSSION The widely held views that inflammation is a key event in atherogenesis and that antioxidants suppress the expression of pro-inflammatory genes imply that administration of antioxidants in vivo should be anti-atherogenic. The results

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of antioxidant trials using animal models of atherosclerosis have generally tended to support this idea. Trials of probucol (and its analogs), butylated hydroxytoluene (BHT), diphenylphenylenediamine, and coenzyme Q have shown that these antioxidants cause large reductions in the extent of arterial lipid deposition (198). The studies in which vitamin E has been administered to hypercholesterolaemic rabbits have shown less consistency. Although several found a protective effect (199 –201), others did not (202) and some showed an increase in the extent and severity of lesions (203). Nevertheless, vitamin E does appear to be effective in reducing lesions in knockout mice, either when used alone (204) or in combination with other antioxidants (205,206). The broadly similar effect of a wide range of structurally unrelated antioxidant compounds suggests that it is their antioxidant activity per se rather than, for example, the lipid-lowering effect of probucol or other as yet undetermined anti-atherogenic properties of these compounds, which accounts for their beneficial effect in animal studies. The influence of dietary antioxidants on human disease is much less clear. A substantial body of epidemiological evidence has shown that antioxidant vitamin intake or supplementation is inversely associated with the risk of cardiovascular disease (207). However, the results of prospective clinical trials using beta carotene, vitamin E, or vitamin E plus other antioxidant vitamins in subjects with coronary heart disease, or at risk of it, have been disappointing. Ten such double-blind, placebo-controlled trials have recently been reviewed by Steinberg and Witztum (208), and 11 by Jialal and Devaraj (209). Of the 11, only 4 gave a positive effect on the primary endpoint and of these one (CHAOS) showed no effect on cardiovascular mortality, another (Transplant Associated Arteriosclerosis Study) was concerned with transplant arteriosclerosis, and a third (SPACE) used subjects with end stage renal disease. The four largest studies were all negative. No trials showed a beneficial influence of beta carotene, and there was evidence in some trials (although not others) for possible adverse effects (210 – 212). A recent review by Padayatty et al. (213) suggests similarly negative results concerning effects of vitamin C on markers of oxidation or clinical benefit. The results of prospective clinical trials therefore seem to contradict the idea that antioxidants could reduce atherosclerosis by inhibiting expression of pro-inflammatory genes. However, proponents of the beneficial effects of antioxidant therapy point to a number of doubts concerning the interpretation of some trials. These views are controversial. Jialal and Devaraj noted that in three of the seven negative studies, plasma antioxidant levels were not measured (and in a fourth, they rose in the placebo group too), and that in four of them there were no biomarkers of oxidative stress. They also note that the form of a-tocopherol might be important. All four positive trials used RRR-a-tocopherol, whereas only two of the seven negative trials did so; the remainder used all racemic a-tocopherol, in which the RRR form is only one of eight stereoisomers. Nevertheless, one of the two negative trials using the RRR form, the HOPE trial,

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was far larger than the four positive ones, and showed no hint of a beneficial effect. Furthermore, uptake by endothelial cells appears stereoisomerindependent (214). Steinberg and Witztum (208) suggest that vitamin E might have been the wrong antioxidant to use in the trials since it reacts only slowly with superoxide, is much less potent than probucol (as judged by effects on LDL oxidation when administered in vivo) and, in the absence of vitamin C or other co-antioxidants, can become a pro-oxidant. They also suggest that subjects should have been selected for the presence of oxidative stress; an analogy is drawn with trials of antihypertensive or lipid-lowering drugs which used subjects selected for hypertension and hyperlipidaemia, respectively. They consider the SPACE trial, which used subjects with end stage renal disease, to be important in this regard since such subjects are known to have high oxidative stress: this trial showed a big reduction in end point. However, the trial was a small one (15 vs. 33 primary endpoints). In the more recent and much larger Heart Protection Study, subjects with high plasma creatinine levels (indicative of renal problems) had more incidents than those with lower levels, but received no benefit from vitamin E. Furthermore, their analogy is not completely convincing since lowering blood pressure and plasma cholesterol levels are beneficial even in subjects who do not have hypertension or hyperlipidaemia. The various hypotheses invoking a role of oxidative stress in atherogenesis have, in any case, regarded this as being a nearly universal process. Finally, Steinberg and Witztum (208) suggest that the typical 5-year duration of a clinical antioxidant trial may be too short to reveal effects on the initial development of lesions, instead indicating only effects (or, rather, a lack of them) on plaque rupture. They argue that the positive results obtained in the even shorter-term animal studies may be misleading because of the rapid disease development in these models and because of the use of endpoints, such as area of lipid deposition, that correspond to early disease. Nevertheless, lipid lowering and blood pressure lowering trials—addressing factors also thought to influence the early stages of atherogenesis—have had positive outcomes over such timescales; it seems unlikely that all these beneficial effects can be explained in terms of pleiotropic effects of the drugs on plaque rupture. Furthermore, their comments were made in the context of using antioxidants to control LDL oxidation. When considering the hypothesis that antioxidants might control inflammation, it is not so plausible to argue that only early stages of the disease should be affected: plaque rupture involves the activation of macrophages, lymphocytes, and mast cells which then degrade extracellular matrix and the fibrous cap (215). It seems inconceivable that inflammation is not involved in the 5 years preceding a clinical event. Hence, the discrepancy between the success of antioxidant trials in animals and the absence of such success in human trials might most economically be explained by the hypothesis that oxidation is more important in animal models of the disease. It is known that smaller animals generate more ROS (which arise mainly from mitochondria) because of their higher metabolic

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rate, and the very high cholesterol levels generally present in the animal models will exacerbate this difference (208). CONCLUSION Antioxidants affect pro-inflammatory transcription factors in vitro, and evidence is emerging for influences in animals, but the effects are not simple and not always related to antioxidant activity per se. Evidence is lacking that antioxidants affect atherosclerosis in people, despite the likely role of inflammation in this disease. Although it is possible that a beneficial effect of antioxidants on atherogenesis, mediated by regulation of pro- and anti-inflammatory genes, could be obtained in the future through the development of suitable compounds and protocols, the data obtained at a cellular level are currently too complex and contradictory, and there are too many doubts about their relevance to in vivo conditions, to be assured of this. ACKNOWLEDGMENTS The support of the BHF, BBSRC, and Gen Foundation, and the assistance of Mrs. J. D. del Rio is gratefully acknowledged. REFERENCES 1. Rubanyi GM. The role of endothelium in cardiovascular homeostasis and diseases. J Cardiovasc Pharmacol 1993; 22:S1 – S14. 2. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993; 362:801 –809. 3. Brown MS, Goldstein JL. Regulation of the activity of the low density lipoprotein receptor in human fibroblasts. Cell 1975; 6:307 – 316. 4. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med 1989; 320:915 – 924. 5. Libby P. Inflammation in atherosclerosis. Nature 2002; 420:868 –874. 6. Kunsch C, Medford RM. Oxidative stress as a regulator of gene expression in the vasculature. Circ Res 1999; 85:753 – 766. 7. Griendling KK, Sorescu D, Lassegue B, Ushio-Fukai M. Modulation of protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology. Arterioscler Thromb Vasc Biol 2000; 20:2175–2183. 8. Brand K, Page S, Rogler G, Bartsch A, Brandl R, Knuechel R, Page M, Kaltschmidt C, Baeuerle PA, Neumeier D. Activated transcription factor nuclear factor-kB is present in the artherosclerotic lesion. J Clin Invest 1996; 97:1715 – 1722. 9. Phal HL. Activators and target genes of Rel/NF-kB transcription factors. Oncogene 1999; 18:6853 –6866. 10. Barnes PJ, Karin M. NF-kB—a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 1997; 336:1066 –1071.

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8 Fatty Acids, Gene Expression, and Coronary Heart Disease (CHD) Anne M. Minihane University of Reading, Reading, UK

Introduction Fatty Acid Structure and Tissue Sources Metabolism of Fatty Acids Absorption Transport as Lipoproteins Intracellular Metabolism Fatty Acid Regulation of Gene Expression PUFA and Hepatic Lipogenesis PUFA Induction of Lipid Oxidation Fatty Acids and Adipocytes Gene Expression Fatty Acid and Arterial Wall Gene Expression PUFA and Their Cellular Mechanisms of Action Transcription Factors Peroxisome Proliferator-Activated Receptors (PPAR) PPAR Ligands Other Families of Transcription Factors that Mediate the PUFA/PUFA Derivative Effect on Gene Expression Sterol Regulatory Element-Binding Protein Hepatic Nuclear Receptor-4 (HNF-4) Nuclear Factor-Y (NF-Y) and Nuclear Factor Kappa B (NF-kB) 181

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INTRODUCTION Over 95% of fat in the diet and in the body is present as fatty acids. In addition to meeting 30 – 40% of total body energy demands in Westernized societies, fatty acids are an integral component of all biological membranes and serves as a precursor for a number of essential compounds in the body such as the hormone-like eicosanoids, which mediate inflammatory and thrombotic processes. Furthermore, in recent years, it has become evident that fatty acids can also act as signalling molecules by serving as ligands for transcriptional factors which modulate gene expression. Research in this area is in its relative infancy, and has for the most part focussed on the expression of hepatic genes directly involved in fatty acid metabolism or lipid transport as lipoproteins. However, data on the ability of fatty acids to modulate gene expression in other tissues such as adipose tissue, endothelial cells, and macrophages are beginning to emerge in the literature. Although findings thus far have provided a valuable insight into the impact of dietary fat on the human genome, it is likely that it only represents the “tip of the iceberg.” Given the vast body of evidence implicating dietary fat composition in the pathology of many chronic diseases including coronary heart disease (CHD), such nutrient –gene interaction information could provide us with valuable insights into how fatty acids changes can be used as a measure to reduce the public health burden of such diseases. An understanding of the tissue specific metabolic effects of fatty acids relies on knowledge of the basic structure and nomenclature of fatty acids, of how they are absorbed and transported in the bloodstream, and upon reaching the target tissue are either stored, incorporated into the membrane bi-layer, or metabolized to active metabolites. Such information is detailed in the earlier part of the chapter. The chapter, which is by no means exhaustive, will then proceed to examine some important fatty acid – gene interactions and their potential impact on cardiovascular health. For a more comprehensive examination of the area please refer to a number of excellent recently published review articles (1 – 5). FATTY ACID STRUCTURE AND TISSUE SOURCES All fatty acids have a common basic structure, consisting of a hydrocarbon chain and a terminal carboxyl group. About 21 are found in significant amounts in food and they are characterized according to the length of the hydrocarbon chain and

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the degree of saturation and the position of the double bonds, if present. The short (SCFA) and medium chain (MCFA) C2 – C14 fatty acids are generally saturated in nature, whereas the C16 – C22, long chain fatty acids (LCFA) may be saturated or unsaturated. The most abundant monounsaturated fatty acid is oleic acid, which is an 18 carbon fatty acid containing one double bond at carbon 9 from the methyl end, and is therefore depicted by the notation C18:1 n-9. Polyunsaturated fatty acids (PUFA), as the name suggests contain two or more double bonds, with the two major PUFA classes the n-3 and n-6 having the first double bond at C3 and C6, respectively. Alpha-linolenic (ALA, C18:3, n-3) and linoleic (LA, C18:2, n-6) acid are the precursors for the n-3 and n-6 fatty acid families, respectively, and are considered essential fatty acids as the mammalian body does not contain the enzymatic machinery to insert double bonds beyond the C9 position, therefore a dietary supply is necessary. The longer chain metabolic derivatives of these fatty acids eicosapentaenoic acid (EPA, C20:5, n-3) and arachidonic acid (AA, C20:4, n-6) often have opposing metabolic effects, in large part attributable to the fact that they give rise to different families of eicosanoid end products. The long chain n-3 PUFAs EPA, docosahexaenoic acid (DHA, C22:6, n-3) and docosapentaenoic acid (DPA, C22:5, n-3) are currently almost exclusively ingested as oily fish or fish oil capsules, although it is likely that genetic engineering will lead to a vegetable oil source within the next 10 years. Recent publications suggest that this series of LCFA may be the most significant fatty acid modulators of gene expression. Tissues EPA and DHA may be synthesized from ALA through a series of elongation and desaturation reactions (Fig. 8.1). However, reaction efficiency is generally low (6), with an average estimated 7 M of ALA required to produce 1 M of EPA. Furthermore, as the n-6 fatty acid metabolic pathways uses the same desaturase enzymes, high dietary LA inhibits EPA formation (Fig. 8.1). In the UK current dietary intakes of 10.0 g LA per day compared with 1.6 g ALA (7) does not favor this metabolic conversion. Therefore, an increased consumption of fatty fish represents a more effective means of increasing total body content of LC n-3 PUFA. As currently fish is almost the exclusive dietary source, nonfish eaters must rely on conversion from the precursor ALA, which is naturally present in certain vegetable oils and green vegetables.

METABOLISM OF FATTY ACIDS Absorption Fatty acids are ingested mainly in the form of triglycerides (TAG), which are hydrolyzed in the gut lumen under the action of lipases. The released fatty acids and monoacyl glycerol are taken up into the enterocyte, where the LCFA are re-esterified to form TAG. As fat is largely insoluble in the aqueous medium of the blood stream the TAG, cholesterol, and phospholipids are packaged into lipid – protein structures called chylomicrons, which contain apoB48

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n-6 PUFA pathway

n-3 PUFA pathway C18:3 (ALA)

C18:2 (LA)

Delta-6 desaturase C18:4

C18:3

El ongase C20:4

C20:3

Delta-5 desaturase C20:5 (EPA)

C20:4 (AA)

El ongase C22:5 (DPA, n-3)

C22:4

El ongase C24:5

C24:4

Delta-6 desaturase C24:6

C24:5

Partial ß oxidation C22:6 (DHA)

C22:6 (DPA, n-6)

Figure 8.1 Long chain polyunsaturated fatty acid (LC-PUFA) biosynthetic pathways. ALA, alpha-linolenic acid; EPA, eicosapentaenoic acid; DPA, docosapentaenoic acid; LA, linoleic acid; AA, arachidonic acid.

as its constitutive protein. Chylomicrons are secreted into the bloodstream via the lymphatic duct. The SCFA and MCFA (C4 –C14) are secreted directly into the bloodstream by the enterocyte and these nonesterified fatty acids (NEFA) are transported in the circulation loosely attached to albumin. Transport as Lipoproteins Once in the circulation, chylomicrons are sequentially hydrolyzed by the action of lipoprotein lipase, found attached to the luminal side of the capillary endothelium, mainly in muscle and adipose tissue [Fig. 8.1(a)]. In muscle, the majority of fatty acids are used as a source of energy for driving muscle action. Adipose tissue serves as a reserve of fatty acids and release from this depot is under strict hormonal control with insulin and catecholamines being centrally involved. In times of need, for example, starvation or intensive exercise, hormone sensitive lipase (HSL) hydrolyzes the TAG contained within the adipocytes fat droplet and fatty acids are released into the circulation. Following several passages through the capillary bed a lipid depleted chylomicron particle results, which is removed by the liver by a receptor mediated

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process. The fatty acid may be directly used by the hepatocyte or re-enter the circulation as very low density lipoproteins (VLDL), the main carrier of lipids processed or synthesized by the liver [Fig. 8.1(b)]. Postprandial lipoprotein metabolism is a highly orchestrated process in part determined by the impact of fatty acids on the hepatic and extra-hepatic expression of genes involved in lipoprotein clearance and VLDL secretion into the circulation (see later). In the postprandial state, hepatic VLDL synthesis and secretion are inhibited as the tissues utilize the dietary fatty acids supplied by the chylomicrons. Postprandial lipoprotein metabolism is a major risk factor for CHD and as will be discussed later the impact of dietary fat composition, in particular PUFA intake, on CHD risk is in part mediated via alterations in this metabolic pathway. In addition to fatty acids of dietary origin, or derived from the elongation and desaturation of essential fatty acids, fatty acids may also be synthesized de novo from glucose, with adipose tissue and liver being the main lipogenic tissues. Fatty acids are themselves important regulators of a number of key lipogenic enzymes as will be described later (8) [Fig. 8.2(a) and (b)].

Figure 8.2 Lipoprotein metabolism pathways: (a) exogenous pathway for the transport of dietary derived lipids and (b; see pg. 186) endogenous pathway for the transport of hepatic derived lipids.
The chylomicrons and VLDL pass through the capillary bed several times until chylomicrons and VLDL remnants remain. Apo, apolipoprotein; LPL, lipoprotein lipase; HDL, high density lipoproteins; VLDL, very low density lipoproteins; IDL, intermediate density lipoproteins; LDL, low density lipoproteins; LCAT, lecithin-cholesterol acyl transferase; LDL-R, low density lipoprotein receptor.

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Continued.

Intracellular Metabolism Fatty acids are thought to gain access to cells by a receptor driven saturable process. Although specific receptors and transporters have not as yet been fully characterized, it is thought that an albumin receptor and one or more fatty acid transporters (FAT) may be involved (9,10). The fatty acids are metabolized into fatty acyl-CoA-thioesters (FA-CoA) and transported intracellularly bound to fatty acid binding proteins (FABPs), where the fatty acids have a number of potential metabolic fats including: (i) peroxisomal or mitochondrial b-oxidation; (ii) incorporated into complex lipids such as phospholipids or TAG; (iii) elongated and/or desaturated to form other fatty acids; (iv) metabolized into lipid derivatives such as eicosanoids; and (v) following release from the FABP, serve as a ligand for transcription factors that subsequently translocate to the nucleus and impact on the expression of target genes. Fatty acids directly released from biological membrane are also thought to serve as ligands for these transcription factors. Furthermore, fatty acid oxidation products such as specific eicosanoids or epoxy- or hydroxyl-fatty acids produced during eicasonoid synthesis in microsomes are also known to interact with transcription factors, often with a higher affinity than the parent PUFA (Fig. 8.3).

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Figure 8.3 Cellular fatty acid metabolism including postulated mechanisms of action on gene expression. FA, fatty acid; PL, phospholipids; TAG, triglycerides; FABP, fatty acid binding protein; FA-CoA, fatty acyl-CoA.

FATTY ACID REGULATION OF GENE EXPRESSION In order to maintain cellular homeostasis and in order to be adaptive to extracellular events, expression of the 30 – 40,000 genes that encode for all cellular proteins needs to be carefully orchestrated. Over the last 30 –40 years, it has become evident that the fatty acid composition within the cell has the ability to either directly or indirectly regulate the expression of numerous genes. As this is reflective of whole body and dietary fat intake, this allows the cells to adapt its metabolic processes in response to fat supply. The overall main effects of PUFA on tissue gene expression is summarized in Fig. 8.4. PUFA and Hepatic Lipogenesis Over 35 years ago, the ability of PUFA to impact on macronutrient metabolism was first recognized. It was observed that the ability of mouse liver to synthesize long chain PUFA de novo from carbohydrate was greatly enhanced, and the concentration of a number of lipogenic enzymes [fatty acid synthetase (FAS), malic enzyme and glucose-6-phosphate dehydrogenase (G6PDH)] increased when the mouse diet was deficient in LA (11). Conversely, inclusion of LA in the diet

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Figure 8.4 Tissue specific effects of polyunsaturated fatty acids (PUFA). LPL, lipoprotein lipase; apo, apolipoprotein; MTP, microsomal transfer protein; TNF, tumour necrosis factor.

decreased fatty acid synthesis following a high carbohydrate diet. No such effects were observed following palmitic acid (C16:0) or oleic acid (C18:1). In a study conducted by Wahle and Radcliffe (12), the feeding of a sunflower enriched diet rich in LA resulted in 40 –50% lower hepatic lipid accretion and steroyl-CoA desaturase (SCD) 1 activity, in genetically obese (fa/fa) rats with an inherent high lipogenic activity, in comparison to animals fed a standard or low sunflower oil diet. A large number of subsequent studies have verified these earlier findings with a range of dietary PUFAs shown to suppress lipogenesis by inhibiting the concentration of enzymes involved in glucose metabolism and fatty acid biosynthesis such as G6PDH, malic enzyme, S14, FAS, acyl co-carboxylase, stearoyl CoA desaturase 1 (SCD1), L -pyruvate kinase (L -PK), glucose transporter-4 and more recently D-5 and D-6 desaturases (1 –5,13 – 15). Early work failed to identify the molecular target of the PUFA effect, with modulation of gene transcription and translation and specific effects on enzyme activity and protein integrity proposed. The cloning of the peroxisome proliferators receptors (PPARs) in 1990 (16), led quickly to the idea that this group of nuclear receptors were the molecular target whereby PUFA co-ordinately suppressed genes involved in lipid biosynthesis, whereas increased expression of

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proteins involved in lipid transport (see following section). More recent studies have identified other important transcription factors such as sterol regulatory element (SREBP) and hepatic nuclear factor-4 (HNF-4) that modulate cellular lipogenesis. The question of whether the PUFAs per se are the regulators of lipogenic gene expression remains controversial. For example, for the S14 gene, eicosanoid inhibitors did not affect gene expression (17) in hepatocytes suggesting that these derivatives of PUFA were not involved. Because the effect of PUFA on lipogenesis is largely localized to hepatic tissue where lipid peroxidation occurs, it has been postulated that lipid peroxidation products may be responsible for the altered lipogenic gene expression (18). However, it is likely that a range of fatty acid derivatives can act as modulators of lipogenesis. PUFA Induction of Lipid Oxidation In addition to their role in lipogenesis, PUFA impact on metabolic fuel repartitioning by increasing hepatic fatty acid oxidation. One of the initial targets for the effect of PUFA is a reduction in the synthesis of hepatic malonyl coenzyme A, which favors fatty acid entry into mitochondria and peroxisomes leading to increased oxidation. Whether such a suppression in malonyl CoA levels is observed in skeletal muscle following PUFA exposure remains controversial, although this proposal will be consistent with the increased fatty acid oxidation observed in human and animals following the feeding of PUFA rich diets (19,20). The increased partitioning of fatty acids is accompanied by a PUFA induced induction of proteins involved in fatty acid oxidation and ketogenesis such as acyl-CoA oxidase (AOX) and CYP4A2 (21). Desaturation of LA and ALA is required to alter metabolic fuel repartitioning (22) and LC n-3 PUFA are thought to be more potent than the n-6 PUFA family. This partitioning effect of PUFA, in particular, n-3 PUFA, has been observed in human as well as animal models (1,19,20,23,24) and it is likely that part of the benefit of fish oil feeding on cardiovascular risk is attributable to an effect of EPA and/or DHA on lipid oxidation (see later). Much of the effect of PUFA on lipid oxidation is mediated by PPARa as will be discussed. Fatty Acids and Adipocytes Gene Expression Advances in our understanding of lipid metabolism have identified the adipose tissue as a key player. Storage and release of fatty acids from adipocytes impact on both hepatic and peripheral lipid and glucose metabolism. Furthermore, although most of the early work on fatty acids as regulators of gene expression focussed on hepatic tissue, it is now evident that the effects of PUFA on macronutrient homeostasis is not tissue exclusive, with PUFA having a significant effect on adipocyte metabolism, in particular, genes involved in adipogenesis and adipose tissue lipogenesis. The molecular action of PUFA, in

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adipocyte maturation and lipid uptake is mediated by PPARg acting in concert with other transcription factor families such as C/EBPs and SREBPs (25 – 27). A number of adipocyte genes are induced by PUFA including adipocyte FABP, fatty acid transport protein (FAT) and CD 36, lipoprotein lipase (LPL), long chain acyl-Co synthase, phosphenoenoylpyruvate carboxykinase (PEPCK) and a specific FABP aP2 (3,5,28,29). The upregulation of such genes is consistent with adipocyte lipid accumulation and removal from the circulation. PPARg is thought to be the main transcription factor that modulates the effects of fatty acids in adipose tissue (30,31). Evidence for a role of PPARg agonists in modulating insulin action is provided by the thiazolidiones series of antidiabetic drugs, potent and selective PPARg-ligands, which effectively improve insulin sensitivity and glucose homeostasis (32,33). In addition to effecting the protein concentration of enzymes that function within the adipocytes, PUFA is also thought to affect the levels of adipocytokines. Upon secretion into the circulation, adipocyte derived signalling molecules act in tissues such as the brain, liver, and skeletal muscles to regulate energy homeostasis. One such molecule is leptin, a product of the ob gene, whose specific site of action is the hypothalamus of the brain, where it binds to the leptin receptor (34,35). This adipocytes derived hormone modulates food intake and energy expenditure and its levels in the blood stream are controlled by hormonal factors such as catecholamine (downregulates) and insulin (upregulates) and nutritional signals such as the fat composition of the diet. In humans, low fat diets and a reduction in the SFA:PUFA ratio of the diet attenuates plasma leptin concentrations (36). Furthermore, in a series of rat models and cell culture experiments, n-3 PUFA, in particular, reduces leptin concentration with an associated reduction in ob mRNA levels (37). The expression of other adipokines such as adiponectin and resistin, which have been shown to be sensitive to the modulating effects of glitazones (34), are also likely to be sensitive to the actions of lipid derived agonists although substantial evidence is currently lacking. Fatty Acid and Arterial Wall Gene Expression The primary pathological features of CHD, atherosclerosis and thrombosis, are now recognized to have a large inflammatory component in their etiology (5,38). Recent evidence suggests that arterial inflammatory processes may be altered by n-3 PUFA and conjugated linoleic acid (CLA, a group of structural isomers of C18:2) (39 – 41). In the arterial wall, the progression of atherogenesis involves the coordinated alteration of the gene expression patterns of endothelial, blood mononuclear, and smooth muscle cells. Various stimuli including damage to the endothelial lining induced by either oxidative stress or hemodynamic blood flow, or locally produced cytokines, induce the increased expression of adhesion molecules on the endothelial surface, which facilitates the recruitment of monocytes into the vascular

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wall. In the intima, monocytes rapidly accumulate oxidized LDL via a number of scavenger receptors, including scavenger receptor A (SR-A), CD36, and CD38, and develop into lipid laden macrophages called foam cells. Macrophages, in addition to accumulating lipid, secrete a range of cytokines that further promote monocyte recruitment, and contribute to the endothelial and smooth muscle cell dysfunction. As mentioned earlier, a number of studies have shown that n-3 PUFA can attenuate the expression of inflammatory cytokines. A decrease in the expression of vascular adhesion molecules such as vascular cell adhesion molecule 1 (vCAM-1), intercellular adhesion molecule 1 (ICAM-1), and E-selectin has been observed following supplementation of endothelial cells in culture with n-3 PUFA, with no effect evident following supplementation with n-6 PUFA (42,43). Furthermore, the feeding of fish oils to human volunteers has been shown to decrease the plasma levels of adhesion molecules such as vCAM-1 (44). Recent evidence indicates that CLA may also have the potential to modulate adhesion molecule levels. In cell culture, a mixture of the two main isomers (cis-9, trans-11, trans-10, cis-12) has been shown to attenuate adhesion molecule mRNA and protein levels in endothelial and smooth muscle cells, respectively (41). Dietary CLA has also been shown to reduce atherogenesis in rabbits fed a highcholesterol atherogenic diet, which has been suggested to be in part attributable to a reduction in adhesion molecule expression (45). However, data from human studies are distinctly lacking. In addition to effects on endothelial gene expression, evidence is now emerging that PUFA may bring about positive changes in macrophages metabolism, such as a reduced expression of scavenger receptors and an increased efflux of cholesterol from the cell.

PUFA AND THEIR CELLULAR MECHANISMS OF ACTION The exact molecular mechanisms by which PUFA modulate the diverse but highly coordinated effects on a number of body tissues is only partly understood and is very much “work in progress.” It is likely that much of the effects are either directly or indirectly modulated at the gene level, rather than a specific effect of the fatty acid on protein integrity or activity. A comprehensive understanding of fatty acid – gene interactions will greatly improve our understanding of how dietary fat intake contributes to CHD pathology and maximizes our understanding of how changes in dietary fat composition can contribute to reduced CHD burden. PUFA have the potential to alter gene expression in a number of ways. 1. Fatty acid changes to cell membranes can alter receptor activity and cell signalling cascades. This in turn can result in alterations (e.g., phosphorylation) to specific transcription factors that are responsive to specific cell surface receptor–ligand interactions and messenger signal cascades (5).

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2.

3.

4.

Native PUFA or their derivatives may impact on the gene expression or mRNA stability of transcription factors or their specific co-factor or inhibitory molecules. PUFA, owing to their unsaturated nature have the potential to decrease the antioxidant activity and increase the redox stress within the cell, which may in turn influence the activity of redox sensitive transcription factors such as nuclear factor kB (NF-kB) and its inhibitor (I-kB). PUFA or PUFA metabolites such as eicosanoids act as ligands and bind with high affinity to specific transcription factors that subsequently translocate to the nucleus and impact on the expression of specific genes. The discovery of the PPAR family, and a subsequent understanding of their function, activation, and nuclear effects has led to the realization that much of the modulating effect of PUFA on cellular metabolism is via this family of transcription factors.

TRANSCRIPTION FACTORS Peroxisome Proliferator-Activated Receptors (PPAR) In 1990, the identification of a ligand induced transcription factor that caused proliferation of peroxisomes in the liver was discovered by Issemann and Green (16) and named the peroxisome proliferator-activated receptor (PPAR). Although this effect on increased peroxisome b-oxidation of fatty acids was found to be mainly confined to rodents, subsequent research demonstrated that PPAR and their ligands were important modulators of other genes involved in both hepatic and extra-hepatic lipid metabolism (46 –48). PPARs have a similar structure to the other members of the steroid/thyroid nuclear receptor superfamily, which has over 2000 members including endocrine receptors such as the estrogen receptor (ER). Similar to other nuclear receptors PPARs possess both a ligand and DNA binding domain (2,5,49). Upon activation by ligands, PPARs translocate to the nucleus and binds to a specific DNA sequence, referred to as a PPAR-response element (PPRE) in the promoter region of target genes (PPRE), resulting in an up- or down-regulation of gene expression. PPREs comprise imperfect direct repeats of AGGTCA separated by a single nucleotide, with 50 extensions portion of AACT (AACTAGGNCAAAGGTCA) (3,5), with PPAR binding to the 50 end of the PPRE and retinoid X receptors (RXR) to the 30 end. These PPRE sites are located on a number of genes involved in lipid metabolism and adipocytes differentiation. The PPAR family consists of three specific receptors, PPARa, PPARd, and PPARg that differ in their tissue distribution and ligand specificity, but possess a common ability to form a heterodimer with the RXR a, b, and g (50). It is the PPAR – RXR complex that is active in altering gene expression (2,28,51).

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PPARa is known to exert its effect mainly in liver and muscle cells where it is centrally involved in glucose and fat homeostasis. PPARg modulates gene expression in tumor and adipose tissues. In adipose tissue, PPARg activation is associated with adipocyte maturation and lipid storage as TAG. Both PPARa and PPARg are expressed in the arterial wall by both endothelial cells and macrophages, and their activation in these tissues inhibits the expression of inflammatory genes and induces the expression of genes involved in cholesterol efflux from macrophages such as apoE and the ABCA1 genes (29). PPARb appears to be expressed in a variety of tissues. However, its specific role is not currently understood. PPAR Ligands Early studies on the effects of PPARs used synthetic agonists, such as the potent hypolipidaemic fibrate drug group, which are PPARa ligands and the thiazolidiones anti-diabetic drugs that are potent PPARg agonists (28,52,53). In 1992, Auwerx (54) demonstrated that fatty acids can serve as PPAR ligands and since that early study it has become apparent that PPARs are activated either directly by PUFA or by a diverse range of PUFA derivatives (5,29,49,55). Numerous competitive assays using labeled ligands have demonstrated the ability of PUFAs, including CLA and the cyclooxygenase and lipoxygenase PUFA oxidation products (specific eicosanoids) to act as PPARa and PPARg agonists (5). The n-3 PUFA, in particular EPA and DHA, are thought to be more potent than the n-6 PUFA as activators of PPARa (1,20), but neither family acts as a particularly strong activator. In contrast, PUFA derivatives, including specific eicosanoids and oxidized lipids such as 8S-hydroxyeicosatetraenoic acid bind PPARa with 1– 2 order of magnitude higher affinity are far more potent activators of PPARa-dependent genes (56). CLAs are also potent PPARa activators being able to displace synthetic PPARa specific synthetic ligand at low concentrations. A 5-fold upregulation of PPARa was evident at CLA concentrations as low as 5 mM in a rat hepatoma cell line (57). CLA is also a recognized PPARg agonist resulting in a decreased PPARg induced inflammatory response in CLA supplemented pigs and RAW264.7 mouse macrophages (5). Other Families of Transcription Factors that Mediate the PUFA/PUFA Derivative Effect on Gene Expression Although PPARs were originally thought as the mediator of all the PUFA effects on gene expression, evidence quickly emerged to suggest that other transcription factors may also be involved. The genes encoding for the delta-5 and delta-6 desaturases are downregulated by PUFA but unregulated by non-PUFA PPAR activators (13,14). PUFA are known to suppress hepatic lipogenesis in PPARa2/2 mice (1,23). These observations have been confirmed by other

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investigators, who observed that n-3 PUFA suppressed the expression of the lipogenic S14 and FAS genes in vivo or in hepatocytes derived from PPARa2/2 mice (4). In addition to PPARs, more recently identified and studied transcription factors include, SREBP, hepatocyte nuclear factor 4 (HNF-4), nuclear factor-Y (NF-Y), and nuclear factor kB (NF-kB). Sterol Regulatory Element-Binding Protein Many genes involved in glucose, cholesterol, and fatty acids metabolism contain a sterol response element (SRE) and their expression is regulated in part by the SREBP family of transcription factors. SREBP-1a, 1c, and 2 regulate genes involved in both cholesterol and fatty acid biosynthesis, with 90% of the total SREBP-1 found in vivo as the 1c form (58). The parent SREBP-1 molecule is a 125 kDa protein attached to the endoplasmic reticulum in the cell. Following proteolytic cleavage, the mature SREBP-1 translocates to the nucleus and binds to the SRE of target genes, where it results in an increase in lipogenic gene expression. Overexpression of SREBP-1 in liver is associated with high rates of fatty acid biosynthesis. In rats, the feeding of diets rich in LA or EPA and DHA were found to reduce hepatic nuclear SREBP-1 content by 60– 70% (59), which was associated with a comparable decrease in the expression of the hepatic FAS gene. Unlike PUFA, SFA or MUFA had no effect (1). The pattern of the response of SREBP-1 to PUFA feeding suggests an effect at both the transcriptional (mRNA) and post-translational (protein) level (2,5). Hepatic Nuclear Receptor-4 (HNF-4) Although PUFA and their metabolites are potent PPAR agonists, the expression of PPAR-a in the human liver is relatively low (46) and it is likely that much of the PUFA effects on hepatic metabolism are mediated through the HNF-4 orphan receptors. In contrast to PPAR, which does not bind fatty acyl-CoA derivatives, HNF-4 does not bind PUFA but is activated by fatty acyl-CoA (2,60). The HNF-4 binds to the DNA promoter region as a homodimer (61) and elicits changes in the gene expression of proteins involved in fatty acid (PEPCK and L -pyruvate kinase) and lipoprotein metabolism [apoC3, apoA1, microsomal transfer protein (MTP)] (5,29). Nuclear Factor-Y (NF-Y) and Nuclear Factor Kappa B (NF-kB) Although the promoter region of the S14 gene (S14 being a primary lipogenic protein) contains SRE and PPRE sequences, the expression of this gene is though to be mainly via the NF-Y transcription factor (2). The transcription factor NF-kB is an important regulator of the expression of inflammatory mediators such as the proinflammatory cytokines. Studies in cell lines have demonstrated strong upregulation of NF-kB translocation following ARA and n-6 PUFA, both not EPA supplementation (62). This divergent effect on NF-kB activity may in part explain the recognized differing effects of n-3 and n-6 PUFA on inflammatory processes (40,63).

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FATTY ACIDS, GENE EXPRESSION, AND THE COORDINATION OF GLUCOSE AND INSULIN HOMEOSTASIS AND LIPOPROTEIN METABOLISM As has been discussed, PUFA and, in particular, the long chain n-3 PUFA, EPA, and DHA impact on lipid, glucose, and insulin metabolism in both hepatic and extra-hepatic tissues. In liver, PUFA modulates numerous genes encoding for proteins that play central roles in lipogenesis, fatty acid oxidation, lipoprotein metabolism, and cholesterol synthesis and secretion as bile salts. The dual action of PUFA on suppressing lipogenic genes such as S14, FAS, and L -pyruvate kinase while increasing the expression of genes involved in fatty acid oxidation such as AOX and FABP, would result in a reduced hepatic fatty acid load. Such reduction in liver fatty acid levels would lead to an improvement in insulin sensitivity and a favorable shift in lipoprotein metabolism. Availability of fatty acid for TAG synthesis is a major factor controlling the secretion of VLDL-TAG by the liver and therefore circulating TAG levels. In addition to plasma TAG levels being an independent marker of CHD risk, high TAG in the circulation results in a pro-atherogenic shift in LDL and HDL metabolism (64). Furthermore, PUFA impacts on lipoprotein metabolism via an effect on a number of proteins involved in lipoprotein secretion, metabolism in the circulation, and subsequent clearance by the liver. PUFA supplementation, and in particular n-3 PUFA, is associated with increased expression of the LPL and apoA-1 genes and decreased expression of the apoC3 gene. These alterations in gene expression would be expected to result in a more effective hydrolysis and clearance of chylomicrons and VLDL from the circulation and in higher HDL concentrations, lipoprotein changes associated with reduced atherogenesis and improved insulin sensitivity. In the adipose tissue, genes such as LPL, FAT, and acyl-co synthase, which modulate lipogenesis, the leptin gene and the genes for insulin receptor substrate 2 (IRS-2), and pyruvate dehydrogenase kinase-4 (PDK-4) that are involved in glucose metabolism are PUFA responsive via PPARg. The coordinated alterations in adipocyte gene expression are thought to result in an increased fatty acid flux into the white adipose tissue and away from muscle and liver. These changes would be predicted to make an important contribution to improved whole body insulin sensitivity by decreasing the exposure of this tissue to fat, thereby increasing muscle cell glucose utilization. Although the benefits of n-3 PUFA on adipose tissue metabolism and whole-body glucose utilization have shown promising results in rodent studies, n-3 PUFA supplementation studies in humans have not as yet demonstrated any benefit with respect to insulin sensitivity (65). However, relatively short supplementation periods and the use of nonsensitive measures of insulin sensitivity may in part explain this apparent lack of effect. Although research is at a relatively early stage, it appears likely that n-3 PUFA also has positive effects on the arterial wall gene expression profile,

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resulting in a reduction in endothelial adhesion molecule expression and scavenger receptor expression in macrophages. Such changes would be predicted to decrease atherogenesis by reducing arterial monocyte recruitment and accumulation of modified lipids by macrophages in the intima. SUMMARY It has long been recognized that the fatty composition of the diet and of body tissue has significant effects on a range of metabolic processes such as lipid synthesis and metabolism, glucose and insulin homeostasis and inflammation. Over the last 30 years, with the discovery of an array of fatty acid sensitive genes and transcription factors the molecular basis of this association is being “unravelled.” Given the large body of epidemiological and clinical evidence linking dietary fatty acid composition and the pathology of major chronic diseases such as coronary heart disease and diabetes (which is currently reaching epidemic levels), a fuller understanding of fatty acid– gene interaction will help target nutritional advise to maximize the benefits of dietary change. REFERENCES 1. Clarke SD. Polyunsaturated fatty acid regulation of gene transcription: a molecular mechanism to improve the metabolic syndrome. J Nutr 2001; 131:1129– 1132. 2. Jump DB, Clarke SD. Regulation of gene expression by dietary fat. Annu Rev Nutr 1999; 19:63 –90. 3. Duplus E, Glorian M, Forest C. Fatty acid regulation of gene transcription. J Biol Chem 2000; 40:30749 – 30752. 4. Sessler AM, Ntambi JM. Polyunsaturated fatty acid regulation of gene expression. J Nutr 1998; 128:923– 926. 5. Wahle KWJ, Rotondo D, Heys SD. Polyunsaturated fatty acids and gene expression in mammalian systems. Proc Nut Soc 2003; 62:349– 360. 6. Burdge GC, Finnegan YE, Minihane AM, Williams CM, Wootton SA. Effect of altered dietary n-3 fatty acid intake upon plasma lipid fatty acid composition, conversion of [13C} a-linolenic acid to longer-chain fatty acids and partitioning towards b-oxidation. Br J Nutr 2003; 90:311 –321. 7. MAFF (Ministry of Agriculture Fisheries and Food) Food Information. Surveillance Sheet 127. Dietary intake of iodine and fatty acids. London: MAFF, 1997a. 8. Frayn KN. Lipoprotein metabolism. In: Frayn KN, ed. Metabolic Regulation: A Human Perspective. 2nd ed. Oxford, UK: Blackwell Science, 2003:253 – 280. 9. Berk PD, Stump DD. Mechanism of cellular uptake of long chain free fatty acids. Mol Cell Biochem 1999; 192:17 – 31. 10. Motojima K, Passilly P, Peters JM, Gonzalez FJ, Latruffe N. Expression of putative fatty acid transporter genes are regulated by peroxisome proliferator-activated receptor alpha and gamma activators in a tissue- and inducer-specific manner. J Biol Chem 1998; 273:16710 – 16714. 11. Allmann DW, Gibson DW. Fatty acid synthesis during early linoleic acid deficiency in the mouse. J Lipid Res 1969; 6:51– 62.

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12. Wahle KWJ, Radcligge JD. Effects of a diet rich in sunflower oil on aspects of lipid metabolism in genetically obese rats. Lipids 1977; 12:109 – 115. 13. Cho HP, Nakamura MT, Clarke SD. Cloning expression and nutritional regulation of the human delta-6 desaturase. J Biol Chem 1999a; 274:471 – 477. 14. Cho HP, Nakamura MT, Clarke SD. Cloning, expression and fatty acid regulation of the human delta-5 desaturase. J Biol Chem 1999b; 274:37335– 37339. 15. Forest C, Franckhauser S, Glorian M, Antras-Ferry J, Robin D, Robin P. Regulation of gene transcription by fatty acids, fibrates and prostaglandins: the phosphoenolpyruvate carboxykinase gene as a model. Prostaglandins Leukot Essent Fatty Acids 1997; 57:47– 56. 16. Issemann I, Green S. Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators. Nature 1990; 347:709 – 710. 17. Jump DB, Clarke SD, MacDougald O, Thelen A. Polyunsaturated fatty acid inhibits S14 gene transcription in liver and cultured hepatocytes. Proc Natl Acad Sci USA 1993; 90:8454 –8458. 18. Duplus E, Glorian M, Forest C. Fatty acid regulation of gene transcription. J Biol Chem 2000; 275:30749 – 30752. 19. Couet C, Delarue J, Fitz P, Antonine JM, Lamisse F. Effects of dietary fish oil on body fat mass and basal fat oxidation in healthy adults. Int Obes J 1997; 21:637– 643. 20. Power GW, Newsholme EA. Dietary fatty acids influence the activity and metabolic control of mitochondrial carnitine palmitoyltransferase in I rat heart and skeletal muscle. J Nutr 1997; 127:2142 – 2150. 21. Karara A, Dishman E, Blair I, Falck JR, Capdevila JH. Endogenous epoxyeicosatrienoic acids. Cytochrome P-450 controlled stereoselectivity of the hepatic arachidonic acid epoxygenase. J Biol Chem 1989; 264:19822– 19827. 22. Nakamura MT, Cho HP, Clarke SD. Regulation of delta-6 desaturase expression and its role in the polyunsaturated fatty acid inhibition of fatty acid synthase gene expression in mice. J Nutr 2000; 130:1561– 1565. 23. Clarke SD. Polyunsaturated fatty acid regulation of gene expression: a mechanism to improve energy balance and insulin resistance. Br J Nutr 2000; 83:S59 – S66. 24. Mori TA, Bao DQ, Burke V, Pudey IB, Watts GF, Beilin LJ. Dietary fish as a major component of a weight-loss diet: effect on serum lipids, glucose and insulin metabolism in overweight hypertensive subjects. Am J Clin Nutr 1999; 70:817 – 825. 25. Lowell BB. PPARgamma: an essential regulator of adipogenesis and modulator of fat cell function. Cell 1999; 99:239 – 242. 26. Morrison RF, Farmer SR. Hormonal signalling and transcriptional control of adipocyte differentiation. J Nutr 2000; 130:3116S– 3121S. 27. Ntambi JM, Young-Cheul K. Adipocyte differentiation and gene expression. J Nutr 2000; 130:3122S– 3126S. 28. Berger J, Moller DE. The mechanism of action of PPAR. Annu Rev Med 2002; 53:409-435. 29. Beisiegel U, Heeren J, Schnieders F. Fatty acids and gene expression. In: Zempleni J, Daniel H, eds. Molecular Nutrition. Wallingford, UK: CABI Publishing, 2003:167– 186. 30. Brun RP, Kim JB, Hu E, Spiegelman BM. Peroxisome proliferator-activated receptor g and the control of adipogenesis. Curr Opin Lipidol 1997; 8:212 –218. 31. Rangwala SM, Lazar MA. Transcriptional control of adipogenesis. Annu Rev Nutr 2000; 20:535– 559.

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32. Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO, Willson TM, Kliewer SA. An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferators-activated receptor gamma (PPAR gamma). J Biol Chem 1995; 270:12953 – 12956. 33. Day C. Thiazolidinediones: a new class of antidiabetic drugs. Diabet Med 1999; 16:179– 192. 34. Walczak R, Tontonoz P. PPARadigms and PPARadoxes: expanding roles for PPARg in the control of lipid metabolism. J Lipid Res 2002; 43:177 – 186. 35. Tartaglia TA. The leptin receptor. J Biol Chem 1997; 272:6093 – 6096. 36. Reseland JE, Anderssen SA, Solvoll K, Hjerman I, Urdal P, Holme I, Drevon CA. Effect of long term changes in diet and exercise on plasma leptin concentrations. Am J Clin Nutr 2001a; 73:240 – 245. 37. Reseland JE, Haugen F, Hollung K, Solvoll K, Halvorsen B, Brude IR, Nenseter MS, Christiansen ER, Drevon CA. Reduction of leptin gene expression by dietary polyunsaturated fatty acids. J Lipid Res 2001b; 42:743– 750. 38. Witztum JL, Steinberg D. The oxidative modification hypothesis of atherosclerosis: does it hold for humans? Trends Cardiovasc Med 2001; 11:93– 102. 39. Calder PC. N-3 polyunsaturated fatty acids and cytokine production in health and disease. Annu Nutr Metab 1997; 41:203 – 234. 40. Wahle KWJ, Rotondo D. Fatty acids and endothelial cell function: regulation of adhesion molecule and redox enzyme expression. Curr Opin Clin Nutr Metab Care 1999; 2:109 –115. 41. Farquharson A, Wu H-C, Grant I, Choung J-J, Eremin O, Heys SD, Wahle KWJ. Possible mechanisms for the putative anti-atherogenic and anti-tumorigenic effects of conjugated polyenoic fatty acids. Lipids 1999; 34:S343. 42. De Caterina R, Libby P. Control of endothelial leukocyte adhesion molecules by fatty acids. Lipids 1996; 31:S57 –S63. 43. Collie Duguid ESR, Wahle KWJ. Inhibitory effect of fish oil n-3 polyunsaturated fatty acids on the expression of endothelial cell adhesion molecules. Biochem Biophys Res Commun 1996; 220:269 – 274. 44. Miles EA, Thies F, Wallace FA, Powell JR, Hurst TL, Newsholme EA, Calder PC. Influence of age and dietary fish oil on plasma soluble adhesion molecule concentrations. Clin Sci 2001; 100:91 –100. 45. Kritchevsky D, Tepper SA, Wright S, Tso P, Czarnecki SK. Influence of conjugated linoleic acid (CLA) on establishment and progression of atherosclerosis in rabbits. J Am Coll Nutr 2000; 19:427S– 477S. 46. Palmer CN, Hsu MH, Griffin KJ, Raucy JL, Johnson EF. Peroxisome proliferator activated receptor-a expression in human liver. Mol Pharmacol 1998; 53:14 – 22. 47. Chawla A, Repa JJ, Evans RM, Mangelsdorf DJ. Nuclear receptors and lipid physiology: opening the X-files. Science 2001; 294:1866– 1870. 48. Kersten S, Desvergne B, Wahli W. Role of PPAR’s in health and disease. Nature 2000; 405:421 – 424. 49. Hihi AK, Michalik L, Wahli W. PPARs: transcriptional effectors of fatty acids and their derivatives. Cell Mol Life Sci 2002; 59:790 – 798. 50. Desvergne B, Wahli W. Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocrine Rev 1999; 20:649– 688. 51. Duplus E, Forest C. Is there a single mechanism for fatty acid regulation of gene transcription? Biochem Pharmacol 2002; 64:893 – 901.

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9 Cell Regulatory Activity of Tocopherols and Tocotrienols Cristina Rota University of Modena and Reggio Emilia, Modena, Italy

Anne M. Minihane University of Reading, Reading, UK

Peter D. Weinberg Imperial College, London, UK

Stefan Weber Universita¨tsklinikum Bonn, Rheinische Friedrich-Wilhelms-Universita¨t, Bonn, Germany

John K. Lodge University of Surrey, Guildford, Surrey, UK

Lester Packer University of Southern California, Los Angeles, California, USA

Gerald Rimbach Christian Albrechts University, Kiel, Germany

Chemistry and Antioxidant Properties of VE Absorption, Transport, and Metabolism Molecular Targets of Alpha Tocopherol Protein Kinase C 201

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Cell Adhesion Proteins, Chemokines, Scavenger Receptors, and a-Tropomyosin Cyclooxygenase NO and Platelet Aggregation VE Sensitive Genes In Vivo Biological Properties of Tocotrienols Anticarcinogenic Properties Neuroprotection and Src Activity References

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Vitamin E (VE) is a potent lipid soluble antioxidant that prevents the propagation of free-radical damage in biological membranes. Tocopherols and tocotrienols are part of an interlinking set of antioxidant cycles, which has been termed the antioxidant network. Besides its antioxidant properties, cell regulatory activities of VE have been found. Advances in molecular and cell biology have led to the discovery of VE sensitive genes and underlying signal transduction pathways. In this review, antioxidant properties, absorption, and transport of VE have been examined. Furthermore, important cell culture and animal studies related to the antiantherogenic, anticarcinogenic and neuroprotective actions of VE, on a molecular level, have been summarized.

CHEMISTRY AND ANTIOXIDANT PROPERTIES OF VE VE occurs in nature in at least eight different isoforms: a-, b-, g-, and d-tocopherols and a-, b-, g-, and d-tocotrienols. The four tocopherol homologs (a-, b-, g-, d-) have a fully saturated 16-carbon phytol side chain, whereas tocotrienols have a similar isoprenoid chain containing three double bonds. Individual tocopherols are named according to the position and number of the methyl groups on the phenol ring, with the a-, b-, g-, and d-vitamins containing three, two, two, and one methyl groups, respectively (Fig. 9.1). These structural differences determine biological activity, a-homologs being the most biologically active. The majority of the functionality of VE is through its role as an antioxidant, maintaining the structural integrity of virtually all cells in the body. Its antioxidant function is mediated through the reduction of free radicals, thus protecting the body against the deleterious effects of such highly reactive oxygen species (ROS) and reactive nitrogen species (RNS), which have been implicated in the pathophysiology of ageing and a number of chronic diseases such as atherosclerosis, cancers, and rheumatoid arthritis (1 – 3). The ROS, which include hydrogen peroxide (H2O2), the superoxide radical (O22), and the highly reactive hydroxy radical ( OH), are by-products of normal aerobic †

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Cell Regulatory Activity of Tocopherols and Tocotrienols R1

tocotrienol isoforms

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H

H

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Figure 9.1 Molecular structure of VE stereoisomers. Tocotrienols consist of a chromanol nucleus and a liphiphilic isoprenoid chain. Tocopherols only differ in the side chain (phytyl). The naturally occurring isoforms a, b, g, and d have methylation patterns as indicated.

metabolism formed during the respiratory and phagocytic processes and during microsomal cytochrome P450 metabolism. The RNS include nitric oxide (NO) and peroxynitrite, formed by the reaction of NO and O22. The polyunsaturated fatty acids (PUFA) of biological membranes are particularly susceptible to free-radical attack owing to their high degree of unsaturation. In brief, the process is initiated by a free-radical such as OH, which extracts hydrogen from PUFA resulting in a PUFA radical. Following molecular rearrangement to form a conjugated diene, the molecule is susceptible to attack by molecular oxygen (O2) resulting in a peroxyl radical (PUFAOO ). Peroxyl radicals are capable of extracting a hydrogen atom from adjacent PUFA, thus propagating a chain reaction. Such auto-oxidation continues, severely affecting the functionality of the tissue, unless the free radicals are scavenged. Owing to its abundance, lipid-solubility and its efficacy with respect to radical quenching, VE is considered to be the most important antioxidant in cell membranes (1,4,5). The antioxidant property of VE is exerted through the phenolic hydroxyl group, which readily donates its hydrogen to the peroxyl radical, resulting in the formation of a stable lipid species. In donating the hydrogen atom, VE becomes a relatively unreactive free radical as the unpaired electron becomes delocalized into the aromatic ring. The efficiency of this protection depends on two factors: first, the mobility of the molecule in membranes, which is determined by the aliphatic tail and second, the number of methyl species on the chromanol ring, with each methyl group conferring additional antioxidant capacity. In addition, the proximity of the methyl species to the hydroxyl group is an important factor. Therefore, a-homologs, which have the greatest number of methyl species, and in which these flank the hydroxyl group, are thought to be more effective than the other homologs. a-Tocotrienol has been shown to be more effective in protecting against lipid peroxidation than a-tocopherol (6,7). A reason suggested for this is the nature of the aliphatic tail. The isoprenoid chain of a-tocotrienol has a stronger disordering effect on membranes than a-tocopherol. This leads to a greater †

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mobility and more uniform distribution within the membrane. Nuclear magnetic resonance studies have also shown that the chromanol ring of a-tocotrienol is situated closer to the membrane surface. These factors contribute to a greater ability of tocotrienols to interact with radicals and allow for quicker recycling of the molecule to its active reduced form (6,7). Possible explanations for the greater in vitro antioxidant activity of a-tocotrienol compared with a-tocopherol are summarized in Fig. 9.2. VE does not work in isolation from other antioxidants; rather it is part of an interlinking set of redox antioxidants, which has been termed the “antioxidant network” (8). It is hypothesized that VE acts catalytically, being efficiently reduced from its free-radical (chromanoxyl) form, that arises after quenching lipid radicals to return back to its reduced native state. This catalysis occurs through the interactions between water- and lipid-soluble substances by both nonenzymatic and enzymatic mechanisms that regenerate VE from its tocotrienoxyl or tocopheroxyl radical back to tocotrienol and tocopherol, respectively. Vitamin C can regenerate VE directly and thiol antioxidants, such as glutathione and lipoic acid, can regenerate VE indirectly via vitamin C. Under conditions where these systems act synergistically to keep the steady-state concentration of VE radicals low, the loss or consumption of VE is prevented.

ABSORPTION, TRANSPORT, AND METABOLISM To date the majority of information available on VE absorption and transport is based on tocopherol (9 –11). In the small intestine, tocopherol esters hydrolyzed to free VE species are incorporated into mixed micelles owing to the action of bile salts and pancreatic juices. Lack of these gastric secretions, as occurs in

Greater in vitro antioxidant activity of α-tocotrienol compared with α-tocopherol results from More uniform distribution in membrane bilayer

Greater recycling activity of 4. chromanoxyl radicals

1.

Recycling 5. activity correlates with inhibition of lipid peroxidation

2. Stronger

disordering of membrane lipids 3. More effective collision with radicals

Figure 9.2 Potential mechanisms by which a-tocotrienols exhibit greater antioxidant activity compared to a-tocopherols.

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individuals with conditions such as pancreatitis, cystic fibrosis, or choleostatic liver disease, leads to VE malabsorption and resultant nutrient deficiency symptoms. The micelles enter the enterocyte via passive diffusion and the tocopherols are packaged into chylomicrons along with the phospholipid, cholesterol, triglyceride, and apolipoproteins moieties. Upon entry into the circulation via the lymphatic system, the chylomicrons are sequentially hydrolyzed due to the action of lipoprotein lipase attached to the capillary endothelium in the target tissue, such as muscle and adipose tissue; a fraction of the tocopherol is released and taken up by the endothelial cells. The resulting chylomicron remnants are taken up by the liver by receptor mediated endocytotic processes. In contrast to vitamins A and D, there does not appear to be a specific carrier protein for VE in the circulation. Instead VE is incorporated into lipoprotein particles in a nonspecific manner. In hepatic cells, tocopherol from chylomicron remnants binds to cytosolic a-tocopherol transfer protein (a-TTP) (12,13), which mediates its transfer to the site of VLDL synthesis (RER and Golgi apparatus). This 32 kDa protein is expressed almost exclusively in the liver, and recent evidence from animal studies suggests that dietary a-tocopherol modulates hepatic a-TTP mRNA levels (14). Unlike tocopherol absorption, which is thought to be nonselective with respect to isomer, a-TTP displays stereoisomer specificity, with almost exclusive incorporation of a-tocopherol into the nascent VLDL particle. Relative affinities of tocopherol analogs for a-TTP, calculated from competition studies, are as follows: a-tocopherol 100%, b-tocopherol 38%, g-tocopherol 9%, and d-tocopherol 2% (13). The majority of non-a isomers are excreted via the bile (15). a-TTP is now recognized to be the primary determinant of plasma tocopherol levels. Mutations of the a-TTP gene lead to reduced plasma and tissue a-tocopherol, which may ultimately lead to a severe condition known as ataxia with VE deficiency (AVED), with associated neuronal and retinal damage (16,17). In a recent study, a-TTP knockout mice (Ttpaþ/2 and Ttpa2/2) were used as a model to examine the association between VE deficiency and atherosclerosis (18). Plasma and tissue a-tocopherols were reduced in a stepwise manner from control through Ttpaþ/2 to Ttpa2/2, with an absence of liver a-TTP in liver homogenates from Ttpa2/2 and a 50% reduction of protein level in the Ttpaþ/2 animals. The VE deficiency was associated with increased lesions in the proximal aorta and increased rates of isoprostanes, a marker of lipid peroxidation. These findings further demonstrate the role of this transfer protein on tocopherol metabolism and, ultimately, on CHD risk. Approximately 50 –70% of total secreted VLDL is hydrolyzed to low density lipoprotein (LDL) with associated transfer of tocopherols into the LDL fraction (19). In the circulation, tocopherol exchanges rapidly between the lipoprotein particles, although .90% is contained within the LDL and HDL fractions (20). The 75 kDa plasma phospholipid transfer protein facilitates tocopherol exchange between HDL and LDL (21). The mechanisms of peripheral cellular uptake of VE are poorly understood although simultaneous uptake of tocopherol, via receptor mediated lipoprotein

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endocytosis, or via fatty acid binding proteins, may be involved. However, recent evidence suggests that specific membrane tocopherol binding proteins (TBPpm) may also mediate tocopherol uptake (22). Information on intracellular tocopherol transport is currently lacking. Owing to its strong hydrophobicity, transfer to cellular sites requires a specific transfer protein. However, it is still unclear how many other a-TBPbm exist and which mechanisms regulate tocopherol transfer within peripheral cells. Recently, a novel binding protein, tocopherol-associated protein (TAP) has been identified (23–26). This 46 kDa protein, which displays significant sequence homology to a-TTP, is ubiquitously expressed although the highest levels have been observed in the liver, brain, and prostate (26). It is suggested that this protein plays a significant general role in intracellular tocopherol metabolism. Structural analysis of TAP suggested that it is a member of the widespread SEC14-like protein family, which plays a role in phospholipid exchange in the cell. Recent ligand competition studies suggest that TAP binds to a-tocopherol but not other tocopherol isomers (23). Although research is at an early stage, it is likely that TAP will prove an important molecule with respect to cellular tocopherol events. Tocopherols are metabolized by cytochrome P450 (CYP) induced omegaoxidation followed by consecutive beta-oxidation yielding carboxyethylhydroxychromans (CEHC) as the final product, which have been found both in the plasma (27) and in the urine (28). CYP4F2 appears to be the primary P450 isoform involved in the oxidation of a, and g-tocopherol (29), but the CYP3A family has also been implicated (30,31). The recent observation shows that VE can activate the pregnane X nuclear receptor (PXR) (32), which leads to expression of CYPs, suggests that VE can regulate its own metabolism. A scheme of VE absorption, transport, and metabolism is given in Fig. 9.3. The antioxidant efficacy of tocotrienols in membranes is higher than that of tocopherols, although its uptake and distribution after oral ingestion is less than that of a-tocopherol. In hamsters fed with a mixture of VE isoforms containing also tocotrienols, a-tocopherol was absorbed preferentially. However, tocotrienols could still be detected in the postprandial plasma of humans and tocotrienols were found in all classes of lipoproteins (33). Even though tocotrienols have a higher radical scavenging activity than tocopherols, they are less bioavailable after oral ingestion. It can be hypothesized that if similar tissue levels can be achieved, tocotrienols would be more effective antioxidants than tocopherols. There is some evidence supporting this hypothesis. When supplementation was carried out in such a way that comparable tissue concentrations of a-tocopherol and a-tocotrienol were reached in rat microsomes and mitochondria, tocotrienolsupplemented heart tissues were more resistant to lipid peroxidation in vitro than the tocopherol-supplemented counterparts (34). However, it has to be taken into account that tocotrienols belong to a family of plant phenolic compounds that have a brief and transient nature with respect to their metabolism, which when compared to a-tocopherol is inferior with regard to tissue retention and half life.

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Figure 9.3 Schematic representation of the delivery and intracellular transport of VE in various tissues. VE, vitamin E; CM, chylomikron; LPL, lipoprotein lipase; TTP, tocopherol transfer protein; TAP, tocopherol-associated protein; PXR, pregnane X receptor; CYP, cytochrome P450 isoforms; CEHCs, carboxyethyl-hydroxychromans.

MOLECULAR TARGETS OF ALPHA TOCOPHEROL Protein Kinase C Since the discovery of tocopherols and tocotrienols, their antioxidant properties have received most study. However, it is now clear, that the role of VE goes beyond its antioxidant function. The first observation of a cell signaling role of VE was the finding by Angelo Azzi’s group that smooth muscle cell (SMC) proliferation and protein kinase C (PKC) activities are inhibited by a-tocopherol. The inhibition of SMC proliferation was specific to a-tocopherol: trolox, phytol, b-tocopherol, and a-tocopherol esters had no effect (35,36). As a-tocopherol and b-tocopherol have very similar free-radical-scavenging activities, the mechanism by which a-tocopherol acts on PKC is not related to its antioxidant properties. Subsequent studies have shown that PKC is inhibited in a number of other cell types, including monocytes (37), neutrophils (38), fibroblasts (39), and mesangial cells (40). Most importantly, this inhibition of PKC by a-tocopherol occurs at concentrations close to those measured in human plasma (41). Antiproliferative effects of VE were not seen for HeLA cells suggesting that there are different cell-specific

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pathways of cellular proliferation in which VE can act (42). In addition, the inhibition of PKC was not related to a direct interaction of a-tocopherol with the enzyme nor with a diminution of its expression. Instead, PKC inhibition by a-tocopherol is linked to the activation of a protein phosphatase 2A, which in turn dephosphorylates PKC-a and thereby inhibits its activity (43,44). An inhibitory effect of a-tocopherol on PKC may be seen only at the cellular level and is not evident with recombinant PKC. Important milestones in experimental VE research are summarized in Fig. 9.4.

Cell Adhesion Proteins, Chemokines, Scavenger Receptors, and a-Tropomyosin Activation of endothelial cells results in release of vascular cytokines such as IL-1 and TNF-a. These cytokines in turn induce the expression of cell surface adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1), which are centrally involved in the endothelial recruitment of neutrophils (45). Focal expression of ICAM-1 and VACM-1 has been reported in arterial endothelium overlying early foam cell lesions in both dietary and genetic models of atherosclerosis in rabbits (46). This expression, together with the activation of monocyte chemoattractant protein-1 (MCP-1) leads to infiltration of mononuclear cells into the wall and, it is widely supposed, to a consequent increase in the oxidation and scavenging of

Figure 9.4

Milestones in experimental VE research.

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LDL, formation of lipid-laden foam cells, and development or progression of atherosclerotic plaques (47). Transcription of ICAM-1, VCAM-1, and MCP-1 is dependent, at least in part, on the activation of NF-kB. Cell culture studies have shown that treatment of endothelial cells with oxidized LDL significantly increases expression of mRNA and proteins levels of ICAM-1 and VCAM-1 (48). However, pretreatment with a-tocopherol reduced cell adhesion protein expression in a dose-dependent manner. Consistent with this finding, adherence of polymorphonuclear leukocytes (PMN) or mononuclear leukocytes (MNC) to endothelial cells activated by oxidized LDL (which is much higher than adherence to unstimulated endothelial cells) was reduced by supplementation of the endothelial cells with a-tocopherol. Furthermore, IL-1 beta-induced production of MCP-1 was dosedependently suppressed by enrichment of human endothelial cells with VE (49). From this and other studies, it is suggested that the putative antiatherogenic effect of a-tocopherol may in part be due to a down-regulation of cell adhesion proteins and chemokines. Despite evidence that VE down-regulates cell adhesion proteins in vitro, in vivo evidence is currently lacking. Ricciarelli et al. (50) have recently demonstrated that the CD36 scavenger receptor, which transports oxidized LDL into the cytosol, is expressed in human SMC. Interestingly, a-tocopherol inhibited the uptake of oxidized LDL by a mechanism involving down-regulation of CD36 mRNA and protein expression. It is hypothesized that beneficial cardiovascular effects of a-tocopherol are at least in part mediated by lowering the uptake of oxidized LDL, which subsequently results in a reduction of foam cell formation. An involvement of tropomyosin in the progression of restenosis has been suggested (51). Early after balloon injury, SMC of the media and those that have migrated into the intima contain decreased amounts of tropomyosin, and late after balloon injury tropomyosin returns toward normal values. In 1999, Aratri et al. (52) discovered the induction of a-tropomyosin expression in rat vascular SMC by a-tocopherol, using differential display techniques (52). No significant changes in mRNA levels were observed when b-tocopherol was used. The authors suggest that the overexpression of tropomyosin induced by a-tocopherol may decrease the contractility of SMC and hence form the molecular basis for the hypotensive effect of VE. Cyclooxygenase Cyclooxygenase has two isoforms, COX-1 and COX-2. COX-1 is constitutively expressed in most cells, whereas COX-2 is regulated by growth factors, tumor promoters, cytokines, glucocorticoids, and lipopolysaccharide (LPS). Cyclooxygenases convert arachidonic acid (AA) into prostaglandin E2 (PGE2), the precursor of thromboxane and eicosanoid synthesis. High levels of COX-2 in epithelial cells are associated with the inhibition of apoptosis, and overexpression of COX-2 has been implicated in the pathogenesis of neoplastic diseases. An upregulation

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of COX-2 transcription has been shown in most human colorectal cancers (53). Interestingly, changes in AA metabolism stimulate cell proliferation via activation of PKC, indicating that PKC might be one of the primary signaling pathways through which certain tumors are initiated or maintained. In recent years, a role of COX-2 in atherogenesis has been identified. Immunocytochemical studies using anti-COX-2 showed that COX-2 is localized to macrophages in atherosclerotic lesions of patients with coronary artery disease (54). In monocytes derived from aged mice, it has been shown that a VE-induced decrease in PGE2 production is mediated via a decreased COX activity (55). However, VE has no effect on COX mRNA and protein levels, indicating a posttranslational regulation of the COX enzyme. Non-a-tocopherol homologs were, like b-tocopherol, effective in inhibiting COX activity, but the degree of inhibition varied in proportion to their antioxidant capacity, suggesting that an antioxidant mechanism may be involved. It has been shown in LPS stimulated RAW264.7 macrophages and IL-1beta treated A549 human epithelial cells that gamma tocopherols inhibited the production of PGE2 owing to a direct inhibition of COX-2 (56). Furthermore, the major metabolite of dietary gamma tocopherols also exhibited an inhibitory effect in these cells. In contrast, a-tocopherol at 50 mM slightly reduced (25%) PGE2 formation in macrophages, but had no effect in epithelial cells. Similar to the previously mentioned study, the inhibitory effect of g-tocopherol and g-CEHC stemmed from their inhibition of COX-2 activity, rather than from affecting protein expression or substrate availability and appeared to be independent of their antioxidant activity. NO and Platelet Aggregation NO produced by the endothelial NO synthase (eNOS) is a pivotal molecule in the regulation of vascular tone. In addition, its production suppresses expression of proinflammatory cytokines, adhesion molecules (57), and MCP-1 (58). It also inhibits platelet adhesion to the endothelium (59), can modify the permeability of the arterial wall (60), suppresses vascular SMC proliferation and migration (61), and can act as an antioxidant (62). The major risk factors for atherosclerosis—age (63), hypercholesterolaemia (64), diabetes (65), hypertension (66), smoking (67), and low birth weight (68)—are all associated with impaired NO activity, often before appreciable disease develops. In rabbits, inhibition of NO production accelerates experimental atherosclerosis (69), whereas increase in NO synthesis reduces it (70). Importantly, NO significantly inhibits NF-kB (71). This may account for its influence on the transcription of genes for adhesion proteins, MCP-1 and others. The postulation of key roles for both NO and NF-kB is therefore not self-contradictory. There is evidence from studies in rabbits that VE reverses the well established deleterious effects of hypercholesterolaemia on NO activity. Stewart-Lee et al. (72) found that relaxation in response to acetylcholine, an

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NO-dependent phenomenon, in the carotid artery was reduced after 4 weeks of diet-induced hypercholesterolaemia but was restored by the addition of 0.2% a-tocopherol acetate to the diet. Andersson et al. (73) obtained a similar result for the coronary circulation. The mechanisms underlying this effect are a matter of controversy. It has been suggested that inactivation of NO by ROS is increased during hypercholesterolaemia and reduced by VE (72,73). However, Bo¨ger et al. (74) found that VE did not reduce ROS release by aortic tissue from cholesterolfed rabbits; instead, they suggested its protective effect on the NO pathway was related to its inhibition of LDL oxidation. Since PKC inhibits NO (75), another possible mechanism arises from the observation that hypercholesterolemia increases PKC levels in rabbit aortic smooth muscle and this is reduced by VE (76). Whatever the mechanism is, protective effects of VE on NO function might be expected to reduce atherosclerosis in hypercholesterolemic rabbits. Although many studies have found such an effect (74,77,78), others have shown an increase in the extent and severity of lesions (79). Keaney et al. (80) obtained an interesting result which may in part account for these discrepancies: although 1000 IU a-tocopherol/kg diet protected against the inhibitory effect of hypercholesterolemia on the NO pathway, 10,000 IU a-tocopherol/kg diet markedly increased it, and also increased the severity of lesions, despite the fact that the oxidizability of LDL was still reduced. Possible mechanisms include pro-oxidant effects of a-tocopherol, or reactions of a-tocopherol with NO to give the tocopheroxyl radical. Li et al. (81) studied the effect of different isoforms of VE on NO activity and platelet aggregation in human platelet rich plasma. All three isoforms tested (a-, b-, or d-tocopherol) markedly decreased ADP-induced platelet aggregation and increased NO release in a dose-dependent manner. The isoforms did not affect cNOS protein expression, but increased cNOS phosphorylation. Furthermore, it has been demonstrated in human subjects that oral supplementation with a-tocopherol (400 – 1200 IU/day) resulted in an increase in platelet tocopherol concentration that correlated with marked inhibition of PMA-mediated platelet aggregation (82). Platelets derived from these subjects also demonstrated apparent complete inhibition of PKC. These findings represent another potential mechanism by which tocopherol could prevent the development of coronary artery disease. Several mechanisms by which VE may prevent coronary artery disease are summarized in Fig. 9.5. VE Sensitive Genes In Vivo Barella et al. (83) monitored differential gene expression in rat liver over a period of 9 months. Similar to findings in cultured cells, VE supplementation down-regulated scavenger receptor CD36. Furthermore, coagulation factor IX and 5-alphasteroid reductase type 1 mRNA levels where down-regulated while hepatic gamma glutamyl–cysteinyl synthetase was significantly up-regulated in response to dietary VE. Measurement of the corresponding biological endpoints such as

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Molecular mechanisms for the anti-atherogenic activity of VE.

activated partial thromboplastin time, plasma dihydrotestosterone, and hepatic glutathione substantiated the gene chip data, which indicated that dietary VE plays an important role in a range of metabolic processes within the liver. Gene-chip technology was also employed to study the effect of dietary VE on gene expression in rat testes (84). Differential gene expression was monitored at five individual time-points over a period of 14 months with all animals individually profiled. Low VE intake resulted in the consistent up-regulation of 7-dehydrocholesterol reductase and GATA binding protein 4, both involved in testosterone synthesis. Cyclin D3, important in cell cycle progression and Wilms tumor 1, related to cancer development, was also up-regulated in the VE deficient animals. Furthermore, in the rat hippocampus, VE exhibited a significant impact on the expression of genes associated with hormones and hormone metabolism (e.g., growth hormone, thyroid hormones, prolactin, and melatonin), nerve growth factor, apoptosis, dopaminergic neurotransmission, and Abeta and AGEs clearance. In particular, VE strongly affected the expression of an array of genes encoding for proteins directly or indirectly involved in the clearance of amyloid beta, changes that are consistent with a protective effect of VE on AD progression, independent of its role as an antioxidant. Gohil et al. (85) explored genome-wide changes in mRNAs from brain cortex and liver of TTP deficient, a genetic model of VE deficiency. Selective inductions of genes regulated by antioxidant response elements were detected in liver of TTP2mice, suggesting increased oxidant stress. Cortex of TTP2 mice revealed repression of genes encoding synaptic proteins, PKC family members, and myelin proteins. A significant decrease in the expression of retinoic acid receptor-related orphan receptor-alpha mRNA indicated staggererlike phenotype including ataxia and deficits of motor coordination of TTP-mice. Overall, advances in microarray technology have enabled us to investigate genes differentially expressed in response to tocopherols in cultured cell and

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in vivo thereby offering the possibility of more insight into the biological properties of VE. DNA arrays are important experimental platforms and might help to address tocoperhols and tocotrienols more specifically with regard to their molecular biological functions. BIOLOGICAL PROPERTIES OF TOCOTRIENOLS Anticarcinogenic Properties Tocotrienols belong to a phytochemical class of isoprenoid molecules. These compounds share a common precursor, mevalonic acid. Tocotrienols are mixed isoprenoids, meaning that only a part, the lipophilic chain is derived via the isoprenoid pathway. Isoprenoids have been shown to exhibit anticarcinogenic properties. When different VE isoforms were analyzed, it could be demonstrated that a-tocopherol and a-tocotrienol inhibited tumor promotion in Raji-cells most effectively (86). Tocotrienols from TRF inhibited the proliferation of human breast cancer cell lines (87,88). The inhibition was found to be independent of the estrogen receptor status of the cell lines (89). Isoprenoids, among them tocotrienols also suppressed the growth of murine B16 melanomas in vitro and in vivo (90). Interestingly, correlations between the late stage tumor-suppressive potency of diverse isoprenoids and their effect of HMG CoA reductase activity approached unity. It is hypothesized that VE might exert antiproliferative properties by interfering with signal transduction events involving PKC. It has been shown that a-tocopherol inhibits the proliferation of SMC by the inhibition of PKC (91). This effect was specific for a-tocopherol as opposed to the isoform b-tocopherol (92). There is no information, however, on the potency of a-tocotrienol on PKC-activity, which shares the structure of the chromanol nucleus with a-tocopherol. Recently, it has been reported that isoprenoids, including tocotrienols, induce cell-cycle arrest in G-1 phase and apoptosis in human and murine tumor cells (93). As these effects can be observed with different isoprenoid, which are not antioxidants, it is possible that the anticarcinogenic effects of tocotrienols are not necessarily related to their antioxidant properties. Neuroprotection and Src Activity Elevated levels of glutamate have been implicated in a wide range of neurological diseases, including epilepsy, cerebral ischemia, Huntington’s disease, and Parkinson’s disease. Receptor-mediated glutamate exicitotoxicity is believed to be a major mechanism of damage in these pathologies and induction of oxidative stress by glutamate has been demonstrated to be the primary cytotoxic mechanisms in cell lines such as C6 glial cells (94), PC-12 neuronal cells (95), immature cortical neuron cells (96), and oligodendroglia cells (97). It has been demonstrated that high glutamate levels block cystine uptake via amino acid transporter Xc2 resulting in a significant depletion of cellular GSH. A glutathione-depleted state impairs cellular antioxidant defenses, followed by an increased vulnerability

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of the cell to ROS. The mitochondrial electron transport chain has been shown to be a source of ROS production during glutamate-induced apoptosis (98). Recently, VE isoforms were tested in a model of neuronal cell death, where HT4 neuronal cells were challenged with glutamate (99). Tocotrienols counteracted glutamate-induced cell death at much lower concentrations than tocopherols. Moreover, tocotrienols effectively inhibited the activation of pp60 c-Src kinase, a kinase that is centrally involved in glutamate-induced cell death. It is hypothesized that these protective effects of tocotrienols are probably independent of their antioxidant activity, because tocopherols were effective only at multi-fold higher concentration (99). The activity of Src kinase has also been shown in the progression of breast cancer (100). Moreover, elevated levels of Src kinase have been found in human skin tumors (101). Owing to the key involvement of Src kinase in neurodegenerative diseases and oncogenesis, inhibition of these kinases would seem to be a likely basis for developing a strategy to create neuroprotective and anticancer drugs. It has been recently shown that tocotrienol modulates 12-lipoxygenase, a key mediator of glutamate-induced neurodegeneration (102). Interestingly, BL15, an inhibitor of 12-LOX, prevented glutamate-induced neurotoxicity. Moreover, neurons isolated from 12-LOXdeficient mice were observed to be resistant to glutamate-induced death. REFERENCES 1. Halliwell B. Antioxidants in human health and disease. Annu Rev Nutr 1996; 16:33– 50. 2. Malins DC, Johnson PM, Wheeler TM, Barker EA, Polissar NL, Vinson MA. Agerelated radical-induced DNA damage is linked to prostate cancer. Cancer Res 2001; 61:6025 –6028. 3. Parthasarathy S, Santanam N, Ramachandran S, Meilhac O. Oxidants and antioxidants in atherogenesis. An appraisal. J Lipid Res 1999; 40:2143– 2157. 4. Brigelius-Flohe R, Traber MG. Vitamin E: function and metabolism. FASEB J 1999; 13:1145 –1155. 5. Ingold KU, Webb AC, Witter D, Burton GW, Metcalfe TA, Muller DP. Vitamin E remains the major lipid-soluble, chain-breaking antioxidant in human plasma even in individuals suffering severe vitamin E deficiency. Arch Biochem Biophys 1987; 259:224 –225. 6. Serbinova E, Kagan V, Han D, Packer L. Free radical recycling and intramembrane mobility in the antioxidant properties of alpha-tocopherol and alpha-tocotrienol. Free Radic Biol Med 1991; 10:263 – 275. 7. Suzuki YJ, Tsuchiya M, Wassall SR, Choo YM, Govil G, Kagan VE, Packer L. Structural and dynamic membrane properties of alpha-tocopherol and alphatocotrienol: implication to the molecular mechanism of their antioxidant potency. Biochemistry 1993; 32:10692 – 10699. 8. Packer L, Weber SU, Rimbach G. Molecular aspects of alpha-tocotrienol antioxidant action and cell signalling. J Nutr 2001; 131:369S – 373S. 9. Cohn W, Gross P, Grun H, Loechleiter F, Muller DP, Zulauf M. Tocopherol transport and absorption. Proc Nutr Soc 1992; 51:179– 188.

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10 Molecular Analysis of the Vitamin A Biosynthetic Pathway Johannes von Lintig University of Freiburg, Freiburg, Germany

Carotenoids and Apocarotenoids: Colors with Functions Vitamin A Functions in Animal Physiology Molecular Identification of b,b-Carotene-15,150 -mono-oxygenases In Vertebrates Three Different VP14-Homologs Exist Molecular Analyses of the Vitamin A Biosynthetic Pathway in Model Organism What Can We Learn from Insects? Molecular Analysis of the Vitamin A Biosynthetic Pathway in Vertebrates Conclusions References

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Elucidating the physiological roles of lipids has long been a major concern in both biology and medicine. Today, we know that lipids are not only a source of energy, but are also essential for the growth and development of the organism. Fat-soluble vitamins and other lipids serve as precursors for ligands that bind to receptors in the nucleus. These receptors, in response to ligand binding, regulate a complex network of gene activities (1). To become biologically active, dietary 221

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lipids must first be absorbed by the intestine and transformed by metabolic enzymes for delivery to their sites of action in the body. The co-ordination of all these processes involves a large number of binding proteins, transporters, and metabolizing enzymes, which contribute to lipid homeostasis. Carotenoids are essential lipids in the human diet. They are not only the natural precursors (provitamin A) for retinoids (vitamin A and its derivatives), but also exert biological function per se. Despite their importance, the molecular components involved in carotenoid metabolism have remained elusive for a long time. This chapter will focus on recent advances in this field of research. The use of model organisms such as the fruit fly Drosophila melanogaster provided first insights into basic principles of this metabolism. The increasing amount of DNA sequence information from different genomes then led to the molecular identification and cloning of homologous genes in vertebrates including human. With this tool in hand, old questions in carotenoid research could finally begin to be addressed definitively on the molecular level, contributing to a mechanistic understanding, for example, the regulation of vitamin A homeostasis or tissue specificity of vitamin A formation, with impact on animal physiology and human health. CAROTENOIDS AND APOCAROTENOIDS: COLORS WITH FUNCTIONS We all are familiar with carotenoids because of the yellow to red colors of fruits, flowers, and vegetables. These compounds are C40 isoprenoids synthesized in plants, certain fungi, and bacteria. Besides pure carbohydrates, this class of compounds comprises hydroxylated and epoxydated derivatives, the so-called xanthophylls (2). The characteristic chemical and physical properties of carotenoids are responsible for light absorption as well as for the inactivation of free radicals, properties which play a crucial role for their diverse biological functions. Carotenoids are essential components of the photosynthetic apparatus by being constituents of the reaction centers as well as of the light-harvesting complexes (3). In addition, these pigments contribute to the color of several plant tissues which accumulate carotenoids to a high extent. Well-known examples are the fruits of pepper and tomato, the flowers of daffodils and dandelions, or the roots of carrots. Among the various classes of pigments found in nature, carotenoids are the most wide spread, with important functions not just restricted to carotenoid-producing organisms. Some animals use dietary carotenoids for coloration. Well-known examples are the feathers of flamingos and the red color of salmon. Because of their antioxidative properties, beneficial effects of carotenoids have been reported in the prevention of coronary heart disease, certain kinds of cancer, and age-related macular degeneration in humans (4). Besides the physiological functions exerted by carotenoids per se, they are the precursors for apocarotenoid synthesis. These wide-spread carotenoidderivatives also exert key biological functions. In plants, they play roles as

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hormones, pigments, flavors, aromas, and defense compounds. Probably, the best known example is the plant hormone abscisic acid (ABA)—a C15 compound derived from the cleavage of the 11,12 double bond of 9-cis-violaxanthin and 9-cis-neoxanthin (5). Plant apocarotenoids are also of some economic value, for example, bixin (annatto) is commonly used as a natural food colorant and crocin is the main pigment in saffron. In animals, retinoids (vitamin A and its derivatives) are C20 apocarotenoids, which can exert vital functions in a panoply of different physiological processes as diverse as vision, pattern formation during embryonic development and metabolic control. The diverse assortment of apocarotenoids found in nature arises from the large number of carotenoids (.600), variations in the cleavage site, and modifications of the primary cleavage products. The pathways for apocarotenoid formation have two characteristics in common: the starting compound is a C40 carotenoid and the first product is an aldehyde (Fig. 10.1). These commonalties suggest that the corresponding enzymes catalyze the oxidative cleavage in a common reaction mechanism. By analyzing the molecular basis of the ABAdeficient phenotype of the maize vp14 mutant, Schwartz et al. (6) cloned for the first time a gene encoding a carotenoid-cleaving enzyme. The heterologously expressed and purified recombinant enzyme catalyzes the oxidative cleavage of 9-cis-epoxycarotenoids such as neoxanthin and violaxanthin to form xanthoxin, the direct precursor of ABA. Maize vp14 belongs to an emerging gene family of putative carotenoid-cleavage enzymes found in animals, plants, and bacteria (7). The genomes of the plant Arabidopsis thaliana, the fruit fly D. melanogaster, and humans were found to encode nine, one, and three homologs, respectively. This led to the hypothesis that some of these homologues also catalyze the oxidative cleavage of carotenoids. Indeed, we showed that the maize vp14 homologs of animals encode carotenoid-cleavage enzymes essentially involved in vitamin A metabolism (8 –10). VITAMIN A FUNCTIONS IN ANIMAL PHYSIOLOGY In humans, vitamin A deficiency (VAD) leads to night blindness in milder forms, whereas more severe progression results in corneal malformations, for example, xerophthalmia. Besides visual defects, this deficiency affects the immune system, leads to infertility, or causes malformations during embryogenesis. The molecular basis for these diverse effects is found in the dual role exerted by vitamin A derivatives in animal physiology. In visual systems, retinal or closely related compounds such as 3-hydroxy-retinal serve as the chromophores of the visual pigments (rhodopsin) (11,12). In vertebrates, the vitamin A derivative retinoic acid (RA) is also a major signal controlling a wide range of biological processes. RA is the ligand of two classes of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs) (13 –16). The active receptor complex, involved in processes as diverse as pattern formation during embryonic development, cell differentiation, and control of metabolic activity, is a

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Figure 10.1 Proposed pathways for apocarotenoid biosynthesis (7 and references cited herein). (A) ABA biosynthesis, (B) zeaxanthin oxidation in saffron styles, and (C) vitamin A biosynthesis. VP14, 9-cis-violaxanthin-oxygenase; BCO, b,b-carotene15,150 -oxygenase; ZCD, zeaxanthin cleavage oxygenase.

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RAR/RXR heterodimer that binds DNA regulatory sequences and regulates gene transcription in response to RA-binding. RXR is not only the heterodimer partner of the RAR receptor, but also an obligate partner for other nuclear receptors (orphan receptors), controlling a wide range of activities in lipid metabolism (1). VAD is a major problem leading to blindness and childhood mortality, particularly in developing countries (17). The demand for this vitamin can be satisfied by the natural content of either animal or plant food sources. This phenomenon was first explained by Moore in 1930 (18). He described a conversion of b-carotene to vitamin A in the small intestine, providing the evidence that a plant-derived carotenoid is the direct precursor for vitamin A in animals. Today, we know that all naturally occurring vitamin A derives from carotenoids which exert provitamin A activity and that the world’s population mainly relies on plant carotenoids from staple foods to satisfy their vitamin A requirement. For vitamin A formation, a central cleavage mechanism at the C-15,C-150 double bond for the conversion of b-carotene to vitamin A was initially proposed by Karrer et al. (19). In 1954, Glover proposed an eccentric cleavage reaction and a stepwise process, leading ultimately to only one mole vitamin A per mole carotene consumed (20). Evidence for this eccentric cleavage was provided by the observation that radioactive b-apocarotenals were converted in mammals to vitamin A esters with the release of “small” radioactive fragments (21). Goodman and Huang (22) and Olson and Hyaishi (23) first described an enzymatic activity in cell-free homogenates from rat’s small intestine which catalyzed provitamin A conversion. These analyses showed that b-carotene is enzymatically cleaved at the central C-15,C-150 double bond to yield two molecules of vitamin A aldehyde (retinal). The enzymatic activity depended on molecular oxygen and thus the enzyme was termed b,b-carotene-15,150 -oxygenase (BCO). It was reported to be soluble, to have a slightly alkaline pH-optimum, and to be inhibited by ferrous iron chelators and by sulfhydryl-binding compounds, indicating that it contains a ferrous iron co-factor (24). Subsequently, this enzyme was also characterized in different mammalian species (25,26) and substrate specificity was determined for different b-carotene stereoisomers (27). Recent analysis dealing with the mode of action of BCO provided strong evidence that oxidative cleavage at the central (15,150 ) double bond is catalyzed in a mono-oxygenase mechanism via a transient carotene epoxide (28). After the description of this enzyme, it was generally assumed that the centric cleavage reaction constitutes the major step in vitamin A synthesis. However, the eccentric cleavage of b-carotene was subsequently also demonstrated in cell-free homogenates of mammals (29,30). Furthermore, it was shown that the resulting long-chain apocarotenoids (.C20) are shortened to RA in a stepwise process which is most probably mechanistically related to the b-oxidation of fatty acids (31,32). Although several endeavors were undertaken, and highly enriched enzyme fractions could be obtained, all attempts to purify these enzymes to homogeneity failed over 40 years of research.

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MOLECULAR IDENTIFICATION OF b,b-CAROTENE15,150 -MONO-OXYGENASES In plants, the first carotenoid cleavage enzyme, VP14, was molecularly identified and characterized by the analysis of an ABA-deficient maize mutant (6). Thus, we pursued the hypothesis that a VP14-related enzyme catalyzes carotenoidcleavage in animals as well. For the cloning of an animal BCO, we employed an expression cloning strategy using Escherichia coli cells genetically engineered to synthesize b-carotene de novo. We searched the entire expression database and found several expressed sequence tags in animals with some weak sequence similarity to plant VP14. After cloning the full-length cDNAs, we expressed the corresponding proteins in the E. coli test system. Upon expression of a VP14 homolog from D. melanogaster, the recipient bacterial strain bleached, thus indicating that retinoids were formed at the expense of orange b-carotene and this was confirmed by HPLC-analyses. The enzymatic properties of the purified recombinant Drosophila enzyme revealed that it exclusively catalyzed the centric cleavage of b-carotene (C40) to yield retinal (C20). Thus, we molecularly identified and functionally characterized for the first time a BCO (8). Confirming that this type of enzyme generally catalyzes in metazoans, the first step in vitamin A metabolism was provided by Wyss et al. (33,34). In an independent approach, they succeeded in the molecular cloning and functional characterization of a BCO from chicken. Their approach relied on partial protein purification and determination of peptide sequences, then using this information to synthesize oligonucleotide primers to generate a partial cDNA and screen a cDNA library derived from small intestine. Amino acid sequence comparison between the Drosophila and chicken BCOs showed an overall similarity with several highly conserved regions and a significant similarity to some domains of the plant carotenoid oxygenase VP14 (35). By sequence similarity to the so far identified genes from Drosophila and chicken, their counterparts from mouse and human were identified and functionally characterized in several laboratories (36 – 39). Using the carotenoidaccumulating E. coli test system or by in vitro assays for enzymatic activity with the purified recombinant protein, it was shown that these mammalian homologs catalyze exclusively the centric oxidative cleavage of b-carotene to yield retinal. Expression of the murine BCO in various carotenoid-accumulating E. coli revealed the cleavage of not only carotenoid substrates such as b- and a-carotene but also lycopene, resulting in the last case in the formation of acyclic retinoids (36). The purified recombinant BCO, however, catalyzed only the cleavage of carotenoid substrates with at least one unsubstituted b-ionone ring, such as b-carotene and b-cryptoxanthin, and there was no significant cleavage of lycopene or zeaxanthin (39). The Km values for b-carotene were estimated to be in the range of 1–10 mM for BCOs from the different species (8,36,37,39). BCO exhibits a slightly alkaline pH-optimum, and enzymatic activity is sensitive to chelating agents such as o-phenanthroline and a,a0 -bipyridyl,

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indicating that it depends on ferrous iron (8,39). Thus, the purified recombinant BCOs share biochemical properties which have been already described for the native BCOs. Purification of the recombinant BCO fusion proteins by affinity chromatography was achieved without the addition of detergents. This characteristic and the predicted amino acid sequences of the various BCOs indicate that we are dealing with hydrophilic, nonmembrane-bound proteins. Indeed, a cytosolic localization of the native BCO was recently demonstrated for its human representative (39). Therefore, in vitro tests for enzymatic activity must be conducted in the presence of detergents to mimic the interaction between the enzyme and its insoluble substrate. However, in vivo, the cytosolic localization of BCO may require specific binding proteins to deliver the carotenoid substrate as well as to pick up the retinoid product, since both are highly lipophilic compounds. On the product side, three different types of cellular retinoid-binding proteins (CRBP I– III) have been characterized (40 and references therein). However, no direct protein –protein interaction between a recombinant murine BCO – GST fusion protein and CRBPs could be detected in pull-down experiments (37). Even though these results argue against a tight protein –protein interaction of CRBP with BCO, it seems likely that CRBPs may facilitate b-carotene cleavage by binding retinal. In mouse testis homogenates, a L -lactate dehydrogenase C was identified and shown to interact specifically with BCO. So far, the exact physiological role of this type of alcohol dehydrogenase is not known, and there is no experimental evidence that this enzyme catalyzes either the oxidation or reduction of aldehydes like retinal. Hence, it remains to be elucidated whether BCO may interact tissue-specifically with a certain subset of proteins involved in retinoid metabolism. Such proteins might control the metabolic flow of the primary cleavage product retinal, either to retinol formation for vitamin A transport and storage, or in the direction of RA formation for retinoid-signaling. To sum up, this recent research led to the molecular identification of BCOs from various metazoan species. The recombinant enzymes share common biochemical properties with the native BCO from tissue homogenates. On the basis of its structural and biochemical properties, BCO from animals belongs to an ancient family of nonheme iron oxygenases, heretofore described only in plants and micro-organisms.

IN VERTEBRATES THREE DIFFERENT VP14-HOMOLOGS EXIST The dual functions of vitamin A in vision (retinal) as well as in development and cell differentiation (RA) indicate that vertebrate retinoid metabolism is quite complex. The need for a close co-ordination of the entire vitamin A metabolism is reflected in the large number of different retinoid-modifying enzymes, such as various retinoid-oxidizing enzymes, as well as intracellular and extracellular retinoid-binding proteins.

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In Drosophila, with vitamin A functions being restricted to vision, only one family member of nonheme iron carotenoid cleavage enzymes is found in the entire genome (10). In vertebrates, however, besides BCO, two additional family members, RPE65 and b,b-carotene-90 ,100 -oxygenase (BCOII), exist (9,41). In humans, bco, rpe65, and bcoII map to the genomic positions 16q21, 1q31, and 11q23, respectively. The existence of these bco homologs indicated that they are related to specific carotenoid/retinoid functions of vertebrates. RPE65 was first described as an abundant protein of the retinal pigment epithelium (RPE) with a molecular mass of 65 kDa (41). This epithelium is crucially involved in vitamin A metabolism of the eyes, namely in a process called the visual cycle. In this multistep pathway, all-trans retinol is converted to 11-cis retinal, which is the photosensitive vitamin A analog of the visual pigments. Mutations in the RPE65 gene are responsible for a severe form of autosomal recessive childhood-onset retinal dystrophy, Leber’s congenital amaurosis (42 – 44). A direct involvement of RPE65 in the visual cycle was indicated by the analysis of Rpe65-deficient mice which accumulate all-trans-retinyl esters in the RPE, as intermediates of the visual cycle. All-trans-retinyl esters are substrates for the isomerohydrolase reaction (45). This molecularly uncharacterized enzyme(s) catalyzes 11-cis retinol formation from all-trans retinyl esters in a combined hydrolase/isomerase reaction (46). Even though RPE65 seems to be crucial for this reaction, the recombinant RPE65 lacks isomerohydrolase activity in vitro. Recent biochemical studies suggest that RPE65 binds stereospecifically all-trans retinyl ester and stimulates the intrinsic isomerohydrolase activity of RPE membranes (47,48). Thus, it was proposed that RPE65 is an all-transretinyl ester binding protein. Since RPE65 shares overall sequence similarities to BCO and other members of this nonheme iron oxygenase family, it remains to be elucidated whether this representative is just a binding protein or an enzymatically active component of the carotenoid/retinoid metabolism in the eyes. Besides RPE65, an additional in silico predicted putative family member, BCOII, was found in the database (9). Upon cloning the full-length cDNA of a murine BcoII, sequence analyses revealed that it encodes a protein of 532 amino acids. The deduced amino acid sequence shares 40% sequence identity with murine BCO. Expression in the E. coli test system revealed that this enzyme specifically catalyzes the cleavage of b-carotene at the C-90 ,C-100 double bond, resulting in the formation of one molecule of b-100 -apocarotenal and one molecule of b-ionone (9). To establish the occurrence of this type of caroteneoxygenase in other vertebrates, we manage to clone cDNAs encoding this BCOII in human and zebrafish. The molecular identification and functional characterization of BCOII in several vertebrate species provide strong evidence that, besides centric (C-15,C-150 ), an additional eccentric (C-90 ,C-100 ) cleavage pathway for b-carotene exists in vertebrates. To sum up, in vertebrates a small gene family of VP14-homolog exists. On the level of the deduced amino acid sequences, all three representatives in a given vertebrate genome possess several common structural features: six histidine

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residues at conserved positions, which may be involved in binding the co-factor ferrous iron; and a well-conserved domain EDDGVVLSSXVVS close to the C-terminus, which can be considered a family signature sequence. Furthermore, sequence comparison revealed that the three different representatives from vertebrates all have a higher degree of similarity to each other than to Drosophila BCO. With the emerging number of sequences for carotenoid cleaving enzymes not only from animals but also from plants now becoming available in the public database, this information can be used to define their catalytic domains and identify their active sites. MOLECULAR ANALYSES OF THE VITAMIN A BIOSYNTHETIC PATHWAY IN MODEL ORGANISM In the past few years, a large number of different components of retinoid metabolism were molecularly identified (49). By reverse genetics, animal models were established with mutations in these genes. This strategy proved to be extremely powerful to learn more about single aspects of the pleiotropic effects of this vitamin, for example, it could be demonstrated that a co-ordinated expression of RA synthesizing and catabolizing enzymes is crucial to fine-tune RA-signaling in the embryo. Furthermore, natural mutations in the genes necessary for the metabolism of vitamin A (visual cycle) in the eyes have recently emerged as an important class of genetic defects responsible for a wide range of retinal dystrophies and dysfunctions in humans (50). The formal first step in retinoid metabolism is the conversion of the provitamin to the actual vitamin. The recent molecular cloning of carotene-oxygenases provided molecular tools to analyze the impact of provitamin A conversion on retinoid metabolism in more functional detail. In the following text, new results from genetically well-defined model organisms will be discussed. The use of these models proved to be a promising strategy to learn more about single aspects of the vitamin A biosynthetic pathway. These analyses demonstrated that BCO catalyzes provitamin A conversion in vivo and revealed the existence of a specific protein-mediated transport mechanism for carotenoids in animals. WHAT CAN WE LEARN FROM INSECTS? The completed human and Drosophila genome projects have revealed that 60% of the genes of Drosophila possess homologs in the human genome. For a long time, the fruit fly has served as a model for functional genomics, and a multitude of investigations led to the identification of genes involved in the visual process and the elucidation of their functions (51). Furthermore, with vitamin A functions being restricted to vision, Drosophila represents an excellent model for the genetic dissection of the pathway leading from dietary carotenoids to vitamin A. Among the various Drosophila mutants affected in their visual performance, the phenotype of five blind mutants could be assigned because of

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their characteristic electroretinograms to the so-called neither inactivation nor after potential (nina) phenotype. This phenotype is caused by a lack of functional visual pigments (rhodopsin) in the compound eyes of these fly mutants (52). Like in all animals, these visual pigments consist of a protein moiety, the opsin, to which an 11-cis-retinal derivative is covalently bound via a Schiff base linkage. The molecular reason for this phenotype was already determined for three (ninaA, ninaC, ninaE) of these five nina-mutants, which all affected different aspects of the synthesis of the protein moiety of the visual pigments (53 – 55). The ninaB and ninaD mutants, however, feature a characteristic not found in the three other nina-mutants. Their visual performance can be rescued by feeding these flies retinal. Thus, the ninaB and ninaD genes were promising candidates to encode molecular components in the synthesis of the visual chromophore from dietary carotenoids, the sole precursor for vitamin A, in Drosophila standard growth medium. The existence of two different mutants with a VAD phenotype indicated that at least two different genes were involved in the formation of vitamin A in insects. The ninaB mutation has been cytologically mapped on chromosome 3 to the position 87E-F in the Drosophila genome (52), coinciding with the physical location of the Drosophila bco gene. We performed detailed molecular analyses of this gene locus and found mutations in the bco gene in two independent ninaB fly stocks, which we showed to abolish the BCO function. Thus, the blind vitamin A deficient phenotype of ninaB flies is caused by mutations in the bco gene, providing the first direct genetic evidence that BCO actually catalyzes vitamin A synthesis in vivo (10). Since only this one representative of the nonheme iron carotenoid-oxygenase gene family is encoded in the entire Drosophila genome, centric cleavage of carotenoids may represent the universal pathway for the synthesis of vitamin A in metazoans. The ninaB gene is expressed exclusively in close spatial vicinity of the photoreceptor cells, indicating that carotenoids must be transported and delivered to BCO-expressing cells for vitamin A synthesis. Carotenoids with provitamin A activity are highly lipophilic molecules, indicating that, as with other lipids, specific binding/transport proteins may exist. In the second vitamin A-deficient Drosophila mutant, ninaD, the carotenoid content was shown to be significantly altered when compared with wild-type flies and ineffective in mediating visual pigment synthesis (56). Our molecular analyses revealed that this phenotype is caused by a defect in the uptake and body distribution of dietary carotenoids (57). The ninaD gene encodes a cellular surface receptor with significant sequence similarity to the mammalian class B scavenger receptor, SR-BI. This receptor plays a key role in HDL-metabolism in mammals (58). In ninaD flies, we found a nonsense mutation in the gene encoding this receptor, thus abolishing its function (57). Direct functional evidence for the role of the ninaD receptor in cellular carotenoid uptake was provided by P-element mediated transformation of flies with a wild-type ninaD allele. Heat-shock induced expression of the wild-type allele in the genetic background of ninaD flies restored carotenoid

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uptake and visual pigment synthesis (57). These analyses provide genetic and functional evidence that carotenoids are distributed to target tissues within the body by protein-mediated transport processes. It has also been reported that in mice deficient for SR-BI, the ninaD homologous receptor, vitamin E metabolism is impaired, resulting in an elevated plasma concentration of this vitamin (59). Together with the results from Drosophila, this finding may indicate that this type of receptor is more generally involved in the metabolism of fat soluble vitamins. Insight into the molecular structure of a cellular carotenoid-binding protein (CBP) comes from the silkworm Bombyx mori. Tabunoki et al. (60) purified a lutein-binding protein from the silk gland of this insect and cloned the corresponding cDNA. This insect CBP has an apparent molecular mass of 33 kDa and binds carotenoids in a 1:1 molar ratio. Sequence comparison revealed that CBP is a new member of the steroidogenic acute regulatory (StAR) protein family. In mammals, these proteins are known as soluble protein carriers mediating the intracellular transport of lipids (61). An example is StAR/StarD1, which delivers cholesterol to mitochondrial P450 side chain cleavage enzymes in steroidogenic cells. The other family members are characterized by the 200 –300 amino acid StAR-related lipid transfer domain with homology to StarD1. MLN64/StarD3 has been also shown in vitro to bind cholesterol, whereas Star2 binds phosphatidylcholine. There are several more family members for which the ligand is so far unknown (62). On the basis of the results from B. mori, these family members are putative candidates for cellular CBPs. These proteins may be needed for delivering the lipophilic carotenoid substrates to BCO and/or for mediating the cellular transport of carotenoids in carotenoidaccumulating tissues. To sum up, these recent results with insects provide molecular insight into basic principles in animal carotenoid metabolism. To become biologically active, dietary provitamin A must be first absorbed, then delivered to the site of action in the body to be metabolically converted to vitamin A (Fig. 10.2). The identification of molecular players acting at the different levels of this process in insects may provide the key toward a better understanding of this metabolism in mammals as well. MOLECULAR ANALYSIS OF THE VITAMIN A BIOSYNTHETIC PATHWAY IN VERTEBRATES In chicken, the tissue-specific expression patterns of BCO were analyzed by a combination of Northern blot and in situ hybridization experiments. Its mRNA was mainly localized in liver, in duodenal villi, as well as in tubular structures of the lung and the kidney (34). In the mouse, BCO mRNA was detectable not only in small intestine and liver but also in kidney, testes, uterine tissues, skin, and skeletal muscle (9,36,37). Analyses of BCO mRNA expression in humans revealed a comparable picture (39). Although Yan et al. (38) reported that

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Insect LDL (lipophorin)

Carotenoids

NinaD

Extra-cellular Target cell

CBP

NinaB

Retinoids

Biological Functions Figure 10.2 Schematic overview of the molecular players involved in insect carotenoid metabolism. The cellular uptake of carotenoids is mediated by the class II scavenger receptor ninaD from circulating lipophorins of the hemolymph. For the cellular transport, specific carotenoid binding proteins (CBP) belonging to the StAR gene family exist. For retinoid synthesis carotenoids are converted by the BCO function encoded by the ninaB gene in Drosophila.

BCO is preferentially expressed in the RPE of the human eye and only at much lower levels in other tissues, more recent results dealing with BCO expression in the eye showed only low mRNA levels in the RPE of humans and monkeys (63). In mammals, a majority of provitamin A carotenoids is already converted to vitamin A in epithelial cells of the intestinal mucosa and then transported to the liver for storage. However, the surprising result of all these current investigations is that BCO steady-state mRNA levels are quite high in peripheral nondigestive tissues. Testes, for example, require retinoids for spermatogenesis, and vitamin A is needed for retinoid-signaling in almost all tissues. Thus, BCO expression in peripheral tissues indicates that, besides an external vitamin A supply via the circulation, the provitamin A may contribute to satisfying local vitamin A demands. This inference is in agreement with the fact that in the circulation of mammals

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significant amounts of provitamin A carotenoids are present in addition to vitamin A derivatives. Direct evidence for such a role for provitamin A as an indispensable precursor for the formation of bioactive retinoids comes from the analysis of BCO function in zebrafish embryos (Danio rerio) which have proven to be valuable for the analysis of complex molecular processes in vertebrate biology. Using this system, we addressed the question whether BCO is needed for embryonic development. First, we demonstrated that BCO is expressed in clearly defined spatial compartments and translated into protein in the zebrafish embryo (64). Additionally, we demonstrated that the egg-yolk of zebrafish contains, besides retinoids, significant amounts of b-carotene. To test whether there is an actual requirement for BCO during zebrafish embryonic development, we performed targeted gene knock-down experiments using morpholino antisense oligonucleotides. Loss of BCO function resulted in abnormalities of the craniofacial skeleton, pectoral fins, and eyes, which are impairments well known from mammalian and zebrafish VAD embryos (64). Comparable impairments have been described in RA-deficient fish, either with mutations in the retinal aldehyde dehydrogenase 2 (raldh2) gene or upon treatment of wild-type embryos with citral, an inhibitor of RA-synthesis (65 – 67). Thus, these analyses suggest that provitamin A conversion is the prerequisite for RA-signaling in several distinct developmental processes in zebrafish embryos and reveal an unexpected vital role of the provitamin in the development of a vertebrate species. The developmental use of the nontoxic provitamin instead of preformed yolk vitamin A for RA-signaling processes may provide an additional control mechanism to finely balance retinoid levels at the cellular level in local tissue environments. Interestingly, embryonic expression of BCO has been also reported in mice, suggesting that a developmental function of provitamin A may exist in mammals as well (36). Unlike vitamin A, high-dose supplementation of b-carotene in humans causes no hypervitaminosis A, indicating that b-carotene cleavage to vitamin A is tightly regulated. Several investigations with animal models showed that the vitamin A status of the individual affects BCO enzymatic activity (68,69). Recent analyses provided evidence that BCO regulation in the small intestine is mediated on the transcriptional level, possibly via a negative feedback regulation mechanism involving RA and its nuclear receptors (70). More detailed analyses of the regulation of the murine Bco gene were provided by promoter analysis by Boulanger et al. (71). These studies provided strong evidence that the Bco promoter contains a PPRE (peroxisome proliferator response element) as a key regulatory switch and is regulated by PPARg (peroxisome proliferator activated receptor). PPARs constitute a subfamily of the steroid hormone superfamily (72). Most of the known naturally occurring ligands of PPARs are dietaryderived fatty acids and their metabolites (73). PPARg activates genes involved in anabolic pathways, particularly in adipose tissues, and is required for placental, cardiac, and adipose tissue development (74). RXR is the obligate heterodimeric partner of the PPAR transcription factors. Promoter analysis showed that Bco

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expression is positively regulated by both the PPAR/RXR heterodimer and the RXR/RXR homodimer, implying that the expression of the key enzyme for vitamin A synthesis can be upregulated by 9-cis RA. A role of retinoid-signaling in the positive regulation of retinoid-metabolizing enzymes on the transcriptional level has also been demonstrated for the lecithin:retinol acyltransferase, LRAT (75). Interestingly, the cellular retinol-binding protein II (CrbpII) gene is the only other gene in carotenoid and retinoid metabolisms yet known to contain a PPRE. As reported for Bco, this gene is not only upregulated by ligands of PPAR, but also by 9-cis RA (76). CRBP-II is expressed in large amounts in the small intestine of adult mammals, the major site of vitamin A synthesis. As described earlier, CRBP-II may act downstream of BCO in binding retinal, the primary cleavage product of provitamin A conversion. LRAT catalyzes the synthesis of retinyl esters, the storage form for vitamin A in the liver. The expression of LRAT is induced by feeding RA. However, details of its gene promoter responsive elements are so far missing. Common mechanisms in the regulation of the genes involved in the vitamin A biosynthetic pathway may contribute to vitamin A homeostasis. The involvement of PPARs may interlink it to the regulation of overall lipid metabolism. The controversial results coming from studies on the Bco promoter level (positive regulation by 9-cis RA) and by analyzing BCO enzymatic activity in the gut (negative regulation by RA) need further elucidation. It may be speculated that the latter is the result of indirect effects due to the influence of RA on additional genes involved in this process. The role of BCO in vitamin A biosynthesis has been well established, whereas the role of the second putative carotene-oxygenase, BCOII, still remains somehow elusive. There has long been a controversy over centric vs. eccentric cleavage of b-carotene in the synthesis of vitamin A. Evidence that eccentric cleavage of carotenoids also occurs was provided by several investigations. Napoli and Race (77), for example, showed that, besides the formation of RA from retinal as the initial product of symmetric b-carotene cleavage, RA is directly formed from b-carotene in cell-free homogenates. Furthermore, it was shown that long-chain apocarotenoids (.C20) are shortened to RA in a stepwise process which is most probably mechanistically related to the b-oxidation of fatty acids (31,22). Thus, BCOII may catalyze the first step in an alternative pathway for RA formation (Fig. 10.3). Direct evidence was provided in the mouse embryo that, besides Raldh1, 2, and 3-dependent pathways, additional ways exist for RA-generation involving so far uncharacterized RA-generating enzymes (78,79). Recently, another Raldh (Raldh4) was molecularly identified in the mouse, but its mRNA was mainly expressed in fetal liver (80). We investigated the expression patterns of the eccentric carotenoidcleaving enzyme in the mouse (9). Here, the BcoII gene was expressed in the same tissues as Bco. The mRNA expression of both types of carotene-oxygenases in the same tissues, for example, small intestine and liver, confirms biochemical investigations and explains the observation of both centric and eccentric cleavage activity in cell-free homogenates of the same tissue. It is not yet clear whether both enzymes are expressed in the same or different cell types of these tissues.

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ROL Adh Sdh RAL Raldh RA

β-Carotene BCOII

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RXR RAR

?

AC

cytochrom P450enzymes

4-Oxo RA 4-OH RA 5,8-Epoxy RA

TAT A RARE

nucleus

Figure 10.3 Schematic overview of the proposed pathways leading to RA signaling in vertebrates. RE, retinyl ester; ROL, retinol; RA, retinoic acid; Adh, alcohol dehydrogenase; Sdh, short chain reductase; Raldh, retinal dehydrogenase; BCO, b,b-carotene-15,150 oxygenase; BCOII, b,b-carotene-90 ,100 -oxygenase; RARE, retinoid receptor responsive element; RAR, retinoic acid receptor; RXR, retinoid X receptor; 4-OXO RA, 4-oxoretinoic acid; 4-OH RA, 4-hydroxy retinoic acid; 5,8-Epoxy RA, 5,8-epoxy retinoic acid.

In addition, low-abundance steady-state mRNA levels of BCOII were present in spleen, brain, lung, and heart (9). In the zebrafish, embryo bcoII expression was also found in the developing heart. In the human fetal heart, BCOII mRNA was found upon analyzing a commercial multitissue RNA panel (unpublished results). These results and the existence of an alternative pathway for RAgeneration in the heart may indicate that BCOII plays a particular role for the development of the cardiovascular system. Biological activities of b-apocarotenoids different from retinoids have also been reported by various studies in animals (81). In vitro, BCOII catalyzes, besides b-carotene cleavage, the oxidative cleavage of lycopene (9). Favorable effects of lycopene, for example, on certain kinds of cancers, have been repeatedly reported (82). Thus, besides being a putative precursor for RA-formation, in the case of b-carotene cleavage, it may be speculated that apocarotenoids deriving from other carotenoids may represent biologically active substances. Much further work needs to be done to understand fully the exact physiological function of BCOII. This must include a detailed biochemical analysis of its enzymatic properties, substrate specificity, subcellular localization, and the elucidation of the fate of the primary cleavage products of the reaction. Furthermore, animal models with mutations in this gene must be established to unequivocally address the impact of this type of carotene-oxygenase on vitamin A metabolism. CONCLUSIONS The molecular identification of the different metazoan carotene-oxygenases established the existence of an ancient family of nonheme iron oxygenases in

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animals. Through these enzymes, animals have access to and can modulate their retinoids as needed for biological processes as diverse as vision, cell differentiation, and development. With the increasing number of carotene-oxygenases in the database, sequence information can be used to predict common structural features and to identify functional domains and active site residues. The identification of proteins involved in the transport of carotenoids in insects demonstrated that this process, as described for other lipids, is protein-mediated. The identification of these genes provides a starting point to characterize analogous genes in mammals. The advanced state of knowledge about the molecular components of the vitamin A biosynthetic pathway gained in the past few years will surely help in the fight against VAD and will open new avenues of research, dealing with biochemical, physiological, developmental, and medical aspects of carotenoids and their numerous derivatives. Furthermore, the identification of genes involved in carotenoid metabolism provides molecular markers to analyze genetic aspects of nutrient interactions and the basis to analyze genetic polymorphism in these genes within the population. The establishment of suitable animal model systems such as knock-out mice in these genes may contribute to elucidating mechanisms underlying the pathogenesis of diseases as well as providing starting points for their prevention. REFERENCES 1. Chawla A, Repa JJ, Evans RM, Mangelsdorf DJ. Nuclear receptors and lipid physiology: opening the X-files. Science 2001; 294:1866– 1870. 2. Pfander H. Key to Carotenoids. Bosten: Birkha¨user Verlag Basel, 1990. 3. Frank HA, Cogdell RK. Carotenoids in photosynthesis. Photochem Photobiol 1996; 63:257– 264. 4. Mayne ST. Antioxidant nutrients and chronic disease: use of biomarkers of exposure and oxidative stress status in epidemiologic research. J Nutr 2003; 133:933– 940. 5. Schwartz SH, Quin X, Zeevaart JA. Elucidation of the indirect pathway of abscisic acid biosynthesis by mutants, genes, and enzymes. Plant Physiol 2003; 131:1591 –1601. 6. Schwartz SH, Tan BC, Gage DA, Zeevaart JA, McCarty DR. Specific oxidative cleavage of carotenoids by VP14 of maize. Science 1997; 276:1872– 1874. 7. Giuliano G, Al-Babili S, von Lintig J. Carotenoid oxygenases: cleave it or leave it. Trends Plant Sci 2003; 8:145– 149. 8. von Lintig J, Vogt K. Filling the gap in vitamin A research. Molecular identification of an enzyme cleaving beta-carotene to retinal. J Biol Chem 2000; 275:11915 – 11920. 9. Kiefer C, Hessel S, Lampert JM, Vogt K, Lederer MO, Breithaupt DE, von Lintig J. Identification and characterization of a mammalian enzyme catalyzing the asymmetric oxidative cleavage of provitamin A. J Biol Chem 2001; 276:14110– 14116. 10. von Lintig J, Dreher A, Kiefer C, Wernet MF, Vogt K. Analysis of the blind Drosophila mutant ninaB identifies the gene encoding the key enzyme for vitamin A formation in vivo. Proc Natl Acad Sci USA 2001; 98:1130 – 1135. 11. Wald G. The molecular basis of visual excitation. Nature 1968; 219:800 –807. 12. Vogt K. Is the fly visual pigment a rhodopsin. Z Naturforsch 1984; 39c:196 –197.

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11 Molecular Mechanisms Underlaying the Health Promoting Activity of Lycopene Estibaliz Olano-Martin University of Reading, Reading, UK

Introduction Antioxidant Activity Modulation of Intracellular Communication Inhibition of Cell Cycle Progression Inhibition of Insulin-Like Growth Factor (IGF-1) Signaling Inhibition of HMG-CoA Reductase Conclusions References

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INTRODUCTION Lycopene (Fig. 11.1) is an acyclic isomer of beta-carotene consisting of a 40-carbon atom open polyisoprenoid chain, which contains 11 conjugated and 2 unconjugated double bonds (1). It is a pigment that imparts red color to tomatoes, rosehip, guava, apricots, watermelon, and pink grapefruit. Lycopene is synthesized by plants and some micro-organisms, but not by animals. It serves as an accessory light-gathering pigment and protects these organisms against the toxic effects of oxygen and light. In plants, it tends to exist in an 241

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Figure 11.1

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Structural formula of lycopene.

all-trans configuration but easily undergoes cis/trans isomerization (1 –3). The exact functions and relative activities of the different isomers are not fully understood and investigations are currently underway to determine whether there are biological differences between them (3). Humans obtain lycopene from their diet and it is the predominant carotenoid in human plasma (4), with a half-life of 2– 3 days. Lycopene levels in blood do not differ significantly between men and women, but its level is affected by age (2) and several biological and lifestyle factors. However, in contrast to other carotenoids, lycopene serum values are not usually reduced by smoking or alcohol consumption (5). Because of its lipophilic nature, lycopene concentrates in low-density and very low-density lipoprotein fractions of the serum and it is present as a 50/50 cis/trans isomeric mixture (2,5). Lycopene is found in higher concentrations in the adrenal, liver, testes, and prostate (6). Unlike beta-carotene, lycopene cannot be converted to vitamin A in the body, since it lacks a beta-ionone ring structure. Therefore it is not an essential nutrient, and it attracted little attention until it was shown in vitro to have a singlet-oxygen-quenching ability twice as high as that of beta-carotene and ten times higher than that of alpha-tocopherol. This made lycopene the most potent antioxidant of the common carotenoids (7). In addition to its antioxidant properties, many epidemiological studies have indicated an association between regular consumption of lycopene-rich foods and decreased risk for a range of common cancers. In 1999, Giovannucci reviewed 72 human studies and found in 57 of these reports an inverse association between tomato consumption or blood lycopene levels and risk of various types of cancer (8). Thirty-five of these associations were significant. Evidence of protective properties of lycopene was highest for cancers of the prostate, lung, and stomach. The fact that the data relating to lycopene and the risk of aggressive prostate cancer are especially compelling (9,10) made this carotenoid one of the most promising chemopreventive agents found in western diets. Indeed, consumption of tomato-based foods has been associated with a 30– 40% lower risk of prostate cancer, and it is lycopene rather than other carotenoids that is responsible for this effect.

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Most of the data in Giovannuccis’ review were from observational studies, and therefore a cause-and-effect relationship could not be firmly established. However, the consistently lower risk of cancer associated with higher consumption of lycopene-containing tomatoes provided a strong foundation for further research on lycopene. Recently, more direct, though still preliminary, evidence has emerged, which suggests not only protective effects of lycopene in prostate cancer, but also in breast cancer (11) and cervical dysplasia (12). In addition to that, researchers have found a statistically significant association between high dietary lycopene and a 48% lower risk of heart disease (13). Studies have also investigated the relationship of lycopene with many other diseases such as cataracts (14), asthma, longevity (15), malaria (16), digestive-tract cancers (17,18), immune modulation (19), Alzheimer’s disease (20), and preclampsia (21). The mechanisms involved in the protective effects of lycopene are still poorly understood, but it is thought that lycopene’s potent antioxidant activity might confer protection against chronic diseases such as cancer, atherosclerosis, and associated coronary heart disease. A number of other non-antioxidant mechanisms have also been suggested.

ANTIOXIDANT ACTIVITY The human body is constantly exposed to oxidative stress. Oxidative stress arises partly from environmental parameters (air pollution, tobacco smoke, and radiation) (22,23) and partly as a natural result of aerobic (oxygen dependent) metabolism. The by-products of oxidation are highly reactive molecules and include free radicals, as well as the highly reactive singlet form of oxygen (24). All these molecules can react with various components of a living cell—proteins, DNA, and lipids—leading to a number of pathological conditions including aging, senile dementia, atherogenesis, ischemia-reperfusion injury, alcohol damage, age-related macular degeneration, and cancer (25). Even if the human body has evolved to fight oxidative stress with endogenous antioxidant defenses, diet-derived antioxidants—including ascorbic acid, alpha-tocopherol, and carotenoids—are still important in protecting against the sum of oxidative stresses challenging the body. In healthy human subjects and in vitro, lycopene has been shown to protect against lipid, lipoprotein, protein, and DNA oxidation (25 – 27), to reduce the susceptibility of lymphocyte DNA to oxidative damage (28,29), to inactivate hydrogen peroxide and nitrogen dioxide (30), and to protect lymphocytes from nitrogen oxide-induced membrane damage and cell death twice as efficiently as beta-carotene (31). Ingestion of a lycopene free diet for 2 weeks resulted in 50% loss of serum lycopene with a 25% increase of in vivo lipid oxidation in healthy human subjects. Low lycopene levels in serum have also been associated with increased risk of coronary heart disease (13,25,32,33) and have been found in patients with cancer.

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1

!

In vitro studies have shown that the lycopene prevents oxidation by quenching singlet oxygen. Among the carotenoids, lycopene exhibits one of the highest physical quenching rate constant (Kq ¼ 31
109 M-l S-1) (7). This protective function is achieved by both physical (energy transfer) and chemical processes [Eqs. (1) and (2)]. The physical quenching rate is much higher than the chemical; and once produced, the triplet excited state of the 3 Lycopene can return to the ground state, dissipating the excess energy as heat (34). Thus, lycopene acts as a catalyst in the deactivation of singlet oxygen. O2  þ Lycopene ! O2 þ 3 Lycopene ! 1

Heat

O2  þ Lycopene ! Lycopene-O2

O2 þ Lycopene

(1) (2)

A second major antioxidant role of carotenoids is the scavenging of free radicals (35). Hydrogen peroxide, nitrogen dioxide, thyi and sulphonyl radicals are some of the reactive oxygen species (ROS) that lycopene has been shown to deactivate. As shown earlier (Fig. 11.1), lycopene has several conjugated double bonds that enable the electrons to be delocalized over the whole system and so be shared by many atoms, making the whole chain relatively electronrich. This means that the delocalized electrons may move around the whole system resulting in an electron cloud, where the electrons do not specifically belong to any one atom. Lycopene will stabilize a nearby free radical by giving up an electron. Lycopene then becomes a free radical itself, but owing to the conjugated bonds, it will be hundreds of times more stable than, for example, hydroxyl radical (Fig. 11.2). Although high concentrations of ROS are cytotoxic, low concentrations are still needed in the cell to keep its intracellular redox state that regulates the activities of a variety of protein tyrosine kinases, protein tyrosine phosphatases, phospholipases, and transcription factors (36–39). As lycopene is a potent antioxidant, it could modulate the intracellular redox status by scavenging ROS and quenching singlet oxygen inside the cell, affecting the expression of redox regulated genes such as NF-kB and AP-1. NF-kB is involved in inflammatory and immune responses, cell survival, and activation, and AP-1 is involved in cell growth, cell proliferation, and differentiation. There are no published data showing the relationship electron cloud (delocalised e– )

· ·

OH Figure 11.2

OH

–

Lycopene free radical scavenging mechanism.

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between lycopene supplementation and changes in the redox potential of the cell, but previous studies have reported the role of oxidants and antioxidants in the regulation of redox-sensitive transcription factors (38,40–43). Therefore, the antioxidant activity of lycopene could protect cells from oxidative damage by preventing arteriosclerosis and/or cancer, but also decrease cell proliferation of cancer cells by interfering with redox-sensitive genes.

MODULATION OF INTRACELLULAR COMMUNICATION Cells connect and communicate via transmembrane channels called gap junctions (Fig. 11.3) that connect the cytoplasms of neighboring cells. The channels consist of two hexamers of specific proteins, which belong to the gene family of connexins. These channels permit small metabolites (,1000 Da), ions, and second messengers to pass from cell to cell. This intercellular signaling mechanism is very important for coordinating biochemical functions in multicellular organisms, and loss of this signaling activity often happens during malignant transformation of cells. Both in vitro (44,45) and in vivo studies (46,47) have demonstrated that inhibition of gap-junctional intracellular communication (GJIC), leading to a long-lasting decrease in cell communication, appears to be a strong tumorpromoting factor. Moreover, Zang et al. reported recently that over-expression of connexin 43 was associated with suppressed proliferation of human osteosarcoma U2OS cells through arresting cell cycle at G1/S phase (48). The GJIC impairment seems to be reversible, and chemicals that increase gap-junctions could be used as chemopreventive agents. Different publications have demonstrated a correlation between the ability of diverse carotenoids to inhibit chemically induced neoplastic transformation and their ability to stimulate gap-communication by increasing connexin 43 levels (49 – 52). In particular, lycopene has been shown to stimulate GJIC in mouse embryonic fibroblast 10T1/2 (52), human dermal fibroblasts (53), human fetal

connexin

Gap junction

Intracellular gap Figure 11.3 Scheme of a gap junction between two adjacent cells. It works as a window between neighboring cells through which ions, current, and cytoplasm can flow.

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skin fibroblast (54), and human oral cavity tumors as seen by scrape-loading dye transfer and electron microscopy (55). In addition to lycopene, its oxidation products such as 2,7,11,-trimethyl-tetradecahexane-1,14dial have been found to have similar activity (56). In many of these studies, an up-regulation of both the transcription and expression of connexin 43 in cells was also observed. In vivo experiments using rats also found enhanced GJIC after treatment with 50 mg/kg lycopene (47). However, this chemopreventive mechanism may be only effective in early stages of cancer as cells loose their growth inhibitory response to lycopene through the selection pressures involved in metastasis. A recent study by Forbes et al. shows how lycopene treatment at a physiological dose (1.0 mM) increased connexin 43 expression and inhibited cell growth in PC-3, an established human prostate cancer cell line, but not in metastatically passaged prostate cells (PC-3MM2) (57). INHIBITION OF CELL CYCLE PROGRESSION In addition, lycopene at physiological concentrations has been shown to inhibit proliferation of several types of human cell lines, including HL-60 leukemic cells (58), human aortic smooth muscle cells (59), endometrial cells (60,61), breast (60 –64), prostate (65 – 68), and lung (60). This is a very important trait since cancer can be very briefly described as uncontrolled cell growth and proliferation (as well as metastasis, or the invasiveness of cancerous cells into other tissues). Therefore, the fact that lycopene can either stop or slow down proliferation makes it a promising chemoprotective agent. In proliferating cells, the cell cycle consists of four phases. Gap 1 (G1) is the interval between mitosis and DNA replication that is characterized by cell growth. The transition that occurs at the restriction point (R) in G1 commits the cell to the proliferative cycle. If the conditions that signal this transition are not present, the cell exits the cell cycle and enters G0, a nonproliferative phase during which growth, differentiation, and apoptosis occur. The G1 phase is characterized by gene expression and protein synthesis and is really the only part of the cell cycle regulated primarily by extracellular stimuli (like mitogens such as growth factors and adhesion). Late G1 phase is marked by transcription of D-cyclins, which bind and activate cyclin kinases (Cdk4 and Cdk6). Once bound, the CDK complex phosphorylates the retinoblastoma protein pRB, which then allows cell cycle progression (Fig. 11.4). Unphosphorylated pRB normally masks E2F transcription activators (actually a family of factors, E2F1-3) so that E2F target genes are not expressed. These target genes are all related to DNA replication and cell division, so pRB acts as a general repressor of cell proliferation (69). It is speculated that lycopene may suppress carcinogen-induced phosphorylation of regulatory proteins, such as p53 and Rb antioncogenes, and stop cell division at the G0 – G1 cell cycle phase. Research has shown (61,68) that

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Figure 11.4 Mitogens drive cell cycle progression by induction of cyclin D and inactivation of the retinoblastoma (Rb) protein. Inactivation of the Rb protein marks the restriction point at which cell-cycle progression becomes independent of mitogens. Inactivated Rb releases E2F transcription factors, which stimulate the expression of downstream cyclins and other genes that are required for DNA synthesis. (Figure reproduced with permission of Dr W. Koch and Expert Reviews in Molecular Medicine.)

2 –5 mM of lycopene supplementation interferes with cell cycle progression at G1 as observed by flow cytometry (Fig. 11.5), reducing the levels of cyclins (mostly cyclin D) and Cdk activity. It is not clear whether lycopene acts directly on cyclin D or interferes with mitogens. Alternatively, the decrease of cyclin D levels may be related to lycopene effects on other sites of the cell cycle machinery. However, this chain of events is responsible for a decrease in pRB phosphorylation and a delay in G1/S transition. Another important protein in regulating the cell cycle is the tumor suppressor protein p53. In fact, p53 is the most frequently disrupted gene in cancer (70), illustrating its importance in this disease, and it has a role in regulating the expression of genes involved in cell cycle arrest and apoptosis. However, there are currently no data that suggest that lycopene might have an impact on p53 levels.

INHIBITION OF INSULIN-LIKE GROWTH FACTOR (IGF-1) SIGNALING Lycopene may also reduce cellular proliferation induced by insulin-like growth factors (IGFs). These growth factors are naturally secreted by the liver, but are also produced in several other tissues (71). They are necessary for normal physiological functions including promotion of cell survival (inhibition of apoptosis), stimulation of metabolism, and proliferation of differentiating cells (72). However, since IGFs are mitogenic and anti-apoptotic, it is thought that high

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Figure 11.5 Summary of the cell cycle analysis by flow cytometry based on DNA content propidium iodide stained cells. When treated with 5 mM of lycopene, cells were arrested at the beginning of S phase. Flow cytometry data were collected for each of the treatments at least 6 times from 6 different experiments.  Indicates a significant difference ( p , 0.05) compared with the control.

levels might enhance cell turnover, increasing the chances of cellular transformation that then could lead to cancer. Indeed, high IGF-1 levels in plasma have been associated with an increased risk of breast cancer in premenopausal women and prostate, colon, and lung cancer (73). Research has shown that lycopene interferes with IGF signaling and cell cycle progression specifically in breast and prostate cancer cells (62,74). Karas et al. examined the effect of physiological concentrations of lycopene (,1 mM) on IGF-1 induced growth of MCF7 mammary cancer cells (62). Lycopene-treated cells significantly reduced the IGF-1 cell growth stimulation (Fig. 11.6), slowing down cell proliferation. These effects were associated with an inhibition of the IGF signaling in the cells and an increase in IGFBs. The suppression of cell proliferation by IGF stimulated growth had been previously reported in endometrial cells (60); and importantly, normal human cells were found to be much less sensitive to lycopene than cancer cells. By interfering with these growth factor-related cancer stimulators, lycopene may reduce both the occurrence and the progression of breast and prostate cancers. The exact mechanism in which lycopene interferes with IGF-1 signaling pathway and slows down cellular growth might be also related with the lycopene’s ability to suppress 3-hydroxy-3 methyl glutaryl coenzyme A (HMG-CoA) reductase activity. It has been found that lycopene can inhibit macrophage HMG-CoA reductase activity (32), which constitutes the key metabolite in the biosynthesis of cholesterol and a variety of sterol and nonsterol

Health Promoting Activity of Lycopene

Figure 11.6

249

Possible effect of lycopene on IGF-1 signal transduction pathway.

isoprenoids such as dolichol. Because dolichol is a crucial isoprenoid during G1 for the expression of IGF-receptors, a decrease in the dolichol levels will then interfere with IGF-1 signaling. Indeed, cells that cannot synthesize dolichol experience an effective deficiency of IGF-1 activity (75,76). Therefore, lycopene might induce a deficit, the isoprenol dolichol, that in turn impairs IGF-1 and cell cycle progression. INHIBITION OF HMG-CoA REDUCTASE As mentioned earlier, it has been reported that lycopene supplementation (10 mM) inhibits the de novo synthesis of cholesterol from [3H]acetate, but not from [14C]mevalonate in the J-774A macrophage cell line (32). This effect has been related with an inhibition of the enzyme HMG-CoA reductase activity by lycopene, and could explain both its cancer prevention and hypocholesterolemia properties. It is well known that mevalonate is required for growth of mammalian cells playing a key role in controlling cell proliferation through the Ras-mediated signal transduction pathway (77 – 79). The Ras oncogenes (HRAS, KRAS, and NRAS) encode 21 kDa proteins called p21s. To initiate eukaryotic cell proliferation, Ras requires the posttranslational attachment of a farnesyl group, an isoprenoid lipid moiety derived from mevalonate, to the carboxyl-terminus of the protein (Fig. 11.7). Therefore, an inhibition of HMG-CoA reductase activity will lead to a mevalonate depletion, which in turn will interfere with the Ras pathway causing G1 arrest in cycling mammalian cells and retarding cancer induction and slowing cellular growth (80,81).

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Figure 11.7 Inhibition of cell cycle progression from G1 to S phase by lycopene by interfering with the Ras signaling pathway.

The effect of lycopene on HMG-CoA reductase activity may also be important in cardiovascular disease. Recent epidemiological studies have reported an inverse association between higher tissue and serum levels of lycopene and the risk of coronary heart disease in women (82). It is thought that lycopene might reduce LDL cholesterol in plasma by inhibiting HMG-CoA reductase (an important step in cholesterol synthesis) and by up-regulating the LDL receptor (32). However, more research is needed in this field since the protective effects of lycopene in cardiovascular disease have been also related to the reduction of homocysteine, platelet aggregation, and blood pressure (83).

CONCLUSIONS There is extensive evidence that regular high consumption of fruits and vegetables decreases the risk of chronic diseases such as cancer or atherosclerosis (84). Among them, tomatoes have gain lots of attention since epidemiological studies have shown that high intake of tomato products were inversely associated with the incidence of certain types of cancer (mostly prostate cancer) (8). To understand the molecular mechanisms involved in the beneficial effects of tomatoes, much research has focused on the carotenoid lycopene—the most abundant phytochemical in tomatoes and the one thought to be responsible of heath-related effects. Research has shown that lycopene interferes in many different pathways, changing the expression of proteins involved in cell to cell communication, cycle, and cell growth. The high antioxidant potential of lycopene might be responsible for some of the effects. However, there is enough evidence that suggests other non-antioxidant mechanisms, such as modulating transcription

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factors might be responsible for the reduction of the risk for chronic diseases of lycopene.

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17. Franceschi S, Bidoli E, La Vecchia C, Talamini R, D’Avanzo B, Negri EC. Tomatoes and risk of digestive-tract cancers. Int J Cancer 1994; 59:181 – 184. 18. De Stefani E, Oreggia F, Boffetta P, Deneo-Pellegrini H, Ronco A, Mendilaharsu M. Tomatoes, tomato-rich foods, lycopene and cancer of the upper aerodigestive tract: a case-control in Uraguay. Oral Oncol 2000; 36:47 – 53. 19. Watzl B, Bub A, Brandstetter BR, Rechkemmer G. Modulation of human T-lymphocyte functions by the consumption of carotenoid-rich vegetables. Br J Nutr 1999; 82:383– 389. 20. Mecocci P, Polidori MC, Cherubini A, Ingegni T, Mattioli P, Catani M, Rinaldi P, Cecchetti R, Stahl W, Senin U, Beal MF. Lymphocyte oxidative DNA damage and plasma antioxidants in Alzheimer disease. Arch Neurol 2002; 59:794 –798. 21. Palan PR, Mikhail MS, Romney SL. Placental and serum levels of carotenoids in preeclampsia. Obstet Gynecol 2001; 98:459– 462. 22. Moller P, Wallin H, Knudsen LE. Oxidative stress associated with exercise, psychological stress and life-style factors. Chem Biol Interact 1996; 102:17– 36. 23. Papas AM. Diet and antioxidant status. Food Chem Toxicol 1999; 37:999 – 1007. 24. Darley-Usmar V, Halliwell B. Blood radicals: reactive nitrogen species, reactive oxygen species, transition metal ions, and the vascular system. Pharm Res 1996; 13:649– 662. 25. Cross CE, Halliwell B, Borish ET, Pryor WA, Ames BN, Saul RL, McCord JM, Harman D. Oxygen radicals and human disease. Ann Intern Med 1987; 107:526 – 545. 26. Agarwal S, Rao AV. Tomato lycopene and low density lipoprotein oxidation: a human dietary intervention study. Lipids 1998; 33:981 – 984. 27. Rao AV, Agarwal S. Bioavailability and in vivo antioxidant properties of lycopene from tomato products and their possible role in the prevention of cancer. Nutr Cancer 1998; 31:199 – 203. 28. Pool-Zobel BL, Bub A, Muller H, Wollowski I, Rechkemmer G. Consumption of vegetables reduces genetic damage in humans: first results of a human intervention trial with carotenoid-rich foods. Carcinogenesis 1997; 18:1847 –1850. 29. Porrini M, Riso P. Lymphocyte lycopene concentration and DNA protection from oxidative damage is increased in women after a short period of tomato consumption. J Nutr 2000; 130:189– 192. 30. Astley SB, Hughes DA, Wright AJ, Elliott RM, Southon S. DNA damage and susceptibility to oxidative damage in lymphocytes: effects of carotenoids in vitro and in vivo. Br J Nutr 2004; 91:53 – 61. 31. Bohm F, Tinkler JH, Truscott TG. Carotenoids protect against cell membrane damage by the nitrogen dioxide radical. Nat Med 1995; 1:98– 99. 32. Fuhrman B, Elis A, Aviram M. Hypocholesterolemic effect of lycopene and betacarotene is related to suppression of cholesterol synthesis and augmentation of LDL receptor activity in macrophages. Biochem Biophys Res Commun 1997; 233:658– 662. 33. Kristenson M, Zieden B, Kucinskiene Z, Elinder LS, Bergdahl B, Elwing B, Abaravicius A, Razinkoviene L, Calkauskas H, Olsson AG. Antioxidant state and mortality from coronary heart disease in Lithuanian and Swedish men: concomitant cross sectional study of men aged 50. BMJ 1997; 314:629– 633. 34. Montenegro MA, Nazareno MA, Durantini EN, Borsarelli CD. Singlet molecular oxygen quenching ability of carotenoids in a reverse-micelle membrane mimetic system. Photochem Photobiol 2002; 75:353 –361.

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35. Mortensen A, Skibsted LH, Truscott TG. The interaction of dietary carotenoids with radical species. Arch Biochem Biophys 2001; 385:13 – 19. 36. Monteiro HP, Stern A. Redox modulation of tyrosine phosphorylation-dependent signal transduction pathways. Free Radic Biol Med 1996; 21:323 –333. 37. Suzuki YJ, Forman HJ, Sevanian A. Oxidants as stimulators of signal transduction. Free Radic Biol Med 1997; 22:269 – 285. 38. Sen CK, Packer L. Antioxidant and redox regulation of gene transcription. FASEB J 1996; 10:709– 720. 39. Schulze-Osthoff K, Los M, Baeuerle PA. Redox signalling by transcription factors NF-kappa B and AP-1 in lymphocytes. Biochem Pharmacol 1995; 50:735 – 741. 40. Carballo M, Marquez G, Conde M, Martin-Nieto J, Monteseirin J, Conde J, Pintado E, Sobrino F. Characterization of calcineurin in human neutrophils. Inhibitory effect of hydrogen peroxide on its enzyme activity and on NF-kappaB DNA binding. J Biol Chem 1999; 274:93 – 100. 41. Furuke K, Shiraishi M, Mostowski HS, Bloom ET. Fas ligand induction in human NK cells is regulated by redox through a calcineurin-nuclear factors of activated T celldependent pathway. J Immunol 1999; 162:1988 –1993. 42. Kelner MJ, Bagnell R, Hale B, Alexander NM. Inactivation of intracellular copper – zinc superoxide dismutase by copper chelating agents without glutathione depletion and methemoglobin formation. Free Radic Biol Med 1989; 6:355 – 360. 43. Rafii B, Tanswell AK, Otulakowski G, Pitkanen O, Belcastro-Taylor R, O’Brodovich H. O2-induced ENaC expression is associated with NF-kappaB activation and blocked by superoxide scavenger. Am J Physiol 1998; 275:L764–L770. 44. Budunova IV, Williams GM. Cell culture assays for chemicals with tumor-promoting or tumor-inhibiting activity based on the modulation of intercellular communication. Cell Biol Toxicol 1994; 10:71 –116. 45. Mehta PP, Bertram JS, Loewenstein WR. Growth inhibition of transformed cells correlates with their junctional communication with normal cells. Cell 1986; 44:187– 196. 46. Krutovskikh VA, Mesnil M, Mazzoleni G, Yamasaki H. Inhibition of rat liver gap junction intercellular communication by tumor-promoting agents in vivo. Association with aberrant localization of connexin proteins. Lab Invest 1995; 72:571 –577. 47. Krutovskikh V, Asamoto M, Takasuka N, Murakoshi M, Nishino H, Tsuda H. Differential dose-dependent effects of alpha-, beta-carotenes and lycopene on gapjunctional intercellular communication in rat liver in vivo. Jpn J Cancer Res 1997; 88:1121– 1124. 48. Zhang YW, Morita I, Ikeda M, Ma KW, Murota S. Connexin43 suppresses proliferation of osteosarcoma U2OS cells through post-transcriptional regulation of p27. Oncogene 2001; 20:4138– 4149. 49. Bertram JS, Pung A, Churley M, Kappock TJ, Wilkins LR, Cooney RV. Diverse carotenoids protect against chemically induced neoplastic transformation. Carcinogenesis 1991; 12:671– 678. 50. Hossain MZ, Wilkens LR, Mehta PP, Loewenstein W, Bertram JS. Enhancement of gap junctional communication by retinoids correlates with their ability to inhibit neoplastic transformation. Carcinogenesis 1989; 10:1743 – 1748. 51. Pung A, Rundhaug JE, Yoshizawa CN, Bertram JS. Beta-carotene and canthaxanthin inhibit chemically- and physically-induced neoplastic transformation in 10T1/2 cells. Carcinogenesis 1988; 9:1533– 1539.

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52. Zhang LX, Cooney RV, Bertram JS. Carotenoids enhance gap junctional communication and inhibit lipid peroxidation in C3H/10T1/2 cells: relationship to their cancer chemopreventive action. Carcinogenesis 1991; 12:2109 – 2114. 53. Zhang LX, Acevedo P, Guo H, Bertram JS. Upregulation of gap junctional communication and connexin43 gene expression by carotenoids in human dermal fibroblasts but not in human keratinocytes. Mol Carcinogenesis 1995; 12:50– 58. 54. Stahl W, von Laar J, Martin HD, Emmerich T, Sies H. Stimulation of gap junctional communication: comparison of acyclo-retinoic acid and lycopene. Arch Biochem Biophys 2000; 373:271– 274. 55. Livny O, Kaplan I, Reifen R, Polak-Charcon S, Madar Z, Schwartz B. Lycopene inhibits proliferation and enhances gap-junction communication of KB-1 human oral tumor cells. J Nutr 2002; 132:3754 – 3759. 56. Aust O, Ale-Agha N, Zhang L, Wollersen H, Sies H, Stahl W. Lycopene oxidation product enhances gap junctional communication. Food Chem Toxicol 2003; 41:1399– 1407. 57. Forbes K, Gillette K, Sehgal I. Lycopene increases urokinase receptor and fails to inhibit growth or connexin expression in a metastatically passaged prostate cancer cell line: a brief communication. Exp Biol Med (Maywood) 2003; 228:967 – 971. 58. Amir H, Karas M, Giat J, Danilenko M, Levy R, Yermiahu T, Levy J, Sharoni Y. Lycopene and 1,25-dihydroxyvitamin D3 cooperate in the inhibition of cell cycle progression and induction of differentiation in HL-60 leukemic cells. Nutr Cancer 1999; 33:105– 112. 59. Carpenter KL, Hardwick SJ, Albarani V, Mitchinson MJ. Carotenoids inhibit DNA synthesis in human aortic smooth muscle cells. FEBS Lett 1999; 447:17 – 20. 60. Levy J, Bosin E, Feldman B, Giat Y, Miinster A, Danilenko M, Sharoni Y. Lycopene is a more potent inhibitor of human cancer cell proliferation than either alpha-carotene or beta-carotene. Nutr Cancer 1995; 24:257 – 266. 61. Nahum A, Hirsch K, Danilenko M, Watts CK, Prall OW, Levy J, Sharoni Y. Lycopene inhibition of cell cycle progression in breast and endometrial cancer cells is associated with reduction in cyclin D levels and retention of p27(Kip1) in the cyclin E-cdk2 complexes. Oncogene 2001; 20:3428 – 3436. 62. Karas M, Amir H, Fishman D, Danilenko M, Segal S, Nahum A, Koifmann A, Giat Y, Levy J, Sharoni Y. Lycopene interferes with cell cycle progression and insulin-like growth factor I signaling in mammary cancer cells. Nutr Cancer 2000; 36:101– 111. 63. Ben-Dor A, Nahum A, Danilenko M, Giat Y, Stahl W, Martin HD, Emmerich T, Noy N, Levy J, Sharoni Y. Effects of acyclo-retinoic acid and lycopene on activation of the retinoic acid receptor and proliferation of mammary cancer cells. Arch Biochem Biophys 2001; 391:295– 302. 64. Prakash P, Russell RM, Krinsky NI. In vitro inhibition of proliferation of estrogendependent and estrogen-independent human breast cancer cells treated with carotenoids or retinoids. J Nutr 2001; 131:1574 – 1580. 65. Pastori M, Pfander H, Boscoboinik D, Azzi A. Lycopene in association with alphatocopherol inhibits at physiological concentrations proliferation of prostate carcinoma cells. Biochem Biophys Res Commun 1998; 250:582 – 585. 66. Kotake-Nara E, Kushiro M, Zhang H, Sugawara T, Miyashita K, Nagao A. Carotenoids affect proliferation of human prostate cancer cells. J Nutr 2001; 131:3303 –3306. 67. Kim L, Rao AV, Rao LG. Effect of lycopene on prostate LNCaP cancer cells in culture. J Med Food 2002; 5:181 – 187.

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12 Cellular Redox Activity and Molecular Functions of Ascorbic Acid John K. Lodge University of Surrey, Guildford, Surrey, UK

Properties of Ascorbic Acid Nutritional Aspects Ascorbic Acid and Redox Status Ascorbate Recycling Ascorbate Recycling and Metabolism Molecular Functions of Ascorbic Acid Ascorbate Modulation of Collagen Formation Ascorbate Modulation of Cell Differentiation Modulation of Transcription Factors Ascorbate Modulation of Nitric Oxide Production Ascorbate-Induced Modulation of DNA Repair Ascorbate-Induced Modulation of Other Genes Conclusion References

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PROPERTIES OF ASCORBIC ACID Ascorbic acid is six carbon lactone structurally related to glucose. Indeed, glucose provides the starting point of ascorbic acid synthesis in those animals 257

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capable of synthesis. Primates are one of the few mammalian species that lack the ability to synthesize ascorbic acid, and must therefore obtain ascorbic acid from the diet. Hence, it is also known as vitamin C. The term ascorbic acid derives from experiments into the causes of scurvy (vitamin C deficiency disease) where a “scorbutic factor” was used to describe a substance, later found to be vitamin C, found in citrus fruits which prevents the onset of scurvy. The structures of ascorbic acid, and its oxidation products, are shown in Fig. 12.1. Owing to the hydroxyl at the C3 position having a pKa of 4.2, at physiological pH ascorbic acid is present as the ascorbate ion and is hence referred to as ascorbate in this text. The loss of one electron results in formation of the ascorbate free radical (AFR), also known as semi-dehydroascorbate. If not recycled back to ascorbate, this will quickly dismute into dehydroascorbate (DHA). Similarly, DHA can be formed from the two-electron oxidation of ascorbate. Thus, as an electron donor, ascorbate can participate in both one and two electron transfer reactions. Both ascorbate and AFR have relatively low one-electron reduction potentials of 282 and 2174 mV, respectively (1), enabling these molecules to react with and reduce a variety of species (2). This also makes ascorbate a powerful reducing agent and most, if not all, of the biochemical functions of ascorbate can be attributed to these reducing properties. Ascorbate oxidation products undergo recycling. However, if not recycled, the lactone ring of DHA breaks down to form first 2,3-diketogulonic acid and finally oxalic acid which accounts for the major metabolite and excretory product of ascorbate. The functions of ascorbate have been reviewed elsewhere (3) and are only summarized here. In mammals, ascorbate is required as an electron donor for eight enzymes, which have either mono-oxygenase or di-oxygenase activity. The mono-oxygenases are dopamine b-hydroxylase, which converts dopamine to noradrenaline, and peptidyl-glycine a-mono-oxygenase, which converts a peptide with a C-terminal glycine to a C-terminal amidated peptide. The di-oxygenases are prolyly 4-hydroxylase, prolyl 3-hydroxylase, and lysyl hydroxylase, which convert proline or lysine residues to the hydroxylated residue during collagen biosynthesis; trimethyllysine hydroxylase and g-butyrobetaine

ascorbic acid ASC

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hydroxylase, which convert trimethyllysine to hydroxythimethylysine and trimethylaminobutyrate to carnitine, respectively; and hydroxyphenylpyruvate dioxygenase, which converts hydroxyphenylpyruvate to homogentisic acid in tyrosine catabolism. Ascorbate acts either as a direct source of electrons (as in the copper containing mono-oxygenases), or to reduce the iron cofactor in the active site of various di-oxygenases which undergo iron oxidation during the catalytic cycle. Ascorbate has a well-characterized antioxidant role in scavenging reactive oxygen species (ROS) and reactive nitrogen species and recycling oxidized a-tocopherol. Briefly, in vitro systems have demonstrated ascorbate as scavenging superoxide, hydroxyl, hydrophilic peroxyl, thiyl, and nitroxide radicals, as well as hypochlorous acid and hydrogen peroxide. These actions have been reviewed in detail elsewhere (4,5). Other functions for ascorbate are in iron metabolism, by maintaining iron in the reduced state ascorbate promotes iron absorption, and can also mobilize iron from ferritin deposits. The classical symptoms of ascorbate deficiency, known as scurvy, can be directly related to these functions of ascorbate and predominantly those in enzyme action. For example, one of the first symptoms is fatigue and this can be explained by a shortage of carnitine, required for shuttling of acyl groups across the mitochondria for b-oxidation and energy production. The most characteristic symptom of scurvy is the poor wound-healing, hemorrhaging, and joint pains which are associated with a lack of collagen. Incomplete hydroxylation of proline and lysine residues disrupts the normal triple-helix formation and cross-linking of collagen fibres. Nutritional Aspects Ascorbate is predominantly found in fruits and vegetables with citrus fruits and berries having the richest sources in fruits, whereas green vegetables such as broccoli, cabbage, sprouts, and others such as peppers have the highest sources in vegetables (6). The recommended daily intake for ascorbate in adult males ranges from 40 mg/d in the United Kingdom to 90 mg/d in the United States and is increased by a further 35 mg/d in cigarette smokers to take into account increased turnover of ascorbate in smokers (7). As one serving of fruit or vegetables can contain 30 mg ascorbate, these intakes can be readily achievable from a healthy diet. DHA is also present in food, accounting 10 –20% of all vitamin C, whereas it can also be formed by oxidation of ascorbate in the GI tract. Absorption of ascorbate occurs primarily by the sodium-dependent vitamin C transporter (SVCT1) in the upper GI tract (8). This is a highly efficient and specific process and requires two sodium ions for the transport of one ascorbate molecule. DHA is absorbed by a sodium-independent process of facilitated diffusion, by an as yet unknown mechanism. Following absorption DHA is presumably reduced to ascorbate in the enterocyte. Using human repletion studies,

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Levine et al. have assessed ascorbate bioavailability at almost 100% at doses between 15 and 200 mg (9,10). However, bioavailability decreases with increasing dose (11) dropping to 50% with doses around 1 g, and 20% with doses of 5 g (12). In a cell culture intestinal model, chronic exposure to ascorbate appeared to reduce the expression of the major intestinal ascorbate transporter SVCT1 in CaCo-2 cells (13), which may help to explain decreased bioavailability at higher doses. It is also likely that high doses of ascorbate will saturate the transport system. Flavonoids such as quercitin have also recently been shown to inhibit ascorbate uptake (14). The bioavailability of ascorbate from food sources appears to be similar to that from the synthetic form (15). Factors which are known to influence bioavailability include glucose which can inhibit ascorbate absorption, but not DHA absorption (16), and in general anything that could destroy ascorbate. There is no data regarding bioavailability of DHA. Following enterocyte absorption, ascorbate is transported across the basolateral membrane into the bloodstream via the sodium-dependent vitamin C transporter 2 (SVCT2) (17). Ascorbate accounts for over 95% of vitamin C in the blood, and in the absence of any transport proteins, is able to travel freely and associate into extracellular spaces. The ascorbate transporters SVCT1 and 2 are also responsible for cellular uptake. Because certain tissues appear to accumulate ascorbate, transport must be against a concentration gradient. This tissue-specific localization reflects the main function of ascorbate in these tissues. Tissues that actively accumulate ascorbate include the adrenals, leucocytes, mesenchymal cells (muscle, cartilage, and bone cells), pituitary, lung, liver, and eye (18). DHA transport is sodium and energy independent and occurs via the glucose transporters GLUT1 and GLUT3 (19). Transport of DHA was found to be at least 10-fold faster than that of ascorbate in neutrophils (20). Once transported, DHA is immediately reduced to ascorbate, thereby preventing reverse transport. Ascorbate release from cells is negligible.

ASCORBIC ACID AND REDOX STATUS Ascorbate Recycling Ascorbate is oxidized to the AFR or DHA (Fig. 12.1). It is well established that ascorbate undergoes recycling from both these oxidation products (21 – 26). Ascorbate recycling is an important process in maintaining ascorbate concentrations. The processes involved have been demonstrated in a number of mammalian cell types, and proceed via different mechanisms either enzymatically or non-enzymatically. The various processes are highlighted in Fig. 12.2. Intracellularly, the AFR is produced following certain free radical scavenging actions and recycling of the tocopheroxyl radical (oxidation product of a-tocopherol formed during lipid peroxidation). Owing to electron delocalization in the lactone ring, the AFR is relatively unreactive and will disproportionate to

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Figure 12.2 Schematic representation of various pathways of ascorbate free radical (AFR) and dehydroascorbate (DHA) recycling in human cell lines. Intracellular AFR is reduced by enzymatic pathways requiring either NADH (e.g., mitochondrial AFR reductase) or NADPH (e.g., TR). Extracellular AFR can also be reduced using electrons donated intracellularly via either an ascorbate- or NADH-dependent AFR reductase. DHA is predominantly reduced via GSH-dependent mechanisms which can be either direct or enzymatic (glutaredoxin). Both these pathways, however, require the recycling of GSH by GSH reductase which consumes NADPH. A decrease in the NADPH:NADPþ ratio stimulates the pentose phosphate pathway.

DHA and ascorbate (27,28). AFR can be reduced back to ascorbate enzymatically by both NADH- and NADPH-dependent mechanisms (23). A cytochrome b5 reductase in the outer mitochondrial membrane of rat liver mitochondria was found to possess NADH-dependent AFR reductase activity (29), and this mechanism presumably accounts for NADH-dependent AFR reductase activity in endothelial cells (23). NADPH-dependent reduction of AFR involves thioredoxin reductase (TR), and this has been demonstrated in rat liver homogenates (21), human erythrocytes (30), and endothelial cells (23). AFR reduction by NADHand NADPH-dependent mechanisms were compared in endothelial cells by May et al. (23). They found that in whole lysates, NADH-dependent reduction predominated, but following removal of particulate matter, NADPH-dependent mechanisms predominanted, thus NADH-dependent AFR reductase activity is largely confined to particulate fractions, whereas NADPH-dependent activity was confined to the cytosol (23). This activity was identified as TR (23).

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Within cytosolic secretory vesicles, the AFR is produced during the ascorbatedependent mono-oxygenase reactions of either dopamine hydroxylase or peptidylglycine mono-oxygenase. AFR is then reduced back to ascorbate utilizing electrons transferred across the vesicle membranes from cytosolic ascorbate via cytochrome b561 (31). Although cytosolic ascorbate is oxidized to AFR in this process, this can then be recycled itself by mechanisms described earlier. Extracellular AFR an also be reduced by various mechanisms that require intracellular donors. It has been demonstrated in erythrocytes that intracellular ascorbate can donate electrons to extracellular AFR via a plasma membrane redox system (32), and that this process causes depolarization of the membrane (33). AFR reductases are present in the plasma membrane of erythrocytes which can potentially reduce AFR using intracellular NADH (34). Intracellular ascorbate can also supply electrons for transmembrane electron transport using ferricyanide as an electron as the extracellular electron acceptor (27,35). An ascorbate-dependent trans-plasma membrane oxidoreductase has been proposed (27); however, more work is required in this area to fully elucidate this activity. DHA is formed either by dismutation of the AFR, or by two electron oxidation of ascorbate by reactive oxidizing species. Efficient AFR reductase activity reduces the amount of AFR available for dismutation into DHA (23,28) and is such an important process. It has been proposed that due to high capacity and affinity of the AFR reductases, in the absence of severe oxidative stress, AFR reduction is probably the most important mechanism of ascorbate recycling (23). Thus, the majority of DHA must come from the antioxidant action of ascorbate following sustained oxidative stress either intra- or inter-cellularly. DHA is formed during the “oxidative burst” of mononuclear cells to destroy pathogens. Indeed, ascorbate recycling in neutrophils can be induced by micro-organisms (24) which initiate the oxidative burst and production of ROS. Similarly, HL-60 promyeloctic tumor cells increased their rate of DHA uptake following activation (36). DHA is also reduced back to ascorbate by enzymatic and non-enzymatic mechanisms. In both circumstances, GSH and NADPH play an important role as the electron donor. Direct chemical reduction by GSH has been demonstrated (37,38); however, some of these experiments were performed in a “test-tube” model. The products of this reaction are ascorbate and GSSG in equimolar amounts, GSSG being formed stoicheometrically (37). Enzymatic reduction of DHA is more efficient. The majority of GSH-dependent enzyme reduction can be attributed (at least in neutrophils) to glutaredoxin (39). Inhibition of this enzyme blocked DHA reduction by up to 80% in neutrophil lysates (39). This reduction is 10 –20-fold faster than direct chemical reduction. In human endothelial cells, DHA reduction was substantial even in the absence of lysate; however, there was a linear increase in DHA reduction with increasing amounts of lysate (23). This demonstrates that in this cell line both enzymatic and non-enzymatic reductions are important. In human erythrocytes, DHA reduction is also GSH-dependent (22,30). Interestingly, in the absence of glucose, DHA reduction consumed NADH as well as NADPH; whereas in the presence of glucose, only NADPH was consumed (22), indicating that cellular

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metabolic pathways must also be taken into account. Although DHA reduction in erythrocytes occurs substantially through a direct chemical reaction with GSH, enzymes such as TR also appear important (30). Other GSH-dependent proteins with DHA reductase activity include protein disulfide isomerase (40), glutathione peroxidase (41), and an as yet unidentified protein isolated from human erythrocytes (42,43). DHA can also be reduced back to ascorbate via a nonGSH-dependent enzyme action. TR, as well as reducing the AFR, can reduce DHA (30,44) and requires NADPH as an electron donor. The contribution of TR though appears to be minor in certain cells, as a 3-fold increase in TR enzyme activity following selenium supplementation did not increase the ability of cultured hepatoma cells to reduce DHA (45). There was also little contribution of TR to DHA reduction relative to that mediated by GSH in human endothelial cells (23). Another NADPH-dependent DHA reductase was identified from rat liver to be 3-a-hydroxysteroid dehydrogenase (46). The importance of GSH-independent DHA reductases was demonstrated in human leukemic cells (HL-60) depleted of GSH with the agent BSO, which were still able to accumulate millimolar concentrations of ascorbate from DHA (47). However, in contrast to these results, human erythrocytes treated with diethylmaleate to deplete GSH showed parallel decreases in the reduction of DHA, and ascorbate-dependent ferricyanide reduction (22). Thus, the major mechanism of DHA reduction may depend on the cell type. The physiological role for DHA reduction is still a matter for debate, at least in the absence of sustained oxidative stress (28). Human cells can been shown to generate ascorbate via recycling of DHA generated extracellularly by activated cells undergoing the oxidative burst, in what was termed a “bystander effect” by the authors (25). Uptake of DHA into cells is obviously a pre-requisite for recycling. However, reduction of DHA by erythrocytes can occur extracellularly by the passage of electrons through the plasma membrane (48). Ascorbate recycling capacity has been investigated in severe deficiency of both young and mature guinea pigs (49). The guinea pig is a good model for study since, like humans, they lack the ability to synthesize ascorbic acid and hence require it in the diet. Ascorbate deficiency resulted in significantly increased recycling capacity of erythrocytes in young but not mature animals, whereas liver homogenate ascorbate recycling was unaffected by age or diet (49). Consistent with this data, a recent human study of ascorbate recycling in cigarette smokers has demonstrated that smoking-induced oxidative stress enhances ascorbate recycling in erythrocytes (50). Taken together these two studies indicate an adaptive mechanism involving induction of DHA reductases during de novo erythrocyte synthesis, in response to oxidative stress (50). Ascorbate Recycling and Metabolism The recycling of ascorbate utilizes reducing equivalents in the form of NAD(P)H. This occurs either directly, via NAD(P)H-dependent reductases, or indirectly via

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GSH-dependent reduction which requires glutathione reductase to recycle the oxidized GSH (GSSG), a reaction which requires NADPH. Ascorbate recycling therefore decreases the intracellular concentration of NAD(P)H and GSH (22), thereby directly influencing intracellular redox status. A consequence of ascorbate recycling is stimulation of the pentose phosphate pathway (22,51). This pathway provides NADPH which is required for synthetic pathways, and maintains GSH concentrations via GSH reductase. Modulation of the pentose phosphate pathway has been demonstrated in various mammalian cell lines (22,51 –53). In Jurkat and human (H9) T-lymphocytes, DHA, both dose-dependently and timedependently, increased the activity of the pentose phosphate pathway enzymes glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase and transaldolase (51). An increase in G6PD protein levels was also observed, indicating a stimulation of gene expression by DHA (51). At the same time, DHA treatment (800 mM for 48 h) increased intracellular GSH levels by over 3-fold (51) and is thus equal, if not more, potent than N-acetyl cysteine in increasing intracellular GSH concentrations. Breakdown products of DHA include the five carbon sugars L -lyxose and L -xylose, which can enter the pentose phosphate pathway (54), thus providing a mechanism for both increased enzyme activity and GSH levels induced by DHA. In ascorbate synthesizing tissues, ascorbate has been shown to be catabolized and recycled through pentose phosphate and gluconeogenic pathways (52). Ascorbate metabolism was investigated further in cells that cannot synthesis ascorbate. Human erythrocytes were shown to effectively metabolize ascorbate to lactate, with DHA being the better substrate (53); whereas in human HepG2 cells, the end product of ascorbate metabolism was glucose instead of lactate, again with DHA being the better substrate (53). The authors suggest that in species that are unable to synthesize ascorbate there exists a catabolic side to an inter-organ ascorbate cycle (53). Experiments in growing cartilage cells demonstrated that ascorbate could maintain the energy status of the cells (55). In ascorbate treated cells, there was a decreased rate of lactate formation, coupled with a maintenance of mitochondrial energy generation (measured by the adenylate energy charge ratio) and isocitrate dehydrogenase (NAD-dependent) activity (55). The authors suggest that ascorbate could inhibit the utilization of pyruvate by anaerobic glycolysis, so promoting mitochondrial oxidative reactions (55). Because ascorbate recycling can deplete glutathione, it has been suggested that this could generate oxidative stress. This has been studied in several systems (56 – 59). Ascorbate/DHA treatment depleted GSH and increased lipid peroxidation in brain slices (56), neuronal cells (57), pancreatic tissue (59), and liver slices (58). These effects were prevented by inhibitors of GLUT transport (57 – 59), implying that the uptake and recycling of DHA induces oxidative stress. The dependence of ascorbate recycling on provision of reducing equivalents via metabolic pathways was shown in experiments with human erythrocytes in the presence or absence of glucose (22). In the presence of glucose, DHA reduction was enhanced and resulted in oxidation of NADPH and activation of

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the pentose phosphate pathway at the G6PD step, whereas GSH and NADH levels were maintained (22). In the absence of glucose, although DHA reduction still occurred, this was associated with a decrease in GSH and NADH levels (22). This implies that in normal metabolizing cells with sufficient metabolic substrate, reduction of DHA occurs via a NADPH- and GSH-dependent cycle, but when metabolism is limited, DHA reduction is switched to a different mechanism involving NADH which does not involve the pentose phosphate pathway.

MOLECULAR FUNCTIONS OF ASCORBIC ACID Ascorbate Modulation of Collagen Formation Maturation of certain mesenchymal tissues including bone and cartilage involves both the synthesis and secretion of collagen forming an extracellular matrix. One of the cellular functions of ascorbate involves the hydroxylation of proline and lysine residues in procollagen. Without this ascorbate-requiring hydroxylation, collagen fibrils cannot bind and form their triple helix structure and thus extracellular matrix formation is impaired. In addition to these posttranslation effects, it is well established that ascorbate can also modify collagen production via pretranslational events. An ascorbate-induced increase in collagen gene expression has been demonstrated in a variety of cell lines including primary avian tendon cells (60), human (61) and porcine (62) skin fibroblasts, human (63) and porcine (62) smooth muscle cells, bovine chondrocytes (64), and even human dermis (65). On the mechanism, studies have demonstrated that ascorbate induces an increase in the rate-constant for procollagen translation and secretion (66) and this involved an enhancement of procollagen gene transcription, accompanied by increased stability of procollagen mRNA from a half-life of 10 to 20 h (67). A stabilization of type I collagen mRNA was also the mechanism of ascorbate-induced collagen expression in porcine smooth muscle cells and fibroblasts (62). Concentrations of ascorbate used typically range from 3 to 300 mM, with effects demonstrated at concentrations as low as 50 mM, and this typically induces a 2 –3-fold increase in levels of procollagen mRNA. Although induction of type I collagen is frequently associated with ascorbate, other forms of collagen have also been investigated. In human skin fibroblasts, types I and II procollagen mRNA are induced by ascorbate (68). In bovine smooth muscle cells, type VI collagen is produced in the absence of ascorbate, but the addition of ascorbate induces type I, decreases type VI, with no change in the levels of type III (69). However, in porcine smooth muscle cells, types I and III are induced by ascorbate (62). In chondrocytes type X collagen (70) and types I, II, and X collagen (64) have been found to be induced by ascorbate. Thus, ascorbate can modulate collagen synthesis not only by hydroxylation of procollagen, but also by increasing both the level and stability of procollagen mRNA. The ability of ascorbate to increase stability of mRNA has been investigated independently. It was found that ascorbate

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(at ascorbate:RNA ratios .1:40) was able to interact directly with both G –C and A –U base pairs of RNA via H-bonding through its anion CO and OH groups (71), providing a potential mechanism of this enhanced stabilization effect. The induction of collagen mRNA by ascorbate appears to occur following a lag period, which appears to depend on the cell type and conditions, but can be from a few hours in L5 skeletal muscle cells (72), to 12 h in avian tendon cells (66) to 1 or 2 days in chondrocytes (70). Such a response is indicative of prior events which are required for transcription. One of these effects may involve the production of lipid peroxides, as ascorbate was found to induce lipid peroxides in fibroblasts (73) and this step was suggested to be necessary for stimulation of collagen gene expression (73). However, one study has demonstrated that the ascorbate-induced generation of lipid peroxides and collagen are coincidental, since in the presence of iron chelators lipid peroxidation was blocked but collagen synthesis was unaffected (74). Also, multiple dosing is often required for these ascorbate-induced effects (62,70), presumably to circumvent the depletion of ascorbate in the culture medium. The use of stable ascorbate analogs may aid in such studies. In preliminary studies reported by Davidson et al. (62), multiple dosing and long exposure times were not necessary when using ascorbate 2-phosphate. The augmentation of procollagen mRNA by ascorbate and the stable analog palmitoyl ascorbate was compared in human intestinal smooth muscle cells (63). The increase in both procollagen mRNA and collagen synthesis was more efficient and sensitive with palmitoyl ascorbate; however, the response was similar with both species at higher concentrations (63). It has been suggested that the requirement for longer incubation times with lower doses of ascorbate may be related to the oxidation of ascorbate in the culture medium (62). However, it is uncertain if it is related to the induction of cellular lipid peroxidation, which can potentially influence gene expression. In opposite to the effect of ascorbate on collagen formation, studies have shown that ascorbate exerts a negative effect on elastin production (62,75 – 77). When both collagen and elastin production were compared together in vascular smooth muscle cells and skin fibroblasts, the differential effects of ascorbate were found to result from a marked stabilization of collagen mRNA over the lesser stabilization of elastin mRNA and the repression of elastin gene transcription (62). Collagen degradation is a symptom of aging. Both aging and UV irradiation can induce levels of matrix metalloproteinases (MMP) which degrade collagen and elastin. In addition to regulating type I and III collagen gene transcription, ascorbate has been shown to downregulate MMP-2 in cultured human amnion-derived cells (78), and reducing activity and expression of MMP-1 and 2 in UV irradiated human keratinocytes (79). Conversely, ascorbate was shown to induce expression of MMP-1 in human periodontal ligament cells but not in MC3T3-E1 osteoblast-like cells (80), whereas MMP-13 expression was upregulated during growth of osteoblastic MC3T3-E1 cells in the presence

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of ascorbate 2-phosphate (81). Although human skin produces both MMP-1 and MMP-8 following UV irradiation, it appears that MMP-1 may play the most important role in collagen degradation (82). Ascorbate concentration has shown to be lower in the epidermis and dermis of photoaged skin (83) and when exposed to UV irradiation, levels of ascorbate (as well as the other antioxidants a-tocopherol and glutathione) are depleted in the skin of humans in vivo (84). These data coupled to the study of Nusgens et al. (65) who demonstrated that topically applied ascorbate enhanced mRNA levels of collagen types I and III as well as the mRNA of the MMP-1 inhibitor TIMP-1 in human dermis (65) indicate that ascorbate treatment can potentially delay the skin aging process. Ascorbate Modulation of Cell Differentiation Ascorbate is required for normal cell growth and differentiation. Through its preand posttranslational modulation of collagen formation, ascorbate is important for growth of mesenchyme-derived connective tissues such as muscle, cartilage, and bone (85). The mechanisms of ascorbate-induced differentiation of collagensecreting cells appear to depend on the cell type. In osteoblasts, the effect of ascorbate is dependent on its collagen-inducing properties (85 – 88); whereas in chondrocytes, ascorbate modulation of gene expression is independent of collagen secretion (70). In muscle cell cultures, ascorbate treatment also appears to induce acetylcholine receptor (AchR) expression (72,89 –91). An investigation into this effect revealed that it was a specific action on the a subunit of AchR and involves mRNA synthesis (91). Induction of AchR expression occurred with a lag of 24 h and followed collagen synthesis. Although collagen secretion is also induced by ascorbate under these conditions, the process of AchR expression and collagen secretion induced by ascorbate were found to be independent processes (72,89). Cell maturation in mineralizing tissues, such as cartilage and bone, is linked to the activity of the enzyme alkaline phosphatase. The activity of this enzyme has been shown to be increased during the ascorbate-induced differentiation of osteoblasts (87,88), chondrocytes (55,92), and ST2 stromal cells (93). This effect also follows a lag period, but the increase in activity is quite dramatic, being over 10-fold higher in chondrocyte cell lines (70,92). In human fibroblasts, ascorbate induced alkaline phosphatase activity, but this was dependent on the presence of an extracellular matrix of fibronectin (94). Collagen matrix formation was also found to be essential for the inductive effects of ascorbate in the differentiation of stromal cells, which included increased alkaline phosphatase activity (93). Ascorbate also appears to be important for growth and differentiation of noncollagen-secreting cells, such as monocytes (95), and keratinocytes (96). It has also been established that ascorbate can stimulate the differentiation of leukemic cells induced by 1a,25-dihydroxyvitamin D3 (97 –100). The ascorbate 2-phosphate induced differentiation of keratinocytes was found to be mediated by

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protein kinase C (PKC), causing activation of PKC by translocating it from the cytosol to the membrane (96). This PKC activation then led to activation of activator protein (AP)-1 (96) which mediates expression of genes associated with cell growth and development. A further interesting observation in this study was that ascorbate treatment also caused the upregulation of the ascorbate transporters SVCT1 and SCVC2, which in turn resulted in accumulation of intracellular ascorbate (96). The involvement of AP-1 has also been demonstrated in the ascorbate stimulation of 1a,25-dihydroxyvitamin D3-induced monocyte differentiation (98). In these experiments, ascorbate increased AP-1 DNA binding in non-induced cells, but inhibited DNA binding in induced cells even though mRNA levels of AP-1 constituents (c-jun, junB, c-fos) were increased (98). The modulation of transcription factors by ascorbate is discussed in the following section. The influence of ascorbate on 1a,25-dihydroxyvitamin D3-induced cell differentiation were caused by addition of ascorbate, but not of DHA or the more stable analog ascorbate 2-phosphate, implying perhaps that oxidation of ascorbate in the medium is a major contributing factor to the effects in such studies. Indeed, in a study in human leukemic cells, AFR, but not DHA or the ascorbate analog isoascorbate, increased cell growth (95). This suggests that ascorbate oxidation and induction of lipid peroxidation are prerequisite for these ascorbate-mediated effects, as in that proposed in collagen synthesis in the previous section, and may therefore be an artifact of the conditions. Modulation of Transcription Factors Ascorbate has been shown to modulate the activities of the nuclear transcription factors nuclear factor-kappa B (NF-kB), and AP-1. Both these factors are redox sensitive and can be modulated by antioxidants (101,102). NF-kB is involved in the regulation of many genes associated with the inflammatory response. In the dormant form, NF-kB, homo- or heterodimers of constituents of the Rel family, is bound to I-kB in the cytosol, but upon activation, I-kB is phosphorylated leading to degradation and the release of NF-kB, which translocates to the nucleus for DNA binding. Modulation of NF-kB activation by ascorbate appears to be dependent on the conditions used. In Jurkat T-cells, ascorbate (0.2 mM) potentiated the activation of NF-kB induced by TNF-a (103). This was evidenced by increased DNA binding of NF-kB, and enhanced I-kB degradation (103). In LPS and IFN-g activated murine macrophages, ascorbate did not induce I-kB-a degradation or NF-kB activation but did prolong the recovery of I-kB, which led indirectly to an enhancement of NF-kB DNA binding (104). NF-kB activation can be induced by UVA irradiation, and in HaCaT keratinocytes, ascorbate (1 mM) was found to potentiate this UVA-induced NF-kB nuclear binding activity (105). Shang et al. investigated the effects of both UVA irradiation and ascorbate on NF-kB activation in melanocytes and keratinocytes and found contrasting results. Although ascorbate (0.1, 0.25 mM) increased NF-kB binding activity in

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HaCaT keratinocytes, ascorbate decreased NF-kB binding in melanocytes (106). Carcamo et al. found that ascorbate inhibited TNF-a-induced activation of NF-kB in the human cell lines HeLa, U937 monocytes, HL-60 myeloid leukemic, breast MCF7 and HUVEC primary endothelial cells (107). Both NF-kB nuclear translocation and I-kB phosphorylation were decreased in ascorbate treated cells (107) and the authors postulated a mechanism whereby ascorbate inhibited the TNF-a-induced activation of NF-kB-inducing kinase (NIK) and I-kB kinases (IKK), independent of p38 MAP kinase (107). Bowie et al. also found that ascorbate (5 – 20 mM) inhibited the TNF-a- or IL-1-induced activation of NF-kB in two endothelial cell lines (primary HUVEC and ECV304) (108). No effect on DNA binding of NF-kB was found but ascorbate was found to inhibit phosphorylation (and therefore degradation) of I-kB through a sustained activation of p38 MAP kinase (108). The p38 MAP kinase mediated this effect by inhibition of IKK (108). It appears then that p38 has a dual role depending on the kinetics of its activation; whereas p38 has a positive regulatory role at the transcriptional level, a rapid and sustained activation can inhibit upstream I-kB degradation and override the positive effect (108). The mechanisms of ascorbate-induced inhibition of NF-kB activation are summarized in Fig. 12.3. Ascorbate has also been shown to modulate NF-kB activation in an animal model of atherosclerosis (109). Antioxidant supplementation (vitamin E 100 IU/kg plus 1 g ascorbic acid per day) in hypercholesterolemic pigs normalized NF-kB activation and preserved endothelial function (109). In contrast to these reports, ascorbate appeared to have no effect on NF-kB activity in HIV infected ACH-2 T cells (110), in TNF-a activated human aortic endothelial cells (111), in LPS or IFN-g activated rat skeletal muscle endothelial cells (112), and in TNF-a activated endothelial cells (113). Downstream products of NF-kB activation have also been investigated. In a cell culture model, ascorbate downregulated IL-1a mRNA expression in both UVA irradiated and non-irradiated cells, whereas IL-6 mRNA expression was unaffected (114). In an animal model, B and T lymphocytes isolated from pigs with hereditary vitamin C deficiency with and without ascorbate supplementation, showed decreased proliferative responses in the depleted group and differential production of IL-2 and IL-6 (115). AP-1 is involved in the expression of genes involved in cellular proliferation and the cell cycle. AP-1 is composed of a mixture of heterodimeric protein complexes derived from the Fos (c-Fos, FosB, Fra-1, Fra-2) and Jun (c-Jun, JunB, and JunD) families. Phosphorylation of these proteins by various kinases, for example, Jun N-terminal Kinase (JNK), is required for transactivation activity. In a macrophage, cell line ascorbate was found to potentiate PMA-induced AP-1 nuclear binding (116), and this involved the amplification of p38 MAP kinase and JNK induction, and thus the authors proposed that ascorbate induces these effects via modulation of critical thiols in the MAP kinase pathway (116). Opposing results were found by Kyaw et al. (117) who demonstrated ascorbate inhibition of endothelin-induced AP-1 DNA binding activity in rat aortic smooth muscle cells. This mechanism involved the inhibition of

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Figure 12.3 Representation of the ascorbate-induced inhibition of nuclear transcription factor DNA binding. Ascorbate treatment has been shown to inhibit TNF-a and/or IL-1 induced activation of NF-kB in endothelial cells by an inhibitory effect on I-kB phosphorylation either by inhibiting I-kB kinase directly (107) or mediated through an activation of p38 MAP kinase (108). Ascorbate 2-phosphate treatment of keratinocytes inhibited AP-1 activation by inhibiting the JNK-mediated phosphorylation of c-Jun (preventing stable AP-1 binding complexes) and by inducing fra-1 which is a negative regulator of AP-1. [This figure reproduced with permission from Catani et al. (118).]

JNK and p38 MAP kinase activation (117). Another investigation found that ascorbate 2-phosphate can interfere with the activity of the JNK and AP-1 pathway in HaCaT keratinocytes (118). In this case, ascorbate 2-phosphate modulation of AP-1 occurred via dual mechanisms. First, ascorbate 2-phosphate was able to inhibit the phosphorylation (and hence activation) of JNK, which in turn prevents phosphorylation of c-Jun (118), preventing the formation of stable AP-1 transcription factor complexes. Secondly, ascorbate 2-phosphate caused the induction of fra-1, which is a negative regulator of AP-1, such that ascorbate was able to inhibit basal and UVB-induced AP-1 activity (118). These pathways are shown in Fig. 12.3. A dual effect of ascorbate on AP-1 binding was demonstrated in HL-60 cells (98). In non-induced cells, ascorbate treatment increased AP-1 DNA binding; however, if the cells were induced to differentiate with 1a,25-dihydroxyvitamin D3 , ascorbate treatment then inhibited AP-1 DNA binding, even though ascorbate treatment increased mRNA levels of c-jun, junB, and c-fos (98), which aid in the formation of stable AP-1 binding complexes.

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The modulation of NF-kB and AP-1 by ascorbate appears to be a matter for controversy since both inhibition and potentiation of these transcription factors has been demonstrated. A critical factor in these observations could be the conditions used, and especially the cell line, mode of activation, and concentration of ascorbate. For example, relatively low concentrations of ascorbate resulted in potentiation of NF-kB activation, whereas millimolar concentrations result in inhibition of activation. This in itself implies multiple mechanisms of ascorbate modulation at various levels of cell signaling. It is interesting that both AP-1 and NF-kB modulation by ascorbate do share a common mechanism in the blunting of phosphorylation reactions by kinases. It is likely that ascorbate influences critical thiols in these kinases, as previously suggested (116). However, further work is required to characterize this effect. In regard to other transcription factors, ascorbate treatment was shown to attenuate activation of the transcription factor IRF-1, which is involved in iNOS expression, in endothelial cells (112), and had no inhibitory effect on IFN-g-induced STAT1 DNA binding in endothelial cells (108). Ascorbate Modulation of Nitric Oxide Production Antioxidants such as ascorbate and vitamin E are known to protect and enhance NO levels (119,120). This forms the basis of studies demonstrating an improvement in endothelium-dependent dilation in patients with CHD (121) and essential hypertension (122) following acute ascorbate supplementation. Various mechanisms have been postulated for the ascorbate prevention of endothelium dysfunction (123). In murine macrophages activated with LPS and IFN-g, a 40% increase in NOx production was induced by ascorbate (124). The authors demonstrated that this effect was due to an 2-fold increase in iNOS mRNA and protein levels in cells in the presence of ascorbate (124). These studies were extended by the same group who investigated the potential mechanism of ascorbateinduced iNOS expression (104). Ascorbate was found to prolong the recovery of I-kB which paralleled the elevated NF-kB binding to DNA (104). In contrast to these effects, ascorbate was found to inhibit iNOS expression in rat skeletal muscle endothelial cells (125) and septic rat skeletal muscle (112). Pretreatment of endothelial cells with ascorbate inhibited the induction of iNOS protein and NOx production induced by LPS and IFN-g (125). This was suggested to be an antioxidant effect via blunting of superoxide and did not involve the NF-kB pathway (125). Another interesting mechanism involves the ascorbate effect on tetrahydrobiopterin (BH4) which acts as a cofactor for eNOS. In porcine aortic endothelial cells, NO production was enhanced by ascorbate by up to 70%, consistent with a stimulation of the catalytic activity (Vmax) of bovine eNOS by up to 50% (126). This effect was mediated through an increase in intracellular BH4 (126). The mechanism for this ascorbate action on BH4 in human umbilical vein endothelial cells was found to involve chemical stabilization (127) since ascorbate increased the half-life of BH4 and decreased biopterin oxidation

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products (127). Thus, the action of ascorbate on eNOS can be seen to be similar to that with its di-oxygenase activity, in maintaining adequate levels of cofactor. Ascorbate-Induced Modulation of DNA Repair Several reports have demonstrated an ascorbate-induced modulation of pathways involved in either DNA repair or apoptosis. Catani et al demonstrated that ascorbate 2-phosphate treatment of HaCaT cells resulted in up-regulation of Mut L homolog-1 (MLH1) expression in spite of UVB irradiation (118) and went on to investigate this regulation further (128). MLH1 allows the removal of mismatches which can occur during DNA replication and is thus part of the DNA repair machinery of the cell. The MLH1 signal transduction pathway involves c-Abl and p73 as downstream targets and is a critical factor in whether the cells undergo either DNA mutation or apoptosis. Ascorbate-induced up-regulation of MLH1 occurred following just 2 h incubation with ascorbate 2-phosphate (1 mM) and also resulted in the up-regulation of p73 gene expression (128). The authors concluded that ascorbate can improve the cellular response to DNA damage, and this was demonstrated by ascorbate increasing cisplatin-induced apoptosis (128). The authors also suggest that both the anticarcinogenic and anti-cancer activities of ascorbate might be explained by this modulation of MLH1 gene expression (128). An ascorbate-induced potentiation of Fas-mediated apoptosis has also been demonstrated in Jurkat and human T cells (129). In this study, DHA was able to enhance cell-surface expression of the Fas receptor (129). However, a recent study has shown an opposite effect. DHA treatment of U937 monocytes or fresh primary human monocytes resulted in inhibition of Fas-mediated apoptosis (130). This effect was associated with an inhibition of caspase-8 activation (130). A role for ascorbate in the regulation of DNA repair enzymes has been previously postulated (131), as significant decreases in specific DNA oxidation products (8-oxodG) of mononuclear cells were found following ascorbate supplementation in humans (131). Taken together these reports demonstrate that ascorbate can protect against oxidative damage both as an antioxidant and as a regulator of DNA repair. Ascorbate-Induced Modulation of Other Genes In a search for ascorbate-regulated genes, Mizutani et al. (132) demonstrated that ubiquitin mRNA levels in guinea pig tissues decreased under ascorbate-deficient conditions and increased under ascorbate-replete conditions. However, the same group failed to demonstrated any ascorbate-induced gene expression of ubiquitin in activated macrophages (104). Ascorbate deficiency in guinea pigs also led to a decreased expression of mRNA for the cytochrome P450 variants CYP1A1 and CYP1A2 (133), leading to the authors to suggest that P450 transcription is regulated by ascorbate in guinea pigs (133). Using cDNA microarray technology, Catani et al. demonstrated that ascorbate 2-phosphate treatment of HaCaT cells resulted in up-regulation of fra-1, glutathione S-transferase Pi (GSTpi) and

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MLH1 expression inspite of UVB irradiation (118). This latter effect was described in the preceding section. Ascorbate treatment of epithelial cells has also been shown to increase mRNA for ferritin by 30% (134), consistent with a close relationship between ascorbate and iron status.

CONCLUSION Ascorbic acid is an essential micronutrient required for a number of cellular functions. The importance of maintaining intracellular ascorbate is demonstrated in the variety of potential mechanisms available to recycle ascorbate from its one and two electron oxidation products AFR and DHA. Ascorbate recycling depletes NAD(P)H and GSH, and in this way ascorbate can influence metabolic pathways. For example, the pentose phosphate pathway is stimulated, which supplies NADPH. It is becoming increasingly evident that ascorbate can also modulate signaling pathways and influence gene expression. Such effects include increasing collagen expression by stabilization of its mRNA, influencing cell maturation and differentiation, modulation of the transcription factors NF-kB and AP-1, and modulating NO formation. Although some of these actions are not well characterized and may be highly dependent on the conditions used, it is clear that ascorbate is more than just a reducing agent.

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13 Cell Signaling Properties of a-Lipoic Acid: Implications in Type 2 Diabetes Hadi Moini and Lester Packer University of Southern California, Los Angeles, California, USA

Kyung-Joo Cho and An-Sik Chung Korea Advanced Institute of Science and Technology, Daejeon, South Korea

Introduction Diabetes and Current Strategies for Its Treatment Targeting the Insulin-Signaling Pathway a-Lipoic Acid Improves Glucose Metabolism in Type 2 Diabetes a-Lipoic Acid Activates the Insulin-Signaling Pathway a-Lipoic Acid Regulates Adipocyte Differentiation Concluding Remarks References

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INTRODUCTION The prevalence of diabetes is on the rise in the world population. It is estimated that 6.2% of the US population is affected by diabetes. The number of diabetics is expected to continue to increase by 4– 5% per year, potentially reaching a total of 220 million cases in 2010 worldwide. Therefore, effective interventions are needed to prevent or cure diabetes and/or attenuate its symptoms and complications. a-Lipoic acid is a disulfide derivative of octanoic acid that forms an 283

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intramolecular disulfide bond in its oxidized form (Fig. 13.1). High electron density resulting from special position of the two sulfur atoms in the 1,2-dithiolane ring confers upon a-lipoic acid a high tendency for reduction of other redox-sensitive molecules according to environmental condition (1). a-Lipoic acid has been long used in the treatment of diabetic neuropathy (2). Recent advances in our understanding of a-lipoic acid actions in muscle and fat cells in vitro and in vivo established that a-lipoic acid enhances glucose disposal and attenuates adipogenesis. These actions of a-lipoic acid are mediated by an array of signaling molecules and transcription factors that now have become attractive targets for design of new pharmacological agents to treat diabetes. Recent findings suggest that a-lipoic acid may emerge as an effective anti-diabetic agent that can be used as an adjunctive treatment in type 2 diabetes. DIABETES AND CURRENT STRATEGIES FOR ITS TREATMENT Diabetes is of two types. Type 1 diabetes is a multifactorial autoimmune disease, which is characterized by T-cell-mediated destruction of the insulin secreting b cells of the Langerhans islets in the pancreas. The destructive process leads to severe insulin depletion, which results in hyperglycemia, owing to hepatic overproduction of glucose and decreased cellular uptake of glucose from the circulation (3). The pathogenesis of type 2 diabetes, however, is not fully understood. The transition from normal glucose tolerance to type 2 diabetes in susceptible individuals is punctuated by insulin resistance, dysregulated hepatic glucose production, impaired glucose tolerance, and declining b cell function. Many obese and insulin-resistant subjects escape diabetes by oversecreting insulin. It is the failure of the pancreatic b cells to compensate for insulin resistance that leads to hyperglycemia. Once type 2 diabetes has occurred, hyperglycemia worsens the pre-existing b cell dysfunction. Approximately, 90 – 95% of individuals diagnosed for diabetes have type 2 diabetes (4). Type 1 and type 2 diabetes are associated with increased risk for development of cardiovascular disease, neuropathy, retinopathy, and nephropathy. Type 1 diabetes requires life-long treatment with exogenous insulin for survival. The therapeutic goals in type 2 diabetes are attenuation of symptoms by normalizing fasting and postprandial blood glucose levels and prevention of acute and long-term complications. It is now clear that aggressive control of hyperglycemia in patients with type 2 diabetes can reduce the development of chronic complications such as retinopathy and nephropathy. Hence, current strategies for

(a) S

COOH S

Figure 13.1

(b) SH

COOH SH

Molecular structure of a-lipoic acid (a) and dihydrolipoic acid (b).

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treatment of type 2 diabetes rely mainly on several approaches aimed to decrease the hyperglycemia itself. These approaches include (i) increasing insulin release from pancreatic islets by sulfonylureas, (ii) attenuating hepatic glucose production by metformin, (iii) enhancing sensitivity of hepatic and peripheral tissues to insulin by peroxisome proliferators-activated receptor-g (PPARg) agonists (thiazolidinediones), (iv) interfering with gut glucose absorption by a-glucosidase inhibitors, and (v) suppressing glucose production and augmenting glucose utilization by administration of exogenous insulin (3,4). TARGETING THE INSULIN-SIGNALING PATHWAY Current therapeutic approaches were largely developed in the absence of a solid understanding of the pathogenesis of type 2 diabetes. However, with the expansion of our understanding of biochemical pathways involved in the development of type 2 diabetes, several mechanistic sites, such as the insulin-signaling pathway (Fig. 13.2), are now considered as a target for new therapeutic interventions. Defects such as decreased tyrosine phosphorylation of the insulin receptor (IR) and insulin receptor substrate-1 or -2 (IRS-1/2), attenuated association of phosphoinositol-3-kinase (PI3-K) with IRS-1/2, and diminished PI3-K activity were repeatedly demonstrated in muscle, fat, or liver tissues from type 2 diabetic patients, individuals with abnormal oral glucose tolerance, or animal models of insulin resistance (7 –13). In contrast, activation of mitogen-activated protein kinases (MAPK) signaling pathway by insulin was not affected in type 2 diabetic patients (14). Hence, it is now clear that type 2 diabetic patients display diminished signaling in the PI3-K axis, which might be overcome by interventions aimed at augmenting activation of the insulin-signaling pathway. One such approach involves developing pharmacological agents that are capable of inhibiting the negative regulator(s) of the insulin-signaling pathway and thereby potentiating actions of insulin. Protein tyrosine phosphatases (PTP) regulate signal transduction pathways involving tyrosine phosphorylation. General inhibition of PTP by nonselective inhibitors such as vanadyl sulfate improved hepatic and peripheral insulin sensitivity in type 2 diabetic patients (15,16). A large body of evidence from cellular, biochemical, and genetic studies have identified protein tyrosine phosphatase 1B (PTP1B) as the major phosphatase responsible for dephosphorylation and inactivation of the IR (17). Deletion of PTP1B gene markedly enhanced insulin sensitivity in mice (18). Furthermore, treatment of insulin-resistant mice with PTP1B antisense oligonucleotide increased activity of the insulin-signaling pathway in liver and fat cells, lowered plasma glucose and insulin levels, and enhanced insulin sensitivity (19,20). These findings established PTP1B as a potential therapeutic target and raised much promise for use of its selective inhibitors in the treatment of type 2 diabetes and obesity (21 –23). The insulin-signaling pathway may be also negatively regulated at the level of IRS-1. Phosphorylation of IRS-1 on serine/threonine residues impairs the

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Figure 13.2 Signaling pathways of insulin. The IR is a tyrosine kinase that undergoes autophosphorylation and catalyzes the phosphorylation of cellular proteins such as members of IRS family, Shc, and Cbl. Upon tyrosine phosphorylation, these proteins interact with signaling molecules through their Src homology 2 (SH2) domain, resulting in activation of diverse series of signaling pathways. Engagement of tyrosine-phosphorylated IRS with the SH2 domains in the p85 regulatory subunit of type I phosphatidylinositol 3-kinase (PI 3-K) activates the catalytic p110 subunit, which catalyzes the phosphorylation of PI 4,5-diphosphate on the 3 position, generating PI 3,4,5-triphosphate. PI 3,4,5triphosphate serves as an allosteric regulator of the phosphoinositide-dependent kinase (PDK), which phosphorylate and activate protein kinase B (Akt) as well as the atypical protein kinase C isoforms, PKCj and PKCl. Although PI 3-K activation was shown to be necessary for insulin-stimulated glucose uptake, the precise identity of the physiologically relevant kinase that triggers translocation of glucose transporter-4 (GLUT4) containing vesicles to plasma membrane is unclear. However, in addition to PI3-K activity other signals seem to be required for insulin-stimulated glucose uptake. This second pathway appears to involve tyrosine phosphorylation of the Cbl proto-oncogene, which is in complex with the adaptor protein CAP. Upon phosphorylation, the Cbl – CAP complex translocates to lipid rafts domains in the plasma membrane and recruits the adaptor protein CrkII through interaction of the SH2 domain of CrkII with phospho-Cbl. CrkII forms a constitutive complex with guanyl nucleotide-exchange protein C3G, which activates the small GTP-binding protein TC10. Once activated, TC10 seems to provide a second signal, in parallel with the activation of the PI3-K pathway, to the GLUT4 containing vesicles. Tyrosine phosphorylated Shc, however, interacts with the adaptor protein Grb2 and recruits the son-of-sevenless exchange protein to the plasma membrane and thereby activates Ras. Once activated, Ras initiates a cascade of serine phosphorylation, which results in activation of Raf, MEK, and MAPK, such as JNK, ERK, and p38 MAPK. p38 MAPK were shown to be involved in enhancing of the intrinsic activity of GLUT4 (5,6).

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ability of IRS-1 to activate downstream PI3-K (24 – 26). Glycogen synthase kinase-3 (GSK-3) and inhibitor kappaB kinase were demonstrated to serine phosphorylate IRS-1 and attenuate insulin signaling (27,28). Selective inhibition of GSK-3 activity lowers blood glucose levels and augments insulin action in insulin-resistant mice, whereas heterozygous deletion of inhibitor kappaB kinase or its inhibition by salicylate prevents fat-induced insulin resistance in rodents (29 – 32). Type-II SH2-domain-containing inositol 5-phosphatase (SHIP2) dephosphorylates key phospholipids that are generated by insulinmediated PI3-K activation and hence negatively regulates the insulin-signaling pathway (33). Heterozygous deletion of SHIP2 increased glucose tolerance and insulin sensitivity, which was accompanied with an elevated recruitment of glucose transporter-4 (GLUT4) and increased glycogen synthesis in mouse skeletal muscles (34). Thus, GSK-3, inhibitor kappaB kinase, and SHIP2 represent potentially novel therapeutic targets for the treatment of type 2 diabetes. An alternate approach to inhibiting the negative regulators would be identifying nonpeptide small molecules that can activate the insulin-signaling pathway. Discovering mimetics of insulin has proven to be difficult. Through extensive screening of over 50,000 mixtures of synthetic compounds and natural products, a small molecule, L-783,281, was identified from a fungal extract (Pseudomassaria sp.) that activated IR in vitro by altering conformation of its kinase domain (35). Oral administration of L-783,281 lowered blood glucose level, improved glucose tolerance, and suppressed elevated plasma insulin levels in animal models of type 2 diabetes (35,36). a-Lipoic acid was also demonstrated to mimic insulin action, activate IR, and enhance glucose uptake into fat and muscle cells. A growing body of evidence suggests that a-lipoic acid administration to type 2 diabetic patients may have potential therapeutic value in lowering elevated glucose levels and suppressing diabetic complications.

a-LIPOIC ACID IMPROVES GLUCOSE METABOLISM IN TYPE 2 DIABETES a-Lipoic acid was first reported to enhance glucose utilization in isolated rat heart and diaphragm (37,38). Subsequent studies demonstrated that chronic a-lipoic acid administration increases GLUT4 protein level in muscle membranes, improves insulin-stimulated 2-deoxyglucose uptake into isolated skeletal muscles, and reduces blood glucose levels in animal models of diabetes (39–41). Furthermore, a-lipoic acid in combination with exercise training additively increased insulinmediated glucose transport in isolated skeletal muscles, decreased the glucose– insulin index, an indication of increased insulin sensitivity, and enhanced oral glucose tolerance in insulin-resistant obese ( fa/fa) Zucker rats (42). This beneficial interactive effect of a-lipoic acid and exercise training, however, was not apparent in insulin-sensitive lean ( fa/2) Zucker rats (43). Oral or intravenous administration of a-lipoic acid also increased insulin sensitivity in individuals

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with type 2 diabetes (44–46). Taken together, these studies show that a-lipoic acid may enhance the capacity of insulin-stimulated glucose transport and utilization. However, a-lipoic acid itself also enhanced glucose uptake into epitrochlearis muscles isolated from both insulin-resistant obese ( fa/fa) and insulin-sensitive lean ( fa/2) Zucker rats (47), suggesting that a-lipoic acid may mimic insulin action on glucose transport and metabolism.

a-LIPOIC ACID ACTIVATES THE INSULIN-SIGNALING PATHWAY Mechanistic studies conducted in L6 myotubes and 3T3-L1 adipocytes as a model of muscle and fat cells in culture revealed that a-lipoic acid rapidly stimulates tyrosine phosphorylation of IR and IRS-1, enhances PI3-K, Akt, and p38 MAPK activities, elevates GLUT4 content in the plasma membranes, and increases glucose uptake into the cells (48 –50). These studies provided strong evidence that stimulation of glucose uptake by a-lipoic acid is due to enhanced translocation and intrinsic activity of GLUT4 through activation of Akt and p38 MAPK, respectively. Subsequent studies investigated the basis for the ability of a-lipoic acid to activate the elements of the insulin-signaling pathway. 3T3-L1 adipocytes were found to have a low capacity to reduce a-lipoic acid to dihydrolipoic acid. Even when added exogenously to the cell culture media, dihydrolipoic acid marginally increased glucose uptake into the cells (51). However, a parallel trend was observed between the intracellular oxidant levels, GSH levels, and glucose uptake following incubation of cells with a-lipoic acid (Fig. 13.3). Glucose uptake was increased by several folds reaching to the peak level at 6 h. Intracellular oxidant levels, measured as 20 ,70 -dichlorofluorescin fluorescence, was also increased in parallel to the changes in the glucose uptake and simultaneously reached to the peak level at 6 h. However, as GSH levels started to rise, the intracellular oxidant levels and the glucose uptake declined (51). These findings suggest that at the early time points (up to 6 h) a-lipoic acid increases glucose uptake by changing the intracellular redox status toward an oxidizing condition, whereas at later time points (.12 h) a-lipoic acid, by increasing GSH levels, shifts the intracellular environment toward more reducing conditions, which lowers the rate of glucose uptake. Intracellular redox status is known to play an important role in the modulation of insulin action. Treatment of IR-transfected Chinese hamster ovary cells with antioxidants such as N-acetyl-cysteine or butylated hydroxyanisole inhibits insulin responsiveness, whereas partial inhibition of glutathione metabolism, which intracellularly induces a mild oxidative stress condition, stimulates IR tyrosine phosphorylation in vitro (52). Moreover, oxidation of critical cysteine residues in the IR b-subunit was found to result in an increase in its intrinsic tyrosine kinase activity, whereas low concentrations of dithiothreitol inactivated the IR kinase, suggesting that the functional activity of the IR can be also modulated by alteration of the redox state of cysteine residues present in IR b-subunit

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Fold increase compared to basal

6 Glucose uptake GSH ∑ DCF fluorescence

5 4 3 2 1 0

0

6

12 18

24 30 36 42 48

-1 -2

Time (h) Figure 13.3 Temporal kinetics of a-lipoic acid-induced changes in glucose uptake, GSH levels, and oxidant levels in 3T3-L1 adipocytes. Glucose uptake, GSH levels, and intracellular oxidant were determined following treatment of 3T3-L1 adipocytes with 250 mM a-lipoic acid for indicated times. Intracellular oxidants were detected by 20 ,70 dichlorofluorescin (DCF ) fluorescence.

(53,54). Furthermore, IR was demonstrated to couple, via Gai2 , to the NADPHdependent H2O2 generating system, which upon insulin stimulation produces H2O2 in 3T3-L1 adipocytes (55). Insulin-dependent H2O2 production was associated with a decreased PTP activity (56) and was also found essential for the activation of PI3-K (57). These findings suggest that redox signals are involved in the regulation of both the early tyrosine phosphorylation cascade and the downstream insulin-signaling events. In parallel to the increase in the intracellular oxidant levels, a-lipoic acid stimulated tyrosine phosphorylation of IR in vivo, which was accompanied by a decrease in the thiol reactivity of IR b-subunit (Fig. 13.4) (58). a-Lipoic acid also directly stimulated tyrosine phosphorylation of immunoprecipitated IR in vitro (51). Furthermore, a-lipoic acid inhibited total PTP activity in vivo, which was accompanied by a decrease in the thiol reactivity of PTP1B, whereas N-acetyl-L -cysteine, a mild thiol antioxidant, increased total PTP activity (Fig. 13.5) (58). The impact of inhibition of oxidant production on IR tyrosine phosphorylation could not be assessed by using inhibitors of phagocyte NADPH oxidase owing to poor characterization of the adipocyte NADPH oxidase. However, these findings strongly suggest that a-lipoic acid directly or indirectly by increasing intracellular oxidant levels may oxidize critical thiol groups of IR b-subunit and PTPs and result in their activation or inactivation,

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Figure 13.4 a-Lipoic acid decreases thiol reactivity of the IR and enhances its tyrosine phosphorylation. Free thiol (a) and phosphotyrosine (b) content of IR b-subunit was determined following treatment of 3T3-L1 adipocytes with a-lipoic acid (500 mM, 30 min) or insulin (100 nM, 10 min).

respectively, and thereby enhance the early tyrosine phosphorylation cascade of the insulin-signaling pathway (58).

a-LIPOIC ACID REGULATES ADIPOCYTE DIFFERENTIATION Obesity is closely correlated with the prevalence of diabetes and cardiovascular disease. Obesity is caused not only by hypertrophy of adipose tissue, but also by adipose tissue hyperplasia, which triggers the transformation of preadipocytes into adipocytes (59). Adipocyte differentiation is a complex process and is regulated by several signaling pathway (60). The PI3-K pathway transduces the proadipogenic effects of insulin by promoting activation of CCAAT element binding protein (C/EBP) b and d and sterol response element binding protein 1. These transcription factors induce the expression and/or activity of PPARg, a pivotal coordinator of adipocyte differentiation. Activated PPARg induces exit from the cell cycle and in cooperation with C/EBPa stimulates the expression of many metabolic genes such as GLUT4, lipoprotein lipase (LPL), and adipocyte-specific fatty acid binding protein (aP2) (61,62), thus constituting a functional lipogenic adipocyte. Besides these integral members of the adipogenesis program, other transcription factors, such as AP-1 and cAMPresponsive element binding protein (CREB), are also known to promote adipogenesis, whereas nuclear factor-kB (NF-kB) suppresses adipocyte differentiation (63 – 65). In contrast to the PI3-K-signaling pathway, MAPK such as extracellular

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Figure 13.5 a-Lipoic acid decreases thiol reactivity of PTPB1 and inactivates total PTP activity. The level of thiol-biotinylated PTP1B (a) or total PTP activity (b) was determined following treatment of 3T3-L1 adipocytes with a-lipoic acid, insulin, N-acetyl-L -cysteine, or H2O2 .

signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) suppress the process of adipocyte differentiation by phosphorylating and thereby attenuating the transcriptional activity of PPARg (66,67). 3T3-L1 preadipocytes initiate their conversion to mature adipocytes 3 days after addition of a hormonal cocktail consisting of insulin, dexamethasone, and 3-isobutyl-1-methylxanthine. By the day 6, the number of fully differentiated adipocytes is increased by several folds. a-Lipoic acid displays a biphasic effect on differentiation of preadipocytes to mature adipocytes (68). At low concentrations (100 mM) a-lipoic acid promotes whereas at higher concentrations (250 mM) it attenuates the hormonal cocktail-, insulin-, or PPARg agonist troglitazone-induced differentiation. The pro- and anti-adipogenic effects of

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Figure 13.6 a-Lipoic acid inhibits differentiation of preadipocytes induced by a hormonal cocktail (insulin, dexamethasone, and 3-isobutyl-1-methylxanthine) or insulin. 3T3-L1 preadipocytes were treated with 10 nM insulin or the hormonal cocktail for 3 days in the absence or presence of indicated concentrations of a-lipoic acid. Cells were then maintained in cell culture media containing a-lipoic acid for additional 3 days in the absence of insulin or the hormonal cocktail. (a) Morphological changes associated with adipogenesis was photographed on the basis of staining cellular triglyceride deposition by Oil Red O. (b) mRNA levels of LPL and aP2 were determined by northern blot analysis and were expressed as fold increase compared with basal.

a-lipoic acid are accompanied with an increase or a decrease, respectively, in proadipogenic proteins such as aP2 and LPL (Fig. 13.6). Analysis of transcription factors involved in the process of adipocyte differentiation demonstrated that a-lipoic acid diminished activities of proadipogenic transcription factors such

Figure 13.7 a-Lipoic acid modulates DNA-binding activities of pro- and antiadipogenic transcription factors in 3T3-L1 adipocytes. DNA-binding activities of NF-kB, AP-1, CREB, C/EBP were analyzed by electrophoretic mobility shift assay following treatment of preadipocytes with 10 nM insulin (I) or the hormonal cocktail (C) in the absence or presence of 500 mM a-lipoic acid for 2 h. Arrows indicate specific binding of nuclear proteins to the labeled DNA.

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as AP-1, C/EBP, CREB, and PPARg while enhancing the activity of the antiadipogenic transcription factor NF-kB (Fig. 13.7) (68). a-Lipoic acid and insulin also display a differential effect on activation of MAPK and PI3-K pathways in preadipocytes primarily due to the differences in the potency and kinetics of the activation of these two pathways (Fig. 13.8). The adipogenic hormone insulin strongly activated PI3-K pathway, which lasted up to several hours but weakly and transiently activated MAPK-signaling pathway. In contrast, a-lipoic acid weakly and transiently activated PI3-K pathway, whereas more strongly activated MAPK-signaling pathway. Importantly, inhibitors of ERK or JNK abolished the anti-adipogenic effect of a-lipoic acid on insulinor the hormonal cocktail-induced adipogenesis (68). These findings demonstrate that insulin and a-lipoic acid oppositely regulate adipocyte differentiation and that the MAPK-signaling pathway mediates actions of a-lipoic acid on adipocytes differentiation by down- or up-regulating activities of the pro- or antiadipogenic transcription factors, respectively. CONCLUDING REMARKS In differentiated adipocytes, a-lipoic acid mimics insulin actions on glucose uptake by modulating activities of several components of the insulin-signaling pathway (Fig. 13.9). a-Lipoic acid may directly interact with IR b-subunit, oxidize its critical thiol groups, and thereby facilitate its autophosphorylation. Alternatively, a-lipoic acid may indirectly oxidize the cysteine residue of IR b-subunit by increasing intracellular levels of oxidants. Whether a-lipoic acid

Figure 13.8 a-Lipoic acid and insulin differentially regulate PI3-K- and MAPKsignaling pathways in preadipocytes.

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Molecular targets of a-lipoic acid in the insulin-signaling pathway.

stimulates adipocyte plasma membrane NADPH oxidase is not known. Nevertheless, the generated oxidants may also oxidize the cysteine residues of PTP such as PTP1B, and inhibit its activity leading to enhancement of IR activation. Furthermore, a-lipoic acid enhances the intrinsic activity of GLUT4 by activating p38 MAPK. Hence, the short-term effect of a-lipoic acid in differentiated adipocytes is the activation of proximal components of the insulin-signaling pathway and the enhancement of glucose uptake. However, long-term incubation of adipocytes with a-lipoic acid increases intracellular GSH, which scavenge H2O2 , and may restore redox states of IR and PTP1B and thereby inactivate the insulin-signaling pathway and attenuate glucose uptake. In preadipocytes, a-lipoic acid and insulin differentially regulate PI3-Kand MAPK-signaling pathways. In contrast to insulin, a-lipoic acid strongly activates MAPK such as JNK and ERK. Activation of JNK and ERK leads to opposite regulation of pro- and anti-adipogenic transcription factors and results in diminished differentiation of preadipocytes. Hence, a-lipoic acid and insulin exert opposite effects on adipocyte differentiation. Decrease in the thiol reactivity of IR and PTPB1 and activation of MAPK following a-lipoic acid treatment of adipocytes are all indicative of a shift in the intracellular redox state toward an oxidizing condition. It is highly likely that a-lipoic acid may directly or indirectly lead to oxidation of other thiol-containing proteins such as Keap1. Thiol groups of Keap1 were recently demonstrated to function as sensors that regulate induction of phase 2 enzymes (69). Oxidation of critical thiol groups of Keap1 resulted in disruption of Keap1 – Nrf2 complex followed by migration of Nrf2 to nucleus where it forms heterodimers

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56. Mahadev K, Zilbering A, Zhu L, Goldstein BJ. Insulin-stimulated hydrogen peroxide reversibly inhibits protein-tyrosine phosphatase 1b in vivo and enhances the early insulin action cascade. J Biol Chem 2001; 276:21938 –21942. 57. Mahadev K, Wu X, Zilbering A, Zhu L, Lawrence JT, Goldstein BJ. Hydrogen peroxide generated during cellular insulin stimulation is integral to activation of the distal insulin signaling cascade in 3T3-L1 adipocytes. J Biol Chem 2001; 276:48662– 48669. 58. Cho KJ, Moini H, Shon HK, Chung AS, Packer L. a-Lipoic acid decreases thiol reactivity of the insulin receptor and protein tyrosine phosphatase 1B in 3T3-L1 adipocytes. Biochem Pharmacol 2003; 66:849 – 858. 59. Caro JF, Dohm LG, Pories WJ, Sinha MK. Cellular alterations in liver, skeletal muscle, and adipose tissue responsible for insulin resistance in obesity and type 2 diabetes. Diabetes Metab Rev 1989; 5:665 – 689. 60. Torti FM, Torti SV, Larrick JW, Ringold GM. Modulation of adipocyte differentiation by tumor necrosis factor and transforming growth factor beta. J Cell Biol 1989; 108:1105– 1113. 61. Schoonjans K, Peinado-Onsurbe J, Lefebvre AM, Heyman RA, Briggs M, Deeb S, Staels B, Auwerx J. PPARalpha and PPARgamma activators direct a distinct tissuespecific transcriptional response via a PPRE in the lipoprotein lipase gene. EMBO J 1996; 15:5336 –5348. 62. Tontonoz P, Hu E, Graves RA, Budavari AI, Spiegelman BM. mPPAR gamma 2: tissue-specific regulator of an adipocyte enhancer. Genes Dev 1994; 8:1224 – 1234. 63. Yang VW, Christy RJ, Cook JS, Kelly TJ, Lane MD. Mechanism of regulation of the 422(aP2) gene by cAMP during preadipocyte differentiation. Proc Natl Acad Sci USA 1989; 86:3629 –3633. 64. Reusch JE, Colton LA, Klemm DJ. CREB activation induces adipogenesis in 3T3-L1 cells. Mol Cell Biol 2000; 20:1008 –1020. 65. Ruan H, Hacohen N, Golub TR, Van Parijs L, Lodish HF. Tumor necrosis factor-alpha suppresses adipocyte-specific genes and activates expression of preadipocyte genes in 3T3-L1 adipocytes: nuclear factor-kappaB activation by TNF-alpha is obligatory. Diabetes 2002; 51:1319 – 1336. 66. Font de Mora J, Porras A, Ahn N, Santos E. Mitogen-activated protein kinase activation is not necessary for, but antagonizes, 3T3-L1 adipocyte differentiation. Mol Cell Biol 1997; 17:6068 – 6075. 67. Camp HS, Tafuri SR, Leff T. c-Jun N-terminal kinase phosphorylates peroxisome proliferator-activated receptor-gamma1 and negatively regulates its transcriptional activity. Endocrinology 1999; 140:392 – 397. 68. Cho KJ, Moon HE, Moini H, Packer L, Yoon DY, Chung AS. Alpha-lipoic acid inhibits adipocyte differentiation by regulating pro-adipogenic transcription factors via mitogen-activated protein kinase pathways. J Biol Chem 2003; 278:34823– 34833. 69. Dinkova-Kostova AT, Holtzclaw WD, Cole RN, Itoh K, Wakabayashi N, Katoh Y, Yamamoto M, Talalay P. Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants. Proc Natl Acad Sci USA 2002; 99:11908 – 11913.

14 Dietary Isoflavones and Coronary Artery Disease—Proposed Molecular Mechanisms of Action Aedin Cassidy University of East Anglia, Norwich, UK

Sonia De Pascual-Teresa Instituto del Frı´o, Consejo Superior de Investigaciones Cientificas, Madrid, Spain

Abstract Introduction Dietary Sources of Phytoestrogens Absorption and Metabolism Estrogen Receptor Mediated Mechanisms of Action Anti-Oxidant Activity Cardiovascular Effects Lipid Metabolism Animal Studies Clinical Studies Blood Pressure Inflammation and Cell Adhesion Platelet Aggregation and Endothelium Reactivity Conclusion References 301

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ABSTRACT Experimental and epidemiological data are available to support the concept that isoflavone-rich diets exert physiological effects in humans; however, to date most research interest has focused on their potential hormonal activities. Dietary isoflavones are one of the major classes of phytoestrogens, and are currently receiving much attention because of their potential role in preventing coronary artery and other chronic diseases. In the overall scheme of cardiovascular protection, isoflavones appear to potentially have a more important role in conditioning the vascular tree than on influencing cholesterol levels. The preferential binding of isoflavones to the ERb and the increasing recognition of the role of this receptor in the endothelial wall provide justification for increasing the awareness of the heart-health effects of diets rich in these phytoestrogens. Furthermore, such effects are not restricted to soy isoflavones but also apply to lignans and many other flavonoid sub-classes, which are abundant in cereals, pulses, fruits, and vegetables. Potential anti-atherogenic effects of isoflavones include a reduction in LDL cholesterol, modulation of pro-inflammatory cytokines, cell adhesion proteins, and nitric oxide (NO) formation, protection of LDL against oxidation, inhibition of platelet aggregation, and an improvement in vascular reactivity. However, further molecular work is required to elucidate the exact mechanisms by which isoflavones affect these processes and to define the physiological relevance of these mechanisms relative to human exposure from these compounds. Although epidemiological data and laboratory studies allude to the possible protective effects of soy isoflavones at specific target tissues, randomized placebo controlled clinical trials are necessary to further address the relative importance of these compounds for cardiovascular health. INTRODUCTION Dietary phytoestrogens are a sub-class of flavonoids that are of particular interest in relation to human health, which embody several groups of nonsteroidal estrogens including isoflavones and lignans that are widely distributed within the plant kingdom (1). Phytoestrogen-rich diets exert physiological effects, and preliminary human studies suggest a potential role for dietary phytoestrogens in hormonedependent disease (2). In particular, a number of cardioprotective benefits have been attributed to dietary isoflavones including a reduction in LDL cholesterol, an inhibition of pro-inflammatory cytokine, cell adhesion protein, and nitric oxide production, potential reduction in the susceptibility of the LDL particle to oxidation, inhibition of platelet aggregation, and an improvement in vascular reactivity. DIETARY SOURCES OF PHYTOESTROGENS In relation to human health, research interest has to date concentrated on the isoflavone and lignan subgroups of phytoestrogens. The isoflavones are the most

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extensively studied of the phytoestrogen class; however, their occurrence in foods is limited largely to soyabeans and a few other legumes (1). Lignans, in contrast, are widely distributed but they have been relatively little studied due in part to difficulties in their isolation and analysis (3,4). The basic structural unit of the isoflavones comprises of two benzene rings, linked via a heterocyclic pyrone ring. The chemical structures of commonly occurring isoflavones are shown in Fig. 14.1. Although nonsteroidal, it is the phenolic ring and in particular the 40 -OH hydroxyl group of isoflavones that are the essential structural component for interaction with estrogen receptors (ER). Although isoflavones have estrogenic activity 100– 1000 times weaker than estradiol (1), some foods and dietary supplements contain comparatively high amounts of these compounds so that plasma levels may exceed endogenous estrogen levels by several orders of magnitude and therefore these compounds have the potential to exert biological effects in vivo. Daily dietary intake of isoflavones in western populations is typically negligible (,1 mg/day), whereas recent estimates indicate intakes of 20 –50 mg/day in Japan, but this may vary between urban and rural areas, and with other lifestyle factors (1). Although all soyabean-derived protein extracts and foods available for human consumption contain significant levels of isoflavones, there is a large variability in concentration and profile among these products as factors such as species, geographical, and environmental conditions, and the extent of industrial processing of the soyabeans and all alter the levels of isoflavones present (5). ABSORPTION AND METABOLISM To date, 12 different isoflavone isomers have been identified. The primary isoflavones in soybeans are the glucosides, genistin, and daidzin and their respective aglycones gensitein and daidzein. Typically, in soybeans and soy foods there are higher gensitein levels than daidzein. Isoflavones are present in plants as O

HO A

O

HO

O

HO

C B

O Daidzein HO

OH

H3CO

O

OH

O

O

Formononetin

O

OH OCH3

OH Glycitein

HO

O

Figure 14.1

OH

OH Genistein

O

Biochanin A

A comparison of the chemical structures of isoflavones.

OCH3

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Gut microflora

Small intestine

malonylglucosides acetylglucosides Glucosidases b-glucosides

daidzein genistein

demethylation dehydroxylation reduction ring cleavage

equol dehydrodaidzein desmethylangolesin p-ethylphenol

Absorption

hepatic conjugation (enterocyclic cycling)

Urinary excretion

Glucuronide conjugates Sulphate conjugates Liver

Figure 14.2

Absorption and metabolism isoflavones in humans.

glycoside conjugates, but following ingestion (Fig. 14.2) they are hydrolyzed by intestinal glucosidases and the resulting aglycones may be absorbed or further metabolized in the large gut to specific metabolites. Interest in gut metabolites has increased in recent years, specifically in the daidzein metabolite, equol, because of its stronger binding affinity to ERs and preliminary evidence to suggest that it is a more potent modulator of hormonal status in healthy young women (6). The role of the intestinal microflora in the metabolism of phytoestrogens has long been established (3,7) with early evidence showing that antibiotic administration blocks metabolism and germ free animals do not excrete the metabolites (7). However, to date it is still unclear what specific bacterial species are responsible for the conversions. Until recently, available data on the absorption and metabolism of dietary phytoestrogens were of a qualitative nature; dietary phytoestrogens are metabolized by intestinal bacteria, absorbed, conjugated in the liver, circulated in plasma, and excreted in urine (Fig. 14.2). Recent studies have addressed quantitatively what happens to isoflavones following ingestion—with pure compound and stable isotope data to compliment recent pharmacokinetic data for soy foods (8,9). Serum genistein and isoflavone levels increase in response to soy or pure compound administration, although not always in a dose dependent manner, and concentrations can readily reach the low micromolar level. Plasma levels in free living Asian subjects are 500 nmol/L when measured after an overnight fast, but because the half life of isoflavones is 6– 9 h, fasting levels are much lower than postprandial levels. Isoflavones circulate in plasma primarily in the conjugated form, mostly bound to glucuronic acid with 3% circulating in the free form. Knowledge of the pharmacokinetics of phytoestrogens is essential prior to making recommendations regarding long-term efficacy in clinical studies, as

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recent research suggests significant differences in bioavailability between foods rich in phytoestrogens and supplements (8). In addition, dose administered, food matrix, and the chemical form of the compound appear to exert effects on the bioavailability (9). Maintenance of a steady state serum level should be optimal for clinical effectiveness of these compounds and on the basis of recent pharmacokinetic data, this would be best achieved by divided doses of the soya food or supplement throughout the day, rather than by a single dose. ESTROGEN RECEPTOR MEDIATED MECHANISMS OF ACTION Isoflavones have a spatial configuration similar to that of mammalian estrogens, bind to ERs and affect estrogen regulated gene products (10 –12). The higher binding affinity of isoflavones for ERb, compared with ERa and the different tissue distributions of these receptors (Fig. 14.3) suggest that these compounds may be tissue selective, exerting estrogenic actions in some tissues such as coronary vessels (13) but not in other tissues such as the endometrium (1,2,14). The estrogenic potency of isoflavones is low compared with 17-b-estradiol, with soy isoflavones having approximately one-third and 1/1000 of the affinity of 17-b-estradiol for the ERb and ERa, respectively (15). Genistein has been shown to have a binding affinity for ERa and ERb of 4% and 87%, respectively, of estrogen, whereas daidzein is less potent having affinities of 0.1% and 0.5%, respectively (15). Since genistein possesses an order of 20 times higher binding affinity for ERb than for ERa, isoflavones

Activating stimuli: Reactive Oxygen Species (ROS) Cytokines

IkB Inactive form IkB kinase IkB Active form Translocation Cytoplasm

Inflamatory and inmune proteins

Transcription NF-kB Target genes

Nucleus

Figure 14.3

Estrogen receptor mediated effects and tissue distribution.

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can be regarded as a type of natural selective estrogen receptor modulator (SERM) for this receptor. However, recent X-ray crystallographic studies examining the interaction of estrogens, raloxifene, and genistein with ERb suggest that the orientation of raloxifene and genistein with ERb is different from that of estradiol, in particular in the interaction with helix 12 of the receptor (1,8). Isoflavones lack specific lipophilic regions, which undoubtedly impact on their ERb binding ability and the subsequent sequence in cellular events triggered by their binding of the agonist. Therefore, isoflavones may be more correctly classified as SERMs than “estrogens,” which suggests that isoflavones may possess the beneficial physiological actions of natural estrogens, without the associated negative effects, in particular in tissues such as the breast (8). Although the reported estrogenic potency of isoflavones is weak, 100– 1000 times less compared with 17-b-estradiol, their biological potential cannot be ignored, as typical circulating levels of isoflavones can exceed endogenous estradiol concentrations by 10,000fold following consumption of a diet containing soy foods (16,17). Genistein has a 100-fold greater binding affinity than daidzein for the mouse uterine cytosolic estrogen receptors. Furthermore, the formation of glucuronide conjugates decreases the relative affinities of isoflavones to estrogen receptors (18). However, binding affinity alone does not determine potency, because the resulting conformational change in the ligand (isoflavone – receptor complex varies among ligand regardless of binding affinity (19). The isoflavone genistein is also .1000-fold more potent at triggering transcriptional activity with ERb than ERa (20) and this difference is far greater than the 30-fold greater binding affinity for ERb than ERa (21). These data therefore suggest that the isoflavone genistein is a potent agonist for ERb and the divergent transcriptional activities of estrogens and isoflavones results not only from their different binding affinities but also from differences in their ability to recruit coregulators and trigger transcriptional functions of ERa and ERb (20). Anti-Oxidant Activity Although there is significant interest in the anti-oxidant properties of isoflavones, to date most of these investigations have focused solely on the anti-oxidant effects of genistein (22). Proposed molecular mechanisms responsible for its anti-oxidant potential include the ability to scavenge radicals, chelate metals, inhibit hydrogen peroxide (H2O2) production, and stimulate anti-oxidant enzymes, including catalase and superoxide dismutase. In a liposomal system, it has been demonstrated that genistein is a more effective anti-oxidant than daidzein, which is likely to be attributable to its third hydroxyl group in the C-5 position. Moreover, the isoflavone precursors biochanin A and formononetin showed very weak anti-oxidant capacities in this in vitro system as they lack the C-40 hydroxyl group, which appears to be an important determinant of the anti-oxidant properties of isoflavones (23). Equol showed superior anti-oxidant actions compared to both the precursor

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molecules and the parent isoflavones, suggesting that the absence of the 2,3double bond in conjunction with a loss of the 4-oxo group enhances antioxidant properties (23). Antioxidant activity, assessed by the trolox equivalent anti-oxidant capacity (TEAC) assay are consistent with these data that equol is a more potent isoflavone compared with genistein and daidzein (24). In an in vitro experimental system, genistein (IC50 ¼ 25 mM) was a more potent inhibitor of the formation of H2O2 by 12-O-tetradecanoylphorbol-13acetate-activated HL-60 cells and the generation of superoxide anions by xanthine/xanthine oxidase compared with daidzein (IC50 ¼ 150 mM), apigenin, and biochanin A (22,25). Activities of anti-oxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase were also significantly increased with gensitein (25). Furthermore, genistein has been shown to enhance antioxidant enzyme activity in murine cells by the suppression of tumor promoterinduced H2O2 formation (22). The free radical-scavenging and anti-oxidant activities of various structurally related isoflavones including genistein, daidzein, biochanin A, and genistin in a cell-free and an endothelial cell model systems were investigated (26). All isoflavones tested had no significant scavenging effects on these radicals at concentrations up to 1.0 mM, suggesting that free radical scavenging activities of some isoflavones may not substantially contribute to their anti-oxidant properties. However, as physiologically achievable concentrations (5 nM) increased intracellular-reduced glutathione levels, this ability may make a more significant contribution to their biological action than their scavenging activities. Genistein and daidzein, the major isoflavone aglycones, have received most attention; however, they undergo extensive metabolism in the gut and liver, which may affect their anti-oxidant properties. Recently, the anti-oxidant activity and, free radical-scavenging properties of the isoflavone metabolites equol, 8-hydroxydaidzein, O-desmethylangiolensin, and 1,3,5 trihydroxybenzene in comparison to their parent aglycones, genistein, and daidzein have been investigated, with electron spin resonance spectroscopy indicating that 8-hydroxydaidzein was the most potent scavenger of hydroxyl and superoxide anion radicals. Isoflavone metabolites also exhibited higher anti-oxidant activity than parent compounds in standard anti-oxidant (FRAP and TEAC) assays indicating that the metabolism of isoflavones affects their free radical scavenging and antioxidant properties (27). Isoflavones have been shown to reduce LDL oxidation. Six healthy volunteers consumed soy protein (60 mg isoflavones per day) for 2 weeks. LDL oxidation, as assessed by the lag time of copper induced oxidation, was shown to be significantly prolonged compared with baseline measurements (28). In vitro data are consistent with these findings, with Kapiotis et al. (29) demonstrating that genistein inhibited the oxidation of LDL in the presence of copper ions or superoxide and NO radicals as measured by thiobarbituric acid-reactive substance formation (TBARS).

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CARDIOVASCULAR EFFECTS Although the mechanisms involved in the development of arteriosclerosis have not been fully established, there is a consensus that the expression by endothelial cells of inflammatory cytokines, adhesion molecules, and chemotactic proteins plays a key role. Epidemiological studies suggest that differences in diet may explain the lower incidence of CVD in Japan compared with other industrialized countries such as the United States or the UK and the wide international variability in intake of dietary isoflavones may play a role (1,30,31). Potential antiatherogenic effects of isoflavones (Table 14.1) include a reduction in LDL cholesterol; modulation of pro-inflammatory cytokines; cell adhesion proteins, and nitric oxide (NO) formation; protection of LDL against oxidation, inhibition of platelet aggregation; and an improvement in vascular reactivity.

LIPID METABOLISM Animal Studies The hypocholesterolemic effect of soy protein has been known for many decades (32). In many animal species, substituting soy protein for dietary animal protein consistently reduces LDL-cholesterol and total cholesterol levels (33). Gerbils fed soy-based diets have significantly lower levels of total cholesterol, LDL þ VLDL cholesterol, and apolipoprotein B concentrations (34). Isoflavone consumption led to a 30% decrease in plasma cholesterol levels and a 50% reduction in atherosclerotic lesion area in a strain of mice with low HDL-cholesterol (35). Table 14.1 Potential Mechanisms by Which Isoflavones Protect Against Arteriosclerosis Antioxidant properties Inhibition of LDL oxidation Stimulation of antioxidant enzymes Induction of GSH synthesis Gene regulatory activity Inhibition of NF-kB dependent signal transduction pathways Inhibition of protein tyrosine kinase activity Inhibition of inducible nitric oxide production in macrophages Down-regulation of cell adhesion and proinflammatory cytokine expression Hypocholesterolaemic effects Increased bile acid secretion Increased LDL receptor activity Reduced cholesterol absorption from gut Platelet function and vascular effects Inhibition of platelet aggregation Improvement of vascular reactivity

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Soy protein containing isoflavones decreased LDL-cholesterol and increased HDL-cholesterol in a group of female monkeys fed a moderately atherogenic diet (36,37) and when a state of menopause was experimentally established in this animal model, by ovariectomy, soy protein consumption, as compared to casein consumption, significantly improved plasma lipids, and lipoprotein concentrations. The key issue of whether the response to soy protein is mediated through the presence of isoflavones has been the focus of much attention. It appears that when isolated soy protein is alcohol-washed and most, but not all, of the isoflavones are removed, there is only a marginal reduction in plasma cholesterol. The hypocholesterolemic effect, however, is restored when the isoflavones are added back to the alcohol-washed isolated soy protein. Furthermore, isoflavones alone have little or no cholesterol-lowering effect in cynomologus monkeys (36,37). Clinical Studies Although the mechanism of action of the cholesterol lowering effect of soya is still poorly understood, clinically it has been effectively used in the therapy of patients with hypercholesterolemia for several decades (33). A meta-analysis of 38 clinical studies concluded that the mean reduction in serum total cholesterol was 9.3%, whereas LDL decreased by 12.9% with soya protein extracts (32). Individuals with the highest initial cholesterol levels experienced the greatest reduction. An intake of 25 g/day of soya protein extract would be associated with a 0.23 mmol/L decrease in serum cholesterol (32). On the basis of this evidence and further clinical studies, the FDA approved a health claim for cholesterol reduction based on an intake of 25 g soya protein per day. This intake is higher than the current daily intake in Japan and it is unknown whether life time exposure to diets rich in these compounds accounts for the lower blood cholesterol and CHD rates in the Asian populations. A Japanese health checkup study of 3596 women observed a strong inverse relationship between daily soy protein intake and serum cholesterol. The average soy protein intake for women was 6.88 g (38), which is calculated as an isoflavone intake of 10 – 30 mg/day. The FDA drew no conclusion regarding the role of isoflavones in the cholesterol lowering effect, but a recent study has shown that isoflavones play a significant role in lowering plasma LDL and their absence from soya renders the food ineffective in reducing cholesterol levels. They showed a linear dose –response relationship between dietary isoflavone content and cholesterol reduction, with no lowering effect observed when isoflavones were removed from the soy protein (39). A group of 156 men and women with moderately elevated total cholesterol and LDLc were randomized to receive a soy protein beverage with differing amounts (0 –58 mg/day) of isoflavones (39) only the isoflavone-containing beverages lowered total cholesterol and LDLc.

310

Cassidy and De Pascual-Teresa

Potter and co-workers (40) randomly assigned 66 hypercholesterolemic, postmenopausal women to one of three diet groups: 40 g milk protein/day, 40 g isolated soy protein/day with 1.39 mg isoflavones/g protein, or 40 g isolated soy protein/ day with 2.25 mg isoflavones/g. The women had been placed on a National Cholesterol Education Program Step I diet, 2 weeks prior to randomization and continued the diet throughout the course of the study. At the end of the 24-week diet period, HDLc had risen and non-HDLc had fallen in the two soy groups, as compared with the milk protein group (40). In a study of 43 postmenopausal women, the HDL cholesterol:total cholesterol level was increased 5.5% by a diet comprising 750 mL of soymilk or 30 g of soy nuts (containing 60 – 70 mg/day isoflavones) (41), whereas the resistance of LDL-cholesterol to oxidation was significantly increased. On the other hand, isoflavones alone (fed as pure compounds or extract) have consistently been found to have no lipid-lowering effect (42 – 44) indicating that the mechanism of action of isoflavones on lipids is complex and probably involves an interaction with the food matrix (Table 14.2). BLOOD PRESSURE The endothelial wall of the blood vessel has been found to have almost equal proportions of ERa and ERb (64,65), and these two receptors play an important role in the vasoreactivity of the blood vessels. ERa rapidly activates endothelial derived nitric oxide synthase (eNOS), the key enzyme responsible for the production of nitric oxide-induced dilatation of the blood vessels (66). This is a nongenomic and rapid event that is reduced in atherosclerotic arteries. Studies of the ERa and ERb knock-out mouse attest to the important role that these two receptors play in vascular events related to cardiovascular disease with data suggesting that ERb deficient mice have several functional abnormalities in vascular smooth muscle cells and blood vessels, providing strong evidence for the essential role for ERb in the regulation of blood pressure and vascular function (67). Therefore, perhaps the greatest benefits of a diet rich in isoflavones may be their effects on improving the quality of blood vessels, rather than effects on blood cholesterol levels per se. Vascular constriction is associated with increased risk of arteriosclerosis and hypertension. The few animal and clinical studies conducted to date suggest that isoflavones can improve vascular compliance (Table 14.3). Isoflavones increase blood vessel dilation and improve blood flow in rhesus monkeys (68). Monkeys fed a diet with isoflavone-containing soy protein isolate had a 6% dilation in lumen diameter, compared with a 6% constriction in monkeys fed soy protein isolate from which isoflavones had been removed. Soy isoflavones were shown to promote arterial dilatation and inhibit constriction in a group of female rhesus monkeys fed an atherogenic diet containing soy isoflavones (69). In vitro platelet aggregation to thrombin and serotonin was also less when compared with female monkeys consuming an atherogenic diet from which soy isoflavones had been removed. Most of these effects have also been confirmed in human

Dietary Isoflavones and Coronary Artery Disease

Table 14.2

Effects of Soy Isoflavones on Serum Lipids

Reference Ashton et al. (45) Blakesmith et al. (46) Cassidy et al. (6)

Crouse et al. (39) Gardner et al. (47)

Gardner-Thorpe et al. (48) Gooderham et al. (49)

Hale et al. (50)

Hodgson et al. (51)

Jayagopal et al. (52)

Jenkins et al. (53)

311

Design of the study

Totalc

HDLc

LDLc

TGA

Placebo-controlled crossover in healthy men (n ¼ 42). 120 mg/day for 1 month Parallel-group in premenopausal women (n ¼ 25). 0 or 86 mg/day for 12 weeks Intervention in normocholesterolemic premenopausal women (n ¼ 6). 45mg/day for 1 month Parallel-group in healthy subjects (n ¼ 156). 0, 3, 27, 37, or 62 mg/day for 9 weeks Parallel-group in postmenopausal women (n ¼ 31). 80 mg/day for 12 weeks Placebo-controlled crossover in healthy men (n ¼ 20). 120 mg/day for 6 weeks Placebo-controlled crossover in healthy men (n ¼ 20). 60 g/day soy protein for 4 weeks Parallel-group in postmenopausal women (n ¼ 29). 80 mg/day for 2 weeks Placebo-controlled crossover in healthy subjects (n ¼ 59, 46 men and 13 postmenopausal women). 55 mg/day for 8 weeks Placebo-controlled crossover in Postmenopausal dietcontrolled type 2 diabetes women (n ¼ 32). 132 mg/day for 12 weeks Placebo-controlled crossover in hyperlipidemic men and postmenopausal women (n ¼ 41). 10 and 73 mg/day for 4 weeks

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312

Table 14.2 Reference Merz-Demlow et al. (54)

Nestel et al. (42) Potter et al. (40)

Ridges et al. (55)

Samman et al. (56)

Sanders et al. (57)

Scheiber et al. (41) Simons et al. (58)

Teede et al. (59) Teixeira et al. (60)

Uesugi et al. (61)

Cassidy and De Pascual-Teresa

Continued Design of the study

Totalc

Placebo-controlled crossover in premenopausal women (n ¼ 13). 10, 65, and 129 mg/day for 12 weeks Placebo-controlled crossover in healthy women (n ¼ 21). 80 mg/day for 5 weeks Parallel-group in postmenopausal women (n ¼ 66). 56 or 90 mg/day for 6 months Hypercholesterolaemic postmenopausal women (n ¼ 18). 45 mg/day for 8 weeks Placebo-controlled crossover in premenopausal women (n ¼ 14). 86 mg/day for 8 weeks Placebo-controlled crossover in healthy subjects (n ¼ 22, 5 men). 2 (control) or 56 mg/day for 17 days Single group of postmenopausal women (n ¼ 42). 60 mg/day for 12 weeks Placebo-controlled crossover in postmenopausal women (n ¼ 20). 80 mg/day for 8 weeks Parallel-group in healthy subjects (n ¼ 179, 96 men). 118 mg/day for 12 weeks Parallel-group in men with moderate hypercholesterolemia (n ¼ 81). 38, 57, 76, and 95 mg/day for 6 weeks Placebo-controlled crossover in perimenopausal women (n ¼ 12). 61.8 mg/day for 4 weeks

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(continued )

Dietary Isoflavones and Coronary Artery Disease

Table 14.2 Reference Wangen et al. (62)

Washburn et al. (63)

313

Continued Design of the study

Totalc

HDLc

LDLc

TGA

Placebo-controlled crossover in postmenopausal women (n ¼ 18). 7.1 (control), 65, and 132 mg/day for 93 days Placebo-controlled crossover in perimenopausal women (n ¼ 51) 34 mg for 6 weeks

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studies. Isoflavones, 80 mg/day, containing 45 mg genistein, improved systemic arterial compliance by 26%, as compared to placebo, in a group of 21 peri- and postmenopausal women (43). A recent study of perimenopausal women fed 34 mg of isoflavones contained in 20 g of soy protein showed a reduction in diastolic blood pressure (63). The mechanism of attenuated contractility in arteries by a low dose of Genistein (2.5 mg/kg b.w.) in rats may be due to the tyrosine kinase inhibitory property of genistein, because at this low dose there was no evidence of estrogenic effects (70). In a study carried out in 41 hyperlipidemic men and women (53), significant differences were observed for systolic blood pressure, which was significantly lower in men following the soy diets than after the control diet. In other human studies, a decrease in both systolic and diastolic blood pressure has been observed in both men and women, following a long period of consumption of soya milk, and with a significant correlation between blood pressure reduction and urinary excretion of genistein (71). However, since the existing data from clinical studies is inconsistent on the effects of isoflavones on blood pressure further studies are required to define the optimal dose and duration for blood pressure lowering effects. INFLAMMATION AND CELL ADHESION The potential anti-inflammatory and cell adhesion properties of isoflavones have been tested in several in vitro model systems. However, to date most of the studies conducted have used concentrations of isoflavones that are unlikely to be achievable at physiologically relevant concentrations. Activation of the endothelium results in the release of vascular cytokines such as interleukin-1 (IL-1b) and tumor necrosis factor alpha (TNF-a). These cytokines in turn induce the cell surface expression of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1), which are centrally involved in the endothelial recruitment of leucocytes (75). Focal expression of ICAM-1 and VCAM-1 has been reported in arterial endothelium overlying early foam cell lesions in both

314

Table 14.3

Cassidy and De Pascual-Teresa Effect of Soy Isoflavones on Blood Pressure

Reference Bloedon et al. (72)

Chiechi et al. (73)

Hale et al. (50)

Hodgson et al. (51)

Jayagopal et al. (52)

Jenkins et al. (53)

Nestel et al. (41)

Rivas et al. (71)

Simons et al. (58)

Teede et al. (59)

Vigna et al. (74)

Washburn et al. (63)

Design of the study

Change of SBP/DBP (mmHg)

Single dose in postmenopausal women (n ¼ 24). 2, 4, 8, or 16 mg/day for 24 h Parallel-group in postmenopausal women (n ¼ 187). 0, 40, or 60 mg/day for 24 weeks

216/213

Parallel-group in postmenopausal women (n ¼ 29). 80 mg/day for 2 weeks Placebo-controlled crossover in healthy subjects (n ¼ 59, 46 men and 13 postmenopausal women). 55 mg/day for 8 weeks Placebo-controlled crossover in postmenopausal diet-controlled type 2 diabetes women (n ¼ 32). 132 mg/day for 12 weeks Placebo-controlled crossover in hyperlipidemic men and postmenopausal women (n ¼ 41). 10 and 73 mg/day for 4 weeks Placebo-controlled crossover in healthy women (n ¼ 21). 80 mg/day for 5 weeks Parallel-group in subjects with hypertension (n ¼ 40). 143 mg/day for 12 weeks Placebo-controlled crossover in postmenopausal women (n ¼ 20). 80 mg/day for 8 weeks Parallel-group in healthy subjects (n ¼ 179, 96 men). 118 mg/day for 12 weeks Parallel-group in postmenopausal women in postmenopausal women (n ¼ 77). 76 mg/day for 12 weeks Placebo-controlled crossover in peri-menopausal women (n ¼ 51). 34 mg for 6 weeks

nc

# ¼ decrease, " ¼ increase, nc ¼ not changed.

23/0

nc

22/21

nc

nc

218/216

nc

23.9/22.4

23/0

0/25

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dietary and genetic models of arteriosclerosis in rabbits (76). This expression, together with the activation of monocyte chemoattractant protein-1 (MCP-1), leads to infiltration of mononuclear cells into the artery wall (77). The uptake of oxidized LDL by these cells leads to the formation of lipid-laden foam cells, and the development or progression of atherosclerotic plaques (78). Transcription of ICAM-1, VCAM-1, and MCP-1 is dependent, at least in part, on the activation of NF-kB, a classical member of the Rel family of transcription factors, and several observations suggest a key role for NF-kB in atherogenesis. In unstimulated cells, NF-kB is inactivated by sequestration to the I-kB family of inhibitor proteins. Agents that activate NF-kB induce degradation of I-kB. Free NF-kB then enters the nucleus and binds to the regulatory regions of its target genes (79) as shown in Fig. 14.4. Its activity is controlled by the redox status of the cell, and generation of reactive oxygen species may be a common step in all of the signaling pathways that lead to I-kB degradation. NF-kB activation is inhibited by several chemically distinct anti-oxidants, including N-acetylcysteine, dithiocarbamates, vitamin E derivatives, GPx activators, and various metal chelators (80) and flavonoids (81). There are now several pieces of evidence which suggest a key role for NF-kB in atherogenesis; activated NF-kB has been identified in situ in human atherosclerotic plaques (82) as well as in an arterial injury model (83), but not in cells of normal vessels devoid of arteriosclerosis. Furthermore, NF-kB is activated by an atherogenic diet and by oxidized LDL (84) and advanced glycosated end products (85). In a human monocyte in vitro model system, lipopolysaccharide (LPS) increased both NF-kB binding to DNA consensus sites and TNF-a release

E2 Diethylstilbestrol 17a-Estradiol 4-OH-Tamoxifen Genistein Coumestrol b-Zearalanol Bisphenol A Methoxychlor

Figure 14.4 isoflavones.

ERa 100 468 58 178 5 94 16 0.05 0.01

ERb 100 295 11 339 36 185 14 0.33 0.13

Brain ERb Vascular ERb Adrenal ERa Kidney ERa Prostate ERb Testes ERa

Breast ER a/b Uterus ER a/b Ovary ER a/b Bladder ERb Bone ERb

The transcription factor NF-kB as a potential molecular target of

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(86). At pharmacological concentrations, genistein (40 mM) attenuated both NF-kB DNA binding and TNF-a release. Genistein is known to inhibit protein tyrosine kinase (87), so these data suggest that LPS-induced NF-kB activation and TNF-a release in human monocytes is protein tyrosine kinase-dependent (86). At these high concentrations, genistein (30 mM) also inhibited NF-kB DNA-protein binding in LPS-stimulated monocytes (RAW 264.7) by 50% (88). Genistein (50 mM), but not daidzein, inhibited TNF-a induced NF-kB activation in cultured human lymphocytes. Furthermore, the consumption of a soy-based dietary supplement containing 50 mg of a isoflavone mixture (genistein:daidzein:glycitin ratio ¼ 1.3:1:0.3) twice daily for 3 weeks was shown to reduce ex vivo NF-kB activation induced by TNF-a in peripheral lymphocytes in healthy male volunteers (89). Following stimulation with the pro-inflammatory cytokines, IL-1 and TNF-a, endothelial cells express the leukocyte adhesion molecules E-selectin, VCAM-1, and ICAM-1. To date several studies have investigated the potential intracellular signaling mechanisms of genistein that would result from expression of these cytokines. Genistein dose-dependently inhibited the maximal E-selectin expression induced by incubation of HUVEC for 4 h with TNF-a (100 U/mL) and IL-1 (100 U/mL). Furthermore, VCAM-1 secretion was inhibited by genistein after stimulation of HUVEC for 24 h with TNF-a or IL-1. In contrast, genistein did not alter ICAM-1 secretion after a 24 h incubation of HUVEC with either of the two cytokines (90). Weber et al. (91) investigated the role of tyrosine phosphorylation in the induction of endothelial leukocyte adhesion molecule 1 (ELAM-1), VCAM-1, and ICAM-1 in HUVECs. Pretreatment of the cells with genistein resulted in a dose-dependent inhibition of TNF-a-induced ELAM-1, VCAM-1, and ICAM-1 surface expression (IC50 ¼ 30 mM). In a further study examining the effects of genistein as a PTK inhibitor of leukocyte (neutrophils, lymphocytes, and monocytes) adhesion to endothelial cells, IL-1 and TNF-astimulated neutrophil and monocyte adhesion to HUVEC was significantly inhibited by genistein compared with stimulated control cells (92). Monocyte-derived macrophages are the principal inflammatory cell in the atheromata. In early stages of artherosclerotic lesion formation, macrophages and endothelial cells interact to trigger a cycle of events that exacerbates endothelial dysfunction, resulting in a loss of homeostatic control (93). Activated macrophages generate large amounts of NO from L -arginine by the action of inducible NO synthase (iNOS) and its overproduction has been associated with oxidative stress and chronic inflammation (94). NO is an important intracellular and intercellular regulator of many biological functions, including macrophage-mediated cytotoxicity (95,96). Cytokines such as IFN-g and other inflammatory stimuli such as bacterial lipopolysaccharide (LPS) regulate the activity of iNOS in macrophages (97,98). Genistein inhibited nitrite production in a dose-dependent manner when rat mesangial cells were activated with IL-1b. This finding suggests a central role for PTK in the signaling pathway of IL-1b, resulting in the activation of iNOS in rat

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mesangial cells (99). Li et al. (100) showed that genistein had an inhibitory effect on NO production (IC50 ¼ 72 mM) in an immortalized astrocyte cell line (DITNC), which were activated using a three-cytokine mixture (TNF-a, IL-1, and IFN-g) designed to maximally induce iNOS. In a further study, Gottstein et al. (101) found that genistein (IC50 ¼ 58 mM) and daidzein (IC50 ¼ 107 mM) significantly inhibited IFN-g plus LPS induced NO production in RAW 264.7 macrophages. iNOS mRNA levels remained unchanged by the isoflavone treatment, which suggests that the inhibitory effect is posttranscriptional and this suppression of LPS activity has also been shown for other plant phenolics (102). Sheu et al. (103) reported an inhibitory effect of genistein and daidzein on LPS-induced expression of the iNOS gene in macrophages, but their co-incubation with LPS and isoflavones may have given rise to this effect. Thus inhibition of iNOS expression may have been caused by a direct interaction of these compounds with the LPS molecule, rather than a direct effect on the cell. In the Gottstein study (101), macrophages were preincubated with genistein and daidzein for 24 h and were then washed twice with PBS before the addition of IFN-g and LPS in order to avoid any direct chemical interaction. Thus, the inhibition of NO production observed in this study may reflect inhibition of TNF-a secretion by genistein and daidzein as it has previously been demonstrated that TNF-a is crucial for the induction of NO synthesis in IFN-g and/or LPS-stimulated macrophages (104,105). MCP-1 is a CC-chemokine consisting of 76 amino acids. This molecule may play a key role in atherogenesis, because it is involved in the recruitment of monocytes and T-cells into the arterial wall. MCP-1 mRNA has been detected in atherosclerotic lesions by in situ hybridization (106,107). Furthermore, a decrease in atherosclerotic lesion size is seen in mice deficient of the MCP-1 receptor CCR-2, and fewer macrophages and monocytes are present in their aortas (108). Therapeutic drugs and dietary factors targeting MCP-1 and/or its receptor may prove useful in the prevention of atherosclerotic lesion development. Recently, it has been shown that genistein (IC50 ¼ 29 mM) and daidzein (IC50 ¼ 37 mM) dose-dependently down-regulated MCP-1 secretion (101), indicating that both of these isoflavones may have the potential to inhibit monocyte infiltration into the arterial wall. It is known that the expression of MCP-1 is regulated at the transcriptional level (77). Therefore, it is hypothesized that genistein and daidzein may regulate TNF-a-induced MCP-1 expression through transcription factors such as nuclear factor kappa B and activator protein-1, present in the promoter region of the MCP-1 gene. PLATELET AGGREGATION AND ENDOTHELIUM REACTIVITY Both genistein and daidzein exert anti-aggregatory activity in human platelets in vitro (101). This finding is consistent with earlier reports that the consumption of soy protein and its isoflavone-enriched fraction lowers platelet aggregation in rats (109). The exact molecular mechanisms by which isoflavones affect platelet

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aggregation are unclear and currently under investigation. Apart from protein tyrosine kinase inhibition (110) within the cyclooxygenase pathway, several other reported molecular effects of flavonoids could have influenced platelet function in the present study. The modification of platelet cyclic-30 ,50 -adenosine monophosphate (cAMP) via the inhibition of phosphodiesterase activity is the most supported pathway for anti-aggregatory effects of flavonoids (111). Inhibition of lipoxygenase activity, as demonstrated principally for myricetin and quercetin (112), is another possible mechanism. Stimulation of adenylate cyclase, leading to increased cAMP levels, has been proposed as a further antiaggregatory signal transduction pathway (113). In addition, the anti-oxidant character of isoflavones may play a role in inhibiting platelet aggregation. Pignatelli et al. (114) showed that collageninduced platelet aggregation was associated with the production of H2O2 , which acts as an important second messenger in platelets, stimulating both the phospholipase C pathway and the arachidonic acid metabolism. Consistent with this finding, platelets primed with nonactivating concentrations of arachidonic acid or collagen were activated by nanomolar concentrations of H2O2 (115). Since isoflavones possess anti-oxidant properties (23,116) and can scavenge radicals, this evidence that reactive oxygen species are involved in platelet stimulation suggests another anti-aggregatory mechanism. In comparison with daidzein, the genistein molecule contains an additional hydroxyl group in the C-5 position, possibly resulting in a higher anti-oxidant activity (25). This might explain why in the present study genistein has been demonstrated to be a more potent inhibitor of platelet aggregation than daidzein. CONCLUSION In the overall scheme of cardiovascular protection, isoflavones appear to potentially have a more important role in conditioning the vascular tree than on influencing cholesterol levels. The preferential binding of isoflavones to the ERb and the increasing recognition of the role of this new receptor in the endothelial wall provides justification for increasing the awareness of the heart-health effects of diets rich in these phytoestrogens. Furthermore, such effects are not restricted to soy isoflavones but also apply to lignans, which are abundant in fruits and vegetables. Potential anti-atherogenic effects of isoflavones include a reduction in LDL cholesterol, modulation of pro-inflammatory cytokines, cell adhesion proteins and nitric oxide formation, protection of LDL against oxidation, inhibition of platelet aggregation, and an improvement in vascular reactivity. To date, most of the published in vitro data have reported beneficial effects only following exposure to pharmacological doses of isoflavones; thus, further research is required to understand the importance of this mechanism following exposure to physiologically relevant levels of isoflavones; and, their corresponding liver and gut metabolites. Although epidemiological data and laboratory studies allude to the possible protective effects of soy isoflavones at specific target

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tissues, randomized placebo controlled clinical trials are necessary to further address the relative importance of these compounds for cardiovascular health.

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93. Tedesco F, Fischetti F, Pausa M, Dobrina A, Sim RB, Daha MR. Complementendothelial cell interactions: pathophysiological implications. Mol Immunol 1999; 36:261– 268. 94. Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 1991; 43:109 – 142. 95. Ignarro LJ. Regulation of cytosolic guanylyl cyclase by porphyrins and metalloporphyrins. Adv Pharmacol 1994; 26:35– 65. 96. Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and the ugly. Am J Physiol 1996; 271:C1424 –C1437. 97. Stuehr DJ, Marletta MA. Induction of nitrite/nitrate synthesis in murine macrophages by BCG infection, lymphokines, or interferon-gamma. J Immunol 1987; 139:518– 525. 98. Narumi S, Finke JH, Hamilton TA. Interferon gamma and interleukin 2 synergize to induce selective monokine expression in murine peritoneal macrophages. J Biol Chem 1990; 265:7036 – 7041. 99. Tetsuka T, Morrison AR. Tyrosine kinase activation is necessary for inducible nitric oxide synthase expression by interleukin-1 beta. Am J Physiol 1995; 269:C55– C59. 100. Li W, Xia J, Sun GY. Cytokine induction of iNOS and sPLA2 in immortalized astrocytes (DITNC): response to genistein and pyrrolidine dithiocarbamate. J Interferon Cytokine Res 1999; 19:121 – 127. 101. Gottstein N, Ewins BA, Eccleston C, Hubbard GP, Kavanagh IC, Minihane AM, Weinberg PD, Rimbach G. Effect of genistein and daidzein on platelet aggregation and monocyte and endothelial function. Br J Nutr 2003; 89:607 – 616. 102. Azumi S, Tanimura A, Tanamoto K. A novel inhibitor of bacterial endotoxin derived from cinnamon bark. Biochem Biophys Res Commun 1997; 234:506 – 510. 103. Sheu F, Lai HH, Yen GC. Suppression effect of soy isoflavones on nitric oxide production in RAW 264.7 macrophages. J Agric Food Chem 2001; 49:1767 – 1772. 104. Green SJ, Crawford RM, Hockmeyer JT, Meltzer MS, Nacy CA. Leishmania major amastigotes initiate the L-arginine-dependent killing mechanism in IFN-gammastimulated macrophages by induction of tumor necrosis factor-alpha. J Immunol 1990; 145:4290 – 4297. 105. Jun CD, Choi BM, Kim HM, Chung HT. Involvement of protein kinase C during taxol-induced activation of murine peritoneal macrophages. J Immunol 1995; 154:6541– 6547. 106. Nelken NA, Coughlin SR, Gordon D, Wilcox JN. Monocyte chemoattractant protein-1 in human atheromatous plaques. J Clin Invest 1991; 88:1121 – 1127. 107. Yla¨-Herttuala S, Lipton BA, Rosenfeld ME, Sarkioja T, Yoshimura T, Leonard EJ, Witztum JL, Steinberg D. Expression of monocyte chemoattractant protein-1 in macrophage-rich areas of human and rabbit atherosclerotic lesions. Proc Natl Acad Sci USA 1991; 88:10143 –10147. 108. Boring L, Gosling J, Cleary M, Charo IF. Decreased lesion formation in CCR22/2 mice reveals a role for chemokines in the initiation of atherosclerosis. Nature 1998; 394:894– 897. 109. Peluso MR, Winters TA, Shanahan MF, Banz WJ. A cooperative interaction between soy protein and its isoflavone-enriched fraction lowers hepatic lipids in male obese Zucker rats and reduces blood platelet sensitivity in male Sprague-Dawley rats. J Nutr 2000; 130:2333– 2342.

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15 Anti-Carcinogenic Properties of Soy Isoflavones Max O. Bingham and Glenn R. Gibson University of Reading, Reading, UK

Introduction Isoflavones Isoflavones and Breast Cancer Isoflavones and Prostate Cancer Isoflavones and Colon Cancer Isoflavones and Cancer—An Overview and the Future References

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INTRODUCTION The incidence and mortality rates of several hormonally related tumors, including breast, prostate, and colon cancer, have until recently been considered low in Asian countries like China, Japan, and Indonesia when compared with western countries (1). Additionally, cancer rates appear to differ between westernized countries. For example, Italy and Finland have substantially lower rates than other western countries. A number of studies have concluded that lifestyle and dietary factors may be important in explaining these differences in incidence rate. Asian diets, which are mainly vegetarian or semivegetarian, differ markedly 327

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from western diets, which are rich in animal proteins and fats. This may affect cancer incidence in a number of ways including alteration of the metabolism and actions of a number of phytochemicals, some of which may have steroidal effects. Of crucial importance is the role of the gut and more specifically the microflora in the gut and its impact in mediating certain effects of diet on disease patterns in western countries (2 –4). The isoflavones, daidzein and genistein, which are a type of phytoestrogen can be included in the group of phytochemicals. These compounds are found in abundance in the plasma and urine of people living in areas of low cancer incidence (5,6). They are either hormone like with inherent estrogenic activities, or converted by the human gut microflora to estrogenic compounds. These compounds have been shown to influence several biological phenomenon, including production, metabolism, and biological activities of sex hormones and also many intra-cellular steroid metabolizing enzymes (3,7,8). Evidence that soy products help to prevent cancer existed many years before it was suggested that isoflavones may be important. Originally, it was thought that components such as protease inhibitors, phytic acid, or b-sitosterol were the active components (9). The subsequent suggestion that isoflavones were more important has led to numerous publications linking isoflavone consumption and cancer incidence. However, the research has not always been positive and recent reports suggest, in animal experiments at least, that isoflavones may have adverse effects with respect to carcinogenesis and other biological functions. Given this conflicting evidence, we review the benefits and risks associated with soy consumption and examine the possible role soy may exert in the mechanisms of carcinogenesis. The main phytoestrogens derived from the diet are genistein, daidzein, and glycitin, which are isoflavones almost exclusively found in soy. They occur as glycosidic conjugates or in unconjugated or conjugated forms in most soy protein products, usually in very high concentrations. The two most estrogenic isoflavones are genistein and equol (10), the latter being a product of daidzein metabolism in the gut by the activities of the human gut microflora. Recent research in estrogen receptors (ERs) has clarified some of the conflicting findings regarding phytoestrogen potency. The application of a wide variety of techniques has tended to generate data indicating different relative potency values. Additionally, since the identification and cloning of a second ER, ERb (11), it has become apparent that isoflavones have different affinities for both ERa and ERb and are found at different concentrations in organ systems within the body. Originally, it was thought that the protective actions of isoflavones were due to anti-estrogenic activities, whereby they blocked ERs to more potent mammalian estrogens such as 17b-estradiol. However, this appears to be an oversimplification, and actions are likely to occur on many different levels. The relationship among health, protection against disease, and isoflavones may in itself be an oversimplification. Anti-carcinogenic properties of soy are more likely to be due to a concerted action of many types of phenolic and other biologically active compounds, including those of isoflavones, lignans,

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and flavonoids. Given the myriad of research that has been completed over recent years in the anti-carcinogenic properties of phenolic compounds in general, it appears likely that the gross impact of such dietary components is more important than the actions of singular compounds. However, to understand the actions, and thus the potential of dietary components to protect against chronic disease development, it is still worthwhile taking the reductionist approach. ISOFLAVONES Isoflavones are non-nutrient plant compounds which belong to the phytoestrogen class, having a similar structure to mammalian estrogens (12). Genistein, one of the predominant soy isoflavones, has been shown to inhibit the growth of cancer cells through modulation of genes that are related to the homeostatic control of cell cycle and apoptosis. It has also been found that genistein inhibits the activation of the nuclear transcription factor, NF-kB, and Akt-signaling pathway, both of which are known to maintain a balance between cell survival and programed cell death (apoptosis). It is known to have antioxidant and estrogenic properties, which target estrogen- and androgen-mediated signaling pathways in the processes of carcinogenesis. Genistein is also found to be a potent inhibitor of angiogenesis and metastasis (13). One of the most important considerations in its ADME sequence is its release from its precursor, genistin. This involves a number of steps, and for a long time it was thought that the human gut microflora was solely responsible for its release and subsequent metabolism. However, recent reports demonstrate that the conversion of genistin to genistein begins in the mouth and then continues in the small intestine suggesting that genistein may be released along the entire length of the alimentary tract (14). However, the mechanisms of genistein in cancer development are not all positive and much disagreement exists within the research environment as to its specific role in cancer development. Many examples have appeared indicating that genistein may help to promote carcinogenesis. Indeed, recent reports have indicated that genistein, daidzein, and biochanin A are capable of achieving chromosome aberrations and aneuploidy, suggesting a possible involvement in the initiation of carcinogenesis (15). Isoflavones and Breast Cancer Soya bean products are regularly consumed in Asian countries such as China, Japan, Korea, and Indonesia. These countries have traditionally had low incidences of breast, prostate, and colon cancer. However, rates are steadily increasing because of changes in dietary habits and lifestyle (5). Traditional diets have been low in fats and red meat, and often rich in fish—all elements that have been independently linked to a decreased cancer risk. However, studies have also indicated that soy bean products may be protective and anti-carcinogenic and this was long before the beneficial effects of isoflavones were recognized (9,16). Thus, it

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is likely that some, if not all, of these purported bioactive compounds from soy, and indeed components from the rest of the diet, may contribute towards this overall epidemiological observation. Specific epidemiological studies of Japanese and White populations who emigrated to Los Angeles, the United States, indicated that the later in life emigration was completed, the lower the risk of breast cancer when compared with those who emigrated earlier in life (17). Additionally, it has been observed that the risk of breast cancer in Asian-American populations was inversely proportional to intake of tofu (a soy protein food that contains high levels of isoflavones) (18). These findings support earlier observations that there is an increased disease risk in populations following emigration from Asia to the United States. Within 6 months of immigration, isoflavones excreted in urine by oriental immigrants were shown to be 10% the amount excreted in the urine of Japanese women—indicating that the Japanese population may have a lower risk of developing diseases such as breast cancer (2). Further evidence for this protective characteristic of isoflavones in breast cancer includes that of some chimpanzees in captivity given experimental breast cancer. These proved very resistant to developing the cancer and excreted high levels of isoflavones, including equol, in urine (19). Moreover, women with breast cancer tend to excrete small amounts of isoflavones, whereas those living in areas of low incidence tend to excrete high levels of isoflavones. Additionally, vegetarians exhibit much higher levels of isoflavones in plasma and urine than omnivores (2,6,20). In summary, epidemiological evidence appears to be supportive for a protective role in isoflavones in breast cancer and indicates that diets high in soy may provide some protection against the development of breast cancer. However, this type of evidence provides no insight into possible mechanisms of action, nor whether isoflavones can conclusively afford protection against cancer. Studies with breast cancer cell lines in culture have shown that phytoestrogens, including daidzein and genistein, stimulate tumor growth at low concentrations but inhibit growth at high concentrations (21). However, the highest concentrations of some of these studies were higher than physiologically relevant levels observed in Japanese women (22 – 24). Recent reports have indicated that genistein alone could inhibit mouse mammary adenocarcinoma, but when this genistein was fed as soy extract, the magnitude of inhibition was much greater, indicating that genistein along with other constituents was important in the suppression of tumor growth in this study (25). In terms of genotoxicity, recent reports suggest that genistein, but not daidzein, could protect against chemically induced breast cancer by inhibiting certain phase-1 cytochrome P450 enzymes involved in initiation (26). The effects of isoflavones on the human breast cancer cell line MCF-7 have been studied and results indicated that they were also capable of significantly inhibiting MCF-7 growth and additionally induced cell apoptosis by regulating iNOS gene expression (27). Subsequent studies have revealed further genes regulated by genistein and involved with breast

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cancer cell proliferation, suggesting that the inhibitory action of genistein appears to be complex and only partially mediated by the alteration of ER-dependent pathways (28,29). The possible association between phytoestrogens or phytoestrogen-rich diets and breast cancer risk has been reviewed (30). Limited evidence suggests that a diet containing soy bean products is chemoprotective. Indeed, a large prospective study in Japan did not show any favorable effect of soy consumption on breast cancer risk (31). The most promising data have originated from studies in rodents on the impact of an isoflavone-rich diet during puberty and adolescence. These demonstrated that an isoflavone-rich diet may only be significant at reducing risks of breast cancer if consumption occurred during this early period of life (32). Later studies (in Japanese women and in rodents) have supported this conclusion (33). Most of the experimental research suggests that prepubertal genistein administration prevented the development of tumors by acting directly upon the mammary gland to enhance maturation and by reducing cell proliferation (34). Early breast-feeding has previously been shown to stimulate breast lobular maturation and is also associated with a reduced risk of premenopausal breast cancer in women (35). Isoflavone-induced breast differentiation could mimic this effect. The production of equol, a secondary metabolite of daidzein, in the gut has been associated with a lower risk of breast cancer (36). It has been shown that equol production tends to be high in people who consume high levels of plant-based carbohydrates, fiber, and protein, but low in those that have a high-fat diet (37,38). People who excrete high levels of equol have an increased ratio of 2-hydroyestrone to 16a-hydroxyestrone in their urine (39), which is thought to lower the risk of breast cancer (40). However, high-equol producers have a slightly higher concentration of sex-hormone binding globulin (SHBG). This increase diminishes the availability of estradiol for 16a-hydroxylation in the liver. Also, an increased load of isoflavones also increases estrogen 2-hydroxylation (41). Thus, this increase in the ratio of 2-hydroyestrone to 16a-hydroyestrone, mediated by levels of SHBG and isoflavone load, appears to be an important issue in explaining a possible protective mechanism of isoflavones in breast cancer. Recent studies support this finding with observations that equol excretion was positively correlated with the ratio of 2-hydroyestrone to 16a-hydroyestrone (42). These observations suggest that the human gut microflora profile associated with equol production may be involved in estrogen metabolism and may therefore also influence breast cancer risk. Interestingly, treatment with the anti-estrogen tamoxifen decreased the ratio of 2-hydroyestrone to 16a-hydroyestrone (43), which according to the theory increases the risk of breast cancer. Other research has suggested negative effects of isoflavones in breast cancer. High prenatal endogenous estrogen concentrations may increase risk in women (34,44). Additionally, experimental evidence from rats supports the view that isoflavones may negatively affect breast cells during pregnancy (34) and increase the number of tumors developing later in life (45). It appears that

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genistein acts as an estrogen agonist and stimulates cell division and proliferation of existing breast cancer cells (46). This has additionally been shown in vivo in a dose-dependent manner and the removal of genistin and genistein from the diet caused tumors to regress (14). Limited evidence also indicates that daidzein may have similar actions to genistein (47). However, given the fact that both Japanese newborns and their mothers have high levels of isoflavones (48), this would indicate that the incidence of breast cancer in this population would be much higher—the opposite is true. Overall, there is little convincing evidence to suggest that isoflavones from soy consumed in adult life are protective against breast cancer for women living in western countries. However, sustained intake throughout life may afford some protection. Moderate intakes of soy products or supplements with isoflavones may be beneficial in terms of cardiovascular health, osteoporosis, and menopause, but negative effects on the breast cannot be ruled out. Isoflavones and Prostate Cancer Incidents of mortality from prostate cancer in western countries is much higher than those in Asia, despite similar incidences of latent, small, or non-infiltrative prostate carcinomas. Prostate cancer is hormone dependent and based upon early studies in Japan, a hypothesis was formed suggesting that diets in countries with low prostate cancer risk may contain high amounts of hormonally active and cancer protective compounds such as isoflavones (2). Plasma concentrations of isoflavones in Japanese men are high (23). However, levels are higher still in prostatic fluid and possibly related to prostate cancer incidence (24). Genistein and its precursor, and biochanin A inhibit cell growth in prostate cancer cells at high concentrations (49). However, the most effective isoflavone is 40 -methylequol and this has additionally been shown to be effective at reducing prostate specific antigen (PSA) (50). This effect is thought to be important because PSA is associated in many cases with a decrease in cell proliferation. A recent case-controlled clinical study (51) was carried out to compare circulating levels of isoflavones in prostate cancer patients and control subjects. Serum levels of genistein, daidzein, and equol were compared and it was found that the group with prostate cancer contained significantly less patients capable of converting daidzein to equol. This may be a significant risk indicator given that it is generally thought that only 30% of western populations have the human gut microflora profile capable of producing equol. Further recent studies have added strength to this argument, because it has been shown that equol was biologically active with potent anti-proliferative effects on benign and malignant prostatic cells at concentrations that can be obtained naturally through dietary soy consumption (52). Animal studies investigating the effects of feeding soy and pure isoflavones have indicated a generally positive response whereby prostate cancer growth was

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inhibited (30). In two different rat models of prostate cancer, soybean isoflavones inhibited the onset of cancer and delayed growth. However, increasing dietary fat intake abolished these effects. In studies of nude mice with transplanted human LNCaP prostate cancer cells, soy products or isoflavone concentrate inhibited cancer growth. Studies of tumors revealed that apoptosis was increased and angiogenesis was inhibited. Genistein is an inhibitor of angiogenesis—a process that is necessary for extended growth and metastatic spread of tumors (7). Studies in humans have revealed a similar relationship. The most recent studies have all indicated that soy intake prevents prostate cancer. For example, a prospective study revealed that consuming soymilk more than once a day was protective against prostate cancer (53). Additionally, a very recent case – control study evaluating the effect of soy food consumption, isoflavone intake, and prostate cancer risk indicated a reduced risk associated with the consumption of soy foods and isoflavones. The promise of subsequent longitudinal follow-up studies will confirm these findings (54). To address issues surrounding potential genistein genotoxicity, Miltyk et al. (55) investigated the potential of this characteristic of genistein to cause genetic damage in men with prostate cancer. They found that while genistein was capable of inducing genetic damage in vitro, a similar effect was not observed in the subjects treated. ERb receptors are present in the prostate and this has lead to increasing interest in how isoflavones may be important in protection from prostate cancer, because many isoflavones bind well to this receptor. The administration of genistein to rats over their lifetime led to a downregulation of both ERs (ERa and ERb) in the prostate (56). This observation indicates that genistein can regulate steroid receptor pathways and may help to prevent prostate cancer via this mechanism. The importance of dietary factors in prostate carcinogenesis has been proven in many epidemiological studies of immigrants from Asia to the United States. However, it appears as though the totality of diet is an important consideration (57,58). A combination of higher meat intakes and the possibility of greater levels of carcinogenic compounds (such as N-nitroso compounds) originating from charcoal cooked red meat and fish increases the potential risk of developing a wide range of cancers including prostate cancer. If this is combined with lower intake of a wide range of protective dietary factors, risks must increase. However, given the extent of factors that have been listed as protective (selenium, zinc, carotenoids, lycopenes, vitamins E and D, and isoflavones to name but a few), it appears as though concentrating upon single dietary factors as protective in prostate cancer would lead to an underestimation of the contribution of the diet as a whole. Indeed, a recent study has indicated that the polyphenols, quercitin and kaempfaerol, and the isoflavones, genistein and biochanin A, originating from both soy and tomato products could inhibit multiple intra-cellular pathways involved in prostate cancer development (59). Furthermore, more holistic research is likely to be needed to understand the complexity of diet and its contribution in the condition.

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Isoflavones and Colon Cancer A previous review about soy foods, isoflavones, and colon cancer concluded that the available data were conflicting and that evidence for the protective effects of soy, in this instance, was limited (60). Since that time, further studies have indicated that soy and isoflavones have either no effect on colon cancer or are mildly procarcinogenic. ERs have been found in normal mucosal and cancer cells in the colon (61), and while low, correspond to that in normal breast tissue (62). Estrogen replacement therapy in postmenopausal women reduces the mortality from colon cancer (63), whereas anti-estrogenic treatments with tamoxifen may enhance risk (64). Unconjugated isoflavone concentrations in gut contents are high and it is thought that they are more than likely to have biological effects on the mucosal cells (7). Animal studies fairly consistently show that soy foods or isoflavones inhibit the formation of aberrant crypt foci, but these did not clearly demonstrate an inhibitory effect on chemically induced colon cancer. Additionally, case –control studies have suggested that soy food consumption may confer a reduced risk of colon cancer, yet the findings are inconsistent (65). Recent reports have indicated a possible role for the Bowman-Birk Inhibitor (BBI) suggesting that it may be important in preventing colon cancer. This is one of the constituents of soy originally thought to be important in observations that soy was protective against cancer, before research interest focused upon isoflavones. Purified BBI and an extract of soy enriched with BBI reduced the incidence and frequency of tumors in rats with chemically induced tumors. However, soy molasses which contained soy isoflavones did not have any effect on the colon carcinogenesis in rats. Additionally, no adverse effects where observed in the rats treated with the purified BBI and the extract enriched with BBI (66). Bacterial fermentation is of considerable importance in the etiology of colon cancer—germ-free animals have much lower rates of incidence of colon cancer (67). However, research to date, although comprehensive, is highly contradictory. Major products of bacterial fermentation include the short chain fatty acids acetate, propionate, and butyrate. These decrease colonic pH and this has been associated with a decreased risk of cancer. There has been considerable interest in the role of butyrate in colon cancer. This is an important source of energy for colonocytes and is thought to have a number of anti-carcinogenic properties, including the ability to inhibit DNA synthesis and reduce cell proliferation (68 –70) fermentation rates in the colon. This may result in decreased ammonia in feces and increases in breakdown and release of lignans (phytoestrogens of similar structure to isoflavones) (71). However, bacterial fermentation has also been shown to produce a wide range of potential carcinogens in the gut. The end products of certain colonic bacteria have been described as carcinogenic or genotoxic: these include nitrosamines, fecapentaenes, secondary bile acids, heterocyclic amines, various

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aglycones of plant origin, phenolic and indolic compounds, nitrated polycyclic aromatic hydrocarbons, diacylglycerol, some azo-based products, and ammonia. Often, these sorts of compounds originate from fermentation of amino acids and proteins or by the activities of microbial enzymes on dietary compounds and xenobiotic compounds from the diet (72). Of course, similar pathways can also release compounds that may be anticarcinogenic. Examples include flavonoids and phytoestrogens such as isoflavones. In general terms, it appears that dietary composition is important in the overall activities and composition of the human gut microflora and this may be important in the development and etiology of colonic cancer. Undoubtedly, the human gut microflora is important in the bioavailability of isoflavones (particularly the secondary metabolite, equol). However, what is far from clear is the role that isoflavones might have in colon cancer. Given the multitude of activities of the human gut microflora and the wide range of products generated that may be important in carcinogenesis, it is vital that the specific gut microflora components involved in colon cancer are identified.

ISOFLAVONES AND CANCER—AN OVERVIEW AND THE FUTURE Many different diets appear to afford protection against cancer and the mechanisms are probably variable. The importance of the role of the human gut microflora in mediation of the effects of plant phytochemicals in humans cannot be overemphasized. Diets rich in fiber and carbohydrate result in increased fermentation rate, which may subsequently impact upon the production of butyrate and secondary plant metabolites, such as equol. This appears to be a function of human gut microflora composition because in certain populations this ability is downregulated or even lacking. In turn, this may be important in modifying the risk of developing breast, prostate, and colon cancer among other chronic diseases. Whether soy isoflavones are important in protection against breast cancer is not currently known. Available evidence suggests that the soybean complex is protective but only in its entirety. Soy and isoflavones appear to be protective in prostate cancer although further work needs to be completed before definitive conclusions may be drawn. In colon cancer, the situation is very unclear, but currently evidence suggests that isoflavones and soy do not afford protection against colon cancer. Parallel processes such as increased butyrate production in the colon appear to be more important protective mechanisms. Given the complexity of the research base currently available and the contradictory evidence of whether isoflavones are anti-carcinogenic or carcinogenic, some difficult questions arise. A significant driving force behind the volume of research completed on these compounds is the potential for developing functional food products that target, for example, cancer. Nutrition research has traditionally focused on single issues (such as reducing the risk of developing particular diseases) in “at risk” individuals. This approach is entirely valid and has provided

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us with many valuable hypotheses. True “gold standard” approaches include characterization of the extent and rate of absorption, tissue dispersal, site-specific targeting of metabolically relevant compounds, and comprehensive studies of time and dose effects—studies which have only recently been completed for some compounds. For completeness, however, we need to address the question of evaluating all the possible effects of specific food components in a genetically heterogeneous population. This is especially important for determining unintended risk as well as intended benefit. Isoflavones clearly fit with this paradigm. Until the impact of these compounds on health and disease is fully established, products developed from these are unlikely to be popular with the consumer even if the smallest risk exists. Therefore, there is a need for research on isoflavones to move toward nutritional genomics, that is, the application of high-throughput genomics tools in nutrition research (73). Careful application of this suite of technologies in nutrition may help an increasing understanding of how nutrition influences metabolic pathways and homeostatic control; how regulation (genomics) is disturbed in early diet-related disease development; the extent to which individual sensitizing genotypes contribute toward such diseases (proteomics and transcriptomics) and the impact on overall metabolism and output in disease status and development (metabonomics). It is likely that the application of these technologies will significantly contribute toward the goal of developing effective dietary intervention strategies on the basis of isoflavones and other phytochemicals and the prevention of diet-related diseases such as hormonally related cancers.

REFERENCES 1. World Cancer Research Fund. American Institute for Cancer Research. Food, nutrition and the prevention of cancer: a global perspective. Washington: American Institute for Cancer Research, 1997. 2. Adlercreutz H. Western diet and western diseases: some hormonal and biochemical mechanisms and associations. Scan J Clin Lab Inv 1990; 50(suppl 201):3– 23. 3. Adlercreutz H. Human health and phytoestrogens. In: Korach KS, ed. Reproductive and Developmental Toxicology. New York: Marcel Dekker Inc., 1998:299 – 371. 4. Adlercreutz H. Evolution, nutrition, intestinal microflora, and prevention of cancer: a hypothesis. Proc Soc Exp Biol Med 1998; 217:241 – 246. 5. Adlercreutz H. Epidemiology of phytoestrogens. In: Adlercreutz H, ed. Phytoestrogens. London: Baillie`re Tindall, 1998:605– 624. 6. Adlercreutz H, Honjo H, Higashi A, Fotsis T, Hamalainen E, Hasegawa T, Okada H. Urinary excretion of lignans and isoflavonoid phytoestrogens in Japanese men and women consuming traditional Japanese diet. Am J Clin Nutr 1991; 54:1093 – 1100. 7. Adlercreutz H, Mazu W. Phytoestrogens and western diseases. Ann Med 1997; 29:95– 120. 8. Kurzer MS, Xu X. Dietary phytoestrogens. Ann Rev Nutr 1997; 17:353– 381. 9. Kennedy AR. The evidence for soybean products as cancer preventative agents. J Nutr 1995; 125(suppl 3):733s– 743s.

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28. Chen WF, Huang MH, Tzang CH, Yang M, Wong MS. Inhibitory actions of genistein in human breast cancer (MCF-7) cells. Biochim Biophys Acta 2003; 1638(2):187–196. 29. Vissac-Sabatier C, Bignon YJ, Bernard-Gallon DJ. Effects of the phytoestrogens genistein and daidzein on BRCA2 tumour suppressor gene expression in breast cell lines. Nutr Cancer 2003; 45(2):247– 255. 30. Adlercreutz H. Phytoestrogens and cancer. Lancet Oncol 2002; 3(6):364 – 373. 31. Key TJ, Sharp GB, Appleby PN, Beral V, Goodman MT, Soda M, Mabuchi K. Soya foods and breast cancer risk: a prospective study in Hiroshima and Nagasaki, Japan. Brit J Cancer 1999; 81:1248 – 1256. 32. Lamartiniere CA. Protection against breast cancer with genistein: a component of soy. Am J Clin Nutr 2000; 71:1705s – 1707s. 33. Shu XO, Jin F, Dai Q, Wen W, Potter JD, Kushi LH, Ruan Z, Gao YT, Zheng W. Soyfood intake during adolescence and subsequent risk of breast cancer among Chinese women. Cancer Epidemiol Biomarkers Prev 2001; 10:483 – 488. 34. Hilakivi-Clarke L, Cho E, Onojafe I, Raygada M, Clarke R. Maternal exposure to genistein during pregnancy increases carcinogen-induced mammary tumorigenesis in female rat offspring. Oncol Rep 1999; 6:1089 – 1095. 35. Newcomb PA, Storer BE, Longnecker MP, Mittendorf R, Greenberg ER, Clapp RW, Burke KP, Willett WC, MacMahon B. Lactation and a reduced risk of premenopausal breast cancer. New Engl J Med 1994; 330:81– 87. 36. Duncan AM, Merz-Demlow BE, Xu X, Phipps WR, Kurzer MS. Premenopausal equol excretor show plasma hormone profiles associated with lowered risk of breast cancer. Cancer Epidemiol Biomarkers Prev 2000; 9:581 –586. 37. Lampe JW, Karr SC, Hutchins AM, Slavin JL. Urinary equol excretion with a soy challenge: influence of habitual diet. Proc Soc Exp Biol Med 1998; 217:335 – 339. 38. Rowland IR, Wiseman H, Sanders TA, Adlercreutz H, Bowey EA. Interindividual variation in metabolism of soy isoflavones and lignans: influence of habitual diet on equol production by the gut microflora. Nutr Cancer 2000; 36:27– 32. 39. Martini MC, Dancisak BB, Haggans CJ, Thomas W, Slavin JL. Effects of soy intake on sex hormone metabolism in premenopausal women. Nutr Cancer 1999; 34:133– 139. 40. Bradlow HI, Telang NT, Sepkovic DW, Osborne MP. 2-hydroyestrone: the ‘good’ estrogen. J Endocrinol 1996; 150(suppl):259s– 265s. 41. Adlercreutz H, Gorbach SL, Goldin BR, Woods MN, Dwyer JT, Hamalainen E. Estrogen metabolism and excretion in oriental and caucasian women—reply. J Natl Cancer Inst 1994; 86:1644– 1645. 42. Atkinson C, Skor HE, Fitzgibbons DE, Scholes D, Chen C, Wahala K, Scwartz SM, Lampe JM. Urinary equol excretion in relation to 2-hydroxyestrone and 16alphahydroxyestrone concentrations: an observational study of young to middle-aged women. J Steroid Biochem 2003; 86(1):71 –77. 43. Lo¨nning PE, Johannessen DC, Lien EA, Ekse D, Fotsis T, Adlercreutz H. Influence of tamoxifen on sex hormones, gonadotrophins and sex hormone binding globulin in postmenopausal breast cancer patients. J Steroid Biochem 1995; 52:491 – 496. 44. Ekbom A, Trichopoulos D, Adami HO, Hsieh CC, Lan SJ. Evidence of prenatal influences on breast cancer risk. Lancet 1992; 340:1015 – 1018. 45. Yang J, Nakagawa H, Tsuta K, Tsubara A. Influence of perinatal genistein exposure on the development of MNU-induced mammary carcinoma in female SpragueDawley rats. Cancer Lett 2000; 149:171 – 179.

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46. Young HJ, Alfred CD, Alfred KF, Karko KL, Doerge DR, Helferich WG. Physiological concentrations of dietary genistein dose-dependently stimulate growth of estrogen-dependent human breast cancer (MCF-7) tumours implanted in athymic nude mice. J Nutr 2001; 131:2957– 2962. 47. de Lomo ML. Effects of soy phytoestrogens genistein and daidzein on breast cancer growth. Ann Pharmacother 2001; 35(9):1118– 1121. 48. Adlercreutz H, Yamada T, Wahala K, Watanabe S. Maternal and neonatal phytoestrogens in Japanese women during birth. Am J Obstet Gynecol 1999; 180:737 – 743. 49. Peterson G, Barnes S. Genistein and biochanin A inhibit the growth of human prostate cancer cells but not epidermal growth factor receptor tyrosine autophosphorylation. Prostate 1993; 22:335 –345. 50. Adlercreutz H, Bartels P, Elomaa V-V, Kang C. Phytoestrogens and prostate cancer. In: Descheemaker K, Debruyne I, eds. Soy and Health 2000: Clinical Evidence— Dietetic Applications. Leuven Apeldoorn: Garant, 2000:61 – 71. 51. Akaza K, Miyanaga NM, Takashima N, Naito S, Hirao Y, Tsukamoto T, Mori M. Is daidzein non-metaboliser a high risk for prostate cancer? A case–controlled study of serum soybean isoflavone concentration. Jpn J Clin Oncol 2002; 32(8):296–300. 52. Hedlund TE, Johannes WU, Miller GJ. Soy isoflavonoid equol modulates the growth of benign and malignant prostatic epithelial cells in vitro. Prostate 2003; 54(1):68– 78. 53. Jacobsen BK, Knutsen SF, Fraser GE. Does high soy milk intake reduce prostate cancer incidence? The adventist health study (United States). Cancer Cause Control 1998; 9:553– 557. 54. Lee MM, Gomez SL, Chang JS, Wey M, Wang RT, Hsing AW. Soy and isoflavone consumption in relation to prostate cancer risk in China. Cancer Epidemiol Biomarkers Prev 2003; 12(7):665– 668. 55. Miltyk W, Craciunescu CN, Fischer L, Jeffcoat RA, Koch MA, Loaczynski W, Mahoney C, Jeffcoat RA, Crowell J, Paglieri J, Zeisel SH. Lack of significant genotoxicity of purified soy isoflavones (genistein, daidzein and glycitein) in 20 patients with prostate cancer. Am J Clin Nutr 2003; 77(4):875 – 882. 56. Lamartiniere CA, Wang J, Fritz WA. Life time genistein in the diet down regulates estrogen receptor-alpha and -beta (ER-alpha and ER-beta) mRNA levels in the rat prostate (abstract). Proc Am Assoc Cancer Res 1999; 40:651. 57. Greenwald P, Milner JA, Anderson DE, McDonald SS. Micronutrients in cancer chemoprevention. Cancer Met Rev 2002; 21(3 – 4):217– 230. 58. Shirai T, Asamoto M, Takahashi S, Imaida K. Diet and prostate cancer. Toxicol 2002; 181 – 182:89– 94. 59. Wang S, DeGroff VL, Clinton SK. Tomato and soy polyphenols reduce insulin-like growth factor-I-stimulated rat prostate cancer cell proliferation and apoptotic resistance in vitro via inhibition of intracellular signalling pathways involving tyrosine kinase. J Nutr 2003; 133(7):2367– 2376. 60. Messina M, Bennink M. Soyfoods, isoflavones and risk of colonic cancer: a review of the in vitro and in vivo data. Bailliere’s Clin Endocrinol Metab 1998; 12:707 – 728. 61. Xu X, Thomas ML. Biphasic actions of estrogen on colon cancer cell growth: possible mediation by high- and low-affinity estrogen binding sites. Endocrine 1995; 3:661– 665. 62. Singh S, Langman ML. Oestrogen and colonic epithelial cell growth. Gut 1995; 37:737– 739.

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63. Calle EE, Miraclemcmahill HL, Thun MJ, Heath CW. Estrogen relplacement therapy and risk of fatal colon cancer in a prospective cohort of postmenopausal women. J Nat Cancer Inst 1995; 87:517– 523. 64. Rutqvist LE, Johanssn H, Signomklao T, Johansson U, Fornander T, Wilking N. Adjuvant tamoxifen therapy for early stage breast cancer and secondary primary malignancies. J Nat Cancer Inst 1995; 87:517 –523. 65. Toyomura K, Kono S. Soybeans, soy foods, isoflavones and risk of colorectal cancer: a review of experimental and epidemiological data. Asian Pac J Cancer Prev 2002; 3(2):125– 132. 66. Kennedy AR, Billings PC, Wan XS, Newberne PM. Effects of Bowman-Birk inhibitor on rat colon carcinogenesis. Nutr Cancer 2002; 43(2):174 – 186. 67. Rumney CJ, Rowland IR, Coutts TM, Randerath K, Reddy R, Shah AB, Ellul A, O’Neill IK. Effects of risk associated human dietary macrocomponents on processes related to carcinogenesis in human-flora-associated (HFA) rats. Carcinogenesis 1993; 14:79– 84. 68. Cummings JH, Gibson GR, Macfarlane GT. Quantitative estimates of fermentation in the hind gut of man. Acta Vet Scand 1989; 86:76– 82. 69. Demigne C, Remesy C, Morand C. Short chain fatty acids. In: Gibson GR, Roberfroid MB, eds. Colonic Microbiota, Nutrition and Health. Dodrecht: Kluwer Academic Publishers, 1999:55 – 69. 70. Kruh J, Defer N, Tichonicky L. Effects of butyrate on cell proliferation and gene expression. In: Cummings JH, Rombeau JL, Sakata T, eds. Physiological and Clinical Aspects of Short Chain Fatty Acids. Cambridge, UK: Cambridge University Press, 1995:275 –288. 71. Bingham SA. Plant cell wall material and cancer protection. In: Waldron KW, ed. Food and Cancer Prevention. Cambridge: Royal Society of Chemistry, 1993:339–347. 72. Goldin BR. In situ bacterial metabolism and colon mutagens. Ann Rev Microbiol 1986; 40:367 –393. 73. Muller M, Kersten S. Nutrigenomics: goals and strategies. Nat Rev Genet 2003; 4(4):315– 322.

16 Effect of Ginkgo biloba Extract EGb 761 on Differential Gene Expression in the Brain Rainer Cermak and Siegfried Wolffram Christian Albrechts University, Kiel, Germany

Chemical Composition of EGb 761 In Vitro Effects of EGb 761 on Differential Gene Expression in Neuronal Cells In Vivo Effects of EGb 761 on Differential Gene Expression in the Brain Conclusion References

341 344 346 349 349

CHEMICAL COMPOSITION OF EGb 761 The leaves of Ginkgo biloba L. contain a variety of chemical compounds, some of which are unique to this ancient tree. Various substances can be found in its leaves, among which flavonoids and terpenoids are probably the most interesting components (1 – 3). In dried Ginkgo leaves, the content of flavonoid glycosides is 0.5– 1% w/w, whereas the content of terpenoids is often ,0.1% w/w (2). The phenolic aglycone moiety of the flavonoid glycosides is made up predominantly of the flavonols quercetin or kaempferol, the occurrence of isorhamnetin being significantly smaller. Other flavonoids are found only in very small 341

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amounts. The glycoside moiety consists of mono- or diglycosides (mainly D -glucose and L -rhamnose units) that are connected to the aglycone via O-b-glycosidic bonds. Some of these glycosides are additionally acylated by p-coumaric acid (Fig. 16.1). These latter compounds are distinctive to Ginkgo biloba leaves and extracts (4,5). In addition to flavonoids, compounds that belong to the group of terpenoids are of particular interest. This group contains the ginkgolides with a diterpenoid structure and the sesquiterpenoid bilobalide, these being unique to Ginkgo biloba (Fig. 16.2). Five different ginkgolides have been isolated, of which ginkgolides A, B, C, and J are found in the leaves; the fifth, ginkgolid M, occurs solely in the roots. Ginkgolides are hexacyclic diterpenes possessing three lactone rings forming electrophilic cage-like structures that enable them to interact with cations and positively charged residues of other molecules. Bilobalide, a C15-trilactone, is considered to be a degradation product of the ginkgolides. Its concentration in Ginkgo biloba leaves is as high as that of all the ginkgolides (1,2,5).

R2 OH R1O

O OH O OH

H3C HO HO

O O

O O HO

O

O OH

OH

Coumaroyl flavonol glycoside

R1

R2 OH

Quercetin 3-O-α-L-[6-p-coumaroyl-(β-D)-glucosyl-(1,2)-rhamnoside]

H

Kaempferol 3-O-α-L-[6-p-coumaroyl-(β-D)-glucosyl-(1,2)-rhamnoside]

H

H

Isorhamnetin 3-O-α-L-[6-p-coumaroyl-(β-D)-glucosyl-(1,2)-rhamnoside]

H

O-CH3

glucoside

OH

glucoside

H

Quercetin 3-O-α-L-[6-p-coumaroyl-(β-D)-glucosyl-(1,2)-rhamnoside]-

7-O-β-D-glucoside Kaempferol 3-O-α-L-[6-p-coumaroyl-(β-D)-glucosyl-(1,2)-rhamnoside]-

7-O-β-D-glucoside

Figure 16.1 Structures of some flavonol glycoside esters with coumaric acid, distinctive compounds of Ginkgo biloba. The coumaroyl esters of the quercetin-rhamnoglucoside and of the kaempferol-rhamnoglucoside are most common in Ginkgo leaves.

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O

A

HO O H3C

O

H

O H

H

H C(CH3)3

R2

H R1

O H

O

O

H H R3

Ginkgolide

R1

R2

R3

A

OH

H

H

B

OH

OH

H

C

OH

OH

OH

J

OH

H

OH

M

H

OH

OH

O

B

O

HO

C(CH3)3 O O

OH

O O

Figure 16.2 Structures of ginkgolides (A) and bilobalide (B) found in leaves of Ginkgo biloba. Ginkgolide M is only present in the roots.

In addition to the preceding substances, a large variety of polysaccharides, long-chain hydrocarbons, alcohols, acids, and so on have been isolated (1,2). The cytotoxic, mutagenic, and allergenic potential of some constituents, especially anacardic or ginkgolic acids, limits the use of a crude leaf extract for therapeutical or prophylactic purposes in humans (6). Thus, several extraction processes have been developed for the removal of toxic substances and concomitant enrichment of desired compounds. The extract EGb 761, formerly patented by Dr. W. Schwabe GmbH & Co, Karlsruhe, Germany, contains 22 – 27% flavone glycosides, 5 –7% terpenoids (2.8 – 3.4% ginkgolides A, B, C and 2.6 – 3.2% bilobalide), and not more than 5 ppm ginkgolic acids (7). This has been the most widely used extract of Ginkgo biloba leaves in clinical studies and is the basis for many commercially available medicinal products of Ginkgo biloba. In Germany and France, EGb 761 is widely prescribed in cases of cerebral insufficiency. In many other countries, it is sold over-the-counter with the objective to improve brain performance and to counteract cerebral insufficiency as a consequence of ageing. EGb 761 is thought to improve the memory of elderly subjects. In addition, several clinical studies claim a therapeutic or prophylactic effect against dementia, especially against Alzheimer’s disease [reviewed in Refs. (8 –12)]. In an effort to search for mechanisms that could explain these clinical observations at a cellular and molecular level, a multitude of effects have been

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described for EGb 761 or for some of its constituents. It is well known that the ginkgolides are high-affinity competitive platelet activator factor (PAF) receptor antagonists. This enables them to potently inhibit PAF-induced platelet aggregation and could explain some of its neuroprotective activity (13,14). Another well described feature of EGb 761 is its antioxidative activity. Several studies demonstrated a protective effect of the extract on neuronal damage induced by oxidative radicals (15 – 18). Although it is thought that the flavonoids are mainly responsible for the antioxidative effects, ginkgolides and bilobalides are also potential radical scavengers (19). In addition, EGb 761 acts vasodilatatory and, thus, increases peripheral and cerebral blood flow, a quality that diminishes ischemic brain damage (20 –23). Various other actions of EGb 761 have been described, among them a prevention of age-related decreases in neurotransmitter density and a decrease in the expression of the peripheral benzodiazepine receptor, which are reviewed elsewhere (24 – 26). In recent years, it has become obvious that the extract and some of its constituents are also able to modify the expression of cellular proteins at the transcriptional level. These observations open a new avenue for possible beneficial actions and future applications of EGb 761 and will be described in the following sections. In this review, only studies suggesting gene modulatory effects of Ginkgo biloba in the brain or in neurons will be considered.

IN VITRO EFFECTS OF EGb 761 ON DIFFERENTIAL GENE EXPRESSION IN NEURONAL CELLS It is widely known that Ginkgo biloba extract EGb 761 has potent antioxidant properties achieved by either scavenging free radicals or chelating prooxidant transition metals (25). As reactive oxygen species (ROS) are involved in the regulation of the expression of a large number of genes, it is likely that EGb 761 modulates genes that are under the control of redox-sensitive transcription factors such as NF-kB, AP-1, and Nrf-1 (27,28). Several studies have reported alterations of gene expression induced by the Ginkgo biloba extract or by some of its constituents in neuronal-like PC12 cells (pheochromocytoma cells differentiated with nerve growth factor). EGb 761 was able to rescue PC12 cells submitted to oxidative stress induced by the toxin 1-methyl-4-phenyl-pyridine (MPPþ) (29). A flavonoid-containing fraction of EGb 761, Cp 202, which is devoid of the terpenoids, was as effective as the whole extract in counteracting MPP þ -induced cytotoxicity. Both EGb 761 and Cp 202 also decreased the protein expression of the high-affinity dopamine transporter, which is responsible for uptake of MPPþ into dopaminergic neurons. Although the reduced protein expression of this transporter can explain the protective effect of EGb 761 and Cp 202 in PC12 cells, the mechanism by which the plant extracts altered its expression remains unknown (29). In this context, it is interesting to note that an inhibitory activity of EGb 761 on the

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uptake of dopamine into synaptosome-enriched fractions of rat brain has been reported (24). EGb 761 also showed anti-apoptotic properties in PC12 cells grown in serum-deprived medium in order to induce apoptosis (30). The extract inhibited several steps of the apoptotic process like mitochondrial cytochrome c release, caspase 3 activation, and DNA fragmentation. A DNA microarray analysis in those cells demonstrated an increased amount of the transcript for an antiapoptotic Bcl-2-like protein after treatment with EGb 761. This was verified on the protein level with Western blots showing that the expression of Bcl-2 protein was significantly up-regulated by the plant extract. Other transcripts of genes involved in apoptosis were also altered in the microarray study. However, these changes were only moderate and not verified by other methods. Thus, at least a part of the anti-apoptotic activity of EGb 761 could be explained by its effects on the transcription of relevant genes. As complex I of the mitochondrial respiratory chain, the enzyme NADH dehydrogenase plays a key role in energy metabolism and oxidative phosphorylation. In differentiated PC12 cells, EGb 761 and bilobalide increased the transcript for complex I (31). In these cells, bilobalide also increased the transcript of another mitochondrial respiratory chain enzyme, namely of cytochrome c oxidase subunit III (32). The latter observation was confirmed in gerbil brains in vivo (discussed subsequently). These results suggest that EGb 761 and bilobalide could counteract defective energy metabolism and ATP depletion by an increased expression of respiratory chain enzymes. This is of special relevance for neurons owing to their high energy demand. In primary cultures of cortical neurons obtained from mice embryos, EGb 761 increased the protein expression of heme oxygenase 1 (33). Heme oxygenase acts as an antioxidant enzyme by degrading heme into iron, carbon monoxide, and biliverdin. Under physiological conditions, iron generated from heme degradation is captured by ferritin. Thus, increasing heme degradation by increased heme oxygenase expression could be a mechanism by which the concentration of prooxidant iron is reduced and the generation of ROS is attenuated. Besides the established antioxidant properties of EGb 761—achieved by scavenging free radicals or chelating prooxidant transition metals like iron (25)—altering the expression of antioxidant enzymes is a rather novel mechanism by which the extract could counteract disturbances of cellular redox homeostasis. An effect of EGb 761 on the expression of enzymes involved in antioxidant defense was also demonstrated in a study investigating differential gene expression in human hNT neurons using a cDNA macroarray (34). Cytosolic superoxide dismutase 1 (CuZn SOD), which catalyzes the dismutation of the oxygen radical superoxide to hydrogen peroxide, was upregulated. Surprisingly, glutathione reductase (which regenerates reduced glutathione from diglutathione) and glutathione S-transferase pi (which neutralizes electrophilic compounds able to generate ROS) were downregulated. In accordance with the macroarray results, the enzyme activities of glutathione reductase and glutathione S-transferase pi

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were also lower after treatment with EGb 761. Whereas the mRNA levels of CuZn SOD were increased, the activity of this enzyme was also lower in EGb 761 treated cells. Thus, antioxidant defense was rather weakened by the Ginkgo biloba extract in hNT neurons. In addition, the macroarray revealed an effect of EGb 761 on genes encoding transcription factors (increase of NF-kB p65 subunit and zinc finger protein 91 mRNAs, decrease of c-myc oncogene transcripts) and genes involved in stress reponses (upregulation of 70 kDa heat shock protein 1). However, the expression of those latter genes was not verified by RT-PCR or at the protein level (34). Compounds of Ginkgo biloba also affected gene expression in glial cells, which account for the majority of cells in the brain. Altered expression of genes in microglia and astrocytes have been described (35,36) Advanced glycation endproducts (AGE), which accumulate under neuroinflammatory conditions, were shown to increase mRNA and protein levels of the inducible nitric oxide synthase (iNOS) in the murine microglial cell line N-11, leading to increased production of nitric oxide. EGb 761 prevented AGE-induced upregulation of iNOS expression and nitric oxide production in these microglial cells. This suggests that the extract might have anti-inflammatory activities in the brain (35). In rat astrocytes, bilobalide induced the expression of glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor (VEGF) at the mRNA and protein level (36). VEGF is able to induce angiogenesis. GDNF has the ability to promote survival of substantia nigra dopamine neurons. Thus, bilobalide may be an interesting drug in the treatment of Parkinson’s disease (37). IN VIVO EFFECTS OF EGb 761 ON DIFFERENTIAL GENE EXPRESSION IN THE BRAIN The earlier mentioned effects were observed after direct treatment of neuronal and glial cells with EGb 761 or its constituents in vitro. One has to bear in mind, though, that these compounds have to cross the blood – brain barrier (BBB) in order to act on central neurons in vivo. At present, hardly any data exist that demonstrate transfer of EGb 761 constituents into brain tissue. Recently, some studies have suggested that certain components of EGb 761, namely flavonoids, might indeed be able to penetrate the BBB (38,39). More importantly, some in vivo studies have demonstrated that EGb 761 has the potential to alter gene expression in central neurons, thereby indirectly proving that its constituents or their metabolites reach the CNS. In an animal model of global brain ischemia, EGb 761 and bilobalide protected hippocampal neurons from death in gerbils (22). EGb 761 (25 – 100 mg/kg) or bilobalide (3 or 6 mg/kg) were administered orally to the animals for 7 days before the gerbils were subjected to ischemia. The treatment with EGb 761 and bilobalide prevented ischemia-induced reductions of cytochrome c oxidase subunit III mRNA. A stimulatory effect of bilobalide on mRNA expression of this enzyme was already observed in PC12 cells (32).

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Thus, orally ingested bilobalide seems to protect neurons by an increased expression of mitochondrial respiratory chain enzymes, thereby probably preventing failure of energy production by oxidative phosphorylation in ischemic brains. Another study suggested that the Ginkgo biloba extract was able to alter the mRNA levels of the glucose transporter subtype GLUT3 in rat hippocampal neurons (40). When rats received 50 mg EGb 761 per day together with their regular diet over a period of 19 days, the transcript of this glucose transporter was upregulated in a subpopulation of rats designated as “poor performers” because of their weak performance in memory tests. This was demonstrated by in situ hybridization of GLUT3 mRNA in hippocampal brain sections. The authors suggested that this effect of EGb 761 could increase the neuronal glucose supply. The question remains open as to whether an improved energy supply via an increased expression of glucose transporters is a mechanism by which EGb 761 may affect hippocampal memory and learning processes. In a study using bulbectomized mice, it was shown that treatment with EGb 761 also affects the expression of proteins involved in neurogenesis (41). In this in vivo model, globose basal cells in the olfactory epithelium start to proliferate after ablation of the olfactory bulb and their progeny differentiates to neurons. Such proliferating neurons increase the expression of the proliferating cell nuclear antigen protein (PCNA). The increase in PCNA expression occurred 2 days sooner in bulbectomized mice treated with EGb 761 in their drinking water (100 mg/kg b.w.) compared with control animals. This accelerated PCNA expression after bulbectomy indicated an earlier onset of proliferation. Three weeks after bulbectomy, protein expression of the growth associated protein 43 (GAP-43), which is a marker for postmitotic, differentiated neurons, was also higher in EGb 761 treated mice. This could be linked to the enhanced proliferation observed in these animals, yielding more cells to enter the differentiation pathway. Thus, EGb 761 appears to modulate the balance between proliferation and differentiation in neurons, which is visible in an altered expression of proteins involved in these processes. Exposure of rats to heat stress leads to leakage of the BBB, brain edema formation, and cell injury with a concurrent increase in the protein expression of constitutive NO synthase (cNOS) and causes expression of iNOS (42). Increased synthesis of NO is thought to be involved in the brain injury after hyperthermia. When EGb 761 (50 mg/kg) or ginkgolide B (2 mg/kg) was orally applied to rats for 5 days prior to heat stress, brain edema formation and BBB permeability were significantly attenuated. In addition, both treatments attenuated the increase of cNOS and iNOS expression in several brain regions (42). To gain further insight into the biochemical effects of Ginkgo biloba in the CNS, a recent study profiled the transcriptional effects of the extract on the brains of mice using oligonucleotide microarrays (43). The effects of EGb 761 on gene transcription were measured in the hippocampus and cerebral cortex of adult mice fed a diet with or without Ginkgo biloba extract (300 mg EGb 761/kg

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diet) over a period of 4 weeks. Plasma concentrations of the flavonols quercetin, kaempferol, and isorhamnetin were measured by HPLC (44,45). In EGb 761 treated mice, the concentrations of these flavonols were significantly higher than those of the control group, confirming the absorption of at least some of the major components of the extract into the systemic circulation. The microarrays used represent all (6000) sequences in the Mouse UniGene database that have been functionally characterized, as well as around 6000 expressed sequence tags (EST) clusters. Of the 12,000 combined genes and ESTs represented on the array, only 10 changed in expression level by a factor of three-fold or more and all were upregulated. In the cortex, mRNAs for neuronal tyrosine/threonine phosphatase 1 and microtubule-associated tau were significantly enhanced. These proteins are both associated with formation –breakdown of intracellular neurofibrillary tangles, a hallmark lesion of Alzheimer’s disease. Hyperphosphorylated tau has been found to be the major protein of these neurofibrillary tangles, possibly because of an imbalance in tau kinase and phosphatase activities in the affected neurons (46). Hyperphosphorylated tau isolated from brains of patients with Alzheimer’s disease has been shown to be efficiently dephosphorylated in vitro by protein phosphatases 1, 2A, and 2B. Thus, upregulation of neuronal phosphatase 1 by EGb 761 could play a neuroprotective role in the brain. Selective inhibition of protein phosphatase 2A by okadaic acid in metabolically competent rat brain slices has been shown to induce a hyperphosphorylation and accumulation of tau like that in Alzheimer’s disease (47,48). The expression of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionicacid-2 (AMPA-2), calcium and chloride channels, prolactin, and growth hormone (GH) were also upregulated in the cortex. Within the past decade, studies have revealed that GH may exert significant effects on the central nervous system. Cognitive impairments are well documented hallmark features of GH deficiency, and clinical studies have reported psychological improvements (in mood and well being) and beneficial effects on certain functions including memory, mental alertness, motivation, and working capacity in adults receiving GH replacement therapy. Moreover, GH therapy in children deficient in this protein has been reported to produce marked behavioral improvement (49). In the hippocampus, only transthyretin mRNA was induced. Transthyretin plays a role in the transport of tyroxine and retinol binding protein in the brain. Thyroid hormones regulate neuronal proliferation and differentiation in discrete regions of the brain during development and are necessary for normal cytoskeletal outgrowth (50). Transthyretin has also been shown in vitro to sequester amyloid beta protein and, thus, to prevent amyloid beta aggregation and amyloid formation (51). Moreover, transthyretin levels in cerebrospinal fluid have been found to be significantly decreased in Alzheimer’s disease patients. Thus, one mechanism whereby EGb 761 may exert neurological effects is the modulation of transthyretin levels. This could have an impact on hormone transport or on amyloid beta sequestration in the brain.

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CONCLUSION The mechanisms responsible for the beneficial effects of the Ginkgo biloba extract EGb 761 are largely unknown. The observation that the extract influences the expression of certain genes is a rather novel finding. Several studies have shown that EGb 761 and some of its constituents affect gene expression in neurons. Moreover, animal studies demonstrated the efficacy of the ingested extract to alter in vivo gene expression in the brain. The consideration of the products of at least some of the genes over-expressed after EGb 761 treatment offer new mechanistic explanations for the clinical effects of the extract. This is of special relevance to certain neurological disorders like ischemic lesions or Alzheimer’s disease. On the basis of these recent findings, future investigations on the gene regulatory activities of Ginkgo biloba will provide a better understanding of the effects of this complex extract at the molecular level. REFERENCES 1. Ho¨lzl J. Die Zusammensetzung von Ginkgo biloba. Pharm Unserer Zeit 1992; 21:215– 223. 2. Sticher O. Quality of Ginkgo preparations. Planta Med 1993; 59:2– 11. 3. van Beek TA. Chemical analysis of Ginkgo biloba leaves and extracts. J Chromatogr A 2002; 967:21 – 55. 4. Tang Y, Lou F, Wang J, Li Y, Zhuang S. Coumaroyl flavonol glycosides from the leaves of Ginkgo biloba. Phytochemistry 2001; 58:1251– 1256. 5. Bedir E, Tatli II, Khan RA, Zhao JP, Takamatsu S, Walker LA, Goldman P, Khan IA. Biologically active secondary metabolites from Ginkgo biloba. J Agric Food Chem 2002; 50:3150 –3155. 6. Hecker H, Johannisson R, Koch E, Siegers CP. In vitro evaluation of the cytotoxic potential of alkylphenols from Ginkgo biloba L. Toxicology 2002; 177:167– 177. 7. Kressmann S, Mu¨ller WE, Blume HH. Pharmaceutical quality of different Ginkgo biloba brands. J Pharm Pharmacol 2002; 54:661 – 669. 8. Kleijnen J, Knipschild PG. Ginkgo biloba for cerebral insufficiency. Br J Clin Pharmacol 1992; 34:352 –358. 9. Oken BS, Storzbach DM, Kaye JA. The efficacy of Ginkgo biloba on cognitive function in Alzheimer disease. Arch Neurol 1998; 55:1409– 1415. 10. Le Bars PL, Kastelan J. Efficacy and safety of a Ginkgo biloba extract. Public Health Nutr 2000; 3:495 – 499. 11. Le Bars PL, Katz MM, Berman N, Itil TM, Freedman AM, Schatzberg AF. A placebocontrolled, double-blind, randomized trial of an extract of Ginkgo biloba for dementia. J Am Med Assoc 1997; 278:1327 – 1332. 12. Kanowski S, Herrmann WM, Stephan K, Wierich W, Horr R. Proof of efficacy of the Ginkgo biloba special extract EGb 761 in outpatients suffering from mild to moderate primary degenerative dementia of the Alzheimer type or multi-infarct dementia. Pharmacopsychiatry 1996; 29:47 –56. 13. Oberpichler H, Sauer D, Rossberg C, Mennel HD, Krieglstein J. PAF antagonist ginkgolide B reduces postischemic neuronal damage in rat brain hippocampus. J Cereb Blood Flow Metab 1990; 10:133– 135.

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14. Smith PF, MacLennan KM, Darlington CL. The neuroprotective properties of the Ginkgo biloba leaf: a review of the possible relationship to platelet-activating factor (PAF). J Ethnopharmacol 1996; 50:131– 139. 15. Xin W, Wei T, Chen C, Ni Y, Zhao B, Hou J. Mechanisms of apoptosis in rat cerebellar granule cells induced by hydroxyl radicals and the effects of EGb761 and its constituents. Toxicology 2000; 148:103 – 110. 16. Bastianetto S, Ramassamy C, Dore´ S, Christen Y, Poirier J, Quirion R. The Ginkgo biloba extract (EGb 761) protects hippocampal neurons against cell death induced by b-amyloid. Eur J Neurosci 2000; 12:1882– 1890. 17. Bastianetto S, Zheng WH, Quirion R. The Ginkgo biloba extract (EGb 761) protects and rescues hippocampal cells against nitric oxide-induced toxicity: involvement of its flavonoid constituents and protein kinase C. J Neurochem 2000; 74:2268– 2277. 18. Wei T, Ni Y, Hou J, Chen C, Zhao B, Xin W. Hydrogen peroxide-induced oxidative damage and apoptosis in cerebellar granule cells: protection by Ginkgo biloba extract. Pharmacol Res 2000; 41:427– 433. 19. Scholtyssek H, Damerau W, Wessel R, Schimke I. Antioxidative activity of ginkgolides against superoxide in an aprotic environment. Chem Biol Interact 1997; 106:183– 190. 20. Zhang WR, Hayashi T, Kitagawa H, Sasaki C, Sakai K, Warita H, Wang JM, Shiro Y, Uchida M, Abe K. Protective effect of Ginkgo extract on rat brain with transient middle cerebral artery occlusion. Neurol Res 2000; 22:517 – 521. 21. Clark WM, Rinker LG, Lessov NS, Lowery SL, Cipolla MJ. Efficacy of antioxidant therapies in transient focal ischemia in mice. Stroke 2001; 32:1000 – 1004. 22. Chandrasekaran K, Mehrabian Z, Spinnewyn B, Drieu K, Fiskum G. Neuroprotective effects of bilobalide, a component of the Ginkgo biloba extract (EGb 761), in gerbil global brain ischemia. Brain Res 2001; 922:282– 292. ¨ zdemir Y, Bolay H, So¨ylemezoglu F, Saribas O, Dalkara T. Chronic ¨ nal I, Gu¨rsoy-O 23. U daily administration of selegiline and EGb 761 increases brain’s resistance to ischemia in mice. Brain Res 2002; 917:174 – 181. 24. DeFeudis FV, Drieu K. Ginkgo biloba extract (EGb 761) and CNS functions: basic studies and clinical applications. Curr Drug Targets 2000; 1:25– 58. 25. Yoshikawa T, Naito Y, Kondo M. Ginkgo biloba leaf extract: review of biological actions and clinical applications. Antioxid Redox Signal 1999; 1:469 – 480. 26. MacLennan KM, Darlington CL, Smith PF. The CNS effects of Ginkgo biloba extracts and ginkgolide B. Prog Neurobiol 2002; 67:235– 257. 27. Saliou C, Valacchi G, Rimbach G. Assessing bioflavonoids as regulators of NF-kB activity and inflammatory gene expression in mammalian cells. Methods Enzymol 2001; 335:380 – 387. 28. Rimbach G, Gohil K, Matsugo S, Moini H, Saliou C, Virgili F, Weber SU, Packer L. Induction of glutathione synthesis in human keratinocytes by Ginkgo biloba extract (EGb761). Biofactors 2001; 15:39 – 52. 29. Gagne´ B, Ge´linas S, Bureau G, Lagace´ B, Ramassamy C, Chiasson K, Valastro B, Martinoli MG. Effects of estradiol, phytoestrogens, and Ginkgo biloba extracts against 1-methyl-4-phenyl-pyridine-induced oxidative stress. Endocrine 2003; 21:89– 95. 30. Smith JV, Burdick AJ, Golik P, Khan I, Wallace D, Luo Y. Anti-apoptotic properties of Ginkgo biloba extract EGb 761 in differentiated PC12 cells. Cell Mol Biol (Noisy-le-grand) 2002; 48:699 – 707.

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31. Tendi EA, Bosetti F, DasGupta SF, Stella AMG, Drieu K, Rapoport SI. Ginkgo biloba extracts EGb 761 and bilobalide increase NADH dehydrogenase mRNA level and mitochondrial respiratory control ratio in PC12 cells. Neurochem Res 2002; 27:319– 323. 32. Chandrasekaran K, Liu LI, Hatanpa¨a¨ K, Drieu K, Rapoport SI. Stimulation of mitochondrial gene expression by bilobalide, a component of Ginkgo biloba extract (EGb 761). In: Packer L, Christen Y, eds. Ginkgo biloba Extract: Lessons from Cell Biology. Paris: Elsevier, 1998:121– 128. 33. Zhuan H, Pin S, Christen Y, Dore´ S. Induction of heme oxygenase 1 by Ginkgo biloba in neuronal cultures and potential implications in ischemia. Cell Mol Biol (Noisy-legrand) 2002; 48:647– 653. 34. Soulie´ C, Nicolle A, Christen Y, Ceballos-Picot I. The Ginkgo biloba extract EGb 761 increases viability of hNT human neurons in culture and affects the expression of genes implicated in the stress response. Cell Mol Biol (Noisy-le-grand) 2002; 48:641 – 646. 35. Wong A, Dukic-Stefanovic S, Gasic-Milenkovic J, Schinzel R, Wiesinger H, Riederer P, Mu¨nch G. Anti-inflammatory antioxidants attenuate the expression of inducible nitric oxide synthase mediated by advanced glycation endproducts in murine microglia. Eur J Neurosci 2001; 14:1961 – 1967. 36. Zheng SX, Zhou LJ, Chen ZL, Yin ML, Zhu XZ. Bilobalide promotes expression of glial cell line-derived neurotrophic factor and vascular endothelial growth factor in rat astrocytes. Acta Pharmacol Sin 2000; 21:151– 155. 37. Lapchak PA, Gash DM, Collins F, Hilt D, Miller PJ, Araujo DM. Pharmacological activities of glial cell line-derived neurotrophic factor (GDNF): preclinical development and application to the treatment of Parkinson’s disease. Exp Neurol 1997; 145:309– 321. 38. Gutmann H, Bruggisser R, Schaffner W, Bogman K, Botomino A, Drewe J. Transport of amentoflavone across the blood – brain barrier in vitro. Planta Med 2002; 68:804– 807. 39. Youdim KA, Dobbie MS, Kuhnle GGC, Proteggente AR, Abbott NJ, Rice-Evans CA. Interaction between flavonoids and the blood – brain barrier: in vitro studies. J Neurochem 2003; 85:180– 192. 40. Lo¨ffler T, Lee SK, No¨ldner M, Chatterjee SS, Hoyer S, Schliebs R. Effect of Ginkgo biloba extract (EGb761) on glucose metabolism-related markers in streptozotocindamaged rat brain. J Neural Transm 2001; 108:1457 – 1474. 41. Didier A, Jourdan F. The Ginkgo biloba extract modulates the balance between proliferation and differentiation in the olfactory epithelium of adult mice following bulbectomy. Cell Mol Biol (Noisy-le-grand) 2002; 48:717 – 723. 42. Sharma HS, Drieu K, Alm P, Westman J. Role of nitric oxide in blood-brain barrier permeability, brain edema and cell damage following hyperthermic brain injury. An experimental study using EGb-761 and ginkgolide B pretreatment in the rat. Acta Neurochir 2000; 76:81– 86. 43. Watanabe CMH, Wolffram S, Ader P, Rimbach G, Packer L, Maguire JJ, Schultz PG, Gohil K. The in vivo neuromodulatory effects of the herbal medicine Ginkgo biloba. Proc Natl Acad Sci USA 2001; 98:6577 – 6580. 44. Ader P, Wessmann S, Wolffram S. Bioavailability and metabolism of the flavonol quercetin in the pig. Free Radic Biol Med 2000; 28:1056 – 1067. 45. Cermak R, Landgraf S, Wolffram S. The bioavailability of quercetin in pigs depends on the glycoside moiety and on dietary factors. J Nutr 2003; 133:2802 – 2807.

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17 Interactions of Flavonoids and Their Metabolites with Cell Signaling Cascades Jeremy P. E. Spencer University of Reading, Reading, UK

Introduction Potential Bioactive Forms of Flavonoids In Vivo Metabolism in the GI Tract and Liver Colonic Metabolism Intracellular Metabolism Modulation of Signaling Cascades by Flavonoids MAP Kinase Signaling and Cell Function Interactions of Flavonoids Within Signaling Pathways Specific Actions of Flavanols Specific Actions of Flavonols Summary References

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Flavonoids have been proposed to act as beneficial agents in a multitude of disease states, including cancer, cardiovascular disease, and neurodegenerative disorders. The biological effect of these polyphenols will ultimately depend on the cellular effects of their circulating metabolites, such as glucuronides and 353

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O-methylated forms and on the extent to which they interact with and/or associate with cells, either by interactions at the membrane or their uptake into the cytosol. Following uptake, flavonoids and their metabolites may affect cells in a number of ways: (i) by regulating the intracellular redox status, (ii) by effecting enzyme function, (iii) by interacting with mitochondria, and (iv) by specific interactions within intracellular signaling cascades vital to cell function. This review will summarize the current knowledge on the role of flavonoids as modulators of cell signaling pathways, such as the MAP kinase pathway, which are vital in determining neuronal survival, regeneration, development, and death. INTRODUCTION Flavonoids have been proposed to act as beneficial agents in a multitude of disease states (1,2), most commonly cancer (3–6), cardiovascular disease (6,7), and neurodegenerative disorders (8 – 12). Epidemiological and dietary intervention studies, in both humans and animals, have suggested that diet-derived phenolics, in particular flavonoids, may play a beneficial role in the prevention of neurodegeneration, age-related cognitive and motor decline (10,11), and brain ischaemia/reperfusion injury (9). Much evidence also exists to support the potential beneficial and neuromodulatory effects of flavonoid-rich Ginkgo biloba extracts, such as EGb 761, in the CNS (13 –15). Clinical trials with EGb 761, which is rich in the flavonoids kaempferol and quercetin, have indicated beneficial effects on brain function, particularly in connection with age-related dementias and Alzheimer’s disease (15,16). Furthermore, the neuroprotective action of one of the green tea flavonoids, epigallocatechin gallate (EGCG), has been shown in both oxidative stress- (17) and Ab-induced (18) neuronal death models. Interestingly, the protective effects observed in both systems were linked to a modulation in signaling through protein kinase C and/or modulation of cell survival/cell cycle genes. Much evidence has linked flavonoids to both beneficial and cytotoxic effects. Such effects may be mediated in a number of ways: (i) by the regulation of the intracellular redox status, (ii) by direct effects on enzyme function, (iii) via the interaction with mitochondria, and (iv) by specific interactions within intracellular signaling cascades vital to cell function. The latter has received much interest over the last few years and this review will attempt to summarize current knowledge on the role flavonoids play as modulators of cell signaling cascades. One such pathway of interest is mitogen activated protein kinase (MAP kinase) pathway, which is vital in determining cellular survival, regeneration, development, and death. In doing so, however, it is essential to consider and integrate knowledge on the potential bioactive forms of flavonoids in vivo. It is now clear that dietary forms of flavonoids are not those found in the circulation and indeed those exposed to tissues. Rather extensive modification of these polyphenols occurs on uptake, which results in significant changes in their chemical and biological properties. These circulating metabolites are thought to be important in

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mediating cellular functions and consequently the initial part of this review will summarize the processes that lead to the formation of in vivo metabolites from the dietary forms.

POTENTIAL BIOACTIVE FORMS OF FLAVONOIDS IN VIVO As mentioned earlier, it has become clear over the last few years that the bioactive forms of flavonoids in vivo are not those forms found in plants, for example, the aglycones or their various glycosides, but instead conjugates and metabolites arising from these on absorption [reviewed in Refs. (19 – 23); Fig. 17.1]. In particular, there is now strong evidence for the extensive phase I de-glycosylation and phase II metabolism of the resulting aglycones such as quercetin, hesperetin, naringenin, and epicatechin to glucuronides, sulfates, and

Neurons glia Dietary Flavonoid Oligomers cleaved

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Figure 17.1 Summary of the formation of gastrointestinal tract and hepatic metabolites and conjugates of flavonoids in humans. Cleavage of oligomeric flavonoids such as procyanidins may occur in the stomach in environments of low pH. All classes of flavonoids undergo extensive metabolism in the jejunum and ileum of the small intestine and resulting metabolites enter the portal vein and undergo further metabolism in the liver. Colonic microflora degrade flavonoids into smaller phenolic acids that may also be absorbed. The fate of most of these metabolites is renal excretion, although, some may enter cells and tissues.

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O-methylated forms during transfer across the small intestine (24) and then again in the liver (Fig. 17.1). Further transformation has been reported in the colon where the enzymes of the gut microflora degrade flavonoids to simple phenolic acids (25), which may also be absorbed and subsequently further metabolized in the liver. Metabolism in the GI Tract and Liver It is now well established that the gastrointestinal tract plays a significant role in the metabolism and conjugation of these polyphenols before entry into the systemic circulation and the liver (19,20,24,26 –28). Enterocytes in the jejunum and ileum of the small intestine transfer flavonoids from the luminal side of the gut to portal vein during which there is removal of sugar groups followed by significant glucuronidation of nearly all flavonoids tested (Fig. 17.2) by the action of b-glucosidase and UDP-glucuronosyltransferase enzymes (19 – 21). In addition, in the case of catechol containing B-ring flavonoids, there is also extensive O-methylation (Fig. 17.2) catalyzed by the action of COMT (24). For example, the flavonoid glycosides, luteolin-7-glucoside, kaempferol-3-glucoside, naringenin-7-glucoside, and quercetin-3-glucoside were all cleaved by rat jejunal or ileal mucosa, before their efflux into the serosal fluid as glucuronides and/or O-methylated metabolites (24,29). A full assessment of uptake and metabolism of flavonoids has been well reviewed previously (19 –23). Interestingly, research with O-methylated metabolites of flavonoids, such as those of epicatechin, provided some of the initial evidence that flavonoids may not simply exert cellular protection through antioxidant mechanisms (30 –32). Here, the protection elicited by 30 -O-methyl epicatechin against oxidative stress-induced cellular damage was not significantly different from that of epicatechin, even though its classical antioxidant nature is greatly reduced. These studies suggested the first time that hydrogen-donating antioxidant activity is unlikely to be the sole mechanism of protection against oxidative cellular damage and led to further mechanistic investigations involving their actions within signaling cascades (33). Colonic Metabolism Evidence suggests that the extent of absorption of dietary polyphenols in the small intestine is relatively low (10 –20%) (24,28,34). The implications of this low absorption in the small intestine means that the majority of ingested polyphenols, including those absorbed and conjugated in the enterocytes and/or the liver and transported back into the lumen, either directly or via the bile (35), will reach the large intestine where they encounter the colonic microflora. The colon contains 1012 microorganisms/cm3, which has an enormous catalytic and hydrolytic potential, and this enzymatic degradation of flavonoids by the colonic microflora results in a huge array of new metabolites. For example, bacterial enzymes may catalyze reactions including hydrolysis, dehydroxylation, demethylation, ring cleavage, and decarboxylation as well as rapid de-conjugation (25).

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A

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Figure 17.2 Structure of epicatechin and its major circulating metabolites: (A) epicatechin, (B) 30 -O-methyl epicatechin, (C) epicatechin-5-O-b-D -glucuronide, (D) epicatechin-7-sulfate, (E) (2)-5-(30 ,40 -dihydroxyphenyl)-g-valerolactone, (F) hippuric acid, (G) 8-glutathionyl quercetin. Glucuronide and sulfate conjugates are formed with the majority of flavonoids in the small intestine and liver, whereas O-methylated forms are only formed where the flavonoid has a catechol B-ring. (B)– (D) represent GI tract and hepatic metabolites, whereas (E) and (F) derive from metabolism by the enzymes of the colonic microflora and (G) may result from intracellular metabolism.

Unlike human enzymes, the microflora catalyze the breakdown of the flavonoid backbone itself to simpler molecules such as phenolic acids. Specific metabolites have been observed in urine after consumption of a variety of phenolics, for example, the glycine conjugate of benzoic acid, hippuric acid (Fig. 17.2). This metabolite is primarily derived from plant phenolics and aromatic amino acids through the action of intestinal bacteria and, consequently, the level of hippuric acid would be expected to increase in the circulation and urine of individuals consuming diets rich in flavanols or polyphenols. The 5,7,3,30 ,40 -hydroxylation pattern of flavanols is believed to enhance ring opening after hydrolysis (25) and metabolism of flavanols by enzymes of

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the microflora of the large intestine results in many metabolites: 3,4-dihydrophenylacetic acid, 3-hydroxyphenylacetic acid, homovanillic acid, and their conjugates derived from the B-ring (25) and phenolic acids from the C-ring. Because flavanols have no C-4 carbonyl group in their structures, they may also be degraded by colonic bacteria to the specific metabolites phenylvalerolactones, such as (2)-5-(30 ,40 -dihydroxyphenyl)-gamma-valerolactone (36) (Fig. 17.2). Indeed, valerolactones derived from colonic metabolism are now believed to be major in vivo metabolites of flavanol or tea ingestion in humans and animals (37 – 41). These colonic metabolites, such as valerolactones, may represent novel bioactive metabolites of flavonoids in vivo, and should be considered as possible mediators of flavonoid action in the body. Thus far, there is no data describing the cellular effects of such metabolites, although interest is growing in these somewhat forgotten in vivo forms. Intracellular Metabolism The cellular effects of flavonoid metabolites will ultimately depend on the extent to which they associate with cells, either by interactions at the membrane or uptake into the cytosol. Information regarding uptake of flavonoids and their metabolites from the circulation into various cell types and whether they are modified further by cell interactions has become increasingly important as attention focuses on the new concept of flavonoids as potential modulators of intracellular signaling cascades vital to cellular function. The uptake of flavonoids and their in vivo metabolites is dependent on cell type (30). However, this is most probably due to a greater level of intracellular metabolism and faster rate of export from some cells rather than simply differing levels of passive diffusion into the cell. Generally, flavonoids may undergo three forms of intracellular metabolism: (i) conjugation with thiols, particularly GSH, (ii) oxidative metabolism, and (iii) P450-related metabolism. For example, the intracellular metabolism of quercetin in human dermal fibroblasts has been shown to involve the formation of intracellular oxidation products, the generation of 20 -glutathionyl quercetin (Fig. 17.2) and the demethylation of O-methylated forms of quercetin (42). In contrast, epicatechin and its O-methylated metabolites entered fibroblasts to a lesser extent and do not undergo measurable cellular metabolism (30). Furthermore, though neurons also accumulate flavonoids and O-methylated forms over time, this uptake is low compared to that in astrocytes where there is also extensive intracellular metabolism detected. Cell-generated metabolites, such as 20 -glutathionyl quercetin are of interest as they may be capable of mediating cellular effects whether beneficial or toxic in cells. Interestingly, no glucuronide metabolites of flavonoids tested thus far, associate with, or are taken up by, cells (30,42), which is most probably because of the hydrophilic nature of these metabolites. This clearly has implications for flavonoid action in vivo as glucuronide metabolites appear as the most abundant following flavonoid ingestion. It therefore appears that glucuronide metabolites

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may only be able to influence cell processes via membrane interactions and not by directly interacting with signaling proteins intracellularly. However, there is the possibility that the glucuronides of flavonoids may be cleaved in vivo under local conditions of neuroinflammation or during inflammatory processes per se and the free aglycone go on to express cellular effects. Indeed, b-glucuronidases are present in a number of tissues within the body (43) and may be released by certain cells. For example, histamine causes rapid exocytosis of b-glucuronidase from lung macrophages (44) and luteolin monoglucuronide is cleaved to free luteolin by b-glucuronidase released from neutrophils stimulated with ionomycin (45,46). MODULATION OF SIGNALING CASCADES BY FLAVONOIDS The precise mechanisms by which flavonoids exert their beneficial or toxic actions remain unclear. However, as mentioned earlier recent studies have speculated that their classical hydrogen donating antioxidant activity (47 – 49) is unlikely to be the sole explanation for cellular effects (30,31,33). This premise is based on a number of lines reasoning. First, as discussed in “Potential Bioactive Forms of Flavonoids In vivo,” flavonoids are extensively metabolized in vivo resulting in a significant alteration in their redox potentials. Indeed, circulating metabolites of flavonoids, such as glucuronides or O-methylated forms, and intracellular metabolites, for example flavonoid –GSH adducts, have a reduced ability to donate hydrogen ions (30) and are less effective scavengers of reactive oxygen and nitrogen species relative to their parent aglycone forms (Spencer et al., unpublished observations). Indeed, studies have indicated that although glucuronides, sulfates, and O-methylated metabolites may participate directly in plasma antioxidant reactions and by scavenging reactive oxygen and nitrogen species in the circulation, their effectiveness is reduced relative to their parent aglycones (50 – 54). Second, concentrations of flavonoids and their metabolite forms accumulated in vivo, for example in the plasma or in organs such as the brain (55) are lower than those recorded for well known small molecule antioxidants such as ascorbic acid and a-tocopherol (56). Consequently, flavonoids are unlikely to express beneficial action in vivo by out-competing antioxidants like vitamin C, which are present at much higher concentrations. Recent experiments have indicated that flavonoids are capable of protecting neurons against oxidative stress more effectively than ascorbate even when the latter was used at 10-fold higher concentrations (32). Third, other biological activities of flavonoids have emerged which suggest additional cellular mechanisms of action. Perhaps most prominently, these include their ability to bind to ATP-binding sites of proteins (57), such as mitochondrial ATPase (58), calcium plasma membrane ATPase (59), protein kinase A (60), protein kinase C (61 –65), and topoisomerase (66). In addition, binding to the benzodiazapine binding sites of GABA-A and adenosine receptors (67,68)

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has been reported and is of interest with regards to their potential abilities to interact within signaling pathways. They may also interact with mitochondria, directly effect enzyme activity (69), interfere with pathways of intermediary metabolism, and down-regulate the expression of adhesion molecules (70 – 72). An example of such action is illustrated by the inhibition of protein kinases by resveratrol and several flavonoids, including the citrus flavanones hesperetin and naringenin (73 – 75). This inhibition is mediated via the binding of the polyphenols to the ATP binding site, presumably causing 3D structural changes in the kinase and consequently its inactivity. Finally, recent evidence suggests that the cellular effects of flavonoids may be mediated by their interactions with specific proteins central to intracellular signaling cascades (8). In particular, investigation has indicated that they may interact selectively within the MAP kinase signaling pathway (76,77) (Fig. 17.3). The next sub-section details the pivotal position the MAP kinase signaling pathway holds in determining the fate of a cell. MAP Kinase Signaling and Cell Function The modulation of the MAP kinase signaling pathway is significant as members of the MAPK family such as the extracellular signal-related kinase (ERK) and Signal

Signal Plasma membrane

Small G-protein MAPKKKs/ MAPKK

PI3-kinase Ca(II) Pro-survival pathway

MAPK p38 JNK ERK

Cytosolic Targets

Akt/PKB

Nuclear Targets

Neuronal Death/Survival

Figure 17.3 Diagrammatic representation of the MAP kinase and Akt/PKB signaling pathways. Extracellular signal-related kinase (ERK) and c-jun amino-terminal kinase (JNK) are involved in signaling to neuronal survival, regeneration, development, and death. ERK and JNK are generally considered as having opposing actions on neuronal apoptosis, with signaling through ERK usually regarded as pro-signaling and JNK proapoptotic. The serine/threonine kinase, Akt/PKB, is one of the main downstream effectors of phosphatidylinositol 3-kinase (PI3-kinase) and a pivotal kinase in neuronal survival.

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c-jun amino-terminal kinase (JNK) are involved in signaling to neuronal survival, regeneration, development, and death (78 –82) (Fig. 17.3). ERK and JNK are generally considered as having opposing actions on neuronal apoptosis (83). ERK1/2 are usually associated with pro-survival signaling (84 – 86) through mechanisms that may involve activation of the cyclic AMP regulatory-binding protein (CREB) (85,87), the up-regulation of the anti-apoptotic protein Bcl-2 and nontranscriptional inhibition of BAD (85,86). On the other hand, JNK has been strongly linked to transcription-dependent apoptotic signaling (81,82), possibly through the activation of c-Jun (88) and other AP-1 proteins including JunB, JunD, and ATF-2 (89). Oxidative stress has a diverse effect of signaling pathways in cells, in particular the MAP kinase cascade (90 –92). Changes in the cellular redox status may result in the activation of pro-apoptotic signaling proteins such as JNK (8,89,93– 95), which may initiate the apoptotic mechanism within cells (96). Additionally, oxidative stress may affect mitochondria by influencing the mitochondrial transition pore (mPT) and/or release of cytochrome c (97,98). There is strong evidence linking the activation of JNK to neuronal loss in response to a wide array of pro-apoptotic stimuli both in developmental and degenerative death signaling (81,89). In the context of oxidative insults in neurons, JNK has been shown to be activated by dopamine (99), by 4-HNE (100 –102) and through reduced expression of SOD1 (103). Alternatively, the modulation of signaling through the serine/threonine kinase, Akt/PKB, one of the main downstream effectors of phosphatidylinositol 3-kinase (PI3-kinase) and a pivotal kinase in neuronal survival (104 – 107), may also be important (Fig. 17.3). Indeed, activation of Akt in some neurons has been shown to lead to an inhibition of proteins central to the cell death machinery, such as the pro-apoptotic Bcl-2 family member, BAD (108), and members of the caspase family (85,104) that specifically cleave poly(ADP-ribose) polymerase (105,109), thus promoting cell survival. BAD itself is regulated by phosphorylation of two serine residues, Ser112 and Ser136 (108), and several studies have revealed that the Ser136 site can be specifically phosphorylated by Akt/PKB (110,111). Interactions of Flavonoids Within Signaling Pathways There are a number of potential sites within signaling pathways where flavonoids, or their metabolites, may interact (Fig. 17.4). Flavonoid-mediated inhibition of oxidative stress-induced apoptosis may occur by preventing the activation of JNK, for example by influencing one of the many upstream MAPKKK activating proteins that transduce signals to JNK (Fig. 17.3). Alternatively, they may act by maintaining calcium homeostasis, which is important in MAP kinase activation (112,113). They may also interact directly with mitochondria, for example by modulating the mPT, which controls cytochrome c release during apoptosis (114,115), or by modulating other mitochondrial associated pro-apoptotic factors such as DIABLO/smac (116,117). Potential interactions with the mPT

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Oxidative Stress

Mitochondrial dysfunction

MAPK Signaling

Apoptosis Necrosis

Degenerative Diseases

Figure 17.4 Relationship between oxidative stress, signaling, mitochondrial function, and cell death/survival. Evidence suggests that flavonoids and/or their in vivo metabolites may protect cells against oxidative stress by influencing the intracellular redox status, by modulating mitochondrial function or by interacting with signaling pathways. Specific actions within signaling pathways are now believed to be partly responsible for both the beneficial and/or cytotoxic effects of flavonoids on cells.

are especially interesting, as the transition pore possesses a benzodiazapinebinding site where flavonoids may bind (67,68) and influence pore opening and cytochrome c release during apoptosis. Interestingly, pharmacological inhibitors of intracellular signaling cascades have similar structures to flavonoids, such as quercetin. For example, analogs of quercetin such as LY294002 have been developed as potent PI3kinase inhibitors (118 –120) and the MEK inhibitor, PD98059, is closely related to the basic flavonoid structure (Fig. 17.5). Quercetin and other flavonoids have also been shown to inhibit PI3-kinase (61,121,122) with inhibition directed at the ATP-binding site of the kinase (118,123). The quercetin analog LY294002 completely and specifically abolishes PI3-kinase activity but not PI4-kinase or other protein and lipid kinases (118). As PI3-kinase appears to be centrally involved with growth factor signal transduction, the inhibition of this kinase may be beneficial in the treatment of proliferative diseases as well as in elucidating the biological role of the kinase in cellular proliferation and growth factor response. Specific Actions of Flavanols Much interest has centered on the beneficial effects of flavanols, such as epicatechin, epicatechin gallate (EGC), and epigallocatechin gallate (EGCG) and there is growing evidence that the cytoprotective nature of these polyphenols is based on their interactions within signaling pathways. For example, epicatechin and one

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OH OH HO

O

O

O

N

OH OH

O

O

Quercetin

LY294002

OH H2N O

O

PD98059

Figure 17.5 Structural similarity between quercetin and the PI3-kinase inhibitor, LY294002, and the MEK inhibitor, PD98059. Both kinase inhibitors show structures that are closely related to the basic flavonoid backbone structure.

of its major in vivo metabolites, 30 -O-methyl epicatechin, have been shown to elicit strong cytoprotective effects against oxidative stress in fibroblasts and neurons (30 – 32). Importantly, the cytoprotective action of these compounds was found to involve both the inhibition caspase-3 activation (31,33) and activation of pro-apoptotic MAPK proteins (33). In particular, both epicatechin and 30 -O-methyl-epicatechin acted to protect neurons against oxLDL-induced activation of JNK, c-jun, and pro-caspase-3. The inhibition of JNK by flavonoids would lead to removal of c-jun/AP-1 mediated regulation of pro-apoptotic proteins, such as Bax, eventual mitochondrial dysfunction and activation of caspases. Interestingly, both epicatechin and its O-methylated metabolite had no effect on the ox-LDL-mediated increase in intracellular oxidative stress and were more protective than 10-fold higher concentrations of ascorbate. These studies strongly suggest that protection in this model is not mediated primarily by antioxidant processes. In another study, the neuroprotective mechanism of another flavanol, EGCG, against oxidative stress-induced cell death was also found to involve modulations of signaling proteins. Here, EGCG caused a stimulation of PKC and a modulation of cell survival/cell cycle genes, such as Bax, Bad, Mdm2, Bcl-2, Bcl-w, and Bcl-x(L) (17). Together, these investigations suggest that protection is likely to be partly mediated through specific actions within signaling pathways, although at this time it remains unclear exactly where such interactions occur within the pathway.

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There has been huge interest in the molecular mechanisms by which flavonoids may affect growth-related signal transduction pathways involved in the progression of cancer (124). Their actions can be divided into two main categories: (i) their ability to stimulate apoptosis of cancer cells but not normal cells (125) and (ii) their inhibition of signaling pathways involved in the progression of carcinogenesis. Many studies have tested for the ability of flavanols such as EGCG to induce apoptosis and recently the mechanisms by which they do this has begun to unravel. For example, the mechanism by which EGCG induces apoptotic cell death in human leukemia U937 cells involves the ASK1, MKK, and JNK/p38 cascade (95) and ERK activation plays an active role in mediating EGCG plus vanadate-induced apoptosis of U937 cells (126). Furthermore, the inhibition of ERK and JNK phosphorylation by EGCG has been proposed to play a role in the inhibition of arsenite-induced apoptosis (127). EGCG and other green tea polyphenols have been found to exert strong inhibition of cell growth and AP-1 activity (128) and activation of ERK and MEK1/2 (129) in cells transfected with a mutant H-ras gene to mimic carcinogenesis in vitro (128). The ras gene mutation, which occurs frequently in many cancer types, perpetually turns on the growth signal transduction pathway and is associated with high endogenous levels of AP-1 (130). Other studies have postulated that the anti-cancer properties of EGCG may be mediated by the selective inhibition of tyrosine phosphorylation of PDGF-Rb (131) or to the downregulation of NF-kappaB inducing kinase (NIK), death-associated protein kinase 1, rhoB, and tyrosine-protein kinase genes and the up-regulation of the gene for retinoic acid receptor alpha1 (132). The treatment of breast cancer cells with epigallocatechin (EGC) has provided evidence that this flavanol may stimulate apoptosis by inducing a decrease in the anti-apoptotic protein, Bcl-2, increases in the pro-apoptotic Bax, and increases in caspase 8 and caspase 10 (125). Here, it was postulated that EGC-triggered apoptosis in breast cancer cells might involve Fas death receptor signaling. In another breast cancer cell line, EGCG was found to induce the phosphorylation of JNK/SAPK and p38, which led to an inhibition of cdc2 phosphorylation, the regulated expression of cyclin A, cyclin B1, and cdk proteins finally and G2 cell cycle arrest. EGCG has also been shown to inhibit the activation of the epidermal growth factor receptor (EGFR) and related signaling pathways in head and neck squamous cell carcinoma (HNSCC) cells (133). Data suggests that EGCG may inhibit both EGFR-related pathways of signal transduction (134) and the activation of HER-2/neu and downstream signaling pathways (135,136) in human head and neck and breast carcinoma cells. These studies provide evidence that EGCG may be useful in treating cases of breast carcinoma, HNSCC, or other cancers where the activation of the signaling pathways plays an important role in tumor survival and growth. Many studies have suggested a role for EGCG and other flavanols in preventing UV-induced skin damage. For example, EGCG has been found to be effective in inhibiting UVB-induced oxidative stress-mediated phosphorylation

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of ERK1/2, JNK, and p38 in normal human epidermal keratinocytes (137) and mouse epidermal cells (138) and to markedly increase AP1 factor-associated responses via a MAPK signaling mechanism (139). More recently, a cream based formulation of green tea polyphenols, or EGCG alone, have been shown to attenuate solar UVB light-induced oxidative stress-mediated ERK1/2, JNK and p38 activation in SKH-1 hairless mouse skin (140). Interestingly, the degree of protection exerted was greater when the flavanols were applied in this topical form than when given orally, presumably because of their greater bioavailability to skin target cells. These data help explain why there are a growing number of commercial skincare products containing flavonoids or flavonoid-extracts even though at this time, neither their mechanism of action has been elucidated nor their beneficial effect has been critically evaluated. In addition to modulations in the MAP kinase pathway, EGCG has also been shown to protect against the adverse effects of UV radiation via modulations in the NF-kappaB pathway (141). This pathway is known to play a critical role in a variety of physiological functions and is involved in inflammation and the development of skin cancer. These observations suggest that EGCG, a possibly other flavanols may be useful in the attenuation of oxidative stress-mediated and MAPK-induced skin disorders such as photo-carcinogenesis in humans. The earlier studies provide evidence that flavanols such as epicatechin and EGCG may exert beneficial and/or cytotoxic actions through their modulation of the MAP kinase pathway and other signaling cascades. It has been postulated that at the activation of pro-survival MAP kinase, signaling proteins may only occur with relatively low concentrations of flavonoids (nM to low mM) and could lead to antioxidant response element (ARE)-mediated gene expression, including that of phase II detoxifying enzymes, such as b-glucuronosyltransferase. Increases in such enzymes could lead to a more rapid and efficient removal of carcinogenic agents in the body. In contrast, higher concentrations of flavonoids, may lead to a sustained activation of MAP Kinases, such as JNK, which would result in an induction of apoptosis (142). These mechanisms together with others, may contribute to the overall chemopreventive function of EGCG and other flavanols in vivo. Specific Actions of Flavonols Although the flavonol quercetin is one of the most frequently researched flavonoids, with both its beneficial (143,144) and deleterious effects (42,145 – 148) on different cell types well described, its mechanism of action remains unclear. Quercetin has been thoroughly investigated for its abilities to express antiproliferative effects (149,150) and induce cell death predominantly by an apoptotic mechanism in cancer cell lines (147,151 –153). For example, it has been observed to induce caspase-3 activation in the malignant cell line HPB-ALL (153) and activate caspase-3, caspase-9 and release cytochrome c in HL-60 cells (152) as well as induce chromatin and nuclear fragmentation in colonic

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cancer cells (150). On the other hand, quercetin treatment has been shown to suppress JNK activity and apoptosis induced by hydrogen peroxide (154) and 4-hydroxy-2-nonenal (155). Furthermore, quercetin may evoke anti-apoptotic effects via the suppression of the peroxide-induced JNK-c-Jun/AP-1 pathway and the ERK-c-Fos/AP-1 pathway in cultured mesangial cells (156). The ability of quercetin to inhibit both AP-1 activation and the JNK pathway (76) has been shown to have relevance in both phorbol 12-myristate 13-acetate (PMA)- and tumor necrosis factor-alpha (TNF-alpha)-induced ICAM-1 expression. As discussed earlier, it has been proposed that low concentrations of quercetin, may activate the MAPK pathway (ERK2, JNK1, and p38) leading to expression of survival genes (c-Fos, c-Jun) and defensive genes (Phase II detoxifying enzymes; glutathione-S-transferase, quinone reductase) resulting in survival and protective mechanisms (homeostasis response). However, increasing the concentrations of these compounds will additionally activate the caspase pathway, leading to apoptosis (77). Quercetin and its O-methylated metabolites have also been shown to be toxic to primary cortical neurons via an inhibition of pro-survival protein kinase cascades (146). Quercetin, and to a lesser extent its O-methylated metabolites, induced neuronal death via a mechanism involving direct inhibition of survival signaling through Akt/PKB and ERK rather than by an induction of the JNK-mediated death pathway. Prior to measurable losses of neuronal viability and membrane integrity, quercetin was observed to stimulate a strong inhibition of basal Akt phosphorylation in cortical neurons that was both time- and concentration-dependent. Furthermore, this inhibition of Akt phosphorylation was apparent at both the regulatory serine 473 and catalytic threonine 308 sites, rendering it inactive. The inhibition of Akt/PKB phosphorylation by quercetin may reflect potential inhibition of its upstream partner PI3-Kinase, as has been described previously (122). The potent inhibition of both Akt/PKB and ERK phosphorylation in this model was accompanied by a reduced phosphorylation of BAD and a strong activation of caspase-3 at later time points. Interestingly, high quercetin concentrations (30 mM) led to sustained loss of Akt phosphorylation and subsequent Akt cleavage by caspase-3, whereas at lower concentrations (,10 mM) the inhibition of Akt phosphorylation was transient and eventually returned to basal levels. In addition, quercetin also triggered CREB activation in neurons where potent inhibition of Akt and ERK and inactivation of BAD was present. This indicates that both pro-apoptotic and potentially anti-apoptotic pathways are activated in neurons in response to quercetin stimulus. However, as overall neuronal death resulted from exposure, it appears that quercetin-induced inhibition of the Akt/Bad survival pathway is dominant here in determining the fate of the neurons. Thus, high concentrations of quercetin may produce a sustained deactivation of Akt/PKB, which leads to extensive caspase-3 activation and subsequent caspase-dependent cleavage of anti-apoptotic Akt/PKB, an event that effectively turns-off the major survival signal and results in the

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acceleration of apoptotic neuronal death. However, at lower concentrations, reversible inhibition of Akt phosphorylation is observed and there is evidence of an attempted survival response reflected in the increase in CREB phosphorylation. These data indicate that although flavonoids are generally regarded as beneficial agents, consideration must be given to their potential toxic actions especially as in vivo concentrations are unclear. SUMMARY Cellular signaling pathways, such as the MAP kinase cascade, are pivotal in the sensing and translation of both extracellular and intracellular signals into specific cellular responses. There is now convincing evidence to suggest that flavonoids and more importantly their in vivo metabolites such as O-methylated forms may interact with such pathways and that these interactions may mediate their cellular effects. This proposal is strengthened by findings indicating that their antioxidant activity is unlikely to be an explanation for their biological actions. Indeed, the metabolism of flavonoids in the gastrointestinal tract, liver, and colon all act to reduce their antioxidant potential and results in plasma concentrations well below that of established physiological antioxidants such as ascorbate and a-tocopherol. It appears that flavonoids are capable of direct modulations of signaling proteins making up important signaling pathways. On the one hand, they may activate pro-apoptotic kinases, such as JNK, and inhibit pro-survival kinases, such as Akt/PKB. On the other hand, they may activate pro-survival kinases, such as ERK, and act to inhibit pro-apoptotic kinases activated by oxidative stress, possibly explaining their beneficial effects against oxidative stress in many cell models. However, the overall cellular effects of flavonoids are still unclear and may be ultimately dependent on the concentration in vivo. To substantiate further a role for flavonoids as beneficial and/or cytotoxic agents in vivo, advances are now needed with regards to the precise molecular targets of action of flavonoids and to which flavonoids and/or metabolites have the most profound effects. REFERENCES 1. Deguchi H, Fujii T, Nakagawa S, Koga T, Shirouzu K. Analysis of cell growth inhibitory effects of catechin through MAPK in human breast cancer cell line T47D. Int J Oncol 2002; 21:1301 – 1305. 2. Havsteen BH. The biochemistry and medical significance of the flavonoids. Pharmacol Ther 2002; 96:67 – 202. 3. Lopez-Lazaro M. Flavonoids as anticancer agents: structure – activity relationship study. Curr Med Chem Anticancer Agents 2002; 2:691 – 714. 4. Le Marchand L. Cancer preventive effects of flavonoids—a review. Biomed Pharmacother 2002; 56:296– 301.

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18 Antioxidant and Gene Regulatory Properties of Procyanidins R. Canali*, R. Ambra*, and F. Virgili National Institute for Food and Nutrition Research (INRAN), Rome, Italy

O. Gulati Horphag Research Ltd., Geneva, Switzerland

Introduction Sources and Bioavailability Antioxidant Capacity Modulation of Gene Expression Cardiovascular Related Genes Cancer Related Genes Inflammation Related Genes Application of cDNA Array Techniques Other Avenues Conclusion References

379 380 382 383 384 384 385 386 389 389 390

INTRODUCTION Polyphenols are attracting growing interest in nutrition and medicine for their anti-inflammatory, anti-viral, or anti-allergic effects and for their protective role in heart diseases, cancer, and different pathologies. Polyphenols are phenolic


Contributed equally to this study.

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compounds, naturally found in vegetables and responsible for their characteristic colors. Nowadays, polyphenols have achieved such a big interest that plants have been engineered for the production of new strains with increased levels of selected polyphenols (1). Initially, polyphenols have been considered molecules without any benefit for humans. Later, nutritional studies have shown that the polyphenol component of vegetables, accounts for most of their antioxidant and free radical-scavenging activities. More recently, their ability to affect enzyme activity and cell response and to modulate gene expression have been reported, providing a novel and different mechanistic perspective underlying their biological activity. This chapter focuses on the recent advances and hypothesis on the antioxidant activity and gene expression effects of procyanidins, a class of polyphenols that is gaining interest within the scientific community.

SOURCES AND BIOAVAILABILITY Chemically, procyanidins are oligomeric or polymeric chains of flavan-3-ols, that is, catechins and epicatechins (Fig. 18.1). These compounds are naturally present in plants as a complex mixture of polymers with an average degree of polymerization between 4 and 10. OH

OH

OH

OH O

HO

O

HO

OH

OH

Catechin

OH

Epicatechin

OH

OH

PROCYANIDIN dimer B4

OH O

HO

OH

OH

OH O

HO

n OH

n = 2 (dimer) to 10 (decamer)

OH OH

Figure 18.1

Structure of flavan-3-ols and procyanidin.

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The main nutritional sources of procyanidins are cocoa, apples, grapes, tea, wine, and strawberries. The average intake, taking into account different characteristics of dietary habits, has been estimated in the range of 30 mg/day (2). The role of procyanidins in human nutrition and disease prevention depends on their bioavailability. The extent of absorption and metabolism of polyphenols is determined largely by their chemical structure and by the rate of glycosation, acylation, conjugation, polymerization, and solubility of the compound. The kinetic of absorption of monomeric units of procyanidins, flavanol3-ols, into plasma has been extensively elucidated (3 – 5). Catechins and epicatechins, usually present in plants in an aglycone form, easily cross-biological membranes, without any deconjugation or hydrolysis (6). Once entered the enterocytes or the liver, monomer units are extensively metabolized to glucuronides, O-methylglucuronides and O-methyl conjugates metabolites. The results concerning the bioavailability of procyanidins polymers are somehow contradictory. Procyanidin polymers, because of their size, are unlikely to be absorbed at the level of the small intestine in their native form (7). However, depolimerization of procyanidin oligomers to monomer and dimer units has been demonstrated to occur at pH 2 (8), allowing the absorption of procyanidin monomer units through the small intestine. The absorption of procyanidin dimer and trimer units has been demonstrated in vitro, in studies with CaCo-2 cells (9) and isolated small intestine perfused with dimer solution (10), and in vivo, in plasma of healthy subjects after consumption of cocoa beverage (11). Hydrolysis of procyanidin polymers to monomers and dimers in the stomach represents a key event, improving procyanidin bioavailability. On the other hand, results obtained by a direct analysis of procyanidin levels in human stomach indicate that procyanidins are not hydrolized into monomers (12). Similarly, Donovan et al. reported that no hydrolysis of oligomers occurred in rat stomach. Moreover, no dimers absorption was detected, suggesting that most of procyanidins ingested reached the small intestine unchanged (7). Procyanidin polymers that are not absorbed in the small intestine are metabolized by the microflora in the large intestine into low molecular weight phenolic acids (13). Bacterial enzyme may catalyze many reactions including hydrolysis, dehydroxylation, demethylation, ring cleavage, and decarboxylation. Such metabolites of procyanidins detected in rats and human urine are mainly derivatives of phenylpropionic, phenylacetic, and benzoic acid (14 – 16). They are easily absorbed and consequently transformed by conjugation with glycine, glucuronic acid, or sulfate groups. The observation that procyanidin polymers are not degraded in the stomach suggests that their healthy effects can be associated to the metabolites generated by the microflora in the colon. Moreover, they may interact directly with intestinal mucosa or interfere with digestion. Procyanidins may contribute in the prevention of various cancers of gastrointestinal tract (17) or influence the bioavailability of metal ions forming stable complexes with them (18).

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Although the mechanisms of procyanidin metabolism and absorption still need some elucidations, most of the in vivo studies support the human health benefits related to procyanidin consumption. ANTIOXIDANT CAPACITY Epidemiological studies have shown inverse association between dietary polyphenols and cardiovascular disease. Polyphenols are associated to the decrease of some risk factors, including reduction of LDL oxidation and modulation of cytokines and eicosanoids involved in the inflammatory response. The antioxidant properties observed in chemical and biological systems associated to polyphenols can contribute to the positive health effects related to polyphenols. Procyanidins, characterized by multiple phenolic groups in their molecular structure, are potentially able to quench free radicals by forming more stable oxidized products. In vitro models showed that procyanidins exhibited scavenging properties toward superoxide anion, hydroxyl radical and lipid peroxyl radicals (19 – 21) and peroxynitrite (22), protecting LDL from oxidation (23,24). Oxidative modification of LDL is, in fact, known to play a key role in the initiation of atherogenesis (25). The antioxidant activity of procyanidins is based on their capacity to trap radicals and to chelate transition metals (21), in contrast long-chain procyanidins lack the capacity to chelate metals, acting only as radical scavengers. The antioxidant potential of procyanidin is influenced by the length of the chain. Catechin monomers resulted to be the most effective in inhibiting iron/ ascorbate-dependent lipid oxidation (26). However, dimers and trimers were associated to the highest protection against 2,20 -azobis(2-amidinopropane) hydrocloride dependent lipid oxidation (27). Moreover, long-chain procyanidins had the strongest effect against the UV-C dependent oxidation (27). Protection of LDL from in vitro oxidation increases with oligomer chain length (28). This high activity is possibly due to the fact that catechol units, the part of the structure responsible for the antioxidant capacity, are in a tight configuration, that is, close one another, therefore increasing the possibility of electron delocalization of the phenoxyl radical formed (29). Accordingly, procyanidin oligomers were more protective against peroxynitrite-dependent damage in comparison with monomers (22). If procyanidins are absorbed and biologically active in vivo, they may prevent free radicals mediated cytotoxicity and lipid peroxidation and protect LDL from oxidation. While catechins and procyanidins have a powerful antioxidant activity in vitro, it is not clear if the extent and the position of the post-absorption modifications can have any influence on their reactive oxygen and nitrogen scavenger activity. Spencer et al. reported that fibroblast protection elicited by 30 -O-methyl epicatechin, the most common epicatechin metabolite in circulation, against hydrogen peroxide damage, was not significantly different from epicatechin. They proposed that metabolites interaction with cell signaling cascades, and not a redox mechanism, is associated with the protective effects (30). Studies in vivo are fundamental to assess the real antioxidant efficacy of

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procyanidins. In vivo treatment with a grape seed procyanidin extract (GSPE, containing 75 – 80% oligomeric proanthocyanidins and 3 –5% monomeric proanthocyanidin) was associated with a decrease of reactive oxygen species production in peritoneal macrophages and hepatic mitochondrial and microsomal lipid peroxidation induced by 12-O-tetradecanoylphorbol-13 acetate (31). Diets rich in catechin and procyanidin, abundant in cocoa, were associated to an increase of plasma antioxidant capacity and a decrease of plasma TBARS (32). LDL isolated from the plasma of subjects consuming cocoa has been reported to be more resistant to ex vivo oxidation processes (28,33). Procyanidins, due to their strong-binding protein properties, may bind LDL protecting them against oxidative challenge. Recently, several studies have focused on the antioxidant activity of Pycnogenolw, a standardized phenolic extract from the bark of French Pinus maritime. Pycnogenolw main constituents are monomers (catechin, epicatechin, and taxifolin) and procyanidin, mainly oligomers of 5 –7 units. Pycnogenolw has been shown to have a fundamental role in the antioxidant network recycling the ascorbic radical and protecting vitamin E against oxidation (34). As shown for isolated procyanidin, we demonstrated that the Pycnogenolw is characterized by a strong-scavenging activity against reactive oxygen species (superoxide anion and hydroxyl radical) measured by ESR techniques and in a dosedependent fashion against nitric oxide (NO). NO was indirectly measured by nitrite generation produced by the reaction of NO (released from sodium nitroprusside decomposition) with oxygen (35). In the same study, we reported that pretreatment of a monocyte – macrophages cell line (RAW 264.7) with Pycnogenolw for 24 h before the administration of lipopolysaccharide (LPS) and interferon g (IFN-g), was associated to a dose dependent decrease of nitrite and nitrate generation (35). Although, this effect could be the result of a direct scavenger activity against NO, we observed a direct inhibitory action on the inducible form of NO-synthase (iNOS) activity and a modulation of iNOS – mRNA expression (35), giving a first indication that gene expression modulatory effects play a key role in the protective mechanism associated to Pycnogenolw. MODULATION OF GENE EXPRESSION Eventhough out of the strict matter of this chapter, one of the first indications of polyphenols ability to modulate gene expression was reported by Nakane and Ono on (2)-epigallocatechin gallate (EGCG), the main green tea polyphenol. The authors reported that EGCG inhibited the activities of cellular DNA and RNA polymerases and reverse transcriptase (36). After this first evidence by Nakane and Ono, many studies have focused on polyphenols ability to modulate gene expression and at present, there are more than 300 medline entries addressing their effects on genome functions. On this line, Hofmann and Sonenshein, utilizing dominant negative mutants, recently demonstrated that apoptosis and

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cell growth inhibition induced by EGCG, in aortic smooth muscle cells (SMC), rely on the tumor suppressor p53 and the redox related transcription factor, nuclear factor-kB (NF-kB) (37). More adherent to the matter of this chapter, procyanidins have been reported to modulate the expression of genes involved in physiological and pathological processes correlated with inflammation, cardiovascular disease, and cancer. Cardiovascular Related Genes Abou-Agag et al. observed that one hour incubation with catechin or epicatechin increased the expression of genes coding for tissue-type plasminogen activator and urokinase-type plasminogen activator in cultured human umbilical vein endothelial cells (HUVECs), suggesting that cardioprotective benefits of red wine could rely on procyanidins ability in regulating fibrinolytic activities (38). Ardevol et al. observed a reduced expression of the hormone-sensitive lipase as a response to wine-procyanidin extracts on 3T3-L1 cells, reporting that the effect was only elicited by polimers and not by monomers (39). Recently, Pal et al. proposed a feedback mechanism in which winepolyphenolics treatment in HepG2 cells lowers intracellular cholesterol levels inducing the expression of hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase and LDL receptor (40). Bagchi and coworkers studied the protective abilities of GSPE (discussed earlier), focusing on its cardioprotective roles. They demonstrated that GSPE inhibited the tumor necrosis factor a (TNF-a)-induced vascular cell adhesion molecule-1 (VCAM-1) expression in HUVEC (41), and increased vascular endothelial growth factor (VEGF) expression in HaCaT human keratinocytes following hydrogen peroxide and TNF-a exposure (42). In a recent comprehensive review of their previous papers on GSPE, Bagchi et al. reported that GSPE inhibits CD36 TNF-a inducible gene expression in HUVEC assayed by microarrays, indicating CD36 as a novel cardioregulatory protein and reinforcing their hypothesis of GSPE as a potential therapeutic tool in promoting cardiovascular health (43). Cancer Related Genes Soleas et al. examined the ability of the main phenolic substances in red wine to modulate the expression of the tumor suppressor protein p53 and found that a 24 h incubation with catechin increased p53 levels in MCF-7 breast cancer cells. Even if at borderline statistical significance, their data may explain the anticancer activities proposed for red wine polyphenols (44). Bagchi et al. observed a protective effect of GSPE against a smokeless tobacco-induced oxidative stress in human mouth keratinocytes, reporting that GSPE exhibited better protection towards apoptotic cell death in comparison with vitamins C and E (45). At molecular level, protection was associated with

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the modulation of the expression of bcl-2 and p53 genes (46). Moreover, GSPE administration protected mice from acetaminophen-induced liver injury and animal lethality. Such protection was associated with increased expression of bcl-XL in the liver (47). Protection against drug-induced cytotoxicity and modulation of apoptosis was reported by Joshi et al. who demonstrated, utilizing a different experimental model, that GSPE modulates the expression of bcl-2, c-myc, and p53 in normal human liver Chang cells (48). Down regulation of pro-apoptotic genes, that is, c-jun and jnk-1, has also been observed by Sato et al. who observed a causative link between beneficial effects of GSPE administration in rats towards ischemia/reperfusion and the ability of GSPE in preventing the expression of anti-death signals such as jnk-1 and c-jun genes (49). Inflammation Related Genes Bito et al. reported that epicatechin was able to modulate the IFN-g-induced intercellular adhesion molecule-1 (ICAM-1) expression in HaCaT cells, although its effectiveness was lower than other polyphenols (50). These results suggest a possible role of procyanidins in modulating cell adhesion processes during inflammation. Cocoa procyanidins have been reported by Mao et al. to affect the transcription of several genes coding for cytokines in mitogen-stimulated human peripheral blood mononuclear cells but not in resting cells (51). Analyzing several procyanidin fractions, they found that the most effective was the larger ones (up to 10 units), inducing increased interleukin-1 (IL-1) b expression and reduced IL-4 and IL-2 transcriptions (51). Different reports addressed the molecular mechanism underlying the anti-inflammatory properties of Pycnogenolw (discussed earlier). As already mentioned, we observed a modulation of iNOS activity and expression by Pycnogenolw in activated RAW 264.7 macrophages (35). We observed that 24 h preincubation of murine macrophages with Pycnogenolw was associated with a significant reduction of NO generation, inhibition of iNOS activity, and iNOS – mRNA expression in response to IFN-g and LPS. Using a dual-luciferase reporter gene assay that reveals the NF-kB-dependent gene expression induced by IFN-g in RAW 264.7, Park et al. demonstrated that 1 h incubation with procyanidins was associated with different effects toward gene expression, depending on the degree of polymerization: Pycnogenolw and trimeric procyanidin enhanced the NF-kB-dependent gene expression, while monomers and dimers repressed it (52). Bito et al. reported that 12 h pretreatment with Pycnogenolw significantly inhibited, in a dose-dependent manner, IFN-g-induced expression of ICAM-1 in the human immortalized keratinocytes cell line HaCaT. This observation pairs with the reported modulatory effects of Pycnogenolw on the adhesion of Jurkat T cells to activated keratinocytes in a co-culture assay (53). Using gel

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mobility shift assays (EMSA), Bito et al. identified Stat1 and IRF-1 as two factors affected by Pycnogenolw, critical for IFN-g-dependent gene activation and signaling pathway. Cho et al. confirmed that Pycnogenolw pretreatment prevented the activation of NF-kB and also of the activator protein-1 (AP-1) in activated RAW 264.7 (54). They also found that Pycnogenolw pretreatment abolished LPS-induced IkB (an inhibitory protein that associates with NF-kB) degradation and down-regulated IL-1b gene expression in RAW 264.7 and IL-2 gene expression in human T-cell line Jurkat E6.1 (55). Pycnogenolw ability to inhibit NF-kB activation and VCAM-1 and ICAM-1 expression was reported by Peng et al. also in TNF-a-treated HUVEC (56). Saliou et al. reported that Pycnogenolw treatment in HaCaT inhibited, in a concentration-dependent manner, UVR-induced NF-kB-dependent gene expression suggesting a role of the extract in protecting human skin against erythema (57). Application of cDNA Array Techniques Complementary DNA (CDNA) arrays are global expression analysis tools that have been recently applied also to nutrition science and are providing insights onto how nutrients act at molecular level, regulating gene functions, and signal-transduction pathways. Application of arrays to nutrigenomics will accelerate discoveries on this field and, if possible, allow the treatment or the prevention of diet-related diseases. cDNA arrays have been applied to the study of the molecular effects of nutrients also in organisms ranging from bacteria (58), plants (59), and Drosophila (60) and have been helpful tools in the identification of pathways influenced by the intake of fatty acids (61), dietary protein (62), short chain fatty acids (63), zinc (64), and heme (65) in mammalian cells. Briefly, cDNA arrays consist in high-density nylon, plastic, or glass supports bearing immobilized DNA sequences of thousands genes. Supports are hybridized with labeled cDNA sequences obtained by reverse transcription of mRNA prepared from cells or tissues and resulting hybridization signals are compared to controls. Rihn et al. reported that 24 h supplementation of Pycnogenolw is associated to a significant modulation of 39 out of the 83 genes detected in a commercially available human cDNA array bearing a total of 588 genes in the human cell keratinocyte line HaCaT (66). Among modulated genes, are to be mentioned two genes coding calgranulins A and B, members of the highly conserved S100 family of low weight calcium-binding proteins, which are known to be accumulated in psoriasis and various dermatitis. Targeting to a better understanding of the anti-inflammatory and a putative anti-cancer properties of Pycnogenolw, we recently applied a genomic analysis method to the human tumor monocytic U937 cell line. U937 cells are model for monocyte to macrophage differentiation studies and are currently used in many laboratories in inflammatory and tumorigenicity studies. U937 cells

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derive from a pleural effusion of a human caucasian histiocytic lymphoma and although they exhibit a lymphoblastoid morphology, they own many of the monocytic-like characteristics exhibited by cells of histiocytic origin. Preliminary, unpublished results of Pycnogenolw effects on U937 cells indicate a significant down regulation of genes encoding for oncogenic hyper-proliferative proteins and an up regulation of genes coding check-point proteins. Moreover, we found that some of the genes affected by Pycnogenolw were modulated in a similar fashion after treatment with purified catechin, demonstrating the contribution of this component to the known biological effects of Pycnogenolw (67). Among others, we found a down regulation of hCDC10 and an up regulation of KOX15 genes in both Pycnogenolw and catechin-treated U937 cells. The KOX15 (or zinc finger protein 22) cDNA was isolated by Wu et al. who reported high expression of the gene in T-leukemia lines but not in nonlymphoid cells, indicating a role in differentiation within lymphoid cells (68). On the other hand, CDC10 is a protein belonging to the septins family which are wellconserved GTPases found in animals and fungi. CDC10 is a structural component of the 10-nm filaments which are assembled in cytoplasmic membrane and are essential for budding of yeasts. By differential display, Nagata et al. found that the expression levels of hCDC10, the human homologue gene, were related to the biological and clinical characteristics of neuroblastoma, that is, high expression levels in tumors are associated with the good prognosis in patients under 1 year of age (69). More recently, using an expression profile analysis, Nishiu et al. identified high expression of hCDC10 in advanced stage malignant lymphomas (70). Gene expression profiling of cancer cell lines challenged by procyanidins is providing clues on the molecular pathways that are related to the anti-cancer properties of Pycnogenolw. It is important to stress that these observations, even though of interest, must be considered preliminary and confirmation is necessary before reaching any final conclusion about the effective role of pine bark procyanidins on gene expression in human cancer cells. We already reported that cardioprotective properties of procyanidins were at least in part associated to their ability to modulate gene expression in HUVEC (38). The study of the proliferative responses of endothelial cells is crucial for the identification of molecular pathways involved in the response to specific diet or nutrient. To this regard, Sartippour recently demonstrated that procyanidins inhibit in vitro HUVEC proliferation and in vivo vessel formation (71,72). They observed that the effects of procyanidins on proliferation are associated to decreased levels of secreted VEGF and that the regulation occurs at the transcriptional level. Oxidized LDL (ox-LDL) induces a proliferative and atherogenic response in endothelial cells including the chemotaxis of inflammatory cells (monocytes/ macrophages) into the subendothelial space. The nonsaturable accumulation of ox-LDL within macrophages, eventually leads to foam cell formation, cytokine expression and fatty streak (73). NF-kB and AP-1 are well-known inflammationrelated transcription factors and have been recently reported to be involved in endothelial dysfunction. As shown in Fig. 18.2, here we report that a 4 h

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Figure 18.2 Effect of Pycnogenolw preincubation on ox-LDL dependent NF-kB activation. HUVEC were treated 4 h with ox-LDL (20 mg protein/mL) and NF-kB activation was assayed by EMSA. Pycnogenolw (5 mg/mL) was preincubated for 12 h and washed out before ox-LDL treatment.

treatment of HUVEC with ox-LDL (20 mg protein/mL) induces a strong activation of NF-kB, assayed by EMSA. If HUVEC are preincubated with Pycnogenolw (5 mg/mL) for 12 h before ox-LDL treatment, a sensible reduction of the NF-kB activation occurs (Fig. 18.2), according to its anti-inflammatory effects (67). To characterize the atherogenic response at the molecular level, we recently applied a global analysis method to cultured HUVEC, obtaining a comprehensive database of candidate genes possibly involved in atherogenesis induced by ox-LDL (74). In the same series of experiments, we studied the effects of purified catechin preincubation on ox-LDL-treated HUVEC for 16 h observing that the monomer is able to prevent or even reverse about one fifth of the changes in gene expression induced by ox-LDL. These data must be considered preliminary, and they need further confirmation of the observations obtained by cDNA array. However, these results can be of interest and provide suggestions for more investigation. We therefore present here some data regarding the genes which displayed the most significant regulation with respect to untreated cells and a brief description of the known function, leaving to the reader the option to get any possible conclusion. Half of the genes modulated by catechin treatment encode transcription factors, whereas others are mainly related to adhesion molecules and matrix metalloproteinases expression. For example, we observed a twofold increase in mRNA levels of the GATA-2 transcription factor in

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ox-LDL-treated HUVEC (74). This expression was switched to a 50% down regulation in catechin-pretreated cells, compared with control untreated cells. GATA-2 has a known function in the control of the expression of adhesion proteins. Accordingly with this function, the messengers of some genes encoding for these class of proteins (e.g., leukocyte adhesion glycoprotein p150, vinculin, and cadherin 16) were altered in catechin preincubated cells, as compared to ox-LDL treated HUVEC. Moreover, catechin preincubation strongly inhibited the previously observed (74) up-regulation of FOS-related antigen (FRA)-2, one of the four members of the FOS gene family, leucine zipper proteins that dimerize with proteins of the JUN family, thereby forming the transcription factor complex AP-1 and playing an important role as regulators of cell proliferation, apoptosis, and differentiation. Other Avenues In addition to their cardioprotective, anti-inflammatory, anti-cancer activities, recent data suggest that polyphenols have anti-HIV effects. A grape seed extract was able to interact with the expression of genes coding for several co-receptors necessary for the internalization of HIV in mononuclear leukocytes, possibly interfering with virus replication (75). Moreover, Ratna and Simonelli demonstrated that intra-muscular injection of catechin in livers of roosters increased the transcript of the estrogen-regulated mRNA stabilizing factor (E-RmRNASF), which is necessary for the estrogendependent stabilization apolipoprotein mRNA, indicating a possible therapeutic use of procyanidins as estrogen-mimicking agents (76). CONCLUSION As shown, results on procyanidin bioavailability are somehow contradictory and need further investigations. Moreover, it is not still clear if the postabsorption modifications can affect their biological effects. However, in vivo studies support the human health benefits of procyanidin consumption and in vitro studies strongly suggest that health improvement depend on the ability of these molecules to activate signaling pathways and modulate gene expression. Figure 18.3 summarizes procyanidin fate in the digestive tract and gene effects reported in this chapter, providing a “nutrigenomic picture” of components of this important group of polyphenols. The effect of diet on and nutrients on gene expression can be studied at different levels of complexity. The first and simplest activity is at the level of single gene expression. In this case, the attention of the investigator is focused to single genes in order to establish whether a gene is differentially modulated by a specific experimental treatment vs. the control. The major proportion of the research addressing the biological activity of procyanidin is actually within this level of complexity. Most of these studies, similar to other diet-genome-related studies, lack corroboration by inverse genetic approaches that unequivocally demonstrate a causative role of a gene/protein as a response to in a dietary manipulation.

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Diet procyanidin fate and summary of effects on gene expression (see text).

On the other hand, the use of gene arrays is enabling the analysis of genomics applied to nutrition at a higher levels. Firstly, at the level of multiple genes, where these are grouped on clusters on the basis of arbitrary choices, usually addressing a common putative or known feature, such as gene co-regulation, similar protein function, or known interactions. For example, genes sharing expression patterns as a response to a treatment with a specific nutrient are possibly co-regulated and participate in functionally related processes. Finally, when all genes are taken in account “in chorus” (considering also unaffected genes), it is possible to reach the ambitious level of “system biology” where all gene fluctuations induced by the treatment with a specific nutrient are taken into account to infer a network picture of the tangled relationships existing between all coded proteins and their genes. Another possibility is simple identification of a “dietgene signature”—that is, a global gene response of a specific cell type or tissue to one nutrient—without considering the gene functions and not even considering their names. Several applications of this signatures can be hypothesized to be utilized for the certification of food or in order to compare different matrixes. The concept of “normal nutrient signatures” in humans could also be used as a tool for the identification and prevention of diet-related diseases. The application of array technology to nutrition will help the comprehension of nutrient effects, as well as the molecular mechanisms underlying them, on human heath and how nutrition influences the normal homeostasis or may restore a pathological condition. REFERENCES 1. Gantet P, Memelink J. Transcription factors: tools to engineer the production of pharmacologically active plant metabolites. Trends Pharmacol Sci 2002; 23:563– 569.

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2. Bravo L. Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance. Nutrition Rev 1998; 56:317 – 333. 3. Richelle M, Tavazzi I, Enslen M, Offord EA. Plasma kinetics in man of epicatechin from black chocolate. Eur J Clin Nutr 1999; 53:22 – 26. 4. Bell JR, Donovan JL, Wong R, Waterhouse AL, German JB, Walzem RL, Kasim-Karakas SE. (þ)-Catechin in human plasma after ingestion of a single serving of reconstituted red wine. Am J Clin Nutr 2000; 71:103– 108. 5. Baba S, Osakabe N, Yasuda A, Natsume M, Takizawa T, Nakamura T, Terao J. Bioavailability of (2)-epicatechin upon intake of chocolate and cocoa in human volunteers. Free Radic Res 2000; 33:635 – 641. 6. Scalbert A, Williamson G. Dietary intake and bioavailability of polyphenols. J Nutr 2000; 130:2073S– 2085S. 7. Donovan JL, Manach C, Rios L, Morand C, Scalbert A, Remesy C. Procyanidins are not bioavailable in rats fed a single meal containing a grape seed extract or the procyanidin dimer B3. Br J Nutr 2002; 87:299 –306. 8. Spencer JP, Chaudry F, Pannala AS, Srai SK, Debnam ES, Rice-Evans C. Decomposition of cocoa procyanidins in the gastric milieu. Biochem Biophys Res Commun 2000; 272:236 – 241. 9. Deprez S, Mila I, Huneau JF, Tome D, Scalbert A. Transport of proanthocyanidin dimer, trimer, and polymer across monolayers of human intestinal epithelial Caco-2 cells. Antiox Redox Signal 2001; 3:957 – 967. 10. Spencer JP, Schroeter H, Shenoy B, Srai SK, Debnam ES, Rice-Evans C. Epicatechin is the primary bioavailable form of the procyanidin dimers B2 and B5 after transfer across the small intestine. Biochem Biophys Res Commun 2001; 285:588– 593. 11. Holt RR, Lazarus SA, Sullards MC, Zhu QY, Schramm DD, Hammerstone JF, Fraga CG, Schmitz HH, Keen CL. Procyanidin dimer B2 [epicatechin-(4beta-8)epicatechin] in human plasma after the consumption of a flavanol-rich cocoa. Am J Clin Nutr 2002; 76:798 – 804. 12. Rios LY, Bennett RN, Lazarus SA, Remesy C, Scalbert A, Williamson G. Cocoa procyanidins are stable during gastric transit in humans. Am J Clin Nutr 2002; 76:1106– 1110. 13. Deprez S, Brezillon C, Rabot S, Philippe C, Mila I, Lapierre C, Scalbert A. Polymeric proanthocyanidins are catabolized by human colonic microflora into low-molecularweight phenolic acids. J Nutr 2000; 130:2733 – 2738. 14. Gonthier MP, Cheynier V, Donovan JL, Manach C, Morand C, Mila I, Lapierre C, Remesy C, Scalbert A. Microbial aromatic acid metabolites formed in the gut account for a major fraction of the polyphenols excreted in urine of rats fed red wine polyphenols. J Nutr 2003; 133:461 – 467. 15. Li C, Lee MJ, Sheng S, Meng X, Prabhu S, Winnik B, Huang B, Chung JY, Yan S, Ho CT, Yang CS. Structural identification of two metabolites of catechins and their kinetics in human urine and blood after tea ingestion. Chem Res Toxicol 2000; 13:177– 184. 16. Rios LY, Gonthier MP, Remesy C, Mila I, Lapierre C, Lazarus SA, Williamson G, Scalbert A. Chocolate intake increases urinary excretion of polyphenol-derived phenolic acids in healthy human subjects. Am J Clin Nutr 2003; 77:912– 918. 17. Carnesecchi S, Schneider Y, Lazarus SA, Coehlo D, Gosse F, Raul F. Flavanols and procyanidins of cocoa and chocolate inhibit growth and polyamine biosynthesis of human colonic cancer cells. Cancer Lett 2002; 175:147– 155.

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19 Cell Signaling Properties of Inositol Hexaphosphate (IP6) Abulkalam M. Shamsuddin The University of Maryland School of Medicine, Baltimore, Maryland, USA

Introduction Inositol Compounds in Cellular Signaling IP3 and Its Receptor IP6 and Its Receptor IP6 as a Signal Molecule In Mammalian Cells Signal Transduction by IP3 and IP6 Along the Evolutionary Tree Interactions of IP6 with Other Proteins and Macromolecules Alterations in Levels of IP5 and IP6 Vesicle Trafficking: Exocytosis and Endocytosis Cell Proliferation and Cell Cycle Normalization of Cell Proliferation DNA Synthesis The Retinoblastoma Protein Protein Kinase C Ras Proteins Induction of Cell Differentiation Programmed Cell Death (Apoptosis) PI 3-Kinase Nuclear Inositol Signaling mRNA Transport 397

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INTRODUCTION Inositol hexaphosphate (IP6, InsP6) is a polyphosphorylated carbohydrate, contained in high concentrations (0.4 –6.4%) in cereals and legumes. IP6 known more commonly as “phytic acid” is the major form of phosphorous in the seeds, wherein it is found as deposits of mixed “phytate” salts of K, Mg, Ca, Mn, and Zn (1 – 3). In the plant kingdom, it is also found in other plant tissues and organs such as pollen, roots, tubers, and turions (4). IP6 accumulates during seed development and is broken down into lower inositol phosphates during germination. The cytosol of almost all mammalian cells contain IP6 and its lower phosphorylated forms (IP1 – 5) as well as the dephosphorylated form, the parent compound—inositol. myo-Inositol is a cyclic alcohol (cyclitol) derivative of glucose. The enzyme myo-inositol(3)P1 synthase (MIPS) converts glucose-6-phosphate to inositol(3)phosphate1 [Ins(3)P1]. In general, IP6 is converted from inositol through the various polyphosphate intermediates via different pathways in different organisms; these pathways are being more similar than different. In addition to being a part of the phospholipids and eventually converted to IP6 and its pyrophosphates, in the plant cells inositol is also utilized in cell wall polysaccharides and other cyclitols. To the surprise of many in the field of nutritional biochemistry, it has been a relatively recent finding that IP6 is not only present, but also is the most abundant of intracellular inositol phosphates in eukaryotes (5). In multicellular organisms, it is imperative that cells communicate with each other and these communications are dependent on external signal molecules, which evoke a “chain reaction” mediated by a host of molecules within the cell. Though there are different families of molecules (such as lipids, glycolipids, carbohydrates, including inositol phosphates) that may serve the latter role in conveying the signals; much of our present day knowledge is on intracellular signaling proteins. At the end of the various signaling pathways are the target proteins; depending on the signals, these proteins carryout specific functions such as metabolism, gene regulation, and structural alterations.

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Signal molecule
! binding with cell surface receptor protein
! intracellular signaling molecules
! target molecules ðand=or
! nuclear signalingÞ
! effects: altered metabolism=gene expression= structure, and so on: As can be seen from the preceding, the first step is for the extracellular signal molecule to bind to cell surface receptors. Interestingly, it appears that there are binding (receptor) proteins for inositol phosphates including IP6, and that they act as signal molecule themselves.

INOSITOL COMPOUNDS IN CELLULAR SIGNALING IP3 and Its Receptor Intracellular signaling proteins behave as switches that turn on or off depending on the signal, the commonest of these proteins are turned activated or inactivated by the addition (by protein kinase) or deletion (by protein phosphatase) of phosphate groups. The other main class of molecular switches is the guanosine 50 -triphosphate (GTP)-binding proteins. Of the two types of GTP-binding proteins, the large trimeric GTP-binding proteins are commonly called G proteins. The G proteins are attached to the cytoplasmic face of the plasma membrane where they serve as relay molecules. A variety of extracellular signals such as vasopressin, acetylcholine, and thrombin activate the inositol –phospholipid signaling pathway by way of G proteins, specifically, Gq via activation of phospholipase C-b (PLC-b), which is also on the plasma membrane. In the inner half of the plasma membrane lipid bilayer is the inositol –phospholipid phosphatidylinositol 4,5-biphosphate (PIP2). The activated PLC-b cleaves PIP2 to generate diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). IP3 leaves the plasma membrane, diffuses rapidly through the cytosol to bind to its receptor protein in the endoplasmic reticulum causing release of Ca2þ stored within the endoplasmic reticulum. This may result in increase of local Ca2þ concentration by 10 – 20-fold and trigger Ca2þ responsive proteins in the cell (6). Inositol 1,3,4,5-tetrakisphosphate (IP4) is also considered to be a signaling molecule, albeit to a lesser extent than its more famous cousin IP3; it has been implicated in the movement of Ca2þ into the cell and/or in maintaining levels of IP3-sensitive Ca2þ pools (6). Interestingly, the level of IP3 itself can be affected during glucose metabolism: an increase in IP3 secondary to an influx of extracellular Ca2þ following membrane depolarization and activation of PLC. PIP2 can also be converted to phosphatidylinositol 3,4,5-triphosphate by PI-3 kinases that also play important roles in regulation of protein trafficking, cell growth and cell survival, cytoskeletal organization, and so on. PIP2 itself

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may also have roles in binding to, and regulation of vinculin, a-actinin, and gelsolin; binding of PIP2 to specific sites on them influences their interactive properties with actin. IP6 and Its Receptor Although much of the research in inositol phosphate-signal transduction centers around IP3 and to a much lesser extent IP4, the role of IP6 as a signal molecule received very little attention till recently. In the late 1980s and early 1990s, IP6 was found to stimulate 45Ca2þ uptake in cultured cerebellar neurons (7) and anterior pituitary cells (8). Coupled with the fact that Law et al. (9) demonstrated that low concentration of IP6 increases both intracellular free-Ca2þ and prolactin secretion in perifused pituitary cells, the stage is set for IP6 to be a signal molecule by its own right. These and other investigators subsequently isolated IP6-binding sites, the IP6 receptor (10 –13). [3H] IP6 bound to specific and saturable recognition sites in membranes prepared from cerebral hemispheres, anterior pituitaries, and cultured cerebellar neurons. Interestingly, the specific binding sites were present in high density in all subcellular fractions including the mitochondria. The presence of IP6-binding sites (or receptor) in the mitochondrial fraction further enhanced the belief that IP6 may act also as an intracellular regulator of Ca2þ homeostasis just like IP3. It appears that the binding affinity for IP6 receptor (or binding sites) reported by different groups is different. For instance, Theibert et al. (12) reported that the IP6 receptor comprises a protein complex of 115, 105, and 50 kDa subunits. On the other hand, the concept that the IP6-binding site in the membrane may not necessarily be protein but nonprotein entities such as phospholipid has been advanced by Poyner et al. (14). IP6 AS A SIGNAL MOLECULE In Mammalian Cells Be that as it may, what does it mean to have IP6-binding sites (receptor) on cell membranes in the brain? The discovery of high-affinity membrane binding sites lends support to the assumption that IP6 may serve as a signal molecule. In the central nervous system, IP6 acts as a potent neural stimulator. Experiments by microinfusion of IP6 into the nucleus tractus solitarius or by iontophoretically applied to the dorsal horn of the spinal cord have demonstrated that IP6 mimics the action of excitatory neurotransmitter glutamate receptor (15,16). However, central nervous system and the anterior pituitary gland are not the only place to have IP6-binding sites (receptors). Since IP6 and IP4 are produced in response to stimulation of cardiac a1-adrenoreceptors, Rowley et al. (17) investigated the IP6-binding sites (receptor) in the rat heart. [3H]-IP6 bound to a single, high affinity site in sections of rat heart (KD ranging from 22 + 1.9 nM in right atria to 35 + 2.6 nM in the interventricular septum).

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The maximal number of binding sites (Bmax) ranged from 5.1 + 0.48 to 12 + 1.8 pmol mg21 protein in left atrium and left ventricle, respectively. Presence of IP6-binding sites in mitochondrial and sarcoplasmic reticulum fractions of heart certainly points to the role(s) of IP6 and IP6-binding sites in the heart muscle. Reports of such IP6-binding sites or receptors in other cells have been steadily emerging. O’Rourke et al. (18) report of IP6-binding sites in a low-density membrane fraction from human platelets, and Kitchen et al. (19) report of specific, albeit low affinity, IP6-binding site in human neutrophil (polymorphonuclear leukocytes) membrane preparations. Interestingly, IP6 plays an important role in the neutrophils. After priming by a number of different agents, human neutrophils may be stimulated to produce a greater respiratory burst than would be elicited by the stimulus alone and some other neutrophil functions may be similarly enhanced by pre-exposure to a priming agent. Eggleton et al. (20) reported that pre-incubation of neutrophils with IP6 alone had no stimulatory effect upon the basal production of reactive oxygen intermediates but the response to a subsequent stimulus by phagocytic particles or N-formyl-Met-Leu-Phe (fMLP) is substantially enhanced (20). An increase in IL-8 secretion by stimulated cells also occurred in the presence of IP6 (21). Thus, the local release of IP6 could have a physiologically important modulatory role on neutrophil functions and confer more effective combat to invading microorganisms in our first line of defense. Signal Transduction by IP3 and IP6 Along the Evolutionary Tree The role of inositol triphosphate as a signaling molecule has been conserved evolutionarily, from the plant through the slime mold up to the mammalian cells. The role of IP3 as a signaling molecule in plants was demonstrated by Samanta et al. (22) when IP6-phytase was added after a definite time of hydrolysis (20 – 30 min) coinciding with the optimal production of Ins(2,4,5)P3 bound to phytase, mobilization of Ca2þ from microsomal/vacuolar fractions was detected. These investigators further demonstrated that Ins(1,4,5)P3 or Ins(2,4,5)P3 – phytase complex constituted in vitro is also effective in releasing Ca2þ. Thus, the IP3 – phytase complex is recognized by putative receptor (or binding site) associated with microsomal fraction resulting in Ca2þ mobilization in plants (23). The preceding has shown the conservation of the signaling role of IP3 through the different organisms. However, the signaling role of IP6 is also conserved. In plants, IP6 plays a role in the processes by which the drought stress hormone abscisic acid induces stomatal closure, conserving water and ensuring plant survival. IP6 levels in Solanum tuberosum stomatal guard cells are elevated in response to abscisic acid, and IP6 inactivates the plasma membrane inward Kþ conductance in a cytosolic calcium-dependent manner. Lemtiri-Chlieh et al. report that guard cell protoplasts release of IP6 from a caged precursor mobilizes calcium. IP6-induced increase in cytoplasmic calcium does not result from

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calcium influx but from IP6-triggered release of calcium from endomembrane stores. These data define IP6 as an endomembrane-acting calcium-release signal in guard cells (24). In an attempt to understand the intricacies of IP6-induced differentiation and inhibition of cell proliferation of cancer cells, pilot studies in our laboratories using HT-29 human colon cancer cell line demonstrated a similar very transient increase in intracellular Ca2þ following IP6 treatment. This increase in Ca2þ level by 3 –4-fold was within 10 s of exposure to IP6. Along with this increased intracellular Ca2þ there was a concomitant downregulation of cyclin E (25). Interactions of IP6 with Other Proteins and Macromolecules Not only have IP6 binding proteins or receptors on cell surface have been identified, but also a variety of proteins within the cytosol may bind IP6 (26). But before we discuss those, let us first see what happens to IP6 levels during the normal physiological activities of various cells. Alterations in Levels of IP5 and IP6 Fluctuations, particularly a substantial increase in the level of IP6 in various cells during different functional or developmental stages have led to investigations into its roles. The levels of IP5 and subsequently IP6 progressively increases in Xenopus laevis oocyte over a 72-h-period (27) during which IP6 remained unmetabolized. During progression of oocyte maturation between stages 3 and 6 of oogenesis, IP6 level rose by 6-fold. Thus, IP6 is intimately associated with, if not directly regulating oogenesis and oocyte maturation. Changes in the cellular mass and concentration of IP5 and IP6 were observed during a complete cell cycle of proliferating rat thymocytes (28). After a very early transient rise in both IP5 and IP6 at the beginning of the cell cycle, a decrease of the intracellular concentration of both compounds occurred. During cell division (between 48 and 72 h) a pronounced increase in IP5 and IP6 was observed indicating a role for these compounds during cell cycle progression. We are beginning to understand how some of these functions may be brought about. The modulation of various cell cycle regulators by IP6 will be discussed later in this chapter. Vesicle Trafficking: Exocytosis and Endocytosis The process by which eukaryotic cells package secretory molecules in plasma membrane and extrude these is exocytosis. On the other hand, cells ingest macromolecules (in specialized cells even other cells) by invagination of the plasma membrane—endocytosis. Exocytosis: A transient and consistent elevation in IP6 concentration in pancreatic insulin secreting b cells has been observed following glucose stimulation and the temporal changes in IP6 concentration correlate well with the initial rise in intracellular Ca2þ (29). This rise in IP6 level is above an extremely

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stable baseline physiological level of IP6 in the unstimulated cells (40 –54 mM); a 10 mM rise in concentration is sufficient to inhibit the serine/threonine protein phosphatase activity and increase the current through voltage-gated L-type Ca2þ channel activity. IP6 has also been demonstrated to stimulate exocytosis in pancreatic b cells and IP6 could recruit insulin secretory granules to the site of exocytosis (30). Interestingly, the increased concentration of IP6 following glucose-stimulation, to bring about this function is most pronounced at the site of exocytosis and the vicinity of Ca2þ channels. Since L-type Ca2þ channels have a key role in various tissues such as the myocardium, smooth muscles, and the brain, this effect of IP6 clearly has a great physiological significance as well as crucial in the pathogenesis of diseases in those. For instance, prolonged opening of L-type Ca2þ channels mediate apoptosis of the b cells; thus, a sustained increase in IP6 concentration could stimulate apoptosis. Indeed, it has been known that a high level of IP6 or prolonged exposure can induce apoptosis in various cancer cell lines (31,32). In the brain, the pyramidal cells of the hippocampus associated with memory possess L-type Ca2þ channels. Longterm disturbance in IP6 metabolism could result in the death of these cells and therefore be instrumental in the pathogenesis of Alzheimer’s disease. It is becoming clear from these experiments that the overall scheme of how the functions of IP6 are concentration dependent: there is a normal physiological level, then the level changes (usually a slight increase), and there are additional functions. Not only the levels in the cells fluctuate, the levels of IP6 in the plasma and other body fluids also vary. The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca2þ sensor for rapid exocytosis. The two fragments of the large cytoplasmic domain of synaptotagmin I are C2A and C2AB. IP6 binds to and induces a conformational change in C2AB in the presence of lysosome. The IP6 binding notably weakens the Ca2þ-dependent C2AB –membrane interaction suggesting that IP6 may act as a modulator of neurotransmitter by altering the state of synaptotagmin – phospholipid interaction (33). Endocytosis: The process of endocytosis involves PIP2, clathrin, and clathrin adapters, the guanosine triphosphatase dynamin I, synaptojanin 1, and the amphiphysin dimer. Dynamin, a high-affinity substrate for calcineurin is a force-generating molecule responsible for membrane fission during endocytosis. IP6 promotes dynamin I-mediated endocytosis in the pancreatic beta cell. This effect of IP6 is dependent on calcineurin-induced dephosphorylation and activation of protein kinase C and inhibition of the phosphoinositide phosphatase synaptojanin (34). Growth factors and receptors: Binding of ligands to the epidermal growth factor receptors (EGFR or erbB) results in rapid disappearance of the receptors from the cell surface. The process of activation of the receptor includes receptor dimerization, activation of intrinsic receptor tyrosine kinase activity, autophosphorylation of the receptor at the c-terminus and tyrosine phosphorylation

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of intracellular signaling molecules such as Shc and PI3K (phosphatidylinositol 3-kinase). Ligand-induced endocytosis and degradation of the erbB receptor cause down-regulation of the receptor. The ligands accelerate the endocytosis of the receptors by promoting clustering of the receptors into clathrin-coated pits on the plasma membrane. This is followed by receptor internalization into clathrin-coated vesicles (35). An important structural component of coated pits is the clathrin lattice anchored to cytoplasmic surface of the membrane by “plasma membrane clathrin-associated protein complex 2” (AP2). Initial binding of erbB receptors with AP2 is necessary for receptor-mediated endocytosis. From a series of very elegant studies done at Professor Agarwal’s laboratory, Zi et al. (36) report that IP6 impairs both receptor-mediated and fluid-phase endocytosis resulting in inhibition of mitogenic signals associated with growth and proliferation of human prostate cancer cell line DU145. IP6 interacts with AP2 and inhibits PI3K. IP6 also inhibits PI-30 -K-AKT signaling pathway as an upstream response in its effects on the inhibition of fluid-phase endocytosis. IP6 completely inhibits transforming growth factor-a [TGFa]-induced binding of activated erbB1 receptor to AP2. IP6 treatment of DU145 human prostate cancer cells also results in a dosedependent increase in levels of activated erbB1 receptor. These are associated with strong inhibition of ligand-induced Shc phosphorylation (36). Interestingly, Voglmaier et al. (37) report that a partial amino acid sequence from the purified IP6 receptor protein and a partial nucleotide sequence from a cDNA clone of the gene are essentially identical to those of the a-subunit of the clathrin assembly protein AP2. The IP6 receptor protein contains a series of subunits, which are the same as those of AP2. In addition, antibodies to AP2 react with the IP6 receptor protein (37). Fibroblast growth factors. Fibroblast growth factors (FGF), which have been implicated in tumor cell growth and angiogenesis, have biological activities that appear to be mediated by both heparin-like extracellular matrix sites and transmembrane tyrosine kinase receptor sites. IP6 inhibits basic FGF (bFGF) binding to heparin. IP6 not only binds to bFGF, but also protects bFGF from degradation by trypsin. In addition, IP6 inhibits the cellular binding of bFGF and other fibroblast growth factor family members such as acidic FGF (aFGF) and K-FGF in a saturable and dose-dependent manner. Concentrations as low as 100 mM IP6 inhibits bFGF-induced DNA synthesis in AKR-2B fibroblasts, as well as the growth of bFGF- and K-FGF-transfected NIH/3T3 cells (38). Insulin-like growth factor. Insulin-like growth factor II/mannose6-phosphate (IGF-II/Man-6-P) receptors participate in the trafficking of lysosomal enzymes. The transduction of the effects of IGF-II is also via transmembrane-anchored receptor protein. As in erbB described earlier, during ligand-induced endocytosis, this receptor interacts with AP2, which can lead to their assembly and subsequent transport in coated vesicles to the lysosomes. Kar et al. (39) discovered that IP6-binding sites in rat brain are similarly distributed to IGF-II sites and that IP6 competes for IGF-II binding sites in the rat brain. Our studies using HT-29 human colon cancer cells showed that the

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precipitous increase in intracellular Ca2þ following IP6 treatment can be blocked by prior pretreatment of cells with IGF-II, indicating a competition with the IGF-II receptor (25). Syndecan-4. Syndecan-4 is a transmembrane heparan sulfate proteoglycan that can regulate cell – matrix interactions and is enriched in focal adhesions (sites where cells form junctions with the extracellular matrix). Its cytoplasmic domain contains a central region with a cationic motif that binds inositol phospholipids. Highest affinity of the syndecan-4 cytoplasmic domain was seen PIP2 and phosphatidylinositol 4-phosphate, and both promoted syndecan-4 oligomerization. IP6 binds with high affinity to the syndecan-4 cytoplasmic domain and is considered a potential down-regulator of syndecan-4 signaling. Cell adhesion experiments showed that IP6 could block syndecan-4-dependent focal adhesion and microfilament bundle formation in fibroblasts (40). Since metastasis of cancer cells to distant sites depend initially on the ability of cancer cells to attach to the extracellular matrix, these results explain at least in part, the molecular basis of decreased cell adhesion to extracellular matrix by IP6 as seen human mammary cancer cell lines (41,42).

CELL PROLIFERATION AND CELL CYCLE Normalization of Cell Proliferation Uncontrolled proliferation is a hallmark of malignant cells, and IP6 reduces the abnormally elevated and uncontrolled rate of cell proliferation of all the different cell lines tested so far (43 –46). Since it does not affect the rate of proliferation of normal cells either in vivo or in vitro, this function can best be called “normalization” of cell proliferation. Although normal cells divide at a controlled and limited rate, malignant cells escape from the control mechanisms that regulate the frequency of cell multiplication and usually have lost the checkpoint controls that prevent replication of defective cells. IP6 can regulate the cell cycle to block uncontrolled cell division and force malignant cells either to differentiate or go into apoptosis. At the present time, aside from normalization of abnormal and uncontrolled cell proliferation, the other fundamental processes are affected by IP6 induction of cellular differentiation. It appears that IP6 also induces apoptosis, albeit in large dosage. It is most likely that execution of these divergent and multiple signaling pathways rather than a single signal and/or pathway brings about these complex processes. Some of the cellular responses to extracellular signals may be dose-dependent and abrupt, whereas other manifestations may not follow any such rule. And the effect of extracellular signal may persist long after the signal has disappeared. Data obtained from in vitro experiments show that the cells’ response to IP6 insofar as cell proliferation and differentiation is concerned follow this last pattern.

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DNA Synthesis Studies in my laboratory showed a suppression of DNA synthesis as measured by H-thymidine incorporation and down-regulation of proliferation marker proliferating cell nuclear antigen (PCNA) by IP6 (47). A marked decrease in the expression of proliferation markers indicated that IP6 disengaged cells from actively cycling. Using dual parameter flow cytometry and combined analysis of the expression of cell cycle-related proteins, we also demonstrated that IP6 controls the progression of the cells through the cell cycle (48). IP6 treatment significantly decreased the S-phase and arrested the human colon and breast cancer cells in the G0 –G1 phase. Interestingly, the intracellular levels of IP6 are high in G1 and G2 – M phases of cell cycle, but drop by 50– 75% during the S-phase (28,49). Studies of human leukemia cells at Professor Lambertenghi-Deliliers’ laboratory at the University of Milan demonstrate that IP6 shows a dose-dependent cytotoxic effect on human leukemia cell lines. The IP6-treated leukemia cells accumulate in G2M phase of cell cycle (as opposed to G0 –G1 phase in breast cancer cells (48)); once again arrest of cells in the cycle, albeit in a different phase (50). cDNA microarray analysis showed an extensive down-modulation of genes involved in transcription and cell cycle regulation (c-myc, HPTPCAAX1, FUSE, cyclin H) and an up-regulation of cell cycle inhibitors such as CKS2, p57, and Id-2. Genes such as STAT-6 and MAPKAP, involved in important signal transduction pathways were also downregulated (50). 3

The Retinoblastoma Protein One of the cell cycle controls disrupted in cancer cells resulting in unrestrained entry into the cell cycle (hence uncontrolled cell proliferation) is the progression of cells through G1 and entry to the S-phase. This progression requires hyperphosphorylation of retinoblastoma protein (pRb). The pRb was originally discovered through studies of an inherited form of eye cancer in children—retinoblastoma. The loss of both copies of the Rb gene results in excessive cell proliferation of the immature retina. Thus, pRb was considered to be important in restraining the cells from uncontrolled cell proliferation. A marked reduction in pRb phosphorylation by IP6 has been reported in human mammary cancer cell line (51). The pRb-related proteins pRb/p107 and pRb2/p130 show considerable homology with pRb and are considered to be a subfamily of pRb family of proteins. They cooperate to regulate cell cycle progression through G1 phase. These proteins bind to and modulate the E2F family of transcription factors that induce the transcription of genes needed for progression to S-phase. pRb2/p130 is the most abundant E2F complex found in quiescent cells in G0 phase. Singh et al. (32) reported that IP6 increases the level of hypophosphorylated Rb-related proteins pRb/107 and pRb2/p130 in DU145 human prostate

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cancer cell line. IP6 moderately decreased E2F4, but increased its binding to both pRb/p107 and pRb2/p130. Protein Kinase C Protein kinase C (PKC) signaling has an important role in diverse cellular processes such as cell proliferation, differentiation, cell death, gene expression, and tumor promotion. Thus, it is no surprise that at least some investigators have directed their attention to the modulation of this family of serine/threonine kinases by IP6. IP6 stimulates insulin secretion and primes Ca2þ-induced exocytosis in pancreatic b cells through activation of PKC (52). Intracellular application of IP6 produces a dose-dependent stimulation of exocytosis, which is dependent on PKC activity. Antisense oligonucleotides directed against specific PKC isoforms reveals the involvement of PKC-1 in IP6-induced exocytosis. Furthermore, expression of dominant negative PKC-1 abolishes IP6-evoked exocytosis, whereas expression of wild-type PKC-1 leads to a significant stimulation of IP6-induced exocytosis (53). Nickel and Belury (54) investigated the effect of IP6 on 12-O-tetradecanoylphorbol -13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity in HEL-30 cells, a murine keratinocyte cell line, and SENCAR mouse skin. TPAinduced ODC activity is an essential event in tumor promotion in mouse skin model. ODC activity was significantly reduced by IP6. When mouse skin was treated in vivo with IP6, ODC induction was also significantly inhibited. The expression of TPA-induced c-mRNA was significantly inhibited by the same IP6 treatments in HEL-30 cells and CD-1 mouse skin. No changes in PKC isoforms a and z expression and phorbol dibutyrate binding due to IP6 treatment were found in HEL-30 cells. These results indicate that IP6 reduces TPAinduced ODC activity independent of PKCa and z expression (54). While Vucenik et al. (51) similarly report that treatment of human breast cancer cells with IP6 resulted in no changes in the expression of PKCa, b, or j isomers. However, there was a 3.1-fold increase in the expression of PKC. Along with the increased activity, there was translocation of the enzyme from the cytosol to the membrane (Vucenik et al., manuscript in preparation). Thus, not only different isomers may be involved in different pathways, but IP6 may also differentially modulate them. Ras Proteins One of the important family of protein molecules that helps broadcast the signal from the cell surface to other parts of the cell is Ras family of monomeric GTPases. There are two subfamilies: that involved in relaying the signal from the cell-surface receptor to the actin cytoskeleton (Rho family) and that involved in regulating the traffic of intracellular transport vesicle (Rab family). As in other GTP-binding proteins, Ras is activated when bound to GTP as opposed to GDP, which renders it inactive. Following activation of Ras, it stimulates various

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signaling proteins downstream along different pathways, one of the most important being serine/threonine phosphorylation cascade. A critical component of this cascade is mitogen-activated protein kinase (MAPK), which when activated enters the nucleus. MAP kinases are usually activated transiently, which peaks at 5 min followed by rapid decline and resulting in (as the name suggests) cell division through activation of genes encoding G1 cyclins. Interestingly, full activation of MAPK requires phosphorylation of both a tyrosine and a threonine residue, done by MAP-kinase – kinase also called MEK. MEK itself needs to be activated via phosphorylation by Map-kinase –kinase – kinase also known as Raf in mammalian system. Since IP6 inhibits cell division, its effect on the cell cycle and these cell cycle regulatory proteins and their genes have been looked at. Using DU145 human prostate cancer cell line, Singh et al. (32) studied the cell cycle progression and apoptosis by flow cytometry. They also investigated the involvement of G1 cell cycle regulators and their interplay, and end point markers of apoptosis. A significant dose- and time-dependent growth inhibition of IP6treated cells was associated with an increase in cells in G1. IP6 strongly increased the expression of cyclin-dependent kinase inhibitors (CDKIs)—Cip1/p21 and Kip1/p27, without any noticeable changes in G1 CDKs and cyclins, except a slight increase in cyclin D2. IP6 inhibited kinase activities associated with CDK2, 4, and 6, and cyclin E and D1. Further studies showed the increased binding of Kip1/p27 and Cip1/p21 with cyclin D1 and E. In down-stream of CDKI–CDK/cyclin cascade, IP6 increased hypophosphorylated levels of Rbrelated proteins, pRb/p107 and pRb2/p130, and moderately decreased E2F4 but increased its binding to both pRb/p107 and pRb2/p130. At higher doses and longer treatment times, IP6 caused a marked increase in apoptosis, which was accompanied by increased levels of cleaved PARP and active caspase 3. IP6 modulated CDKI – CDK – cyclin complex, and decreases CDK –cyclin kinase activity, possibly leading to hypophosphorylation of Rb-related proteins and an increased sequestration of E2F4. Higher doses of IP6 could induce apoptosis and that might involve caspases activation. Induction of Cell Differentiation In general, cell differentiation depends on changes in gene expression resulting in synthesis and accumulation of different sets of RNA and consequently protein molecules; the latter often represent the differentiated features. Examples of such markers of differentiation include hemoglobin for mature red blood cells, prostate specific acid phosphatase for prostatic epithelial cells, lactalbumin for mammary cells, and myoglobin for muscle cells. There are many steps in the pathway leading from DNA to protein; in principle, all of them can be regulated, affecting gene expressions. These steps include: transcriptional control—when and how often a given gene is transcribed; RNA processing control—regulating how the RNA transcript is spliced; RNA transport and

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localization control—selecting the mRNAs to be exported from the nucleus to the cytoplasm and where in the cytoplasm they are to be localized; translational control—which mRNAs in the cytoplasm are translated; mRNA degradation control—selectively inactivating certain mRNA molecules in the cytoplasm; or protein activity control. For most genes, transcriptional control is the critical one. These alterations in gene expressions can be (and often are) responses to external cues or stimuli; the signal(s) switching the regulatory regions of DNA near the site where transcription begins or by activating the gene regulatory proteins that turn genes on or off. IP6 has been demonstrated to induce differentiation of malignant cells of divergent origins to the normal phenotype. It was first demonstrated in K-562 human erythroleukemia cells, which showed increased hemoglobin production following IP6 treatment (55). Similar induction of tissue specific differentiation was reported for human colon carcinoma HT-29 cells (47,56), prostate cancer cells (57), breast cancer cells (58), and rhabdomyosarcoma cells (59). The molecular mechanisms involving these IP6-induced differentiation will be fascinating to study. It is, however, known that PI 3-K (phosphoinositide 3-kinase) plays an important role in granulocytic differentiation of HL-60 leukemia cells (60). Interestingly, the intracellular concentration of IP6 (and IP5) is elevated by about two orders of magnitude during chemotactic stimulation of HL-60 cells (61). Clearly, an elevated level of IP6 plays a yet to be determined role in these differentiated functions. Programmed Cell Death (Apoptosis) At concentrations up to 2 – 5 mM, IP6 inhibits cell proliferation of cancer cell lines with concomitant induced differentiation, but without a substantial increase in cell death in most cell lines. However, at higher dosage, or on prolonged treatment it induces apoptosis or programmed cell death (32). HeLa cells on the other hand appear to be more sensitive, undergoing apoptosis at IP6 concentrations where very little apoptosis was observed in other cell lines (31). Be that as it may, treatment of HeLa cells with tumor necrosis factor or insulin stimulates the Akt-nuclear factor kB (NFkB) pathway. This is a cell survival signal involving the phosphorylation of Akt and IkB, nuclear translocation of NFkB, and NFkB-luciferase transcription activity. IP6 blocks all these cellular events and also causes mitochondrial permeabilization, followed by cytochrome c release setting in motion the activation of the apoptotic machinery: caspase 9, caspase 3, and poly(ADP-ribose) polymerase (PARP). PI 3-Kinase As one can gather from the preceding discussion, the enzyme PI 3-K has been involved in a variety of cellular processes, including those affected by IP6; indeed it is a crucial molecule in cellular signal transduction. PI 3-K causes

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phosphorylation of the D-3 position of the inositol ring of phosphoinositides to produce phosphatidylinositol-3-phosphate. In mouse epidermal cell line JB6, IP6 markedly blocks epidermal growth factor-induced PI 3-kinase activity in a dose-dependent manner. This blocking of PI 3-K activity by IP6 profoundly impairs epidermal growth factor- or phorbol ester-induced JB6 cell transformation and extracellular signal-regulated protein kinases activation, as well as the transcription factor activator protein 1 (AP-1) activation (62).

NUCLEAR INOSITOL SIGNALING mRNA Transport Contrary to the fact that the genetic machinery of the cell lies in the nucleus, detailed investigations of its structure has been relatively scanty. So it is no surprise that the nuclear inositol signaling was virtually ignored, except for a notable few (63). The nuclear skeleton, also called nuclear matrix or scaffold is a complex structure of fibrogranular network and is contiguous with the intermediate filaments of cytoskeleton. And then there are intranuclear channels, which derive from both the layers of the nuclear envelope terminate at the nucleoli or pass through the cytoplasm. Because these intranuclear channels are continuous with the cytoplasm they have been speculated to be involved in nucleocytoplasmic transport and that they would make the contact between the nucleoli and the nuclear pores possible. There are no reasons to believe that external stimuli working on the cell membrane would not influence the nucleus; insulin-like growth factor (IGF), when bound to its receptor in the plasma membrane rapidly activates nuclear PLC-b1. It was Cocco et al. (64) who first demonstrated that the envelope-deprived nuclei could synthesize both phosphatidyl 4-phosphate and phosphatidyl 2-phosphate (PIP2). Thanks to this pioneering work, it has been known (at least to a few investigators!) for some time now that almost all, if not all of the enzymes and inositol derivatives that have been identified in the cytoplasm have also been found in the nucleus (63). So what do they do there? It should not come as a surprise if they are involved in regulation of gene expression. The transcription of DNA to messenger RNA (mRNA) is carried out within the nucleus, being segregated from the cytoplasm by the nuclear envelope; the mRNA is then transported by a complex series of events through pores into the cytoplasm. York et al. (65) demonstrated that the enzyme phospholipase C and two proteins that influence the generation of IP6 are required for proper and efficient export of mRNA from the nucleus to the cell. They identified three genes in the yeast that are involved in the inositol signaling pathway: PLC1 encoding phospholipase C (which converts PIP2 to IP3 and DAG), IPK1 encoding the inositol polyphosphate kinase Ipk1p (which converts IP5 to IP6), and IPK2 encoding Ipk2p (which converts IP3 to IP4 and IP5). A mutation in

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any of these genes blocks export of mRNA from the nucleus to the cytoplasm. IP6 being the end product of this metabolic pathway is therefore the most likely effector molecule controlling the mRNA export. The IPK2 gene is identical to the yeast gene ARG8, which encodes Arg82p, a pleiotropic kinase that regulates processes as diverse as response to stress, sporulation, and mating. Incidentally, in yeast, stress increases IP6 level (66) causing increased export of certain mRNAs that when translated into proteins would counteract the stressful stimuli. Arg82 has a predicted molecular mass similar to the yeast IP3 – IP4 kinase activity. Along with Arg80, Arg81, and Mcm1, Arg82p is also an essential component of ArgR – Mcm1 transcription complex essential for proper transcriptional control that activates or represses genes involved in arginine metabolism. Odom et al. (67) now show that this arginine production and breakdown is dependent on the kinase activity of Ipk2p and the generated IP4 and IP5. Chromatin Remodeling The packaging of DNA into chromatin in eukaryotic cells limits its access to DNA-binding proteins. Chromatin remodeling is therefore essential for efficient transcription of eukaryotic genes, which use ATP-dependent chromatin remodeling complexes to regulate gene expression. The SWI2/SNF2 family of ATPdependent chromatin remodeling complexes is used to regulate DNA accessibility for transcription. Four related classes of protein complexes use the energy of ATP hydrolysis to alter nucleosome architecture. Shen et al. (68) and Steger et al. (69) report that mutations in genes encoding inositol polyphosphate kinases that produce IP4, IP5, and IP6 impair transcription in vivo, providing link between inositol polyphosphates, chromatin remodeling, and gene expression. NF-kB (Nuclear Transcription Factor-kB) NF-kB proteins are gene regulatory proteins that also play an important role in intercellular signaling during normal vertebrate development. There are five NF-kB proteins in mammals (RelA, RelB, c-Rel, NF-kB1, and NF-kB2). They form a variety of homodimers and heterodimers each of which activates different sets of genes, the most abundant dimer is a p50/p65 heterodimer that binds to DNA. In unstimulated cells, inhibitory proteins called IkB bind tightly to the dimers and hold NF-kB in an inactive state within large protein complexes in the cytoplasm preventing the latter (NF-kB) from localizing to the nucleus and binding to the DNA. Inflammatory cytokines such as tumor necrosis factor a (TNF-a) and interleukin-1 (IL-1) trigger a signaling pathway resulting in phosphorylation, ubiquitylation, and finally degradation of IkB by 26S proteasome complex. This degradation of IkB exposes a nuclear localization signal on the NF-kB proteins allowing them now to move into the nucleus and stimulate the transcription of specific genes such as those involved in cell cycle regulation, cell adhesion, and programmed cell death (apoptosis).

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Since NF-kB is constitutively active in advanced and androgen-independent human prostate cancer cell line DU145 cells, Agarwal et al. (70) assessed whether IP6 inhibits this constitutively active NF-kB and associated upstream effectors. They also investigated whether such an affect of IP6 has biological relevance in causing inhibition of proliferation and induction of apoptosis in DU145 cells. Treatment of cells with 1 and 2 mM IP6 resulted in a strong inhibition of NF-kB activation. This finding was consistent with a decrease in nuclear levels of NF-kB subunit proteins p65 and p50. IP6-treated cells also showed a strong inhibition in phospho-IkBa protein levels concomitant with a significant increase in total IkBa levels. This effect of IP6 also correlated with a significant inhibition in IkBa kinase (IKKa) activity; and all of these correlating with a strong inhibition in cell proliferation and induction of apoptosis in IP6-treated cells (70). In a similar manner, in mouse epidermal JB6 cells, IP6 strongly blocked UVB-AP-1 and NF-kB transcriptional activities in a dose-dependent fashion. IP6 also suppressed UVB-induced AP-1 and NFkB –DNA binding activities and inhibited UVB-induced phosphorylation of extracellular signal-regulated protein kinases (Erks) and c-Jun NH2-terminal kinases (JNKs). IP6 also blocked UVB-induced phosphorylation of IkB-alpha, which is known to result in the inhibition of NF-kB transcriptional activity. Contrary to the epidermal growth factor-induced PI 3-K activity (62), IP6 does not block UVB-induced PI 3-K activity, suggesting that the inhibition of UVB-induced AP-1 and NkF-B activities by IP6 is not mediated through PI-3 kinase (71). Zinc-Finger Motif A group of DNA-binding motifs consisting of an a-helix and a b-sheet held together by one or more zinc atoms serves as an important regulator of transcription. These are often found as a cluster with additional zinc fingers arranged one after the other. This arrangement allows the a-helix of each zinc finger to come in contact with the major groove of DNA for a considerable length along the groove. Though there has not been any experimental demonstration of removal of the zinc atoms from the zinc-finger motifs, it has nonetheless been speculated by some that IP6 could in theory bind the zinc atoms and in turn affect the ability of zinc-finger motifs (now without the zinc) to bind to the DNA. It has also been speculated that IP6 by removing zinc could inhibit thymidine kinase, an enzyme essential for DNA synthesis (72). DNA Repair There are other ways by which IP6 could influence the various activities within the cells. For instance, repair of double-strand breaks in DNA is essential for maintaining the stability of the genome; failure to repair may result in loss of genetic information, chromosomal translocation, and even cell death. Two mechanisms for this repair has been described—homologous recombination or non-homologous endjoining, IP6 has been demonstrated to stimulate nonhomologous end-joining; it has been proposed to be brought about by the binding of IP6 to the DNA-dependent

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protein kinase DNA–PKcs (73). A more recent study reports that it is not DNA– PKcs (a large protein of 3500 amino acids, Mw 465 kDa), but the DNA end binding protein Ku (consists of Ku70—70 kDa, and Ku86—83 kDa) that binds to IP6 (74). Be that as it may, these studies, in spite of their differences in their specific findings clearly show a very important role of IP6 in DNA repair mechanism. Once the assault on the cell has gone past the scope of DNA repair, the otherwise heretofore normal cell is likely to transform to a malignant (cancer) cell. Insofar as the transformation of cells from normal to malignancy is concerned, there are various models and pathways, one of these pathways is the activation of transcription factors activating protein-1 (AP-1) and nuclear factor NF-kB via phosphatidylinositol 3-kinase (PI-3 kinase). Using tumor promoter-induced cell transformation of human skin JB6 cells, Huang et al. (62) have demonstrated that IP6 blocks epidermal growth factor-induced PI-3 kinase and AP-1 activity. Zi et al. (36) reported similar results on DU145 human prostate cancer cells along with concomitant inhibition of cell growth. Upstream of these pathways lies the MAPK, which are serine/threonine kinases that are rapidly activated upon extracellular stimulation. This family of kinases include extracellular signal-regulated kinases (Erks), c-Jun N-terminal kinases (JNKs), and p38 kinases. IP6 inhibited the activities of Erks and JNKs, but not of the p38 kinases in human skin, prostate, and breast cancer cells (36,62,75,76). Thus, given the commonality shared by these three divergent cell types, the blocking of this cellular to nuclear signaling pathway appears as an important mechanism of anticancer action of IP6. ROLE OF IP6 IN ENERGY TRANSDUCTION Back in 1963, Morton and Raison (77) first suggested a link between IP6 and ATP regeneration and its importance in seed development and germination. As usual, there was considerable skepticism to such “outlandish” ideas. It was not till 1978 that the hypothesis appeared more plausible: an IP6 –ADP phosphotransferase described by Biswas et al. (78) could use IP6 as a phosphate donor for the conversion of ADP to ATP by transferring a phosphate group from 2-position of IP6 to ADP in developing mung bean (Phaseolus aureus) seeds (78). Additional support came from Phillippy et al. (79) who isolated a inositol 1,3,4,5,6-pentakisphosphate 2-kinase, which is involved in both formation of IP6 in maturing seeds and catalyze the conversion of ATP from ADP in germinating seeds, in this case the soybean. It should not be an “outlandish” idea to think that in mammalian cells too, IP6 may be crucial in providing the phosphates so necessary in the conversion of ADP ! ATP. ANTICANCER ACTION OF IP6: FROM THE LABORATORY TO THE CLINIC Since the first reports from my laboratory in the early 1987 (80 –82), we and subsequently others have established the role IP6 as a broad-spectrum anticancer

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agent, effective against cancers of different experimental models (43 – 45). myo-Inositol itself was also demonstrated to have anticancer function, albeit modest; however, the combination of inositol þ IP6 produced the best results, being significantly better in different cancers than either one alone (43 – 46). This was based on my working hypothesis that the anti-proliferative action of IP6 is via the lower inositol phosphates and that adding inositol to IP6 would increase the mass of these molecules through the activities of the kinases and phosphatases as: Inositol þ IP6
! 2IP3 : In addition, since the lower inositol phosphates (IP3 in particular) are the signal transduction molecules (as you have gathered from the preceding discussions) common to most forms of life, the anticancer effect would be seen in a wide variety of tissues and organs. But that is where the ideal evolution of hypothesis-driven scientific exercise ends as I only had a hypothesis and the burden of proof was upon me. You will note from the bibliography that all the various pathways of signal transduction have been described since my demonstration of anticancer action in the later part of 1980s. In essence, my colleagues and I were fortunate to have observed some fascinating phenomena in the form of striking anticancer action of IP6 and we are now beginning to understand some of the molecular mechanisms. Science would like to have it the other way round: mechanisms first, demonstrate the effect (if any) later. Perhaps (in part) owing to the lack of this knowledge as to the mechanism of the function of IP6, there has not been the expected enthusiasm to translate these rather amazing laboratory findings to the clinic. Belated though it may be, following availability of IP6 þ inositol, since 1998, pilot clinical trials have been started, which indeed confirm the broad-spectrum anticancer action in the human (83,84). An enhanced anticancer activity without compromising the patient’s quality of life was demonstrated in a pilot clinical trial of patients with advanced colorectal cancer (Dukes C and D) with multiple liver and lung metastasis (84). IP6 þ inositol was given as an adjuvant to chemotherapy according to Mayo protocol. One patient with liver metastasis who refused chemotherapy was treated only with IP6 þ inositol; her control ultrasound and abdominal computed tomography scan after 14 months showed a significantly reduced growth rate. A reduced tumor growth rate was noticed overall and in some cases a regression of lesions was noted. Additionally, when IP6 þ inositol was given in combination with chemotherapy, side effects of chemotherapy (drop in leukocyte and platelet counts, nausea, vomiting, alopecia) were diminished and patients were able to perform their daily activities (84). Notwithstanding the politics of medical economics, it is hoped that reports such as this will bring the benefit of IP6 þ inositol to the doors of cancer patients and to the population at large for cancer prevention.

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OTHER BIOLOGICAL EFFECTS OF IP6 IP6 is not only virtually safe and has nontoxic effects, but it also has many other beneficial health effects such as inhibition of kidney stone formation and reduction in risk of developing cardiovascular disease. IP6 was administered orally either as the pure sodium salt or in a diet in order to reduce hypercalciuria and to prevent formation of kidney stones, and no evidence of toxicity was reported (85). A potential hypocholesterolemic effect of IP6 may be very significant in the clinical management of hyperlipidemia and diabetes (86). IP6 inhibits agonist-induced platelet aggregation (87) and efficiently protects myocardium from ischemic damage and reperfusion injury (88), both of which are important for the prevention and management of cardiovascular diseases.

CONCLUSION I have attempted to give a somewhat comprehensive up-to-date review of the role of IP6 and other inositol phosphates in cellular signal transduction. Beyond reasonable doubt, IP6 plays a crucial role in cellular signaling. Likewise, its anticancer actions and other health benefits are also unquestionable. In addition, this is a compound that is present in physiological concentrations in various cells, tissues, and in our body fluid, the level of which changes with intake and deficiency (89,90). Hopefully, some day, it may join the list of compounds as a vitamin!

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9. Law GJ, Pachther JA, Dannies P. Inositolhexakisphosphate increases cytosolic free Ca2þ and prolactin secretion from perifused pituitary cells. J Gen Physiolo 1988; 92:P8A– P9A. 10. Copani A, Bruno V, Cavallaro S, Fiore L, Sortino MA, Canonico PL, Nicoletti F. Receptors for inositolhexakisphosphate in neurons and anterior pituitary cells. Pharmacol Rese 1990; 22(suppl 1):83 –84. 11. Nicoletti F, Bruno V, Cavallaro S, Caponi A, Sortino MA, Canonico PL. Specific binding sites for inositolhexakisphosphate in brain and anterior pituitary. Mol Pharmacol 1990; 37:689 – 693. 12. Theibert AB, Estevez VA, Ferris CD, Danoff SK, Barrow RK, Prestwich AD, Snyder SH. Inositol 1,3,4,5-tetrakisphosphate and inositol hexakisphosphate receptor proteins: isolation and characterization from rat brain. Proc Natl Acad Sci USA 1991; 88:3165– 3169. 13. Huisaman B, Lochner A. Inositol polyphosphates and their binding proteins—a short review. Mol Cell Biochem 1996; 157:229– 232. 14. Poyner DR, Cooke F, Hanley MR, Reynolds JM, Hawkins PT. Characterization of metal ion-induced [3H]inositol hexakisphosphate binding to rat cerebellar membranes. J Biol Chem 1993; 268:1032 – 1038. 15. Vallejo M, Jackson TR, Lightman S, Hanley MR. Occurrence and extracellular actions of inositol pentakis- and hexakisphosphate in mammalian brain. Nature 1987; 330:656 – 658. 16. Hanley MR, Jackson TR, Vallejo M, Patterson SI, Thastrup O, Lightman S, Rogers J, Henderson G, Pini A. Neural function: metabolism and actions of inositol metabolites in mammalian brain. Philos Trans R Soc Lond Biol Sci 1988; 320:381– 398. 17. Rowley KG, Gundlach AL, Cincotta M, Louis WJ. Inositol hexakisphosphate binding sites in rat heart and brain. Br J Pharmacol 1996; 118:1615 – 1620. 18. O’Rourke F, Matthews E, Feinstein MB. Isolation of InsP4 and InsP6 binding proteins from human platelets: InsP4 promotes Ca2þ efflux from inside-out plasma membrane vesicles containing 104 kDa GAP1IP4protein BP. Biochem J 1996; 315:1027 –1034. 19. Kitchen E, Condliffe AM, Rossi AG, Haslett C, Chilvers ER. Characterization of inositol hexakisphosphate (InsP6)-mediated priming in human neutrophils: lack of extracellular [3H]-InsP6 receptors. Br J Pharmacol 1996; 117:979 –985. 20. Eggleton P, Penhallow J, Crawford N. Priming action of inositol hexakisphosphate (InsP6) on the stimulated respiratory burst in human neutrophils. Biochim Biophys Acta 1991; 1094:309 – 316. 21. Eggleton P. Effect of IP6 on human neutrophil cytokine production and cell morphology. Anticancer Res 1999; 19:3711– 3715. 22. Samanta S, Dalal B, Biswas S, Biswas BB. Myoinositol tris-phosphate-phytase complex as an elicitor in calcium mobilization in plants. Biochem Biophys Res Commun 1993; 191:427 – 434. 23. Dasgupta S, Dasgupta D, Sen M, Biswas S, Biswas BB. Interaction of myoinositoltrisphosphate-phytase complex with the receptor for intercellular Ca2þ mobilization in plants. Biochemistry 1996; 35:4994 – 5001. 24. Lemtiri-Chlieh F, MacRobbie EA, Webb AA, Manison NF, Brownlee C, Skepper JN, Chen J, Prestwich GD, Brearley CA. Inositol hexakisphosphate mobilizes an endomembrane store of calcium in guard cells. Proc Natl Acad Sci USA 2003; 100: 10091– 10095.

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25. Cole KE, Smith M, Xu J-F, Vucenik I, Shamsuddin AM. Modulation of the intracellular calcium signal in human colon cancer cells by the novel antineoplastic agent inositol hexaphosphate. Anticancer Res 1997; 17:4070 – 4071. 26. Fukuda M, Mikoshiba K. The function of inositol high phosphate binding proteins. Bioessays 1997; 19:593– 603. 27. Ji H, Sandberg K, Baukal AJ, Catt KJ. Metabolism of inositol pentakisphosphate to inositol hexakisphosphate in Xenopus laevis oocytes. J Biol Chem 1989; 264: 20185– 20188. 28. Guse AH, Greiner E, Emmrich F, Brand K. Mass changes of inositol 1,3,4,5,6pentakisphosphate and inositol hexakisphosphate during cell cycle progression in rat thymocytes. J Biol Chem 1993; 268:7129 – 7133. ˚ , Carlqvist H, Mitchell RH, Bertorello A, Nilsson T, 29. Larsson O, Barker CJ, Sjo¨holm A Honkanen RE, Mayr GW, Zwiller J, Berggren P-O. Inhibition of phosphatases and increased Ca2þ channel activity by inositol hexakisphosphate. Science 1997; 278:471– 474. 30. Efanov AM, Zaitsev SV, Berggren P-O. Inositol hexakisphosphate stimulates nonCa2þ -mediated and primes Ca2þ-mediated exocytosis of insulin by activation of protein kinase C. Proc Natl Acad Sci USA 1997; 94:4435 – 4439. 31. Ferry S, Matsuda M, Yoshida H, Hirata M. Inositol hexakisphosphate blocks tumor cell growth by activating apoptotic machinery as well as by inhibiting the Akt/NFkB-mediated cell survival pathway. Carcinogenesis 2002; 23:2031 – 2041. 32. Singh RP, Agarwal C, Agarwal R. Inositol hexaphosphate inhibits growth, and induces G1 arrest and apoptotic death of prostate carcinoma DU145: modulation of CDKI – CDK– cyclin and pRb-related protein– E2F complexes. Carcinogenesis 2003; 24:555– 563. 33. Lu YJ, He Y, Sui SF. Inositol hexakisphosphate (InsP6) can weaken the Ca(2þ)dependent membrane binding of C2AB domain of synaptotagmin I. FEBS Lett 2002; 527:22– 26. 34. Hoy M, Efanov AM, Bertorello AM, Zaitsev SV, Olsen HL, Bokvist K, Leibiger B, Leibiger IB, J Zwiller J, Berggren P-O, Gromada J. Inositol hexakisphosphate promotes dynamin I-mediated endocytosis. Proc Natl Acad Sci USA 2002; 99:6773– 6777. 35. Lamaze C, Baba T, Redelmeier TE, Schmid SL. Recruitment of epidermal growth factor and transferrin receptors into coated pits in vitro: differing biochemical requirements. Mol Biol Cell 1993; 4:715 – 727. 36. Zi X, Singh RP, Agarwal R. Impairment of erbB1 receptor and fluid phase endocytosis and associated mitogenic signaling by inositol hexaphosphate in human prostate carcinoma DU145 cells. Carcinogenesis 2000; 21:2225 –2235. 37. Voglmaier SM, Keen JH, Murphy JE, Ferris CD, Prestwich GD, Snyder SH, Theibert AB. Inositol hexakisphosphate receptor identified as the clathrin assembly protein AP-2. Biochem Biophys Res Commun 1992; 187:158– 163. 38. Morrison RS, Shi E, Kan M, Yamaguchi F, McKeehan W, Rudnicka-Nawrot M, Palczewski K. Inositolhexakisphosphate (InsP6): an antagonist of fibroblast growth factor receptor binding and activity. In Vitro Cell Dev Biol Anim 1994; 30A:783– 789. 39. Kar S, Quirion R, Parent A. An interaction between inositol hexakisphosphate (IP6) and insulin-like growth factor II receptor binding sites in the rat brain. Neuroreport 1994; 5:625– 628.

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40. Couchman JR, Vogt S, Lim ST, Lim Y, Oh ES, Prestwich GD, Theibert A, Lee W, Woods A. Regulation of inositol phospholipid binding and signaling through syndecan-4. J Biol Chem 2002; 277:49296 – 49303. 41. Tantivejkul K, Vucenik I, Shamsuddin AM. Inositol hexaphosphate (IP6) inhibits key events of cancer metastases: I. In vitro studies of adhesion, migration and invasion of MDA-MB 231 human breast cancer cells. Anticancer Res 2003; 23. 42. Tantivejkul K, Vucenik I, Shamsuddin AM. Inositol hexaphosphate (IP6) inhibits key events of cancer metastases: II. Effects on integrins and focal adhesions. Anticancer Res 2003; 23:3681– 3690. 43. Vucenik I, Shamsuddin AM. Cancer inhibition by inositol hexaphosphate (IP6) and inositol: from laboratory to clinic. J Nutrition 2003; 133:3778S–3784S. 44. Shamsuddin AM. Anti-cancer function of phytic acid. Int J Food Sci Technol 2002; 37:769– 782. 45. Shamsuddin AM, Vucenik I, Cole KE. IP6: a novel anti-cancer agent. Life Sci 1997; 61:343– 354. 46. Shamsuddin AM, Ullah A, Chakravarthy A. Inositol and inositol hexaphosphate suppress cell proliferation and tumor formation in CD-1 mice. Carcinogenesis 1989; 10:1461 – 1463. 47. Yang G-Y, Shamsuddin AM. IP6-induced growth inhibition and differentiation of HT-29 human colon cancer cells: involvement of intracellular inositol phosphates. Anticancer Res 1995; 15:2479– 2488. 48. El-Sherbiny Y, Cox MC, Ismail ZA, Shamsuddin AM, Vucenik I. G0/G1 arrest and S phase inhibition of human cancer cell lines by inositol hexaphosphate (IP6). Anticancer Res 2001; 21:2393– 2404. 49. Barker CJ, Wright J, Kirk CJ, Michell RH. Inositol 1,2,3 trisphosphate is a product of InsP6 dephosphorylation in WRK-1 rat mammary epithelial cells and exhibit transient concentration changes during the cell cycle. Biochem Soc Trans 1995; 23:169S. 50. Deliliers GL, Servida G, Fracchiolla NS, Ricci C, Borsotti C, Colombo G, Soligo D. Effects of inositol hexaphosphate (IP6) on human normal and leukaemic hematopoietic cells. Brit J Haematology 2002; 117:577 – 587. 51. Vucenik I, Tantivejkul K, Ramakrishna G, Anderson LM, Ramljak D. Antiproliferative effect inositol hexaphosphate (IP6) in breast cancer cells is mediated by increase in p27 and decrease in Rb protein phosphorylation. Proc Amer Assoc Cancer Res 2000; 41:339. 52. Efanov AM, Zaitsev SV, Berggren P-O. Inositol hexakisphosphate stimulates nonCa2þ-mediated and primes Ca2þ-mediated exocytosis of insulin by activation of protein kinase C. Proc Natl Acad Sci USA 1997; 94:4435 – 4439. 53. Hoy M, Berggren PO, Gromada J. Involvement of protein kinase C-epsilon in inositol hexakisphosphate-induced exocytosis in mouse pancreatic beta-cells. J Biol Chem 2003; 278:35168 – 35171. 54. Nickel KP, Belury MA. Inositol hexaphosphate reduces 12-O-tetradecanoylphorbol13-acetate-induced ornithine decarboxylase independent of protein kinase C isoform expression in keratinocytes. Cancer Lett 1999; 140:105 – 111. 55. Shamsuddin AM, Baten A, Lalwani ND. Effect of inositol hexaphosphate on growth and differentiation in K562 erythroleukemia cell line. Cancer Lett 1992; 64:195– 202. 56. Sakamoto K, Venkatraman G, Shamsuddin AM. Growth inhibition and differentiation of HT-29 cells in vitro by inositol hexaphosphate (phytic acid). Carcinogenesis 1993; 14:1815– 1819.

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57. Shamsuddin AM, Yang G-Y. Inositol hexaphosphate inhibits growth and induces differentiation of PC-3 human prostate cancer cells. Carcinogenesis 1995; 16:1975–1979. 58. Shamsuddin AM, Yang G-Y, Vucenik I. Novel anti-cancer functions of IP6: growth inhibition and differentiation of human mammary cancer cell lines in vitro. Anticancer Res 1996; 16:3287 – 3292. 59. Vucenik I, Kalebic T, Tantivejkul K, Shamsuddin AM. Novel anticancer function of inositol hexaphosphate (IP6): inhibition oh human rhabdomyosarcoma in vitro and in vivo. Anticancer Res 1998; 18:1377– 1384. 60. Bertagnolo V, Neri LM, Marchisio M, Mischiati C, Capitani S. Phosphoinositide 3-kinase activity is essential for all-trans-retinoic acid-induced granulocytic differentiation of HL-60 cells. Cancer Res 1999; 59:542– 546. 61. Pittet D, Schlegel W, Lew DP, Monod A, Mayr GW. Mass changes in inositol tetrakisand pentakisphosphate isomers induced by chemotactic peptide stimulation in HL-60 cells. J Biol Chem 1989; 264:18489 – 18493. 62. Huang C, Ma W-Y, Hecht SS, Dong Z. Inositol hexaphosphate inhibits cell transformation and activator protein 1 activation by targeting phosphatidylinositol-30 kinase. Cancer Res 1997; 57:2873– 2878. 63. Cocco L, Martelli A, Barnabe O, Manzoli FA. Nuclear inositol lipid signaling. Advan Enzyme Regul 2001; 41:361 – 384. 64. Cocco L, Gilmour RS, Ognibene A, Manzoni FA, Irvine RF. Synthesis of polyphosphoinositides in nuclei of friend cells. Evidence of phosphoinositide metabolism inside the nucleus which changes with cell differentiation. Biochem J 1987; 248:765–770. 65. York JD, Odom AR, Murphy R, Ives EB, Wente SR. A phospholipase C-dependent inositol polyphosphate kinase pathway required for efficient messenger RNA export. Science 1999; 285:96– 100. 66. Ongusaha PP, Hughes PJ, Davey J, Michell RH. Inositol hexakisphosphate in Schizosaccharomyces pombe: synthesis from Ins(1,4,5)P3 and osmotic regulation. Biochem J 1998; 335:671 –679. 67. Odom AR, Stahlberg A, Wente SR, York JD. A role for nuclear inositol 1,4,5trisphosphate kinase in transcriptional control. Science 2000; 287:2026 – 2029. 68. Shen X, Xiao H, Ranallo R, Wu W-H, Wu C. Modulation of ATP-dependent chromatin remodeling complexes by inositol polyphosphates. Science 2003; 299:112– 114. 69. Steger DJ, Haswell ES, Miller AL, Wente SR, O’Shea EK. Regulation of chromatic remodeling by inositol polyphosphates. Science 2003; 299:114 – 116. 70. Agarwal C, Dhanalakshmi S, Singh RP, Agarwal R. Inositol hexaphosphate inhibits constitutive activation of NF-kB in androgen-independent human prostate carcinoma DU145 Cells. Anticancer Research 2003; 23:3855 – 3861. 71. Chen N, Ma WY, Dong Z. Inositol hexaphosphate inhibits ultraviolet B-induced signal transduction. Mol Carcinog 2001; 31:139 – 144. 72. Thompson LU, Zhang L. Phytic acid and minerals: effect of early markers of risk for mammary and colon carcinogenesis. Carcinogenesis 1991; 12:2041– 2045. 73. Hanakahi LA, Bartlet-Jones M, Chappell C, Pappin D, West SC. Binding of inositol phosphate to DNA-PK and stimulation of double-strand break repair. Cell 2000; 102: 721 – 729. 74. Ma Y, Lieber MR. Binding of inositol hexaphosphate (IP6) to Ku but not to DNAPKcs. J Biol Chem 2002; 277:10756 – 10759. 75. Chen N, Ma W-Y, Dong Z. Inositol hexaphosphate inhibits ultraviolet B-induced signal transduction. Mol Carcinog 2001; 31:139 – 144.

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76. Vucenik I, Ramakrishna G, Tantivejkul K, Lynch M, Song H, Weghorst C, Shamsuddin A, Anderson L, Ramljak D. Inositol hexaphosphate (IP6) differentially modulates the expression of PKC in MCF-7 and MDA-MB 231 cells. Proc Am Assoc Cancer Res 1999; 40:653. 77. Morton RK, Raison JK. A complete intracellular unit for incorporation of amino-acid into storage protein utilizing adenosine triphosphate generated from phytate. Nature 1963; 200:429 – 433. 78. Biswas S, Maity IB, Chakrabarti S, Biswas BB. Purification and characterization of myo-inositol hexaphosphate-adenosine diphosphate phosphotransferase from Phaseolus aureus. Arch Biochem Biophys 1978; 185:557 –566. 79. Phillippy BQ, Ullah AH, Ehrlich KC. Purification and some properties of inositol 1,3,4,5,6-pentakisphosphate 2-kinase from immature soybean seeds. J Biol Chem 1994; 269:28393 – 28399. 80. Elsayed A, Chakravarthy A, Shamsuddin A. Inositol hexaphosphate from corn decreased the frequency of colorectal cancer in azoxymethane-treated rats. Seventysixth Annual Meeting of the U.S. – Canadian Division of the International Academy of Pathology, Chicago, IL, Mar 9 – 13, 1987. Lab Investig 1987; 56:21A. 81. Elsayed A, Ullah A, Shamsuddin A. Post-initiation dietary supplementation with corn derived inositol hexaphosphate (IP6) inhibits large intestinal carcinogenesis in F-344 rats. Seventy-first Annual Meeting of the Federation of American Societies for Experimental Biology, Washington, DC, Mar 29 –Apr 3, 1987. Fed Proc 1987; 46:585. 82. Shamsuddin AM, Elsayed AM, Ullah A. Suppression of large intestinal cancer in F-344 rats by inositol hexaphosphate. Carcinogenesis 1988; 9:577 – 580. 83. Sun AS, Yes HC, Wang LH, Huang YP, Maeda H, Pivazyan A, Hsu C, Bruckner HW, Fasy TM. Pilot study of a specific dietary supplement in tumor bearing mice and stage IIIB and IV non-small cell lung cancer patients. Nutr Cancer 2001; 39:85 –95. 84. Druzijanic N, Juricic J, Perko Z, Kraljevic D. IP-6 & inositol: adjuvant to chemotherapy of colon cancer. A pilot clinical trial. European Association of Cancer Research Annual Meeting EACR 17, Granada, Spain, Jun 8 – 11, 2002. Rev Oncologı´a 2002; 4(suppl 1):171. 85. Grases F, March JG, Prieto RM, Simonet BM, Costa-Bauza´ A, Garcia-Raja A, Conte A. Urinary phytate in calcium oxalate stone-formers and healthy people. Dietary effects on phytate excretion. Scand J Urol Nephrol 2000; 34:162–164. 86. Jariwalla RJ, Sabin R, Lawson S, Herman ZS. Lowering of serum cholesterol and triglycerides and modulations by dietary phytate. J Appl Nutr 1990; 42:18– 28. 87. Vucenik I, Podczasy JJ, Shamsuddin AM. Antiplatelet activity of inositol hexaphosphate (IP6). Anticancer Res 1999; 19:3689 – 3693. 88. Rao PS, Liu XK, Das DK, Weinstein GS, Tyras DH. Protection of ischemic heart from reperfusion injury by myo-inositol hexaphosphate, a natural antioxidant. Ann Thorac Surgery 1991; 52:908 – 912. 89. Grases F, Simonet BM, Vucenik I, Perello J, Prieto RM, Shamsuddin AM. Effects of exogenous inositol hexakisphosphate (InsP6) on the levels of InsP6 and of inositol trisphosphate (InsP3) in malignant cells, tissues and biological fluids. Life Sci 2002; 71:1535– 1546. 90. Grases F, Simonet BM, Vucenik I, Prieto RM, Costa-Bauza A, March JG, Shamsuddin AM. Absorption and excretion of orally administered inositol hexaphosphate (IP6 or phytate) in humans. Biofactors 2001; 15:53 –61.

20 Modulation of Gene Expression by Dietary Iron Paul Sharp King’s College, London, UK

Introduction Dietary Iron Absorption Iron-Dependent Regulation of Intestinal Iron Absorption Iron Regulatory Proteins Iron Responsive Elements Regulation of DMT1 Regulation of IREG1 Regulation of Dcytb and Hephaestin Hypoxia The Role of Hepcidin in Regulating Body Iron Metabolism Hemochromatosis Iron: A Pro-oxidant Conclusions References

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INTRODUCTION Iron is an essential trace metal in our diet and plays a key role in a plethora of biochemical processes in the body including the binding and release of oxygen 421

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in hemoglobin and electron transfer in mitochondrial oxidative phosphorylation by the cytochromes. These processes can be compromised if dietary iron supply is reduced with obvious consequences for human health. However, despite being an essential nutrient, iron is a powerful pro-oxidant, and in excess can lead to oxidative damage to cells and tissues through the formation of free radicals (1). Consequently, body iron levels must be regulated within strictly defined limits to avoid pathologies associated with iron deficiency and overload. The major iron-related diseases are iron deficiency anemia, which affects up to 2 billion people and is regarded as the most prevalent nutritional deficiency disorder worldwide, and hereditary hemochromatosis, a genetic disease that predisposes 1 person in every 200 of northern European decent to body iron loading. To maintain homeostatic control over iron nutriture, a number of regulatory mechanisms have evolved to match dietary iron absorption to the body’s physiological requirements. This chapter will review the current knowledge regarding the major iron-sensitive signals involved in the homeostatic regulation of body iron metabolism. DIETARY IRON ABSORPTION Most western diets contain a mixture of heme (found exclusively in animal tissues) and nonheme iron (not only found extensively in cereals and vegetables, but also in meat). Heme accounts for 5– 10% of the daily iron intake in industrialized countries (2); whereas in vegetarian diets and in developing countries, the heme iron intake is negligible. The main form of iron in all diets is nonheme iron. These different forms of iron are absorbed by totally independent transport mechanisms that reside on the apical membrane of the duodenal enterocytes (Fig. 20.1). Heme is removed from hemoglobin and myoglobin in the intestinal lumen and is absorbed intact through an as yet uncharacterized heme transporter. Inside the cell, iron is removed from the porphyrin shell by the action of heme oxygenase 1 (3). Nonheme iron in the diet is present largely in its ferric form, which is nonbioavailable and must therefore be reduced to ferrous prior to uptake into the duodenal enterocytes. This is achieved by the action of a number of reducing agents in the diet, including ascorbic acids and small peptides containing cysteinyl and histidyl residues. In addition, the apical membrane of duodenal enterocytes contains ferric reductase activity in the form of the recently characterized Dcytb enzyme (Duodenal cytochrome b) (4). The relatively low pH of the duodenal lumen together with the acid microclimate that covers the apical surface of enterocyte (5,6) stabilizes iron in the ferrous form and provides the inwardly directed proton gradient that drives iron uptake through the apical membrane iron transporter DMT1 (7,8). Nonheme iron together with iron liberated from heme enter a common pool inside the duodenal enterocytes and this iron can either be stored in the form of ferritin inside the cells or can be transferred across the basolateral membrane via IREG1 (9) before being re-oxidized back to Fe3þ by

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Fe 3+ ferritin ascorbate etc.

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Figure 20.1 Duodenal iron transport. Nonheme iron is present in the diet largely in the less bioavailable ferric or Fe3þ form. To facilitate absorption, iron must first be reduced to the Fe2þ or ferrous state and this is achieved by the presence of dietary reducing agents, such as ascorbate, in the duodenal lumen and by the endogenous ferric reductase Dcytb, which resides on the apical membrane of enterocytes. Fe2þ is readily transported into enterocytes through DMT1 via a proton-dependent uptake mechanism. Heme enters the cell intact through an as yet uncharacterized transporter (HT) and the iron contained within the porphyrin ring is excised under the action of heme oxygenase (HO). Iron derived from both heme and nonheme sources enters a common pool inside the enterocytes and can either be targeted for storage in ferritin, if body iron requirements are low, or can enter a labile iron pool (LIP) from where it is processed for onward transport out of the cell. Efflux across the basolateral membrane occurs through IREG1 as Fe2þ, though iron is rapidly oxidized to Fe3þ in the presence of the ferrioxidase activity of hephaestin (Hp) and picked up by transferrin (Tf ) for transport to its sites of utilization in the body.

hephaestin (10). Ferric iron leaving the cell is picked up by transferrin for onward delivery to the iron stores in the liver and to the utilizing tissues. IRON-DEPENDENT REGULATION OF INTESTINAL IRON ABSORPTION All of the key components of the intestinal iron transport machinery identified earlier are subject to tight regulation by the dietary iron load, the state of the body iron stores, and the iron requirements for erythrocyte production. Regulation of these intestinal genes by iron utilizes several mechanisms. All cells and tissues must maintain a homeostatic balance between receiving an adequate iron supply while avoiding the build up of free iron inside the cell that might lead to permanent cellular damage through the generation of reactive oxygen species (ROS). In most cells, iron supply is regulated by the number of

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transferrin receptors (TfR) expressed on the cell surface; whereas inside the cell, excess iron is sequestered in ferritin. The expression of both TfR and ferritin is directly related to intracellular iron levels through posttranscriptional mechanisms that involve interactions between cytosolic iron regulatory proteins (IRP) and stem loop structures, known as iron responsive elements (IRE), that exist in either the 50 or 30 untranslated region (UTR) of several target mRNA species. Iron Regulatory Proteins Two cytosolic IRP, IRP-1 and IRP-2, are known to exist in most cells. Both of these proteins can bind to IRE structures within RNA sequences when cellular iron levels are depressed. However, under iron replete conditions, RNA binding is quickly inactivated by either posttranslational modification of IRP-1 (there is no decrease in total IRP-1 levels) or degradation of IRP-2. The underlying mechanism behind the iron-dependent posttranslational inactivation of IRP-1 has been studied extensively. Structurally, IRP-1 is remarkably similar in its amino acid sequence to the mitochondrial acontitase (11) that converts citrate to isocitrate in the tricarboxylic acid cycle. Under iron replete conditions, IRP-1 can act as a cytoplasmic aconitase and this dual function is controlled by the presence or absence of an iron sulfur cluster (Fig. 20.2). When cellular iron is high, a 4Fe– 4S cluster is inserted into IRP-1 and is held in place by three conserved cysteine residues (also present in mitochondrial aconitase). Under these conditions, IRP-1 has a closed conformation and cannot bind IREs. The fourth position iron in the cluster is highly labile and is readily removed when cellular iron levels are low, leading to disassembly of the Fe – S cluster which permits the apoprotein to bind IRE sequences (12 – 14). IRP-2 is less abundant in cells than IRP-1, and is subject to de novo synthesis when cellular iron levels are low and proteosomal degradation when iron levels are high (15). IRP-2 contributes significantly to the total IRP RNA binding activity in several tissues but particularly in the brain (16) and intestine (17). Both IRP-1 and IRP-2 bind successfully to the consensus IRE sequence; however, recent evidence suggests that IRP-2 may be able to recognize exclusively a specific subset of IRE sequences (18,19). Iron Responsive Elements The IRE is a stem loop structure within the UTRs of several mRNA species. A single IRE was first identified in the 50 UTR of ferritin mRNA (20 –23), and shortly afterwards five similar sequences were identified in the 30 UTR of the TfR mRNA (24). The sequence of the hairpin loop 50 -CAGUGN-30 is highly conserved and there is a further C six bases upstream of this central motif that is unpaired and forms a bulge in the stem (Fig. 20.2). However, the sequence within the stem is variable and can give rise to several different predicted secondary structures.

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G

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U G N N-N N-N N-N N-N C N-N N-N N-N N-N N-N N-N 5’ – N-N – 3’

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aconitase activity

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Figure 20.2 IRE/IRP interactions regulate the expression of a number of iron responsive mRNA species. IREs are stem loop structures present within the UTR of a number of mammalian mRNA species (a). A number of variations in the structure of individual IREs have been observed but they are characterized by an asymmetrical bulge within the RNA stem, due to the presence of an unpaired C, and the consensus sequence 50 -CAGUGN-30 which forms the loop. Under low cellular iron conditions, IRE interact with cytosolic IRP (b). However, this binding is lost in iron replete cells due to the insertion of a cubic 4Fe–4S cluster that is held in place by three conserved cysteine residues. In the case of IRP-1, the presence of the iron–sulfur cluster converts the RNA binding protein into one that exhibits cytoplasmic aconitase activity. IRP binding to IRE in the 50 UTR (e.g., in ferritin) prevents the binding of the eIF4F complex to the 43S entry site that is required protein translation (c)—hence under low cellular iron conditions, ferritin protein is decreased. TfR contains five consensus IRE sequences in its 30 UTR (d). Binding of IRP in this scenario blocks endonuclease-mediated mRNA degradation, thus increasing mRNA half-life.

Under iron replete conditions, ferritin mRNA is efficiently translated to form of the cell’s major storage protein. However, when cellular iron levels decrease, ferritin protein levels are also lowered. This decrease in ferritin is directly attributable to the position of the IRE in the 50 UTR. The IRE in both ferritin H and L chains (and other iron responsive with 50 IRE sequences such as mitochondrial aconitase and erythroid 5-ALA-synthase) is less than 40 bases from the AUG site, and binding of IRP to these IRE sequences prevents the binding of the eukaryotic initiation factor (eIF4F) complex to the 43S ribosomal subunit that is necessary for protein translation (25). The regulation of the TfR by iron is directly opposite to the control of ferritin. Under iron replete conditions, TfR protein levels are low; whereas,

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when cellular iron is low, the expression of TfR protein is elevated due to activated IRP binding to all or a number of the five IRE sequences present in the 30 UTR. It is believed that IRP/IRE binding does not alter the rate of TfR protein translation but rather promotes mRNA stability by protecting it from endonucleolytic degradation, thus increasing TfR mRNA half-life (24,26,27). Regulation of DMT1 The overall regulation of DMT1 by iron is a complex issue. Analysis of the DMT1 gene has revealed evidence that it encodes at least four variants through two alternate splicing events—one at the 50 end of the gene leads to two separate initiation sites in exon 1A or exon 1B, repectively, both of which are in frame with exon 2 (28) and an alternate splice site in exon 16 that is in frame with exon 16A or exon 17 (29). The exon 16A variant leads to the transcription of mRNA containing a single IRE in the 30 UTR, whereas the exon 17 transcript lacks this IRE. With regard to the exon 1A and exon 1B variants, it has been suggested that DMT1A is predominantly found in epithelial tissues, whereas DMT1B is ubiquitously expressed (28). The relative abundance of these four possible variants (i.e., 1A/16A, 1A/17, 1B/16A, or 1B/17) and their function in terms of iron uptake in various tissues are still unclear. Currently, there is a great deal of confusion regarding the role of the 30 IRE in DMT1 regulation by iron. Initial studies in animal models of iron deficiency demonstrated that DMT1 mRNA was regulated appropriately by iron status for mRNA containing an IRE in the 30 UTR, that is, an increase under iron-deficient conditions (7). Furthermore, in vitro binding studies established that the 30 IRE recognizes and binds cytosolic IRP (30). However, recent data suggest that despite binding IRP, the DMT1 IRE might not be functionally involved in regulating mRNA levels (31). This is an area that clearly needs to be resolved. Mechanisms other than IRE/IRP interactions are also implicated in the regulation of DMT1. Our own work and that of others (28) has shown that the expression of non-IRE containing DMT1 transcripts is regulated by both iron deficiency and iron loading. This suggests that the promoter region of the gene may play an important role in the overall regulation of DMT1 expression. To this end, several putative transcription factor binding sites have been identified upstream of the exon 1B variants (29,32). Using a DMT1 construct in which 1.5 kb of the DMT1 1B promoter was cloned upstream of a luciferase reporter gene, we have demonstrated that several transition metals (iron, zinc, and copper) can induce luciferase activity (33). At this stage, the nature of the metal-sensitive transcription factors activated in these studies remains elusive. Regulation of IREG1 IREG1—also known as ferroportin 1 (34) and MTP1 (35)—mRNA also contains an IRE sequence, though in contrast to DMT1 it resides in the 50 UTR. In general, binding of IRP to a 50 IRE is thought to lead to a decrease in protein translation

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(e.g., ferritin). However, in our studies using the intestinal Caco-2 cell line, we do not see any change in IREG1 mRNA or protein expression or alterations in cellular iron efflux following iron loading (36). Indeed, several studies have reported that duodenal IREG1 expression is upregulated in iron-deficient human and animals as well as in hemochromatotic patients (9,37). These findings, taken together, question the role of the IRE in regulating IREG1 expression. Previous studies have shown that IRE/IRP interactions that take place at a distance of greater than 67 nucleotides from the transcription start site cannot repress protein translation (38). In the duodenal IREG1 transcript, the distance from the transcription start site to the base of the IRE is somewhat greater than 67 bases (9,35) suggesting that the IRE may not be functional. Intriguingly, IRE-mediated regulation of IREG1 does take place in hepatocytes (39,40) and in a number of other tissues (41) suggesting that IREG1 mRNA processing is tissue-specific and is designed to match the function of those tissues in body iron metabolism. Regulation of Dcytb and Hephaestin Efficient transepithelial transport of iron in the intestine relies on two key redox reactions, reduction of ferric iron to ferrous to allow entry into the enterocyte across the apical membrane and oxidation of ferrous iron leaving the cell to ferric to permit loading onto transferrin for onward transport in the plasma. These processes are governed by Dcytb, the recently characterized ferrireductase (4), and hephaestin, a ceruloplasmin homolog that acts as a ferrioxidase in enterocytes (10), respectively. Dcytb in particular is strongly iron-regulated, expression is increased in iron deficiency and by hypoxia (4). However, despite the crucial role played by hephaestin in iron efflux, highlighted by sla mice that have a profound anemia due to a hephaestin mutation, there is still some question as to whether it is regulated by iron. In rodent models, dietary iron manipulation has no discernable effect on hephaestin expression (42), whereas in patients with iron deficiency, hephaestin levels are upregulated (43). Importantly, this latter study demonstrated that the expression of DMT1, Dcytb, IREG1, and hephaestin were positively related to each other independently of iron status, suggesting that a co-ordinated mechanism exists for the transfer of dietary iron across the intestine and into the blood. However, unlike the transporters themselves, Dcytb and hephaestin do not contain IRE sequences in their UTRs indicating perhaps that iron status regulates these genes at the transcriptional level. HYPOXIA The development of iron deficiency anemia is a multistage process, initiated by a decrease in body iron stores (characterized by reduced serum ferritin levels), progressing onto iron-deficient eryrthropoiesis (when the stores are empty) that, if untreated, ultimately leads to the development of a microcytic hypochromic

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anemia. The decrease in hemoglobin production seriously compromises the body’s ability to distribute oxygen to the tissues resulting in hypoxic stress. Hypoxia itself has long been recognized as an important regulator of body iron metabolism. Approximately 1% of the circulating red blood cell population is removed and degraded by the reticuloendothelial macrophages every day and replaced with new erythrocytes. The rate of erythropoiesis is controlled by the hormone erythropoietin, produced in cells residing in the kidney and liver, and the primary stimulus for its production is tissue hypoxia (44,45). The trigger for induction of erythropoietin production by hypoxia is the transcription factor HIF-1 (hypoxia inducible factor-1) (46,47), which is formed of two subunits (a and b) that combine and bind to hypoxia responsive elements—DNA sequences in the flanking regions of a number of hypoxia-sensitive genes (48). Hypoxia has been shown to upregulate duodenal iron absorption in rats (49) and in humans (50) presumably to satisfy the increased requirement for iron in erythropoiesis. The cellular mechanisms involved in this adaptation are emerging and include an increased electrical driving force for iron uptake (51), an increased expression of proteins involved in iron transport including Dcytb (4), IREG1 (9) and a TfR (52). In TfR, the effect of hypoxia is mediated by the presence of a hypoxia responsive element consensus sequence (52). Interestingly, analysis of the DMT1 promoter identified two potential hypoxia responsive sequences suggesting that it too may be a hypoxia-regulated gene (29). However, it is unknown whether similar sequences exist in the flanking regions of other key components of the iron transport machinery. A further link between hypoxia and iron metabolism has been identified recently. In mice subjected to hypobaric pressure and in hepatoma cell lines maintained under low oxygen conditions, the expression of hepcidin, an important peptide involved in the regulation of body iron metabolism (see later), was significantly decreased (53). This gives rise to the possibility that hypoxic stimuli could also exert effects on genes that do not contain classical DNA sequences recognizing the HIF-1 transcription factor. THE ROLE OF HEPCIDIN IN REGULATING BODY IRON METABOLISM The nature of the signals generated by the body iron stores has been the subject of much speculation. Recently, a candidate peptide that might explain the link between iron requirements and the regulation of intestinal absorption has been identified. Hepcidin is predominantly expressed in the liver and its mRNA encodes a 25 amino acid peptide with significant antimicrobial activity (54,55). In addition to this antimicrobial action, hepcidin expression is associated with body iron status and is dramatically upregulated when liver iron is high and downregulated when the stores are depleted (56). A role for hepcidin in iron metabolism was subsequently established using knockout mice in which the

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USF2 transcription factor had been deleted. These animals developed a severe iron overload, strikingly similar to that found in human hemochromatosis and in the Hfe 2/2 mouse (57). Subsequent examination of the Usf 2 2/2 mice revealed that the hepcidin gene had also been disrupted (the two mouse genes are only 1240 bp apart) (56). An alternative gene targeting methodology confirmed that it was the disruption of the hepcidin gene and not USF2 that resulted in the iron overloading phenotype (58). Interestingly, recent studies have demonstrated a link between hepcidin expression and the regulation of human iron metabolism. Two families have been identified who have mutations in the hepcidin gene that led to a novel variant of juvenile hemochromatosis, a particularly severe form of the iron loading disease that typically affects people in their late teens and early twenties (59). Recently, transgenic mice over expressing hepcidin have been generated (58). These animals have severe body iron deficiency and microcytic hypochromic anemia suggesting a reciprocal relationship between hepcidin expression and iron accumulation. Furthermore, studies in humans have demonstrated that inappropriate expression of hepcidin is associated with the anemia of chronic disease (53,60). In one of these studies (60), two patients with severe iron deficiency were identified who had large hepatic adenomas. Analysis of the tumors showed that hepcidin mRNA was massively over expressed, a situation that would lead to increased circulating hepcidin levels. Resection of the tumors reversed the clinical anemia suggesting that changes in hepcidin expression provide the link for the perturbations in iron metabolism observed in chronic disease. Further evidence for this link is afforded by recent work demonstrating that hepcidin mRNA is also increased in animal models of inflammation (53) and following exposure of human hepatocytes to monokines and cytokines (61). Hepcidin expression following administration of an iron-deficient diet is inversely correlated with intestinal iron transporter expression (62). In this model, it is proposed that changes in dietary iron levels affect the degree of transferrin saturation by iron. Consequently, the changes in transferrin saturation are detected by the liver, and hepcidin expression is regulated accordingly— decreased when transferrin saturation is low, increased when transferrin saturation is high. However, to add a further level of intrigue to the role of hepcidin in the regulation of body iron metabolism, recent studies have shown that exposure of isolated hepatocytes (61) and hepatoma cells (63) to iron down regulated hepcidin mRNA. In current models regarding the regulation of intestinal iron absorption, hepcidin is thought to be the major modulator involved. Elevated levels of hepcidin are believed to act directly on the intestinal epithelium and act as a repressor of iron absorption. However, at this time the precise intestinal site of action of hepcidin is unclear with the arguments focusing on whether it contributes to the programing of the duodenal crypt cells or acts directly on the mature enterocytes (Fig. 20.3).

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Figure 20.3 Regulation of duodenal iron transport: roles of HFE/TfR and hepcidin. Circulating transferrin (Tf ) binds to transferrin receptors (TfR) located on the serosal surface of the duodenal crypt cells. HFE is also localized to this membrane due to its interaction with b2-microglobulin and binds to TfR regulating the rate of receptor recycling. This level of control is lost in hemochromatosis. The HFE/Tf/TfR complex is endocytosed and iron dissociates from Tf in the acidified environment of the endosome and is transported into the cytosol via DMT1. Changes in the cellular iron content influence the binding of IRP to IRE in the UTR of a number of mRNA species (including DMT1, TfR, IREG1, and ferritin) and alters protein translation accordingly. HFE/TfR interactions also, in part, regulate iron accumulation by hepatocytes. Hepcidin, a 25 amino acid peptide, is synthesized and released from the hepatocyte when body iron levels are high and is thought to act directly on the intestinal epithelium as a negative regulator of dietary iron absorption. The intestinal site of action of hepcidin is still unclear. It could bind within the crypts and contribute to the overall programing of duodenal iron transport by regulating the transcription of a number of genes involved in cellular iron metabolism. Alternatively, hepcidin could act directly on the mature enterocytes to influence the cell surface expression of the essential components of the iron transport machinery.

HEMOCHROMATOSIS Hereditary hemochromatosis is a relatively common inborn error of iron metabolism (1:200 people mainly of northern European decent are affected) that is characterized by excess iron accumulation and deposition within several tissues, especially the liver, which can lead to cell and tissue damage (e.g., fibrosis and

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cirrhosis in the liver). The most common form of hemochromatosis arises from an autosomal recessive mutation that leads to the substitution of tyrosine for cysteine at amino acid 282 of the HFE protein (64). Subsequent analysis of the hfe gene has demonstrated that a number of other less pathogenic (in terms of body iron status) mutations also exist in addition to C282Y (65). The involvement of HFE in iron metabolism was confirmed following the production of a knockout mouse, which subsequently developed iron overload and resembled the human hereditary hemochromatosis phenotype (66). The HFE protein is localized to a number of tissues that have major roles in body iron metabolism including the duodenum (where it is found exclusively in the crypts of Lieberku¨hn) (67), the liver (in Kupffer cells and hepatocytes) (68,69), and in tissue macrophages and circulating monocytes (70). HFE is a member of the MHC class 1 family of molecules, and not an iron transport protein as first predicted, and as such it needs to be associated with b2microglobulin for normal intracellular processing and cell surface expression (71 – 73). b2-Microglobulin itself is known to play an essential role in iron metabolism since deletion of this gene leads to a progressive iron overload similar to that seen in hemochromatosis patients (74,75). In addition to its interaction with b2-microglobulin, HFE also binds to TfR regulating the rate at which transferrin-bound iron can enter the cell (76,77). Normally, iron uptake by the duodenum from the plasma is directly proportional to the plasma iron concentration. However, in hfe knockout mice duodenal iron uptake from plasma transferrin is significantly lower than in control mice, supporting the hypothesis that HFE plays a crucial role in regulating the uptake of transferrin-bound iron (78). The HFE protein is thought to be a major regulator of intestinal iron absorption (Fig. 20.3) through its interaction with TfR since this establishes the prevailing cellular iron concentration within the duodenal crypt cells, which ultimately determines the level of expression of the proteins involved in iron absorption (i.e., DMT1 and Dcytb at the apical membrane and IREG1 plus hephaestin at the basolateral surface) in enterocytes as they migrate along the crypt-villus axis. Since only the mature enterocytes, located on the upper third of the villus, participate in translocation of dietary iron from the intestinal lumen to the blood, this essentially means that transporter protein levels are pre-programed in the cells leaving the crypt and that re-programing in response to changes in body iron status would take a further 2– 3 days to be established (i.e., the time taken from crypt stem cell division to arrival of the mature enterocyte at the villus tip). Given that the C282Y mutant protein does not interact with TfR, it has been postulated that in hemochromatotic patients as well as knockout mouse models of the disease, dietary iron absorption proceeds in a less regulated fashion and is therefore inappropriately high in relation to the body iron stores. Several pieces of evidence suggest that HFE/TfR interaction cannot be the only regulatory pathway controlling intestinal iron absorption. First, in studies employing hfe knockout mice, levels of the iron transporters and ferrireductase activity are not compromised compared with wild type littermates (79).

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Furthermore, these animals still respond appropriately to dietary iron deficiency or loading even in the absence of functional HFE protein (80 – 83). In nonphlebotomized patients with hemochromatosis, levels of DMT1 and IREG1 also were unaltered compared with iron replete subjects (84). This apparent paradox can perhaps be explained by the recent observation that HFE status can regulate hepatic hepcidin production. In hemochromatotic patients, and in hfe knockout mice, hepcidin mRNA levels are inappropriately low given the degree of iron loading (85,86), which might in turn account for the relatively high intestinal iron absorption that characterizes the disease. Furthermore, when hfe knockout mice were crossed with transgenic mice that constitutively expressed hepcidin intestinal, iron absorption was significantly reduced (87) adding credence to the hypothesis that hepcidin and not HFE is the master controller of body iron metabolism. IRON: A PRO-OXIDANT Despite its essential role in metabolism, iron is also a prospective pro-oxidant and is therefore potentially harmful. Excess iron promotes lipid peroxidation and tissue damage in vitro (1) raising the possibility that, via these pro-oxidant effects, disturbances in iron metabolism may play a pathogenic role in a number of diseases. Iron participates in the Fenton reaction with hydrogen peroxide (a by-product of cellular oxygen metabolism) to produce the highly damaging hydroxyl radical. Fe2þ þ H2 O2 ¼ Fe3þ þ OH
þ OH† Hydroxyl radicals once generated react with virtually all molecules in living cells, resulting, for example, in the peroxidation of membrane lipids or DNA strand breakages leading to cell aging and death. Iron chelated in low molecular weight complexes in particular has been shown to lead to the generation of ROS that in turn impact on the modulation of cellular signal transduction pathways (e.g., those leading to NFkB activation) with downstream consequences for gene transcription (88). Furthermore, iron-dependent generation of lipid peroxidation products is associated with altered expression of a number of genes (89). The central nervous system in particular appears to be very sensitive to damage by ROS and there is an increasing body of evidence that suggests that ROS generated through metal-dependent processes might lead to neurodegenerative diseases such as Alzheimer’s disease, Friedriech’s ataxia, and Parkinson’s disease (90). Iron plays an essential role in neural function as it is a cofactor for the production of the neurotransmitters dopamine, noradrenaline, and 5-HT (91). An intriguing link between iron and Alzheimer’s disease has emerged with the discovery that the amyloid precursor protein (APP) contains a type II IRE in its 50 UTR (92) in a region containing mutations associated with familial

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Alzheimer’s disease (93 – 95). Studies have shown that IRP binding to the APP 50 UTR is reduced after treatment of cells with the iron chelator desferrioxamine, suggesting a significant role for iron in the metabolism of APP as well as highlighting a possible therapeutic role for iron chelators in the treatment of Alzheimer’s disease (92). The IRE/IRP system per se may be very important in regulating brain iron metabolism. Studies in mice with a targeted disruption of the IRP2 gene have revealed that the mutant mice have disregulated iron metabolism in the CNS and ultimately develop movement disorders characterized by ataxia, bradykinesia, and tremor that are associated with iron accumulation in the white matter tracts and nuclei (96). In certain situations, excess cellular iron is associated with cancer, for example, death from hepatocellular carcinoma is increased several hundred fold in patients diagnosed with hereditary hemochromatosis (97). The link between iron and cancer rests on the fact that proliferating cells have an absolute requirement for iron to catalyze a number of key reactions involved in oxygen sensing, energy metabolism, and DNA synthesis (in the absence of iron, cells cannot proceed from G1 to S phase of the cell cycle). Indeed iron chelators are being increasingly used as anticancer agents since they can exert antiproliferative effects on cancer cells (98). CONCLUSIONS The studies reviewed here highlight the numerous pathways by which iron can influence gene expression and physiological function. These range from the well-characterized interactions between cytosolic IRP and IRE sequences present in the UTR of a number of genes through to the influences of novel peptides (hepcidin) and ROS on the cellular signals that control the expression of key components of the body’s iron regulatory system. The role of hepcidin in particular is an area attracting much current research interest since is appears to act as the common pathway that relays the information from both the main site of iron storage (the liver) and the iron utilization (the bone marrow) that is necessary to match intestinal iron absorption in line with the body’s physiological requirements. Perturbations in body iron metabolism do not just lead to the development of iron deficiency anemia or iron loading disorders but also impact on a number of other multifactorial disease processes including neurodegeneration and cancer. One thing that is clear from this survey is that many of the iron-sensitive mechanisms involved in the regulation of gene expression are incompletely understood at present and this rapidly developing area of nutigenomics is ripe for further extensive study. REFERENCES 1. Halliwell B, Gutteridge JM. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem J 1984; 219:1– 14.

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Sharp patients with hereditary hemochromatosis, reveals unique pattern of expression in gastrointestinal tract. Proc Natl Acad Sci USA 1997; 94:2534 – 2539. Bastin JM, Jones M, O’Callaghan CA, Schimanski L, Mason DY, Townsend AR. Kupffer cell staining by an HFE-specific monoclonal antibody: implications for hereditary hemochromatosis. Br J Haematol 1998; 103:931– 941. Zhang AS, Xiong S, Tsukamoto H, Enns CA. Localization of iron metabolism-related mRNAs in rat liver indicate that HFE is predominantly expressed in hepatocytes. Blood (DOI 10.1182/blood-2003-07-2378) 2003. Parkkila S, Parkkila AK, Waheed A, Britton RS, Zhou XY, Fleming RE, Tomatsu S, Bacon BR, Sly WS. Cell surface expression of HFE protein in epithelial cells, macrophages, and monocytes. Haematologica 2000; 85:340 – 345. Feder JN, Tsuchihashi Z, Irrinki A, Lee VK, Mapa FA, Morikang E, Prass CE, Starnes SM, Wolff RK, Parkkila S, Sly WS, Schatzman RC. The hemochromatosis founder mutation in HLA-H disrupts b2-microglobulin interaction and cell surface expression. J Biol Chem 1997; 272:10425– 10428. Waheed A, Parkkila S, Zhou XY, Tomatsu S, Tsuchihashi Z, Feder JN, Schatzman RC, Britton RS, Bacon BR, Sly WS. Hereditary hemochromatosis: effects of C282Y and H63D mutations on association with b2-microglobulin, intracellular processing, and cell surface expression of the HFE protein in COS-7 cells. Proc Natl Acad Sci USA 1997; 94:12384 – 12389. Waheed A, Grubb JH, Zhou XY, Tomatsu S, Fleming RE, Costaldi ME, Britton RS, Bacon BR, Sly WS. Regulation of transferrin-mediated iron uptake by HFE, the protein defective in hereditary hemochromatosis. Proc Natl Acad Sci USA 2002; 99:3117– 3122. Santos M, Schilham MW, Rademakers LH, Marx JJ, de Sousa M, Clevers H. Defective iron homeostasis in beta 2-microglobulin knockout mice recapitulates hereditary hemochromatosis in man. J Exp Med 1996; 184:1975 –1985. Rothenberg BE, Voland JR. b2 knockout mice develop parenchymal iron overload: a putative role for class I genes of the major histocompatibility complex in iron metabolism. Proc Natl Acad Sci USA 1996; 93:1529– 1534. Parkkila S, Waheed A, Britton RS, Bacon BR, Zhou XY, Tomatsu S, Fleming RE, Sly WS. Association of the transferrin receptor in human placenta with HFE, the protein defective in hereditary hemochromatosis. Proc Natl Acad Sci USA 1997; 94:13198 –13202. Feder JN, Penny DM, Irrinki A, Lee VK, Lebron JA, Watson N, Tsuchihashi Z, Sigal E, Bjorkman PJ, Schatzman RC. The hemochromatosis gene product complexes with the transferrin receptor and lowers its affinity for ligand binding. Proc Natl Acad Sci USA 1998; 95:1472 – 1477. Trinder D, Olynyk JK, Sly WS, Morgan EH. Iron uptake from plasma transferrin by the duodenum is impaired in the Hfe knockout mouse. Proc Natl Acad Sci USA 2002; 99:5622– 5626. Dupic F, Fruchon S, Bensaid M, Borot N, Radosavljevic M, Loreal O, Brissot P, Gilfillan S, Bahram S, Coppin H, Roth MP. Inactivation of the hemochromatosis gene differentially regulates duodenal expression of iron-related mRNAs between mouse strains. Gastroenterology 2002; 122:745 – 751. Ajioka RS, Levy JE, Andrews NC, Kushner JP. Regulation of iron absorption in Hfe mutant mice. Blood 2002; 100:1465 – 1469.

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81. Lebeau A, Frank J, Biesalski HK, Weiss G, Srai SK, Simpson RJ, McKie AT, Bahram S, Gilfillan S, Schumann K. Long-term sequelae of HFE deletion in C57BL/6  129/O1a mice, an animal model for hereditary hemochromatosis. Eur J Clin Invest 2002; 32:603– 612. 82. Simpson RJ, Debnam ES, Laftah AH, Solanky N, Beaumont N, Bahram S, Schumann K, Srai SK. Duodenal nonheme iron content correlates with iron stores in mice, but the relationship is altered by Hfe gene knock-out. Blood 2003; 101:3316– 3318. 83. Simpson RJ, Debnam E, Beaumont N, Bahram S, Schumann K, Srai SK. Duodenal mucosal reductase in wild-type and Hfe knockout mice on iron adequate, iron deficient, and iron rich feeding. Gut 2003; 52:510 –513. 84. Stuart KA, Anderson GJ, Frazer DM, Powell LW, McCullen M, Fletcher LM, Crawford DH. Duodenal expression of iron transport molecules in untreated haemochromatosis subjects. Gut 2003; 52:953 – 959. 85. Bridle KR, Frazer DM, Wilkins SJ, Dixon JL, Purdie DM, Crawford DH, Subramaniam VN, Powell LW, Anderson GJ, Ramm GA. Disrupted hepcidin regulation in HFE-associated hemochromatosis and the liver as a regulator of body iron homoeostasis. Lancet 2003; 361:669– 673. 86. Ahmad KA, Ahmann JR, Migas MC, Waheed A, Britton RS, Bacon BR, Sly WS, Fleming RE. Decreased liver hepcidin expression in the Hfe knockout mouse. Blood Cells Mol Dis 2002; 29:361 – 366. 87. Nicolas G, Viatte L, Lou DQ, Bennoun M, Beaumont C, Kahn A, Andrews NC, Vaulont S. Constitutive hepcidin expression prevents iron overload in a mouse model of hemochromatosis. Nat Genet 2003; 34:97– 101. 88. Boldt DH. New perspectives on iron: an introduction. Am J Med Sci 1999; 318:207–212. 89. Barisani D, Meneveri R, Ginelli E, Cassani C, Conte D. Iron overload and gene expression in HepG2 cells: analysis by differential display. FEBS Lett 2000; 469:208– 212. 90. Thompson KJ, Shoham S, Connor JR. Iron and neurodegenerative disorders. Brain Res Bull 2001; 55:155 – 164. 91. Youdim MBH. Neuropharmacological and neurobiochemical aspects of iron deficiency. In: Dobbing J, ed. Brain, Behavior, and Iron in the Infant Diet. London: Springer-Verlag, 1990:83 – 106. 92. Rogers JT, Randall JD, Cahill CM, Eder PS, Huang X, Gunshin H, Leiter L, McPhee J, Sarang SS, Utsuki T, Greig NH, Lahiri DK, Tanzi RE, Bush AI, Giordano T, Gullans SR. An iron-responsive element type II in the 50 -untranslated region of the Alzheimer’s amyloid precursor protein transcript. J Biol Chem 2002; 277:45518– 45528. 93. Chartier-Harlin MC, Crawford F, Houlden H, Warren A, Hughes D, Fidani L, Goate A, Rossor M, Roques P, Hardy J, Mullan M. Early-onset Alzheimer’s disease caused by mutations at codon 717 of the beta-amyloid precursor protein gene. Nature 1991; 353:844– 846. 94. Goate A, Chartier-Harlin MC, Mullan M, Brown J, Crawford F, Fidani L, Giuffra L, Haynes A, Irving N, James L, Mant R, Newton P, Rooke K, Roques P, Talbot C, Pericak-Vance M, Roses A, Williamson R, Rossor M, Owen M, Hardy J. Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer’s disease. Nature 1991; 349:704 – 706.

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95. Murrell J, Farlow M, Ghetti B, Benson MD. A mutation in the amyloid precursor protein associated with hereditary Alzheimer’s disease. Science 1991; 254:97 – 99. 96. LaVaute T, Smith S, Cooperman S, Iwai K, Land W, Meyron-Holtz E, Drake SK, Miller G, Abu-Asab M, Tsokos M, Switzer R III, Grinberg A, Love P, Tresser N, Rouault TA. Targeted deletion of the gene encoding iron regulatory protein-2 causes misregulation of iron metabolism and neurodegenerative disease in mice. Nat Genet 2001; 27:209– 214. 97. Bradbear RA, Bain C, Siskind V, Schofield FD, Webb S, Axelsen EM, Halliday JW, Bassett ML, Powell LW. Cohort study of internal malignancy in genetic hemochromatosis and other chronic nonalcoholic liver diseases. J Natl Cancer Inst 1985; 75:81– 84. 98. Le NTV, Richardson DR. The role of iron in cell cycle progression and the proliferation of neoplastic cells. Biochim Biophys Acta 2002; 1603:31– 46.

21 Dietary Selenium and Gene Expression Alexandra Fischer University of Oxford, Oxford, UK

Josef Pallauf Justus-Liebig-University of Giessen, Giessen, Germany

Introduction Selenium Metabolism Symptoms of Dietary Selenium Deficiency Regulation of Selenoprotein Expression Dietary Selenium and Selenoprotein Expression Dietary Selenium and Differential Gene Expression References

441 442 443 444 447 449 452

INTRODUCTION The element selenium (Se34) was discovered in 1817 by the Swedish chemist Jacob J. Berzelius. Historically, selenium was regarded as a naturally occurring toxic agent, but this perspective has undergone a radical transformation in the past 50 years. In 1973, it was established that selenium is an essential micronutrient in mammals (1,2). It exerts a number of important health benefits including protection against oxidative stress, cancer, AIDS, inflammatory diseases, and male infertility. On the molecular level, selenium shows extraordinary behavior, as it is incorporated into proteins as selenocysteine, for which the UGA codon is 441

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transformed from one that signals translation termination to one specific for selenocysteine.

SELENIUM METABOLISM The absorption of selenium from relevant organic and inorganic resources is usually very high (70 – 95%). In contrast to many other micronutrients, the absorption is not influenced by the selenium status of the organism (3), so that selenium-homeostasis is believed to be regulated by excretion via the urine (4). The amount and the actual transport through the intestinal border are dependent on the selenium compound. For selenate a sodium cotransport, as well as an anion exchange with hydroxylanion, has been found (5). Selenite very likely reacts with membrane-bound thiols (6) to form aminoacid-like products, for example, selenodicysteine. These products can be transported via aminoacid transporters, which are also believed to be responsible for the transport of selenoaminoacids, such as selenomethionine and selenocysteine. Throughout the intermediate metabolism of selenium, mammals synthesize several different metabolites in the course of converting inorganic selenium to organic forms and vice versa (Fig. 21.1). Hydrogen selenide (H2Se) is a key metabolite, formed from inorganic sodium selenite (oxidation state þ4) via selenodiglutathione (GSSeSG) through reduction by thiols and NADPH-dependent

selenate

selenite

selenocysteine

4 GSH

selenomethionine

nonspecific proteins (e.g. albumin)

GSSG

GSSeSG NADPH

Se±0

+

NADP + GSSG

specific selenoproteins (e.g. GPx,TrxR)

tRNASEC

H2Se

+CH3

CH3SeH +CH3

(CH3)2Se

+CH3

(CH3)3Se+

Figure 21.1

Intermediate selenium metabolism, modified according to Combs (63).

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reductases. Furthermore, H2Se can be released from selenocysteine by lyase action (7). It provides selenium for the synthesis of selenoproteins after activation to selenophosphate. The reduction from selenate to selenite is believed to be analogous to the reduction of sulfur in the body (8). Nonspecific incorporation of selenium into proteins occurs through substitution of selenomethionine for methionine, especially under high selenomethionine intake (9). Selenium can be excreted either via the lungs or via the urine after methylation from selenide to methylselenocysteine, dimethylselenide, or trimethylselenonium. Almost all selenium found in the feces represents the nonabsorbed selenium from the diet (10). The dose and status of selenium influence the amount and form of excretion. In rats given orally 16 mg Se/kg body weight, only 10% of the total selenium excreted in urine consisted of trimethylselenonium, whereas in animals which received doses of 1500 mg Se/kg body weight, it was the main excretion form (65%) in urine (11). Injections in pharmacological doses (5 mg/kg body weight) led 24 h later to excretion of selenium via the lungs as dimethlyselenide in rats (12). SYMPTOMS OF DIETARY SELENIUM DEFICIENCY Dietary selenium deficiency has been implicated as a factor in Keshan disease, a cardiomyopathy that affects young women and children in certain regions of China that have selenium-poor soil. This disease can be prevented by selenium supplementation, as can Kashin-Beck disease, a deforming osteoarthritis also found in China. Although these selenium deficiency diseases have been recognized for sometime, evidence is mounting that less-overt deficiency can also cause health problems and furthermore that supra-nutritional levels of selenium may give additional protection from disease. Whereas deficiency has an adverse effect on immunocompetence, selenium supplementation appears to enhance the immune response. Selenium appears to be a key nutrient in counteracting certain viral infections; thus, in a selenium-deficient host, the benign coxsackie virus becomes virulent causing heart damage (13), the influenza virus causes more serious lung pathology (14), and HIV infection progresses more rapidly to AIDS (15). Furthermore, moderate selenium insufficiency may contribute to male infertility (16) and aging (17). Findings have been equivocal in linking selenium to cardiovascular disease risk, although other conditions involving oxidative stress and inflammation have shown some association with selenium status (18). There is growing evidence that higher selenium intakes are associated with reduced cancer risk. The results of animal experiments, epidemiological studies, and clinical intervention trials support the hypothesis that dietary selenium reduces the risk of certain types of cancers (19). These effects are at least partly due to the function of selenium as an essential factor in the selenoproteins, whose expression is reduced when selenium is limited. It could be demonstrated that the function of one or more of these selenoproteins is essential for life, as a knockout of the selenium-specific tRNA gene

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in mice resulted in early embryonic lethality (20). In these proteins, selenium is present in the form of selenocysteine (Sec). The estimates of the actual number of selenoproteins vary greatly. Recently, Kryukov et al. (21) have identified selenoprotein genes in sequenced mammalian genomes by searching for selenocysteine insertion RNA structures, the coding potential of UGA codons, and the presence of cysteine-containing homologs. By this method, they have identified 25 selenoproteins in the human genome, but the functions of the encoded proteins are largely unknown (Table 21.1). Selenoproteins with known functions play critical roles in a variety of biological processes, and several of them are involved in antioxidant defense. Two of the glutathione peroxidases (GPx), the cellular GPx and the plasmatic GPx, protect cells against peroxidative damage by reducing hydrogen peroxide and free fatty acid hydroperoxides (22). An antioxidant function has also been proposed for the plasma protein, selenoprotein P (SelP) (23). Another GPx family member, phosopholipid hydroperoxide glutathione peroxidase (PHGPx), reduces phospholipids, cholesterol, and cholesteryl ester hydroperoxides, thereby protecting cells against membrane lipid peroxidation (24). PHGPx also plays a structural role in the mitochondrial capsule of mature spermatozoa where the protein becomes oxidatively cross-linked and inactive (16). This noncatalytic function of PHGPx may be responsible for the male infertility seen in selenium deficiency. Further selenoproteins in mammals are three thioredoxin reductases, which function in cellular redox homeostasis by reducing thioredoxin and other substrates. This could be the link to the protective role of selenium in inflammatory diseases (25). Other oxido-reductases that contain Sec include the family of diodinases, which are involved in thyroid hormone metabolism (26), and selenophosphate synthetase 2 (SPS2), which synthesizes the selenium donor for Sec biosynthesis. This enzyme is unique in that it is the only selenoprotein expressed in both prokaryotes and eukaryotes (27). Several selenoproteins have no known enzymatic activity, including SelW, which is expressed in cardiac and skeletal muscles (28), and Sep15, which is implicated in preventing prostate cancer (29). In addition, recent studies have revealed that one of the new selenoproteins, SelR, is a zinc-containing methionine sulfoxide reductase (30). With the discovery of this enzyme, a possibility is raised that selenium is also involved in aging as an enzyme catalyzing a complementary reaction (MsrA) has been implicated in antioxidant defense and the life-span of mammals (31).

REGULATION OF SELENOPROTEIN EXPRESSION Given that the nutritional requirement for selenium is partly related to its function in the selenoproteins, the synthesis of this class of proteins is important. In prokaryotes, archaebacteria, and eukaryotes, Sec is encoded by a UGA codon. The incorporation of Sec into protein requires a novel Sec-charged tRNA that

Dietary Selenium and Gene Expression

Table 21.1

445

Mammalian Selenocysteine-Containing Proteins, Modified According to

Refs. (21,40)

Selenoprotein Cytosolic GPx Gastrointestinal GPx Plasma GPx

Phospholipid hydroperoxide GPx 0

Common abbreviations

Sec location in protein (length of protein)

cGPx, GPx1 GI-GPx, GPx2

47 (201) 40 (190)

Protection of oxidation Local redoxprotection?

pGPx, GPx3

73 (226)

PHGPx, GPx4

73 (197)

GPx6

73 (221)

Redoxbuffer?, regulation of prostanoidmetabolism? Redoxregulation, sperm maturation Unknown

0

Function

5 -Deiodinase, type I

5 DI-I

126 (249)

50 -Deiodinase, type II

50 DI-II

133 (265)

50 -Deiodinase, type III

50 DI-III

144 (278)

Thioredoxin reductase

TR1, TrxR

498 (499)

Thioredoxin reductase 2 Mitochondrial thioredoxin reductase

TR2

655 (656)

Reduction of thioredoxin, DNA-synthesis, thioldisulfide balance Unknown

TR3

522 (523)

Unknown

Selenophosphate synthetase-2

SPS2

60 (448)

Selenoprotein synthesis

15 kDa selenoprotein Selenoprotein H Selenoprotein I Selenoprotein K Selenoprotein M Selenoprotein N Selenoprotein O

Sep15

93 (162)

SelH SelI SelK SelM SelN SelO

44 (122) 387 (397) 92 (94) 48 (145) 428 (556) 667 (669)

Unknown, proteinfolding? Unknown Unknown Unknown Unknown Unknown Unknown

T3-synthesis: T4 þ 2e2 þ Hþ ! T3 þ I2; degradation of T3 and T4 T3-synthesis: T4 þ 2e2 þ Hþ ! T3 þ I2 Degradation of T3 and T4

(continued )

446

Table 21.1

Fischer and Pallauf

Continued

Selenoprotein

Common abbreviations

Selenoprotein P

SelP

Methionine sulfoxide reductase Selenoprotein Selenoprotein Selenoprotein Selenoprotein

SelR

S T V W

SelS SelT SelV SelW

Sec location in protein (length of protein) 59, 300, 318, 330, 345, 352, 367, 369, 376, 378 (381) 95 (116)

188 36 273 13

(189) (182) (346) (87)

Function Unknown, redoxprotection? Antioxidant defense? Unknown Unknown Unknown Unknown, redoxprotection?

contains the anticodon UGA. As UGA normally signals the termination of protein synthesis, how does the ribosome cope with a codon that encodes two different functions? The underlying mechanisms in eukaryotes are poorly understood and are under intensive investigation, but some insights can be drawn from the genetic and biochemical studies of selenoprotein synthesis in Escherichia coli carried out by Bo¨ck and co-workers (32). Four genes (selA –selD) were identified as being involved in this process. The gene products have been identified as a selenocysteine synthase (SELA), a selenocysteine-specific elongation factor (SELB), a selenocysteine-specific tRNA (tRNASec, selC gene product), and a selenophosphate synthetase (SELD). tRNASec is first charged with serine by seryltRNA synthetase. Selenocysteine synthase then catalyzes the conversion of seryl-tRNASec to selenocysteyl-tRNASec using selenophosphate as the active selenium donor (33). Selenophosphate synthetase catalyzes the synthesis of selenophosphate from selenide and ATP. Recoding of UGA as a selenocysteine codon requires a mRNA secondary structure called selenocysteine-insertion sequence (SECIS), which is localized in eukaryotes in the 30 -nontranslated region and thus decoding from an appreciable distance is necessary (33). For the incorporation, at least two additional proteins are involved, the elongation factor eEFsec (SELB homolog) and SBP2 (34). SECIS elements function by recruiting SBP2 to form a tight SECIS– SBP2 complex. An RNA-binding domain was identified in the C-terminal sequence of SBP2 and an additional domain was identified that was required for Sec insertion. Besides binding to SECIS elements, SBP2 binds eEFsec which in turn recruits tRNASec and inserts Sec into nascent polypeptides in response to UGA codons (35).

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This mechanism shows that Sec is dramatically different from any other of the 20 protein aminoacids in the mode of its incorporation and basic biosynthetic steps. It is the only aminoacid that directly requires a structural element in mRNA in addition to the information specified by the genetic code. It is synthesized on its own tRNA, whereas free Sec is not a substrate for selenoprotein synthesis. The Sec biosynthetic machinery is strikingly different from that of other aminoacids and employs additional Sec-specific components. These unique features of Sec biosynthesis and insertion favor the view that Sec was added to the already existing genetic code to take advantage of the unique chemistry of selenium to counteract environmental stress and/or evolve new functions (36). This is underlined by the fact that most of the functions of selenoenzymes identified to date catalyze oxido-reduction reactions in which the selenocysteine residue exists in the active site. Substitution of the structurally related aminoacid, cysteine, for selenocysteine produced functional enzymes, but significantly decreased the Kcat , indicating that the presence of selenocysteine confers specific biochemical properties. DIETARY SELENIUM AND SELENOPROTEIN EXPRESSION The biosynthesis of selenoproteins is primarily dependent on the bioavailability of selenium and consequently on the formation of tRNASec, which regulates the level of all selenoproteins. In selenium deficiency, UGA is interpreted as a stop-codon, which results in a premature chain termination at the UGA codon. The decrease in selenoprotein synthesis is further accompanied by a fall in the levels of the respective mRNAs. This is not due to a lower transcription rate but due rather to a loss of stability, for example, enhanced degradation of the mRNA (37). This occurs by the surveillance pathway, designated nonsensemediated decay, where the UGA Sec codon is recognized as nonsense (38,39). For an optimum translation of a selenoprotein apart from selenium supply, its mRNA appears to require an optimum UGA location, an optimum UGA context, and the SECIS element to optimize selenocysteine incorporation. Surprisingly, individual selenoproteins vary in their specific responses to selenium deprivation, a phenomenon referred to as “the hierarchy of selenoproteins” (40). Although some of the selenoproteins disappear rapidly in selenium deprivation and are slowly resynthesized when resupplemented, others decline gradually in the presence of a limited selenium supply and rebound immediately at resupplementation. After 8– 10 weeks of selenium deficiency, cGPx activity in the liver of pigs (41), rats (42), and rabbits (43) was reduced to 3– 10% when compared with selenium supplemented control animals. In other organs (e.g., heart and skeletal muscles), the extent of the loss of activity was considerably less given an inherent generally lower cGPx activity in these organs (41,43). The greater sensitivity of cGPx activity to selenium deficiency has been attributed largely to an increased turnover in mRNA (44). The position of the UGA Sec codon relative to the sole, downstream intron in cGPx mRNA determines whether the mRNA is subject to nonsense-mediated decay (45). However,

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other selenoprotein mRNAs, such as 50 DI-I, PHGPx, and SelP, are not as sensitive as cGPx during selenium deprivation despite the presence of introns downstream of their UGA codons (46). SBP2 has also been reported as preferentially recognizing SECIS elements in specific selenoprotein mRNAs suggesting a mechanism which accounts, at least in part, for selenoprotein expression hierarchy during selenium deficiency (47). In selenium-deficient rats and rabbits, cGPx mRNA was found to be downregulated by 85– 90% after 8 –10 weeks of selenium deficiency (42,48). Irrespective of the mechanisms involved, the hierarchy in the biosynthesis of selenoproteins is considered to reflect their biological importance. The following ranking has been suggested: GI-GPx . TrxR, SelP, 50 DI-I . cGPx Furthermore, a characteristic of selenium deficiency in mammals is that a hierarchy exists with respect to the retention of selenium in different organs. During selenium deprivation in the diets of rats and mice, the amounts of this element were substantially reduced in the liver and kidney, whereas the brain and testes retained most of their selenium (49 – 51). The same finding is reflected in selenoprotein activities. In rats made selenium-deficient for two generations, total GPx activity was below 5% in liver, kidney, lung, heart, adrenal gland, pancreas, and muscle, below 10% in male thymus and testes, but only decreased to 50% in the brain and to 25% in the ovaries and female thymus (52). Interestingly, transgenic mice expressing i6-deficient Sec tRNA(Ser)Sec had reduced levels of selenium in their tissues and a hierarchy of selenoprotein activities similar to that observed with selenium-deficient mice (53). From these results, it can be concluded that mechanisms must exist that guide selenium to a specific enzyme within a particular tissue, probably via tissuespecific regulation of mRNA stability. cGPx and PHGPx are preferentially retained under selenium-limiting conditions in the brain, reproductive organs, and endocrinological tissues. Brigelius-Flohe´ (54) proposes the ranking order of tissue-specific stabilities, which do not reflect absolute activities, for cGPx as follows: brain
thymus . thyroid . heart . liver, kidney, lung For some selenoproteins, a hormone-dependent regulation could be observed. Human TrxR is rapidly induced in monocytes through 1,25-dihydroxy vitamin D3 and in fetal osteoblast-like cells through certain cytokines and growth factors (55). The three deiodinase isoenzymes are regulated by thyroid hormones, retinoids, sexual hormones, gluco- and corticosteroids, and a series of growth factors and cytokines (56). For PHGPx, a sexual hormone-dependent expression has been suggested. An induction of GPx genes by oxidative stress was often postulated, but has never been convincingly demonstrated in vivo. The induction of cGPx through an oxygen-responsive element has only been described in vitro (57).

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DIETARY SELENIUM AND DIFFERENTIAL GENE EXPRESSION Apart from the direct influence of dietary selenium intake and selenium status on selenoprotein expression, selenium seems to have a major impact on overall gene expression as well. With the introduction of array technology to measure differential expression of thousands of genes simultaneously, new insights into selenium-dependent gene expression in rodents have been revealed in recent years. The results from these studies, which will be elucidated in more detail in the following section, are summarized in Table 21.2. Rao et al. (58) used Affymetrix high density oligonucleotide arrays (Gene Chipw MU6500) representing 6347 murine genes to determine the transcriptional profile associated with low selenium status in the intestines of C57BI/6J mice. Mice were fed with either a torula yeast, selenium-deficient diet (,0.01 mg Se/kg), or the same diet supplemented with a high level of dietary selenium (1 mg Se/kg, as seleno-L -methionine) for 90 days. Total RNA for the expression profiles was obtained from the middle segment of the small intestine corresponding to the jejunum after 111 days of feeding. A comparison between mice fed with the selenium-deficient diet or the high selenium diet revealed that selenium status was associated with alterations in specific mRNA levels, which may reflect changes in gene expression, mRNA stability, or both. Of the 6347 genes surveyed, 48 displayed a greater than 2-fold decrease in expression in response to low selenium status, whereas 84 displayed a greater than 2-fold increase in expression. These genes were consistent with a state of DNA damage, genetic instability, and oxidative stress. These included alterations in expression of the cell cycle arrest/DNA damage inducible genes GADD34, GADD45ß, GADDg, and XP-E, as well as the molecular chaperones hsp27 and hsp40.

Table 21.2 Global View of Transcriptional Changes Induced by Dietary Selenium Deficiency in Rodents #Selenoproteins Reduced antioxidant capacity "Apoptosis/cell cycle control/oncogenesis Induction of: Protein phosphorylation Signal transduction Angiogenesis Cell adhesion Intestine specific changes #Xenobiotic metabolism #Lipid metabolism Tumorigenic stress specific changes #Decreased apoptotic ability

"Stress response/inflammation Induction of: Heat shock response DNA damage-inducible genes Oxidative stress-inducible genes Liver specific changes "Xenobiotic metabolism

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Also induced were the mitogen- and stress-activated protein kinase AMPKg, metallothionein-I, a free radical scavenger implicated in oxidative damage protection, and MDM2, an oncogene. Low selenium status was also associated with changes in the expression of genes involved in cell proliferation, such as M-phase inducer phosphatase 2, G2/mitotic-specific cyclin B2, cyclin-dependent kinase 1 and PAC-1. Furthermore, of the 48 genes that decreased in expression in mice of low selenium status, 15% were genes that participate in lipid metabolism, especially in lipid transport, including apolipoprotein AI, AIV, CIII, APOBEC-1, nonspecific lipid-transfer protein, and fatty acid binding protein. Apo-AI is the major determinant of the capacity of HDL particles to promote cholesterol efflux and is associated with the inhibition of atherosclerosis. Zeng et al. (59) investigated the protective role for selenium enriched broccoli in tumorigenesis, thereby a gene profile in the liver of multiple intestinal neoplasi (Min) mice was conducted. Mice were fed with either 0.11 mg Se/kg control diet or 2.1 mg Se/kg selenobroccoli diets for 10 weeks. Expression profiles were obtained in mouse liver samples with mouse pathways finder-1 GEArray membrane (Superarry, Bethesda, MD, USA), which contained 23 sequence-verified known marker genes. Furthermore, the authors applied DNA mobility shift assay to define the transcriptional response in Min mouse liver. When compared with the low selenium control diet, selenium-enriched broccoli upregulated the mRNA levels of ikBa, hsp86, and gadd45. In addition, the analysis of binding of liver nuclear proteins to 32P-labeled probes demonstrated that seleniumenriched broccoli enhanced the binding of the transcription factors p53, NF1B, and AP-1 to their cis-acting elements. ikBa and NF1B are main mediators of the cellular response to a variety of extracellular stress stimuli, such as initiating apoptosis. Similarly, the expression of hsp86 and gadd45 genes correlated with induction of apoptosis. These results suggest that dietary selenium intake can activate pro-apoptotic gene expression and enhance the DNA binding of pro-apoptotic transcription factors in response to tumorigenic stress. Christensen et al. (60) compared the expression in rat liver of genes for transferrin, transferring receptor, ferritin light and heavy chains, and ironregulatory proteins in selenium adequacy and deficiency. Weanling male Sprague– Dawley rats were fed with torula yeast selenium-deficient diets supplemented with either 0 or 0.15 mg Se/kg diet as sodium selenite for 15 weeks. To examine differential gene expression, a multiplex relative reverse transcriptase –polymerase reaction method was applied. Three of the six genes examined showed modest, but consistent upregulation in selenium deficiency. Transferrin mRNA was 30% more abundant in deficient liver than in selenium-adequate liver. For the transferrin receptor, the difference was 32%, and for iron regulatory protein 1, it was 63%. The authors concluded a possible role for dietary selenium in moderating iron metabolism. To examine the molecular events associated with selenium deficiency in rats, we applied cDNA array technology to define the transcriptional response in rat liver after 7 weeks on a selenium deficient torula yeast based diet

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(,0.01 mg Se/kg), compared with rats fed the control diet (200 mg Se/kg as sodium selenite) (42). AtlasTM Rat cDNA Toxicology Array II from Clontech was used to monitor simultaneously the expression of 465 genes, whereby a fold-change of two or more was considered as significant. Besides a 13.9-fold downregulation of the cGPx gene, selenium deficiency was accompanied by an increase in the expression of UDP-glucuronosyltransferase 1 and bilirubin UDP-glucuronosyltransferase isoenzyme 2. These two enzymes are known to have an important function in the detoxification of xenobiotics in liver. Likewise, rat liver cytochrome P450 4B1, also involved in xenobiotic metabolism and inducible by glucocorticoids, was induced 2.3-fold. The mRNA levels of arachidonate 12-lipoxygenase (ALOX 12) were 2.4-fold higher in selenium deficient animals when compared with controls. It has been shown that ALOX12 and PHGPx are opposite enzymes balancing the intracellular concentration of hydroperoxy lipids (61), whereby an inhibition of PHGPx activity increases the enzymatic catalysis of ALOX 12 (62). These results provide evidence that selenium deficiency has an impact on selenoprotein expression and probably as a secondary effect on induction of a stress response, for example, modulation of inflammatory and cell cycle dependent genes. This response may be due to oxidative stress, DNA damage, or both, and could be related to a reduction in the activity of selenoproteins and detoxification enzymes. Because some of the selenium-dependent enzymes, such as GPx, thioredoxin reductase, and selenoprotein P, function as antioxidants, it seems plausible that low selenium status is associated with some forms of oxidative stress. However, differences can be seen between the different animal models and/or the different tissues investigated. In the selenium-deficient mouse, no upregulation in expression of any gene involved in drug detoxification could be observed, whereas in the liver of selenium-deficient rats, genes encoding for xenobiotica metabolizing proteins, such as P450, were significantly induced. This induction may be liver specific, or may be mediated at the protein level in the intestine as opposed to increases in mRNA abundance. Taken as a whole, these results suggest that suboptimal intake of a single trace mineral can induce multiple transcriptional pathways suggestive of oxidative stress, DNA damage, and alterations in cell cycle progression and thereby can have widespread effects on gene expression patterns, providing a framework for understanding the multiple roles of selenium in human health. The overall screening for the mRNAs of genes with altered expression due to dietary selenium status has yielded new avenues of investigation and confirmed previous observations associated with dietary selenium status. Efforts to identify nutrient-regulated genes will significantly enhance the value and usefulness of the genome and EST databases by providing a physiological response that can be associated with the new genes. In addition, functional genomic techniques, such as array technology and two-dimensional protein gel electrophoresis, will play an increasing role in linking physiological perturbations to the molecular and cellular mechanisms.

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REFERENCES 1. Flohe´ L, Gunzler WA, Schock HH. Glutathione peroxidase: a selenoenzyme. FEBS Lett 1973; 32:132 – 134. 2. Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG. Selenium: biochemical role as a component of glutathione peroxidase. Science 1973; 179:588 – 590. 3. Wolffram S. Der Stoffwechsel erna¨hrungsphysiologisch relevanter anorganischer und ¨ bers Tiererna¨hr 2000; 28:65 – 94. organischer Selenverbindungen. U 4. Sunde RA. Selenium. In: O’Dell BL, Sunde RA, eds. Handbook of Nutritionally Essential Mineral Elements. New York: Marcel Dekker, 1997:493 –556. 5. Ardu¨ser F, Wolffram S, Scharrer E. Active absorption of selenate by rat ileum. J Nutr 1985; 115:1203 – 1208. 6. Mykkanen HM, Wasserman RH. Relationship of membrane-bound sulfhydryl groups to vitamin D-stimulated uptake of [75Se]selenite by the brush border membrane vesicles from chick duodenum. J Nutr 1990; 120:882 – 888. 7. Ganther HE. Selenium metabolism, selenoproteins and mechanisms of cancer prevention: complexities with thioredoxin reductase. Carcinogenesis 1999; 20:1657 –1666. 8. Axley MJ, Stadtman TC. Selenium metabolism and selenium-dependent enzymes in microorganisms. Annu Rev Nutr 1989; 9:127– 137. 9. Whanger PD, Butler JA. Effects of various dietary levels of selenium as selenite or selenomethionine on tissue selenium levels and glutathione peroxidase activity in rats. J Nutr 1988; 118:846– 852. 10. Burk RF. Biological activity of selenium. Annu Rev Nutr 1983; 3:53– 70. 11. Nahapetian AT, Janghorbani M, Young VR. Urinary trimethylselenonium excretion by the rat: effect of level and source of selenium-75. J Nutr 1983; 113:401 – 411. 12. Hassoun BS, Palmer IS, Dwivedi C. Selenium detoxification by methylation. Res Commun Mol Pathol Pharmacol 1995; 90:133– 142. 13. Beck MA, Esworthy RS, Ho YS, Chu FF. Glutathione peroxidase protects mice from viral-induced myocarditis. FASEB J 1998; 12:1143 – 1149. 14. Nelson HK, Shi Q, Van Dael P, Schiffrin EJ, Blum S, Barclay D, Levander OA, Beck MA. Host nutritional selenium status as a driving force for influenza virus mutations. FASEB J 2001; 15:1846– 1848. 15. Chen C, Zhou J, Xu H, Jiang Y, Zhu G. Effect of selenium supplementation on mice infected with LP-BM5 MuLV, a murine AIDS model. Biol Trace Elem Res 1997; 59:187– 193. 16. Ursini F, Heim S, Kiess M, Maiorino M, Roveri A, Wissing J, Flohe L. Dual function of the selenoprotein PHGPx during sperm maturation. Science 1999; 285:1393 – 1396. 17. Martin-Romero FJ, Kryukov GV, Lobanov AV, Carlson BA, Lee BJ, Gladyshev VN, Hatfield DL. Selenium metabolism in Drosophila: selenoproteins, selenoprotein mRNA expression, fertility, and mortality. J Biol Chem 2001; 276:29798 – 29804. 18. Rayman MP, Rayman MP. The argument for increasing selenium intake. Proc Nutr Soc 2002; 61:203– 215. 19. Duffield-Lillico AJ, Reid ME, Turnbull BW, Combs GF Jr, Slate EH, Fischbach LA, Marshall JR, Clark LC. Baseline characteristics and the effect of selenium supplementation on cancer incidence in a randomized clinical trial: a summary report of the Nutritional Prevention of Cancer Trial. Cancer Epidemiol Biomarkers Prev 2002; 11:630– 639.

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20. Bo¨sl MR, Takaku K, Oshima M, Nishimura S, Taketo MM. Early embryonic lethality caused by targeted disruption of the mouse selenocysteine tRNA gene (Trsp). Proc Natl Acad Sci USA 1997; 94:5531– 5534. 21. Kryukov GV, Castellano S, Novoselov SV, Lobanov AV, Zehtab O, Guigo R, Gladyshev VN. Characterization of mammalian selenoproteomes. Science 2003; 300:1439– 1443. 22. Arthur JR. The glutathione peroxidases. Cell Mol Life Sci 2000; 57:1825 – 1835. 23. Hill KE, Burk RF. Selenoprotein P: recent studies in rats and in humans. Biomed Environ Sci 1997; 10:198 –208. 24. Maiorino M, Coassin M, Roveri A, Ursini F. Microsomal lipid peroxidation: effect of vitamin E and its functional interaction with phospholipid hydroperoxide glutathione peroxidase. Lipids 1989; 24:721 – 726. 25. Flohe´ L, Brigelius-Flohe R, Saliou C, Traber MG, Packer L. Redox regulation of NF-kappa B activation. Free Radic Biol Med 1997; 22:1115– 1126. 26. Ko¨hrle J, Brigelius-Flohe´ R, Bo¨ck A, Gartner R, Meyer O, Flohe´ L. Selenium in biology: facts and medical perspectives. Biol Chem 2000; 381:849 – 864. 27. Guimaraes MJ, Peterson D, Vicari A, Cocks BG, Copeland NG, Gilbert DJ, Jenkins NA, Ferrick DA, Kastelein RA, Bazan JF, Zlotnik A. Identification of a novel selD homolog from eukaryotes, bacteria, and archaea: is there an autoregulatory mechanism in selenocysteine metabolism? Proc Natl Acad Sci USA 1996; 93:15086–15091. 28. Yeh JY, Vendeland SC, Gu Q, Butler JA, Ou BR, Whanger PD. Dietary selenium increases selenoprotein W levels in rat tissues. J Nutr 1997; 127:2165 – 2172. 29. Hu YJ, Korotkov KV, Mehta R, Hatfield DL, Rotimi CN, Luke A, Prewitt TE, Cooper RS, Stock W, Vokes EE, Dolan ME, Gladyshev VN, Diamond AM. Distribution and functional consequences of nucleotide polymorphisms in the 30 -untranslated region of the human Sep15 gene. Cancer Res 2001; 61:2307 – 2310. 30. Kryukov GV, Kumar RA, Koc A, Sun Z, Gladyshev VN. Selenoprotein R is a zinccontaining stereo-specific methionine sulfoxide reductase. Proc Natl Acad Sci USA 2002; 99:4245 –4250. 31. Moskovitz J, Bar-Noy S, Williams WM, Requena J, Berlett BS, Stadtman ER. Methionine sulfoxide reductase (MsrA) is a regulator of antioxidant defense and lifespan in mammals. Proc Natl Acad Sci USA 2001; 98:12920 – 12925. 32. Ehrenreich A, Forchhammer K, Tormay P, Veprek B, Bo¨ck A. Selenoprotein synthesis in E. coli. Purification and characterisation of the enzyme catalysing selenium activation. Eur J Biochem 1992; 206:767 –773. 33. Low SC, Berry MJ. Knowing when not to stop: selenocysteine incorporation in eukaryotes. Trends Biochem Sci 1996; 21:203– 208. 34. Copeland PR, Driscoll DM. Purification, redox sensitivity, and RNA binding properties of SECIS-binding protein 2, a protein involved in selenoprotein biosynthesis. J Biol Chem 1999; 274:25447 – 25454. 35. Fagegaltier D, Hubert N, Yamada K, Mizutani T, Carbon P, Krol A. Characterization of mSelB, a novel mammalian elongation factor for selenoprotein translation. EMBO J 2000; 19:4796 –4805. 36. Hatfield DL, Gladyshev VN. How selenium has altered our understanding of the genetic code. Mol Cell Biol 2002; 22:3565 – 3576. 37. Bermano G, Arthur JR, Hesketh JE. Selective control of cytosolic glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase mRNA stability by selenium supply. FEBS Lett 1996; 387:157 – 160.

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38. Moriarty PM, Reddy CC, Maquat LE. Selenium deficiency reduces the abundance of mRNA for Se-dependent glutathione peroxidase 1 by a UGA-dependent mechanism likely to be nonsense codon-mediated decay of cytoplasmic mRNA. Mol Cell Biol 1998; 18:2932 – 2939. 39. Weiss SL, Sunde RA. Cis-acting elements are required for selenium regulation of glutathione peroxidase-1 mRNA levels. RNA 1998; 4:816– 827. 40. Flohe´ L, Andreesen JR, Brigelius-Flohe´ R, Maiorino M, Ursini F. Selenium, the element of the moon, in life on earth. IUBMB Life 2000; 49:411– 420. 41. Lei XG, Dann HM, Ross DA, Cheng WH, Combs GF, Roneker KR. Dietary selenium supplementation is required to support full expression of three selenium-dependent glutathione peroxidases in various tissues of weanling pigs. J Nutr 1998; 128:130–135. 42. Fischer A, Pallauf J, Gohil K, Weber SU, Packer L, Rimbach G. Effect of selenium and vitamin E deficiency on differential gene expression in rat liver. Biochem Biophys Res Commun 2001; 285:470 – 475. 43. Mu¨ller AS, Pallauf J, Most E. Parameters of dietary selenium and vitamin E deficiency in growing rabbits. J Trace Elem Med Biol 2002; 16:47– 55. 44. Christensen MJ, Burgener KW. Dietary selenium stabilizes glutathione peroxidase mRNA in rat liver. J Nutr 1992; 122:1620 – 1626. 45. Sun X, Moriarty PM, Maquat LE. Nonsense-mediated decay of glutathione peroxidase 1 mRNA in the cytoplasm depends on intron position. EMBO J 2000; 19:4734– 4744. 46. Lei XG, Evenson JK, Thompson KM, Sunde RA. Glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase are differentially regulated in rats by dietary selenium. J Nutr 1995; 125:1438 –1446. 47. Low SC, Grundner-Culemann E, Harney JW, Berry MJ. SECIS-SBP2 interactions dictate selenocysteine incorporation efficiency and selenoprotein hierarchy. EMBO J 2000; 19:6882 – 6890. 48. Mu¨ller AS, Pallauf J. Down-regulation of GPx1 mRNA and the loss of GPx1 activity causes cellular damage in the liver of selenium-deficient rabbits. J Anim Physiol Anim Nutr (Berl) 2002; 86:273– 287. 49. Bermano G, Nicol F, Dyer JA, Sunde RA, Beckett GJ, Arthur JR, Hesketh JE. Tissuespecific regulation of selenoenzyme gene expression during selenium deficiency in rats. Biochem J 1995; 311:425 – 430. 50. Behne D, Wolters W. Distribution of selenium and glutathione peroxidase in the rat. J Nutr 1983; 113:456– 461. 51. Hill KE, Lyons PR, Burk RF. Differential regulation of rat liver selenoprotein mRNAs in selenium deficiency. Biochem Biophys Res Commun 1992; 185:260 –263. 52. Thompson KM, Haibach H, Sunde RA. Growth and plasma triiodothyronine concentrations are modified by selenium deficiency and repletion in second-generation selenium-deficient rats. J Nutr 1995; 125:864 –873. 53. Moustafa ME, Carlson BA, El Saadani MA, Kryukov GV, Sun QA, Harney JW, Hill KE, Combs GF, Feigenbaum L, Mansur DB, Burk RF, Berry MJ, Diamond AM, Lee BJ, Gladyshev VN, Hatfield DL. Selective inhibition of selenocysteine tRNA maturation and selenoprotein synthesis in transgenic mice expressing isopentenyladenosine-deficient selenocysteine tRNA. Mol Cell Biol 2001; 21:3840– 3852. 54. Brigelius-Flohe´ R. Tissue-specific functions of individual glutathione peroxidases. Free Radic Biol Med 1999; 27:951 – 965.

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55. Schu¨tze N, Fritsche J, Ebert-Dumig R, Schneider D, Ko¨hrle J, Andreesen R, Kreutz M, Jakob F. The selenoprotein thioredoxin reductase is expressed in peripheral blood monocytes and THP1 human myeloid leukemia cells—regulation by 1,25-dihydroxyvitamin D3 and selenite. Biofactors 1999; 10:329 – 338. 56. Ko¨hrle J. The deiodinase family: selenoenzymes regulating thyroid hormone availability and action. Cell Mol Life Sci 2000; 57:1853– 1863. 57. Cowan DB, Weisel RD, Williams WG, Mickle DA. Identification of oxygen responsive elements in the 50 -flanking region of the human glutathione peroxidase gene. J Biol Chem 1993; 268:26904 – 26910. 58. Rao L, Puschner B, Prolla TA. Gene expression profiling of low selenium status in the mouse intestine: transcriptional activation of genes linked to DNA damage, cell cycle control and oxidative stress. J Nutr 2001; 131:3175– 3181. 59. Zeng H, Davis CD, Finley JW. Effect of selenium-enriched broccoli diet on differential gene expression in min mouse liver. J Nutr Biochem 2003; 14:227 – 231. 60. Christensen MJ, Olsen CA, Hansen DV, Ballif BC. Selenium regulates expression in rat liver of genes for proteins involved in iron metabolism. Biol Trace Elem Res 2000; 74:55– 70. 61. Schnurr K, Borchert A, Kuhn H. Inverse regulation of lipid-peroxidizing and hydroperoxyl lipid-reducing enzymes by interleukins 4 and 13. FASEB J 1999; 13:143–154. 62. Chen CJ, Huang HS, Lin SB, Chang WC. Regulation of cyclooxygenase and 12-lipoxygenase catalysis by phospholipid hydroperoxide glutathione peroxidase in A431 cells. Prostaglandin Leukot Essent Fatty Acids 2000; 62:261 – 268. 63. Combs GF. Chemopreventive mechanisms of selenium. Med Klin 1999; 94:18 – 24.

22 Modulation of Gene Expression by Dietary Zinc Raymond K. Blanchard and Robert J. Cousins University of Florida, Gainesville, Florida, USA

Introduction Zinc as an Antioxidant Ionic Zinc MT as an Antioxidant Molecular Mechanisms of Zinc-Regulated Gene Expression Expression Profiling of Zinc-Regulated Genes References

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INTRODUCTION Zinc is defined in classical literature as a redox neutral atom. This is in contrast to other nutrient trace elements, such as iron and copper, which change ionic charge during biological functions. Functions of zinc have been categorized as catalytic, structural, and regulatory (1). In each of these functional categories, zinc plays a role more analogous to calcium, perhaps related to exchange rates with a ligand center, in comparison with the redox metals such as copper and iron (2). Consequently, the properties of zinc that are considered as antioxidant in nature are indirect and do not involve direct interaction with reactive oxidant species. 457

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Literature on the antioxidant properties of zinc is voluminous and involves descriptions of the effects of cellular defense against oxidative and nitrosative stresses (3 –5). Therefore, our comments will be focused on the nutritional genomics of zinc as an antioxidant. Much of this available information was generated with in vitro systems, where individual molecules interact depending on redox potentials, or with isolated cells perturbed to induce a change in redox state and/or initiate a stress response. Evidence with integrative systems occasionally supports the participation of zinc in one or more antioxidant functions. The purpose of this review is to discuss evidence on zinc-regulated genes and induction of zinc-responsive networks that have antioxidant properties. ZINC AS AN ANTIOXIDANT Ionic Zinc Galvanizing, the industrial coating of iron-containing metal objects with zinc to prevent oxidation (rust formation), has been in practice for over a century. Zinc is a strong Lewis acid (electron acceptor) that binds strongly to thiolates and amines (2). For most functions of zinc, it must undergo intracellular trafficking much like calcium with concentrations regulated by transporters. These concepts were initially developed by Williams and colleagues (2). Estimates of the free Zn2þ concentration are placed at or below the nanomolar range (2), but may actually be in the order of only a few atoms per cell (6). The trafficking rate of zinc may be the most important factor so that, while only a few atoms may be “free,” the flux rate through that “free” pool may be tremendous. Quantitation of the “free” zinc pool size, though important, is less relevant to the concept of zinc trafficking than is the Zn2þ exchange rate from ligands of specific binding complexes. Exchange rates are more a function of secondary/ tertiary protein structure and folding energies than the binding constant of a specific motif for zinc. Zinc exchange rates for metalloproteins range from minutes, in the case of metallothionein (MT), to days (alkaline phosphatase) (2). Relevant to antioxidant properties is that, when zinc is bound to a redoxactive species, for example, a protein thiol, oxidation can result in Zn2þ release (4), which may involve a transition into the “free” zinc pool. That release would be followed by rebinding of Zn2þ to another ligand or transport into a vesicle. MT as an Antioxidant A number of important findings converge to strongly suggest MT, with its high cysteine-thiol content and ability to bind seven atoms of Zn2þ, has a role in cellular redox. Paramount among these is the inducibility of the MT gene by zinc and specific hormones and cytokines [reviewed in Ref. (7)]. Other issues include: (i) correlation of cytoprotection and MT induction; (ii) altered cellular response to oxidative stress in MT-null mice; (iii) evidence for MT in protective

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mechanisms in both oxidative and nitrosative stress (5); (iv) indications that some interactions between MT and specific redox-active molecules [e.g., nitric oxide (NO)] may have signaling roles in normal physiology, including antimicrobial activity [reviewed in Ref. (7)]. An antioxidant function for MT was first advanced in 1985 by Thornalley and Vasak (8). Using in vitro radical-generating systems, they observed that MT was particularly effective in quenching OH† radicals. Subsequent findings by numerous laboratories have implicated MT in antioxidant roles for reactive oxygen species [ROS (including OH† and superoxide)], carbon-centered radicals (e.g., CCl3† ), and nitrogen species [e.g., nitric oxide (NO) and peroxynitrite] (3 – 5,7,9,10). Confounding a definitive relationship has been the difficulty in separating the effects of zinc from those produced by MT (11). This was particularly an issue where zinc is capable of causing MT induction in cell and animal models. Further complicating interpretations of zinc and MT antioxidant effects are the concurrent effects of other nutrient antioxidants (e.g., vitamins C and E) and cellular antioxidant systems, including the glutathione (GSH – GSSG) couple, catalase, glutathione S-transferases, superoxide dismutase, and glutathione peroxidases (12). In addition, effects of zinc nutritional status (both deficient and excessive) in animal models, those of zinc treatment of cells, and the MT induction in cells and animals by oxidants all present difficulties in delineating unequivocal zinc/MT antioxidant effects. These problems are not necessarily circumvented with the use of transgenic knockout or MT-overexpressing mice or cells, as compensatory mechanisms are frequently induced, which can influence interpretation of the results. As will be discussed subsequently, the spectrum of zinc-regulated genes is such that many yet uncharacterized proteins may influence zinc-related antioxidant effects. A theoretical breakthrough to explain the antioxidant properties of MT was produced by Maret and colleagues. Specifically, their data suggest that MT should be viewed as a reduced species that, upon interaction with an oxidant, results in sulfhydryl oxidation and Zn2þ release (4). Mobilization of Zn2þ from metalloproteins by the oxidant hypochlorous acid (HOCl) had previously been demonstrated. HOCl is produced by neutrophils to inactivate cellular proteins during host defense. Zn2þ released by HOCl was advanced as a mechanism to explain oxidant-induced tissue injury during inflammation (13). In contrast, Pitt and coworkers proposed S-nitrosylation of MT sulfhydryls by NO stress regulates the MT – thionein redox pair through Zn2þ release (9). NMR evidence suggests NO selectively releases Zn2þ from the amino-terminal (b domain) metal cluster of MT, which coordinates three zinc atoms (14). The b domain of MT is believed to have a lower binding affinity for Zn2þ, suggesting that it may function in homeostasis. NO appears to preferentially release those zinc ions. It has been suggested that NO may interact with bacterial zinc finger proteins involved in growth, causing Zn2þ release as well as inhibition of bacterial DNA replication (15). Intracellular pathogens may be killed by NO-producing phagocytic cells through such a mechanism. NO-induced

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release of Zn2þ from cytoplasmic and nuclear pools was actually documented a decade ago (16). Additionally, intracellular NO, produced by inducible NOS (iNOS) in response to cytokines, causes an MT-dependent release of nuclear Zn2þ in endothelial cells (17). Therefore, it is not surprising that exogenous NO causes induction of MT gene expression through release of intracellular Zn2þ in these cells. Of note in this context is that dietary zinc deficiency produces an upregulation of intestinal iNOS mRNA stimulated by IL-1a and is associated with intestinal injury that can lead to diarrhea in a rat model (18,19). Downregulation of intestinal iNOS expression upon zinc repletion in zinc-deficient rats is rapid, suggestive of a direct link among Zn2þ, NO, and iNOS gene expression (19). A possible mechanism for the involvement of MT against carbon-centered oxidative damage is less clear. Using carbon tetrachloride (CCl4) as a hepatotoxin, isolated cultured rat hepatocytes were protected from damage when cellular zinc and MT levels were elevated by zinc added to the medium or by stimulation of MT expression with dexamethasone and IL-6 (10). A protective effect of zinc against CCl4 in rat liver was first observed three decades ago (20). Subsequently, this protective effect was ascribed to MT as a zinc-inducible antioxidant [reviewed in Ref. (7)]. When such protective effects were tested in MT-null and MT-overexpressing mice, results were less conclusive (21). Specifically, CCl4 clearly produced hepatic oxidative damage in the MT-null mice. However, MT overexpression did not have a beneficial effect. In contrast to these findings, ROS- and NO-induced damage to the pancreas was prevented in MT-overexpressing mice (22). This protective action of MT was viewed as potentially advantageous for pancreatic islet survival during early phases of transplantation. The murine pancreas is very sensitive to dietary zinc status (23), which suggests that this organ may be particularly susceptible to zincrelated oxidative effects. In that regard, it must be kept in mind that the pancreas is also a target of zinc toxicity [reviewed in Ref. (1)]. The cytoprotective role of zinc and the involvement of MT have not been fully defined. On a molecular basis, MT is clearly a reductant that, when acted upon by an oxidant, undergoes oxidative release of Zn2þ with disulfide formation in the protein (Fig. 22.1). In this context, zinc or MT should be considered as an “antioxidant.” The fate of Zn2þ could be one that activates the metal-responsive transcription factor (MTF-1) and/or other transcription factors and thus signals a cascade of modulated genes including those beneficial for health. Oxidative Zn2þ release from MTF-1 or other transcription factors could similarly influence genes that are beneficial or lead to cell death through apoptosis. MOLECULAR MECHANISMS OF ZINC-REGULATED GENE EXPRESSION Zinc-modulated gene expression has been studied most thoroughly for the MT family of genes. MT protein, with its variable abundance in many cell

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O2 Cytokines

iNOS cNOS NO Cl –

O2• – SOD MPO

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Antioxidant Effects

Figure 22.1 A hypothetical phagocytic cell responding to activation by lipopolysaccharide (LPS) and/or cytokines (e.g., IL1, IL6, or IFNg). Oxidative release of Zn2þ from the b metal-binding domain of MT by ROS (e.g., hydroxyl radical, OH† , and hypochlorite anion, ClO2) or reactive nitrogen species (e.g., nitric oxide, NO) is shown. Zn2þ-stimulated activation of the MTF-1 and zinc-responsive gene activation and decrease in activation of other genes by Zn2þ are also shown. Principal enzymes, nitric oxide synthases (iNOS and cNOS), superoxide dismutase (SOD), and myeloperoxidase (MPO) are indicated. ROS also interact with ARE transcription factors (ARE-TF ) to induce oxidative stress response genes, including the MT gene.

types, unusually high cysteine content, and tremendous zinc-binding capacity, quickly evoked questions about its cellular function as well as regulatory interactions between the protein and the essential micronutrient. Radioisotopes of zinc were used to examine the kinetics and physiological compartmentalization of the metal, but the regulation of MT gene expression awaited the advent of molecular biology. The earliest reports showing dietary zinc as a regulator of

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MT gene expression were in 1975 when Richards and Cousins (24) demonstrated that induction in both intestine and liver could be blocked by actinomycin D and therefore required RNA synthesis. Later, Shapiro and Cousins (25) were able to demonstrate similar results from isolated polyribosomes, indicating a direct increase in the amount of mRNA encoding MT. As northern blotting and other expression analysis tools have been developed, the MT genes have become the prototypical zinc-responsive gene model due in part to the large magnitude and rapid rate of change observed in both mRNA and protein. After the cloning of the MT-I gene, Mayo et al. found that the gene retained its metal responsiveness after transfection into cells (26,27). Thus began the search for the cis-acting elements within the gene that are responsible for imparting zinc, as well as cadmium, inducibility. Sequence analysis of the proximal promoters for the human and mouse MT-I and -II genes identified several conserved sequence elements including one that was implicated in metal regulation. Deletion analysis using reporter gene constructs showed that multiple 12 bp elements were involved in zinc responsiveness and these were named metal regulatory elements, or MREs (28). Each element could individually confer, with varying degrees of magnitude, metal responsiveness to an unrelated, nonresponsive promoter. Insertion of an MRE consensus element into the upstream proximal promoter of a gene was subsequently demonstrated to be necessary and sufficient to confer zinc responsiveness. The human MT-I and -II genes have seven and eight MREs, respectively, within 500 bp of the transcription start site (29), designated MREa through MREh. They are found in both directional orientations and, while the core element of the MRE is tightly conserved, the surrounding bases confer differing magnitudes of response; therefore, all MREs are not equivalent. Although MREs act synergistically, with more copies allowing greater maximal induction, different consensus variants were identified and the MT-I MREd was determined to be the most responsive (30). Another development that has confounded the characterization of MREs is the interaction of other promoter elements adjacent to or overlapping with MRE sequences. This was not surprising for MT, which was known to respond to a variety of stress stimuli in addition to toxic metals. Promoter element interactions initially came to light during the detailed mapping of a mouse MRE where a potential SP1 transcription factor binding site was found to be overlapping the MRE (31). Subsequent reports have identified glucocorticoid response elements, antioxidant response elements (AREs), as well as USF and NFI transcription factor binding sites within promoters as active elements regulating MT expression (32 –35). Additional examination of the SP1 element associated with the hMT-II MREb indicates that the SP1 transcription factor is a negative regulator of MT expression (36). Zinc-regulated gene expression has long been linked to oxidative stress responses. For example, induction of MT protein in isolated rat liver cells was observed upon treatment with the free-radical-generating compounds, t-butyl hydroperoxide and 3-methylindole (11). Additional linkage of MT promoter

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MREs to antioxidant responses was demonstrated subsequently (35). While functionally defining an ARE element in the proximal mouse MT-I promoter, Dalton et al. reported that even with the ARE deleted there was still some MT induction by H2O2 . Further analysis demonstrated that MREs by themselves were sufficient for H2O2 induction of MT. Having identified the promoter element responsible for zinc responsiveness, the search began for transcription factors that bind this sequence. A variety of DNA-binding methodologies used to analyze nuclear protein extracts have identified several candidates by their ability to specifically bind the MRE consensus sequence [reviewed in Ref. (30)]. The zinc-inducible MRE-binding MTF-1 was the first MRE-binding factor characterized and cloned (37,38). The cDNA revealed that the N-terminal half of the 675 AA protein contains six consecutive zinc finger motifs, whereas the C-terminal half has three distinct transcriptional activation domains (39). Further characterization revealed significant functional heterogeneity in the zinc fingers. Fingers 2 –4 constitute the DNA/MRE-binding core, whereas the most N-terminal finger, #1, appears to function as a unique zinc-binding sensor (40). Fingers 5 and 6 also have characteristics that may function as parts of the zinc sensor. Thus, MTF-1 not only functions as a transcriptional inducer but also as an intracellular zinc detector. In addition, one or more zinc-binding domains of MTF-1 may be susceptible to oxidant-induced zinc release. Since its discovery, several features of MTF-1 have been identified that may play significant roles in further understanding its function. First, MTF-1 levels may be regulated by zinc. Moreover, immunolocalization studies have demonstrated that MTF-1 is translocated from the cytoplasm to the nucleus after treatment of the cells with zinc, cadmium, or other stressors such as heat shock or H2O2 (41,42). Perhaps, most interesting is the fact that a transgenic knockout mouse model for MTF-1 displays embryonic lethality for the homozygous mutant by day 14 of gestation (43). This indicates an essential role in cellular biology beyond MTF-1’s ability to induce MT, because the homozygous MT-I and -II knockout mouse is viable and appears to develop normally (44). Other reports add to the evidence that MTF-1 serves a role beyond metal regulation of gene expression. In one report, a search conducted to identify additional targets for MTF-1 yielded a diverse panel of candidates including a-feto protein, C/EBPa transcription factor, and tear lipocalin (45). Another indicates that MTF-1 may partner with p53 to transcriptionally regulate the synthesis of the ribosomal S25 protein (46). Additionally, MTF-1 appears to be directly involved in responding to oxidative stress produced by H2O2 and tert-butylhydroquinone, which activate MTF-1 DNA-binding activity (47). Further evidence suggests that this activation occurs via an increase in the level of intracellular zinc released from MT after oxidative damage (48). Thus, it appears that the roles of zinc-regulated gene expression and cellular antioxidant response are intricately linked at multiple levels.

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EXPRESSION PROFILING OF ZINC-REGULATED GENES By the early 1990s, the number of genes being reported in the literature as zinc responsive in a variety of different metabolic pathways from different tissues led to a growing interest in identifying as many of these genes as possible. As interest in this functional role for zinc expanded, so did an interest in relating expression of specific genes to the pathology of nutritional zinc deficiency. Although essentiality of this micronutrient in animals, including humans, has been known for decades, specific biochemical defects that could explain the plethora of clinical findings, including tissue peroxidation, have not emerged. Prior to the development of genomics and the sequencing of the human genome, few techniques were available to clone the cDNAs of unknown differentially expressed genes. The first article to report the results of differential zinc expression utilized differential hybridization cDNA library screening. Shay and Cousins (49) identified, and confirmed by northern blot analysis, nine differentially expressed mRNAs from rat small intestine after 16 days of dietary zinc deficiency. Among the mRNAs identified in this report were intestinal fatty acid-binding protein, aldolase B, calbindin D-28K, apolipoprotein A-I, ubiquitin, and cytochrome c oxidase, which is the first screening evidence of zincmodulated genes involved in cellular oxidative status. These moderately to highly abundant genes encompassed a broad spectrum of metabolic pathways, a precedent that has been consistently seen in all subsequent analyses. Though the field of genomics was getting started in the mid-1990s, there was still a paucity of genes represented in the DNA sequence databases; therefore, the most robust methods for identifying differentially expressed mRNAs relied on de novo cloning and screening of cDNAs. The next report of zincregulated expression screening utilized the PCR-based method of mRNA differential display (DD). Using the same rat small intestinal model with an additional zinc-supplemented dietary treatment group, we identified another 13 dietary zincregulated mRNAs (50). However, as the amplification by PCR allowed for the detection of much rarer mRNA species, and the genome databases were far less complete than they are today, only three could be matched to genes of known function and four to expressed sequence tags (ESTs), whereas six sequences were completely novel. These genes, which were suppressed by excess dietary zinc and induced by zinc deficiency, included two peptide hormones, cholecystokinin and uroguanylin, and ubiquinone oxidoreductase, a mitochondrial electron transport component. Interestingly, though both of the hormones serve very different physiological functions, their prohormones are proteolytically activated extracellularly by zinc metallopeptidases. Of particular interest is that each of these dysregulated genes could be linked to clinical signs of zinc deficiency, specifically, cholecystokinin (anorexia), uroguanylin (intestinal fluid secretion and diarrhea), and mitochondrial oxidoreductase (apoptosis). Subsequent experiments showed, at the proteome level, that upregulated uroguanylin mRNA levels

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were concurrent with increased immunoreactive uroguanylin peptide levels in a zinc-dependent fashion (19). Given the human health issues associated with the immunodeficiency resulting from clinical zinc deficiency, the focus on identification of zinc-responsive genes was expanded to an immune organ (the thymus). Moore et al. (51) used DD to examine such genes in the mouse thymus after 3 weeks of dietary zinc excess and deficiency. This was also the first zinc-based expression profiling study to theoretically complete a survey of an entire transcriptome. Approximately 265 differentially displayed cDNA bands were identified out of 48,000 cDNA bands examined and, of 42 selected for follow-up, all but one were identified with known genes or ESTs in GenBank. Among this list, were those for four ribosomal subunit proteins and the mitochondrial 16S ribosomal RNA indicating a significant effect of zinc status on the protein synthesis pathways within thymocytes. In addition, four distinct heat shock proteins showed decreased expression due to zinc deficiency. Of the immunologically relevant genes identified, the T cell cytokine receptor, which was induced by zinc deficiency, and the H2-Aa subunit of the MHC class II receptor, which was suppressed in deficiency, were confirmed by quantitative reverse transcriptase real-time PCR (Q-PCR). Together, these indicate a zinc status effect at the level of cytokine signaling pathways that control T cell development. Those findings support a role for zinc in immune cell development and function advanced by Fraker and Prasad (52,53). The foundations of genomics had been well established by the late 1990s, when the first applications to nutrition, or nutrigenomics, were reported using the new resources available. The first two reports of zinc-regulated gene expression used macroarrays containing 1200 unique cDNA probes. In the first, with zincdeficient rat small intestine, 32 differentially expressed genes were reported, and represented a variety of different cellular systems (54). Six of these expression changes were confirmed using Q-PCR. Perhaps most interesting, in regard to zinc antioxidant capabilities, is the large proportion of glutathione S-transferases and other redox-associated genes that were modulated by zinc deficiency. Three different glutathione S-transferase mRNAs were depressed by zinc deficiency, which suggests that thiol status as well as redox status is disturbed. In addition, depression of ATP synthases and intracellular ATPases point towards decreased overall cellular energy level, which correlates with stunted growth clinically observed during zinc deficiency. The correlation between clinical observations and types of genes affected by zinc deficiency is also seen in the multiple immune-related genes with altered expression that were reported. The other macroarray paper reported, and subsequently confirmed by Q-PCR, four additional mouse thymus genes that were modulated by 3 weeks of zinc deficiency (23). As expected of the thymus, three of these, the myeloid cell leukemia sequence-1, the laminin receptor, and the lymphocyte-specific protein tyrosine kinase (lck) are immune-related genes, whereas the fourth, RAD23B, is a DNA damage repair and recombination protein. Lymphocyte-specific protein kinase was of particular interest because of the documented use of a zinc ion to

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interact with its companion protein, the CD4 receptor (55). This highlights an area of zinc functionality that is not often considered, the role of zinc to mediate structural or regulatory interactions between two different proteins. Most of the structural aspects of zinc in protein biochemistry have focused on medium to high affinity binding sites within a single protein molecule; yet, a growing number of reports suggest that lower affinity binding of zinc may play a crucial role in a variety of protein–protein interactions of regulatory significance. It should be noted that, in these two studies, the difference in number of genes detected is generally a reflection of tissue type. Intestinal RNA gave detectable signals for 30– 40% of the cDNAs on the array, whereas it was only 19% for thymus RNA. This is due to a bias towards metabolic genes in the choice cDNAs to be spotted on these limited content arrays rather than tissue-specific differences in gene expression between the intestine and the thymus. As arrays become more comprehensive in their gene content, the bias has decreased until it is simply a reflection of biological difference between the number of genes required for different physiological pathways. A commercial cDNA macroarray of 1200 probes, as well as a glass oligonucleotide microarray of 1353 probes, were used to examine expression changes for genes in rat liver after a zinc-deficient diet was fed for 11 days (56). Of 1550 mRNAs detected, 31 were found to be upregulated and 35 downregulated by zinc deficiency. A similar wide distribution of functional classes was shown and included the previously identified genes, fatty acid-binding protein, and glutathione S-transferases. Of particular note was the zinc-deficient suppression of three cytochrome P450s and the NADPH-cytochrome P450 reductase, which may result in altered hepatic metabolism of xenobiotics. Recently, high density oligonucleotide microarrays such as Affymetrix GeneChips were used to examine zinc-regulated human gene expression. Using the human U133A GeneChipw composed of 22,000 gene elements, we surveyed changes due to zinc deficiency as well as supplementation in the human monocytic cell line, THP-1 (57). This system serves as an in vitro model for circulating PBMCs, where Cao et al. (58) demonstrated similar zinc responses for MT and the zinc transporter Zip2 between THP-1 cells and human PBMCs. Treatment with the cell permeable zinc chelator TPEN at 10 mM for 4 h was used to create a severe, acute zinc deficiency while supplementation was at 40 mM zinc for the same time. By setting up zinc concentrations on both sides of normal culture conditions, the genes that are directly zinc responsive should display a continuity of regulation across the treatments, that is, the gene would either be downregulated in deficiency and upregulated in supplementation or the reverse. Of 19,000 genes detected on triplicate GeneChips for each treatment, 1045 were identified under high statistical significance (P , 0.0001) as changed in at least one condition. Of these, only 283 were significantly different from normal in both treatments, and K-means clustering was used to group them according to similar responses. Of that group, 104 genes positively correlated with zinc levels, whereas 86 inversely correlated with zinc levels across all

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three treatments. After breaking down the members of each correlation group by functional category, 30 – 40% of each group were uncharacterized gene products, which was not entirely unexpected given that a major portion of the gene elements on the array have no annotated function. Of those in the positive correlation group, the next most highly represented functional group was nucleic acidbinding proteins, followed equally by genes involved in metabolism and signal transduction. In the inverse correlation group, metabolism genes were most highly represented, followed by signal transduction and then genes that influence immune/cytokine function. Interestingly, the prototypical zinc-responsive gene family, MT gene, does not show up in either of these groups owing to a combination of small fold change in the downregulation in zinc deficiency and moderate variance that did not meet the stringent statistical analysis used. Strikingly, the mRNA for tristetraprolin (TTP) showed the greatest magnitude of change with a 14-fold decrease in zinc deficiency and an almost two-fold increase in supplementation. TTP is a zinc finger protein that binds to TNF-a mRNA and accelerates its degradation (59). Alterations of TNF-a gene expression are commonly transduced by the NFkB transcription factor; therefore, it was very striking to find prominently positioned in the list of zinc-modulated genes both inhibitory proteins of NFkB, IkB-a and IkB-b. These findings provide additional mechanisms to support the correlation between zinc status and immune function, especially in the TNF cytokine signal transduction pathway. An additional report analyzes zinc-deficient changes in gene expression with a stress and aging-specific low-density microarray of 204 probes. In diversifying from previous reports, the human lung fibroblast cell line, IMR90, was chosen because the lung is chronically exposed to oxidative conditions and, therefore, potentially very sensitive to zinc depletion. These cells were evaluated for antioxidant and DNA repair gene expression changes under two different conditions of zinc deficiency (60). The first zinc-deficient treatment was accomplished by growing cells for 5 days in medium prepared from zinc-depleted FBS, whereas the second used 40 mM TPEN for 2 h. Given the differences in treatment times and deficiency models, as well as a conservative two-fold change cut-off, out of the 30 genes changed by TPEN treatment and the 32 changed by depleted media, there were only three found to be in common. These mRNAs encoded the proteins glutathione peroxidase, proteasome 26S subunit, and chromosome segregation 1. Nonetheless, when functional grouping of modulated genes were examined, they showed similar types of changes in both types of deficiency, which supports the theory that zinc-deficient cells are more susceptible to oxidative DNA damage. By way of comparison, array analysis in the context of micronutrients and antioxidants can be found in two reports from the area of selenium nutrition. Macroarrays were used to evaluate selenium deficiency and its interaction with vitamin E in rat liver, whereas microarrays were used to evaluate low selenium status in mouse intestine (61,62). Both found that selenium deficiency, like zinc, affects a diverse population of genes and this includes a significant number that are involved in cellular redox maintenance.

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Though dietary zinc-modulated genes are found in a wide variety of cellular pathways, all of the expression profiling to date shows that the percentage of altered genes is still a small fraction of the transcriptome population. As expression profiling provides information about multiple genes in the same experiment, one of the questions raised is whether observed changes are due to direct effects of the nutrient upon a gene or whether changes are a response to the altered expression of direct responding genes. Often, this cannot be answered in the context of the experimental system being profiled, as it requires very precise manipulation of the cellular environment, which is not possible to control in whole animal models with their extensive homeostatic mechanisms. Therefore, classical reporter gene experiments in a cell culture system or RNA interference techniques are desirable alternatives to determine whether nutrients are acting directly on gene promoter elements. The various methods utilized for expression profiling have different advantages and disadvantages and most have been brought to bear upon the question of zinc-responsive genes. DD of mRNA and serial analysis of gene expression (SAGE) have been available the longest and have the advantage of requiring minimal genomic sequence information for the system being evaluated. And although SAGE analysis has not yet been applied to zinc-regulated expression profiling, the increasing availability of SAGE libraries may soon change that fact. Indeed, one of the benefits to applying multiple methods of detection is that inherent bias in each technique may prejudice the selection of genes identified. This is especially the case for detecting low abundance genes and reflects part of the growing conundrum that, while low or even transiently expressed gene products perform physiologically relevant functions, they present a challenge to our current analytical capabilities. In addition, different methods of detection often present differing magnitudes of change although they generally agree on the direction of the change. In all reports to date, there have been appreciable inconsistencies among changes detected by array analysis vs. those from northern analysis or Q-PCR. The latter techniques tend to indicate a greater magnitude of change than observed on arrays, but there is still a strong concordance regarding the direction of change. The application of large-scale mRNA expression profiling has provided new understanding as well as new questions regarding the antioxidant properties of zinc. Therefore, though nutrigenomics has seen increased application within the nutritional science community, it will need to be followed by the application of proteomics to fully understand the ramifications of zinc as an antioxidant and regulator of gene expression. REFERENCES 1. Cousins RJ. Zinc. In: Filer LJ, Ziegler EE, eds. Present Knowledge in Nutrition. 7th ed. Washington: International Life Sciences Institute Nutrition Foundation, 1996:293 –306.

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2. da Silva JJR, Williams RJP. The Biological Chemistry of the Elements: The Inorganic Chemistry of Life. Oxford: Clarendon Press, 1991:299 –318. 3. Powell SR. The antioxidant properties of zinc. J Nutr 2000; 130:1447S– 1454S. 4. Maret W. The function of zinc metallothionein: a link between cellular zinc and redox state. J Nutr 2000; 130:1455S– 1458S. 5. Klotz LO, Kroncke KD, Buchczyk DP, Sies H. Role of copper, zinc, selenium and tellurium in the cellular defense against oxidative and nitrosative stress. J Nutr 2003; 133:1448S– 1451S. 6. Outten CE, O’Halloran TV. Femtomolar sensitivity of metalloregulatory proteins controlling zinc homeostasis. Science 2001; 292:2488 – 2492. 7. Davis SR, Cousins RJ. Metallothionein expression in animals: a physiological perspective on function. J Nutr 2000; 130:1085 – 1088. 8. Thornalley PJ, Vasak M. Possible role for metallothionein in protection against radiation-induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals. Biochim Biophys Acta 1985; 827:36 – 44. 9. St Croix CM, Wasserloos KJ, Dineley KE, Reynolds IJ, Levitan ES, Pitt BR. Nitric oxide-induced changes in intracellular zinc homeostasis are mediated by metallothionein/thionein. Am J Physiol Lung Cell Mol Physiol 2002; 282:L185 – L192. 10. Schroeder JJ, Cousins RJ. Interleukin 6 regulates metallothionein gene expression and zinc metabolism in hepatocyte monolayer cultures. Proc Natl Acad Sci USA 1990; 87:3137– 3141. 11. Coppen DE, Richardson DE, Cousins RJ. Zinc suppression of free radicals induced in cultures of rat hepatocytes by iron, t-butyl hydroperoxide, and 3-methylindole. Proc Soc Exp Biol Med 1988; 189:100 – 109. 12. Gutteridge JM, Halliwell B. Free radicals and antioxidants in the year 2000. A historical look to the future. Ann NY Acad Sci 2000; 899:136 – 147. 13. Fliss H, Menard M. Hypochlorous acid-induced mobilization of zinc from metalloproteins. Arch Biochem Biophys 1991; 287:175 – 179. 14. Zangger K, Oz G, Haslinger E, Kunert O, Armitage IM. Nitric oxide selectively releases metals from the amino-terminal domain of metallothioneins: potential role at inflammatory sites. FASEB J 2001; 15:1303 – 1305. 15. Schapiro JM, Libby SJ, Fang FC. Inhibition of bacterial DNA replication by zinc mobilization during nitrosative stress. Proc Natl Acad Sci USA 2003; 100:8496–8501. 16. Kro¨ncke KD, Fehsel K, Schmidt T, Zenke FT, Dasting I, Wesener JR, Bettermann H, Breunig KD, Kolb-Bachofen V. Nitric oxide destroys zinc –sulfur clusters inducing zinc release from metallothionein and inhibition of the zinc finger-type yeast transcription activator LAC9. Biochem Biophys Res Commun 1994; 200:1105 –1110. 17. Spahl DU, Berendji-Grun D, Suschek CV, Kolb-Bachofen V, Kro¨ncke KD. Regulation of zinc homeostasis by inducible NO synthase-derived NO: nuclear metallothionein translocation and intranuclear Zn2þ release. Proc Natl Acad Sci USA 2003; 100:13952 – 13957. 18. Cui L, Takagi Y, Wasa M, Sando K, Khan J, Okada A. Nitric oxide synthase inhibitor attenuates intestinal damage induced by zinc deficiency in rats. J Nutr 1999; 129:792– 798. 19. Cui L, Blanchard RK, Cousins RJ. The permissive effect of zinc deficiency on uroguanylin and inducible nitric oxide synthase gene upregulation in rat intestine induced by interleukin 1alpha is rapidly reversed by zinc repletion. J Nutr 2003; 133:51– 56.

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20. Chvapil M, Ryan JN, Elias SL, Peng YM. Protective effect of zinc on carbon tetrachloride-induced liver injury in rats. Exp Mol Pathol 1973; 19:186 –196. 21. Davis SR, Samuelson DA, Cousins RJ. Metallothionein expression protects against carbon tetrachloride-induced hepatotoxicity, but overexpression and dietary zinc supplementation provide no further protection in metallothionein transgenic and knockout mice. J Nutr 2001; 131:215 – 222. 22. Li X, Chen H, Epstein PN. Metallothionein protects islets from hypoxia and extends islet graft survival by scavenging most kinds of reactive oxygen species. J Biol Chem 2004; 279:765 – 771. 23. Moore JB, Blanchard RK, McCormack WT, Cousins RJ. cDNA array analysis identifies thymic LCK as upregulated in moderate murine zinc deficiency before T-lymphocyte population changes. J Nutr 2001; 131:3189 – 3196. 24. Richards MP, Cousins RJ. Mammalian zinc homeostasis: requirement for RNA and metallothionein synthesis. Biochem Biophys Res Commun 1975; 64:1215 – 1223. 25. Shapiro SG, Cousins RJ. Induction of rat liver metallothionein mRNA and its distribution between free and membrane-bound polyribosomes. Biochem J 1980; 190:755– 764. 26. Durnam DM, Perrin F, Gannon F, Palmiter RD. Isolation and characterization of the mouse metallothionein-I gene. Proc Natl Acad Sci USA 1980; 77:6511– 6515. 27. Mayo KE, Warren R, Palmiter RD. The mouse metallothionein-I gene is transcriptionally regulated by cadmium following transfection into human or mouse cells. Cell 1982; 29:99– 108. 28. Stuart GW, Searle PF, Chen HY, Brinster RL, Palmiter RD. A 12-base-pair DNA motif that is repeated several times in metallothionein gene promoters confers metal regulation to a heterologous gene. Proc Natl Acad Sci USA 1984; 81:7318–7322. 29. Stuart GW, Searle PF, Palmiter RD. Identification of multiple metal regulatory elements in mouse metallothionein-I promoter by assaying synthetic sequences. Nature 1985; 317:828 – 831. 30. Samson SL, Gedamu L. Molecular analyses of metallothionein gene regulation. Prog Nucleic Acid Res Mol Biol 1998; 59:257– 288. 31. Culotta VC, Hamer DH. Fine mapping of a mouse metallothionein gene metal response element. Mol Cell Biol 1989; 9:1376 – 1380. 32. Kelly EJ, Sandgren EP, Brinster RL, Palmiter RD. A pair of adjacent glucocorticoid response elements regulate expression of two mouse metallothionein genes. Proc Natl Acad Sci USA 1997; 94:10045 – 10050. 33. Datta PK, Jacob ST. Activation of the metallothionein-I gene promoter in response to cadmium and USF in vitro. Biochem Biophys Res Commun 1997; 230:159– 163. 34. Majumder S, Ghoshal K, Gronostajski RM, Jacob ST. Downregulation of constitutive and heavy metal-induced metallothionein-I expression by nuclear factor I. Gene Expr 2001; 9:203 –215. 35. Dalton T, Palmiter RD, Andrews GK. Transcriptional induction of the mouse metallothionein-I gene in hydrogen peroxide-treated Hepa cells involves a composite major late transcription factor/antioxidant response element and metal response promoter elements. Nucl Acids Res 1994; 22:5016 – 5023. 36. Ogra Y, Suzuki K, Gong P, Otsuka F, Koizumi S. Negative regulatory role of Sp1 in metal responsive element-mediated transcriptional activation. J Biol Chem 2001; 276:16534 – 16539.

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37. Westin G, Schaffner W. A zinc-responsive factor interacts with a metal-regulated enhancer element (MRE) of the mouse metallothionein-I gene. EMBO J 1988; 7:3763– 3770. 38. Radtke F, Heuchel R, Georgiev O, Hergersberg M, Gariglio M, Dembic Z, Schaffner W. Cloned transcription factor MTF-1 activates the mouse metallothionein I promoter. EMBO J 1993; 12:1355– 1362. 39. Radtke F, Georgiev O, Muller HP, Brugnera E, Schaffner W. Functional domains of the heavy metal-responsive transcription regulator MTF-1. Nucl Acids Res 1995; 23:2277– 2286. 40. Bittel DC, Smirnova IV, Andrews GK. Functional heterogeneity in the zinc fingers of metalloregulatory protein metal response element-binding transcription factor-1. J Biol Chem 2000; 275:37194 – 37201. 41. Smirnova IV, Bittel DC, Ravindra R, Jiang H, Andrews GK. Zinc and cadmium can promote rapid nuclear translocation of metal response element-binding transcription factor-1. J Biol Chem 2000; 275:9377 –9384. 42. Saydam N, Georgiev O, Nakano MY, Greber UF, Schaffner W. Nucleo-cytoplasmic trafficking of metal-regulatory transcription factor 1 is regulated by diverse stress signals. J Biol Chem 2001; 276:25487 – 25495. 43. Gunes C, Heuchel R, Georgiev O, Muller KH, Lichtlen P, Bluthmann H, Marino S, Aguzzi A, Schaffner W. Embryonic lethality and liver degeneration in mice lacking the metal-responsive transcriptional activator MTF-1. EMBO J 1998; 17:2846 – 2454. 44. Michalska AE, Choo KH. Targeting and germ-line transmission of a null mutation at the metallothionein I and II loci in mouse. Proc Natl Acad Sci USA 1993; 90:8088–8092. 45. Lichtlen P, Wang Y, Belser T, Georgiev O, Certa U, Sack R, Schaffner W. Target gene search for the metal-responsive transcription factor MTF-1. Nucl Acids Res 2001; 29:1514– 1523. 46. Adilakshmi T, Laine RO. Ribosomal protein S25 mRNA partners with MTF-1 and La to provide a p53-mediated mechanism for survival or death. J Biol Chem 2002; 277:4147– 4151. 47. Dalton TP, Li Q, Bittel D, Liang L, Andrews GK. Oxidative stress activates metalresponsive transcription factor-1 binding activity. Occupancy in vivo of metal response elements in the metallothionein-I gene promoter. J Biol Chem 1996; 271:26233– 26241. 48. Zhang B, Georgiev O, Hagmann M, Gunes C, Cramer M, Faller P, Vasak M, Schaffner W. Activity of metal-responsive transcription factor 1 by toxic heavy metals and H2O2 in vitro is modulated by metallothionein. Mol Cell Biol 2003; 23:8471–8485. 49. Shay NF, Cousins RJ. Cloning of rat intestinal mRNAs affected by zinc deficiency. J Nutr 1993; 123:35– 41. 50. Blanchard RK, Cousins RJ. Differential display of intestinal mRNAs regulated by dietary zinc. Proc Natl Acad Sci USA 1996; 93:6863 – 6868. 51. Moore JB, Blanchard RK, Cousins RJ. Dietary zinc modulates gene expression in murine thymus: results from a comprehensive differential display screening. Proc Natl Acad Sci USA 2003; 100:3883 – 3888. 52. Shankar AH, Prasad AS. Zinc and immune function: the biological basis of altered resistance to infection. Am J Clin Nutr 1998; 68:447S – 463S. 53. Fraker PJ, King LE, Laakko T, Vollmer TL. The dynamic link between the integrity of the immune system and zinc status. J Nutr 2000; 130:1399S– 1406S.

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54. Blanchard RK, Moore JB, Green CL, Cousins RJ. Modulation of intestinal gene expression by dietary zinc status: effectiveness of cDNA arrays for expression profiling of a single nutrient deficiency. Proc Natl Acad Sci USA 2001; 98:13507 – 13513. 55. Huse M, Eck MJ, Harrison SC. A Zn2þ ion links the cytoplasmic tail of CD4 and the N-terminal region of Lck. J Biol Chem 1998; 273:18729– 18733. 56. Dieck HT, Doring F, Roth HP, Daniel H. Changes in rat hepatic gene expression in response to zinc deficiency as assessed by DNA arrays. J Nutr 2003; 133:1004 – 1010. 57. Cousins RJ, Blanchard RK, Popp MP, Liu L, Cao J, Moore JB, Green CL. A global view of the selectivity of zinc deprivation and excess on genes expressed in human THP-1 mononuclear cells. Proc Natl Acad Sci USA 2003; 100:6952 – 6957. 58. Cao J, Bobo JA, Liuzzi JP, Cousins RJ. Effects of intracellular zinc depletion on metallothionein and ZIP2 transporter expression and apoptosis. J Leukoc Biol 2001; 70:559 –566. 59. Carballo E, Lai WS, Blackshear PJ. Feedback inhibition of macrophage tumor necrosis factor-alpha production by tristetraprolin. Science 1998; 281:1001 – 1005. 60. Ho E, Courtemanche C, Ames BN. Zinc deficiency induces oxidative DNA damage and increases p53 expression in human lung fibroblasts. J Nutr 2003; 133:2543 – 2548. 61. Fischer A, Pallauf J, Gohil K, Weber SU, Packer L, Rimbach G. Effect of selenium and vitamin E deficiency on differential gene expression in rat liver. Biochem Biophys Res Commun 2001; 285:470 – 475. 62. Rao L, Puschner B, Prolla TA. Gene expression profiling of low selenium status in the mouse intestine: transcriptional activation of genes linked to DNA damage, cell cycle control and oxidative stress. J Nutr 2001; 131:3175 –3181.

Index

Anticarcinogenic properties soy isoflavones, 327 – 336 tocotrienols, 213 Antioxidant AP-1 mediated gene expression, 148 – 152 applications, 160 – 164 atherogenesis, DNA methylation, 158 caspase-3 proteins, 129 cellular viability, 125 chromatin structure, transcription, 157 – 160 differential gene expression, 5 – 6 endothelial cells, 141 –167 immune function and, 110 – 112 inflammation, 106 molecular mechanism, 134 – 137 NF-kB mediated gene expression, endothelium, oxidants, 143 – 148 pro-inflammatory genes, 164 – 167 vitamin E, 152 – 157 zinc, 458 – 460 Antioxidant defenses immune function, 97 – 117 infection/injury, 102 – 103 Antioxidant properties procyanidins, 379 – 390 VE, chemistry, 202 –204

Adipocyte, fatty acid regulation of, 189 – 190 Adipocyte differentiation, a-lipoic acid, 290 – 294 Aging inflammatory stress effects on, 103 low-grade inflammation mechanisms, 105– 106 oxidative stress effects on, 103 Alanine (ALA), 16 a-lipoic acid adipocyte differentiation, 290 – 294 cell signaling properties, 283 – 295 glucose metabolism, diabetes, type 2, 287– 288 insulin-signaling pathway, 288 a-tocopherol, 208– 210 molecular targets, 206 NO, platelet aggregation, 210– 211 protein kinase C, 206– 208 VE sensitive genes, 211– 213 Animal physiology b,b-caroteine-15,150 mono-oxygenases, 226–227 vitamin A, 223– 225 VP14-homologs, 227– 229 Animal studies, isoflavones, 308 – 309 473

474 AP-1 mediated gene expression, endothelium, 148– 152 Apoptosis cell death, 409–410 gene expression, 125– 131 PI 3-kinase, 409– 410 Arterial wall gene expression, fatty acid regulation, 190 –191 Arteriosclerosis, potential mechanisms, 308 Ascorbate modulation cell differentiation, 267–268 collagen formation, 265– 267 nitric oxide production, 271–272 Ascorbate recycling, metabolism, 263– 265 Ascorbate-induced modulation DNA repair, 272 molecular functions, 272– 273 Ascorbic acid ascorbate recycling, 260–263 cellular redox activity, 257– 273 collagen formation, 265– 267 immune function, anti-oxidants, effects on, 111 molecular functions, 265– 273 cell differentiation, 267– 268 DNA repair, 272 nitric oxide production, 271 –272 transcription factor modulation, 268– 271 nutritional aspects, 259– 260 redox status, 260– 265 Asthma, GSTP1, health conditions, 23 Atherogenesis DNA methylation, 158 pro-inflammatory genes, 164– 167 Atherosclerosis, vitamin E, 153

b,b-caroteine-15,150 -mono-oxygenases, carotenoids, 226– 227 Blood pressure isoflavones, 310, 313 Breast cancer, isoflavones, 329– 333

Index Caenorhabditis elegans, 68 DAF-16 transcription target, 76 – 77 insulin/IGF-1 signaling, 70 – 73 stress resistance, 73 – 76 lifespan regulation, 69 – 81 mitochrondrial electron transport, 78 –81 nervous system, 78 reproduction system, 77 –78 Caloric restriction (CR), 68 Cancer related genes, procyanidins, 384 – 385 Cancer, GSTM1 biomarkers, 21 Cancer, GSTT1 biomarkers, 24 Cancer, MnSOD biomarkers,17– 18 Capase-3 proteins, 129 Cardiovascular effects, isoflavones, 308 Cardiovascular related genes, procyanidins, 384 Carotenoids b,b-caroteine-15,150 -monooxygenases, 226 – 227 color function, 222 – 223 Catalase (CAT), 27 GST genes, 27 – 28 cDNA array techniques complementary DNA, 386 procyanidins, 386 –389 Cell adhesion proteins, 208 – 209 Cell cycle progression inhibition, lycopene, 246 – 247 Cell cycle, IP6 , cell proliferation, 405 – 410 Cell differentiation ascorbic acid, 267 – 268 IP6, 408 – 409 Cell functions, MAP kinase signaling, 360 – 361 Cell proliferation normalization cell cycle, 405 – 410 IP6, 405 – 408 ras proteins, 407 – 408 retinoblastoma protein, IP6, 406 – 407 Cell regulatory activity, tocopherols/ tocotrienols, 201 – 214

Index Cell signaling properties a-lipoic acid, 283– 295 IP6, 397 –415 Cell survival, apoptosis, 125– 131 Cellular redox activity, ascorbic acid, 257 – 273 Cellular signaling inositol compounds, 399– 400 IP3 receptor, inositol compounds, 399 – 400 IP6, signal transduction, 401– 402 Chemokines, scavenger receptors, a-tropomyosin, alpha tocopherol, cell adhesion proteins, 208– 209 Chromatin remodeling, IP6, nuclear inositol signaling, 411 Chromatin structure, antioxidants, transcription, 157–160 Class discovery, microarray data analysis hierarchical trees, 53 k-means clustering, 50– 52 principal component analysis, 53 self-organizing maps, 52– 53 Class prediction methods, microarray data analysis cross-validation, 60 supervised learning methods, 59– 60 Clustering, hierarchical trees, 53 Collagen formation, ascorbic acid, 265– 267 Colon cancer, isoflavones, 334– 335 Colonic metabolism, flavonoids in vivo, 356 – 358 Complementary DNA arrays (cDNA), 386 Copper, zinc superoxide dismutase (SOD3), 18– 19 Coronary heart disease (CHD), 181 fatty acids, gene expression 181– 186 isoflavones diets, 310– 319 Cross-validation, microarray, class prediction methods, 60 Cyclic strain, 160 Cyclooxygenase, alpha tocopherol, 209– 210 Cytokines adverse effects, 101 infection/injury, 99– 101

475 DAF-16 transcription target, 76 – 77 Data analysis, 47 – 60 class prediction methods, 59 – 60 cross-validation, 60 expression data, fold changes, 48 – 50 hierarchical trees, 53 k-means clustering, 50 – 52 principal component analysis, 53 self-organizing maps, 52 – 53 Diabetes a-lipoic acid, 283 – 295 insulin-signaling pathway, 285 – 287 treatment strategies, 284 – 285 type 2, a-lipoic acid, 287 –288 Dietary iron, gene expression modulation, 421 – 433 Dietary selenium gene expression, 441 – 451 selenoprotein expression, 447 – 448 Dietary zinc, 457 – 468 Differential gene expression dietary selenium, 449 – 451 Ginkgo biloba extract Egb 761, 341 – 349 neuronal cells, 344 – 346 oxidants, antioxidants, 5– 6 DMT1 regulation, iron, intestinal absorption, 426 DNA methylation, atherogenesis, 158 DNA repair ascorbate-induced modulation, 272 nuclear inositol signaling, 411 DNA synthesis, cell proliferation, 406 Drosophila, lifespan regulation, 81 – 83 Egb 761 Ginkgo biloba chemical composition, 341 – 344 differential gene expression brain 346 –348 neuronal cells, 344 – 346 Endocytosis, IP6 interactions, 402 – 405 Endothelial cells, antioxidants, 141 – 167 Endothelial gene expression, vitamin E, 152 – 157

476 Endothelial nitric oxide synthase. (eNOS), 25 dietary interventions, 26–27 health conditions, 26 smokers, 26 Endothelium oxidants AP-1 mediated gene expression, 148 –152 NF-kB mediated gene expression, 143 –148 Endothelium reactivity, platelet aggregation, 317– 318 Estrogen receptor isoflavones, 305 mechanisms of action, 306 Exocytosis/endocytosis, vesicle trafficking, 402– 405 Expression data, fold changes, 48– 50 Fatty acid regulation adipocytes, 189– 190 arterial wall gene expression, 190 –191 coronary heart disease, 181– 196 lipoprotein metabolism, 195– 196 PPAR ligands, 193 PUFA induction hepatic lipogenesis, 187–189 lipid oxidation, 189 PUFA mechanisms, 191–192 transcription factors, 192– 194 peroxisome proliferators-activator receptors, 192– 193 Fatty acids, metabolism, 183– 187 absorption, 183 lipoproteins, 184– 185 Fatty acid structure, tissue sources, 182– 183 Flavanols, signaling cascade modulations, 362–365 Flavonoids bioactive forms, 355– 359 colonic metabolism, 356– 358 intracellular metabolism, 358– 359 metabolism, GI tract and liver, 356 metabolites, 353– 367 signaling cascade modulations, 359 –367

Index flavonols, 362 – 365 MAP kinase signaling, 360 –361 Fold changes, microarray data analysis, 48 – 50 Folic acid, immune function, 115 Gene clusters, hierarchical trees, 56 – 57 Gene expression modulation, 457 – 468 cancer related genes, 384 – 385 cardiovascular related genes, 384 cDNA array techniques, 386 – 389 dietary iron, 421 – 433 inflammation related genes, 385 – 386 procyanidins, 383 –389 Gene expression adipocytes, 189 –190 apoptosis, 125 – 131 arterial wall gene expression, 190 – 191 coronary heart disease, 181 – 196 dietary selenium, 441 – 451 endothelial cells, 141 –167 fatty acid regulation, 187 –191 glucose insulin homeostasis, 195 – 196 microarray data analysis, 43 – 62 neutrigenomics, 1 – 9 NF-kB, 143 oxidants/antioxidants, 3 – 4 oxidative stress, 68– 86 profiling, 464 – 468 PUFA induction, lipid oxidation, 189 PUFA mechanisms, fatty acid regulations, 191 – 192 PUFA, hepatic lipogenesis, fatty acid regulation, 187 – 189 transcription factors fatty acid regulations, 192 – 194 peroxisome proliferators-activator receptors, 192 – 193 PPAR ligands, 193 zinc, 460 – 463 GI tract, flavonoids metabolism, 356 Ginkgo biloba extract Egb 761 chemical composition, 341 – 344 differential gene expression, 341 – 349

Index Glucose insulin homeostasis, gene expression, 195– 196 Glucose metabolism, diabetes type 2, 287– 288 Glutathione, anti-oxidants, effects on, 111– 112 Glutathione peroxidase (GPX1), 29 polymorphisms, 29 Glutathione-s-transferase M1, (GSTM1), 20 oxidative stress, 19– 20 Glutathione-s-transferase P1 (GSTP1), 22– 24 asthma, 23 ile allele associations, 23 val allele associations, 23 Glutathione-s-transferase T1 (GSTTI), 24 biomarkers, 24 cancer, 24 GSH, immune function, 112– 114 GST genes catalase, 27– 28 endothelial nitric oxide synthase, 25–27 glutathione peroxidase, 29 glutathione-s transferase M1, 20 gluthathione-s-transferase P1, 22– 24 gluthathione-s-transferase T1, 24 GSTM1 biomarkers, 21 myeloperoxidase, 30 NADPH dehydrogenase, quinine 1, 31 NADPH oxidase, 28 oxidative stress, 20– 24 GSTM1 biomarkers GST genes, 21, 23 glutathione-s-transferase M1, 20 cancer, 21 lung disease, 21– 22 other disorders, 22 Hemocromatosis, iron, 430– 432 Hepatic lipogenesis, PUFA, 187–189 Hepatic nuclear receptor-4(HNF-4), transcription factors, 194 Hepcidin, iron, metabolism, 428– 429 Hephaestin regulation, iron, intestinal absorption, 427

477 Hierarchical trees (clustering), 53 class discovery, 53 experiment, gene clusters, 56 – 57 measurements, 54 – 55 QT clustering, 57 – 59 HMG-CoA reductase, lycopene, 249 – 250 Hormesis, lifespan regulation, C. elegans, 69 – 70 Human stress varations glutathione-s-transferases, 19 – 20 manganese superoxide dismutase, 15 – 18 MnSOD health conditions, 17 –18 MnSOD polymorphisms, 16 – 17 oxidative stress, 13 – 32 SOD3, 18 – 19 GST genes, 20 – 24 Hypoxia, iron, 427 – 428 Ile allele associations, GSTP1, 23 Immune function antioxidant defenses, 101 – 102 antioxidant effects on, 110 – 112 ascorbic acid, 111 glutathione, 111 – 112 vitamin E, 110 – 111 antioxidant modulation, 97 – 117 cytokines, infection, injury, 99 – 101 folic acid, 115 GSH, 112 – 114 infection injury, 98 oxidants, 106 pro-inflammatory, adverse effects, 101 taurine, 115 – 116 vitamin B6, 114 – 115 Infection injury anti-oxidant defenses, 102 – 103 cytokines, 99 –101 inflammatory agents, 98 Inflammation, immune function, effects on, 106 Inflammation mechanisms, low-grade, 105 – 106 Inflammation-related genes, procyanidins, 385 – 386 Inflammatory agents, infection injury, 98

478 Inflammatory genes, vitamin E, 155 –156 Inflammatory stress, aging, effect on, 103 Injury, infection anti-oxidant defenses, 102– 103 immune function, cytokines, 99– 101 Inositol compounds cellular signaling, 399– 400 IP3 receptor, 399–400 IP6 signal molecule, 400– 405 protein macromolecules, 402– 405 Inositol hexaphosphate (IP6), 397– 415 Insects, vitamin A, 229– 230 Insulin/IGF-1 signaling C. elegans, 70 – 73 lifespan regulation, 73– 76 Insulin-like growth factor (IGF-1) signaling, lycopene, 247– 249 Insulin-signaling pathway a-lipoic acid, 288 diabetes, 285– 287 Intracellular communication modulation, lycopene, 245– 246 Intracellular metabolism fatty acids, 186 flavonoids in vivo, 358– 359 Ionic zinc, 458 IP3 receptor, inositol compounds, 399– 400 IP3/IP6, inositol compounds, 401– 402 IP5/IP6 interactions, 402 IP6 anticancer agent, 413– 414 cell differentiation induction, 408– 409 cell proliferation normalization, 405 –408 cell cycle, 405– 410 DNA synthesis, 406 protein kinase C, 407 ras proteins, 407– 408 retinoblastoma protein, 406– 407 energy transduction, 413 nuclear inositol signaling, 410– 413 chromatin remodeling, 411 DNA repair, 411– 412 mRNA transport, 410– 411 NF-kB, 411– 412 zinc-finger motif, 411– 412

Index programmed cell death, apoptosis, 409 – 410 signal transduction, cellular signaling, IP3, 401 – 402 IP6 interactions cellular signaling, 402 –405 vesicle trafficking, exocytosis/ endocytosis, 402 – 405 IREG1 regulation, iron, intestinal regulation, 426 – 427 Iron, dietary absorption, 422 – 423 gene expression modulation, 421 – 433 Iron, hemocromatosis, 430 – 432 Iron, hypoxia, 427 – 428 Iron, intestinal absorption, 423 –427 Dcyth regulation, 427 DMT1 regulation, 426 hephaestin regulation, 427 IREG1 regulation, 426 – 427 regulatory proteins, 424 responsive elements, 424 – 426 Iron, metabolism, hepcidin, 428 – 429 Iron, pro-oxidant, 432 – 433 Isoflavone, 329 – 336 absorption, metabolism, 303 – 305 arteriosclerosis, 308 blood pressure, 310, 313 breast cancer, 329 – 332 cardiovascular effects, 308 colon cancer, 334 – 335 diets, mechanisms of action, 301 – 319 estrogen receptor, mechanisms of action, 305 anti-oxidant activity, 306 inflammation, cell adhesion, 313, 315 – 317 lipid metabolism, 308 – 310 animal studies, 308 –309 clinical studies, 309 – 310 phytoestrogens, 302 –303 platelet aggregation, endothelium reactivity, 317 – 318 prostate cancer, 332 – 333 soy blood pressures, 314 serum lipids, 311 – 312

Index k-Means clustering, microarray data analysis, 50– 52 Late-onset neurological disorders, 17 Lifespan, replicative, 85– 86 Lifespan regulation, C. elegans, 69 – 81 DAF-16 transcription target, 76– 77 insulin/IGF-1 signaling, 70– 73 stress resistance, 73– 76 mitochrondrial electron transport, 78– 81 nervous system, 78 reproduction system, 77– 78 stress, hormesis, 69– 70 Lifespan regulation, drosophila, 81 – 83 Lifespan regulation, mammals, 83– 85 Linear, microarray, normalization methods, 45 Lipid metabolism isoflavones, 308– 310 animal studies, 308– 309 clinical studies, 309– 310 Lipid oxidation, PUFA induction, 189 Lipoprotein metabolism fatty acids, 184– 185 glucose insulin homeostasis, 195 – 196 Lung disease, GSTM1 biomarkers, 21–22 Lycopene cell cycle progression inhibition, 246– 247 HMG-CoA reductase, inhibition of, 249– 250 insulin-like growth factor (IGF-1) signaling, inhibition of, 247 – 249 intracellular communication modulation, 245 –246 molecular mechanisms, health benefits, 241 – 251 oxidative stress, 243– 245 Macromolecules, proteins, IP6 interactions, 402– 405 Mammalian cells, cellular signaling, 400 – 401 Mammals, lifespan regulation, 83– 85

479 Manganese superoxide dismutase (MnSOD), 15 oxidative stress, 15 – 18 MAP kinase signaling, signaling cascade modulations, 360 – 361 Mechanisms of action estrogen receptors, 306 isoflavones, 305 Metabolism absorption, fatty acids, 183 ascorbate recyling, 263 – 265 fatty acids, 183 – 187 flavonoids in vivo, 356 hepcidin, iron, 428 – 429 intracellular metabolism, fatty acids, 186 isoflavones, 303 – 305 lipoproteins, 184 – 185 selenium 442 – 443 Microarray, gene expression data organization (MGED), 47 Microarray, normalization methods, 44 – 47 linear, 46 per array, 46 – 47 per gene, 46 –47 specific samples, 46 – 47 Microarray data, gene expression, 43 – 62 Microarray data analysis, 47– 60 class discovery, 50 – 53 hierarchical trees, 53 k-means clustering, 50 – 52 principal component analysis, 53 self-organizing maps, 52 – 53 class prediction methods, 59 – 60 cross-validation, 60 expression data, fold changes, 48 – 50 software, 61 – 62 validation, 60 –61 in silico, 60 – 61 laboratory based, 61 Minimal information about a microarray experiment (MIAME), 9, 47

480 Mitochrondrial electron transport, C. elegans, 78 – 81 MnSOD health conditions cancer, 17– 18 neurological disorders, 17 oxidative stress, 17– 18 Model organism, molecular analyses, 229 Molecular analyses vertebrates, 231– 235 vitamin A biosynthetic pathway, 229 Molecular functions ascorbic acid, 265–273 DNA repair, 272 nitric oxide production, 271– 272 ascorbate-induced modulation, 272 –273 cellular redox activity, 257– 273 transcription factor modulation, 268 –271 Molecular mechanisms lycopene, 241– 251 zinc, gene expression, 460–463 MRNA transports, nuclear inositol signaling, 410– 411 Myeloperoxidase (MPO), 30 NADPH health conditions, 28– 29 polymorphisms, 28 NADPH dehydrogenase quinine 1 (NQO1), 31 GST genes, 31 polymorphisms, 31 NADPH oxidase, GST genes, 28 Nervous system, C. elegans, 78 Neuronal cells, differential gene expression, 344–346 Neuroprotection, tocotrienols, 213 –214 NF-kB mediated gene expression, 143 endothelium, oxidants, antioxidants, 143 –148 IP6, nuclear inositol signaling, 411 nuclear transcription factor-kB, 411 –412 proteins regulated by, 145

Index Nitric oxide production, ascorbate modulation, 271 – 272 NO, platelet aggregation, alpha tocopherol, 210 – 211 Normalization methods, microarray, 44 – 47 linear, 45 per array, 45 – 46 per gene, 46 – 47 specific samples, per gene, 46 – 47 Nuclear factor kappa B (NF-kB), transcription factors, 194 Nuclear inositol signaling chromatin remodeling, IP6, 411 DNA repair, IP6, 411 IP6, 410 – 413 mRNA transports, IP6, 410 – 411 Nutrigenomics definition, 1 –2 methods, applications, 4, 7 – 9 oxidants, antioxidants, gene expression, 1 – 9 Oxidants/antioxidants differential gene expression, 3 – 6 nutrigenomics, 1 –9 endothelium, 148 – 152 immune function, effects on, 106 NF-kB mediated gene expression, 143 – 148 Oxidative stress aging, effect on, 103 gene expression, 68 – 86 human stress variation, 13 – 32 manganese superoxide dismutase, 15 – 18 MnSOD health conditions, 17 – 18 MnSOD polymorphisms, 16 – 17 glutathione-s-transferases, 19 – 20 GST genes, 20 – 24 SOD3, 18 – 19 lycopene, 243 – 245 replicative lifespan, 85 – 86 PCA (principal component analysis), 53 Peroxisome proliferators-activated receptors (PPAR), 192 transcription factors, 192 – 193

Index Phytoestrogens, isoflavones, 302– 303 PI 3-kinase, apoptosis, 409– 410 Platelet aggregation alpha tocopherol, NO, 210– 211 endothelium reactivity, isoflavones, 317 – 318 Polymorphisms CAT, 27 GPX1, 29 MPO, 30 – 31 NADPH, 28 NQO1, 31 SOD3, 19 Polyphenols, procyanidins, 380 Polyunsaturated fatty acids (PUFA), 203 epatic lipogenesis, 187– 189 induction, 189 mechanisms, 191– 192 polyunsaturated fatty acids, 203 PPAR ligands, transcription factors, 193 Procyanidins antioxidant properties, 379–390 gene expression modulation, 383 – 389 cancer related genes, 384– 385 cardiovascular related genes, 384 cDNA array techniques, 386– 389 inflammation related genes, 385 – 386 polyphenols, 380 sources, bioavailability, 380–382 Programmed cell death, IP6, 409– 410 Pro-inflammatory genes, antioxidants, 164 – 167 Pro-inflammatory, cytokines, immune function, 101 Pro-oxidant, iron, 432– 433 Prostate cancer, isoflavones, 332–333 Protein(s), macromolecules IP6 interactions, 402– 405 Protein kinase C alpha tocopherol, 206– 208 IP6, cell proliferation normalization, 407 QT clustering, hierarchical trees, 57 – 59

481 Ras proteins, cell proliferation normalization, 407 – 408 Reactive oxygen species (ROS), 68 Redox status ascorbate recycling, 260 – 263 ascorbic acid, 260 – 265 Replicative lifespan, oxidative stress, 85 – 86 Reproduction system, C. elegans, 77 – 78 Retinoblastoma protein, cell proliferation normalization, 406 – 407 Scavenger receptors, chemokines, 208 – 209 Selenium deficiency, symptoms, 443 – 444 gene expression, 441 – 451 metabolism, 442 – 443 selenocysteine-containing proteins, 445 – 446 selenoprotein expression, 447 – 448 Selenocysteine-containing proteins, 445 – 446 Self-organizing maps (SOM), 52 class discovery, 52 – 53 Serum lipids, soy isoflavones, 311 – 312 Shear stress, 161 – 164 Signaling cascade modulations, 362 –365 flavonoids, 359 – 367 flavonols, 365 –367 MAP kinase signaling, 360 – 361 Single nucleotide polymorphisms (SNPS), 14 Smoking, eNOS health conditions, 26 SOD2, MnSOD, 15 SOD3 copper, zinc superoxide dismutase, 18 – 19 oxidative stress, humans stress variations,18 –19 polymorphisms, 19 Software, microarray analysis, 61 – 62 Soy isoflavones anti-carcinogenic properties, 327 – 336 blood pressure, 314 serum lipids, 311 – 312

482 Sterol regulatory element-binding protein, transcription factors, 194 Stress hormesis, C. elegans, 69– 70 Stress resistance, insulin/IGF-1 signaling, 73– 76 Stress-induced premature senescense (SIPS), 85 Supervised learning methods, 59– 60 Taurine, immune function, 115– 116 Tocopherol, alpha, molecular targets, 206 Tocopherols/tocotrienols, cell regulatory activity, 201– 214 Tocotrienols biological properties, 213– 214 anticarcinogenic properties, 213 neuroprotection, Src activity, 213 –214 cell regulatory activity, tocopherols, 201 –204 Transcription factors ascorbic acid, molecular functions, 268 –271 fatty acid regulations, gene expression, 192 –194 hepatic nuclear receptor-4(HNF-4), 194 nuclear factor kappa B (NF-kB), 194 peroxisome proliferators-activator receptors, 192– 193 PPAR ligands, 193 sterol regulatory element-binding protein, 194 Transcriptomics, applications, techniques, 7 Type 2 diabetes, a-Lipoic acid, glucose metabolism, 287– 288 Val allele associations, health conditions, 23 Validation, microarray analysis, 60– 61

Index in silico, 60 – 61 laboratory based, 61 Valine (VAL), 16 Vesicle trafficking, IP6 interactions, 402 – 405 Vitamin A biosynthetic pathway, molecular analyses model organism, 229 vertebrates, 231 – 235 carotenoids b,b-caroteine-15,150 -monooxygenases, 226 – 227 color function, 222 – 223 deficiency (VAD), 223 functions, 223 –225 insects, 229 – 230 molecular analysis, 221 – 236 Vitamin A deficiency (VAD), 223 Vitamin E (VE), 202 absorption, transport, metabolism, 204 – 206 atherosclerosis, 153 chemistry, antioxidant properties, 202 – 204 endothelial gene expression, 152 – 157 immune function, effects on, 110 – 111 inflammatory genes, 155 – 156 VP14-homologs, animal physiology, 227 – 229 Zinc as antioxidant, 458 – 460 ionic, 458 MT, 458 – 460 dietary, gene expression modulation, 457 – 468 expression profiling, 464 –468 molecular mechanisms, 460 – 463 Zinc-finger motif, IP6, nuclear inositol signaling, 411