Food Factors for Health Promotion
Forum of Nutrition Vol. 61
Series Editor
Ibrahim Elmadfa
Vienna
Food Factors for Health Promotion Volume Editor
Toshikazu Yoshikawa Kyoto Prefectural University of Medicine, Kyoto
53 figures, 1 in color and 12 tables, 2009
Basel · Freiburg · Paris · London · New York · Bangalore · Bangkok · Shanghai · Singapore · Tokyo · Sydney
Toshikazu Yoshikawa Molecular Gastroenterology and Hepatology Kyoto Prefectural University of Medicine Graduate School of Medical Science Kyoto, Japan
Library of Congress Cataloging-in-Publication Data International Conference on Food Factors for Health Promotion (2007 : Tokyo, Japan) Food factors for health promotion / volume editor, Toshikazu Yoshikawa. p. ; cm. -- (Forum of nutrition, ISSN 1660-0347 ; v. 61) Includes bibliographical references and index. ISBN 978-3-8055-9097-6 (hard cover : alk. paper) 1. Functional foods--Congresses. I. Yoshikawa, Toshikazu. II. Title. [DNLM: 1. Food--Congresses. 2. Nutritive Value--Congresses. 3. Metabolic Phenomena--Congresses. 4. Nutrition Therapy--Congresses. 5. Nutritional Physiological Phenomena--Congresses. W1 BI422 v.61 2009 / QU 145.5 I615f 2009] QP144.F85I58 2009 613.2--dc22 2009010218
Bibliographic Indices. This publication is listed in bibliographic services, including Current Contents® and PubMed/MEDLINE. Disclaimer. The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publisher and the editor(s). The appearance of advertisements in the book is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements. Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. © Copyright 2009 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) www.karger.com Printed in Switzerland on acid-free and non-aging paper (ISO 9706) by Reinhardt Druck, Basel ISSN 1660–0347 ISBN 978–3–8055–9097–6 e-ISBN 978–3–8055–9098–3
Contents
VII XI
List of Contributors Preface Yoshikawa, T. (Kyoto) Genomics
1 10
25
Genomics for Food Functionality and Palatability Abe, K. (Tokyo) Lipid Metabolism and Nutrigenomics – Impact of Sesame Lignans on Gene Expression Profiles and Fatty Acid Oxidation in Rat Liver Ide, T.; Nakashima, Y.; Iida, H.; Yasumoto, S.; Katsuta, M. (Tsukuba) Genome Science of Lipid Metabolism and Obesity Takahashi, N.; Goto, T.; Hirai, S.; Uemura, T.; Kawada, T. (Kyoto) Proteomics
39
Oxidative Stress-Induced Posttranslational Modification of Proteins as a Target of Functional Food Naito, Y.; Yoshikawa, T. (Kyoto) Bioavailability and Safety
55 64
75
Absorption and Function of Dietary Carotenoids Nagao, A. (Tsukuba) Metabolism of Flavonoids Wang, Y.; Ho, C.-T. (Brunswick, N.J.) β-Carotene Degradation Products –Formation, Toxicity and Prevention of Toxicity Siems, W. (Bad Harzburg); Salerno, C.; Crifò, C. (Rome); Sommerburg, O. (Heidelberg); Wiswedel, I. (Magdeburg)
V
Antioxidants 87
Dietary Flavonoids as Antioxidants Terao, J. (Tokushima) Life-style Related Diseases
95
104 117 129
136 147
Inflammatory Components of Adipose Tissue as Target for Treatment of Metabolic Syndrome Yu, R.; Kim, C.-S.; Kang, J.-H.; (Ulsan) Soybean Isoflavones in Bone Health Ishimi, Y. (Tokyo) Probiotics in Primary Prevention of Atopic Dermatitis Ji, G.E. (Seoul) Astaxanthin Protects Neuronal Cells against Oxidative Damage and Is a Potent Candidate for Brain Food Liu, X.; Osawa, T. (Nagoya) Function of Marine Carotenoids Miyashita, K. (Hakodate) Exercise and Food Factors Aoi, W. (Kyoto) Chemoprevention and Cancer
156 170 182
193
204
217 218
VI
Molecular Basis for Cancer Chemoprevention by Green Tea Polyphenol EGCG Tachibana, H. (Fukuoka) Chemoprevention by Isothiocyanates: Molecular Basis of Apoptosis Induction Nakamura, Y. (Okayama) Ginger-Derived Phenolic Substances with Cancer Preventive and Therapeutic Potential Kundu, J.K.; Na, H.-K.; Surh, Y.-J. (Seoul) Chemoprevention with Phytochemicals Targeting Inducible Nitric Oxide Synthase Murakami, A. (Kyoto) Chemoprevention of Tocotrienols: The Mechanism of Antiproliferative Effects Wada, S. (Kyoto) Author Index Subject Index
Contents
List of Contributors
Professor Keiko Abe
Professor Chi-Tang Ho
Department of Applied Biological Chemistry Graduate School of Agricultural and Life Sciences The University of Tokyo 1-1-1 Yayoi Bunkyo-ku Tokyo, Japan
Department of Food Science Rutgers University 65 DudleyRoad New Brunswick, USA
Dr. Wataru Aoi Laboratory of Health Science Graduate School of Life and Environmental Sciences Kyoto Prefectural University 1-5 Hangi-cho Shimogamo Sakyo-ku Kyoto, Japan
Dr. Takashi Ide National Food Research Institute Kannondai 2-1-12 Tsukuba, Japan
Dr. Hiroshi Iida National Food Research Institute Kannondai 2-1-12 Tsukuba, Japan
Dr. Yoshiko Ishimi
Department of Biochemical Sciences University of Rome La Sapienza Piazzale Aldo Moro 5 Rome, Italy
Project for Bio-Index Nutritional Epidemiology Program, National Institute of Health and Nutrition 1-23-1 Toyama Shinjuku-ku Tokyo, Japan
Dr. Tsuyoshi Goto
Professor Geun Eog Ji
Laboratory of Molecular Functions of Foods Division of Food Science and Biotechnology Graduate School of Agriculture Kyoto University Uji Kyoto, Japan
College of Human Ecology Seoul National University Department of Food and Nutrition San 56-1 Shillim-Dong Kwanak-Ku Seoul, Korea
Professor Dr. Carlo Crifò
Dr. Shizuka Hirai Laboratory of Molecular Functions of Foods Division of Food Science and Biotechnology Graduate School of Agriculture Kyoto University Uji Kyoto, Japan
Dr. Ji-Hye Kang Department of Food Science and Nutrition University of Ulsan Mugeo-dong Ulsan, South Korea
VII
Dr. Masumi Katsuta
Dr. Akihiko Nagao
National Institute of Crop Science Kannondai 2-1-18 Tsukuba, Japan
National Food Research Institute National Agriculture and Food Research Organization 2-1-12 Kannondai Tsukuba Ibaraki, Japan
Professor Teruo Kawada Laboratory of Molecular Functions of Foods Division of Food Science and Biotechnology Graduate School of Agriculture Kyoto University Uj Kyoto, Japan
Dr. Chu-Sook Kim Department of Food Science and Nutrition University of Ulsan Mugeo-dong Ulsan, South Korea
Dr. Joydeb Kumar Kundu National Research Laboratory of Molecular Carcinogenesis and Chemoprevention College of Pharmacy Seoul National University Shillim-dong Kwanak-gu Seoul, South Korea
Dr. Xuebo Liu Laboratory of Food and Biodynamics Graduate School of Bioagricultural Science Nagoya University Furo-cho Nagoya, Japan
Dr. Kazuo Miyashita Graduate School of Fisheries Sciences Hokkaido University Hakodate, Japan
Professor Yuji Naito Molecular Gastroenterology and Hepatology Kyoto Prefectural University of Medicine 456 Kajii-cho Kamigyo-ku Kyoto, Japan
Dr. Yoshimasa Nakamura Faculty of Agriculture Okayama University 1-1-1 Tsushima-naka Okayama, Japan
Dr. Yasutaka Nakashima National Food Research Institute Kannondai 2-1-12 Tsukuba, Japan
Dr. Toshihiko Osawa Laboratory of Food and Biodynamics Graduate School of Bioagricultural Science Nagoya University Furo-cho Nagoya, Japan
Professor Costantino Salerno Laboratory of Clinical Biochemistry University of Rome La Sapienza Via dei Sardi 58 Rome, Italy
Dr. Werner Siems Dr. Akira Murakami Division of Food Science and Biotechnology Graduate School of Agriculture Kyoto University Kyoto, Japan
Research Institute of Physiotherapy & Gerontology, KortexMed Institute of Medical Education Hindenburgring 12 A Bad Harzburg, Germany
Dr. Hye-Kyung Na
Dr. Olaf Sommerburg
National Research Laboratory of Molecular Carcinogenesis and Chemoprevention College of Pharmacy Seoul National University Shillim-dong Kwanak-gu Seoul, South Korea
University Children´s Hospital Department III University of Heidelberg Im Neuenheimer Feld 153 Heidelberg, Germany
VIII
List of Contributors
Professor Young-Joon Surh
Dr. Sayori Wada
National Research Laboratory of Molecular Carcinogenesis and Chemoprevention College of Pharmacy Seoul National University Shillim-dong Kwanak-gu Seoul, South Korea
Laboratory of Health Science Kyoto Prefectural University 1-5 Hangi-cho Shimogamo Sakyo-ku Kyoto, Japan
Dr. Hirofumi Tachibana Division of Applied Biological Chemistry Department of Bioscience and Biotechnology Faculty of Agriculture Kyushu University Higashi-ku Hakozaki 6-10-1 Fukuoka, Japan
Dr. Nobuyuki Takahashi Laboratory of Molecular Functions of Foods Division of Food Science and Biotechnology Graduate School of Agriculture Kyoto University. Uji Kyoto, Japan
Dr. Yu Wang Massachusetts Institute of Technology 77 Massachusetts Avenue Room 56-731 Cambridge, USA
Dr. Ingrid Wiswedel Department of Pathological Biochemistry Institute of Clinical Chemistry and Pathological Biochemistry Otto-von-Guericke University Magdeburg Leipziger Str. 44 Magdeburg, Germany
Dr. Satoko Yasumoto National Agricultural Research Center Kannondai 3-1-1 Tsukuba, Japan
Professor Junji Terao
Professor Toshikazu Yoshikawa
Department of Food Science Graduate School of Nutrition and Bioscience The University of Tokushima 18-15 Kuramoto-cho 3 Tokushima, Japan
Molecular Gastroenterology and Hepatology Kyoto Prefectural University of Medicine 456 Kajii-cho Kamigyo-ku Kyoto, Japan
Dr. Taku Uemura
Dr. Rina Yu
Laboratory of Molecular Functions of Foods Division of Food Science and Biotechnology Graduate School of Agriculture Kyoto University Uji Kyoto, Japan
Department of Food Science and Nutrition University of Ulsan Mugeo-dong Ulsan, South Korea
List of Contributors
IX
Preface
Food factors are considered to be critical for human health promotion. It was a great pleasure for me to host the International Conference on Food Factors for Health Promotion (ICoFF 2007) that took place from November 27 to December 1, 2007, in Kyoto, Japan. The ICoFF2007 was organized mainly by the Japanese Society for Food Factors, which leads basic and clinical research in the field of functional food. The theme of the ICoFF2007 was ‘Food Factors for Health Promotion’. We thought that this was appropriate because food factors may function as the frontline in the prevention of lifestyle-related disProf. Toshikazu Yoshikawa eases as well as in health promotion. Prevention of lifestyle-related disease is listed as one of the priority research subjects not only in Japan but also in Western countries. Multiple factors are involved in the development of these diseases. A key future challenge is to clarify these factors, invent a method of detecting any change in the initial phase and establish a diagnostic approach that applies to prevention studies involving food factors. Although the function of food factors has been the main focus of this conference, other areas of interest in recent basic and clinical research also include bioavailability, metabolism, and safety of foods and their ingredients. This meeting provided an important opportunity to promote the development of food factor science. We invited several opinion leaders and young researchers from the USA, EU, Korea and other countries.
XI
This book includes an introductory overview of food factors and perspectives on bioavailability, chemistry and biomarkers that were presented during the ICoFF2007. We thank all the authors for their contributions and efforts in the preparation of this book. We also extend our sincere gratitude to the many scientists who reviewed the chapters found herein. Prof. Toshikazu Yoshikawa, Kyoto
XII
Yoshikawa
Genomics Yoshikawa T (ed): Food Factors for Health Promotion. Forum Nutr. Basel, Karger, 2009, vol 61, pp 1–9
Genomics for Food Functionality and Palatability Keiko Abe The University of Tokyo, Graduate School of Agricultural and Life Sciences, Tokyo, Japan
Abstract In the 1980s, Japan proposed the termiology of ‘functional food’ and its concept [1], and since then the importance of conducting basic and applied studies on food functionality has been emphasized globally. Functional foods in particular as well as common foods in general are constituted with a variety of components including functional factors, and it has been recognized as difficult to evaluate their functionalities by usual chemical, biochemical and physiological methodologies [2]. Against this backdrop, nutrigenomics came into being as a new method of evaluating functional foods, as well as nutrients, in a holistic manner. Meanwhile the endowed chair, Functional Food Genomics, was established at the University of Tokyo with the aegis of 32 food companies in Japan. This academia-industry collaboration has been working well to disclose why and how some particular functional foods elicit their effects in the body. These include soy protein isolate, cocoa polyphenol, sesamin as a lignan of sesame origin, and many others. On the other hand, food safety has been gaining public attention, and we applied genomics for assessment of the wholesomeness of newly developed hypoallergenic wheat flour compared with normal flour. The application of this way of holistic evaluation suggested that the new product was basically the same as the normal product in terms of all-gene expression profiles. The same method was applied to a new sweet protein, neoculin, which resembled toxic lectins in conformation. The result indicated that neoculin had lost its lectin activity, possessing no particular toxic effect. It is thus likely that genomics can be applied to a variety of foods in general for the Copyright © 2009 S. Karger AG, Basel purpose of simultaneously assessing their functionality.
In the 1980s, Prof. Soichi Arai, Department of Agricultural Chemistry at The University of Tokyo took the initiative of successfully launching, with the help of his colleagues, including myself, the Priority Area Research Project on Functional Food supported by the Grant-in-Aid from the Ministry of Education, Science, Sports and Culture. Food has traditionally been thought of as having two functions: the primary function of providing nutrition to the body and the secondary function of providing culinary enjoyment. The research team demonstrated that there exists a third function which contributes to reducing the risk of developing lifestyle-related diseases. The team declared to the world that it had named the type of food with this function, ‘functional food’ [1].
This basic and applied research opened the new field of food studies and attracted tremendous attention from inside and outside Japan, triggering a boom of research activities in this area. The advent of the aging society has accelerated this trend. The Department of Agricultural Chemistry had, as its traditional research principle, the extension of studies in life sciences from basic to applied research. It is a very natural consequence that the Department of Applied Biological Chemistry, a successor of this idea, has been leading functional food science to launch this endowed chair. What is more, given that cooperative research projects between industry and academia are today being promoted by the government, functional food science may even serve as a pioneering model. The other reason stems from the characteristics of food studies. Functional foods have some elements that help reduce the risk of disease, but unlike pharmaceuticals, each of them is a complex system consisting of multiple elements. Functional food factors may be affected by other ingredients contained in the same food, and their functions could be increased or decreased. In addition, we continuously consume a large amount of food every day, and the compounds resulting from metabolism in the body are therefore plentiful both in quantity and in quality. An excessive intake may exert adverse effects on physical health. It might cancel the positive effect of functional food factors. Even if any benefit is evident, the part that receives the benefit varies extensively: the digestive tract, the liver, kidneys, blood, muscles, the brain and others. Also, the duration of the effect is not even. What we need to do is comprehensively verify the overall effect of eating a single kind of functional food (table 1). In this sense, the traditional styles of physiology, biochemistry, molecular and cell biology, etc. are so much aimed at the deep investigation of individual matters that they are not appropriate for food research. This is where genomics comes in as a new science for analyzing the information of all genes. In 2002, nutrigenomics was established as applied genomics in the science of nutrition. Behind this event was a major global progress in functional food science as a new domain of the nutrition sciences [3]. The notable aspect of this progress is the attempt to measure the effects of ingested functional foods by means of simple indicators such as biomarkers prior to examining those by human intervention tests. The emergence of nutrigenomics came to represent these efforts. At the beginning, transcriptomics was the mainstay [4]. Lee et al. [5] found that old mice have a stronger expression of inflammatory and stress genes and a weaker expression of protein metabolism- and growth-related genes than young mice, and regarded the genes as indicators of aging. They also verified that the fluctuations in genes serving as markers of age can be controlled by limiting the daily calorie intake by 30%. In other words, the hypothesis that restricting calorie intake slows aging has been substantiated at the genetic level. This was the first ever nutrigenomics research report. The present author had introduced a DNA microarray system to start research before the term nutrigenomics was established in Europe. There was the belief that
2
Abe
Table 1. Characteristics of food compared with drug Item
Food
Drug
Chemistry Composition Source Structure Reactivity
multiple natural heterogeneous high
simple mostly designed homogeneous low
Intake Purpose Time Period Daily consumption
for nutrition usual lifelong large
for remedy when needed generally short trace
Physiology Taste Absorption Remaining time Target organ or tissue Efficiency/efficacy Secondary effect Metabolite Synergy Individual difference
important various various non-specific slow impossible multiple large large
unimportant easy short specific quick possible simple small large
this exhaustive analytical approach would be indispensable in the overall elucidation of the diverse range of effects that the consumption of food, which is a multicomponent complex system, has on living bodies (fig. 1). From the perspective of the industry, the approaches to food development are fundamentally different from the methods for developing pharmaceutical products. Simply put, the former is centrifugal while the latter is concentric (fig. 1). Nutrigenomics is significant to analysis in a wide range of centrifugal studies.
Functional Food Genomics
In the second half of 2003, about 30 member companies of a nonprofit organization called the International Life Sciences Institute of Japan (ILSI Japan) made a joint investment in the launch of an Endowed Chair of Functional Food Genomics run by a guest associate professor, Dr. Ichiro Matsumoto, Graduate School of Agricultural and Life Sciences, in which the author herself works to initiate studies on
Genomics for Food Functionality
3
Target tissue/cell
Functional food
Genes
Proteins
$$ %%
Physiological functions • Improved lipid metabolism • Resistance to obesity • Resistance to infection • Antioxidation and antiinflammation • Intestinal modulation and antiinfection • Immunotolerance and hypoallergenicity
&&
Analysis
Evaluation
Data
Fig. 1. Nutrigenomics-based evaluation of functional food.
nutrigenomics in cooperation with academia and industry. Major research achievements are as follows.
Soy Protein Functioning to Improve Lipid Metabolism In joint research with Fuji Oil Co., Ltd., the gene expression in rat liver was analyzed after a long-term intake of soy protein isolate (SPI) in order to determine what is behind the improvement in dislipidemia and hypercholesteremia as one of the multiple functions performed by soy protein (fig. 1) [6]. It confirmed that the concentrations of cholesterol and triglyceride in the blood were lower in the group with a 2-month intake of SPI than in the control group of casein intake. A DNA microarray analysis of the liver found that rats fed SPI had higher expression of genes for the antioxidant and sterol metabolism systems and lower expression of genes for cell proliferation, structural protein, amino acid metabolism and sterol metabolism systems than casein-fed rats. A majority of genes with at least 50% difference are concerned with cholesterol metabolism, fatty acid metabolism and antioxidation. The SPI intake was confirmed to have the most significant impact on fat metabolism and antioxidation systems. The genes involved included those relating to the fatty acid synthesis systems, which are considered to have reduced the concentration of triglyceride in the blood. On the other hand, the blood cholesterol concentration was significantly
4
Abe
reduced while the gene expression in the sterol synthesis system was significantly increased. Considering the significant rise in the excretion of acid steroid (bile acid) and neutral steroid (cholesterol), it is assumed that SPI exhibited a function of facilitating steroid excretion in a short time and that this effect lasted for a long time to eventually up-regulate sterol synthesis genes for the purpose of constantly controlling the level of cholesterol in the body.
Antiobesity Function of Cocoa Polyphenol Morinaga & Co., Ltd., has found that the group of rats fed with a high-fat diet (27% lard) and 12.5% cocoa polyphenol had a lower rate of weight increase and a lower weight of visceral fat with statistical significance than rats fed with a cocoa substitute (control) [7]. Also, the high cocoa group tended to have a lower level of serum triacylglycerol. To elucidate the reason for this by examining gene expression in the liver and fat cells, this study performed a DNA microarray analysis of the liver and mesenteric white fat cells. It has confirmed that (1) the antiobesity effect on the liver is caused by the decline in the blood triacylglycerol concentration after down-regulation of fatty acid biosynthesis, and that (2) the same effect on fat cells is produced by inhibition of the fatty acid transport system after down-regulation of PPARγ, inhibition of the fatty acid synthesis system after down-regulation of SREBP-1c, and the decline in cumulative fat after combustion of fatty acid following up-regulation of uncoupling proteins that act as exothermic factors.
Function of Sesamin to Regulate β-Oxidation and to Boost Alcohol Metabolism Sesamin is a principal lignan contained in sesame seeds. Under joint research with Suntory Limited, an analysis was conducted of gene expression changes in the liver of a rat fed with sesamin for 3 days to observe whether its intake up-regulated the expression of genes for various fat metabolism enzymes associated with β-oxidation and lipogenesis [8]. More interestingly, in the light of the up-regulation of genes for aldehyde dehydrogenase (ALDH) acting on alcohol catabolism, it was demonstrated that sesamin exhibits the function of regulating alcohol metabolism as activated ALDH accelerates the decomposition of acetaldehyde.
Royal Jelly Functioning to Facilitate Osteogenesis At the initiative of Nagaragawa Research Center of API Co., Ltd., an analysis was conducted of the osteogenetic effect of royal jelly (RJ) [9]. An increase in the mineral weight of the neck bone was confirmed in a mouse administered with RJ for 2
Genomics for Food Functionality
5
months. An analysis of gene expression in a femur observed a significant up-regulation of some 300 genes in the RJ-fed group in comparison with the non-RJ-fed group. Seventy percent of these genes were upregulated in the group receiving subcutaneous administration of 17β-estradiol (E2). This implies that RJ produces an estrogenic effect.
Safety Assessment of Low Allergen Wheat As a nutrigenomic case study on food safety assessment, an analysis of hypoallergenic wheat flour was carried out [10]. A team led by Dr. Hisanori Kato, Associate Professor, Graduate School of Agricultural and Life Sciences, The University of Tokyo, examined the gene expression in the livers and small intestines of rats fed a 12% protein diet containing hypoallergenic wheat flour or ordinary wheat flour. It was found that very few genes had changes in fold and that none of these genes were concerned with any function adverse to living bodies, such as toxicity. Today, increasing attention is focused on food safety. This study has confirmed that DNA microarray analysis can be used in the safety assessment. Just recently, nutrigenomics incorporated proteomics and metabolomics into transcriptomics. In other words, it is necessary to use these three in a manner where they are coordinated. Known as ‘coordinative genomics,’ it is now considered of indispensable significance to research on the overall effect of a complicated heterosystem, namely food, on living bodies. In the future, it is necessary to perform a study on personalized functional food in consideration of the variations among individuals. It is thought from a molecular perspective that the individual difference results from single nucleotide polymorphisms generated by the partial variation of individual genes. It is anticipated that we are sure to see tailor-made functional foods in the near future. Steps towards second-generation nutrigenomics are already being taken at a steady pace.
Food Safety Genomics with Special Reference to Neoculin as a New Sweet Protein and Plant Lectins – A Pilot Study
While the majority of sweet substances are of low molecular weight, there are six proteins, brazzein, thaumatin, mabinlin, monellin, neoculin (NCL) and pentadin, which elicit sweetness to humans. These substances as nonglycemic sweeteners will provide a potential use for people with obesity, diabetes and other metabolic syndromes. NCL occurring in the tropical fruits of Curculigo latifolia is currently the only protein that has both sweetness and a taste-modifying activity to convert sourness into sweetness [11]. The strong sweetness of NCL makes it possible to use this substance as a tool for basic taste signaling research as well. However, it has remained unclear how NCL induces this unique sensation.
6
Abe
Neoculin
180°
Garlic lectin
Fig. 2. X-ray crystallography.
10,000
PHA-E
1,000 100 10 1 1
100
10,000
10,000
WGA
1,000 100 10 1 1
100
10,000
Neoculin
10,000 1,000 100 10 1 1
100 10,000 Control
Neoculin
WGA
Fig. 3. DNA microarray analysis.
Genomics for Food Functionality
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Recently, we quantitatively evaluated the acid-induced sweetness of NCL by a cellbased assay [12]. In brief, human sweet taste receptor hT1R2-hT1R3 was functionally expressed, together with chimeric G␣, in cultured cells. The cells responded to NCL pH dependently under acidic conditions. The pH-response relationship reflected a sigmoidal, imidazole titration curve, suggesting the involvement of histidine residues in the acid-induced sweetness [13]. Actually, an NCL variant in which all the five histidine residues were replaced with alanine elicited strong sweetness at neutral as well as acidic pH. The His →Ala variant is apparently a novel sweet protein genetically engineered. Both the primary and overall tertiary structures of NCL resemble those of monocot mannose-binding lectins [14]. This study investigated differences in biochemical properties between NCL and the lectins. Structural comparison between the mannose-binding site of lectins and the corresponding regions of NCL showed that there is at least one amino acid substitution at each site in NCL, suggesting a reason for the lack of its mannose-binding ability (fig. 2). This was consistent with hemagglutination assay data demonstrating that NCL had no detectable agglutinin activity. DNA microarray analysis indicated that NCL had no significant influence on gene expression in the Caco-2 cell, whereas kidney bean lectin (Phaseolus vulgaris agglutinin) greatly influenced various gene expressions [15] (fig. 3). These data strongly suggest that NCL has no lectin-like properties, encouraging its practical use in the food industry.
References 1 Swinbanks D, O’Brien J: Japan explores the boundary between food and medicine. Nature 1993;364: 180. 2 Arai S, Yasuoka A, Abe K: Functional food science and food for specified health use policy in Japan: state of the art. Curr Opin Lipidol 2008;19: 69–73. 3 Roberfroid MB: Global view on functional foods: European perspectives. Brit J Nutr 2002;88:S133– 138. 4 Müller M, Kersten S: Nutrigenomics: goals and strategies. Nat Rev 2003;4:315–322. 5 Lee C-K, Klopp RG, Weindruch R, Prolla TA: Gene expression profile of aging and its retardation by caloric restriction. Science 1999;285:1390–1393. 6 Tachibana N, Matsumoto I, Fukui K, Arai S, Kato H, Abe K, Takamatsu KJ: Intake of soy protein isolate alters hepatic gene expression in rats. Agric Food Chem 2005;53, 4253–4257.
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7 Matsui N, Ito R, Nishimura E, Yoshikawa M, Kato M, Kamei M, Shibata H, Matsumoto I, Abe K, Hashizume S: Ingested cocoa can prevent high-fat diet-induced obesity by regulating the expression of genes for fatty acid metabolism. Nutrition 2005;21: 594–601. 8 Tsuruoka N, Kidokoro A, Matsumoto I, Abe K, Kiso Y: Modulating effect of sesamin, a functional lignan in sesame seeds, on the transcription levels of lipidand alcohol-metabolizing enzymes in rat liver: a DNA microarray study. Biosci Biotechnol Biochem 2005;69:179–188. 9 Narita Y, Nomura J, Ohta S, Inoh Y, Suzuki KM, Araki Y, Okada S, Matsumoto I, Isohama Y, Abe K, Miyata T, Mishima S: Royal jelly stimulates bone formation: physiologic and nutrigenomic studies with mice and cell lines. Biosci Biotechnol Biochem 2006;70:2508–2514. 10 Narasaka S, Endo Y, Fu ZW, Moriyama M, Arai S, Abe K, Kato, H: Safety evaluation of hypoallergenic wheat flour by using a DNA microarray. Biosci Biotechnol Biochem 2006;70, 1464–1470.
Abe
11 Shirasuka Y, Nakajima K, Asakura T, Yamashita H, Yamamoto A, Hata S, Nagata S, Abo M, Sorimachi H, Abe K: Neoculin as a new taste-modifying protein occurring in the fruit of Curculigo latifolia. Biosci Biotechnol Biochem 2004;68:1403–1407. 12 Nakajima K, Asakura T, Oike H, Morita Y, ShimizuIbuka A, Misaka T, Sorimachi H, Arai S, Abe K: Neoculin, a taste-modifying protein, is recognized by human sweet taste receptor. Neuroreport 2006;17: 1241–1244. 13 Nakajima K, Morita Y, Koizumi A, Asakura T, Terada T, Ito K, Shimizu-Ibuka A, Maruyama J, Kitamoto K, Misaka T, Abe K: Acid-induced sweetness of neoculin is ascribed to its pH-dependent agonistic-antagonistic interaction with human sweet taste receptor. FASEB J 2008;22:2323–2330.
14 Shimizu-Ibuka A, Morita Y, Terada T, Asakura T, Nakajima K, Iwata S, Misaka T, Sorimachi H, Arai S, Abe K: Crystal structure of neoculin: insights into its sweetness and taste-modifying activity. J Mol Biol 2006;359:148–158. 15 Shimizu-Ibuka A, Nakai Y, Nakamori K, Morita Y, Nakajima, K, Kadota K, Watanabe H, Okubo S, Terada T, Asakura T, Misaka T, Abe K: Biochemical and genomic analysis of neoculin compared to monocot mannose-binding lectins. J Agric Food Chem 2008;56:5338–5344.
Professor Keiko Abe Department of Applied Biological Chemistry Graduate School of Agricultural and Life Sciences The University of Tokyo 1-1-1 Yayoi, Bunkyo-ku Tokyo 113-8657 (Japan) Tel. + 81 3 5841 5129, Fax +81 3 5841 8006, E-Mail
[email protected]
Genomics for Food Functionality
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Genomics Yoshikawa T (ed): Food Factors for Health Promotion. Forum Nutr. Basel, Karger, 2009, vol 61, pp 10–24
Lipid Metabolism and Nutrigenomics – Impact of Sesame Lignans on Gene Expression Profiles and Fatty Acid Oxidation in Rat Liver Takashi Idea ⭈ Yasutaka Nakashimaa ⭈ Hiroshi Iidaa ⭈ Satoko Yasumotob ⭈ Masumi Katsutac a National Food Research Institute, bNational Agricultural Research Center, and cNational Institute of Crop Science, Tsukuba, Japan
Abstract The impact of sesamin, episesamin and sesamolin (sesame lignans) on hepatic gene expression profiles was compared with a DNA microarray. Male Sprague-Dawley rats were fed experimental diets containing 0.2% sesamin, episesamin or sesamolin, and a control diet free of lignans for 15 days. Compared to a lignan-free diet, a diet containing sesamin, episesamin and sesamolin caused more than 1.5- and 2-fold changes in the expression of 128 and 40, 526 and 152, and 516 and 140 genes, respectively. The lignans modified the mRNA levels of not only many enzymes involved in hepatic fatty acid oxidation, but also proteins involved in the transportation of fatty acids into hepatocytes and their organelles, and in the regulation of hepatic concentrations of carnitine, CoA and malonylCoA. It is apparent that sesame lignans stimulate hepatic fatty acid oxidation by affecting the gene expression of various proteins regulating hepatic fatty acid metabolism. The changes in the gene expression were generally greater with episesamin and sesamolin than with sesamin. In terms of amounts accumulated in serum and the liver, the lignans ranked in the order sesamolin, episesamin and sesamin. The differences in bioavailability among these lignans appear to be important to their divergent physiological activities. We also confirmed that dietary sesame seed affected the expression of genes related to fatty acid oxidation in a manner similar to isolated lignan compounds. Copyright © 2009 S. Karger AG, Basel
Sesame seeds contain compounds known collectively as lignans. Sesamin and sesamolin are fat soluble, and sesame seeds and their oil contain these two lignans at a ratio of about 2:1 [1]. Another major lignan of sesame is sesaminol which exists as glucosides [2], and is not extractable in oil. In the refining of sesame oil, sesamin is epimerized to form episesamin, and most of the sesamolin is degraded [3]. The lignan preparation obtained as a by-product of the refining of edible sesame oil therefore consists of sesamin and episesamin at a ratio of 1:1. This preparation has
been tested for its physiological activity by many investigators. We previously demonstrated that this preparation strongly increased the activity and gene expression of enzymes involved in fatty acid oxidation in the rat liver [4]. This may account for its serum lipid-lowering effect [4–6]. We subsequently demonstrated that episesamin [7] and sesamolin [8], compared to sesamin, are much stronger at increasing the activity and gene expression of enzymes involved in fatty acid oxidation. These results indicate that large differences exist among various lignans in their effect on hepatic fatty acid oxidation. However, no study has simultaneously compared the physiological activities of these three lignans. The DNA microarray is a powerful tool for genome-wide analyses of gene expression patterns. Using DNA microarrays containing about 8,000 probes, Tsuruoka et al. [9] confirmed our previous finding [4] that a lignan preparation containing equivalent amounts of sesamin and episesamin strongly increased the mRNA expression of various enzymes involved in fatty acid oxidation in the rat liver. In addition, they showed that it increased the gene expression of aldehyde dehydrogenase 1 family members (Aldh1a1 and 1a7). They suggested that upregulation of these enzymes may be responsible for the preventive effect of the lignan preparation on the ethanol-induced liver damage observed previously [10]. As they used a lignan preparation containing both sesamin and episesamin in their experiment, information on different effects of various lignans affecting hepatic gene expression profiles is still lacking. The recent development of DNA microarray technology has enabled the examination of more widespread changes in gene expression profiles. Here, we compared the physiological activity of dietary sesamin, episesamin and sesamolin in affecting gene expression profiles in the rat liver using a microarray containing more than 30,000 oligonucleotide probes. In addition, we also examined the effect of sesame seeds on hepatic gene expression.
Methods Animals and Diets Male Sprague-Dawley rats obtained from Charles River Japan, Kanagawa, Japan, at 5 weeks of age were divided into 4 groups with equal mean body weights consisting of 7 animals each and fed either a diet free of lignan or diets containing 0.2% lignan (sesamin, episesamin or sesamolin) for 15 days (experiment 1). In a second experiment (experiment 2), three groups of rats consisting of 7 animals each were fed either a diet containing 10 or 20% sesame seed powder or a diet without sesame for 15 days. We used a sesame line rich in lignan developed by Sirato-Yasumoto et al. [1] in this experiment. Diets containing 10 and 20% sesame seed powder had 0.165 (0.116% as sesamin and 0.049% as sesamolin) and 0.33% (0.232% as sesamin and 0.098% as sesamolin) lignan, respectively. The basal composition of the experimental diet was the same as described previously [1, 8]. Upon termination of the experimental period, animals were anesthetized using diethyl ether and killed by bleeding from the abdominal aorta, after which livers were excised. This study was approved by the review board of animal ethics of our institute, and we followed the institute’s guidelines in the care and use of laboratory animals.
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Affymetrics GeneChip and GeneSpring Analyses RNA extracted from the livers of 5 and 6 rats from each group was subjected to microarray analyses in experiments 1 and 2, respectively. Rats with the highest and lowest body weights in experiment 1, and the animals with the lowest body weight in experiment 2 in each group at the time of killing were eliminated from the microarray analyses. RNA was processed using kits supplied by Affymetrix (Santa Clara, Calif., USA) to prepare fragmented biotinylated cRNA for hybridization to the Rat Genome 230 2.0 Array. Analyses of the DNA microarray data were performed using GeneSpring GX v7.3 software (Agilent Technologies Inc., Santa Clara, Calif., USA).
Real-Time PCR Quantification of Hepatic mRNA The quantification of mRNA by real-time PCR was performed as detailed previously [11]. mRNA abundance was calculated as a ratio to the mRNA level of β-actin in each cDNA sample and expressed as fold change, assigning a value of 1 for rats fed a diet free of lignan (experiment 1) or sesame seed (experiment 2).
Analyses of Lignans and Carnitine Concentrations of lignans in the liver and serum were analyzed by HPLC as detailed previously [12]. The hepatic concentration of carnitine was analyzed by the method of Pearson et al. [13].
Results
Impact of Sesame Lignans (Sesamin, Episesamin, and Sesamolin) and Sesame Seed on Gene Expression Profile in Rat Liver In total, 679 genes were found to be significantly (p < 0.05) up- or downregulated more than 1.5-fold by either sesamin, episesamin or sesamolin (experiment 1). Compared with a lignan-free diet, a diet containing sesamin caused changes more than 1.5- and 2-fold in the expression of 128 and 40 genes, respectively. More of the genes were affected by episesamin and sesamolin. Episesamin and sesamolin caused changes more than 1.5-fold in the expression of 526 and 516 genes, respectively. The numbers of genes up- or downregulated by dietary lignans more than 2-fold are shown as Venn diagrams (fig. 1). The diagram in figure 1a shows that many genes were commonly upregulated by various lignans. Actually, 97% of genes upregulated more than 2-fold by sesamin were also upregulated by one or both of the other lignans. This was the same for episesamin and sesamolin. Therefore, lignans resembled each other with respect to affecting genes whose expression was upregulated. The proportions were much lower for the genes downregulated by lignans. However, detailed analyses indicated that the situation was similar to that for the upregulated genes. A subsequent Tukey’s test revealed that 93 and 90% of the genes downregulated more than 2-fold by episesamin and sesamolin, respectively, were also significantly down-regulated by
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Episesamin
Sesamin
30 55
2
33
3
0
21
3
a
Sesamin
1
0
8
Episesamin
Sesamolin
4 0
28
b
Sesamolin
Fig. 1. Venn diagrams of genes up- or downregulated in the livers of rats fed various sesame lignans. Number of genes upregulated (a) and downregulated (b) more than 2.0-fold. Each of the circles represents the genes affected by the respective lignan. The numbers in the spaces between overlapping circles represent the number of genes that were affected by the lignan species represented by the circles. The numbers in the outer portion of each circle represent the number of genes that were exclusively affected by the specific lignan represented by that particular circle.
one or both of the other lignans, although the extent of the changes was not necessarily greater than 2-fold. This evaluation was rather difficult for sesamin because only 9 genes were downregulated more than 2-fold by this lignan. The functions of genes significantly up- or downregulated more than 1.5-fold by treatment with various lignans were clarified using annotations and information supplied by GeneSpring and several databases (Rat Genome Database, Entrez Gene, and PubMed). Miscellaneous genes with diverse functions were up- or downregulated by lignans. Previous studies showed that dietary lignans profoundly affect hepatic fatty acid oxidation [4–9, 12]. Therefore, genes related to fatty acid oxidation whose expression was up- or downregulated more than 1.5-fold were selected and are listed in table 1. Dietary lignans increased the gene expression of many mitochondrial and peroxisomal enzymes related to fatty acid oxidation, including those involved in acylcarnitine biosynthesis (Crat, Crot and Cpt1b), the conversion of acylcarnitine to acylCoA (Cpt2), β-oxidation (Ehhadh, Ech1, Acaa1 and 2, Acox1, Hadha, Hadhb and Acadvl), the auxiliary pathway of β-oxidation (Dci, and Decr1 and 2), ω-oxidation (Cyp4a1; Cyp4a10 and Cyp4a3), and ketogenesis (Acat1 and Hmgcl). In addition, the genes for Pex11a, presumed to play a role in peroxisome membrane biogenesis [14], and for Fxc1, which mediates the import and insertion of hydrophobic membrane proteins into the mitochondrial inner membrane [15], were activated by lignans. The three lignans decreased the expression of Acaca involved in the biosynthesis of malonyl-CoA to a similar level. In contrast, they increased the expression of Mlycd, which catalyzes the breakdown of malonyl-CoA. The increases were greater with episesamin and sesamolin than with sesamin. Episesamin and sesamolin, but not sesamin, significantly increased the mRNA expression of Mgll, which may be involved in the hydrolysis of intracellular triacylglycerol [16], and Pank1, which catalyzes a rate-limiting step in CoA biosynthesis [17].
Sesame Lignans and Gene Expression
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Table 1. Microarray analyses of genes for proteins involved in the regulation of fatty acid oxidation and those involved in the biogenesis of mitochondria and peroxisomes whose expression was affected (>1.5fold) by either sesamin, episesamin or sesamolin (experiment 1) or by a diet containing 10 or 20% sesame seed (experiment 2). Accession number
Gene symbol
Gene name
Enzymes involved in fatty acid oxidation AI411979
Crat
Carnitine acetyltransferase
NM_133606
Ehhadh
Enoyl-coenzyme A, hydratase/3- hydroxyacyl coenzyme A dehydrogenase
J02844
Crot
Carnitine O-octanoyl transferase
NM_017306
Dci
Dodecenoyl- coenzyme A delta isomerase
NM_031987
Crot
Carnitine O-octanoyl transferase
NM_022594
Ech1
Enoyl coenzyme A hydratase 1, peroxisomal
NM_013200
Cpt1b
Carnitine palmitoyl transferase 1β, muscle isoform
NM_016999
Cyp4a1; Cyp4a10
Cytochrome P450, family 4, subfamily a, polypeptide 1; 10
NM_ 012489
Acaa1
Acetyl-coenzyme A acyltransferase 1, peroxisomal
AA899304
Acat1
Acetyl-coenzyme A acyltransferase 1, mitochondrial
NM_012930
Cpt2
Carnitine palmitoyl transferase 2
NM_057197
Decr1
2,4-dienoyl CoA reductase 1, mitochondrial
NM_017340
Acox1
Acyl-coenzyme A oxidase 1, palmitoyl
AF044574
Decr2
2,4-dienoyl CoA reductase 2, peroxisomal
NM_133618
Hadhb
Hydroxyacyl-coenzyme A dehydrogenase/3-ketoacyl-coenzyme A thiolase/enoyl-coenzyme A hydratase (trifunctional protein), β-subunit
M33936
Cyp4a3
Cytochrome P450, family 4, subfamily a, polypeptide 3
AA893326
Cyp4a3
Cytochrome P450, family 4, subfamily a, polypeptide 3
AA800240
Hadha
Hydroxyacyl-coenzyme A dehydrogenase/3-ketoacyl-coenzyme A thiolase/enoyl-coenzyme A hydratase (trifunctional protein), α-subunit
NM_130433
Acaa2
Acetyl-coenzyme A acyltransferase 2 (mitochondrial 3-oxoacylcoenzyme A thiolase)
D13921
Acat1
Acetyl-coenzyme A acyltransferase 1, mitochondrial
NM_024386
Hmgcl
3-hydroxy-3-methylglutaryl-coenzyme A lyase
NM_012891
Acadvl
Acyl-coenzyme A dehydrogenase, very long chain
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Experiment 1
Experiment 2
fold change (dietary lignans)
fold change (dietary sesame)
lignan-free
sesamin
episesamin
sesamolin
0%
10%
20%
1.0 ± 0.3a
3.7 ± 0.7a
13 ± 2b
12 ± 3b
1.0 ± 0.1a
2.1 ± 0.2b
4.5 ± 0.6c
1.0 ± 0.1a
2.9 ± 0.3b
8.5 ± 0.7c
8.2 ± 0.9c
1.0 ± 0.1a
3.3 ± 0.4b
8.4 ± 0.4c
1.0 ± 0.1a
3.1 ± 0.4b
6.6 ± 1.0c
6.9 ± 0.7c
1.0 ± 0.1a
3.0 ± 0.3b
5.3 ± 0.5c
1.0 ± 0.1a
3.8 ± 0.3b
6.4 ± 0.4c
6.6 ± 0.4c
1.0 ± 0.1a
3.6 ± 0.2b
4.5 ± 0.2c
1.0 ± 0.1a
3.3 ± 0.2b
5.3 ± 0.4c
5.5 ± 0.3c
1.0 ± 0.0a
2.0 ± 0.1b
2.5 ± 0.1c
1.0 ± 0.1a
3.0 ± 0.2b
5.2 ± 0.2c
4.9 ± 0.3c
1.0 ± 0.1a
3.5 ± 0.1b
4.9 ± 0.1c
1.0 ± 0.1a
1.1 ± 0.1a
3.2 ± 0.6b
4.2 ± 0.6b
1.0 ± 0.0a
1.2 ± 0.1a
2.7 ± 0.5b
1.0 ± 0.0a
2.6 ± 0.1b
3.7 ± 0.1c
4.0 ± 0.2c
1.0 ± 0.1a
3.6 ± 0.2b
5.3 ± 0.2c
1.0 ± 0.1a
1.6 ± 01b
3.3 ± 0.2c
3.5 ± 0.2c
1.0 ± 0.0a
2.0 ± 0.2b
3.3 ± 0.1c
1.0 ± 0.1a
1.9 ± 0.2b
2.4 ± 0.3bc
2.7 ± 0.3c
1.0 ± 0.1a
1.8 ± 0.1b
2.5 ± 0.1c
1.0 ± 0.1a
1.8 ± 0.1b
2.9 ± 0.2c
2.8 ± 0.1c
1.0 ± 0.1a
2.0 ± 0.2b
2.7 ± 0.2c
1.0 ± 0.0a
1.8 ± 0.1b
2.6 ± 0.1c
2.6 ± 0.2c
1.0 ± 0.1a
1.6 ± 0.1b
2.0 ± 0.1c
1.0 ± 0.0a
1.5 ± 0.0b
2.5 ± 0.1c
2.4 ± 0.1c
1.0 ± 0.0a
1.5 ± 0.1b
1.9 ± 0.1c
1.0 ± 0.0a
1.8 ± 0.1b
2.3 ± 0.2c
2.3 ± 0.2c
1.0 ± 0.0a
1.9 ± 0.1b
2.5 ± 0.1c
1.0 ± 0.0a
1.5 ± 0.1b
2.2 ± 0.1c
2.1 ± 0.1c
1.0 ± 0.0a
1.6 ± 0.1b
2.0 ± 0.1c
1.0 ± 0.1a
1.7 ± 0.1b
2.0 ± 0.1bc
2.1 ± 0.2c
1.0 ± 0.1a
1.3 ± 0.1b
1.6 ± 0.1b
1.0 ± 0.1a
1.3 ± 0.1b
1.3 ± 0.1b
1.5 ± 0.1b
1.0 ± 0.1a
1.3 ± 0.1b
1.6 ± 0.1b
1.0 ± 0.0a
1.4 ± 0.0a
1.9 ± 0.1b
1.9 ± 0.1b
1.0 ± 0.1a
1.4 ± 0.1b
1.9 ± 0.0c
1.0 ± 0.1a
1.2 ± 0.1a
1.9 ± 0.1b
1.8 ± 0.1b
1.0 ± 0.0a
1.3 ± 0.0b
1.6 ± 0.1c
1.0 ± 0.1a
1.5 ± 0.1b
1.8 ± 0.1c
1.8 ± 0.1bc
1.0 ± 0.1a
1.5 ± 0.1b
1.9 ± 0.1c
1.0 ± 0.1a
1.2 ± 0.1a
1.6 ± 0.1b
1.5 ± 0.1b
1.0 ± 0.0a
1.4 ± 0.1b
1.8 ± 0.1c
1.0 ± 0.0a
1.3 ± 0.1b
1.6 ± 0.1c
1.5 ± 0.1c
1.0 ± 0.0a
1.3 ± 0.1b
1.5 ± 0.0c
Sesame Lignans and Gene Expression
15
Table 1. Continued Accession number
Gene symbol
Gene name
U88294
Cpt1a
Carnitine palmitoyltransferase 1a, liver
BI296347
Acad11
Acyl-coenzyme A dehydrogenase family, member 11
Proteins involved in biogenesis of mitochondria and peroxisomes NM_053487
Pex11a
Peroxisomal biogenesis factor 11A
BI273703
Pex11a
Peroxisomal biogenesis factor 11A
AW141617
Pex19
Peroxisomal biogenesis factor 19
AF061242
Fxc1
Fractured callus expressed transcript 1
Enzymes and transporters involved in the regulation of fatty acid oxidation NM_022193
Acaca
Acetyl-coenzyme A carboxylase-α
NM_053477
Mlycd
Malonyl-CoA decarboxylase
AY081195
Mgll
Monoglyceride lipase
AI713204
Mgll
Monoglyceride lipase
BG372713
Mgll
Monoglyceride lipase
AA850195
Pank1
Pantothenate kinase 1
NM_019269
Slc22a5
Solute carrier family 22 (organic cation transporter), member 5
NM_053965
Slc25a20
Solute carrier family 25 (carnitine/acylcarnitine translocase), member 20
NM_012804
Abcd3
ATP-binding cassette, subfamily D (ALD), member 3
AF072411
Cd36
Cd36 antigen
Values represent means ± SE for 5 and 6 rats for experiments 1 and 2, respectively. Values with different superscripts differ significantly at p < 0.05.
Lignans affected the gene expression of transporters involved in lipid metabolism. All the lignans significantly increased the mRNA expression of Slc22a5, which mediates high-affinity sodium-dependent carnitine transport [18]. The increases were stronger with episesamin and sesamolin than with sesamin, and comparable between the two former compounds. Episesamin and sesamolin, but not sesamin, significantly increased the mRNA levels of Slc25a20, which transports acylcarnitine into the mitochondrial matrix. Episesamin and sesamolin increased the mRNA expression of Cd36, a long-chain fatty acid transporter located in the plasma membrane. However, sesamin did not cause a significant increase in this parameter. Episesamin and sesamolin, but not sesamin, significantly increased the gene expression of Abcd3, which
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Experiment 1
Experiment 2
fold change (dietary lignans)
fold change (dietary sesame)
lignan-free
sesamin
episesamin
sesamolin
0%
10% a
20% b
1.3 ± 0.1
1.8 ± 0.2c
–
–
–
–
1.0 ± 0.1
–
–
–
–
1.0 ± 0.1a
1.4 ± 0.1b
1.9 ± 0.2c
1.0 ± 0.1a
4.0 ± 0.6b
15 ± 2c
13 ± 3c
1.0 ± 0.2a
6.0 ± 0.7b
11.5 ± 1.4c
1.0 ± 0.1a
2.9 ± 0.3a
7.0 ± 0.6b
6.2 ± 1.3b
1.0 ± 0.1a
3.5 ± 0.4b
5.2 ± 0.4c
–
–
–
–
1.0 ± 0.1a
1.4 ± 0.1b
1.9 ± 0.2c
1.0 ± 0.1a
1.1 ± 0.0a
1.6 ± 0.1b
1.7 ± 0.1b
1.0 ± 0.0a
1.2 ± 0.0b
1.7 ± 0.1c
1.0 ± 0.2b
0.61 ± 0.06a
0.61 ± 0.04a
0.54 ± 0.04a
1.0 ± 0.0b
0.56 ± 0.07a
0.49 ± 0.06a
1.0 ± 0.1a
1.3 ± 0.0b
1.7 ± 0.0c
1.6 ± 0.1c
–
–
–
a
1.0 ± 0.1
1.6 ± 0.1
2.4 ± 0.2
3.2 ± 0.5
1.0 ± 0.1
1.7 ± 0.2
2.5 ± 0.3c
1.0 ± 0.1a
1.4 ± 0.1a
2.3 ± 0.2b
3.1 ± 0.4c
1.0 ± 0.1a
1.5 ± 0.2a
2.1 ± 0.2b
1.0 ± 0.1a
1.4 ± 0.1a
2.3 ± 0.1b
2.9 ± 0.4b
1.0 ± 0.1a
1.6 ± 0.2b
2.1 ± 0.2b
1.0 ± 0.1a
1.2 ± 0.1a
1.6 ± 0.1b
1.7 ± 0.2b
1.0 ± 0.0a
1.4 ± 0.1b
1.6 ± 0.1c
1.0 ± 0.0a
2.2 ± 0.2b
4.3 ± 0.3c
3.8 ± 0.5c
1.0 ± 0.1a
2.1 ± 0.1b
3.2 ± 0.3c
1.0 ± 0.1a
1.3 ± 0.1a
2.0 ± 0.1b
2.1 ± 0.2b
1.0 ± 0.0a
1.6 ± 0.1b
2.1 ± 0.1c
1.0 ± 0.1a
1.1 ± 0.1a
1.6 ± 0.1b
1.6 ± 0.2b
–
–
–
a
a
b
b
1.0 ± 0.1
ab
1.4 ± 0.3
bc
3.6 ± 0.6
c
3.9 ± 1.0
a
a
1.0 ± 0.1
b
a
1.2 ± 0.1
4.1 ± 0.5b
is involved in the import of fatty acids and/or fatty acyl-CoAs into peroxisomes [19]. The sesamin-dependent change was not significant. In experiment 2, a diet containing 10% sesame seed, compared with a control diet free of sesame seed, caused changes more than 1.5- and 2-fold in the expression of 135 (91 genes were up- and 44 downregulated) and 55 genes (43 genes were upand 12 downregulated), respectively. The number of genes affected approximately doubled with a diet containing 20% of sesame seed. This diet caused changes more than 1.5- and 2-fold in the expression of 302 (212 genes were up- and 90 downregulated) and 123 genes (101 genes were up- and 22 downregulated), respectively. As expected, sesame seed dose-dependently increased the mRNA expression of many
Sesame Lignans and Gene Expression
17
hepatic enzymes involved in fatty acid oxidation (table 1). The magnitude of the increases observed with a diet containing 20% sesame seed (this diet had 0.232% sesamin and 0.098% sesamolin) was comparable to that observed with a diet containing 0.2% episesamin or sesamolin in experiment 1. We also observed that sesame seed increased mRNA levels of Pex11a and Fxc1 which are involved in the biogenesis of peroxisomes and mitochondria, respectively. In addition, sesame seed dosedependently increased the mRNA expression of Pex19 involved in the proliferation of peroxisomes [20]. As observed with dietary lignans, sesame seed increased mRNA levels of Mgll, Pank1, Slc22a5, Slc25a20, and Cd36, but decreased the mRNA level of Acaca. Sesame-dependent changes in the mRNA expression of Mlycd and Abcd3 were attenuated compared with those obtained with sesame lignans. The mRNA level of Mlycd was comparable between rats fed a control diet (1.0 ± 0.1-fold) and those fed a diet containing 10% sesame seed (1.1 ± 0.1-fold). The value was slightly but significantly higher in rats fed a diet containing 20% sesame seed (1.3 ± 0.1-fold) than in the animals fed a control diet. However, no significant differences were observed in the mRNA expression of Abcd3 among the groups. Effect of Sesame Lignans and Sesame Seed on the Serum and Liver Concentrations of Lignans, Liver Concentrations of Carnitine, and Liver mRNA Levels Sesamin, episesamin or sesamolin, but no other lignan, was detected in rats fed the respective lignan (experiment 1; table 2). Lignans were detected neither in serum nor in liver in rats fed a lignan-free diet. Serum episesamin and sesamolin concentrations in rats fed the corresponding lignan were 4.4- and 11.9-fold higher, respectively, than the sesamin concentration in rats given sesamin. Also, the serum sesamolin concentration was 2.7-fold the concentration of episesamin in rats fed the respective lignan. Sesamolin compared with both episesamin and sesamin, and episesamin compared with sesamin, accumulated more in the liver as well. Hepatic levels of episesamin and sesamolin were 3.5- and 6.6-fold greater, respectively; the levels of sesamin and sesamolin were 1.9-fold that of episesamin. Sesamin and sesamolin were detected in serum and liver in rats fed diets containing sesame seed (experiment 2). Although the concentration of sesamin in sesame seed was twice that of sesamolin, the sesamolin concentration greatly exceeded the sesamin concentration in both serum and liver. Sesame seed dose-dependently increased the serum and liver concentrations of these lignans. Sesame seed greatly increased the hepatic concentration of carnitine (experiment 2). Values were about 3 and 5 times higher in rats given diets containing 10 and 20% sesame seed, respectively, than in the animals fed a control diet. We also analyzed mRNA levels of Crot, Acox1, Ehhadh, Cpt2, Hadha and Hadhb involved in hepatic fatty acid oxidation by real-time PCR in both experiments. The results obtained using this methodology were consistent with those obtained with the DNA microarray (table 1). In addition, real-time PCR analyses in experiment 2 confirmed the findings of the microarray analysis that sesame seed increased the mRNA
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expression of Mgll, Slc22a5, Slc25a20 and Cd36. All these changes ranged within the expected values, as observed using the microarray.
Discussion
Using DNA microarray technology, we have confirmed previous findings [4, 7–9, 12] that various lignans increased the gene expression of hepatic enzymes involved in fatty acid oxidation. Moreover, we showed that the physiological activities enhancing the gene expression of hepatic enzymes involved in fatty acid oxidation were much stronger with episesamin and sesamolin than with sesamin, and comparable between the former lignans. As lignans increased mRNA levels of Cyp4a1, Cyp4a10 and Cyp4a3, they may also stimulate ω-oxidation of fatty acids [21, 22]. Since lignans, especially episesamin and sesamolin, upregulated the gene expression of Fxc1 and Pex11a, they may also stimulate the proliferation of mitochondria and peroxisomes. We observed that lignans, particularly episesamin and sesamolin, affected the expression of not only genes for the enzymes involved in fatty acid oxidation, but also those for various proteins related to fatty acid metabolism, as summarized in figure 2. The upregulation of the mRNA expression of Cd36 by lignans indicated that the compounds increase the hepatic uptake of fatty acid from the blood stream to supply the substrate for fatty acid oxidation. Dietary lignans also increased the gene expression of mitochondrial acylcarnitine transporter (Slc25a20) [23] and peroxisomal fatty acid/acyl-CoA transporter (Abcd3) [19]. These changes should facilitate the delivery of fatty acids into these organelles. It has been suggested that Mgll is involved in complementing the action of lipoprotein lipase and hepatic lipase in degrading triacylglycerol from lipoproteins in the liver [16]. The upregulation by lignans of Mgll expression may increase the supply of free fatty acid as a substrate for β-oxidation. All the lignans significantly increased the mRNA expression of Slc22a5, and the increases were greater with episesamin and sesamolin than with sesamin. Upregulation in the liver of the expression of this protein is expected to increase the availability of carnitine and hence stimulate mitochondrial transport of fatty acid to be oxidized in this organelle [18]. Episesamin and sesamolin, but not sesamin, caused a significant increase in the expression of Pank1. Therefore, it is expected that these lignans also increase the availability of CoA in the liver to activate fatty acids [17]. All these observations indicated that sesame lignans, especially episesamin and sesamolin, not only upregulate the gene expression of various enzymes involved in hepatic fatty acid oxidation, but also coordinately modify the expression of various proteins involved in regulating fatty acid transport into hepatocytes and their organelles, as well as the availability of substances required for fatty acid oxidation (carnitine and CoA) to stimulate hepatic fatty acid oxidation. A reduction in hepatic lipogenesis appears to be an alternative mechanism for the lipid-lowering effect of lignans. As various lignans decreased the mRNA expression
Sesame Lignans and Gene Expression
19
Table 2. Effect of sesame lignans (experiment 1) and sesame seed (experiment 2) on the serum and liver concentrations of lignans, liver concentrations of carnitine, and liver mRNA levels Experiment 1
Experiment 2
Dietary lignans
Dietary sesame
lignan-free
sesamin
episesamin
sesamolin
0%
10%
20%
Sesamin
ND
11.2 ± 3.9
ND
ND
ND
1.11 ± 0.18a 2.13 ± 0.40b
Episesamin
ND
ND
49.3 ± 1.6
ND
ND
ND
ND
Sesamolin
ND
ND
ND
132 ± 14
ND
19.5 ± 4.1a
35.9 ± 5.7b
Total
ND
11.2 ± 3.9a
49.3 ± 1.6b
132 ± 14c
ND
20.6 ± 4.2a
38.1 ± 6.0b
Sesamin
ND
2.26 ± 0.30
ND
ND
ND
0.463 ± 0.101a
1.13 ± 0.18b
Episesamin
ND
ND
6.17 ± 0.99
ND
ND
ND
Sesamolin
ND
ND
ND
11.8 ± 1.4
ND
1.16 ± 0.23a 1.96 ± 0.28b
Total
ND
2.26 ± 0.30a 6.17 ± 0.99b 11.8 ± 1.4c
ND
1.63 ± 0.33a 3.09 ± 0.38b
–
–
–
95.5 ± 4.2a
305 ± 15b
495 ± 14c
Lignans Serum, μg/dl
Liver, μg/g
Liver carnitine, nmol/g mRNA level in the liver (fold change) Crot
1.0 ± 0.0a
2.6 ± 0.2b
6.7 ± 0.4c
6.4 ± 0.6c
1.0 ± 0.1a
2.6 ± 0.1b
4.2 ± 0.4c
Acox1
1.0 ± 0.1a
1.6 ± 0.1b
3.7 ± 0.2c
3.3 ± 0.2c
1.0 ± 0.1a
1.6 ± 0.1b
2.5 ± 0.1c
Ehhadh
1.0 ± 0.1a
2.9 ± 0.4b
12 ± 1d
9.8 ± 0.8c
1.0 ± 0.1a
3.0 ± 0.4b
8.2 ± 1.1c
Cpt2
1.0 ± 0.0a
1.8 ± 0.1b
3.0 ± 0.1c
3.0 ± 0.2c
1.0 ± 0.1a
2.0 ± 0.2b
2.7 ± 0.2c
Hadha
1.0 ± 0.0a
1.4 ± 0.1b
2.0 ± 0.1c
2.0 ± 0.1c
1.0 ± 0.1a
1.5 ± 0.0b
1.7 ± 0.1c
Hadhb
1.0 ± 0.0a
1.6 ± 0.1b
3.0 ± 0.2c
2.6 ± 0.2c
1.0 ± 0.0a
1.7 ± 0.1b
1.9 ± 0.1c
Mgll
–
–
–
–
1.0 ± 0.1a
1.7 ± 0.2b
2.3 ± 0.3b
Slc22a5
–
–
–
–
1.0 ± 0.0a
1.8 ± 0.1b
2.4 ± 0.2c
Slc25a20
–
–
–
–
1.0 ± 0.0a
1.9 ± 0.1b
2.2 ± 0.3b
Cd36
–
–
–
–
1.0 ± 0.1a
1.6 ± 0.0b
3.7 ± 0.6c
Values represent means ± SE for 7 rats. Values with different superscripts differ significantly at p < 0.05. ND = Not detected.
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Ide · Nakashima · Iida · Yasumoto · Katsuta
Plasma membrane
Citric acid
Mitochondria
Ketone bodies Acat1 Hmgcl
TCA cycle Acetyl-CoA Mlycd
Acetyl-CoA
Citric acid
Acaca
-Oxidation Malonyl-CoA
Slc22a5
Acyl-CoA Carnitine
Carnitine
Acyl-carnitine Cpt1a Cpt1b Slc25a20
Cpt2 Acyl-carnitine
Acadvl, Hadha, Hadhb, Acaa2, Decr1, Dci Downregulation
Acyl-CoA Pantothenic acid
Slc25a20
Pank1
Upregulation Acyl-carnitine
CoA Fatty acid
Crot
Abcd3
Cd36 Fatty acid Mgll Monoacylglycerol
Acyl-CoA
Acyl-CoA
Acyl-CoA -Oxidation (medium chain)
Acox1, Ech1, Ehhadh, Acaa1, Decr2 Peroxisomes
Triacylglycerol
Fig. 2. Sesame lignan-dependent changes in the expression of genes involved in the regulation of hepatic fatty acid oxidation.
of Acaca to a similar level, it is thought that they decreased the production of malonyl-CoA as the substrate for lipogenesis to a similar extent. In addition, we observed that lignans increased the mRNA level of Mlycd, which catalyzes the breakdown of malonyl-CoA [24]. We found that the increases were stronger with episesamin and sesamolin than with sesamin. Therefore, it is likely that the availability of malonylCoA as a substrate for lipogenesis is lower in rats fed episesamin and sesamolin than in those fed sesamin. Apart from its role as a precursor for fatty acid biosynthesis, malonyl-CoA plays an important role in regulating hepatic fatty acid oxidation as a potent inhibitor of carnitine palmitoyltransferase I [24]. Therefore, the lignan-dependent changes in the gene expression of Acaca and Mlycd may stimulate mitochondrial transport of fatty acids as substrates for β-oxidation. Observations of hepatic gene expression profiles in the present study support the idea that various sesame lignans are the natural agonists for peroxisome proliferatoractivated receptor-α (PPARα). Studies have showed that various enzymes involved in fatty acid oxidation located in peroxisomes and mitochondria are upregulated by PPARα [25]. More recently, information suggests that PPARα agonists affect the
Sesame Lignans and Gene Expression
21
expression of diverse genes in addition to the genes related to fatty acid oxidation. Some of the changes may be the result of secondary metabolic events occurring after the activation of PPARα. In the present study, we observed that lignans not only upregulated the gene expression of enzymes involved in fatty acid oxidation, but also affected the expression of many genes involved in lipid and carbohydrate metabolism. The mRNA expression of appreciable numbers of these genes has also been observed to be affected in a similar fashion by various synthetic PPARα agonists in the liver of experimental animals or in cultured hepatocytes [14, 17, 18, 21, 24, 26, 27]. Among the three lignans tested in the present study, episesamin and sesamolin were generally stronger than sesamin in altering the expression of various genes. The binding affinity for, and hence the ability to activate PPARα may be greater for episesamin and sesamolin than for sesamin. However, the observation that liver and serum sesamin levels were much lower than episesamin and sesamolin levels in rats fed the respective lignan raises the possibility that a difference in bioavailability is a crucial factor responsible for the divergent effects of lignans on hepatic gene expression. However, even though twice as much sesamolin as episesamin accumulated in the liver, the impact on hepatic gene expression was comparable between these compounds. Therefore, it is expected that episesamin has a more profound effect on gene expression than sesamolin if these compounds are indistinguishable in terms of bioavailability. As expected, diets containing lignans in the form of sesame seeds also strongly increased the mRNA expression of many enzymes involved in hepatic fatty acid oxidation. In addition, sesame seed affected the gene expression of various proteins related to the regulation of hepatic fatty acid metabolism in a manner similar to that observed with sesame lignans in experiment 1. In addition, we observed that the sesame seed-dependent increase in the gene expression of a protein involved in carnitine transport across the plasma membrane (Slc22a5) in the liver was associated with a large increase in the concentration of carnitine in this tissue. A diet containing 20% sesame seed had about 0.2% sesamin and 0.1% sesamolin. However, concentrations of lignans in the liver and serum in rats fed sesame seed appeared considerably lower than those expected from the results obtained among the animals fed various lignans. In fact, serum and liver concentrations of sesamin in rats given a diet containing this compound were about 5 and 2 times higher, respectively, than the values in the animals fed a diet containing 20% sesame seed despite the fact that these diets contained comparable amounts of sesamin. Also, although sesamolin concentration was about 0.1% in the diet containing 20% sesame seed, serum and liver concentrations of this compound in the animals fed this diet were merely 27 and 17% of those observed with a diet containing 0.2% sesamolin. Therefore, it is suggested that lignans were less absorbable when supplied in the diet as sesame seed than when the purified compounds were added to diets. Despite the fact that the accumulation of lignans in serum and liver was attenuated in the rats fed sesame seed compared to the animals fed purified lignan compounds, the increases in the gene expression of various hepatic fatty acid oxidation enzymes in rats fed a diet containing 20% sesame
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Ide · Nakashima · Iida · Yasumoto · Katsuta
seed were comparable to those obtained with the diet containing 0.2% episesamin or sesamolin. Therefore, there is a possibility that compound(s) other than sesamin and sesamolin are also involved in the sesame seed-dependent increase in the gene expression of enzymes related hepatic fatty acid oxidation. In conclusion, the DNA microarray analysis showed that dietary sesame lignans (sesamin, episesamin and sesamolin) profoundly affect gene expression profiles in the liver. The analysis not only confirmed the previous finding that lignans increased the gene expression of enzymes involved in hepatic fatty acid oxidation, but also showed that they modified the expression of proteins involved in the transportation of fatty acids into hepatocytes and their organelles, and regulated hepatic concentrations of carnitine, CoA and malonyl-CoA. All these changes should facilitate the oxidation of fatty acids in hepatocytes. The changes were generally greater with episesamin and sesamolin than with sesamin. The differences in bioavailability among these lignans appear to be important in terms of the divergent physiological activity of these compounds. The diets containing sesame seed also affected the gene expression of proteins related to fatty acid oxidation in a manner similar to that observed with diets containing purified sesame lignans. However, analyses of serum and liver concentrations of sesamin and sesamolin in rats fed sesame seed raised the possibility that compound(s) other than these lignans are also involved in the sesame seeddependent increase in the gene expression of hepatic fatty acid oxidation enzymes.
References 1 Sirato-Yasumoto S, Katsuta M, Okuyama Y, Takahashi Y, Ide T: Effect of sesame seeds rich in sesamin and sesamolin on fatty acid oxidation in rat liver. J Agric Food Chem 2001;49:2647–2651. 2 Katsuzaki H, Kawakishi S, Osawa T: Sesaminol glucosides in sesame seeds. Phytochemistry 1994;35: 773–776. 3 Fukuda Y, Nagata M, Osawa T, Namiki M: Contribution of lignan analogues to antioxidative activity of refined unroasted sesame seed oil. J Am Oil Chem Soc 1986;63:1027–1031. 4 Ashakumary L, Rouyer IA, Takahashi Y, Ide T, Fukuda N, Aoyama T, Hashimoto T, Mizugaki M, Sugano M: Sesamin, a sesame lignan, is a potent inducer of hepatic fatty acid oxidation in the rat. Metabolism 1999;48:1303–1313. 5 Hirose N, Inoue T, Nishihara K, Sugano M, Akimoto K, Shimizu S, Yamada H: Inhibition of cholesterol absorption and synthesis in rats by sesamin. J Lipid Res 1991;32:629–638. 6 Hirata F, Fujita K, Ishikura Y, Hosoda K, Ishikawa T, Nakamura H: Hypocholesterolemic effect of sesame lignan in humans. Atherosclerosis 1996;122:135– 136.
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7 Kushiro M, Masaoka T, Hageshita S, Takahashi Y, Ide T, Sugano M: Comparative effect of sesamin and episesamin on the activity and gene expression of enzymes in fatty acid oxidation and synthesis in rat liver. J Nutr Biochem 2002;13:289–295. 8 Lim JS, Adachi Y, Takahashi Y, Ide T: Comparative analysis of sesame lignans (sesamin and sesamolin) in affecting hepatic fatty acid metabolism in rats. Br J Nutr 2007;97:85–95. 9 Tsuruoka N, Kidokoro A, Matsumoto I, Abe K, Kiso Y: Modulating effect of sesamin, a functional lignan in sesame seeds, on the transcription levels of lipidand alcohol-metabolizing enzymes in rat liver: a DNA microarray study. Biosci Biotechnol Biochem 2005;69:179–188. 10 Akimoto K, Kigawa Y, Akamatsu T, Hirose N, Sugano M, Shimizu S, Yamada H: Protective effect of sesamin against liver damage caused by alcohol or carbontetrachloride in rodents. Ann Nutr Metab 1993;37:218–224. 11 Ide T: Interaction of fish oil and conjugated linoleic acid in affecting hepatic activity of lipogenic enzymes and gene expression in liver and adipose tissue. Diabetes 2005;54:412–423.
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12 Kushiro M, Takahashi Y, Ide T: Species differences in the physiological activity of dietary lignan (sesamin and episesamin) in affecting hepatic fatty acid metabolism. Br J Nutr 2004;91:377–386. 13 Pearson DJ, Chase JFA, Tubbs PK: The assay of (-)-carnitine and its O-acyl derivatives. Method Enzymol 1969;14:612–622. 14 Schrader M, Reuber BE, Morrell JC, JimenezSanchez G, Obie C, Stroh TA, Valle D, Schroer TA, Gould SJ: Expression of PEX11β mediates peroxisome proliferation in the absence of extracellular stimuli. J Biol Chem 1998;273:29607–29614. 15 Rothbauer U, Hofmann S, Mühlenbein N, Paschen SA, Gerbitz KD, Neupert W, Brunner M, Bauer MF: Role of the deafness dystonia peptide 1 (DDP1) in import of human Tim23 into the inner membrane of mitochondria. J Biol Chem 2001;276:37327– 37334. 16 Karlsson M, Contreras JA, Hellman U, Tornqvist H, Holm C: cDNA cloning, tissue distribution, and identification of the catalytic triad of monoglyceride lipase. Evolutionary relationship to esterases, lysophospholipases, and haloperoxidases. J Biol Chem 1997;272:27218–27223. 17 Ramaswamy G, Karim MA, Murti KG, Jackowski S: PPARα controls the intracellular coenzyme A concentration via regulation of PANK1α gene expression. J Lipid Res 2004;45:17–31. 18 Luci S, Geissler S, König B, Koch A, Stangl GI, Hirche F, Eder K: PPARα agonists up-regulate organic cation transporters in rat liver cells. Biochem Biophys Res Commun 2006;350:704–708. 19 Wanders RJ, Visser WF, van Roermund CW, Kemp S, Waterham HR: The peroxisomal ABC transporter family. Pflugers Arch 2007;453:719–734. 20 Sacksteder KA, Jones JM, South ST, Li X, Liu Y, Gould SJ: PEX19 binds multiple peroxisomal membrane proteins, is predominantly cytoplasmic, and is required for peroxisome membrane synthesis. J Cell Biol 2000;148:931–944.
21 Hardwick JP, Song BJ, Huberman E, Gonzalez FJ: Isolation, complementary DNA sequence, and regulation of rat hepatic lauric acid ω-hydroxylase (cytochrome P-450LAω). Identification of a new cytochrome P-450 gene family. J Biol Chem 1987;262: 801–810. 22 Kimura S, Hardwick JP, Kozak CA, Gonzalez FJ: The rat clofibrate-inducible CYP4A subfamily. II. cDNA sequence of IVA3, mapping of the Cyp4a locus to mouse chromosome 4, and coordinate and tissue-specific regulation of the CYP4A genes. DNA 1989;8:517–525. 23 Indiveri C, Iacobazzi V, Giangregorio N, Palmieri F: The mitochondrial carnitine carrier protein: cDNA cloning, primary structure and comparison with other mitochondrial transport proteins. Biochem J 1997;321:713–719. 24 Lee GY, Kim NH, Zhao ZS, Cha BS, Kim YS: Peroxisomal-proliferator-activated receptor α activates transcription of the rat hepatic malonyl-CoA decarboxylase gene: a key regulation of malonylCoA level. Biochem J 2004;378:983–990. 25 Schoonjans K, Staels B, Auwerx J: Role of the peroxisome proliferator-activated receptor (PPAR) in mediating the effects of fibrates and fatty acids on gene expression. J Lipid Res 1996;37:907–925. 26 Motojima K, Passilly P, Peters JM, Gonzalez FJ, Latruffe N: Expression of putative fatty acid transporter genes are regulated by peroxisome proliferator-activated receptor α and γ activators in a tissue- and inducer-specific manner. J Biol Chem 1998;273: 16710–16714. 27 Cherkaoui-Malki M, Meyer K, Cao WQ, Latruffe N, Yeldandi AV, Rao MS, Bradfield CA, Reddy JK: Identification of novel peroxisome proliferator-activated receptor α (PPARα) target genes in mouse liver using cDNA microarray analysis. Gene Expr 2001;9:291–304.
Dr. Takashi Ide Laboratory of Nutritional Function, Division of Food Functionality, National Food Research Institute Kannondai 2-1-12 Tsukuba 305-8642 (Japan) Tel. +81 29 838 8083, Fax +81 29 838 7996, E-Mail
[email protected]
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Genomics Yoshikawa T (ed): Food Factors for Health Promotion. Forum Nutr. Basel, Karger, 2009, vol 61, pp 25–38
Genome Science of Lipid Metabolism and Obesity Nobuyuki Takahashi ⭈ Tsuyoshi Goto ⭈ Shizuka Hirai ⭈ Taku Uemura ⭈ Teruo Kawada Laboratory for Molecular Function of Food, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
Abstract Abnormalities in lipid metabolism cause obesity leading to metabolic syndrome. Thus, improvement of the abnormalities is significant for the treatment of metabolic syndrome. Nuclear receptors activated by specific ligands regulate lipid metabolism at the genomic level. The expression of lipid metabolism-related enzymes is increased or decreased by the activity of various nuclear receptors. The regulation of enzyme expression is mediated by specific response elements to each nuclear receptor in promoters of target genes. Many food factors acting as agonists or antagonists control the activities of nuclear receptors. Here, we provide several examples of food factors acting as agonists or antagonists, which are useful for the management of obesity accompanied by lipid metaboCopyright © 2009 S. Karger AG, Basel lism abnormalities.
Recently, metabolic syndrome has become a severe health and social problem. The syndrome is accompanied by obesity with lipid metabolism abnormalities which are often due to daily excess energy intake. Therefore, improvement of these abnormalities is significant for the treatment of obesity and the metabolic syndrome. Lipid metabolism is regulated by various factors such as glucose availability, energy expenditure rate, and stored lipid amounts. Short-term regulation of lipid metabolism (in terms of seconds and minutes) is mediated by changes in enzymatic activities. On the other hand, in the middle- and long-term ranges (in terms of hours and days), the increase or decrease in the mRNA expression of genes encoding lipid metabolism-related enzymes is regulated. Many transcriptional factors are involved in the regulation of mRNA expression. The most significant factor is the nuclear receptor superfamily (table 1). The nuclear receptors have common structures and functions. The transcriptional factors are activated by small hydrophobic molecules acting as ligands, which the plasma membrane is permeable to, and then positively or negatively regulate
Table 1. Nuclear receptor superfamily Steroid hormone receptors Estrogen receptor Androgen receptor Progesterone receptor Glucocorticoid receptor Mineralocorticoid receptor Homodimer orphan receptors Retinoid X receptor EAR Hepatocyte nuclear factor Monomer orphan receptors Receptor tyrosine kinase-like orphan receptor Estrogen receptor-related receptor Rev-erb Retinoid X receptor heterodimer receptors PPAR LXR FXR PXR Retinoic acid receptor Thyroid hormone receptor Vitamin D receptor Constitutive androstane receptor
mRNA expression of target genes. Members of the superfamily have two distinct and conserved functional domains, an N-terminal DNA-binding domain (DBD) and a C-terminal ligand-binding domain (LBD). The structure of the N-terminal DBD (approximately 80 amino acids) is similar among the members (fig. 1). This domain is composed of two typical Zn finger motifs recognizing specific sequences with six nucleotides. Most nuclear receptors generally form a hetero- or homodimer, so that a nuclear receptor complex binds to two copies of the sequence in a promoter region. LBD is a C-terminal region with approximately 250 amino acids containing 12 α-helixes, which contributes to not only the binding of ligands but also the formation of a dimer and the interaction of other regulatory proteins. The nuclear receptors require other accessory proteins for the regulation of the target gene expression. After a ligand binds to a nuclear receptor, various cofactors are recruited to the ligand-nuclear receptor complex. For the induction of target gene expression, coactivators should interact with the nuclear receptor (fig. 2). The association of coactivators is dependent on ligand binding to a nuclear receptor (therefore, the
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Takahashi · Goto · Hirai · Uemura · Kawada
DBD About 80 amino acids
LBD About 250 amino acids
Binding to coactivators
Binding to coactivators
Binding to a response element Dimerization Nuclear translocation
Binding to a ligand Dimerization Binding to cofactors
Fig. 1. Common structure of nuclear receptors. Nuclear receptors have two distinct domains: DBD and LBD. Other regions that are significant for nuclear receptor activation are shown.
Coactivators
Ligands
Coactivators RNA polymerase complex
Target gene expression
Response element
Promoter (i.e. TATA box)
Fig. 2. Nuclear receptor complex. Ligand-bound nuclear receptors bind to a response element on the target gene promoter with various cofactors such as coactivators. Some cofactors mediate the recruitment of the RNA polymerase complex. Then, the expression of target genes is induced.
association is used to examine whether a compound serves as a ligand for nuclear receptors: ‘coactivator pull-down assay’). General coactivators bind to many nuclear receptors, which contribute to the chromatin remodeling due to histone acetylation and the association to other transcriptional complexes such as RNA polymerase. The general coactivators include CREB-binding protein (CBP)/p300, steroid receptor coactivator (SRC), and transcriptional intermediary factor-1 (TIF1). Other coactivators associate only to restricted nuclear receptors. For example, peroxisome proliferator-activated receptor-γ (PPARγ) coactivator-1 (PGC1), which was originally identified as a PPARγspecific coactivator, interacts with only a few nuclear receptors such as PPARα and EER. These coactivators have distinct in vivo expression patterns. Thus, nuclear receptors interact with different coactivators in different types of cell. This might be the reason why nuclear receptors induce the expression of different genes in different cells. The ligands of nuclear receptors are numerous, including sterol-, fatty acid-, and carotenoid-related compounds. For example, PPARγ is activated by fatty acids, a
Genome Science of Lipid Metabolism and Obesity
27
prostaglandin J2 derivative, isoprenols, and several terpenoids. Moreover, fatty acids with different carbon chains and/or saturations show different activities as the PPARγ ligand (generally, long-chain and/or unsaturated fatty acids are more highly active than short-chain and/or saturated ones). Thus, nuclear receptors sense various indicators (ligands) and respond at the cellular level to changes in the indicators. Moreover, the ligands are not only endogenous but also exogenous compounds including food factors, which have structures similar to those of endogenous ligands. Therefore, such food factors can regulate nuclear receptors as agonists or antagonists. In this review, the genomic regulation of lipid metabolism by various food factors in vitro and in vivo is described.
Lipid Metabolism Regulation by Nuclear Receptor Activity
Many nuclear receptors are involved in the regulation of lipid metabolism. Nuclear receptors are involved in the changes in cellular gene expression. Particularly, the expression of rate-limiting enzymes is often controlled by nuclear receptors. These expression controls are important targets of food factors to improve the lipid metabolism abnormalities. Here, we focus on lipid metabolism-related enzymes and explain how the enzyme expression is regulated by nuclear receptors in vitro and in vivo.
Fatty Acid and Triglyceride Synthesis (SREBP1, FAS, ACC and SCD) Sterol response element-binding protein-1 (SREBP1) is a key transcriptional factor in de novo fatty acid synthesis. Many target genes of SREBP are involved in the de novo fatty acid synthesis. The SREBP1 protein is firstly expressed as a membrane-integrated precursor. When activated, specific proteases cleave the precursor to release a cytoplasmic domain serving as an active transcriptional factor to induce expression of target genes. SREBP1 stimulates the expression of fatty acid synthase (FAS) and acyl-CoA carboxylase (ACC), which are important enzymes for de novo fatty acid synthesis. Functional FAS complex is a homodimer of two identical subunits with multiple catalytic properties encoded by a single-copy gene. A substrate of the FAS-mediated fatty acid synthesis is a malonyl-CoA derived from acetyl-CoA. This reaction to produce malonyl-CoA is catalyzed by ACC. Thus, both FAS and ACC are indispensable enzymes for the de novo fatty acid synthesis. Palmitic acid (C16:0), an end product of the synthesis, is then elongated by a specific enzyme to produce stearic acid (C18:0). Finally, these saturated fatty acids are desaturated to produce oleic acid (C18:1). This is mediated by stearoyl-CoA desaturase-1 (SCD1). This enzymatic activity is significant for the maintenance of fatty acid composition.
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Liver X receptor (LXR) and pregnane X receptor (PXR) stimulate de novo fatty acid synthesis in the liver. The LXR/PXR-dependent stimulation is mediated by SREBP through LXR/PXR-dependent induction of hepatic SREBP1 expression. The SREBP1 promoter has both an LXR response element (LXRE) and a PXR response element [1]. Therefore, the activation of both nuclear receptors induces SREBP1 expression. The effects of LXR and PXR on the accumulation of hepatic triglyceride (TG) are mainly mediated by SREBP1 activity. The induced SREBP1 increases the mRNA levels of FAS via SREBP response elements in promoter regions of the FAS gene [2]. The FAS promoter also has an LXRE, indicating that LXR directly stimulates FAS expression [3]. In addition, FAS expression is also induced by farnesoid X receptor (FXR), which is activated by bile acid in the liver [4]. This is mediated via LXRE because FXR can bind to LXRE in many gene promoters. Although the physiological significance of this is yet unclear, the FXRdependent regulation of FAS expression is very interesting in the context of crosstalk between lipogenesis and cholesterol homeostasis in the liver. Finally, PPARγ also induces FAS expression in adipocytes, although the FAS promoter has no typical PPAR response element (PPRE) [5]. The mechanism of the PPARγ-dependent regulation is unknown. However, FAS activity is required for TG accumulation in adipocytes and actually increases during adipocyte differentiation. ACC is also an SREBP1 target gene in the liver. The ACC promoter has a sterol response element (SRE) [6], indicating that ACC expression is induced by SREBP1. This suggests the possibility that LXR and PXR could stimulate ACC expression via SREBP1 induction. Actually, it has been reported that hepatic ACC expression is stimulated by LXR activation via SREBP1 induction, although PXR-dependent stimulation of ACC expression has not yet been reported. Moreover, ACC is directly regulated by LXR through LXRE in the promoter region, as well as FAS [7]. However, ACC activity is controlled by phosphorylation at the enzyme activity level rather than at the mRNA transcription level. Therefore, the nuclear receptor-mediated regulation of ACC might be an accessory-regulating system in total ACC activity. Expression of SCD1 is induced by SREBP1 through SRE of the SCD1 promoter. Therefore, LXR and FXR regulate SCD1 expression via SREBP1 induction as well as FAS and ACC expressions [8]. On the other hand, it has been well known that polyunsaturated fatty acids (PUFAs) suppress SCD1 expression in the liver. This suggests that PPARα, which is activated by PUFAs in the liver, might be involved in the hepatic regulation of SCD1 expression. Actually, treatment with WY-14643 (a synthetic PPARα agonist) decreased the mRNA level of the SCD1 gene in the liver [9]. However, in vitro analysis of the SCD1 promoter demonstrated that a PPRE of the SCD1 promoter is distinct from a PUFA response element which is responsible for the PUFA-dependent suppression of SCD1 expression [10]. This suggests that the PUFA-dependent suppression of SCD1 expression is independent of PPARα activity in hepatocytes. At least it is clear that PPARα activation suppresses SCD1 expression in the liver, although the details remain unclear.
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Lipolysis (HSL and ATGL) Adipocytes release free fatty acid (FFA) derived from accumulated TG to supply energy to other tissues such as cardiac and skeletal muscles. The liver absorbs the adipocyte-derived FFA to metabolize it and releases ketone bodies for neurons, which can use ketone bodies under hypoglycemic conditions. Therefore, lipase activity to hydrolyze TG into FFA and glycerols is indispensable for the maintenance of systemic energy balance. Adipocyte lipolysis is mediated by two distinct lipases: hormone-sensitive lipase (HSL) and adipocyte-specific triglyceride lipase (ATGL). HSL is activated through activation of adrenergic receptors to induce hormone-induced lipolysis. ATGL is concerned with adipocyte basal lipolysis to regulate the size of lipid droplets. Both lipases are controlled at mRNA levels together with enzymatic activity levels. HSL is activated by hormones like noradrenalin to increase the FFA blood level under fasting conditions. Adipocyte differentiation induces this lipase expression, suggesting that HSL expression is regulated by PPARγ in adipocytes. Actually, thiazolidinedione (TZD) treatment increases the mRNA expression level of HSL in adipocytes [11]. However, this induction is not direct. Transcription factor specificity protein-1 (Sp1) through the GC box of the HSL promoter mediates the induction of HSL in adipocytes. Deletion of the GC box or inhibition of Sp1 DNA-binding activity markedly reduces the PPARγ-dependent induction of HSL in adipocytes. Therefore, although PPARγ requires the involvement of Sp1, HSL is a PPARγ target gene in adipocytes. Another lipase in adipocytes, ATGL, is also a PPARγ target gene, which is a TG-specific lipase that is induced during adipogenesis and remains highly expressed in mature adipocytes. TZD induces ATGL mRNA [12], although it is still unknown whether the promoter of the ATGL gene has PPRE. Because ATGL is an important enzyme that interacts with adipocyte lipid droplets and controls the volume of the droplets, the promoter of the ATGL gene should be analyzed as soon as possible to elucidate the mechanism of ATGL transcriptional regulation in adipocytes.
Fatty Acid Oxidation (CPT1 and ACO) FFA as a fuel molecule is metabolized via the hepatic β-oxidation pathway to generate NADH used for oxidative phosphorylation in mitochondria. Absorbed FFA is activated to acyl-CoA by acyl-CoA synthase (ACS) as described later. Then, acyl-CoA is transported into mitochondria where the metabolic pathway occurs. Because the mitochondrial membrane is impermeable to acyl-CoA, the fatty acyl-CoA should be transformed to a mitochondrial membrane-permeable form to enter mitochondria directly for oxidation. This transformation is mediated by carnitine palmitoyl transferase-1 (CPT1), an outer mitochondrial membrane-integrated enzyme that catalyzes the transfer of long-chain acyl groups from CoA to carnitine. Newly produced acyl-
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carnitine can enter the mitochondrial matrix, where acyl-CoA is reproduced through the opposing reaction mediated by CPT2, another isoform of CPT. Mitochondrial fatty acid oxidation is limited by the CPT1-mediated step. On the other hand, mammalian cells have another organelle for fatty acid oxidation: peroxisomes. In hepatocytes, peroxisomes are considered to contribute up to 50% of the total fatty acid oxidative activity of the liver under average conditions. The fatty acid oxidation in peroxisomes is slightly different from that in mitochondria. In the peroxisomal pathway, the rate-limiting enzyme is acyl-CoA oxidase (ACO) that catalyzes the first step of the pathway. Therefore, CPT1 and ACO expressions are important for the stimulation of mitochondrial and peroxisomal fatty acid oxidations. The expressions of both genes are known to be regulated by nuclear receptors. Expressions of CPT1 and ACO are stimulated by PPAR activation. In hepatocytes, fibrates, PPARα agonists, induce the mRNA expressions of both genes in a manner of PPARα expression [13, 14]. Although the induction is mediated by a functional PPRE in the promoter of each gene, PPARγ cannot induce their expressions in adipocytes. This might be partly because PPRE has isoform specificity and because PPAR activity shows cell specificity dependent on cell-specific cofactors. The difference in target genes between PPAR isoforms results in the difference in fatty acid utilization between hepatocytes and adipocytes, in which fatty acid is degraded into acyl-CoA and utilized for TG synthesis, respectively. In addition, CPT1 expression is suppressed by PXR activation in the liver [15]. Because PXR activation causes hepatic TG accumulation, it is reasonable to suppress hepatic CPT1 expression by PXR activity.
Others (LPL, Fatty Acid Transporters and ACS) Circulating TG in lipoproteins should be hydrolyzed by a specific lipase before cellular uptake via fatty acid transporters. The indispensable step is mediated by lipoprotein lipase (LPL). The expression of this lipase is upregulated during adipocyte differentiation, suggesting that PPARγ is involved in the regulation in adipocytes. Actually, TZD and other PPARγ agonists induce LPL expression in adipocytes. This regulation is mediated through PPRE in the promoter region [16]. In addition, expression of LPL is enhanced by LXR activation [17]. LXR agonist-fed mice exhibit a significant increase in LPL gene expression in the liver and macrophages, but not in adipose tissues and muscle. The LXR-dependent regulation is also mediated by LXRE in the LPL promoter region. The mechanism of the PPAR- and LXR-dependent induction of LPL is necessary for the accumulation of lipid in adipocytes and hepatocytes, respectively. Hydrolyzed FFAs are absorbed into cells by both passive and facilitated diffusions. The facilitated diffusion is mediated by fatty acid transporter proteins. Thus, two distinct proteins are identified as fatty acid transporter proteins: CD36/FAT (fatty acid translocase) and FATPs (fatty acid transport proteins). CD36/FAT is a multifunctional protein that has been reported as a scavenger receptor (an oxidized-LDL receptor)
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and a thrombospondin receptor. This gene expression is regulated by PPAR activity in various tissues including liver, skeletal muscle, and adipose tissues [18], whereas several reports have shown PPAR-independent expression of the CD36/FAT gene in vitro and in vivo. Therefore, regulation of this gene is very complicated. Actually, the CD36/FAT gene has 3 distinct promoters in the 5⬘-noncoding region and the intron between exons 1 and 2 [19]. However, the promoter of CD36/FAT has a PPRE in the mouse [20]. In our experiments, TZD treatment induces CD36/FAT gene expression by adipocytes in vitro and in vivo. Thus, the differences in the control of this gene expression might be due to the specificity of cells and/or experimental conditions. Recently, it has been reported that LXR and PXR also control the expression of CD36/ FAT in hepatocytes [21]. The PPAR activation induces the expression of another fatty acid transporter, FATP, in adipocytes, liver, and intestine [22]. This transporter is significant in fatty acid uptake in intestinal epithelial cells. Thus, it is appropriate that PPAR, which is activated by dietary fatty acids, regulates FATP in the intestine. Thus far, there is no report that the expression of the FATP gene is regulated by LXR and/ or PXR. Whether LXR/PXR also upregulates FATP gene expression as well as CD36/ FAT is a very interesting question. In addition, ACS is a key enzyme in the metabolism of absorbed fatty acid. The absorbed fatty acid should be transformed into acyl-CoA to be metabolized for both TG synthesis and β-oxidation. This first step in the cellular utilization of the absorbed fatty acid is mediated by ACS. Regulation of ACS expression is regulated by PPAR in hepatocytes and adipocytes. The promoter region of this gene has typical PPRE [22]. It is likely that the PPAR-dependent induction of ACS contributes to β-oxidation in hepatocytes (PPARα dependent) and to TG synthesis in adipocytes (PPARγ dependent). Interestingly, fatty acyl-CoA such as stearoyl-CoA and palmitoyl-CoA serves as a PPAR antagonist [23]. Therefore, the antagonistic effect of acyl-CoA on PPAR activity might be feedback regulation to prevent excess cellular utilization of fatty acid.
Food Factors That Regulate Nuclear Receptor Activity
Agonistic Effects of Food Factors Thus far, our and other groups have identified various agonistic food factors that regulate the activity of nuclear receptors to control dysfunctions of lipid metabolism [24–28]. Particularly, many food factors with PPAR agonistic effects have been found. PPARα is involved in fatty acid oxidation in the liver. Therefore, application of the food factors with PPARα agonist activity enhances the uptake and oxidation of fatty acid in hepatocytes. As a result, hepatic and circulating TG amounts decrease. Therefore, the food factors with PPARα agonist activity are valuable for the treatment of obesity-related hyperlipidemia. On the other hand, PPARγ agonists such as TZD
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improve insulin resistance to decrease blood glucose level. This is because they promote adipocyte differentiation to increase the relatively high number of insulin-sensitive small adipocytes. Recently, the combinational application of PPARγ and PPARα agonists has been useful for the treatment of both hyperglycemia and hyperlipidemia. Although PPARγ agonists often cause body weight gain, PPARα activity suppresses the gain through the increase in hepatic lipid consumption. Thus, dual agonists to activate both PPARs are valuable. Example 1: Isoprenols for PPARs Farnesol (a C15 branched-chain fatty alcohol), an isoprenol, serves as a dual agonist for PPARα and PPARγ in ‘in vitro’ experiments [24]. In luciferase ligand assay for PPARs, farnesol activated both PPARα and PPARγ, although the activity of PPARα was more potent than that of PPARγ. Farnesol-related isoprenols such as geranylgeraniol (C20) also activated PPARs, whereas geraniol, another isoprenol (C10), activated PPARγ but not PPARα. Next, to examine the effect of farnesol and geranylgeraniol on the mRNA expression of PPAR target genes, the compounds were added to HepG2 hepatocytes or 3T3-L1 adipocytes. In PPARα-expressing HepG2 cells, both farnesol and geranylgeraniol induced the mRNA expression of PPARα target genes such as ACO and CPT1A. Moreover, the expression of PPARγ target genes in differentiated 3T3-L1 adipocytes (aP2 and LPL) was induced by the addition of farnesol and geranylgeraniol. These results indicate that isoprenols such as farnesol and geranylgeraniol serve as PPAR dual agonists in vitro. Then, to examine the in vivo effects of the isoprenols, we performed animal experiments using diabetic obese KK-Ay mice. In the experiments, we used farnesol, the most potent effect to activate PPARs. Farnesol was fed for 4 weeks with HFD. Body weight and organ weights showed no difference between the farnesol-fed and control HFD-fed mice. Although it had been expected that farnesol is effective on both adipose tissues and liver, only hepatic PPARα target genes were induced in the farnesol-fed mice [Goto and Kawada, unpubl. data]. In the liver, farnesol increased the expression levels of ACO and CPT1A mRNAs and decreased the hepatic TG content, whereas farnesol did not increase the expression levels of aP2 and LPL mRNAs in adipose tissues. However, the blood glucose level during fasting was decreased by the farnesol feeding. The improvement of hyperglycemia might be caused by the increase in the fatty acid oxidation level in the liver to reduce insulin resistance in other tissues. The reason why farnesol was effective only in the liver, but not in adipose tissues, might be due to the degradation of farnesol in the liver so that farnesol itself could not be delivered to adipose tissues. Branched fatty acids and alcohols including farnesol and geranylgeraniol are metabolized via the microsomal α-oxidation pathway. Hepatocytes show very high activity of this pathway to degrade substrates very fast. Of course, we cannot deny the possibility that farnesol has no effect on PPARγ activation in vivo. Further investigations are necessary to answer this question.
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Example 2: Phytol and Phytanic Acid In the previous section on farnesol, it has been mentioned that the effect of hepatic metabolism might be important for the functions of food factors in vivo. In this context, phytol and phytanic acid are very interesting food factors. We have reported that phytol (a component of chlorophyll) activates PPARα in the luciferase ligand assay and induces mRNA expression of PPARα target genes in hepatocytes [25]. Although phytol showed PPARγ activation activity, the activity was very moderate in comparison with that of PPARα activation. Phytol is metabolized to phytanic acid in the liver. The metabolite can be detected in the blood. Therefore, phytanic acid is a circulating form of phytol after the hepatic metabolism. Interestingly, it has been reported that phytanic acid activates PPARγ and retinoid X receptor-α [29]. In those reports, PPARα activation by phytanic acid is very weak in comparison with PPARγ activation. On the basis of these facts, we can propose an attractive model of functions of phytol as a food factor. First, phytol activates PPARα to induce the expression of its target genes and enhance fatty acid oxidation in the liver to decrease the hepatic and circulating lipid levels. Next, phytol itself is metabolized to phytanic acid to be delivered to adipose tissues, in which phytanic acid induces the expression of PPARγ target genes leading to the improvement of insulin resistance. Interestingly, it has been shown that the gene expression of enzymes involved in the transformation of phytol to phytanic acid is upregulated by PPARα activity in the liver [30]. Therefore, phytol enhances its own transformation to phytanic acid. This model is very attractive because the consideration of metabolites expands the possibility of food factors as PPAR regulators, but we should perform some additional experiments to prove the model.
Antagonistic Effects of Food Factors Agonistic activity of food factors for PPARα is very significant for the management of hyperlipidemia and hyperglycemia as described above. On the other hand, it has been suggested that hyperactivation of PPARγ causes hypertrophy of adipose tissues to induce obesity and obesity-related insulin resistance [31]. Therefore, PPARγ agonists such as TZD should be used together with dietary control to restrict energy uptake for the suppression of obesity. This indicates that the PPARγ activation is not beneficial when there is a high concentration of agonists, whereas the activation is very useful when PPARγ agonists are restricted. This concept is confirmed by the fact that PPARγ hetero-knockout mice (PPARγ+/–) show resistance to HFD-induced hyperglycemia [31]. In the PPARγ hetero-knockout mice, a decrease in PPARγ gene expression causes a decrease in PPARγ activity. The decrease in PPARγ activity suppresses the HFD-induced adipocyte hypertrophy to improve insulin resistance. Therefore, it is likely that PPARγ antagonists are also useful for long-term treatment in the presence of endogenous PPARγ agonists.
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LXR also has significant effects on lipid and glucose metabolism, particularly hepatic metabolism. It has been reported that hyperglycemia is improved by LXR activation. Although this mechanism is unknown, this might be partly because LXR activation induces adipocyte differentiation to increase insulin-sensitive small adipocytes, as TZD shows. On the other hand, the hepatic LXR activation induces lipogenesis to cause hepatic TG accumulation (hepatic steatosis). This is due to the LXR-dependent induction of SREBP1 regulating the expression of lipogenesis-related genes in the liver. The hepatic induction of SREBP1 by LXR leads to an increase in circulating lipid content. Therefore, when we focus on the improvement of hyperlipidemia, it is beneficial to suppress the LXR activity in the liver. Example 1: β-Cryptoxanthine for PPARs Dietary foods contain various carotenoids such as carotenes and xanthophylls. β-Cryptoxanthine is a major carotenoid in citrus fruits. We have recently found that this carotenoid serves as a PPARγ antagonist in vivo and in vitro [Ohyama and Kawada, unpubl. data]. Diabetic obese KK-Ay mice were fed HFD containing β-cryptoxanthine for 4 weeks. The β-cryptoxanthine-fed mice showed no significant difference in body and organ weights. However, their fasting blood glucose level decreased. In addition, oral glucose tolerance test demonstrated improvement in insulin resistance after 4 weeks of feeding with β-cryptoxanthine. The number of small adipocytes increased and that of large ones decreased in the adipose tissues of the β-cryptoxanthine-fed mice in comparison with those of control HFD-fed mice. Interestingly, the gene expression of enzymes involved in fatty acid oxidation in the liver was upregulated in the β-cryptoxanthine-fed mice, such that the hepatic TG content of the β-cryptoxanthine-fed mice was lower than that of the control mice. These data of the β-cryptoxanthine-fed mice were very similar to those of the PPARγ hetero-knockout mice. The reason for the hepatic TG decrease may be the increase in the level of circulating leptin secreted by adipocytes. Although the details of the mechanism are yet unknown, the β-cryptoxanthine feeding caused increases in both mRNA and protein levels of leptin in adipose tissues. Actually, the TG content of skeletal muscle also decreased in the β-cryptoxanthine-fed mice accompanied by the increase in the expression of fatty acid oxidation-related enzymes. These results indicate that β-cryptoxanthine inhibits adipocyte hypertrophy to maintain insulin sensitivity and affects the release of adipocytokines such as leptin to improve the insulin resistance of other tissues. To investigate the mechanism of the β-cryptoxanthine effects on the improvement in lipid metabolism, we performed in vitro experiments using culture cells. Luciferase ligand assay showed β-cryptoxanthine-dependent inhibition of TZD-induced PPARγ activation in a dose-dependent manner. Moreover, the β-cryptoxanthine treatment suppressed lipid accumulation and GPDH enzyme activity (a differentiation marker enzyme involved in TG synthesis) 10 days after differentiation induction in 3T3-L1 adipocytes. The expression of PPAR target genes was also inhibited
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in 3T3-L1 adipocytes by the β-cryptoxanthine treatment. These data suggest that β-cryptoxanthine serves as an antagonist of PPARγ. Although more details should be elucidated for both in vivo and in vitro effects of β-cryptoxanthine, it appears that PPARγ antagonists such as β-cryptoxanthine are valuable for the management of obesity-related insulin resistance. Example 2: Diosgenin for LXRs Diosgenin is the main aglycon of saponin in fenugreek, which is a spice used in the Eastern Mediterranean region to Central Asia and Ethiopia and is widely cultivated in India, Pakistan, and China. Thus far, diosgenin and fenugreek have been reported as food factors that improve hyperglycemia and hyperlipidemia. However, their detailed mechanisms remain unknown. Thus, we examined the effects of fenugreek or diosgenin on lipid metabolism in diabetic obese mice and HepG2. Diabetic obese KK-Ay mice fed fenugreek for 4 weeks inhibited HFDinduced hepatic steatosis in comparison with control HFD-fed mice. This is due to decreased mRNA levels of lipogenesis-related genes such as SREBP1, FAS, and SCD1 in hepatocytes. These results indicate that fenugreek suppressed SREBP1 expression in the liver to inhibit hepatic lipogenesis leading to the improvement of the HFD-induced hepatic steatosis. Interestingly, the fenugreek-fed mice showed significant improvement in insulin resistance in OGTT. Therefore, it is likely that the hepatic suppression of steatosis by fenugreek is sufficient to affect other tissues in terms of insulin sensitivity. SREBP1 is regulated by LXR in the liver, suggesting the possibility that fenugreek containing diosgenin affects LXR activation in hepatocytes. Thus, LXR ligand assay on HepG2 hepatocytes was performed using diosgenin. As expected, diosgenin inhibited LXR activation in a dose-dependent manner in HepG2 cells. This inhibitory effect of diosgenin was sufficient to suppress LXR agonistinduced SREBP1 expression in HepG2 cells. The diosgenin treatment also inhibited SREBP1-dependent expression of the lipogenesis-related genes such as FAS and SCD1 in HepG2 cells. LXR activation increases cellular TG content in HepG2 cells. However, the diosgenin treatment decreased the cellular TG content in the presence of an LXR agonist. These data indicate that diosgenin inhibits LXR activation to suppress the lipogenesis-related gene expression in hepatocytes and the LXR-dependent increase in the hepatic TG content. It is suggested that fenugreek containing diosgenin is useful for the treatment of hyperlipidemia and hepatic steatosis.
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Acknowledgements The authors thank Kana Ohyama, Chikako Ando, Noriko Mizoguchi, Michihiro Takada, Aki Teraminami, Tomoya Sakamoto, and Rino Kimura for their support in the preparation of the manuscript, and to Sayoko Shinotoh for her secretarial support. The works described in this review were supported by the Research and Development Program for New Bio-industry Initiation.
References 1 Repa JJ, Liang G, Ou J, Bashmakov Y, Lobaccaro JM, Shimomura I, Shan B, Brown MS, Goldstein JL, Mangelsdorf DJ: Regulation of mouse SREBP-1c by oxysterol receptors, LXRalpha and LXRbeta. Genes Dev 2000;14:2819–2830. 2 Latasa MJ, Moon YS, Kim KH, Sul HS: Nutritional regulation of the fatty acid synthase promoter in vivo: sterol regulatory element binding protein functions through an upstream region containing a sterol regulatory element. Proc Natl Acad Sci USA 2000;97:10619–10624. 3 Joseph SB, Laffitte BA, Patel PH, Watson MA, Matsukuma KE, Walczak R, Collins JL, Osborne TF, Tontonoz P: Direct and indirect mechanisms for regulation of fatty acid synthase gene expression by liver X receptors. J Biol Chem 2002;277:11019–25. 4 Zhou J, Zhai Y, Mu Y, Gong H, Uppal H, Toma D, Ren S, Evans RM, Xie W: A novel pregnane X receptor-mediated and sterol regulatory element-binding protein-independent lipogenic pathway. J Biol Chem 2006;281:15013–15020. 5 Schadinger SE, Bucher NL, Schreiber BM, Farmer SR: PPARgamma2 regulates lipogenesis and lipid accumulation in steatotic hepatocytes. Am J Physiol Endocrinol Metab 2005;288:E1195–E1205. 6 Lopez JM, Bennett MK, Sanchez HB, Rosenfeld JM, Osborne TF: Sterol regulation of acetyl coenzyme A carboxylase: a mechanism for coordinate control of cellular lipid. Proc Natl Acad Sci USA 1993;93:1049– 1053. 7 Talukdar S, Hillgartner FB: The mechanism mediating the activation of acetyl-coenzyme A carboxylase-alpha gene transcription by the liver X receptor agonist T0-901317. J Lipid Res 2006;47:2451–2461. 8 Tabor DE, Kim JB, Spiegelman BM, Edwards PA: Identification of conserved cis-elements and transcription factors required for sterol-regulated transcription of stearoyl-CoA desaturase 1 and 2. J Biol Chem 1999;274:20603–20610. 9 Miller CW, Ntambi JM: Peroxisome proliferators induce mouse liver stearoyl-CoA desaturase 1 gene expression. Proc Natl Acad Sci USA 1996;93:9443– 9448.
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10 Ntambi JM: Regulation of stearoyl-CoA desaturase by polyunsaturated fatty acids and cholesterol. J Lipid Res 1999;40:1549–1558. 11 Deng T, Shan S, Li PP, Shen ZF, Lu XP, Cheng J, Ning ZQ: Peroxisome proliferator-activated receptor-gamma transcriptionally up-regulates hormonesensitive lipase via the involvement of specificity protein-1. Endocrinology 2006;147:875–884. 12 Kershaw EE, Schupp M, Guan HP, Gardner NP, Lazar MA, Flier JS: PPARgamma regulates adipose triglyceride lipase in adipocytes in vitro and in vivo. Am J Physiol Endocrinol Metab 2007;293:E1736– E1745. 13 Napal L, Marrero PF, Haro D: An intronic peroxisome proliferator-activated receptor-binding sequence mediates fatty acid induction of the human carnitine palmitoyltransferase 1A. J Mol Biol 2005; 354:751–759. 14 Tugwood JD, Issemann I, Anderson RG, Bundell KR, McPheat WL, Green S: The mouse peroxisome proliferator activated receptor recognizes a response element in the 5⬘ flanking sequence of the rat acyl CoA oxidase gene. EMBO J 1992;11:433–439. 15 Nakamura K, Moore R, Negishi M, Sueyoshi T: Nuclear pregnane X receptor cross-talk with FoxA2 to mediate drug-induced regulation of lipid metabolism in fasting mouse liver. J Biol Chem 2007;282: 9768–9776. 16 Auwerx J, Schoonjans K, Fruchart JC, Staels B: Regulation of triglyceride metabolism by PPARs: fibrates and thiazolidinediones have distinct effects. J Atheroscler Thromb 1996;3:81–89. 17 Zhang Y, Repa JJ, Gauthier K, Mangelsdorf DJ: Regulation of lipoprotein lipase by the oxysterol receptors, LXRalpha and LXRbeta. J Biol Chem 2001; 276:43018–43024. 18 Sato O, Kuriki C, Fukui Y, Motojima K: Dual promoter structure of mouse and human fatty acid translocase/CD36 genes and unique transcriptional activation by peroxisome proliferator-activated receptor alpha and gamma ligands. J Biol Chem 2002;277:15703–15711.
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19 Sato O, Takanashi N, Motojima K: Third promoter and differential regulation of mouse and human fatty acid translocase/CD36 genes. Mol Cell Biochem 2007;299:37–43. 20 Teboul L, Febbraio M, Gaillard D, Amri EZ, Silverstein R, Grimaldi PA: Structural and functional characterization of the mouse fatty acid translocase promoter: activation during adipose differentiation. Biochem J 2001;360:305–312. 21 Zhou J, Febbraio M, Wada T, Zhai Y, Kuruba R, He J, Lee JH, Khadem S, Ren S, Li S, Silverstein RL, Xie W: Hepatic fatty acid transporter Cd36 is a common target of LXR, PXR, and PPARgamma in promoting steatosis. Gastroenterology 2008;134:556–567. 22 Cha BS, Ciaraldi TP, Carter L, Nikoulina SE, Mudaliar S, Mukherjee R, Paterniti JR Jr, Henry RR: PPARgamma and RXR agonists have complementary effects on glucose and lipid metabolism in human skeletal muscle. Diabetologia 2001;44:444– 452. 23 Murakami K, Ide T, Nakazawa T, Okazaki T, Mochizuki T, Kadowaki T: Fatty-acyl-CoA thioesters inhibit recruitment of steroid receptor co-activator 1 to alpha and gamma isoforms of peroxisome-proliferator-activated receptors by competing with agonists. Biochem J 2001;353:231–238. 24 Takahashi N, Kawada T, Goto T, Yamamoto T, Taimatsu A, Kimura K, Saitoh M, Hosokawa T, Miyashita K, Fushiki T: Dual action of isoprenols to activate both PPARγ and PPARα in 3T3-L1 adipocytes and HepG2 hepatocytes. FEBS Lett 2002;514: 315–322. 25 Goto T, Takahashi N, Kato S, Egawa K, Ebisu S, Moriyama T, Fushiki T, Kawada T: Phytol, a phytochemical compound, activates PPARα and regulates lipid metabolism in PPARα-expressing cells. Biochem Biophys Res Commun 2005;337:440–445.
26 Kuroyanagi K, Kang MS, Goto T, Kusudo T, Hirai S, Yu R, Yano M, Sasaki T, Takahashi N, Kawada T: Citrus auraptene acts as an agonist for PPARs and enhances adiponectin production and MCP-1 reduction in 3T3-L1 adipocytes. Biochem Biophys Res Commun 2008;366:219–225. 27 Kang MS, Hirai S, Goto T, Kuroyanagi K, Ezaki Y, Takahashi N, Kawada T: Dehydroabietic acid, herbal terpenoid, acts as ligands for PPARs in macrophages to regulate inflammation. Biochem Biophys Res Commun 2008;369:333–338. 28 Takahashi N, Kawada T, Goto T, Kim CS, Taimatsu A, Egawa K, Yamamoto T, Jisaka M, Nishimura K, Yokota K, Yu R, Fushiki T: Abietic acid activates PPARγ in RAW264.7 macrophages and 3T3-L1 adipocytes to regulate gene expression involved in inflammation and lipid metabolism. FEBS Lett 2003;550:190–194. 29 Zomer AW, van Der Burg B, Jansen GA, Wanders RJ, Poll-The BT, van Der Saag PT: Pristanic acid and phytanic acid: naturally occurring ligands for the nuclear receptor peroxisome proliferator-activated receptor alpha. J Lipid Res 2000;41:1801–1807. 30 Gloerich J, van den Brink DM, Ruiter JP, van Vlies N, Vaz FM, Wanders RJ, Ferdinandusse S: Metabolism of phytol to phytanic acid in the mouse, and the role of PPARalpha in its regulation. J Lipid Res 2007;48:77–85. 31 Kubota N, Terauchi Y, Miki H, Tamemoto H, Yamauchi T, et al: PPARgamma mediates high-fat diet-induced adipocyte hypertrophy and insulin resistance. Mol Cell 1999;4:597–609.
Professor Teruo Kawada Laboratory of Molecular Functions of Food Division of Food Science and Biotechnology Graduate School of Agriculture Kyoto University, Uj Kyoto 606-8502 (Japan) Tel. +81 75 753 6262, Fax +81 75 753 6264, E-Mail
[email protected]
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Proteomics Yoshikawa T (ed): Food Factors for Health Promotion. Forum Nutr. Basel, Karger, 2009, vol 61, pp 39–54
Oxidative Stress-Induced Posttranslational Modification of Proteins as a Target of Functional Food Yuji Naito ⭈ Toshikazu Yoshikawa Molecular Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine, Kyoto, Japan
Abstract In lifestyle-related diseases including metabolic syndrome, atherosclerosis, and cancer, oxidative stress is indicated by several markers, among which are lipid peroxides, aldehydes, and nitrotyrosine. We hypothesized that identification of proteins that are posttranslationally modified due to oxidative stress would lead to a greater understanding of some of the potential molecular mechanisms involved in degeneration and inflammation in these disorders. Proteomics is an emerging method for identification of proteins and their modification residues, and its application to food factor science is just beginning. Especially, we can obtain several monoclonal antibodies to detect specifically oxidized proteins, which can be applied to analysis by immunostaining or immunoblot. In this review, we present the use of these monoclonal antibodies in several diseases, from which new insights have emerged into mechanisms of metabolism and inflammation in these disorders that are Copyright © 2009 S. Karger AG, Basel associated with oxidative stress.
It is well known that oxidative stress is involved in the pathogenesis of lifestyle-related diseases, including atherosclerosis, hypertension, diabetes mellitus, ischemic diseases, and malignancies. Oxidative stress, which refers to a state of elevated levels of reactive oxygen species (ROS), occurs form a variety conditions that stimulate either ROS production or a decline in antioxidant defenses. During oxidative stress, the oxidation of cellular components results in the modification of DNA, proteins, lipids, and carbohydrates [1]. In the case of proteins, numerous posttranslational modifications have been characterized as resulting either from direct oxidation of amino residues or through the formation of reactive intermediates by the oxidation of other cellular components. The oxidative stress-induced posttranslational modification (OPTM) of 20 kinds of basic amino acids takes an important role in the manifestation of the function of many proteins. OPTM may be subdivided into two general forms: reversible OPTM and irreversible OPTM (fig. 1). The oxidation of cysteine to sulfenic, sulfinic,
Oxidative stress
Inflammatory cells Drugs Ischemia Hyperglycemia Metals
Detection of OPTM proteins
Functional analysis of OPTM proteins
Association with diseases
Oxidative stress-specific modification HNE, HEL addition Nitration Chlorination Bromonation Necrosis Apoptosis Innate immune reaction Irreversible OPTM Decrease in defense system • Degeneration • Chronic inflammation • Cytokine storm • Carcinogenesis
Oxidative stress NO stress AGEs Target cells Reversible OPTM
Redox regulation Signal modification Mitochondrial dysfunction Abnormal cell proliferation
Oxidation of cystein residues S-thiolation Glutationation Nitrosylation
Fig. 1. Induction of oxidative stress, detection and functional analysis of OPTM proteins, and their association with lifestyle-related diseases. AGEs = Advanced glycation end products.
and sulfonic acids has been shown to occur frequently and sulfenic and sulfinic acids often can be reduced enzymatically. Some of the lipid peroxidation products exhibit a facile reactivity with proteins, generating a variety of intra- and intermolecular covalent adducts. Furthermore, especially in the situation of inflammation, nitration by reactive nitrogen species (RNS), chlorination by hypochlorous acid (HOCl), and bromonation by hypobromous acid of the targeted protein are frequently detected. In this review, we focused on the detection of OPTM proteins in lifestyle-related diseases and their animal models, and introduced several investigations for functional foods by using OPTM proteins as their target.
Oxidative Stress-Induced Posttranslational Modification
As the relationship between oxidative stress and various diseases becomes clear, OPTM proteins are now attracting attention. The transduction of an oxidant signal into a biological response can be mediated in several ways, but one principal mechanism involves the oxidation of protein cysteine residues. The thiol (-SH) moiety on the side chain of the amino acid cysteine is particularly sensitive to redox reactions
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and is an established redox sensor. As for cysteine residue-specific OPTM of the protein (S-thiolation), it is found to act as a switch regulating biological function like phosphorylation-dephosphorylation [2]. For example, oxidative modification of free reactive thiols (S-thiolation) on the small G protein Ras increases Ras activity and thereby promotes ROS-dependent hypertrophic signaling in cardiac myocytes [3]. There are an increasing number of proteins that are known to be regulated by S-thiolation. Proteins regulated by cysteine oxidation include ion translocators, structural proteins, metabolic enzymes, DNA isomerases, and signaling proteins. Signal transduction proteins that are directly regulated by oxidative modification of cysteine residues include protein phosphatases, protein kinases, G-proteins, and membrane receptors [4]. Recent studies used biotinylated cysteine (biotin-cysteine) as a probe for proteins that are S-thiolated during oxidative stress. Biotin-cysteine rapidly crosses the plasma membrane and can be used to detect, quantify, purify, and identify proteins susceptible to oxidation in cells. Using this method, Ishii et al. [5] have identified 26 proteins including glycolytic enzymes, cytoskeletal proteins, redox enzymes, and stress proteins as substrates for reversible cysteine-targeted oxidation when human neuroblastoma SH-SY5Y cells were exposed to 15-deoxydelta12,15-prostaglandin J2. OPTM of amino acid residues in a peptide may lead to structural changes, ranging from a slight conformational change to a severe denaturation accompanied by fragmentation. It may lead to either activation or inhibition of the protein activities. Mild oxidative stress can induce modification of cysteine such as reversible S-thiolation, disulfide formation, glutathionylation, and S-nitrosylation. Although OPTM of cysteine residue inactivates protein targets, such S-thiolation can also result in a gain of function for certain stress-responsive signaling systems. For example, transcriptional activation of antioxidant-responsive element (ARE)-containing genes is upregulated by S-thiolation. Several genes containing ARE are activated by the activation of Nrf-2-Keap 1 system by oxidative stress. The S-thiolation of Keap 1, the cytoplasmic inhibitor of Nrf2, by ROS or electrophiles results in the dissociation of the Keap 1-Nrf2 complex. Once freed from inhibition by Keap 1, Nrf2 translocates to the nucleus and activates the expression of ARE-containing genes. In addition to these reversible OPTM, it becomes clear that irreversible OPTM for histidine, lysin, and cysteine residues means generation of abnormal protein associated with the etiology of lifestyle related diseases. Moderate oxidation of amino acid side chains can give rise to a number of adducts. Many reactions result in the formation of direct oxidation of arginine and proline residues (semialdehyde formation), sulfoxide and nitrosylation formation, oxo-, hydroxy-, nitro-, and chloroderivatives of amino acids, and finally covalent cross-links within and between protein molecules [6]. Furthermore, lipid peroxidation products can diffuse across cellular membranes, allowing the reactive aldehyde-containing lipids to covalently modify proteins localized throughout the cell and relatively far away from the initial site of ROS formation. The most reactive aldehydes generated from polyunsaturated
Oxidative Stress-Induced Posttranslational Modification of Proteins
41
fatty acid oxidation are 4-hydroxy-2-nonenal (HNE), 4-oxo-2-nonenal, and acrolein. OPTM proteins have been shown to have a wide variety of effects on cells in vitro depending upon the concentration utilized, and as such, interpretation of experimental results must be considered cautiously. Because the side chains of cysteine, histidine, and lysine are often used in catalysis, the most common effect of OPTM is enzyme inactivation. Lipid peroxidation products can inactivate or modify the function of several proteins (Na+-K+-ATPase [7], glucose transporter [8], adipocyte fatty acid-binding protein [9, 10], NADP+-dependent isocitrate dehydrogenase [11], thioredoxin and thioredoxin reductase [12–14], glutathione peroxidase [15], and heat shock protein 90 [16]). A large body of evidence supports OPTM via endogenous and exogenous ROS compounds as an important factor in the induction of autoimmunity [17–19]. OPTM epitope causes the failure of acquired tolerance for autoantigen by what is shown as a new antigen. Recent studies suggest that OPTM protein is a cause of autoimmune disease and inflammatory disorder [19–21]. OPTM proteins could be the targets of B cell-mediated immune responses and induce T cell responses and add the potential of certain aldehydes to induce autoimmunity by breaking the B cell tolerance to nonmodified proteins. It has been shown that the OPTM of self-proteins by lipid peroxidation products indeed results in a break of a tolerance to self-proteins. The presence of autoantibodies against OPTM protein (glutamic acid decarboxylase) in patients with type I diabetes as well as in patients with Stiffman syndrome [22] has also been demonstrated. Recently, Toyoda et al. [19] have reported that the HNEspecific epitopes can be a triggering antigen of anti-DNA response, and the monoclonal antibodies against HNE-specific epitopes can bind to two structurally distinct antigens (i.e. native DNA and 4-oxo-2-nonenal-modified proteins). Their findings provide the evidence to suspect an etiologic role for the lipid peroxidation in autoimmune disease. One of the most important processes for maintaining homeostasis is the ability of proteolytic systems to eliminate OPTM proteins. Cells degrade OPTM proteins via the proteasome pathway and the lysosomal pathway. The ubiquitin-proteasome system is considered the major pathway responsible for the degradation and elimination of mildly oxidized proteins, and is involved in regulating proteins involved in several cellular activities like cell cycles. Interestingly, a proteomic analysis of OPTM proteins shows that the major intracellular target of protein carbonylation is one of the regulatory subunits in 26S proteasome, S6 ATPase [23]. Removal of OPTM proteins is crucial for cell survival. Indeed, if OPTM proteins are not eliminated either through proteasome or lysosomal pathways, they are able to begin to accumulate and potentially aggregate. These aggregated proteins can alter cell functions and lead to necrosis or apoptosis. Furthermore, a recent study clearly showed the possibility that the decrease in autophagy function may cause the autoimmune inflammation in the intestine, because autophagy is a primary mechanism to resolve these OPTM proteins [24].
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4-Hydroxy-2-Nonenal
A growing body of evidence suggests that many of the effects of cellular dysfunction under oxidative stress are mediated by products of the peroxidative degradation of polyunsaturated fatty acids. When unsaturated fatty acid is exposed to oxidative stress, a lipid peroxidation response progresses like a chain reaction, and various kinds of degradation products by lipid peroxidation responses are generated. Many past studies showed that aldehyde molecules, an end product of lipid peroxidation, are implicated as causative agents in cytotoxic processes initiated by the exposure of biological systems to oxidizing agents. In contrast to free radicals, aldehydes are relatively stable and can diffuse within or even escape from the cell and attack targets far from the site of the original event [25]. HNE (fig. 2), among the reactive aldehydes, is believed to be largely responsible for the cytopathological effects observed during oxidative stress. The most common and reliable approach for detection of HNE adducts is the use of antibodies which recognize HNE bound to amino acid sidechains of proteins. In 1995, Toyokuni et al. [26] raised a monoclonal antibody directed at the HNEmodified protein. This monoclonal antibody, which is now commercially available, has been attested to be specific for the HNE-histidine Michael adduct. The development of specific antibodies against protein-bound HNE has made it possible for us to obtain highly probable evidence for the occurrence of oxidative stress in vivo. Toyokuni et al. [27] demonstrated the presence of HNE-modified proteins in vivo in the iron-nitrolotriacetate (Fe3+-NTA)-induced renal carcinogenesis, which is one of the best characterized in vivo models of oxidative stress. The immunoreactive HNE adducts were markedly generated in the renal proximal tubules with degeneration several hours after administration of Fe3+-NTA. The HNE-modified proteins have also been detected in other animal and human studies, including hyperglycemia injury of pancreatic β-cells [28], ischemia-reperfusion injury [29], carbon tetrachloride-induced liver injury [30], ischemic heart or renal failure [31, 32], inflammatory bowel disease [33], chronic hepatitis type C [34], nonalcoholic steatohepatitis [35], atherosclerotic lesion [36], Alzheimer’s disease [37], Parkinson’s disease [38], amyotrophic lateral sclerosis [39], exercise-induced muscular injury [40]. Collectively, the results of these studies document the association of HNE-derived epitopes with chemical-induced oxidative stress or disease states linked directly or indirectly with chronic inflammation. However, the mechanistic role of HNE-modified proteins in the cellular damage accompanying oxidative stress is not well delineated. To examine the relationship between oxidative stress, HNE-protein modification, and cellular signaling, we treated cultured human mesangial cells with high (25 mM)concentration glucose and identified proteins modified by HNE using immunoblotting [41]. Interestingly, specific targets with estimated molecular masses of 60, 80, 85, and 105 kDa were strongly stained by anti-HNE antibody in mitochondrial fraction of high glucose-treated mesangial cells (fig. 3). In addition, fluorescent intensity of
Oxidative Stress-Induced Posttranslational Modification of Proteins
43
-6 PUFAs
R
R’ Peroxidation
Fig. 2. Formation/detection of HNE-modified proteins. HNE is generated during lipid peroxidation of ω–6 polyunsaturated fatty acids (PUFAs). The Michael type addition of HNE to protein is formed in vivo. X represents the side chain of nucleophilic amino acids, such as cysteine, histidine, and lysine. Protein-bound HNE can be detected by immunochemical methods and mass spectrometry using an anti-HNE antibody.
OH O
HNE
Protein
O HNE-specific modification of proteins
OH Anti-HNE Ab
X Protein
RedoxSensor CC-1 was increased in high glucose-exposed mesangial cells and the merged images with MItoTracker Green FM clearly indicated that mitochondria are the major source of ROS production in high glucose-exposed mesangial cells. It is conceivable that these specific targets identified in vitro are involved in the mechanism of HNE-induced cell signaling and would be of interest in identifying the proteins. Moreover, these specific targets may be candidates for biomarkers to evaluate the antioxidative properties of food factors. We have found that astaxanthin, a carotenoid, reduced the increases of these HNE-modified proteins in mitochondrial fraction as well as inhibited the ROS production from the mitochondria in high glucose-exposed mesangial cells. These molecular-based data strongly support an in vivo evidence that the treatment with astaxanthin inhibits diabetic nephropathy via reducing oxidative stress in a mouse model [42].
Nε-(Hexanonyl)lysine
Nε-(hexanonyl)lysine (HEL) has been found in the reaction between linoleic hydroperoxide and lysine moiety. It has been shown that the formation of HEL is a good marker for oxidative modification by oxidized ω–6 fatty acids such as linoleic acid and arachidonic acid [43]. Presence of HEL is reported by the immunostaining by using monoclonal and polyclonal antibodies against HEL. Kato et al. [44] evaluated muscular oxidation injury by excessive exercise using an anti-HEL antibody and reported that the functional food factor flavonoid is useful in reduction of this oxidation injury by showing that this compound clearly reduced the HEL-positivity in
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WB: MnSOD (25 kDa)
25 kDa
WB: HNE (4-hydroxy-2-nonenal)
105 kDa
75 kDa
D-Glucose ASX (10–6 M)
5 –
25 5 25 – – +
Mitochondria
5 25 5 25 + – + + Cytosol
Fig. 3. Astaxanthin inhibited high glucose-induced production of HNE-modified proteins in mitochondria of normal human mesangial cells. Representative Western blotting image out of three independent experiments is shown. Anti-HNE antibody was used to detect protein adducts in mitochondrial and cytosolic fractions of high glucose (25 mM)-treated normal human mesangial cells. Anticytochrome C oxidase complex IV subunit antibody was used as a mitochondrial protein marker. Arrows indicate bands stained with anti-HNE antibody. M = Marker. Data are from Manabe et al. [41].
muscular tissues. Fukuchi et al. [45] have reported that HEL is detected in foam cellrich areas in atherosclerotic specimens obtained from New Zealand White rabbits, where they are primarily colocalized with C-reactive protein (CRP)-positive cells, suggesting that the generation of oxidative stress marker may be mediated by CRP in atherosclerotic lesions. Osakabe et al. [46] have demonstrated that rosmarinic acid, a major polyphenolic component of Perilla frutescens, reduces lipopolysaccharideinduced liver injury in d-galactosamine-sensitized mice, and also demonstrates that its cytoprotective action may be derived from antioxidative action of the ingredient by showing the decrease in HEL production in liver as a biomarker of lipid peroxidation. The antioxidative action of cacao liquor proanthocyanidin polyphenols has been also shown by the in vivo investigation using several biomarkers including nitrotyrosine, HEL, and HNE adducts in mice lung [47]. We have recently demonstrated that astaxanthin (1) increased the utilization of lipids as an energy substrate during exercise, and (2) improved muscle lipid metabolism in exercise via inhibitory effect of oxidative carnitine parmitoyltransferase I (CPT I) modification by HEL [48] (fig. 4). A rate-limiting step of lipid metabolism in myocytes is the entry of long-chain fatty acids into mitochondria. CPT I located on the mitochondrial membrane plays an important role in the entry of fatty acid. Recent studies have shown that FAT/CD36 is associated with CPT I on the mitochondrial membrane and elevates its function. Our study has shown that an increase in the interaction between CPT I and FAT/CD36 in muscle during exercise is facilitated by
Oxidative Stress-Induced Posttranslational Modification of Proteins
45
IB:HEL
IB:FAT/CD36 *
2.5
*
*
2.0
Normalized ratio
Normalized ratio
*
2.5
*
1.5 1.0 0.5
*
2.0 1.5 1.0 0.5 0
0
CONT
a
AST
Sedentary
CONT
CONT
AST
Running
*
b
AST
Sedentary
CONT
AST
Runing
Fig. 4. Amount of fatty acid translocase (FAT/CD36) that coimmunoprecipitated with carnitine palmitoyltransferase I (CPT I; a) and HEL-modified CPT I (b) in skeletal muscle. A single session of exercise was performed at 30 m/min for 30 min on the final day of the experiment. Lysate protein from the muscle collected immediately after running was immunoprecipitated with CPT I antibody. Immunoprecipitates were separated by SDS-PAGE and membranes probed for FAT/CD36 (a) or HEL (b). Values are mean ± SE obtained from 6 mice. * p < 0.05. Data are from Aoi et al. [40].
astaxanthin, which would be one of mechanisms involved in the promotion of lipid metabolism. Exercise-induced oxidative stress is mainly derived from mitochondrial production of ROS associated with ATP generation and thus CPT I located on the mitochondria membrane is easily exposed to oxidative stress. We found that astaxanthin limits the modification of CPT I by HEL during exercise. Modification of CPT I by HEL may alter the colocalization of CPT I with FAT/CD36 by changing the CPT I molecule. These data indicate that an increase in fatty acyl-CoA uptake into the mitochondria via CPT I during exercise may be involved in the promotion of lipid metabolism by antioxidant activity of astaxanthin. Moreover, our data show the possibility that the HEL modification of CPT I may be a good biomarker for the evaluation of antioxidative properties of food factors in vivo.
Neutrophil-Dependent Oxidative Stress
In activated neutrophils, NADPH oxidase in cell membranes becomes activated, and an electron transfer takes place from NADPH in cells to oxygen inside and outside cells, and the oxygen that received electrons becomes superoxide radicals (O2–˙), which is rapidly converted to hydrogen peroxides (H O ) by spontaneous dismutation or enzymatic SOD, and hydroxyl radicals (˙OH), which are formed nonenzymatically in the presence of Fe2+ as a secondary reaction [1, 49]. On the other hand, the toxicity of H O is enhanced by the activity of myeloperoxidase (MPO). MPO is abundant in primary azurophilic granules of leukocytes including neutrophils and monocytes/ 2
2
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2
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macrophages, and secreted into the phagolysosomal compartment following phagocyte activation. In combination with H2O2, MPO can oxidize the halides and the pseudohalide thiocyanate (SCN–) to their corresponding hypohalous acids. H2O2 + HX → HOX + H2O (X = Cl–, Br–, I–, or SCN–)
Owing to its high concentration in biological fluids, Cl– is the major substrate for MPO; consequently, HOCl is a major oxidation product. It has several known targets. One of these targets is the molecule ‘tyrosine’ in both its free and bound forms. Upon reaction with HOCl, tyrosine is converted into the 3-chlorotyrosine molecule (fig. 5). This molecule could also cause tissue damage or dysfunction in many inflammatory conditions. In addition, 3-chlorotyrosine is unique because it is heat stable and is not readily formed by an artificial mechanism, which makes it an excellent marker, a ‘molecular fingerprint’, for MPO-induced oxidation [50]. Recent in vivo studies indicate that assessment of 3-chlorotyrosine protein adduct formation by immunohistochemistry could be a useful marker of neutrophil-induced cell injury in druginduced liver injury [51], inflammatory neuronal degeneration [52], atherosclerosis [53, 54], and cystic fibrosis [54]. If we want to evaluate the anti-inflammatory effect, especially in neutrophil-associated inflammation, these chlorotyrosine molecules may be a good marker for the function of food factors. Recently, Kunitomo et al. [55, 56] have demonstrated that the administration of coenzyme Q10 or corosolic acid, a constituent of banaba leaves, significantly attenuates the increase in 3-chlorotyrosine in serum, reduces the elevated serum insulin levels and elevated blood pressure, and finally improves endothelial dysfunction in the mesenteric arteries in a rat model of metabolic syndrome.
Nitrosative Protein Tyrosine Modification
Nitration and nitrosylation of tyrosine residues are mediated by RNS produced during inflammation, aging, and oxidative stress. Increases in RNS production result from excess or deregulating nitric oxide (NO) reacting with reactive oxygen species (ROS). NO, as a signal molecule, is generated during inflammation by neutrophils and phagocytes, and it reacts with superoxide to generate RNS, including peroxynitrite (ONOO–) and NO itself, which in turn reacts with tyrosyl radicals to add –NO2 or –NO to tyrosine residues, forming 3-nitrotyrosine or 3-nitrosotyrosine residues, respectively. As one of the most important mediators of MPO, nitrotyrosine plays a key role in the process of oxidation seen early in inflammatory conditions including ulcerative colitis and rheumatoid arthritis. One important aspect about the formation of nitrotyrosine that must be noted is that it is not solely generated by peroxynitrite (ONOO), a product of the interaction between NO and O2˙–. MPO can also independently
Oxidative Stress-Induced Posttranslational Modification of Proteins
47
HOOC
OH
NH2
OH Cl
CH2
Cl
Cl
OH CH2 HO
H2N
CH2
COOH
H2N
COOH
CH2 H2N
COOH
HOCl
OH
ROS MPO NO
CH2 H2N ONOO
COOH
HOBr
–
OH
OH
OH
NO2
Br
CH2
CH2 H2N
Br
Br
COOH
H2N
COOH
CH2 H2N
COOH
Fig. 5. Neutrophil-dependent modification of tyrosine residues of proteins.
oxidize nitrite (NO2–), a stable end product of NO metabolism, to form nitrogen dioxide (NO2). NO is also a RNS that in turn can nitrogenate tyrosine (fig. 6). A third pathway utilizes MPO-generated HOCl, which also oxidizes NO2– to nitryl chloride (NO2Cl), which is also a RNS. Several recent investigations validate the importance of nitrotyrosine (table 1). For example, protein immunoprecipitation and Western blotting revealed MnSOD as a target of 3-nitrotyrosine formation in traumatic brain injury [57], adriamycininduced neurotoxicity [58], and Alzheimer’s disease [59]. During inflammatory response, ONOO– reacts with the metal center of MnSOD to generate reactive free radicals. The production of such nitrating agents, which is close to Tyr34 in the active site of the enzyme, facilitates site-specific nitration [60, 61]. In addition to the transitional metal centers, protein tyrosine nitration is affected by several conditions: the proximity to the site of nitrating agent generation, the acidity, the near presence of amino acids (tryptophan, cysteine, methionine) that compete for nitrating agents, and the location of tyrosine residue [62]. Heme oxygenase 1 (HO-1) protein may be one of target of ONOO–. The HO system is the rate-limiting enzymatic step that catalyzes the breakdown of heme into equimolar amounts of biliverdin, an antioxidant rapidly converted to bilirubin, and carbon monoxide, an antiapoptotic vasodilator, with the release of its iron moiety. Kruger et al. [63] have demonstrated that ONOO– generates 2
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Classical pathway NO˙ ONOO– NO2˙
O2˙–
OH NO2
MPO-dependent pathway H2O2
MPO
NO2– NO2Cl
HOCl
H2N
CH2 COOH 3-Nitrotyrosine
MPO NO˙
NO2–
NO2˙
Fig. 6. Formation of nitrotyrosine by the classical and MPO-dependent pathway.
3-nitrotyrosine within an HO-1 and HO-1 immunoprecipitate and decreases HO activity, supporting their hypothesis of HO-1 inactivation by ONOO–. Serum levels of nitrotyrosine may be a good stable marker for NO stress in vivo. In a clinical field, Musso et al. [64] have compared the serum nitrotyrosine levels and many clinical parameters in 64 nonobese nondiabetic patients with nonalcoholic fatty liver disease (NAFLD) and 74 control subjects. Persons with NAFLD had greater systemic nitrosative stress than did control subjects, but the two groups did not differ significantly in any other features. Nitrotyrosine and adiponectin concentrations and vitamin A intakes independently predicted alanine aminotransferase concentrations in NAFLD patients and liver histology in a subgroup of 29 subjects with biopsy-proven nonalcoholic steatohepatitis. Devaraj et al. [65] have demonstrated that γ-tocopherol supplementation alone and in combination with α-tocopherol alters biomarkers of oxidative stress, especially nitrotyrosine in serum, and inflammation in subjects with metabolic syndrome. These data may indicate the need to test γ-tocopherol in prospective clinical trials to elucidate its utility in cardiovascular disease prevention, although prospective clinical trials with α-tocopherol have not yielded positive results.
Conclusion
Identification of OPTM proteins in lifestyle diseases allows one to determine which proteins are more affected by oxidation in these diseases and, consequently, more prone to inactivation, and thus represents a significant step in linking well-established inflammation, degeneration, or carcinogenesis with oxidative events at a protein level.
Oxidative Stress-Induced Posttranslational Modification of Proteins
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Table 1. Target proteins of 3-nitrotyrosine formation Cell/tissue
Target proteins
Tyrosine number
Function
First author
Cancer cells/ tissue
tissue inhibitor of MMP-4
Y114, Y195, Y188, Y190
enzyme inactivation
Donnini, 2008
Muscle
carbonic anhydrase III
aging
Chen, 2008
Retina
TrkA receptor
impairment of NGF signaling
Ali, 2008
Brain
MnSOD
decrease in mitochondrial respiration
Tangpong, 2008; Bayir, 2007; Anantharaman, 2006; Pittman, 2002
Brain
ferric-human neuroglobin
scavenge/ neuroprotection
Nicolis, 2007
Muscle
desmin
neurodegeneration
Janue, 2007
Mesothelioma
HIF-1α, p53
enzyme inactivation Thomas, 2006
Heart/tissue
human myoglobin
Y103, Y146
scavenge/little effect on enzyme
Macrophage
Indoleamine 2,3-dioxygenase
Y15 (Y345,Y353)
enzyme inactivation Fujigaki, 2006
Aorta
heme oxygenase-1
PC12 cell
α-tubulin
Y161, Y357
neuronal differentiation
Recombinant protein
cytochrome P450 2B1
Y190
enzyme inactivation Lin, 2005, 2003
Muscle
creatine kinase
Y82, (Y14, Y20)
physiological?
Kanski, 2005
Muscle
α-enolase
Y43
interference of phosphorylation
Casoni, 2005
Cardiac myocyte
α-actinin
contractile dysfunction
Borbely, 2005
Atheroma
apolipoprotein A-I
proatherogenic HDL Zheng, 2004
Lung
MnSOD
enzyme inactivation Gray, 2004
Macrophage
iron regulatory protein-1
self-protecting
Gonzalez, 2004
Heart
carnitine palmitoyl transferase I
endotoxin toxicity
Fukumoto, 2004
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enzyme inactivation Kruger, 2006 Tedeschi, 2005
Naito · Yoshikawa
Table 1. Continued Cell/tissue
Target proteins
Hippocampus
Tyrosine number
Function
First author
synaptophysin
impairment of ACh release
Tran, 2003
Blood
hemoglobin
major target of peroxynitrite in blood
Pietraforte, 2003
Brain
Na,K-ATPase
undefined marker
Golden, 2003
PC12 cell
α-tubulin
NGF-induced differentiation
Cappelletti, 2003
Isolated protein
glutathione S-transferases
enzyme inactivation Wong, 2001
Heart
myofibrillar creatine kinase
heart failure
Mihm, 2001
Glioma cell
p53
loss of p53 DNA binding ability
Cobbs, 2001
Endothelial cell
extracellular matrix
endothelial transcytosis of MPO
Baldus, 2001
Blood
oxyhemoglobin
physiological scavenger
Minetti, 2000
Pancreatic cancer
c-Src kinase
enhanced tyrosine kinase signaling
MacMillan-Crow, 2000
Mitochondria
cytochrome c
changes in redox properties
Cassina, 2000
Yeast
GAPDH
enhancement of chaperone expression and ubiquitination
Buchczyk, 2000
Neuroblastoma
p130cas
toxicity target
Saeki, 1999
Y67
NGF = Nerve growth factor; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; HDL = high-density lipoprotein; MnSOD = manganese superoxide dismutase; MMP = matrix metalloproteinase; HIF-1α = hypoxiainducible factor 1α.
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14 Park YS, Misonou Y, Fujiwara N, et al: Induction of thioredoxin reductase as an adaptive response to acrolein in human umbilical vein endothelial cells. Biochem Biophys Res Commun 2005;327:1058– 1065. 15 Park YS, Koh YH, Takahashi M, et al: Identification of the binding site of methylglyoxal on glutathione peroxidase: methylglyoxal inhibits glutathione peroxidase activity via binding to glutathione binding sites Arg 184 and 185. Free Radic Res 2003;37:205– 211. 16 Carbone DL, Doorn JA, Kiebler Z, et al: Modification of heat shock protein 90 by 4-hydroxynonenal in a rat model of chronic alcoholic liver disease. J Pharmacol Exp Ther 2005;315:8–15. 17 Ahsan H, Ali A, Ali R: Oxygen free radicals and systemic autoimmunity. Clin Exp Immunol 2003;131: 398–404. 18 Kovacic P, Jacintho JD: Systemic lupus erythematosus and other autoimmune diseases from endogenous and exogenous agents: unifying theme of oxidative stress. Mini Rev Med Chem 2003;3:568– 575. 19 Toyoda K, Nagae R, Akagawa M, et al: Proteinbound 4-hydroxy-2-nonenal: an endogenous triggering antigen of antI-DNA response. J Biol Chem 2007;282:25769–25778. 20 Chang MK, Binder CJ, Miller YI, et al: Apoptotic cells with oxidation-specific epitopes are immunogenic and proinflammatory. J Exp Med 2004;200: 1359–1370. 21 Chou MY, Hartvigsen K, Hansen LF, et al: Oxidation-specific epitopes are important targets of innate immunity. J Intern Med 2008;263:479–488. 22 Trigwell SM, Radford PM, Page SR et al: Islet glutamic acid decarboxylase modified by reactive oxygen species is recognized by antibodies from patients with type 1 diabetes mellitus. Clin Exp Immunol 2001;126:242–249. 23 Ishii T, Sakurai T, Usami H, et al: Oxidative modification of proteasome: identification of an oxidationsensitive subunit in 26 S proteasome. Biochemistry 2005;44:13893–13901. 24 Saitoh T, Fujita N, Jang MH et al: Loss of the autophagy protein Atg16L1 enhances endotoxininduced IL-1beta production. Nature 2008;456:264– 268. 25 Uchida K: Protein-bound 4-hydroxy-2-nonenal as a marker of oxidative stres. J Clin Biochem Nutr 2005;36:1–10. 26 Toyokuni S, Miyake N, Hiai H, et al: The monoclonal antibody specific for the 4-hydroxy-2-nonenal histidine adduct. FEBS Lett 1995;359:189–191.
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27 Toyokuni S, Uchida K, Okamoto K, et al: Formation of 4-hydroxy-2-nonenal-modified proteins in the renal proximal tubules of rats treated with a renal carcinogen, ferric nitrilotriacetate. Proc Natl Acad Sci USA 1994;91:2616–2620. 28 Ihara Y, Toyokuni S, Uchida K, et al: Hyperglycemia causes oxidative stress in pancreatic beta-cells of GK rats, a model of type 2 diabetes. Diabetes 1999;48:927–932. 29 Yamagami K, Yamamoto Y, Kume M, et al: Formation of 8-hydroxy-2⬘-deoxyguanosine and 4-hydroxy-2-nonenal-modified proteins in rat liver after ischemia-reperfusion: distinct localization of the two oxidatively modified products. Antioxid Redox Signal 2000;2:127–136. 30 Hartley DP, Kroll DJ, Petersen DR: Prooxidantinitiated lipid peroxidation in isolated rat hepatocytes: detection of 4-hydroxynonenal- and malondialdehyde-protein adducts. Chem Res Toxicol 1997;10:895–905. 31 Blasig IE, Grune T, Schonheit K, et al: 4-Hydroxynonenal, a novel indicator of lipid peroxidation for reperfusion injury of the myocardium. Am J Physiol 1995;269:H14–H22. 32 Eschwege P, Paradis V, Conti M, et al: In situ detection of lipid peroxidation by-products as markers of renal ischemia injuries in rat kidneys. J Urol 1999; 162:553–557. 33 Nair J, Gansauge F, Beger H, et al: Increased ethenoDNA adducts in affected tissues of patients suffering from Crohn’s disease, ulcerative colitis, and chronic pancreatitis. Antioxid Redox Signal 2006;8: 1003–1010. 34 Kageyama F, Kobayashi Y, Kawasaki T, et al: Successful interferon therapy reverses enhanced hepatic iron accumulation and lipid peroxidation in chronic hepatitis C. Am J Gastroenterol 2000;95: 1041–1050. 35 Serviddio G, Bellanti F, Tamborra R, et al: Uncoupling protein-2 (UCP2) induces mitochondrial proton leak and increases susceptibility of non-alcoholic steatohepatitis (NASH) liver to ischaemia-reperfusion injury. Gut 2008;57:957– 965. 36 Uchida K, Toyokuni S, Nishikawa K, et al: Michael addition-type 4-hydroxy-2-nonenal adducts in modified low-density lipoproteins: markers for atherosclerosis. Biochemistry 1994;33:12487–12494. 37 Reed T, Perluigi M, Sultana R, et al: Redox proteomic identification of 4-hydroxy-2-nonenal-modified brain proteins in amnestic mild cognitive impairment: insight into the role of lipid peroxidation in the progression and pathogenesis of Alzheimer’s disease. Neurobiol Dis 2008;30:107–120.
38 Selley ML: (E)-4-hydroxy-2-nonenal may be involved in the pathogenesis of Parkinson’s disease. Free Radic Biol Med 1998;25:169–174. 39 Perluigi M, Fai Poon H, Hensley K, et al: Proteomic analysis of 4-hydroxy-2-nonenal-modified proteins in G93A-SOD1 transgenic mice–a model of familial amyotrophic lateral sclerosis. Free Radic Biol Med 2005;38:960–968. 40 Aoi W, Naito Y, Sakuma K, et al: Astaxanthin limits exercise-induced skeletal and cardiac muscle damage in mice. Antioxid Redox Signal 2003;5:139– 144. 41 Manabe E, Handa O, Naito Y, et al: Astaxanthin protects mesangial cells from hyperglycemiainduced oxidative signaling. J Cell Biochem 2008; 103:1925–1937. 42 Naito Y, Uchiyama K, Aoi W, et al: Prevention of diabetic nephropathy by treatment with astaxanthin in diabetic db/db mice. Biofactors (Oxford) 2004; 20:49–59. 43 Kato Y, Mori Y, Makino Y, et al: Formation of Nepsilon-(hexanonyl)lysine in protein exposed to lipid hydroperoxide. A plausible marker for lipid hydroperoxide-derived protein modification. J Biol Chem 1999;274:20406–20414. 44 Kato Y, Miyake Y, Yamamoto K, et al: Preparation of a monoclonal antibody to N(epsilon)-(Hexanonyl) lysine: application to the evaluation of protective effects of flavonoid supplementation against exercise-induced oxidative stress in rat skeletal muscle. Biochem Biophys Res Commun 2000;274: 389–393. 45 Fukuchi Y, Miura Y, Nabeno Y, et al: Immunohistochemical detection of oxidative stress biomarkers, dityrosine and N(epsilon)-(hexanoyl)lysine, and C-reactive protein in rabbit atherosclerotic lesions. J Atheroscler Thromb 2008;15:185–192. 46 Osakabe N, Yasuda A, Natsume M, et al: Rosmarinic acid, a major polyphenolic component of Perilla frutescens, reduces lipopolysaccharide (LPS)induced liver injury in d-galactosamine (d-GalN)sensitized mice. Free Radic Biol Med 2002;33: 798–806. 47 Yasuda A, Takano H, Osakabe N, et al: Cacao liquor proanthocyanidins inhibit lung injury induced by diesel exhaust particles. Int J Immunopathol Pharmacol 2008;21:279–288. 48 Aoi W, Naito Y, Takanami Y, et al: Astaxanthin improves muscle lipid metabolism in exercise via inhibitory effect of oxidative CPT I modification. Biochem Biophys Res Commun 2008;366:892–897. 49 Naito Y, Takano H, Yoshikawa T: Oxidative stressrelated molecules as a therapeutic target for inflammatory and allergic diseases. Curr Drug Targets 2005;4:511–515.
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50 Winterbourn CC, Kettle AJ: Biomarkers of myeloperoxidase-derived hypochlorous acid. Free Radic Biol Med 2000;29:403–409. 51 Gujral JS, Hinson JA, Jaeschke H: Chlorotyrosine protein adducts are reliable biomarkers of neutrophil-induced cytotoxicity in vivo. Comp Hepatol 2004;3(suppl 1):S48. 52 Ryu JK, Tran KC, McLarnon JG: Depletion of neutrophils reduces neuronal degeneration and inflammatory responses induced by quinolinic acid in vivo. Glia 2007;55:439–451. 53 Heinecke JW: The HDL proteome: a marker – and perhaps mediator – of coronary artery disease. J Lipid Res 2008; Epub ahead of print. 54 Shao B, Oda MN, Bergt C, et al: Myeloperoxidase impairs ABCA1-dependent cholesterol efflux through methionine oxidation and site-specific tyrosine chlorination of apolipoprotein A-I. J Biol Chem 2006;281:9001–9004. 55 Kunitomo M, Yamaguchi Y, Kagota S, et al: Beneficial effect of coenzyme Q10 on increased oxidative and nitrative stress and inflammation and individual metabolic components developing in a rat model of metabolic syndrome. J Pharmacol Sci 2008;107:128–137. 56 Yamaguchi Y, Yamada K, Yoshikawa N, et al: Corosolic acid prevents oxidative stress, inflammation and hypertension in SHR/NDmcr-cp rats, a model of metabolic syndrome. Life Sci 2006;79: 2474–2479. 57 Bayir H, Kagan VE, Clark RS, et al: Neuronal NOSmediated nitration and inactivation of manganese superoxide dismutase in brain after experimental and human brain injury. J Neurochem 2007;101:168– 181. 58 Tangpong J, Cole MP, Sultana R, et al: Adriamycinmediated nitration of manganese superoxide dismutase in the central nervous system: insight into the mechanism of chemobrain. J Neurochem 2007; 100:191–201.
59 Anantharaman M, Tangpong J, Keller JN, et al: Beta-amyloid mediated nitration of manganese superoxide dismutase: implication for oxidative stress in a APPNLH/NLH X PS-1P264L/P264L double knock-in mouse model of Alzheimer’s disease. Am J Pathol 2006;168:1608–1618. 60 MacMillan-Crow LA, Crow JP, Thompson JA: Peroxynitrite-mediated inactivation of manganese superoxide dismutase involves nitration and oxidation of critical tyrosine residues. Biochemistry 1998; 37:1613–1622. 61 Yamakura F, Taka H, Fujimura T, et al: Inactivation of human manganese-superoxide dismutase by peroxynitrite is caused by exclusive nitration of tyrosine 34 to 3-nitrotyrosine. J Biol Chem 1998;273:14085– 14089. 62 Yeo WS, Lee SJ, Lee JR, et al: Nitrosative protein tyrosine modifications: biochemistry and functional significance. BMB Rep 2008;41:194–203. 63 Kruger AL, Peterson SJ, Schwartzman ML, et al: Up-regulation of heme oxygenase provides vascular protection in an animal model of diabetes through its antioxidant and antiapoptotic effects. J Pharmacol Exp Ther 2006;319:1144–1152. 64 Musso G, Gambino R, De Michieli F, et al: Nitrosative stress predicts the presence and severity of nonalcoholic fatty liver at different stages of the development of insulin resistance and metabolic syndrome: possible role of vitamin A intake. Am J Clin Nutr 2007;86:661–671. 65 Devaraj S, Leonard S, Traber MG, et al: Gammatocopherol supplementation alone and in combination with alpha-tocopherol alters biomarkers of oxidative stress and inflammation in subjects with metabolic syndrome. Free Radic Biol Med 2008; 44: 1203–1208.
Professor Yuji Naito Molecular Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine Kamigyo-ku Kyoto 602-8566 (Japan) Tel. +81 75 251 5519, Fax +81 75 251 0710, E-Mail
[email protected]
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Bioavailability and Safety Yoshikawa T (ed): Food Factors for Health Promotion. Forum Nutr. Basel, Karger, 2009, vol 61, pp 55–63
Absorption and Function of Dietary Carotenoids Akihiko Nagao National Food Research Institute, National Agriculture and Food Research Organization, Tsukuba, Japan
Abstract Carotenoids are highly hydrophobic pigments with yellow to red color and their major dietary sources are fruits and vegetables. They have an essential physiological function as a vitamin A precursor and also have antioxidant, anticancer, immune enhancement and antiobesity activities related to prevention of degenerative diseases. The release of carotenoids from food matrix, their dispersion within the digestive tract, and their solubilization in mixed micelles are important steps for carotenoid bioaccessibility. Solubilized carotenoids are taken up by epithelial cells of the small intestine by simple diffusion and/or transporter-mediated processes and then secreted to lymph as chylomicron. Carotenoids accumulated in tissues are thought to be metabolized to small molecules by enzymatic cleavage and/or chemical oxidation with active oxygen species at conjugated double bonds. The hydroxyl group of xanthophylls can be oxidatively metabolized to carbonyl group. Carotenoids with long chain of conjugated double bonds physically quench singlet oxygen and scavenge oxygen radicals, particularly under low oxygen pressure, and thereby they have been thought to work as lipophilic antioxidants for human health. In addition to antioxidant activities, each carotenoid has characteristic functions such as cell cycle inhibition, induction of cell differentiation and apoptosis, and enhancement of gap-junctional communication. However, the detailed mechanisms of these biological actions have not been fully revealed yet and deserve future studies. Copyright © 2009 S. Karger AG, Basel
Carotenoid usually consists of eight units of isoprene and has a symmetrical skeleton of 40 carbon atoms with a long chain of conjugated carbon double bonds (fig. 1). The conjugated double bonds endow carotenoid molecules with a characteristic yellow to red color and antioxidant activities. They are synthesized by plants and microorganisms, and can be found at high concentration in photosynthetic organelle. Animals accumulate carotenoids in their tissues through dietary intake of carotenoids. Carotenoids play essential physiological roles in harvesting light energy and preventing oxidative stress in photosynthetic tissues. Metabolites of carotenoids such as abscisic acid and trisporic acid work as hormones in plants and fungi. In vertebrates,
-Carotene
␣-Carotene
Lycopene
HO -Cryptoxanthin OH
HO Lutein
Fig. 1. Major carotenoids present in human plasma.
some carotenoids are the precursors of vitamin A, which is involved in cell differentiation, growth and vision. In addition to these essential physiological roles, carotenoids have been thought to have beneficial effects on human health due to their biological actions [1]. Many epidemiological studies have shown that intake of carotenoid-rich fruits and vegetables or carotenoid level in serum is negatively correlated with incidence of degenerative diseases. Animal studies as well as in vitro studies indicate that carotenoids have antioxidant, anticancer, immune-enhancing activities. In this chapter, recent studies on intestinal absorption of dietary carotenoids, their metabolism and functions are described.
Bioaccessibility and Intestinal Absorption
Bioavailability of dietary carotenoids is much lower than that of triacylglycerols. Solubilization in gastrointestinal tract and absorption by epithelial cells of small
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Fruits and vegetables Release from food matrix
Emulsion Fats and oils
Epithelial cells Lipolytic enzymes
Phosphatidylcholine Bile acids
Mixed micelles
Chylomicron
Solubilization Intestinal absorption
Fig. 2. Absorption of dietary carotenoids.
intestine largely affect bioavailability of carotenoids. Dietary carotenoids are solubilized as follows: release from food matrix, dispersion in gastrointesinal tract, and subsequent solubilization in mixed micelles. Solubilized carotenoids are taken up by the epithelial cells and secreted to lymph after incorporation into the chylomicron (fig. 2) [2]. Mechanical destruction of food matrix and heating during cooking and processing enhance the release of carotenoids from foods. In particular, carotenoids in vegetables are hard to release because they have rigid cell walls. For example, ingestion of tomato paste showed 2.5-fold higher lycopene concentration in human plasma than that of fresh tomato [3]. After carotenoids are released from food matrices, they interact with digestive fluids in gastrointestinal tract. As carotenoids are insoluble in aqueous medium due to their high hydrophobicity, their dispersion in digestive tract is attained by emulsification with dietary lipids and bile containing phospholipids and bile acids. After hydrolysis of dietary and biliary lipids, carotenoids are solubilized in mixed micelles, which consist of bile acid, fatty acid, monoacylglycerol, cholesterol and phospholipid. The mixed micelle is a disc-like particle, with a diameter of 40–200 nm. Carotenoids solubilized in the micelles are thought to be accessible to epithelial cells of the small intestine. Thus, the ratio of the amount of carotenoids in the mixed micelles to that present in food ingested represents the bioaccessibility.
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Dietary fats and oils are involved in the carotenoid solubilization processes as described above. Firstly, they facilitate dispersion of carotenoids during cooking and gastrointestinal digestion, although solubility of carotenoids in fats and oils is limited: 0.112–0.141% in case of β-carotene [4]. Solubilization of spinach β-carotene (ca. 4 mg/100 g fresh weight) needs at least 2.8–3.6 g oil/100 g fresh weight. Secondarily, hydrolysis products of fats and oils serve as components of the mixed micelles. Moreover, intake of fats and oils induces the secretion of bile and pancreatic lipases, which are essential for the formation of the mixed micelles. Thus, dietary fats and oils enhance the bioaccessibility of dietary carotenoids. After solubilization in the mixed micelles, carotenoids become accessible to the epithelial cells of the jejunum. Carotenoid transfer from the mixed micelles to the epithelial cells has been thought to occur by simple diffusion across the phospholipid bilayers of the cytoplasmic membrane. Uptake of 14C-labelled β-carotene by epithelial cells was linearly proportional to its extracellular concentration and independent of temperature. The incubation with excess unlabelled β-carotene did not inhibit the uptake [5]. Uptake of various carotenoids from the mixed micelles by Caco-2 human intestinal cells was correlated with their lipophilicity [6]. Lysophosphatidylcholine in the mixed micelles enhanced the carotenoid uptake by Caco-2 cells, suggesting that the physical perturbation of membrane integrity by lysophosphatidylcholine favors the transfer of carotenoids across the plasma membrane [7]. These results support the simple diffusion mechanism for carotenoid uptake by the epithelial cells of the intestine. However, recent studies have suggested that transporters are involved in intestinal absorption of carotenoids. Plasma lycopene level in mice overexpressing scavenger receptor class B type I (SR-B1) increased 10-fold in comparison with wildtype mice [8]. Treatment of Caco-2 cells with anti-SR-B1 antibody and an SR-B1 inhibitor partially inhibited the cellular uptake of lycopene and lutein. SR-B1 knockout mouse showed lower intestinal absorption of β-carotene than wild-type mouse [9]. However, impairment of SR-B1 did not completely block carotenoid absorption. Therefore, it is most likely that one part of carotenoids is absorbed by simple diffusion and the other part by transporter-mediated mechanism through SR-B1 as well as other unknown transporters. After uptake of carotenoids by the epithelial cells, intracelluar trafficking of carotenoids and incorporation into the chylomicron occur before secretion to lymph. The chylomicron is assembled with apolipoproteins, phospholipids and triacylglycerols resynthesized from digests of fats and oils. Therefore, dietary fats and oils would affect the secretion of carotenoids to lymph as well as the solubilization process described above. However, details of secretion process after carotenoid uptake by epithelial cells has not been clarified yet. It has been well known that accumulation of carotenoids in tissues is largely varied among mammal species. Humans accumulate both carotenes and xanthophylls, whereas cows accumulate only carotenes and rodents accumulate little carotenoids. It has not been revealed yet whether these variations are due to the differences in intestinal absorption or metabolism in the body.
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Metabolic Transformation
Provitamin A carotenoids are oxidized at the central double bond to two molecules of retinal by 15,15⬘-dioxygenase in the intestinal epithelium [10]. Retinal is reduced to retinol and then converted to retinyl esters, which are transported to the liver after incorporation into the chylomicron. The dioxygenase is present in several tissues other than the intestine and would work to produce vitamin A directly from carotenoids accumulated in the peripheral tissues. Recently, a gene encoding another dioxygenase which can oxidize 9⬘,10⬘-double bond of carotenoids was discovered [11]. The enzyme expressed in Escherichia coli converted lycopene as well as provitamin A carotenoids to the corresponding carbonyl compounds. However, in vivo formation of metabolites derived by this asymmetric cleavage enzyme reaction has not been confirmed yet. In addition to these enzymatic cleavage reactions, chemical oxidation of carotenoids in vitro are known to produce carbonyl compounds, which are produced by cleavage at any position of conjugated double bonds [12]. Thus, cleavage reaction might occur in vivo whether it is mediated by enzyme or reactive oxygen species. Oxidation products of astaxanthin, which might be formed by cleavage at 9, 10-double bond, were found in the human plasma after intake of astaxanthin [13]. Moreover, 3-hydroxy-β-ionone and 3-hydroxy-14⬘-apocarotenal were detected as cleavage products of zeaxanthin in the human macula. These cleavage products found in human tissues indicate that the cleavage reaction would occur in vivo. Various carotenoid metabolites have been found in human plasma and breast milk. Khachik et al. [14] found 34 carotenoids including geometrical isomers, among which 9 were thought to be metabolites because they were not present in foods. 2,6-cyclolycopene-1,5-diol was identified as a metabolite of lycopene and suggested to be formed via 2,6-cyclolycopene-1,5-epoxide from lycopene-1,2-epoxide, an oxidation product of lycopene, by enzymatic or nonenzymatic hydrolysis [14]. One of the major metabolites of lutein, anhydrolutein, was found in human plasma and thought to be formed by dehydration under acidic condition in the stomach. Other metabolites of lutein in human plasma were 3-hydroxy-β,ε-carotene-3⬘-one, 3⬘-hydroxy-ε,ε-carotene-3-one, ε,ε-carotene-3,3⬘-dione, and 3⬘-epilutein [15]. These metabolites as well as zeaxanthin were thought to be formed from lutein by repeated oxidation, reduction and isomerization. Moreover, canthaxanthin and capsanthone were found in human plasma after intake of 4, 4’-dimethoxy-β-carotene [16] and capsanthin [17], respectively. Amarouciaxanthin A was found in plasma and liver of mouse fed fucoxanthin [18], a major xanthophyll in brown algae. Fucoxanthin was hydrolyzed to fucoxanthinol, and then oxidatively converted to amarouciaxanthin A by NAD-dependent dehydrogenase present in liver microsomes. These results suggest that mammals have a metabolic activity to oxidize the secondary hydroxyl group to carbonyl group in xanthophylls. Crocetin, a C20 apocarotenoid, is present in saffron stigmas and gardenia fruits and has been used as yellow food colorings. It has a characteristic structure with carboxyl
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groups at both ends of the carbon chain, which is shorter than usual carotenoids. In mice, crocetin was rapidly absorbed into the blood circulation and metabolized to glucuronide conjugates, which would be excreted to urine [19]. This metabolism suggests that carotenoids are finally eliminated from the body as glucuronide conjugates after cleavage to apocarotenoids. Thus, in mammals, carotenoids are transformed to various derivatives with C40 carbon chain as well as smaller molecules. However, biological actions of these metabolites remain largely unknown except for vitamin A.
Functions
Provitamin A carotenoid present in foods is an important source of vitamin A, which plays essential roles in cell differentiation, growth and vision. This physiological function is not described here, because it is beyond the scope of this chapter. Most carotenoids have a long chain of conjugated double bonds, which endow these molecules with antioxidant activities. Carotenoids that have more than 9 conjugated double bonds can quench singlet oxygen by physically receiving its energy, and then, the excited carotenoids turn to ground state by releasing energy as heat. Thus, one molecule of the carotenoids can quench singlet oxygen repeatedly [20]. The reactivity of the carotenoids with singlet oxygen is much higher than those of vitamin E and C, which quench singlet oxygen by chemical reaction. Therefore, carotenoids are excellent singlet oxygen quenchers among dietary antioxidants and have been thought to protect tissues from singlet oxygen, which is generated in skin exposed to UV irradiation through the endogenous photosensitizers and in macrophage phagocytosis through myeloperoxidase reaction. Carotenoids work as antioxidants by scavenging oxygen radicals as well as quenching singlet oxygen. Lipid peroxy radicals make a stable adduct to conjugated double bonds of carotenoids. Under the low oxygen pressure, the carbon radical adduct is stabilized by the conjugation system. However, reactivity of carotenoids with lipid peroxy radicals is lower than that of vitamin E, which is present in tissues at higher concentration than carotenoids. Therefore, carotenoid is not a major antioxidant to scavenge oxygen radicals in lipid peroxidation in vivo. On the other hand, carotenoid was found to scavenge peroxynitrite in isolated LDL, suggesting the protection of blood lipoproteins from peroxynitrite and prevention of atherosclerosis initiation by carotenoids [21]. These antioxidant properties of carotenoids as well as vitamin E and C are thought to prevent oxidative lesions which would lead to cancer and cardiovascular diseases. In addition to antioxidant and provitamin A activities, individual carotenoids have been reported to have various biological activities. In animal models, β-carotene, α-carotene, lycopene, β-cryptoxanthin and lutein have been reported to suppress carcinogenesis of the liver, colon, skin, and lung initiated with chemicals. Feeding rats with β-carotene reduced the number of aberrant crypt foci and incidence of colon cancer induced by azoxymethane.
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In the cell culture system, carotenoids have been reported to suppress propagation of cancer cells by cell cycle inhibition, induction of differentiation and apoptosis, and enhancement of gap-junctional communication, since Murakosi et al. [22] first reported that α-carotene suppressed the propagation of human neuroblastoma cells in 1989. Lycopene inhibited cell growth of androgen-independent human prostate cancer cells at the G0/G1 phase and of normal epithelial cells of prostate by suppressing expression of cyclin D [23]. These biological activities of lycopene would support the epidemiological observation that lycopene intake is associated with lowered incidence of prostate cancer. β-Carotene inhibited propagation of several colon cancer cells and the inhibition might be mediated by suppression of cyclooxygenase 2 expression and formation of prostaglandin E2. Crocetin, crocin, lutein, β-carotene, and lycopene inhibited propagation of human promyelocytic leukemia cells by inducing cell differentiation. Certain carotenoids cause apoptosis induction, which is an important function in prevention and treatment of cancer. In 1995, Muto et al. [24] first reported that β-carotene inhibited cell growth of human cervical dysplastic cells by inducing apoptosis, which is mediated by suppression of EGF receptor protein level. β-Carotene as well as retinoic acid induced apoptosis in DU145 human prostate cancer cells, and lycopene at high concentration induced apoptosis in LNCaP human prostate cancer cells by enhancing permeability of the mitochondrial membrane. Indeed, intake of tomato products containing lycopene by subjects of prostate cancer and hyperplasia increase the ratio of apoptotic cells in the prostate [25]. Polar xanthophylls, neoxanthin and fucoxanthin, which have a characteristic allenic bond and are present in green leafy vegetables and edible brown algae, respectively, also induced apoptosis of human prostate cancer cells as well as colon cancer cells [26]. Gap-junctional communication among cells is involved in maintaining tissue homeostasis. In cancer tissues, cell propagation is not properly regulated due to the reduced communication caused by a decreased gap junction. β-Carotene, canthaxanthin, lutein, lycopene and retinoic acid suppressed transformation of mouse fibroblast C3H/10T1/2 treated with methylcholanthrene by increasing gap-junctional communication [27], which was mediated by increased expression of connexin 43 encoding a gap-junction protein. The increased gap junction was also found in the liver of rats fed α-carotene, β-carotene, and lycopene. In humans, mRNA level of connexin 43 increased in the colon mucosa of subjects who ingested β-carotene. Immune enhancement by carotenoid intake has been thought to be an important biological activity in cancer prevention, in particular, for aged people, who have less immune ability and high risk of cancer. Intake of β-carotene by aged people for a long period enhanced activity of natural killer cells engaged in cell-mediated immune response [28]. After administration of β-carotene to nonsmoking healthy men, monocytes expressing MHC class II (HLA-DR) and adhesion molecules (ICAM-1 and LFA-3) increased, and secretion of TNF-α by the isolated monocytes was enhanced, indicating enhancement in antigen presentation [29]. As lycopene,
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lutein, canthaxanthin, and astaxanthin as well as β-carotene have been reported to show immune enhancement, it is not attributed to vitamin A activity, but to intrinsic properties of each carotenoid. The detailed mechanism of immune enhancement by carotenoids has not been revealed yet. The carotenoids as antioxidants are thought to protect immune cells, which are vulnerable to oxidative stress, and to modulate formation of arachidonate metabolites, which suppress immune functions. In addition to immune enhancement, carotenoids may have suppressive effect on allergic reaction. Simultaneous intake of vitamin E and β-carotene was reported to suppress IgE production in mice [30]. Carotenoids have been reported to show several biological activities other than those mentioned above. Inhibition of platelet aggregation, growth inhibition of human aortic smooth muscle cells, and suppression of adhesion molecule expression in human aortic endothelial cells were reported. These effects as well as antioxidant activity of carotenoids are thought to be involved in prevention of cardiovascular diseases. In a recent study, fucoxanthin was found to reduce fat accumulation in mice. Thus, carotenoids showed various biological activities and are expected to have beneficial effects on human health. However, the exact mechanisms have not been fully revealed and deserve future studies.
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8 Moussa M, Landrier JF, Reboul E, Ghiringhelli O, Comera C, Collet X, Frohlich K, Bohm V, Borel P: Lycopene absorption in human intestinal cells and in mice involves scavenger receptor class B type I but not Niemann-Pick C1-like 1. J Nutr 2008;138: 1432–1436. 9 van Bennekum A, Werder M, Thuahnai ST, Han CH, Duong P, Williams DL, Wettstein P, Schulthess G, Phillips MC, Hauser H: Class B scavenger receptor-mediated intestinal absorption of dietary carotene and cholesterol. Biochemistry 2005;44: 4517–4525. 10 Nagao A, During A, Hoshino C, Terao J, Olson JA: Stoichiometric conversion of all trans-beta-carotene to retinal by pig intestinal extract. Arch Biochem Biophys 1996;328:57–63. 11 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. 12 Kim SJ, Nara E, Kobayashi H, Terao J, Nagao A: Formation of cleavage products by autoxidation of lycopene. Lipids 2001;36:191–199.
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13 Kistler A, Liechti H, Pichard L, Wolz E, Oesterhelt G, Hayes A, Maurel P: Metabolism and CYP-inducer properties of astaxanthin in man and primary human hepatocytes. Arch Toxicol 2002;75:665–675. 14 Khachik F, Spangler CJ, Smith JC Jr, Canfield LM, Steck A, Pfander H: Identification, quantification, and relative concentrations of carotenoids and their metabolites in human milk and serum. Anal Chem 1997;69:1873–1881. 15 Khachik F, de Moura FF, Zhao DY, Aebischer CP, Bernstein PS: Transformations of selected carotenoids in plasma, liver, and ocular tissues of humans and in nonprimate animal models. Invest Ophthalmol Vis Sci 2002;43:3383–3392. 16 Zeng S, Furr HC, Olson JA: Metabolism of carotenoid analogs in humans. Am J Clin Nutr 1992;56: 433–439. 17 Etoh H, Utsunomiya Y, Komori A, Murakami Y, Oshima S, Inakuma T: Carotenoids in human blood plasma after ingesting paprika juice. Biosci Biotechnol Biochem 2000;64:1096–1098. 18 Asai A, Sugawara T, Ono H, Nagao A: Biotransformation of fucoxanthinol into amarouciaxanthin A in mice and HepG2 cells: formation and cytotoxicity of fucoxanthin metabolites. Drug Metab Dispos 2004;32:205–211. 19 Asai A, Nakano T, Takahashi M, Nagao A: Orally administered crocetin and crocins are absorbed into blood plasma as crocetin and its glucuronide conjugates in mice. J Agric Food Chem 2005;53:7302– 7306. 20 Di Mascio P, Kaiser S, Sies H: Lycopene as the most efficient biological carotenoid singlet oxygen quencher. Arch Biochem Biophys 1989;274:532– 538. 21 Panasenko OM, Sharov VS, Briviba K, Sies H: Interaction of peroxynitrite with carotenoids in human low density lipoproteins. Arch Biochem Biophys 2000;373:302–305. 22 Murakoshi M, Takayasu J, Kimura O, Kohmura E, Nishino H, Iwashima A, Okuzumi J, Sakai T, Sugimoto T, Imanishi J, Iwasaki R: Inhibitory effects of alpha-carotene on proliferation of the human neuroblastoma cell line GOTO. J Natl Cancer Inst 1989;81:1649–1652.
23 Obermuller-Jevic UC, Olano-Martin E, Corbacho AM, Eiserich JP, van der Vliet A, Valacchi G, Cross CE, Packer L: Lycopene inhibits the growth of normal human prostate epithelial cells in vitro. J Nutr 2003;133:3356–3360. 24 Muto Y, Fujii J, Shidoji Y, Moriwaki H, Kawaguchi T, Noda T: Growth retardation in human cervical dysplasia-derived cell lines by beta-carotene through down-regulation of epidermal growth factor receptor. Am J Clin Nutr 1995;62:1535S-1540S. 25 Kim HS, Bowen P, Chen LW, Duncan C, Ghosh L, Sharifi R, Christov K: Effects of tomato sauce consumption on apoptotic cell death in prostate benign hyperplasia and carcinoma. Nutr Cancer 2003;47: 40–47. 26 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. 27 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. 28 Santos MS, Meydani SN, Leka L, Wu D, Fotouhi N, Meydani M, Hennekens CH, Gaziano JM: Natural killer cell activity in elderly men is enhanced by beta-carotene supplementation. Am J Clin Nutr 1996;64:772–777. 29 Hughes DA, Wright AJ, Finglas PM, Peerless AC, Bailey AL, Astley SB, Pinder AC, Southon S: The effect of beta-carotene supplementation on the immune function of blood monocytes from healthy male nonsmokers. J Lab Clin Med 1997;129:309– 317. 30 Bando N, Yamanishi R, Terao J: Inhibition of immunoglobulin E production in allergic model mice by supplementation with vitamin E and beta-carotene. Biosci Biotechnol Biochem 2003;67:2176–2182.
Dr. Akihiko Nagao National Food Research institute, National Agriculture and Food Research Organization Kannondai 2–1-12 Tsukuba, Ibaraki, 305-8642 (Japan) Tel. +81 298 838 8039, Fax +81 298 838 7996, E-Mail
[email protected]
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Bioavailability and Safety Yoshikawa T (ed): Food Factors for Health Promotion. Forum Nutr. Basel, Karger, 2009, vol 61, pp 64–74
Metabolism of Flavonoids Yu Wang ⭈ Chi-Tang Ho Department of Food Science, Rutgers University, New Brunswick, N.J., USA
Abstract Flavonoids, the most abundant polyphenolic compounds in foods, can be classified into flavanols, flavones, flavonols, flavanones, isoflavones and anthocyanidins. They have been demonstrated to possess strong antioxidant and disease-preventing properties especially for various degenerative diseases such as cancers and cardiovascular diseases in in vitro and in vivo models. However, flavonoids undergo metabolic transformation such as methylation, sulfation and glucuronidation, and consequently changes of their structures and biological activities. In order to reveal a relationship between parent compounds and their metabolites, the changes of structure and bioactivity of variCopyright © 2009 S. Karger AG, Basel ous flavonoids are reviewed in the vitro and vivo models.
Flavonoids are a class of plant secondary metabolites widely distributed in the leaves, seeds, bark and flowers of plants. They have attracted great deal of attention in the past few years, because they have been demonstrated to occur ubiquitously and play potentially protective roles in human health. Flavonoids are benzo-γ-pyrone derivatives consisting of phenolic and pyrane rings, and are classified into flavanols, flavones, flavonols, flavanones, isoflavones and anthocyanidins. Flavonols are the most abundant flavonoids in foods. Quercetin and kaempferol, which are the major representatives of flavonols, are very rich in onions (up to 1.2 g/kg fresh weight), curly kale, leeks, broccoli and blueberries [1]. Those compounds generally occur in the glycosylated forms, which are preferable to glucose and rhamnose. Compared to flavonols, flavones are found much less in fruits and vegetables, and the most common edible source of flavones is parsley and celery which mainly contain glycosides of luteolin and apigenin. However, polymethoxyflavones (PMF), a general term for flavones, bear four or more methoxy groups. PMFs exist almost exclusively in citrus plants [2]. Isoflavones are one class of phytoestrogens. Due to their structural similarly to estradiol, they can bind to estrogen receptors. The major sources of isoflavones in the human diet are soya and its products such as soy flour, miso, tofu and soy milk which contain three major compounds, genistein, daidzein, and glycitein at a concentration ratio of 1:1:0.2 [3]. Flavanones mostly occur as the glycosylation
with the disaccharides, which may impact bitter taste. However, the aglycones of flavanones could be found such as naringenin in grapefruit, hesperetin in oranges, and eriodictyol in lemons [4]. Flavanols are discussed in their monomers (catechins) and polymers (proanthocyanidins). Apricots contain 250 mg catechins/kg fresh weight and are the fruits containing the highest level of catechins; they are followed by cherries, grapes and apples. However, green tea and chocolate, which contain 800 and 610 mg catechins/kg fresh weight, respectively, are by far its richest sources, particularly gallocatechin (GC), epigallocatechin (EGC), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG) in tea [1, 5, 6]. Compared with other classes of flavonoids, flavanols are not glycosylated in foods. Proanthocyanidins are dimers, oligomers and polymers of catechins which are condensed together at positions 4 and 8 (or 6). Anthocyanins exist in different chemical structures which show various colors according to pH. Aglycones of anthocyanins are sensitive to light, pH or oxygen, but they can be stabilized by glycosation generally with glucose at position 3, or esterification with organic acids and phenolic acids. Anthocyanins are abundant in foods with intensive color such as blackcurrants or blackberries (4,000 mg/kg fresh weight) [7]. Flavonoids have been demonstrated to be protective against degenerative diseases such as cancers, cardiovascular diseases and neurodegenerative disorders in in vitro and in vivo models. Studies show that their beneficial effects are due to their ability to scavenge free radicals, enhance or inhibit some specific enzymes, induce apoptosis in tumor cells, and regulate the immune system. Quercetin has been shown to be a potent antioxidant and anti-inflammatory agent that protects blood vessels, cells and their structures from the harmful effects produced by free radicals. Quercetin also modulates phase 1 and phase 2 enzymes and inhibits azoxymethane-induced colorectal carcinogenesis. Kaempferol causes G2/M arrest and induces apoptosis in human esophageal adenocarcinoma cells, and inhibits STAT1 (signal transducer and activator of transcript 1) and NK-κB (nuclear factor κB) activation in activated macrophages. Green tea catechins have been the most studied health-promoting flavonoids in recent years. Tea is one of the most widely consumed beverages in the world. Green tea and its catechin constituents have been extensively studied both in vitro and in animal models of carcinogenesis. Cyanidin, an anthocyanidin present in cherry and strawberry, exhibited significant decrease in CCl4-induced lipid and protein peroxidation and induced G2/M arrest and apoptosis in U937 cells. Delphinidin, an anthocyanidin present in dark fruit, is believed to contribute to the inhibition of cyclooxygenase-2 expression by blocking mitogen-activated protein kinase signaling and nuclear factor-κB, activator protein-1 and C/EBPδ nuclear translocation. Health benefits of soybean and its products have been recognized in recent years. Genistein, which is an isoflavone, is considered to be the main nutraceutical in soybeans. As a phytoestrogen, genistein lacks estrogenic activity and exhibits antiestrogenic activity. Genistein induced apoptosis by activation of calpain-caspase and ASK-1 signaling pathway in human breast cancer MCF-7 cells [8].
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Considering health benefits of flavonoids, it becomes of importance to understand their metabolism and bioavailability which may provide sufficient information on how these compounds are utilized. Bioavailability is an overall effect of absorption, biotransformation (metabolism), distribution, and excretion. One of the most important factors of bioavailability is metabolism, which is a detoxification process and can be grouped into phase I and phase II enzyme systems. Xenobiotic substances are transformed into more hydrophilic molecules by those enzymes so that they can be easily excreted from the body. Phase I enzymes involve oxidation and reduction reactions as well as hydrolysis of esters, amides and ether linkages, and in turn introduce stronger hydrophilic groups such as –OH, –NH2, –SH, and –CO2H. Phase II enzymes, also called transferases or conjugation enzymes, link the hydrophilic groups described above to even stronger hydrophilic groups, such as glucuronic acid or sulphate. Consequently, the polarity and hydrophilicity of thus formed molecules are significantly increased and the tendency of these molecules to be easily absorbed and excreted is also greatly increased [9]. It has been indicated that the biological activities of flavonoids in vivo depend on bioavailability and their metabolites generated during metabolism. Although native flavonoids have been described as powerful beneficial agents, these compounds undergo extensive metabolism in liver, small intestine and colon, leading to biotransformation such as methylation, glucuronidation, sulfation as well as degradation into phenolic acids or other compounds. Biotransformation can dramatically alter the biological properties. Therefore, it is still hard to attribute these health effects to native forms of flavonoids, and characterization of metabolism and verification of metabolites should be accomplished systematically. This review summarizes current knowledge on the metabolic fate of flavonoids in in vitro and in vivo models, and provides insight into biotransformation of parent compounds and biological activities of metabolites.
Metabolism of Flavonoids
After oral administration, flavonoids are extensively metabolized during absorption in the small intestine, and subsequently go though liver metabolism, which includes conjugates of O-methylation, sulfation and glucuronidation. The nonabsorbed fraction, particularly flavonoid glycosides, reach the colon, degraded by colonic microflora, and through the circulation extensively metabolized in the liver. However, during the metabolism of flavonoids, there are some exceptional cases. EGCG can be hydrolyzed to EGC in human saliva by the esterase [10]. Luteolin-7-glucoside, kaempferol-3-glucoside and quercetin-3-glucoside are hydrolyzed and absorbed in the small intestine, but rhamnoglucoside and diglucosides cannot be cleaved, indicating that the activity of β-glucosidase in the small intestine is dependent on the location and structure of sugar moiety [11]. In addition, anthocyanin glycosides can be absorbed intact [12]. All those metabolites are eliminated through biliary or urinary routes.
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Large conjugates are more likely to be excreted through the biliary way, whereas small conjugates preferentially follow the urinary route. Based on this evidence, metabolism of flavonoids is grouped into three categories: (1) phase І biotransformation of flavonoids including hydroxylation and demethylation, (2) flavonoid conjugates, and (3) colonic metabolism. All these metabolites are different from their parent compounds in the structure and bioactivity.
Phase І Biotransformation of Flavonoids
The metabolism of flavonoids, particularly hydroxylation and demethylation is considered to be mediated by cytochrome 450. Human liver microsomes transformed the soy isoflavone daidzein (7,4⬘-dihydroxyisoflavone) to three monohydroxylated and three dihydroxylated metabolites namely, 6,7,4⬘-trihydroxyisoflavone, 7,3⬘,4⬘trihydroxyisoflavone, 7,8,4⬘-trihydroxyisoflavone as well as 7,8,3⬘,4⬘-tetrahydroxyisoflavone, 6,7,8,4⬘-tetrahydroxyisoflavone, and 6,7,3⬘,4⬘-tetrahydroxyisoflavone. Genistein (5,7,4⬘-trihydroxyisoflavone) was metabolized by human liver microsomes into six hydroxylation products. The main metabolites were the three aromatic monohydroxylated products 5,6,7,4⬘-tetrahydroxyisoflavone, 5,7,8,4⬘-tetrahydroxyisoflavone and 5,7,3⬘,4⬘-tetrahydroxyisoflavone [13]. The human liver microsomal study of flavonoid metabolism showed that galangin (3,5,7-trihydroxyflavone) and kaempferide (3,5,7-trihydroxy-4⬘-methoxyflavone) could be hydroxylated and demethylated, respectively, to kaempferol (3,5,7,4⬘-tetrahydroxyflavone) [14]. Demethylation of 3⬘-O-methylquercetin has been observed to generate quercetin in the fibroblasts [15]. In fact, P450-related metabolism of flavonoids especially demethylation is mainly focused on the subclass of PMFs. The interaction between PMFs and liver cytochrome P-450 isozymes has been observed. For example, early studies showed that the activity of some cytochrome P450 enzymes, such as 7-ethoxyresorufin-O-deethylase (classified as CYP 1A) and nifidifine oxidase (CYP 3A4) in human liver microsomes, was inhibited by tangeretin in a noncompetitive manner. Cytochrome P450 (CYP) is the key enzyme system involved in the metabolism of PMFs and is capable of catalyzing hydroxylation and demethylation reactions. The metabolic pathway of PMFs is considered to be identical across the species. The 3⬘ and 4⬘ positions on the B ring of PMFs are the primary site of biotransformation. The number and position of the hydroxyl and methoxy groups on the B ring of PMFs have a great influence on the metabolism of PMFs [2]. In an in vitro experiment, when tangeretin was incubated with Aroclor-induced rat liver microsomes, the major metabolites were identified as 4⬘-demethyltangeretin, 3⬘,4⬘-dihydroxy-5,6,7,8-tetramethoxyflavone and 5,4⬘-didemethyltangeretin or 5,4⬘- dihydroxy-6,7,8-trimethoxyflavone; whereas in the in vivo biotransformation study of tangeretin where rats were submitted to repeated gavage, the major metabolites found in rat urine and feces were characterized as 4⬘-demethyltangeretin and
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3⬘,4⬘-dihydroxy-5,6,7,8-tetramethoxyflavone, though other metabolites with intact 4⬘-methoxy group and demethylation at various positions on the A ring were also observed. Hence, it can be concluded that the 4⬘-methoxy group of tangeretin is the primary site for demethylation and the 3⬘ position is the most vulnerable site for hydroxylation or oxidation by phase I enzymes (fig. 1). Also, urine analysis determined that 38% of the tangeretin metabolites were excreted as conjugates of glucuronates and sulfates [2]. In the in vitro biotransformation study of nobiletin, nobiletin was treated with rat liver S-9 mixture for 24 h, and 3⬘-demethylnobiletin was identified as the major metabolite. It has also been found that the demethylation rate of nobiletin is slow with a half-life of >24 h, suggesting that the half-life of nobiletin in an in vivo system might be considerably long. The in vivo metabolic study of nobiletin in male SpragueDawley rats identified the dominant metabolite as 3⬘-demethylnobiletin with two other mono-demethylated nobiletins and two di-demethylnobiletin products. Also in this in vivo experiment, 3⬘-demethylnobiletin was the only metabolite that was detected in serum. In a different nobiletin biotransformation study on male SpragueDawley rats, 4⬘-demethylnobiletin was isolated and characterized with two other minor metabolites. A recent metabolic study of nobiletin in CD-1 mice identified three metabolites in urine and plasma: 3⬘-demethylnobiletin, 4⬘-demethylnobiletin and 3⬘,4⬘-didemethylnobiletin (or 3⬘,4⬘-dihydroxy-5,6,7,8-tetramethoxyflavone) [2].
Flavonoid Conjugates
The P450-mediated metabolism of flavonoids has been shown in liver microsomes, but the metabolism of conjugates is thought to be the competitor to the phase І metabolism. Methylation and conjugation with glucuronic acid or sulfate are the major forms of conjugated metabolites of flavonoids. Because of the catechol and pyrogallol structures, flavonoids are metabolized primarily to the 3⬘-O-methyl and 4⬘-O-methyl derivatives, respectively. Quercetin is metabolized to 3⬘-O-methylquercetin as the primary metabolite and 4⬘-O-methylquercetin as the secondary metabolite [16]. Likewise, anthocyanins show the same trend. The metabolite of delphinidin 3-O-βd-glucopyranoside is 4⬘-O-methyldelphinidin 3-O-β-d-glucopyranoside, indicating that methylation of the 4⬘-OH on the flavonoid B-ring is a common metabolic way for flavonoids carrying the pyrogallol [17]. EGC and EGCG can be methylated to 4⬘-O-methyl-EGC and 4⬘⬘-O-methyl-EGCG or 4⬘,4⬘⬘-O-dimethyl-EGCG, respectively, by the catechol-O-methyltransferase (COMT) which shows the higher activity in rat liver cytosol than in human and mouse liver cytosol [10]. Moreover, the dimethylated metabolite is preferable at lower concentrations of EGCG, whereas at higher concentrations, monomethylation is dominant. Besides methylation, glucuronidation and sulfation are also common conjugated metabolites. Glucuronidation of flavanols, flavones, flavonols, flavanones has been
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OMe
OMe MeO
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Fig. 1. Biotransformation pathway of tangeretin in rat.
observed. The number of the available hydroxyl groups may influence the extent of glucuronidation. Flavonoids containing catechol or pyrogallol B ring are susceptible to glucuronidation, whereas those containing monohydroxylated B ring are less susceptible to glucuronidation [18]. EGCG-4⬘⬘-O-glucuronide and EGC-3⬘-Oglucuronide are the major metabolites formed in human, rat and mouse microsomes [10]. However, EC undergoes sulfation in the human liver and intestine. In addition, conjugated EGCG (methylated, glucuronated, or sulfated) can be further conjugated with methylation, glucuronidation and/or sulfation to form mixed EGCG metabolites [10]. Glucuronide and/or sulfate conjugates of quercetin have been reported in rat and human models after administration of quercetin [19, 20]. The affinity of each position (4⬘-, 3⬘-, 7-, 3- and 5-) to the UDP-glucuronosyltransferases has been demonstrated following the order 4⬘- <3⬘- <7- <3-. The glucuronidation at the 7 position shows the maximum rate of formation, and there is no conjugation formed at the 5 position [19]. The major metabolites of quercetin in the small intestine are quercetin-3 and 7-glucuronides, which are further metabolized in the liver through two pathways: (1) methylation of catechol functional group of both quercetin glucuronides and (2) hydrolysis of the glucuronide then followed by sulfation [20]. Proanthocyanidins are oligomers or polymers of flavan-3-ols. The procyanidin oligomer is not depolymerized into monomeric flavan-3-ols during passage through stomach and gastrointestinal (GI) tract [21]. It is interesting that the type of isoflavone conjugates has been found to be gender dependent. Unchanged daidzein, daidzein sulfate, daidzein glucuronide are observed in the urine of male rats, whereas only unchanged daidzein and daidzein glucuronide are obtained in the female rat [22]. Likewise, glucuronides are the main isoflavone metabolites in women [23]. In addition, glucuronidated anthocyanins have also been obtained [24]. Flavonoid-thiol conjugates such as flavonoid glutathione and flavonoid cysteine are barely studied. EGCG-2⬘⬘-cysteine and EGCG-2⬘-cysteine can be detected in the urine after administration of 1,500 mg EGCG/kg mice, because EGCG can be oxidized to a quinone or semiquinone which then reacts with the sulfhydryl group of cysteine [10]. EGCG conjugated with glutathione and N-acetylcysteine has not been observed. However, quercetin quinones have been demonstrated to interact with
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glutathione and N-acetylcysteine. In vitro, cysteine shows a faster reaction with quercetin than glutathione and N-acetylcysteine; however, due to the high concentration of glutathione in vivo, glutathione-quercetin conjugates are the preferential pathways of metabolism [25]. In addition, cysteine or N-acetylcysteine conjugates could be derived from the degradation of glutathione conjugates [26]. When glutathione quercetin is excreted into the bile, glutamic acid and glycine can be sequentially removed by the enzymes in the bile or the bacteria in the GI tract leading to the formation of cysteine quercetin. Then cysteine quercetin can be reabsorbed by the GI tract, and excreted in the urine with the addition of an acetyl group in the kidney. In the flavonoid-thiol conjugate pathway, either EGCG or quercetin may generate quinone forms, and then be followed by the adduction of the sulfhydryl or the glutathionyl group. In other words, certain flavonoids particularly with a di-hydroxylated or catechol B ring, could be oxidized into their quinone forms during metabolism [10, 25].
Colonic Metabolism
Bacterial metabolism of flavonoids mainly in the colon has long been studied in animal and human models as a rate-limiting step in the elimination of flavonoids. The colonic microflora is responsible for the hydrolysis of flavonoid glycosides as well as flavonoid glucuronides and sulfates. Apart from the hydrolysis, colonic microflora could cause the heterocyclic oxygen ring split to form numerous phenolic and carboxylic acids. Three types of ring fission have been described according to the flavonoid structure [27]. Flavonols are degraded into phenylacetic acids and phenylpropionic acids, whereas flavones and flavanones mainly produce phenylpropionic acids. Naringin and rutin were incubated with human colonic microflora. The major phenolic end products were 3-(4-hydroxylphenyl)-propionic acid and 3-phenylpropionic acid for naringin, and 3-hydroxyphenylacetic acid and 3-(3-hydroxylphenyl)-propionic acid for rutin [28]. After administration of quercetin-3-rutinoside in healthy human subjects with the intact colon, phenylacetic acids were identified as the major metabolites: 3-hydroxyphenylacetic acid (36%), 3-methoxy-4-hydroxyphenylacetic acid (8%) and 3,4-dihydroxyphenylacetic acid (5%). These phenylcarboxylic acids are subjected to further bacteria degradation and enzymatic transformation. Phenylpropionic acids can be degraded into benzoic acid either by colonic microflora or by β-oxidation in the liver through the process of dehydroxylation, and then conjugated to glycine in the liver and kidney [29]. In contrast to the cleavage of heterocyclic C ring of other flavonoids, flavan-3-ols (catechins) reveal a different structure of diphenylpropan-2-ols as the precursors of the lactones. After ingestion of 20 mg/kg decaffeinated green tea, the compounds 5-(3⬘,4⬘,5⬘-trihydroxyphenyl)-γ-valerolactone, 5-(3⬘,4⬘-dihydroxyphenyl)γ-valerolactone and 5-(3⬘,5⬘-trihydroxyphenyl)-γ-valerolactone were detected in the human urine and plasma [10]. These specific colonic metabolites of catechins are also observed in their oligo- and polymeric forms, procyanidins. The colonic metabolism
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of isoflavones varies among individuals. After anaerobic incubation of bacteria with daidzein for 2 weeks, only 4 of 10 samples had metabolized daidzein; among them, one sample contained dihydrodaidzein, another sample dihydrodaidzein and equol, and equol and O-desmethylangolensin were found in the remaining 2 samples. Anthocyanidin glycosides can be hydrolyzed by the colonic microflora. The protocatechuic acid is the major metabolite of anthocyanins by the colonic microflora [30].
Biological Activities of Flavonoid Metabolites
Generally, the health benefits of flavonoids depend on the bioactivities of their metabolites. For example, administration of anthocyanins is related to the prevention of diseases, but during ingestion, 60–90% of anthocyanins disappear. The major metabolite is protocatechuic acid, accounting for about 72% of ingested anthocyanins. Therefore, the health benefit of anthocyanins is attributed to protocatechuic acid [30]. Native flavonoids undergo metabolism in the small intestine, liver or colon, which causes changes in their structure. Chemical structures may influence their redox potential and/or their antioxidant capacity. Metabolism pathways are typically regarded as a means of inactivating biologically active compounds and facilitating their excretion. Metabolites are less effective at inhibiting cancer cell growth than EGCG. The methylated metabolites of EGCG decrease the inhibition of growth and proapoptotic activity compared with EGCG against murine osteoclasts [10]. Indeed, in some cases, metabolic pathways result in metabolites that are more active than their parent compounds. For example, EGCG-2⬘⬘-cysteine is more redox active as measured by production of H2O2 [10]. Three metabolites of nobiletin, 3⬘-demethylnobiletin, 4⬘-demethylnobiletin and 3⬘,4⬘-didemethylnobiletin have much stronger inhibitory effects on LPS-induced iNOS and cyclooxygenase-2 expression than their parent compound [2].
Conclusions
Apart from the liver, which is the major metabolism site, small intestine, colon and even kidney play important roles in the metabolism of flavonoids. Here EGCG is used as an example to reveal a whole scheme of metabolism pathways (fig. 2). EGCG can be degraded into EGC in the mouth by the saliva, and then in the small intestine EGCG and EGC are absorbed into the circulation. During the passage through the small intestine, only a very small portion of EGCG can be metabolized. The liver containing phase ІІ enzymes degrades the parent compounds into their conjugated forms. Meanwhile, quinone may react with cysteine to form EGCG-thiol conjugates. The nonabsorbed fraction in the small intestine goes directly to the colon, and is metabolized by the colonic microflora to their lactones. Metabolism of EGCG to methylated, glucuronide and ring fission products mostly reduces its biological activity.
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OH OH HO
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Fig. 2. Metabolic fate of EGCG.
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The biological activity of flavonoids definitely depends on their metabolism. In this review, we have shown that biological activity can be affected by the recovery of parent compounds as well as metabolites in the blood, and the rate of excretion in urine. Most flavonoids undergo a slow metabolism with a low recovery, showing that metabolism may alter the cellular response of biological effects. Therefore, studies only focusing on the native forms become unrealistic. In the future, much more effort should be made to investigate the biological activity of conjugated and microbial metabolites of flavonoids. This may give us insight into the contribution of flavonoid metabolites to health promotion and development of a new diet for optimal health benefits.
References 1 Hollman PCH, Arts ICW: Flavonols, flavones and flavanols – nature, occurrence and dietary burden. J Food Sci Agric 2000;80:1081–1093. 2 Li S, Pan MH, Lo CY, Tan D, Wang Y, Shahidi F, Ho CT: Chemistry and health effects of polymethoxyflavones and hydroxylated polymethoxyflavones. J Funct Foods 2009;1:2–12. 3 Coward L, Barnes NC, Setchell KDR, Barnes S: Genistein, daidzein, and their beta-glycoside conjugates: antitumor isoflavones in soybean foods from American and Asian diets. J Agric Food Chem 1993; 41:1961–1967. 4 Rousseff RL, Martin SF, Youtsey CO: Quantitative survey of narirutin, naringin, heperidin, and neohesperidin in Citrus. J Agric Food Chem 1987;35: 1027–1030. 5 Arts ICW, van de Putte B, Hollman PCH: Catechin contents of foods commonly consumed in The Netherlands. 1. Fruits, vegetables, staple foods, and processed foods. J Agric Food Chem 2000;48:1746– 1751. 6 Arts IC, van de Putte B, Hollman PC: Catechin contents of foods commonly consumed in The Netherlands. 2. Tea, wine, fruit juices, and chocolate milk. J Agric Food Chem 2000;48:1752–1757. 7 Mazza G, Maniati E: Anthocyanins in Fruits, Vegetables, and Grains. Boca Raton, CRC Press, 1993. 8 Pan MH, Ho CT: Chemopreventive effects of natural dietary compounds on cancer development. Chem Soc Rev 2008;37:2558–2574. 9 Yan Z, Caldwell GW: Metabolism profiling, and cytochrome P450 inhibition and induction in drug discovery. Curr Top Med Chem 2001;1:403–425. 10 Lambert JD, Sang S, Yang CS: Biotransformation of green tea polyphenols and the biological activities of those metabolites. Mol Pharm 2007;4:819–825.
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11 Spencer JPE, Chowrimootoo G, Choudhury R, Debnam ES, Srai SK, Rice-Evans C: The small intestine can both absorb and glucuronidate luminal flavonoids. FEBS Lett 1999;458:224–230. 12 Cao G, Prior RL: Anthocyanins are detected in human plasma after oral administration of an elderberry extract. Clin Chem 1999;45:574–576. 13 Kulling SE, Honig DM, Metzler M: Oxidative metabolism of the soy isoflavones daidzein and genistein in humans in vitro and in vivo. J Agric Food Chem 2001;49:3024–3033. 14 Otake Y, Walle T: Oxidation of the flavonoids galangin and kaempferide by human liver microsomes and CYP1A1, CYP1A2 and CYP2C9. Drug Metab Dispos 2001;30:103–105. 15 Spencer JPE, Rice-Evans C, Williams RJ: Modulation of pro-survival Akt/protein kinase B and ERK1/2 signaling cascades by quercetin and its in vivo metabolites underlie their action on neuronal viability. J Biol Chem 2003;278:34783–34793. 16 de Boer VC, Dihal AA, van der Woude H, Arts IC, Wolffram S, Alink GM, et al: Tissue distribution of quercetin in rats and pigs. J Nutr 2005;135:1718– 1725. 17 Ichiyanagi T, Rahman MM, Kashiwada Y, Ikeshiro Y, Shida Y, Hatano Y, Matsumoto H, Hirayama M, Tsuda T, Konishi T: Absorption and metabolism of delphinidin 3-O-β-d-glucopyranoside in rats. Free Radic Biol Med 2004;36:930–937. 18 Prior RL, Wu X, Gu L: Perspective flavonoid metabolism and challenges to understanding mechanics of health effects. J Sci Food Agric 2006;86:2487– 2491. 19 Day AJ, Bao Y, Morgan MRA, Williamson G: Conjugation position of quercetin glucuronides and effect on biological activity. Free Radic Biol Med 2000;29:1234–1243.
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20 O’Leary KA, Day AJ, Needs PW, Mellon FA, O’Brien NM, Williamson G: Metabolism of quercetin-7- and quercetin-3-glucuronides by an in vitro hepatic model: the role of human β-glucuronidase, sulfotransferase, catechol-O-methyltransferase and multiresistant protein 2 (MRP2) in flavonoid metabolism. Biochem Pharmacol 2003;65:479–491. 21 Tsang C, Auger C, Mullen W, Bornet A, Rouanet JM, Crozier A, Teissedre PL: The absorption, metabolism and excretion of flavan-3-ols and procyanidins following the ingestion of a grape seed extract by rats. Br J Nutr 2006;95:847. 22 Bayer T, Colnot T, Dekant W: Disposition and biotransformation of the estrogenic isoflavone daidzein in rats. Toxicol Sci 2001;62:205–211. 23 Zhang Y, Hendrich S, Murphy PA: Glucuronides are the main isoflavone metabolites in woman. J Nutr 2003;133:399–404. 24 Kay CD, Mazza GJ, Holub BJ: Anthocyanins exist in the circulation primarily as metabolites in adult men. J Nutr 2005;135:2582–2588.
25 Awad HM, Boersma MG, Boeren S, van Bladeren PJ, Vervoort J, Rietjens IM: Quenching of quercetin quinone/quinone methides by different thiolate scavengers: stability and reversibility of conjugate formation. Chem Res Toxicol 2003;16:822–831. 26 Spencer JPE, Mohsen MMAE, Rice-Evans C: Cellular uptake and metabolism of flavonoids and their metabolites: implications for their bioactivity. Arch Biochem Biophys 2004;423:148–161. 27 Hollman PCH: Absorption, bioavailability, and metabolism of flavonoids. Pharm Biol 2004;42:74–83. 28 Rechner AR, Smith MA, Kuhnle G, Gibson GR, Debnam ES, Srai SKS, Moore KP, Rice Evans CA: Colonic metabolism of dietary polyphenols: influence of structure on microbial fermentation products. Free Radic Biol Med 2004;36:212–225. 29 Peppercorn MA, Goldman P: Caffeic acid metabolism by bacteria of the human gastrointestinal tract. J Bacteriol 1971;108:996–1000. 30 Galvano F, Vitaglione P, Volti GL, Giacomo CD, Gazzolo D, Vanella L, Fauci LL, Fogliano V: Protocatechuic acid: the missing human cyanidins’ metabolite. Mol Nutr Food Res 2008;52:386–387.
Professor Chi-Tang Ho Department of Food Science, Rutgers University 65 Dudley Road New Brunswick, NJ 08901 (USA) Tel. +1 732 932 9611, ext. 235, Fax +1 732 932 6776, E-Mail
[email protected]
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Bioavailability and Safety Yoshikawa T (ed): Food Factors for Health Promotion. Forum Nutr. Basel, Karger, 2009, vol 61, pp 75–86
β-Carotene Degradation Products – Formation, Toxicity and Prevention of Toxicity Werner Siemsa ⭈ Costantino Salernod ⭈ Carlo Crifòe ⭈ Olaf Sommerburgb ⭈ Ingrid Wiswedelc a
Research Institute of Physiotherapy and Gerontology, KortexMed Institute of Medical Education, Bad Harzburg, Department III, University Children’s Hospital, University of Heidelberg, Heidelberg, and cDepartment of Pathological Biochemistry, Institute of Clinical Chemistry and Pathological Biochemistry, Otto-von-Guericke University Magdeburg, Magdeburg, Germany; dLaboratory of Clinical Biochemistry, and eDepartment of Biochemical Sciences, University of Rome La Sapienza, Rome, Italy b
Abstract Carotenoids are widely used as important micronutrients in food. Furthermore, carotenoid supplementation has been used in the treatment of diseases associated with oxidative stress such as various types of cancer, inflammatory diseases or cystic fibrosis. However, in some clinical studies harmful effects have been observed, e.g. a higher incidence of lung cancer in individuals exposed to extraordinary oxidative stress. The causal mechanisms of harmful effects are still unclear. Carotenoid breakdown products (CBPs) including highly reactive aldehydes and epoxides are formed during oxidative attacks in the course of antioxidative action. We investigated the formation of CBPs by stimulated neutrophils (and at further conditions), tested the hypothesis that CBPs may exert mitochondriotoxicity and tried to prevent toxicity in the presence of members of the antioxidative network. Stimulated neutrophils are able to degrade β-carotene and to generate a number of CBPs. Concerning mitochondriotoxicity, we found that CBPs strongly inhibit state 3 respiration of rat liver mitochondria at concentrations between 0.5 and 20 μM. This was true for retinal, β-ionone, and for mixtures of cleavage/breakdown products. The inhibition of mitochondrial respiration was accompanied by a reduction in protein sulfhydryl content, decreasing GSH levels and redox state, and elevated accumulation of malondialdehyde. Changes in mitochondrial membrane potential favor functional deterioration in the adenine nucleotide translocator as a sensitive target. The presence of additional antioxidants such as α-tocopherol, ascorbic acid, N-acetyl-cysteine or others could mitigate mitochondriotoxicity. The findings reflect a basic mechanism of increasing the risk of cancer Copyright © 2009 S. Karger AG, Basel induced by carotenoid degradation products.
Carotenoids are known as biologically important micronutrients with a large number of functions. Of all known carotenoids around fifty display provitamin A activity [1]. Carotenoids are also precursors of retinoids. It has been suggested that the antioxidant potency of β-carotene is transformed by scavenging oxygen radicals, thus protecting against cancer [2, 3]. Therefore, the intake of β-carotene was recommended,
especially in the form of supplements. Carotenoid supplementation has been further used for prevention and treatment of diseases with oxidative stress [4], such as cancer, UV-mediated skin diseases, neurodegenerative diseases, and cystic fibrosis. The majority of epidemiological studies consistently showed that increased consumption of food rich in β-carotene is associated with a reduced risk of lung and some other types of cancer [5]. A similar relationship has been found between levels of β-carotene in blood plasma and risk of cancer [5, 6]. In contrast, the ATBC and CARET studies indicated that supplementation of β-carotene and/or vitamin A in individuals at high risk of lung cancer increased the incidence of lung cancer [7, 8]. Pro-oxidant activity of β-carotene and procarcinogenic action in the case of preexisting premalignant lesions were discussed as possible reasons for these unexpected effects [7–14]. Murata and Kawanishi [15] reported that low concentrations of retinal (vitamin A aldehyde) and retinol (vitamin A) cause cellular DNA cleavage and induction of 8-oxo-7,8-dihydro-2-deoxyguanosine formation in HL-60 and HP100 cells. Superoxide radical anions generated by autoxidation of carotenoid derivatives were dismutated to H O , which was responsible for DNA damage. Retinal has a pro-oxidant capability, which could lead to carcinogenesis. β-Carotene supplementation seems to be absolutely necessary for several diseases such as cystic fibrosis [16]. Patients with cystic fibrosis were recommended to take a daily dose of about 1 mg β-carotene per kilogram body weight, which is much higher than that given to the patients in the CARET trial. Therefore, the causal mechanism of increased cancer risk mediated by β-carotene intake has to be elucidated to establish safe conditions for supplementation. In a previous work, we provided evidence that carotenoid cleavage products (CCPs) inhibit Na+-K+-ATPase activity [17]. Interestingly, CBPs were stronger inhibitors of Na+-K+-ATPase activity than the endogenous major lipid peroxidation product 4-hydroxynonenal (HNE) [17]. Here, we investigate the questions (a) whether CBPs are generated not only in the presence of hypochlorite and under other – more or less – artificial conditions, but also by stimulated neutrophils, (b) which mitochondriotoxic effects CBPs have, and (c) whether the presence of other members of the antioxidative network such as α-tocopherol, ascorbic acid etc. can mitigate or even prevent toxic effects of CBPs. We argue that pro-oxidative actions of CBPs – especially in mitochondria and nucleus – are responsible for the harmful effects of high-dosage supplementation of β-carotene in patients suffering from extraordinary oxidative stress (due to cigarette smoking or working with asbestos). In our opinion, those conditions may also exist if other inflammatory conditions exist in the lungs (or other tissues). Impairment of mitochondrial function including changes in calcium homeostasis [18] can cause an increase in the formation of superoxide, thus promoting oxidative stress resulting in the oxidation of lipids, proteins and DNA molecules. DNA damage seems to be possible also by direct reactions of CBPs with DNA molecules. Oxidative DNA damage is 2
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a hallmark of cancerogenesis. We have found that CBPs inhibit state 3 respiration in mitochondria. Furthermore, we provide evidence that CBPs increase oxidative stress in mitochondria, which is characterized by increased malondialdehyde (MDA) formation and decreases in mitochondrial GSH and protein SH [13].
Methods Preparation and Formation of CBPs
The generation and analysis of the CBPs was performed as previously described [19]. Mixtures of breakdown products from β-carotene, retinal, and β-ionone were produced by mixing samples of a methanolic stock solution of these compounds at a concentration of 0.04 mm, each with 1 mm hypochlorous acid at room temperature. CBP collection was carried out after bleaching reaction was finished, indicated by a stable color of the stock solution. The extraction was carried out twice with hexane. The hexane phases were combined and evaporated with nitrogen until dry. The residue was redissolved in aliquots of hexane, adjusted to 1 or 0.5 mm stock solutions of CCPs, which were stored at –80°C.
Incubation of Carotenoids with Stimulated Neutrophils Leukocytes were purified from heparinized human blood, freshly drawn from healthy donors according to a method of Ferrante and Thong [20, 21]. Leukocyte preparation containing 90–98% of polymorphonuclear leucocytes (PMLs) and apparently free of contaminating erythrocytes was obtained by a one-step procedure involving centrifugation of blood samples layered on FicollHypaque medium. Cells were suspended in 0.5 mm Ca2+-containing phosphate-buffered saline at 37°C, pH 7.4, and stimulated in the presence of 1 μg/ml phorbal myristate acid. After 5 min of preincubation, β-carotene was added to the medium, leading to a final concentration of 1 μm for the degradation experiments and of 100 μm and 10 mm for the identification experiments, respectively. For the degradation experiments at time points 0 and 30 min, fractions of the culture media were extracted for measurements of absorption spectra. Similar experiments without phorbal myristate acid stimulation of the cells were carried out as controls. For calculation of the degradation rate, further experiments were carried out in which degradation of β-carotene (initial concentration 1 μm) by PMLs (10 × 106 cells/ml) was measured after 10, 30, and 60 min. For the identification experiments, fractions of the culture media were extracted after 0 and 30 min, and the resulting fractions were analyzed by HPLC and gas chromatography mass spectrometry (GCMS). HPLC and GCMS procedures have been described in detail in the study by Sommerburg et al. [21].
Preparation of Mitochondria For the experiments, freshly isolated liver mitochondria from 180- to 220-gram male Wistar rats were used. The mitochondria were prepared in ice-cold medium containing 250 mm sucrose, 1 mm EGTA and 1% (w/v) bovine serum albumin at pH 7.4 (isolation medium) using a standard procedure. After the initial isolation, Percoll was used for purifying mitochondria from a fraction containing some endoplasmic reticulum, Golgi apparatus, and plasma membranes. The mitochondria were well coupled, as indicated by a respiratory control index greater than 5 with glutamate plus malate as substrates.
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Incubation of Mitochondria and Measurement of Mitochondrial Respiration For measurements of mitochondrial respiration, aliquots of CBP solutions (retinal, β-ionone, retinal breakdown products, β-ionone breakdown products, and β-carotene breakdown products) were transferred into reaction vials and evaporated with argon until dry. Afterwards, 2 ml of the incubation medium (containing 10 mm sucrose, 120 mm KCl, 15 mm NaCl, 20 mm Tris, 2 mm MgCl2, 5 mm NaH2PO4, pH 7.4) was added to dissolve CBPs. The solution was transferred into a thermostat-controlled chamber equipped with a Clark-type electrode. Then the mitochondrial suspension (final concentration 1 mg protein/ml) was added. After 3 min of preincubation, 5 mm glutamate and 5 mm malate were added, and the state 4 respiration was measured. State 3 respiration was adjusted by adding 200 μm ADP. Uncoupled respiration was accomplished by addition of 0.1 μm carbonyl cyanide p-(tri-fluoromethoxy) phenylhydrazone to the mitochondrial suspension in the presence of hydrogen-supplying substrates. For control, the incubation medium without any carotenoid breakdown products was transferred into the incubation chamber followed by preincubation of mitochondria (1 mg/ml) for 3 min. The addition of substrates and the time schedule were identical to incubations in the presence of CBPs. Furthermore, incubations of up to 20 min in the presence and absence of CBPs were carried out, and samples were withdrawn for GSH, GSSG, MDA and protein SH measurements. For experiments on possible prevention of toxicity exerted by CBPs, all incubations were repeated in the absence and in the presence of various antioxidants (α-tocopherol 1, 5, 10, 20, 100 μm; ascorbic acid 10, 50, 100, 250 μm; urate 20, 100 μm, NAC 100 μm; DHLA 10 μm; SOD, catalase, ebselen 100 μm, and various combinations).
Assays for GSH, GSSG, Protein SH and MDA GSH and GSSG were analyzed by a microtiter plate assay according to Baker et al. [22]. The content of protein SH was determined according to Ellman [23]. TBA-MDA conjugates were measured using an HPLC-based method [24].
Mitochondrial Membrane Potential The dissipation of the mitochondrial membrane potential was followed at 30°C in a thermostatcontrolled chamber equipped with a tetra phenyl phosponium cation-sensitive electrode [25].
Statistical Analysis Significant differences were determined using Student’s t test. A probability of p < 0.05 was accepted as significant. Data are presented as mean ± SE.
Results
Degradation of β-Carotene by Activated Neutrophils (PMLs) It could be demonstrated that β-carotene was degraded in culture medium of activated PMLs, but not in medium of nonactivated PMLs. The degradation rate, calculated
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Table 1. CBPs identified after oxidation of β-carotene in the presence of hypochlorite and after incubation with primary cultures with activated human PMLs Apocarotenals, long-chain products
Short-chain products
Apo-4⬘-carotenal
β-Ionone
Apo-8⬘-carotenal
Ionene
Apo-12⬘-carotenal
β-Cyclocitral
Retinal (Apo-15⬘-carotenal)
β-Ionone-5,6-epoxide
Hexanedioic acid, mono(2-ethylhexyl)ester
Dihydroactinidiolide 4-oxo-β-ionone 1,5,5-trimethyl-6-acetomethyl-cyclohexene 3,7,7-trimethyl-1-penta-1,3-dienyl-2oxabicyclo[3.2.0]hept-3-ene 2,6,6-trimethyl-1-cyclo-hexene-1-acetaldehyde 4,6,6-trimethyl-2-(3-methylbuta-1,3-dienyl)3-oxatricyclo[5.1.0.0(2,4)] octane
Apocarotenals were identified by HPLC, short-chain products by GCMS.
from separate experiments in which the time dependence was investigated, was 730 pmol/ml × (10 × 106 cells) × h after addition of 1 nmol/ml of β-carotene.
Identification of CBPs After treatment of β-carotene with hypochlorite and after its incubation with activated primary cultures of human PMLs, various CBPs could be identified by HPLC (apocarotenals) and GCMS (so-called short-chain products of carotenoids). Those CBPs are listed in table 1 [see also 21].
Inhibition of Mitochondrial State 3 Respiration All types of CBPs investigated strongly inhibited the state 3 respiration in a dosedependent manner (fig. 1). State 4 respiration was hardly affected [13]. The presence of 20 μm CBPs led to a 30–50% decrease. Also low concentrations of CBPs, such as 1 μm and even 0.5 μm, exerted clear inhibitions. The inhibition by retinal was 12.4 ± 0.5% at 1 μm and 6.3 ± 2.9% at a concentration of 0.5 μm. The ranges of inhibition for the different CCPs were 5–12% at 1 μm and 3–6% at 0.5 μm.
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Inhibition of ADP-stimulated respiration (% of control)
Retinal 50
Retinal CP Ionone
40
Ionone CP Carotene CP
30
20
10 0 0.5
1
5
20
Concentration CCPs (μM)
Fig. 1. Influence of carotenoid cleavage/breakdown products on respiration. Rat liver mitochondria were incubated at 30°C in a medium containing 10 mM sucrose, 120 mM KCl, 15 mM NaCl, 20 mM Tris, 2 mM MgCl2, 5 mM NaH2PO4, pH 7.4 (incubation medium). Five different types of cleavage products were used: retinal, β-ionone, mixtures of retinal (retinal CP) or β-ionone (ionone CP) or β-carotene cleavage products (carotene CP). Inhibition of respiration is presented as a decrease in the difference of respiration after and before ADP addition (state 3 minus state 4 respiration) in percent of complete inhibition. 100% inhibition corresponds to a decrease in respiration of 53.4 ± 3.5 nmol O2/mg/ min, which is the ADP-induced increase in respiration of controls.
Adenine Nucleotide Translocator as a Sensitive Target of Oxidation by CBPs The dissipation of the mitochondrial membrane potential was measured in order to distinguish between inhibition of the adenine nucleotide translocator and inhibition of the F0F1-ATPase by the decomposition products of carotenoids. After blocking the electron transport within the respiratory chain by cyanide, the F0F1-ATPase splits ATP to ADP and inorganic phosphate, paralleled by pumping protons into the extramitochondrial space. Therefore, inhibition of the F0F1-ATPase results in the acceleration of depolarization. For CBPs which inhibited significantly the ADP-induced increase in respiration (state 3 minus state 4), it was shown that they had no effect on the kinetics of membrane depolarization [13]. This observation supports the suggestion that the inhibition of respiration by degradation products of carotenoids is mainly caused by impairing adenine nucleotide translocation.
Changes in GSH, GSSG, Protein SH, and MDA Concentrations The mitochondrial GSH content rapidly decreased in the presence of retinal and other β-carotene derivatives. Strongest decreases were observed in the presence of retinal
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and retinal breakdown products. 20 μm CBPs led to a GSH decrease from 6.2 ± 0.5 nmol/mg protein (control) to 1.7 ± 0.3 (retinal) and 2.51 ± 0.44 (retinal breakdown products). Parallel to this, GSSG content increased. The GSSG increase in combination with GSH decrease resulted in increasing ratios of GSSG/total GSH. 20-min incubation of mitochondria in the presence of 20 μm retinal led to a threefold increased ratio compared with controls. Moreover, most of CBPs caused a decrease in total GSH pool (GSH + GSSG) due to GSSG efflux through mitochondrial membranes. Loss of protein SH was found during the 20-min incubation with either of the CBP mixtures at a concentration of 20 μm. The mitochondrial protein SH content decreased from 86 ± 4.6 (control) to 68 ± 1.9, and 78 ± 7.7 nmol SH/mg protein in the presence of 20 μm of retinal breakdown products and retinal, respectively. Even when only a small share of protein SH of mitochondria is lost (usually less than 20% of total), the absolute protein SH loss is markedly higher than the absolute loss of GSH. In the presence of 20 μm of retinal breakdown products, the total SH loss was 22 ± 2.8 nmol/mg protein. Taking into account the final protein concentration of about 1 mg mitochondrial protein/ml of suspension, the bulk of retinal breakdown products may be bound to SH groups. The levels of MDA are enhanced more than tenfold after 20 min of incubation in the presence of 20 μm CBPs in comparison with control incubations.
Discussion
Formation of β-Carotene Degradation Products (CBPs) There are various conditions for CBP formation (table 2). Some of these conditions were studied by our research group [13, 14, 17, 21, 26]. It was clearly shown that β-carotene is degraded by stimulated PMLs in vitro. This gave the pathophysiological meaning to our experiments on formation and identification of CBPs by hypochlorous acid (table 1). While formation of apocarotenals under these conditions has been studied before [19, 27–29], this was not the case for short-chain products. When performing gas chromatography mass spectrometry, the CBPs 5,6-epoxi-β-ionone, ionene, β-cyclocitral, β-ionone, dihydroactinidiolide, and 4-oxo-β-ionone were found to be formed during degradation of β-carotene by hypochlorous acid. This may be of biological and medicinal relevance because many CBPs are carbonyls and epoxides and highly reactive and, therefore, potentially toxic.
Toxicity of CBPs: CBPs Exert Pro-Oxidant Effects Many of the CBPs were identified as aldehydes. Retinal, the different apocarotenals and also a certain number of newly identified short-chain derivatives are of aldehydic
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Table 2. Conditions leading to β-carotene degradation and formation of CBPs Endogenous enzymatic degradation steps Radical reactions (e.g. AIBN, AMVN) UV light (artificial light, sunlight) Heat Cigarette smoke Phagocytosing cells Hypochlorite Autoxidation in dependence on pO2 All physiological antioxidative reactions
nature. It is well known that aldehydes react rapidly with sulfhydryl groups and lysyl and histidine residues even at low cellular levels. Recently, we demonstrated the inhibition of the Na+-K+-ATPase by a mixture of β-carotene cleavage products derived from hypochlorite treatment [17]. We found that CBPs exerted a much higher in vitro toxicity than HNE, nonenal, and nonanal, which are also aldehydes and react with nucleophilic groups. This means that CBPs are very reactive and that they may be of particularly high relevance under pathophysiological conditions. Thus, the depletion of mitochondrial protein SH and GSH after exposure of isolated rat liver mitochondria may be caused by direct reactions of aldehydes with mitochondrial sulfhydryl groups. The data presented further demonstrate that the decrease in SH groups under the influence of CBPs was paralleled by the inhibition of state 3 respiration (predominantly the ADP-induced increase in respiration) due to impairment of adenine nucleotide translocation. This could be expected since the adenine nucleotide translocator has been shown to be sensitive to fatty acid CoA derivatives and to lipid peroxidation products such as HNE [30]. Inhibition of electron transfer by the respiratory chain due to the inhibition of adenine nucleotide transport leads to a rise in superoxide radical anion production by the respiratory chain and subsequently to formation of H2O2 and hydroxyl radicals [31]. Accordingly, an increase in oxidative stress is induced in mitochondria. This suggestion is in line with our observation that CBPs caused additional MDA accumulation.
Biological Impact of CBPs Several conditions result in the rapid nonenzymatic oxidative cleavage of β-carotene, i.e. heavy oxidative stress (smoking, asbestos) and photoirradiation in the skin and eyes [32]. The same should be true for hypochlorite-mediated carotenoid cleavage in the neighborhood of activated PMLs. Hypochlorite released by phagocytic cells
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is, at least temporarily, present at high concentrations ranging from 5 to 50 μm in the tissue [33]. These levels are high enough to initiate the nonenzymatic cleavage of β-carotene. Carotenoid levels are markedly higher than 1 nmol/g in human tissues. Therefore, these concentrations are high enough for CBP formation leading to toxic effects. Additionally, due to the lipophilic properties of carotenoids, even significantly higher concentrations can be measured in mitochondrial and plasma membranes in comparison with the values in total tissue [34]. Furthermore, higher concentrations of CBPs may be expected in mitochondria because of their capability to produce oxygen free radicals themselves [35]. Supplementation of β-carotene in combination with heavy oxidative stress (smoking, inflammation) may further increase the in vivo concentrations of CBPs. Therefore, CBP concentrations in mitochondria which correspond to the concentrations used in our in vitro experiments (0.5–20 μm) are very likely in such situations. Our data provide evidence that CBPs deplete mitochondrial sulfhydryl groups and impair oxidative phosphorylation in mitochondria at the level of adenine nucleotide translocation. Oxidative stress resulting from the impairment of the mitochondrial energy metabolism in the presence of CBPs and indicated by enhanced MDA formation may induce oxidative damage to DNA molecules in the mitochondria and nucleus. Mitochondrial DNA has a pronounced susceptibility to oxidative stress because of the absence of histones and a lower capacity of DNA repair in comparison with the nucleus. Marques et al. [36] have shown the DNA-damaging potential of CBPs. Treatment of calf thymus DNA with CBPs significantly increased levels of 1,N2-etheno-2⬘-deoxyguanosine and 8-oxo-7,8-dihydro-2⬘-deoxyguanosine, known mutagenic DNA adducts. Furthermore, formation of apo-14-carotenal augments inflammatory response mediated by cytokines such as TNF-α. If apo-14-carotenal binds to peroxisome proliferator-activated receptor-α (PPARα), the PPARα cannot inhibit NFκB-mediated inflammation anymore. Additionally, retinaldehyde and retionoid acid signal functions in carotenoid breakdown under oxidative stress are under discussion [37, 38]. Oxidative DNA damage, in general, increases the risk of cancer. Thus, our data may indicate a basic mechanism of the harmful effects of carotenoids in situations of increased oxidative stress (fig. 2). Under conditions of mild oxidative stress, antioxidative effects of β-carotene supplementation will dominate. But under conditions of extraordinary oxidative stress, there exists a potential risk of cancerogenesis due to the rapid accumulation of pro-oxidative-acting CBPs (fig. 2).
Prevention of CBP-Induced Mitochondriotoxicity by Antioxidants All antioxidants used were able to reduce β-carotene degradation by human neutrophils and, therefore, the rapid formation of β-carotene breakdown products. In the presence of antioxidants, the impairment of mitochondrial respiration and mitochondrial SH system by CBPs was drastically reduced, and in some cases almost completely
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Mild oxidative stress
Heavy oxidative stress (cigarette smokers, asbestos workers)
ROS OCl–
-carotene
Oxidative breakdown products (retinal, shortened carbonyls, aldehydes, epoxides...)
Antioxidant activity
ADP ATP
Pro-oxidant activity
Protection against DNA oxidation
MDA
SH
O2·–, H2O2 DNA oxidation
Prevention of tumors
Potential risk of tumors
Beneficial
Harmful
Fig. 2. β-Carotene supplementation. Antioxidant and pro-oxidant activities under different conditions. Predominant antioxidant and tumor-preventing activity of β-carotene supplementation under physiological conditions and conditions of mild oxidative stress, but dominating pro-oxidative effects of such supplementation under conditions of extraordinary oxidative stress.
prevented. α-Tocopherol was the most effective member of the antioxidative network under those conditions.
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5 Ziegler RG, Mayne ST, Swanson CA: Nutrition and lung cancer. Cancer Causes Control 1996;7:157– 177. 6 Peto R, Doll R, Buckley JD, Sporn MB: Can dietary beta-carotene materially reduce human cancer rates? Nature 1981;290;201–208. 7 Omenn GS, Goodman GE, Thornquist M, Balmes J, Cullen MR, Glass A, Keogh JP, Meyskens FL, Valanis B, Williams JH, Barnhart S, Hammar S: Effects of a combination of β-carotene and vitamin A on lung cancer and cardiovascular disease. N Engl J Med 1996;334:1150–1155.
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8 Albanes D, Heinonen OP, Taylor PR, Virtamo J, Edwards BK, Rautalahti M, Hartman AM, Palmgren J, Freedman LS, Haapakoski J, Barrett MJ, Pietinen P, Malila N, Tala E, Liippo K, Salomaa ER, Tangrea JA, Teppo L, Askin FB, Takinen E, Erozan Y, Greenwald P, Huttunen JK: Alpha-Tocopherol and beta-carotene supplements and lung cancer incidence in the alpha-tocopherol, beta-carotene cancer prevention study: effects of base-line characteristics and study compliance. J Natl Cancer Inst 1996;88: 1560–1570. 9 Mayne ST, Handelman GJ, Beecher G: BetaCarotene and lung cancer promotion in heavy smokers – a plausible relationship? J Natl Cancer Inst 1996;88:1513–1515. 10 Mayne ST: Beta-carotene, carotenoids, and disease prevention in humans. FASEB J 1996;10:690–701. 11 Wang X-D, Russell RM: Procarcinogenic and anticarcinogenic effects of beta-carotene. Nutr Rev 1999;57:263–272. 12 Young AJ, Lowe GM: Antioxidant and prooxidant properties of carotenoids. Arch Biochem Biophys 2001;385:20–27. 13 Siems W, Wiswedel I, Schild L, Augustin W, Langhans C-D, Sommerburg O: β-Carotene cleavage products induce oxidative stress in vitro by impairing mitochondrial respiration. FASEB J 2002; 16;1289–1291. 14 Siems W, Wiswedel I, Salerno C, Crifò C, Augustin W, Schild L, Langhans C-D, Sommerburg O: Betacarotene breakdown products may impair mitochondrial functions – potential side effects of high-dose beta-carotene supplementation. J Nutr Biochemistry 2005;16:385–397. 15 Murata M, Kawanishi S: Oxidative DNA damage by vitamin A and its derivative via superoxide generation. J Biol Chem 2000;275:2003–2008. 16 Homnick DN, Cox JH, DeLoof MJ, Ringer TV: Carotenoid levels in normal children and in children with cystic fibrosis. J Pediatr 1993;122:703– 707. 17 Siems WG, Sommerburg O, van Kuijk FJGM: Carotenoid oxidative degradation products inhibit Na+-K+-ATPase. Free Radic Res 2000;33:427–435. 18 Schild L, Keilhoff G, Augustin W, Reiser G, Striggow F: Distinct Ca2+ thresholds determine cytochrome c release or permeability transition pore opening in brain mitochondria. FASEB J 2001;15:565–567. 19 Handelman GJ, van Kuijk FJGM, Chatterjee A, Krinsky NI: Characterization of products formed during the autoxidation of beta-carotene. Free Radic Biol Med 1991;10:427–437.
20 Ferrante A, Thong YH: A rapid one-step procedure for purification of mononuclear and polymorphonuclear leukocytes from human blood using a modification of the Hypaque-Ficoll technique. J Immunol Methods 1978;24:389–393. 21 Sommerburg O, Langhans C-D, Arnhold J, Leichsenring M, Salerno C, Crifò C, Hoffmann GF, Debatin K-M, Siems WG: beta-Carotene cleavage products after oxidation mediated by hypochlorous acid – a model for neutrophil-derived degradation. Free Radic Biol Med 2003;35:1480–1490. 22 Baker MA, Cerniglia GJ, Zaman A: Microtiter plate assay for the measurement of glutathione and glutathione disulfide in large numbers of biological samples. Anal Biochem 1990;190:360–365. 23 Ellman GL: Tissue sulfhydrylgroups. Arch Biochem Biophys 1959;82:70–77. 24 Chirico S: High-performance liquid chromatography-based thiobarbituric acid test. Methods Enzymol 1994;233:314–318. 25 Kamo N, Muratsugu M, Hongoh R, Kobatake Y: Membrane potential of mitochondria measured with an electrode sensitive to tetraphenyl phosphonium and relationship between proton electrochemical potential and phosphorylation potential in steady state. J Membr Biol 1979;49:105–121. 26 Hurst JS, Contreras JE, Siems WG, van Kuijk FJGM: Oxidation of carotenoids by heat and tobacco smoke. Biofactors 2004;20:23–35.. 27 Mader I: Beta-carotene: thermal degradation. Science 1964;144:533–534. 28 Mordi RC, Walton JC, Burton GW, Hughes L, Ingold KU, Linday DA, Moffatt DJ: Oxidative degradation of β-carotene and β-apo-8´-carotenal. Tetrahedron 1993;49:911–928. 29 Krinsky NI: The antioxidant and biological properties of the carotenoids. Ann NY Acad Sci 1998;854; 443–447. 30 Chen JJ, Bertrand H, Yu BP: Inhibition of adenine nucleotide translocator: by lipid peroxidation products. Free Radic Biol Med 1995;19:583–590. 31 Cadenas E, Davies KJ: Mitochondrial free radical generation, oxidative stress, and aging. Free Radic Biol Med 2000;29:222–230. 32 Ribaya-Mercado JD, Garmyn M, Gilchrest BA, Russell RM: Skin lycopene is destroyed preferentially over beta-carotene during ultraviolet irradiation in humans. J Nutr 1995;125:1854–1859. 33 Davies JM, Horwitz DA, Davies KJ: Potential roles of hypochlorous acid and N-chloroamines in collagen breakdown by phagocytic cells in synovitis. Free Radic Biol Med 1993;15:637–643.
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34 Bianchi-Santamaria A, Stefanelli C, Cembran M, Gobbi M, Peschiera N, Vannini V, Santamaria L: Hepatic subcellular storage of beta-carotene in rats following diet supplementation. Int J Vitam Nutr Res 1999;69:3–7. 35 Quiles JL, Huertas JR, Manas M, Ochoa JJ, Battino M, Mataix J: Oxidative stress induced by exercise and dietary fat modulates the coenzyme Q and vitamin A balance between plasma and mitochondria. Int J Vitam Nutr Res 1999;69:243–249. 36 Marques SA, Loureiro APM, Gomes OF, Garcia CCM, Di Mascio P, Medeiros MHG: Induction of 1,N2-etheno-2´-deoxyguanosine in DNA exposed to beta-carotene oxidation products. FEBS Lett 2004;560:125–130.
37 Ziouzenkova O, Plutzky J: Retinoid metabolism and nuclear receptor responses: new insights into coordinated regulation of the PPAR-RXR complex. FEBS Lett 2008;582:32–38. 38 Ziouzenkova O, Orasanu G, Sukhova G, Lau E, Berger JP, Tang G, Krinsky NI, Dolnikowski GG, Plutzky J: Asymmetric cleavage of beta-carotene yields a transcriptional repressor of retinoid X receptor and peroxisome proliferator-activated receptor responses. Mol Endocrinol 2007;21:77–88.
Werner Siems, MD, PhD Research Institute of Physiotherapy and Gerontology, KortexMed Institute of Medical Education Hindenburgring 12 A DE–38667 Bad Harzburg (Germany) Tel. +49 5322 55898 224, Fax +49 5322 55898 228, E-Mail
[email protected]
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Antioxidants Yoshikawa T (ed): Food Factors for Health Promotion. Forum Nutr. Basel, Karger, 2009, vol 61, pp 87–94
Dietary Flavonoids as Antioxidants Junji Terao Department of Food Science, Graduate School of Nutritional and Bioscience, University of Tokushima, Tokushima, Japan
Abstract Flavonoids are ubiquitously present in fruits and vegetables. They have attracted much attention in relation to prevention of degenerative diseases such as atherosclerosis. Their antioxidant activity should be at least partly responsible for such prevention. The mechanism of antioxidant activity of flavonoids can be characterized by direct scavenging or quenching of oxygen free radicals or excited oxygen species as well as inhibition of oxidative enzymes that generate these reactive oxygen species. The essential part of the free radical-scavenging activity of flavonoids is attributed to the o-dihydroxyl group in the B ring (catechol group) in their diphenylpropane structure. Catechol type flavonoids therefore possess powerful antioxidant activity. Conjugation of glucuronide/sulfate during intestinal absorption attenuates their antioxidant activity, but some metabolites containing an o-dihydroxyl structure, such as quercetin 3-O-β-D-glucuronide (Q3GA), retain considerable antioxidant activity. Q3GA was found to be effective in the inhibition of lipid hydroperoxide-induced oxidative stress in the nerve cell model PC-12. Our in vivo study using high cholesterol-fed rabbits also showed accumulation of quercetin metabolites in aortic tissue, and inhibition of deposition of cholesteryl ester hydroperoxide. It is evident that quercetin metabolites are distributed in human atherosclerotic lesions, particularly the macrophage-derived foam cell. The specific target should therefore be taken into account when evaluating the antioxidant activity of dietary flavonoids Copyright © 2009 S. Karger AG, Basel in vivo.
Flavonoids are plant secondary metabolites derived from malonyl-coenzyme-A (malonyl-CoA) and p-coumaroyl-CoA. Hydroxyl groups are bound to their basic diphenylpropane structure. The reducing property of the resulting phenolic hydroxyl group is usually responsible for their free radical-scavenging activity. More than 4,000 species of flavonoids have been discovered in the plant kingdom and are recognized for protection against ultraviolet light, pigmentation, stimulation of nitrogen-fixing nodules, phytoalexin, and as substances for allelopathy [1]. Flavonoids are mainly present as their glycosides, in which monosaccharides (e.g. glucose and galactose) or di- or oligo-saccharides (e.g. rutinose) are bound to the hydroxyl group by a β-glucoside linkage. Aglycones are released from their glycosides by hydrolytic
enzymes having β-glucosidase activity. Flavonoids can be classified into several subclasses: flavone, flavonol, flavan-3-ol (catechin), isoflavone, flavanone, and anthocyanidin. Dihydroflavonols, flavan-3,4-diol, coumarin, chalcone, dihydrochalcone and aurone are also flavonoids. Flavonoids in the diet have a bitter or astringent taste and inhibit digestive enzymes. Recent studies strongly suggest that dietary flavonoids may have a favorable role in human health through their antioxidant activity [2]. Oxidative stress is frequently referred to as an essential cause initiating and/or promoting degenerative diseases such as atherosclerosis. Much attention has therefore been paid to the antioxidant activity of dietary flavonoids from the viewpoint of food factors that modulate oxidative stress [3]. Dietary flavonoids seem to participate in the antioxidant network together with vitamin E, vitamin C and other biological antioxidants in the human body [4]. Recent studies also suggest that dietary flavonoids can exert various effects by a mechanism different from classical antioxidant activity: regulation of the activity and protein expression of specific enzymes [5].
Structure-Activity Relationship for the Antioxidant Activity of Flavonoids
Flavonoids can scavenge free radical species such as superoxide (O2•–), the hydroxyl radical (•OH) and the lipid peroxyl radical (LOO•) by donating an electron or hydrogen atom [3]. The phenolic hydroxyl group responsible for scavenging these radicals is dissociated to its anion form depending on the medium pH. The radicalscavenging activity of flavonoids is therefore elevated with an increase in medium pH. Bors et al. [6] were the first to claim three partial structures contributing to the radical-scavenging activity of flavonoids: (a) o-dihydroxyl structure in the B ring (catechol structure) as a radical target site; (b) 2,3-double bond with conjugation to 4-oxo group which is necessary for delocalization of an unpaired electron from the B ring, and (c) hydroxyl groups at the 3 and 5 position which are necessary for enhancement of radical-scavenging activity (fig. 1). The catechol group is essential for the radical-scavenging activity of flavan-3-ols and flavones lacking 2, 3-double bonds. Quercetin and myricetin are suggested to exert the strongest radical-scavenging activity among flavonoids because these two flavonols possess all the partial structures contributing to radical-scavenging activity. The galloyl group present in gallate type flavan-3-ols such as epicatechin gallate (ECG) and epigallocatechin gallate (EGCG) also participates in the radical target site because gallate comprises o-dihydroxyl structures as a form of the pyrogallol group. Gallate type flavan-3-ols are therefore more effective than nongallate type flavan-3-ols as free radical scavengers [7]. We measured 1,1-diphenyl-2-picrylhydrazyl radical-scavenging capacity of several flavones, flavonols and flavone-3-ols to verify the structure-activity relationship of flavonoids as free radical scavengers (table 1) [8]. Data clearly demonstrated the validity of the hypothesis of Bors et al. [6]. The 3 position at the C ring also contributes to the radical-scavenging activity of flavonols because luteolin (which
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OH 3’ B HO
O A
Fig. 1. Structure of quercetin. The circles indicate partial structures contributing to the free radical-scavenging activity of flavonoids.
2
C 4
2,3-double bond with conjugation to 4-oxo group
3 OH
5 OH
o-dihydroxyl structure (catechol group)
OH 4’
O
hydroxyl group at the 3 and 5 posiiton
Table 1. Structure-activity relationship of flavonoids in 1,1-diphenyl-2-picrylhydrazyl radical-scavenging capacity 3’ 3’
3’ O
7
3 5
O
7
5’
5’
O
5’
OH
5
O
5
O
OH
5
O
HO Flavan-3-ol
Flavonol
Flavone
4’ 5’
4’ 7
O
O
7
3’
4’
4’
OH OH
Flavonoid
3
5
7
3⬘
4⬘
5⬘
Scavenged radicals mol/mol
Flavone Apigenin Luteolin
– –
OH OH
OH OH
– OH
OH OH
– –
0 3.9±0.4
Flavonol Fisetin Kaempferol Quercetin Myricetin
OH OH OH OH
– OH OH OH
OH OH OH OH
OH – OH OH
OH OH OH OH
– – – OH
6.1±0.8 1.9±0.1 6.6±0.5 7.1
Flavan-3-ol (–)-Epicatechin (–)-Epigallocatechin (–)-ECG (–)-EGCG
OH OH O-gallate O-gallate
OH OH OH OH
OH OH OH OH
OH OH OH OH
OH OH OH OH
– OH – OH
4.9±0.4 6.8±0.6 6.4±0.7 9.3±1.4
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possesses the catechol group and 2,3-double bond but lacks a hydroxyl group at the 3 position) showed lower radical-scavenging capacity as compared with quercetin. The physical nature of flavonoids is also a determinant for their radical-scavenging activity in heterogeneous systems such as biological fluids and tissues. Lipid peroxidation occurring in biomembranes can be initiated by attack of aqueous radicals from the extracellular fluid or cytosol followed by lipid peroxyl radical-mediated radical chain oxidation within phospholipid bilayers. The diphenylpropane structure of flavonoids is lipophilic and therefore assumed to interact with phospholipid bilayers or insert into the interior of bilayers. Uekusa et al. [9] demonstrated that EGCG has a strong interaction with liposomal model membranes because of its high lipophilicity. Localization between water and the organic phase expressed by log P may be a suitable index for determining the antioxidant activity of each flavonoid in biomembranes. We found that quercetin and epicatechin protect the aqueous radical-induced lipid peroxidation in unilamellar liposomes more effectively than the lipophilic antioxidant vitamin E [10].
Antioxidant Activity of Quercetin Metabolites in a Nerve Cell Model
We focused on quercetin as a typical flavonoid possessing strong antioxidant activity. We tried to estimate its effect on oxidative stress using in vitro cultured cells. Quercetin is completely converted into its conjugated metabolites during intestinal absorption, and thus neither quercetin aglycone nor quercetin glycosides are present in human plasma [11]. Day et al. [12] demonstrated that several conjugated metabolites including glucuronide and sulfate conjugates with or without O-methylation to the hydroxyl group accumulate in circulating human plasma. In general, metabolic conversion of flavonoids reduces or extinguishes their antioxidant activity because the hydroxyl group responsible for the activity disappears by conjugation or O-methylation [13]. We discovered that one of the major metabolites, quercetin 3-O-β-d-glucuronide (Q3GA), retains appreciable antioxidant activity because the o-dihydroxyl structure in the B ring is unchanged even after conjugation [14]. We then tried to estimate the protective effect of Q3GA against oxidative stress in a nerve cell model. Central nervous disorders such as dementia and Alzheimer’s disease, as well as decline in cognitive function, have been closely associated with oxidative stress in nerve cells. Quercetin intake has been expected to exert a neuroprotective effect, resulting in a lower risk of central nervous disorders [15]. We introduced 13-hydroperoxyoctadecadienoate (13-HPODE), a primary product of lipid peroxidation, as an inducer of oxidative stress in differentiated PC-12 cells [16]. Q3GA significantly suppressed reactive oxygen species (ROS) formation in cells when cells were preincubated with Q3GA and then treated with 13-HPODE. We concluded that antioxidative metabolites of quercetin can protect nerve cells from attack by lipid hydroperoxides. It is uncertain if quercetin metabolites accumulate in the target site of brain tissues by crossing the blood-brain barrier. Recent studies using rodents clarified that dietary
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Fig. 2. Proposed mechanism for the prevention of LDL oxidation by flavonoid metabolites circulating in the bloodstream. (1) Free inactive metabolite is released from plasma albumin. It permeates into the intima when endothelial cells are exposed to oxidative stress. (2) Aglycone is formed from glucuronide metabolites by the enhanced activity of β-glucuronidase. (3) Aglycone effectively scavenges ROS, resulting in the prevention of formation of oxidative LDLs.
Inactive metabolites Monocyte
Inactive metabolites (1)
Adhesion Endothelial cell
Macrophage
Deconjugation (2) Active aglycone
Albumin
LDL NADPH oxidase Lipoxygenase MPO (3) ROS
Phagocytosis
Oxidized LDL
Foam cell
quercetin could accumulate as its metabolites in brain tissue, although its level was much lower than those found in other organs such as the lung and liver [17].
Antioxidant Activity of Quercetin Metabolites in the Vascular System
The ‘oxidized low-density lipoprotein (LDL) hypothesis’ has been frequently used to explain an initial event of atherosclerosis in which oxidized LDL is specifically incorporated into macrophages via scavenger receptors, resulting in formation of foam cells [18]. Antioxidative flavonoids are expected to act as antiatherosclerotic food factors by inhibiting LDL oxidation in the vascular system. In vitro studies clearly demonstrated that catechol type flavonoids can act as efficient antioxidants against LDL oxidation. Catechol type flavonoids are mainly present as inactive metabolites in plasma at a lower concentration than that expected to exert effective inhibition [19]. It is therefore unlikely that the intake of flavonoids contributes to antioxidant defense in plasma [20]. We found that consumption of quercetin-rich food failed to enhance the antioxidant activity of the albumin fraction against aqueous radical-induced LDL oxidation, even though quercetin metabolites are mainly distributed in the albumin fraction of human plasma [21]. This result strongly suggests that the antioxidant effect of quercetin metabolites works not in the plasma but in the target site of the vascular system, where they accumulate selectively. In a study using high cholesterol-fed rabbits, we found that quercetin metabolites accumulated in aorta tissue selectively, and suppressed deposition of cholesteryl
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ester hydroperoxides [22]. This is the first evidence that dietary flavonoids target blood vessels as antioxidants in an animal model of atherosclerosis. Quercetin metabolites may transfer into blood vessels from plasma albumin when the vessel is abnormally loaded with cholesterol. We recently demonstrated that the antioxidative quercetin metabolite Q3GA accumulates in human atherosclerotic plaques [23]. This metabolite was found to be concentrated in macrophage-derived foam cells. ECG was also selectively detected in macrophage-derived foam cells [24]. Antioxidative flavonoids may be incorporated into activated macrophages as the terminal target in the vascular system. The endothelial cell seems to be an alternative target for antioxidative flavonoids because Q3GA was found to effectively inhibit endothelial cell-dependent LDL oxidation [23]. NADPH oxidase activity which generates O2•– and/or lipoxygenase activity that can induce LDL lipid peroxidation may have a critical role in endothelial cell-derived LDL oxidation. Antioxidative flavonoids are expected to approach the site of ROS attack, thereby inhibiting LDL oxidation. Myeloperoxidase (MPO) is an oxidative enzyme in neutrophils and is activated in inflammation. It may also participate in the ROS generation responsible for LDL oxidation in the vascular system. MPO is inhibited by quercetin aglycone and Q3GA [25]. The catechol group may be required for the inhibition of MPO activity. β-Glucuronidase-mediated deconjugation of flavonoid metabolites may occur in inflammation [26]. Release of active aglycone containing an o-dihydroxyl structure from inactive metabolites may be an alternative route for dietary flavonoids to exert their antioxidant activity in the vascular system (fig. 2).
Conclusion
Flavonoids were recently suggested to act as pro-oxidants which induce the expression of antioxidant enzymes such as superoxide dismutase by enhancing the cellular redox signaling pathway [5]. The pro-oxidant activity of flavonoids means ROS generation via autocatalytic oxidation or peroxidase reaction of the catechol group to semiquinone radicals [27]. This event may contribute to the oxidative damage of cells [28]. The final product of catechol type flavonoids after free radical scavenging, o-quinine, is highly reactive with the protein thiol group, resulting in toxic effects [29, 30]. Such toxic effects of catechol type flavonoids have been observed in in vitro culture cells, but not in in vivo studies. Metabolic conversion to their conjugated and O-methylated derivatives substantially attenuates their physicochemical and biological properties [12]. Accumulation of flavonoid metabolites in the appropriate target site is probably required to exert their antioxidant activity. Conversion of inactive metabolites to active aglycones via a deconjugation reaction in the target site may be a key process for efficient exertion of their antioxidant activity (fig. 3). The significance of dietary flavonoids as antioxidants in vivo is much more complicated than that expected from in vitro assays.
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Quinone reductase (?) Active o-semiquinone radical
Catechol e, H+
OH
O• R
Conjugation UGT
O
e, H+
OH
OH R
o-quinone
Deconjugation -glucuridase
OH R’-S
OH
O R
R H2O2 generation Cellular redox signaling pathway
OH O-glucuronic acid
Protein-SH
Arylation of protein thiols Toxic effect
Expression of antioxidative enzymes
R Metabolite Inactive
Cellular oxidative damage
Fig. 3. Possible mechanism of pro-oxidants and the toxic effect of catechol type flavonoids. Their toxic effect is found in only in a cell culture system. Catechol type flavonoids are mostly converted into noncatechol type metabolites by conjugation or O-methylation to the o-dihydroxyl group during absorption in the digestive tract. The ratio of active aglycone to inactive metabolites in the target site is regulated by the balance between conjugation and deconjugation reaction. Regulation of deconjugation reaction may be crucial for potential antioxidant activity of dietary flavonoids in vivo. UGT = Uridine-5’-diphosphoglucuronosyltransferase.
References 1 Crozier A, Jaganath IB, Cliford MN: Phenols, polyphenols and tannins: an overview; in Crozier A, Clifford MN, Ashihara H (eds): Plant Secondary Metabolites. Oxford, Blackwell, 2006, pp 1–24. 2 Hooper L, Kroon PA, Rimm EB, Cohn JS, Harvey I, Le Cornu KA, Ryder JJ, Hall WL, Cassidy A: Flavonoids, flavonoid-rich foods, and cardiovascular risk: a meta-analysis of randomized controlled trials. Am J Clin Nutr 2008;88:38–50. 3 Rice-Evans CA, Miller NJ, Paganga G: Structureantioxidant activity relationships of flavonoids and phenolic acids. Free Radic Biol Med 1996;20:933– 956. 4 Terao J: Dietary flavonoids as antioxidants in vivo: conjugated metabolites of (-)epicatechin and quercetin participate in antioxidative defense in blood plasma. J Med Invest 1999;46:159–168.
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5 Virgili F, Marino M: Regulation for cellular signals from nutritional molecules; a specific role for phytochemicals, beyond antioxidant activity. Free Radic Bol Med 2008;45:1205–1216. 6 Bors W, Heller W, Michel C, Saran M: Flavonoids as antioxidants: determination of radical-scavenging efficiencies; in Packer L, Glazer AN (eds): Methods in Enzymology. San Diego, Academic Press, 1990, vol 186, pp 343–355. 7 Nanjo F, Goto K, Seto R, Suzuki M, Sakai M, Hara Y: Scavenging effects of tea catechins and their derivatives on 1,1-diphenyl-2-picrylhydrazyl radical. Free Radic Biol Med 1996;21:895–902. 8 Murakami A, Ashida H, Terao J: Multitargeted cancer prevention by quercetin. Cancer Lett 2008;269: 315–325.
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9 Uekusa Y, Kamihara M, Nakayama T: Dynamic behavior of tea catechins interacting with lipid membranes as determined by NMR spectroscopy. J Agric Food Chem 2007;55:9986–9992. 10 Terao J, Piskula M: Flavonoid as inhibitors of lipid peroxidation in membranes; in Rice-Evans CA, Packer L (eds): Flavonoids in Health and Disease. New York, Marcel Dekker, 1997, pp 277–294. 11 Scalbert A, Williamson G: Dietary intake and bioavailability of polyphenols. J Nutr 2000;130; 2073S–2085S. 12 Day AJ, Mellon F, Barron D, Sarrazin G, Morgan MRA, Williamson G: Human metabolism of dietary flavonoids: identification of plasma metabolites of quercetin. Free Radic Res 2001;35:941–952. 13 Williamson G, Barron D, Shimoi K, Terao J: In vitro biological properties of flavonoid conjugates found in vitro. Free Radic Res 2005;39:457–459. 14 Moon JH, Tsushida T, Nakahara K, Terao J: Identification of quercetin 3-O-β-glucuronide as an antioxidative metabolite in rat plasma after oral administration of quercetin. Free Radic Biol Med 2001;30:1274–1285. 15 Youdin KA, Shukitt-Hale B, Joseph JA: Flavonoids and the brain: interactions at the blood-brain barrier and their physiological effects on the central nervous system. Free Radic Biol Med 2004;37:1683– 1693. 16 Shirai M., Kawai Y, Yamanishi R, Kinoshita T, Chuman H, Terao J: Effect of a conjugated quercetin metabolite, quercetin 3-glucuronide, on lipid hydroperoxide-dependent formation of reactive oxygen species in differentiated PC-12 cells. Free Radic Res 2005;40;1047–1059. 17 de Boer VC, Dihal AA, van den Wounde H, Arts IC, Wolffram S, Alink GM, Rietjens IMCM, Keijer J, Hollman PCH: Tissue distribution of quercetin in rats and pigs. J Nutr 2005;135:1718–1725. 18 Glass CK, Witztum JL: Atherosclerosis: the road ahead. Cell 2001;104:503–516. 19 Moon J-H, Nakata R, Oshima S, Inakuma T, Terao J: Accumulation of quercetin conjugates in blood plasma after the short-term ingestion of onion by women. Am J Physiol 2000;279:R461–R467. 20 Lotito SB, Frei B: Consumption of flavonoid-rich foods and increased plasma antioxidant capacity in humans: cause, consequence, or epiphenomenon? Free Radic Biol Med 2006;41:1727–1746.
21 Murota K, Hotta A, Ido H, Kawai Y, Moon J-H, Sekido K, Hayashi H, Inakuma T, Terao J: Antioxidant capacity of albumin-bound quercetin metabolites after onion consumption in humans. J Med Invest 2007;54:370–374. 22 Kamada C, da Silva EL, Ohnishi-Kameyama M, Moon J-H, Terao J: Attenuation of lipid peroxidation and hyperlipidemia by quercetin glucoside in the aorta of high cholesterol-fed rabbit. Free Radic Res 2005;39:185–194. 23 Kawai Y, Nishikawa T, Shiba T, Saito S, Murota K, Shibata N, Kobayashi M, Kanayama M, Uchida K, Terao J: Macrophage as a target of quercetin glucuronides in human atherosclerotic arteries. J Biol Chem 2008;283:9424–9434. 24 Kawai Y, Tanaka H, Murota K, Naito M, Terao J: (–)-Epicatechin gallate accumulates in foamy macrophages in human atherosclerotic aorta: implication in the anti-atherosclerotic actions of tea catechins. Biochem Biophys Res Comm 2008;374: 527–532. 25 Shiba Y, Kinoshita T, Chuman H, Taketani Y, Takeda E, Kato Y, Naito M, Kawabata K, Ishisaka A, Terao J, Kawai Y: Flavonoids as substrates and inhibitors of myeloperoxidase: molecular actions of aglycone and the metabolites. Chem Res Toxicol 2008;21:1600– 1609. 26 Shimoi K, Saka N, Nozawa R, Sato M, Amano I, Nakayama T, Kinae N: Deglucuronidation of a flavonoid, luteolin monoglucuronide, during inflammation. Drug Metab Dispos 2001;29:1521–1524. 27 Gouphua C, Sofic E, Prior RL: Antioxidant and prooxidant behavior of flavonoids: structure-activity relationships. Free Radic Biol Med 1997;22:749– 760. 28 Halliwell B: Are polyphenols antioxidants or prooxidants? What do we learn from cell culture and in vivo studies? Arch Biochem Biophys 2008;476:107– 112. 29 Boots A, Haenen GRMM, Bast A: Health effects of quercetin: from antioxidant to nutraceutical. Eur J Pharmacol 2008;585:325–337. 30 Metodiewa D, Jaiswal AK, Cenas N, Dickancaite E, Segura-Aguilar J: Quercetin may act as a cytotoxic prooxidant after its metabolic activation to semiquinone and quinoidal products. Free Radic Biol Med 1999;26:107–116.
Professor Junji Terao Department of Food Science, Graduate School of Nutritional and Bioscience, University of Tokushima Tokushima 770-8503 (Japan) Tel. +81 88 633 7081, Fax +81 88 633 1089, E-Mail
[email protected]
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Inflammatory Components of Adipose Tissue as Target for Treatment of Metabolic Syndrome Rina Yu ⭈ Chu-Sook Kim ⭈ Ji-Hye Kang Department of Food Science and Nutrition, University of Ulsan, Ulsan, South Korea
Abstract Obesity is an independent risk factor in the etiology of various metabolic diseases such as insulin resistance, type 2 diabetes and cardiovascular diseases. In this chapter, we discuss obesity-induced inflammation as a potential link with obesity-related metabolic syndrome, and discuss how obesityrelated inflammatory components such as immune cells, and cytokines/chemokines and adipocytokines, provoke obesity-related pathologies. In particular, we focus on the hypothesis that anti-inflammatory food factors/phytochemicals may be useful for inhibiting the initiation and development of obesity-induced inflammation and metabolic syndrome. Copyright © 2009 S. Karger AG, Basel
Obesity is a low-grade systemic chronic inflammatory condition. Obesity-induced inflammation depends on a set of inflammatory components similar to those involved in classical inflammation and is characterized by increased infiltration of immune cells such as macrophages and T cells into adipose tissue, abnormal cytokine/chemokine production, increased levels of inflammatory mediators, and the activation of inflammatory signaling pathways (fig. 1) [1, 2]. Obesity-induced inflammation, which is also referred to as metaflammation (metabolically triggered inflammation) [2], is considered a potential mechanism linking together obesity-induced metabolic pathologies such as insulin resistance, type 2 diabetes, fatty liver disease, atherosclerosis, some immune disorders, and several types of cancer [1, 2]. This chapter focuses on obesity-related inflammatory components including adipose immune cells, cytokines/chemokines, adipocytokines, and inflammatory crosstalk between adipose tissue cells, and their link to the obesity-related pathologies. In addition, the potential modulatory activities of food factors/phytochemicals targeting obesity-related inflammatory components will be considered.
Chronic inflammatory condition Metaflammation Obesity
Inflammation
Insulin resistance Type 2 diabetes mellitus Cardiovascular disease
Monocyte/Macrophage Cytokines/Chemokines/adipocytokines (TNF␣ , IL-6, MCP-1 etc.) Complement, ROS, NO, Inflammatory signaling pathway (IKK, NF-B, JNK), TLRs
Fig. 1. Inflammatory components of adipose tissue and metabolic syndrome.
Adipose Tissue Macrophages
Adipose tissue is a heterogeneous population of cells consisting of adipocytes and stromal vascular cells comprising various cell types including preadipocytes, immune cells (macrophages, T and B lymphocytes, etc.) and endothelial cells. Obesity alters the profile of immune cells, and the changes that take place are directly attributable to the enhanced inflammatory response in the adipose tissue. Macrophages are thought to be the key immune cells responsible for adipose tissue inflammatory responses. Accumulation of adipose macrophages is prominent in both obese humans and rodents, and the number of macrophages in adipose tissue correlates with body mass index, adipocyte size and total body fat [3, 4]. Proinflammatory molecules, such as inducible nitric oxide synthase, cytokines/chemokines such as TNF-α and monocyte chemoattractant protein-1 (MCP-1) are prominently unregulated in adipose tissue macrophages in obesity [3–5], and it appears that the adipose tissue macrophages are the primary source of the circulating inflammatory molecules found in the obese state [6]. Accumulation of the macrophages in adipose tissue and activation of these cells amplify inflammatory responses by releasing inflammatory mediators such as cytokines/chemokines, and contribute not only to a chronic low grade inflammatory milieu in obesity but also to the obesity-related complications. Moreover, the macrophage-derived inflammatory cytokines inhibit adipocyte differentiation, leading to adipocyte hypertrophy and misregulation of the production of adipocytokines, which are believed to cause the development of both insulin resistance and type 2 diabetes. Macrophages display heterogeneity of function/activation, in the form of the alternative states of polarization, M1 and M2 [7], and it has been suggested that phenotypic changes among the adipose tissue macrophages are key components of the obesityinduced inflammatory microenvironment and may contribute to the development of obesity-related pathologies. M1 or ‘classically activated’ macrophages are induced by proinflammatory mediators (LPS and IFN-γ); they produce increased amounts
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of proinflammatory cytokines (TNF-α, IL-6, IL-12), and generate reactive oxygen species such as NO via iNOS. M2 or ‘alternatively activated’ macrophages are characterized by low proinflammatory cytokine expression but release high levels of the anti-inflammatory cytokine IL-10. M1 macrophages are thought to induce inflammatory responses, whereas M2 macrophages participate in inhibition of these responses and in promotion of tissue repair. Interestingly, recent studies have shown that the number of M1 or ‘classically activated’ inflammatory macrophages increases in obese adipose tissue [8]. Lumeng et al. [8] demonstrated that adipose tissue macrophages from obese mice fed a high-fat diet displayed increasing expression of genes such as TNF-α and iNOS that are characteristic of M1 macrophages, while macrophages from the adipose tissue of lean mice expressed genes such as Ym1, arginase 1 and IL-10, characteristic of M2 macrophages. These findings indicate that obesity leads to a shift in the activation state of adipose tissue macrophages from the M2-polarized state to the M1 proinflammatory state.
Adipose Tissue-Derived Inflammatory Molecules
Adipose tissue not only stores energy as fat but also secretes various biologically active proteins that regulate cell functions and energy metabolism via a network of autocrine and paracrine signaling pathways. Adipose tissue-derived biologically active proteins called adipocytokines play an important role in regulating energy balance, food intake, lipid and glucose metabolisms, insulin sensitivity, and the vascular microenvironment. They include inflammatory cytokines/chemokines (e.g. TNF-α, IL-6, MCP-1, etc.) and adiponectin, which has anti-inflammatory properties. Misregulation of the adipocytokines accompanying abnormal expansion of adipose tissue is a hallmark of obesity, and thus is implicated not only in altering the inflammatory microenvironment in obesity, but also in triggering the development of obesity-related pathologies.
Tumor Necrosis Factor-α TNF-α, a major proinflammatory cytokine involved in both acute and chronic inflammatory responses, is a representative adipocytokine related to obesity-induced metabolic syndrome. TNF-α acts as a powerful inducer of other proinflammatory adipocytokines such as IL-6, MCP-1, leptin, and PAI-1, and thus contributes to inflammatory conditions linked to obesity. Chronic exposure to TNF-α decreases the expression of the insulin-sensitive glucose transporter 4 and insulin receptor substrate-1 (IRS-1), suppresses tyrosine phosphorylation of IRS-1, and enhances serine phosphorylation of IRS-1, thereby impairing insulin signaling [9, 10]. The level of TNF-α in adipose tissue increases in obese rodents as well as in obese patients, and
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the increased TNF-α was shown to be directly associated with obesity-induced insulin resistance [11]. It was subsequently demonstrated that neutralization of TNF-α and/or TNF-α gene deficiency can counteract obesity-induced insulin resistance [9, 12]. These findings provided the basis for linking obesity to inflammation and/or obesity-induced insulin resistance.
Interleukin-6 IL-6 is a multifunctional cytokine that regulates the immune response, hematopoiesis, the acute phase response and inflammation, and plays a role in the pathogenesis of insulin resistance. IL-6 inhibits insulin signal transduction in hepatocytes by affecting the suppressor of cytokine signaling-3 pathway [13]. Circulating IL-6 levels increase in human obesity and insulin resistance [14], and weight loss results in a decrease in IL-6 in both adipose tissue and serum [15]. IL-6 enhances the release of adhesion molecules by the endothelium, and the hepatic release of fibrinogen, and exerts procoagulant effects on platelets [16]. As with TNF-α, IL-6 has a role in the development of obesity-related pathologies such as insulin resistance and atherosclerosis.
Monocyte Chemoattractant Protein-1 Chemokines such as MCP-1, a member of the CC chemokine superfamily, play pivotal roles in the trafficking and activation of monocytes/macrophages and are thus implicated in various inflammatory conditions. We and others have shown that MCP-1 release from adipocytes is significantly greater in obese mice than in nonobese mice, and is markedly increased when adipocytes are cocultured with macrophages [5, 17, 18]. When we examined various fat depots (e.g. mesenteric, epididymal, renal, and subcutaneous adipose tissue), we found that the levels of MCP-1 mRNA and protein and the amounts of MCP-1 protein released were significantly greater in the adipose tissues of obese mice than in that from nonobese mice [5]. Moreover, medium conditioned by mesenteric adipose tissue induced the highest extent of macrophage migration and strongly induced macrophage production of proinflammatory mediators such as nitric oxide and TNF-α. Neutralization of the MCP-1 in the conditioned medium significantly inhibited these effects. These findings suggest that MCP-1 plays a crucial role in the adipose tissue inflammatory response by inducing monocyte/macrophage trafficking and by activating cells in the adipose tissues, and may be closely associated with visceral obesity-related complications. In addition, it appears that MCP-1 from the hypertrophic adipocytes in obese adipose tissue can also trigger infiltration of macrophages into the adipose tissue and then induce the macrophages to release inflammatory mediators [5]. In this regard, hypertrophic adipocytes themselves play a major role in initiating inflammatory responses in obese adipose tissue, whereas
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macrophages promote the inflammatory condition via crosstalk with the adipocytes involving inflammatory mediators such as MCP-1 [5, 19]. MCP-1 may therefore be a useful therapeutic target for modulating visceral obesity-related diseases.
Adiponectin Adiponectin is a representative adipocytokine which is exclusively expressed in adipocytes/adipose tissue and released into circulation. It has various metabolic functions: it enhances fat oxidation, and thus decreases the intracellular triglyceride content of the liver and muscles; it increases insulin sensitivity by upregulating expression of the insulin receptor and IRS-1 in skeletal muscle, as well as activation of 5⬘-AMPactivated protein kinase and phosphorylation of acetyl-CoA carboxylase [20]; it also protects against the development of atherosclerosis, and is implicated in the modulation of the expression of adhesion molecules and nuclear factor-kB signaling in vascular endothelial cells [20]. In human and rodents, plasma adiponectin concentrations decrease with obesity, and their levels are positively correlated with whole body insulin sensitivity.
Functional Food Factors/Phytochemicals Targeting Obesity-Related Inflammatory Components
As already mentioned, misregulation of cytokine/chemokine and adipocytokine release from adipose tissue and/or adipocytes/macrophages causes obesity-induced inflammation and subsequently triggers the development of obesity-related pathologies such as insulin resistance, type 2 diabetes, and cardiovascular diseases. Hence, food factors and/or phytochemicals that can suppress the inflammatory responses of adipose cells may be useful in preventing or reducing the development of obesityrelated pathologies (fig. 2). Capsaicin, a spicy ingredient of hot peppers, elicits anti-inflammatory activities. It inhibits the development of carrageen-induced paw inflammation and adjuvantinduced arthritis [21, 22], as well as ethanol-induced inflammation, by inhibiting the release of proinflammatory mediators [23]. It also reduces the inflammatory responses of macrophages by inhibiting IκB-α degradation [24]. When we investigated if it inhibited the activities of adipose tissue macrophages, we found that it reduced macrophage migration in response to MCP-1 and to medium conditioned by mesenteric adipose tissue, as well as macrophage production of inflammatory mediators such as nitric oxide, TNF-α, and MCP-1 in response to the conditioned medium, indicating that it has the potential to suppress the inflammatory response amplified by adipose tissue macrophages in obese individuals. We further examined whether capsaicin modulates the production of adipocytokines (i.e. IL-6, MCP-1,
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Macrophage
MCP-1 IL-6
TNF␣ NO Adiponectin Preadipocyte
Large adipocyte
Food factors Phytochemicals
Metabolic syndrome Insulin resistance Type 2 diabetes mellitus Atherosclerosis
Fig. 2. Functional food factors/phytochemicals targeting obesity-related inflammatory components.
and adiponectin) by obese adipose tissues and isolated adipocytes, and whether it alters the inflammatory responses of obese adipose tissue macrophages. Interestingly, it decreased the amounts of IL-6 and MCP-1 released from obese fat tissues and fat cells, and increased the amounts of adiponectin released [25]. This dual action may be favorable for treatment of obesity-induced insulin resistance or atherosclerosis. We have tested whether capsaicin regulates inflammatory responses at the transcriptional level. It has been shown that PPARγ ligands such as thiazolidinediones, which are used as antidiabetic drugs, suppress expression of TNF-α, IL-6, and MCP-1 genes and release of the corresponding proteins, and enhance adiponectin release [26], and that PPARγ ligand inhibits monocyte/macrophage chemotaxis and activation [27, 28]. Moreover, capsaicin can act as a PPARγ ligand, resulting in inhibition of TNF-α production by LPS-stimulated macrophages [27]. Like thiazolidinediones, capsaicin significantly inhibits MCP-1 and IL-6 production, and enhances adiponectin production by adipocytes, indicating that its dual effect on adipocytokine release from adipocytes and on macrophage responses may result from its acting as a ligand of PPARγ. We previously showed that capsaicin inhibits the NF-κB pathway by inhibiting IκB degradation in LPS-stimulated macrophages, thus leading to suppression of the release of proinflammatory mediators [24]: it inhibited NF-κB activation in 3T3-
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L1 adipocytes treated with mesenteric adipose tissue-derived or macrophage-derived conditioned media. These findings strongly suggest that capsaicin affects the release of inflammatory adipocytokines such as IL-6 and MCP-1 by modulating the proinflammatory transcription factor NF-κB as well as PPARγ [25]. We have also confirmed that the beneficial dual action of capsaicin in vitro is relevant in vivo. That is, we found that intraperitoneal injection of capsaicin (2 mg/kg, body weight) significantly decreased transcripts of MCP-1 and IL-6 in the adipose tissue of obese mice, and increased adiponectin transcripts [25], and that macrophage infiltration into the adipose tissue of the obese mice was significantly decreased. Furthermore, we demonstrated that the capsaicin treatment markedly countered not only the obesity-induced inflammation but also the impaired glucose tolerance of obese mice [25]. Taken together, these findings suggest that capsaicin may be a useful phytochemical for attenuating obesity-induced inflammation and obesity-related complications such as insulin resistance. We have also tested whether other naturally occurring phytochemicals with antiinflammatory properties could be used against obesity-induced inflammation. We found that active components of spices (i.e. diallyl disulfide, allyl isothiocyanate, piperine, zingerone, curcumin) markedly suppressed the migration of macrophages and activation of cells induced by mesenteric adipose tissue-conditioned medium [29]. Allyl isothiocyanate, zingerone, and curcumin inhibited the production of proinflammatory mediators such as TNF-α and nitric oxide, and release of MCP-1 from 3T3-L1 adipocytes. Our findings suggest that the spice-derived components suppress obesityinduced inflammatory responses by inhibiting the accumulation of macrophages in adipose tissue or their activation, and by inhibiting MCP-1 release by adipocytes [30], indicating that these components also have the potential to improve chronic inflammatory conditions associated with obesity. In addition to the spice-derived components, we found that phytochemicals such as naringenin chalcone, citrus auraptene, and anthocyanins also suppressed inflammatory responses in obese adipose tissue by inhibiting MCP-1 and macrophage infiltration [30–32], indicating that such food factors also counteract obesity-induced inflammatory responses and obesity-related pathologies.
Conclusion
Obesity-induced inflammation plays an important role in the development of obesity-related pathologies such as insulin resistance, type 2 diabetes, atherosclerosis, and certain types of cancer. The inflammatory components of adipose tissue consist of immune cells, cytokines/chemokines, adipocytokines, and inflammatory signaling molecules, and it appears that the crosstalk between the components plays a crucial role in the development of the pathologies. The obesity-associated inflammatory components may provide attractive targets for therapy, and food-derived factors/
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phytochemicals may be useful in the prevention and/or management of obesityrelated inflammatory pathologies.
References 1 Shoelson SE, Lee J, Goldfine AB: Inflammation and insulin resistance. J Clin Invest 2006;116:1793– 1801. 2 Hotamisligil GS: Inflammation and metabolic disorders. Nature 2006;444:860–867. 3 Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Ferrante AW Jr: Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest 2003;112:1796–1808. 4 Xu H, Barnes GT, Yang Q, et al: Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance. J Clin Invest 2003; 112:1821–1830. 5 Yu R, Kim CS, Kwon BS, Kawada T: Mesenteric adipose tissue-derived monocyte chemoattractant protein-1 plays a crucial role in adipose tissue macrophage migration and activation in obese mice. Obesity (Silver Spring) 2006;14:1353–1362. 6 Heilbronn LK, Campbell LV: Adipose tissue macrophages, low grade inflammation and insulin resistance in human obesity. Curr Pharm Des 2008;14: 1225–1230. 7 Gordon S, Taylor PR: Monocyte and macrophage heterogeneity. Nat Rev Immunol 2005;5:953–864. 8 Lumeng CN, Bodzin JL, Saltiel AR: Obesity induces a phenotypic switch in adipose tissue macrophage polarization. J Clin Invest 2007;117:175–184. 9 Hotamisligil GS, Shargill NS, Spiegelman BM: Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science 1993;259:87–91. 10 Hotamisligil GS: Inflammatory pathways and insulin action. Int J Obes Relat Metab Disord 2003; 27(suppl 3):S53–S55. 11 Hotamisligil GS, Arner P, Caro JF, Atkinson RL, Spiegelman BM: Increased adipose tissue expression of tumor necrosis factor-alpha in human obesity and insulin resistance. J Clin Invest 1995;95: 2409–2415. 12 Uysal KT, Wiesbrock SM, Marino MW, Hotamisligil GS: Protection from obesity-induced insulin resistance in mice lacking TNF-alpha function. Nature 1997;389:610–614. 13 Senn JJ, Klover PJ, Nowak IA, et al: Suppressor of cytokine signaling-3 (SOCS-3), a potential mediator of interleukin-6-dependent insulin resistance in hepatocytes. J Biol Chem 2003;278:13740–13746.
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14 Vozarova B, Weyer C, Hanson K, Tataranni PA, Bogardus C, Pratley RE: Circulating interleukin-6 in relation to adiposity, insulin action, and insulin secretion. Obes Res 2001;9:414–417. 15 Bastard JP, Jardel C, Bruckert E, et al: Elevated levels of interleukin 6 are reduced in serum and subcutaneous adipose tissue of obese women after weight loss. J Clin Endocrinol Metab 2000;85:3338–3342. 16 Yudkin JS, Kumari M, Humphries SE, MohamedAli V: Inflammation, obesity, stress and coronary heart disease: is interleukin-6 the link? Atherosclerosis 2000;148:209–214. 17 Bruun JM, Lihn AS, Pedersen SB, Richelsen B: Monocyte chemoattractant protein-1 release is higher in visceral than subcutaneous human adipose tissue (AT): implication of macrophages resident in the AT. J Clin Endocrinol Metab 2005;90: 2282–2289. 18 Fain JN, Madan AK, Hiler ML, Cheema P, Bahouth SW: Comparison of the release of adipokines by adipose tissue, adipose tissue matrix, and adipocytes from visceral and subcutaneous abdominal adipose tissues of obese humans. Endocrinology 2004;145: 2273–2282. 19 Suganami T, Nishida J, Ogawa Y: A paracrine loop between adipocytes and macrophages aggravates inflammatory changes: role of free fatty acids and tumor necrosis factor alpha. Arterioscler Thromb Vasc Biol 2005;25:2062–2068. 20 Yamauchi T, Hara K, Kubota N, et al: Dual roles of adiponectin/Acrp30 in vivo as an anti-diabetic and anti-atherogenic adipokine. Curr Drug Targets Immune Endocr Metabol Disord 2003;3:243–254. 21 Ahmed M, Bjurholm A, Srinivasan GR, et al: Capsaicin effects on substance P and CGRP in rat adjuvant arthritis. Regul Pept 1995;55:85–102. 22 Marsh D, Dickenson A, Hatch D, Fitzgerald M: Epidural opioid analgesia in infant rats II: responses to carrageenan and capsaicin. Pain 1999;82:33–38. 23 Park JS, Choi MA, Kim BS, Han IS, Kurata T, Yu R: Capsaicin protects against ethanol-induced oxidative injury in the gastric mucosa of rats. Life Sci 2000;67:3087–3093. 24 Kim CS, Kawada T, Kim BS, et al: Capsaicin exhibits anti-inflammatory property by inhibiting IkB-a degradation in LPS-stimulated peritoneal macrophages. Cell Signal 2003;15:299–306.
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25 Kang JH, Kim CS, Han IS, Kawada T, Yu R: Capsaicin, a spicy component of hot peppers, modulates adipokine gene expression and protein release from obese-mouse adipose tissues and isolated adipocytes, and suppresses the inflammatory responses of adipose tissue macrophages. FEBS Lett 2007; 581: 4389–4396. 26 Ghanim H, Dhindsa S, Aljada A, Chaudhuri A, Viswanathan P, Dandona P: Low dose rosiglitazone exerts an anti-inflammatory effect with an increase in adiponectin independently of free fatty acid (FFA) fall and insulin sensitization in obese type 2 diabetics. J Clin Endocrinol Metab 2006;91:3553– 3558. 27 Park JY, Kawada T, Han IS, et al: Capsaicin inhibits the production of tumor necrosis factor alpha by LPS-stimulated murine macrophages, RAW 264.7: a PPARgamma ligand-like action as a novel mechanism. FEBS Lett 2004;572:266–270. 28 Tanaka T, Fukunaga Y, Itoh H, et al: Therapeutic potential of thiazolidinediones in activation of peroxisome proliferator-activated receptor gamma for monocyte recruitment and endothelial regeneration. Eur J Pharmacol 2005;508:255–265.
29 Woo HM, Kang JH, Kawada T, Yoo H, Sung MK, Yu R: Active spice-derived components can inhibit inflammatory responses of adipose tissue in obesity by suppressing inflammatory actions of macrophages and release of monocyte chemoattractant protein-1 from adipocytes. Life Sci 2007;80:926– 931. 30 Kuroyanagi K, Kang MS, Goto T, et al: Citrus auraptene acts as an agonist for PPARs and enhances adiponectin production and MCP-1 reduction in 3T3-L1 adipocytes. Biochem Biophys Res Commun 2008;366:219–225. 31 Hirai S, Kim YI, Goto T, et al: Inhibitory effect of naringenin chalcone on inflammatory changes in the interaction between adipocytes and macrophages. Life Sci 2007;81:1272–1279. 32 Choe MR, Kang JH, Yoo H, Choe SY, Yang CH, Kom MO, Yu R: Cyanidin and cyanidin-3-O-b-Dglucoside suppress the inflammatory responses of obese adipose tissue by inhibiting the release of chemokines MCP-1 and MRP-2. J Food Sci Nutr 2007;12:148–153.
Dr. Rina Yu Department of Food Science and Nutrition, University of Ulsan Mugeo-dong, Nam-ku Ulsan 680-749 (South Korea) Tel. +82 52 259 2372, Fax +82 52 259 1698, E-Mail *
[email protected]
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Life-style Related Diseases Yoshikawa T (ed): Food Factors for Health Promotion. Forum Nutr. Basel, Karger, 2009, vol 61, pp 104–116
Soybean Isoflavones in Bone Health Yoshiko Ishimi Project for Bio-index, Nutritional Epidemiology Program, National Institute of Health and Nutrition, Tokyo, Japan
Abstract Soybean isoflavones are structurally similar to estrogen, bind to estrogen receptors, and exhibit weak estrogenic activity. It has been reported that isoflavones play an important role in the prevention of hormone-dependent diseases, including osteoporosis, cardiovascular diseases, cancer, and postmenopausal syndrome. There are many researches indicating isoflavones prevent bone loss caused by estrogen deficiency in animal models. Furthermore, it has been demonstrated that a combination of isoflavone treatment and exercise cooperatively prevented bone loss in the estrogendeficient status. Epidemiological studies demonstrated the relationship between the lower incidence of osteoporosis in Asian women and a diet rich in soy foods. Although a number of observational studies confirm the findings from the animal studies, the results from intervention studies are still controversial. One of the potential reasons for these inconsistencies could be individual differences in the isoflavone metabolism. Recently, it has been suggested that the clinical effectiveness of isoflavones might partly depend on the ability to produce equol, a gut bacterial metabolite of daidzein showing stronger estrogenic activity than the predominant isoflavones. Several candidate bacteria responsible for equol production have been suggested, for example Lactococcus 20-92 strain. From these findings, food factors enhancing equol production have received great deal of attention recently. On the other hand, safety assessment of isoflavones has been conducted by the Japanese Food Safety Commission. Further studies are required to address the numerous questions on the potential benefits, mechanisms of action, and safety of isoflavones. Copyright © 2009 S. Karger AG, Basel
Soybeans have been consumed in Asia since ancient times. Compared with Caucasians, the low incidence of heart disease, reproductive cancers, hip fracture, and climacteric symptoms in Asians has been considered to be associated with their high intake of soy foods. Recent growing interest in health and diet has led to an increased focus on soy foods and their functional components, e.g. isoflavones. Soybean isoflavones are structurally similar to estrogen, bind to estrogen receptors, and exhibit weak estrogenic activity. Isoflavones exert beneficial health effects by acting as antioxidants, tyrosine kinase and topoisomerase inhibitors as well as estrogenic activity. It has been reported that they play an important role in the prevention of chronic diseases, including osteoporosis, cardiovascular diseases, hormone-dependent cancer, and postmenopausal syndrome [1–5].
Osteoporosis is a skeletal disorder in which bone strength is compromised by the loss of bone density and bone quality. It is the leading cause of increased morbidity and functional loss in the elderly. Particularly postmenopausal women suffer from osteoporosis, which is part of the postmenopausal syndrome [6]. Although the treatment of postmenopausal osteoporosis is hormone replacement therapy, the reported side effects, such as development of hormone-dependent breast and uterine cancers [7], have prompted the use of alternative therapies. Soybean isoflavones have received considerable attention as alternatives to hormone replacement therapy, since the risk for side effects of isoflavone treatment seem to be low compared with hormone replacement therapy [8]. Epidemiological studies indicate that women who have high soy intake have less risk for osteoporosis than women who consume a typical Western diet [8]. A recent meta-analysis of 10 randomized controlled trials (RCTs) indicated that isoflavone intervention significantly attenuated bone loss in postmenopausal women [9]. However, the results from human studies are still controversial [10]. One of the potential reasons for these inconsistencies could be individual differences in isoflavone metabolism. Recent studies suggest that the clinical effectiveness of isoflavones on bone metabolism might be due to their ability to produce the metabolite equol in the intestine [11]. This chapter focuses on the current topics of research on isoflavones and their relationship to bone health.
Isoflavones and Their Metabolites
The major isoflavones contained in soy-based food products are daidzein, genistein, and glycitein (fig. 1). They exist mainly in the glycoside, acetyl glycoside, or malonyl glycoside forms in soy foods; hence, it becomes necessary to hydrolyze the glycosidic bonds for intestinal absorption to enable physiological activities. These glycosidic bonds are hydrolyzed by glycosidase produced by intestinal microflora such as Lactobacilli, Bacteroides and Bifidobacteria [12]. Furthermore, intestinal microflora affects the metabolism, wherein isoflavones are metabolized to equol, or O-desmethylangolensin (O-DMA) from their precursor daidzein (fig. 1). Recent studies suggest that the clinical effectiveness of isoflavones might be due to their ability to produce metabolites, such as dihydrodaidzein, tetrahydrodaidzein, equol and O-DMA in the intestine [13]. Particularly, equol, a metabolite of daidzein, has received considerable attention because its biological activities differ from those of its precursor [11]. The metabolites of genistein and glycitein were also primarily found in human urine (genistein: dihydrogenistein, 6⬘-OH-O-DMA [13], 4-hydroxyphenyl-2-propionic acid and phloroglucinol; glycitein: dihydroglycitein, 5⬘-methoxy-O-DMA and 6-methoxy-equol). However, the physiological activities of these metabolites are still unclear.
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O
HO
OH
O
OH
HO
Genistein HO
O
HO
O
O Equol
O
OH
Daidzein
HO
O
OH
Dihydrodaidzein HO
O
H3CO O
OH
OH
O OH
Glycitein
OH
O-DMA
Fig. 1. Molecular structures of isoflavone aglycone and daidzein metabolites.
Equol has higher estrogenicity, stronger antioxidative efficacy, and exhibits antiandrogenic properties [11]. Moreover, equol is a chiral molecule, which exists as enantiomers R (+)-equol and S (–)-equol. In humans, the metabolism of daidzein to equol results in the production of only S-equol. Interindividual variability in equol production may be unique to humans; all the animals including rats, mice, and chimpanzees, tested systematically excrete equol. Although O-DMA was found in 80–90% of a human population, equol was found in only 30–50% of the population [14]. This is because of individual differences in the intestinal microbiota responsible for equol production. Intestinal bacteria play a key role in isoflavone metabolism; young infants with undeveloped gut microflora do not produce equol, while germ-free animals also do not produce equol or O-DMA. Since some reports suggested a lower disease risk for equol producers than for nonproducers [11], there is a growing interest in certain bacterial strains that can produce equol. A number of strains involved in daidzein metabolism were identified. However, the identification of equol-producing bacteria is complicated. To date, only one lactic acid bacterium (Lactococcus 20-92 homologous to Lactococcus garvieae) has been identified that produces equol directly from daidzein without producing O-DMA [15]. Interestingly, the strain Lactococcus 20-92 can also cleave glycosidic bonds of daidzin; Uchiyama et al. [15] detected L. garvieae in the Italian cheese Toma Piemontese.
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Effects of Isoflavone on Bone Metabolism: Animal Studies
Estrogen deficiency is associated with increased bone turnover and acceleration of bone loss, which lead to an increased susceptibility to bone fracture. A number of studies have previously reported that soybean isoflavones, genistein and daidzein, dosedependently inhibited bone loss in both female and male osteoporotic animal models without causing notable effects on the reproductive organs [16]. Ishimi et al. [17] also determined the selective effects of genistein on B-lymphopoiesis and bone loss caused by estrogen deficiency in ovariectomized (OVX) mice. In this study, genistein (0.7 mg/ day s.c.) inhibited bone loss due to OVX without any uterine hypertrophy, which has been thought to be a side effect of estrogen administration. The dose which induced uterine hypertrophy was 10 times higher than that which inhibited bone loss. This suggests that genistein may be a natural selective estrogen receptor modulator (SERM). Interestingly, isoflavones inhibited bone loss in androgen-deficient male mice, indicating that soybean isoflavones prevent bone loss due to androgen deficiency in males. Similarly, equol could also inhibit bone loss due to OVX. Fujioka et al. [18] reported that administration of equol (0.5 mg/day s.c.) inhibited bone loss of the whole body and femur in OVX mice. Although E2 administration (0.03 μg/day s.c.) prevented OVX-induced bone loss from all regions, uterine hypertrophy occurred in E2-administered OVX mice [18]. These results suggest that similar to SERMs, isoflavones including equol inhibit bone loss apparently without estrogenic activity in the reproductive organs in estrogen-deficient animals. It is now recognized that one of the mechanisms by which estrogen deficiency causes bone loss is the stimulation of osteoclast formation, a process enhanced by several inflammatory cytokines, such as tumor necrosis factor-α and interleukin-1β. Furthermore, Nakamura et al. [19] recently reported a critical role for the osteoclastic estrogen receptor-α (ERα) in mediating estrogen-dependent bone maintenance in female mice. They selectively ablated ERα in differentiated osteoclasts [ERα(DeltaOc/ DeltaOc)] and found that ERα(DeltaOc/DeltaOc) females, but not males, exhibited trabecular bone loss, similar to the osteoporotic bone phenotype in postmenopausal women. Furthermore, estrogen induced apoptosis and upregulation of Fas ligand expression in osteoclasts of the trabecular bones of wild type but not ERα(DeltaOc/ DeltaOc) mice. The expression of ERα was also required for the induction of apoptosis by tamoxifen and estrogen in cultured osteoclasts. These results support a model in which estrogen regulates the life span of mature osteoclasts via the induction of the Fas/Fas ligand system, thereby providing an explanation for the osteoprotective function of estrogen as well as SERMs including isoflavones. On the other hand, isoflavones have been reported to inhibit bone resorption via other nonhormonal effects, including antioxidant activity, inhibition of tyrosine kinase and 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). In fact, statins, cholesterol-lowering agents that inhibit the activity of HMG-CoA reductase, induce bone formation and inhibit bone resorption both in vitro and in vivo.
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Akelel I: soy protein/4.4 mg Iso, 6M
Akelel I
Akelel II: soy protein/80.4 mg Iso, 6M
Akelel II
Anderson: soy protein/90 mg Iso,12M
Anderson
Arjmandi: soy protein/60 mg Iso, 12M
Arjmandi
Harkness I: soy protein/110 mg Iso, 6M
Harkness I
Harkness II: soy protein/50 mg Iso, 6M
Harkness II
Kreijkamp: soy protein/99 mg Iso, 12M
Kreijkamp-kaspers
Olsen I: soy milk/70 mg Iso, 24M
Olsen I
Potter I: soy protein/90 mg Iso, 6M
Potter I
Potter II: soy protein/55.6 mg Iso, 6M
Potter II
Uesugi: Iso 61.8 mg, 3M
Uesugi
Y-B Ye I: Iso 87 mg, 6M
Y-B Ye I
Y-B Ye II: Iso 126 mg, 6M
Y-B Ye II
Y-P Gao I: Iso 60 mg, 6M
Y-P Gao I
Y-P Gao II: Iso 90 mg, 6M
Y-P Gao II
Y-P Gao III: Iso 150 mg, 6M
Y-P Gao III
Total
Total
–300
–200
–100
0
100
200
300
Weighted mean differences with 95% CI
Fig. 2. Meta-analysis of 10 RCTs of isolated isoflavone intervention for BMD of lumbar spine in postmenopausal women [9].
Effects of Isoflavone on Bone Metabolism: Observational and Intervention Studies
Observational studies indicate that women who have high soy intake have less risk for osteoporosis than women who consume a typical Western diet [8, 16]. Recently, increasing numbers of RCTs have tested the effects of soy isoflavones on bone mineral density (BMD) in postmenopausal women. So far, one meta-analysis of RCTs has evaluated the effect on spine BMD of soy isoflavones (fig. 2). This included 10 trials testing both extracted soy isoflavones and isolated soy protein containing isoflavones, and revealed a significantly beneficial effect of soy isoflavone intake on BMD in lumbar spine at more than 90 mg/day [9]. However, the results from human studies are still controversial. Recently, Brink et al. [10] reported that consumption of isoflavone-enriched products (110 mg/day of isoflavone aglycone equivalents) for 1 year did not affect BMD and bone turnover in apparently healthy early postmenopausal white women. Potential reasons for these inconsistencies could include different races, the timing of exposure to isoflavone, duration of intervention, and difference in diet and isoflavone intake from foods between the isoflavone and placebo groups.
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Alternatively, interindividual differences in isoflavone metabolism could be a contributing factor. Recent studies suggest that the clinical effectiveness of isoflavones on bone metabolism might be due to their ability to produce the metabolite equol in the intestine [11].
Combined Effects of Isoflavones and Exercise on Bone Metabolism in Estrogen-Deficient Status
It has been shown that running exercise partially prevented bone loss induced by estrogen deficiency. Frost [20] showed that estrogen deficiency increased the ‘set point’ for the skeleton to respond to loading, causing the skeleton to be less sensitive to mechanical force and decreasing its bone mass. In this regard, it is likely that phytoestrogens can influence the set point of the mechanical loading that affects bone mass. Wu et al. [21] assessed the combined effects of isoflavones and exercise on BMD in postmenopausal Japanese women as well as in OVX mice. The combined intervention of moderate exercise and the submaximal dose of genistein administration showed a cooperative effect in preventing bone loss in OVX mice. Furthermore, they recruited 136 subjects (average age was 55 years), who were postmenopausal within 5 years of natural menopause, and randomly assigned them to four groups: placebo; walking combined with placebo (3 times/week, 6 km/h); isoflavone intake (75 mg conjugates/ day; equivalent to 47 mg of aglycone; Fujicco Co. Ltd., Kobe, Japan) in addition to the normal diet, and isoflavone combined with walking exercise [21]. After 1-year intervention, 108 subjects completed the study. BMD of the lumber spine, left hip and subwhole body was assessed by DXA using Hologic QDR-4500 (Hologic Inc., Waltham, Mass., USA) at baseline and after 1 year. Average daily intake of isoflavone aglycone from soy foods was around 28 mg per day in the subjects in 4 groups at baseline and after 1 year. There were no significant differences in daily intake of isoflavones and other nutrients among the groups at baseline, and between baseline and after 1 year in each group. Of the percent change in BMD, walking showed significant main effects on the preservation of BMD in the total hip region after 1 year. Interventions with isoflavones or walking showed significant main effects on the preservation of BMD at Ward’s triangle in the hip after 1 year. Combined intervention of isoflavone intake and walking exercise for 1 year showed a trend for a greater effect on BMD at total hip and Ward’s triangle regions than either intervention alone [21]. Since the effect of isoflavone alone was modest compared with those in the Westerners [21], the subjects in the placebo and isoflavone intervention groups were stratified based on their fecal equol production, and serum equol concentrations were measured in order to investigate whether any difference exists in the effects of isoflavone on BMD between equol producers and nonproducers in postmenopausal Japanese women [22].
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Possible Role of Equol Status in the Effects of Isoflavones on Bone Health
Equol is one of the main metabolites of the isoflavone daidzein. Equol production depends on the individual’s intestinal flora, and research has shown that approximately 30–50 % of individuals in the population studied are capable of producing equol from daidzein [11]. Setchell et al. [11] reported that compared with the control group, equol producers showed a 2.4% increase in their lumbar spine BMD, whereas no significant change in this BMD was observed in the nonproducers after a 2-year isoflavone intervention. Wu et al. [22] assessed the effects of equol-producing activity on BMD in postmenopausal Japanese women. Fifty-four women (29 in the placebo, 25 in the isoflavone group) completed the 1-year intervention, and their data were used for equol analysis. The percentage of equol producers in our subjects was 55% by assessment of equol production in fecal suspension incubated with daidzein under anaerobic condition. The number of equol producers and nonproducers was 15 and 14 in the placebo group, and 15 and 10 in the isoflavone group, respectively. Serum daidzein, which was determined by reverse-phase high-performance liquid chromatography, dramatically increased in the isoflavone group after 1 year. However, there was no difference in serum daidzein between equol producers and nonproducers. Conversely, serum equol was increased only in equol producers in the isoflavone group after 1 year. Serum equol did not change in equol producers in the placebo group. The percent change in bone loss at total hip and intertrochanter of the hip of equol producers was significantly lower than that of equol nonproducers in the isoflavone group, as assessed by Student’s t test (fig. 3). However, none of the differences between producers and nonproducers was observed in the placebo group. From these results, the effects of isoflavone on bone mass might depend on the equol-producing activity in postmenopausal Japanese women [22].
Isolation of Equol-Producing Bacteria from Human Feces
Intestinal bacteria play a key role in isoflavone metabolism; young infants with undeveloped gut microflora do not produce equol, while germ-free animals also do not produce equol or O-DMA. A number of strains involved in daidzein metabolism were identified. However, the identification of equol-producing bacteria is complicated. Several candidate bacteria responsible for daidzein metabolism have been suggested; for example, a Clostridium sp. and Eubacterium ramulus metabolized daidzein to O-DMA in vitro, and equol was found in soymilk fermented with some strains of Bifidobacterium. Uchiyama et al. [15] and Ishimi et al. [23] were the first to find, mainly four, equol-producing bacteria in human feces; they concluded that the Lactococcus 20-92 strain (Lc. 20-92) as homologous to L. garvieae is the most appropriate bacteria for food usage because we have dietary habit of L. garvieae.
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Equol producers Equol nonproducers
a
0.0 –0.5 –1.0 –1.5 –2.0 –2.5 –3.0 –3.5 –4.0
Placebo
Isoflavone
BMD change
BMD change
Isolated isoflavone: 47 mg/day, 12 months
b
0 –0.5 –1.0 –1.5 –2.0 –2.5 –3.0 –3.5 –4.0 –4.5
Placebo
Isoflavone
*p < 0.05
0
Placebo
Isoflavone
Placebo
–3.0 –4.0
BMD change
BMD change
–1.0 –2.0
–5.0
2.0 0.0 –2.0 –4.0 –6.0
–6.0
c
Isoflavone
4.0
d
*p < 0.05
Fig. 3. Percent change in BMD of the whole body and hip analyzed by equol status in postmenopausal Japanese women [22]. a Whole body. b Total hip. c Femoral neck. d Intertrochanter. Statistical differences as assessed by Student’s t test.
Ishimi et al. [23] detected Lc. 20-92 in the feces of 133 of postmenopausal Japanese women using real-time PCR using the particular primer for L. garvieae. The bacteria were detected in 47 of 133 samples (35.3%) [23]. Interestingly, the people with L. garvieae were not always equol producers, suggesting that some other bacteria and several factors such as hydrogen gas and short-chain fatty acids, which can affect the environmental conditions in the colon, might be also important for equol production in humans.
Food Factors Affecting Equol Production
It has been suggested that food factors contribute to the ability to produce equol; however, contradictory results were obtained from association studies. For example, Adlercreutz et al. [24] reported a positive association between urinary equol concentration and intake of fat and meat in a Japanese population, whereas in a Western population, Rowland et al. [25] reported that equol producers consumed significantly
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less energy as fat and significantly more energy as carbohydrates than equol nonproducers. In another cross-sectional study, equol-producing women had, on average, a higher intake of dietary fiber than nonproducers. However, in a feeding study, it was not possible to induce equol production by the diets of nonproducers with highfiber wheat bran cereal or soy protein [26]. Bolca et al. [27] suggested that persons with a higher polyunsaturated fatty acid and alcohol intake were more likely to be strong equol producers, and no differences were found in the intake of dietary fiber or the use of pre-, pro- or symbiotic preparations among 100 healthy postmenopausal women. Fructooligosaccharides (FOS), a mixture of indigestible and fermentable sugars, are known prebiotics that enhance calcium, magnesium and iron absorption in the large intestine. Uehara et al. [28] reported that FOS improves or prolongs the bioavailability or enterohepatic circulation of daidzein and genistein in rats administered with isoflavone glycoside conjugates (a single dose, 100 mg/kg body weight, via stomach tube). In a similar study, the equol concentration started to increase in the central venous blood at 12 h after the administration of isoflavone glycoside conjugates with FOS feeding and was significantly higher in the FOS-fed group than in the control group at 48 and 72 h. Furthermore, in OVX mice, the combination of dietary isoflavones and FOS has been shown to be more efficient than when administered alone in the prevention of bone loss, thus correlating with increased equol production [29]. In an in vitro study, however, FOS inhibited equol production. There is a discrepancy between the results of the in vivo and in vitro studies. In French postmenopausal women, FOS did not increase urinary equol production. However, racial differences might exist with regard to isoflavone metabolism. Therefore, further human studies involving Asian subjects are required. Several factors such as animal species, race, sex, age, and genetic background, including individual variation in intestinal microflora and diet, should be considered with regard to isoflavone metabolism and metabolite production.
Safety Evaluation of Isoflavones in Japan
Soybean isoflavone was approved in 2001 as the principle ingredient in Food for Specified Health Uses (FOSHU) by the Japanese Ministry of Health, Labour and Welfare and is aimed at individuals concerned about bone health. There are tea, soymilk and soft drinks containing 40 mg of isoflavone conjugates, which is equivalent to 25 mg of aglycone form. In 2004, applications of a tablet containing soy isoflavone aglycones as its principal ingredient, and a fermented food containing isoflavone aglycone in amounts exceeding the usual content in FOSHU were filed for approval. Foods with fortified or condensed isoflavones had not been consumed before. And there is a possibility that tablets and capsules would be excessively consumed. Accordingly, the Japanese Food Safety Commission of the Cabinet conducted
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Table 1. Effects of 1 year isoflavone intake on serum sex and thyroid hormone concentrations in postmenopausal Japanese Women Placebo
Isoflavone
(n = 29)
(n = 25)
Placebo vs Iso
Estradiol (pg/mL)
Baseline After 1 year % change
12.66 (4.03) 12.98 (7.31) 6.09 (57.71)
12.32 (3.34) 11.98 (2.94) 1.20 (23.80)
NS NS NS
FSH (U/L)
Baseline After 1 year % change
70.36 (26.02) 60.00 (19.83)* −12.36 (8.40)
68.19 (18.66) 58.12 (17.86)* −14.05 (9.01)
NS NS NS
LH (U/L)
Baseline After 1 year % change
26.68 (13.87) 22.43 (11.12)* −12.16 (15.98)
27.70 (9.32) 22.33 (7.90)* −19.10 (14.44)
NS NS NS
Progesterone (ng/mL)
Baseline After 1 year % change
0.27 (0.11) 0.21 (0.10)* −24.54 (29.47)
0.29 (0.16) 0.24 (0.12)* −14.07 (18.08)
NS NS NS
T3 (ng/ml)
Baseline After 1 year % change
1.13 (0.16) 1.10 (0.16) −1.60 (60.11)
1.08 (0.13) 1.03 (0.16) −3.92 (7.96)
NS NS NS
T4 (μg/dL)
Baseline After 1 year % change
8.57 (1.12) 8.46 (1.13) −1.85 (7.46)
8.19 (1.28) 7.54 (1.42)* −4.48 (6.23)
NS NS NS
TSH (mU/L)
Baseline After 1 year % change
2.34 (1.10) 2.75 (2.81) 12.71 (69.59)
2.30 (0.74) 2.25 (1.10) −9.22 (32.74)
NS NS NS
* Significantly different from baseline by paired t-test, p <0.05
a safety evaluation on soy isoflavones, and issued a Notice: ‘Basic approaches to evaluating the safety of FOSHU containing soy isoflavones’ in 2006. The main contents of the report are summarized in 3 points. Firstly, the upper limit of isoflavone aglycone intake from FOSHU was set at 30 mg/day for additional consumption with a normal diet. This limit represented half of the 57.3 mg soy isoflavones (aglycone equivalent) in the soymilk that was given daily to the premenopausal Japanese women in whom estrogen level tended to decrease and menstrual cycles tended to be longer over 2–3 cycles. Secondly, the maximum recommended level for safe isoflavone aglycone intake in a daily diet is 70–75 mg/day at the present time. This limit was selected on the basis of two findings. One was the National Nutrition Survey in 2002 in Japan that showed
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the 95th percentile intake of soy isoflavones at 64–76 mg/day. The other was the result of an experiment in which postmenopausal Italian women taking a 150-mg soy isoflavone aglycone tablet daily showed no effects after 3 years, but showed a significant increase in the occurrence of endometrial hyperplasia after 5 years as compared with a control group. One half of that amount was thus selected as the maximum recommended level, allowing for individual and experimental variations. Thirdly, it was not recommended that pregnant women, infants and children take soy isoflavone from FOSHU [30]. These criteria were also adapted to the so-called Health Foods fortified with isoflavones. In order to examine the effects of isoflavone intake on hormone levels in postmenopausal women, Ishimi et al. [23] evaluated serum concentrations of estrogen, FSH, LH, progesterone and thyroid hormones in their participants. There were no significant differences in estrogenic hormone levels between the placebo and isoflavone treatment groups (table 1). The same was observed with thyroid hormone levels. These results suggest that additional isoflavone intake with a normal diet (total intake was about 75 mg of aglycone equivalent a day) for a year did not affect serum hormone levels in postmenopausal Japanese women [23]. Marini et al. [31] recently reported that 54 mg/day of genistein supplementation for 3 years exhibited a promising safety profile in breast and endometrium with positive effects on bone formation in a cohort of osteopenic postmenopausal women. It is well known that consuming soy foods has many benefits. For example, they are a good source of protein, calcium; soy protein decreases serum cholesterol, and isoflavones maintain bone health in postmenopausal women. Therefore, the Japanese Ministry of Health, Labour and Welfare set the goal at 100 g/day of beans intake in the Health Japan 21 Program in 2000. Soybeans have been consumed since ancient times in Asia, and there has been no report of any problems even in cases of excessive consumption. Soy isoflavone intake from soy foods in a normal daily diet is therefore considered safe.
Conclusions
Firstly, several factors such as race, age, diet, time of exposure, and individual variations in genetics and intestinal microflora affect the effects of isoflavones on bone health. Secondly, preventive effects of daidzein on bone loss in postmenopausal women might depend on the capacity of an individual to produce equol. Thirdly, 47–54 mg/day of isoflavone supplementation with a normal diet for 1–3 years has not shown any adverse effects at least in postmenopausal women. Further studies are required to address the numerous questions regarding the potential benefits, mechanisms of action and safety of isoflavones.
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References 1 Adlercreutz H, Mazur W: Phyto-oestrogens and Western diseases. Ann Med 1997;29:95–120. 2 Adlercreutz H: Phyto-oestrogens and cancer. Lancet Oncol 2002;3:364–373. 3 Lampe JW: Isoflavonoid and lignan phytoestrogens as dietary biomarkers. J Nutr 2003;133:956S–964S. 4 Magee RJ, Rowland IR: Phyto-oestrogens, their mechanism of action: current evidence for a role in breast and prostate cancer. Br J Nutr 2004;91:513–531. 5 Messina M, Kucuk O, Lampe JW: An overview of the health effects of isoflavones with an emphasis on prostate cancer risk and prostate-specific antigen levels. J AOAC Int 2006;89:1121–1134. 6 Inzerillo AM, Zaidi M: Osteoporosis: trends and intervention. Mt Sinai J Med 2002;69:220–231. 7 Beral V, Bull D, Reeves G: Endometrial cancer and hormone-replacement therapy in the Million Women Study. Lancet 2005;365:1543–1551. 8 Messina MJ: Soy foods and soy isoflavones and menopausal health. Nutr Clin Care 2002;5:272–282. 9 Ma DF, Qin LQ, Wang PY, Katoh R: Soy isoflavone intake increases bone mineral density in the spine of menopausal women: meta-analysis of randomized controlled trials. Clin Nutr 2008;27:57–64. 10 Brink E, Coxam V, Robins S, Wahala K, Cassidy A, Branca F: Long-term consumption of isoflavoneenriched foods does not affect bone mineral density, bone metabolism, or hormonal status in early postmenopausal women: a randomized, double-blind, placebo controlled study. Am J Clin Nutr 2008;87: 761–770. 11 Setchell KDR, Brown NM, Lydeking-Olsen E: The clinical importance of the metabolite equol-a clue to the effectiveness of soy and its isoflavones. J Nutr 2002;132:3577–3584. 12 Xu X, Harris KS, Wang HJ, Murphy PA, Hendrich S: Bioavailability of soybean isoflavones depends upon gut microflora in women. J Nutr 1995;125:2307– 2315. 13 Heinone S, Wähälä K, Adlercreutz H: Identification of isoflavone metabolites dihydrodaidzein, dihydrogenistein, 6⬘-OH-O-dma, and cis-4-OH-equol in human urine by gas chromatography-mass spectroscopy using authentic reference compounds. Anal Biochem 1999;274:211–219. 14 Frankenfeld CL, McTiernan A, Tworoger SS, Atkinson C, Thomas WK, Stanczyk FZ, Marcovina SM, Weigle, DS, Weiss NS, Holt VL, Schwartz SM, Lampe JW: Serum steroid hormones, sex hormonebinding globulin concentrations, and urinary hydroxylated estrogen metabolites in post-menopausal women in relation to daidzein-metabolizing phenotypes. J Steroid Biochem Mol Biol 2004;88: 399–408.
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15 Uchiyama S, Ueno T, Suzuki T: Identification of a newly isolated equol-producing lactic acid bacterium from the human feces. J Intestinal Microbiol (Tokyo) 2007;21:217–220. 16 Setchell KD, Lydeking-Olsen E: Dietary phytoestrogens and their effect on bone: evidence from in vitro and in vivo, human observational, and dietary intervention studies. Am J Clin Nutr 2003;78(suppl):593S– 609S. 17 Ishimi Y, Miyaura C, Ohmura M, Onoe Y, Sato T, Uchiyama Y, Ito M, Wang X, Suda T, Ikegami S: Selective effects of genistein, a soybean isoflavone, on B-lymphopoiesis and bone loss caused by estrogen deficiency. Endocrinology 1999;140:1893–1900. 18 Fujioka M, Uehara M, Wu J, Adlercreutz H, Suzuki K, Kanazawa K, Takeda K, Yamada K, Ishimi Y: Equol, a metabolite of daidzein, inhibits bone loss in ovariectomized mice. J Nutr 2004;134:2623–2627. 19 Nakamura T, Imai Y, Matsumoto T, Sato S, Takeuchi K, Igarashi K, Harada Y, Azuma Y, Krust A, Yamamoto Y, Nishina H, Takeda S, Takayanagi H, Metzger D, Kanno J, Takaoka K, Martin TJ, Chambon P, Kato S: Estrogen prevents bone loss via estrogen receptor alpha and induction of Fas ligand in osteoclasts. Cell 2007;130:811–823. 20 Frost HM: On our age-related bone loss: insights from a new paradigm. J Bone Miner Res 1997;12: 1539–1546. 21 Wu J, Oka J, Tabata I, Higuchi M, Toda T, Fuku N, Ezaki J, Sugiyama F, Uchiyama S, Yamada K, Ishimi Y: Effects of isoflavone and exercise on BMD and fat mass in postmenopausal Japanese women: a 1-year randomized placebo-controlled trial. J Bone Miner Res 2006;21:780–789. 22 Wu J, Oka J, Ezaki J, Ohtomo T, Ueno T, Uchiyama S, Toda T, Uehara M, Ishimi Y: Possible role of equol status in the effects of isoflavone on bone and fat mass in postmenopausal Japanese women: a double-blind, randomized, controlled trial. Menopause 2007;14:866–874. 23 Ishimi Y, Oka J, Tabata I, Ohtomo T, Ezaki J, Ueno T, Uchiyama S, Toda T, Uehara M, Higuchi M, Yamada K, Wu J: Effects of soybean isoflavones on bone health and its safety in postmenopausal Japanese women J Clin Biochem Nutr 2008;43(suppl 1):48–52. 24 Adlercreutz H, Honjo H, Higashi A, Fotsis T, Hämäläinen E, Hasegawa T, Okada H: Urinary excretion of lignans and isoflavonoid phytoestrogens in Japanese men and women consuming a traditional Japanese diet. Am J Clin Nutr 1991;54: 1093–1100.
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25 Rowland IR, Wisemen H, Sanders TAB, 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. 26 Lampe JW, Skor HE, Li S, Wähälä K, Howald WN, Chen C: Wheat bran and soy protein feeding do not alter urinary excretion of the isoflavan equol in premenopausal women. J Nutr 2001;131:740–744. 27 Bolca S, Possemiers S, Herregat A, Huybrechts I, Heyerick A, De Vriese S, Verbruggen M, Depypere H, De Keukeleire D, Bracke M, De Henauw S, Verstraete W, Van de Wiele T: Microbial and dietary factors are associated with the equol producer phenotype in healthy postmenopausal women. J Nutr 2007;137:2242–2246. 28 Uehara M, Ohta A, Sakai K, Suzuki K, Watanabe S, Adlercreutz H: Dietary fructooligosaccharides modify intestinal bioavailability of a single dose of genistein and daidzein and affect their urinary excretion and kinetics in blood of rats. J Nutr 2001; 131:787–795.
29 Ohta A, Uehara M, Sakai K, Takasaki M, Adlercreutz H, Morohashi T, Ishimi Y: A combination of dietary fructooligosaccharides and isoflavone conjugates increases femoral bone mineral density and equol production in ovariectomized mice. J Nutr 2002;132: 2048–2054. 30 The Japanese Food Safety Commission of the Cabinet: Basic approaches to evaluating the safety of Food for Specified Health Uses containing soy isoflavones (in Japanese). 2006; http://www.fsc.go.jp/ hyouka/hy/hy-singi-isoflavone_kihon.pdf. 31 Marini H, Bitto A, Altavilla D, Burnett BP, Polito F, Di Stefano V, Minutoli L, Atteritano M, Levy RM, D’Anna R, Frisina N, Mazzaferro S, Cancellieri F, Cannata ML, Corrado F, Frisina A, Adamo V, Lubrano C, Sansotta C, Marini R, Adamo EB, Squadrito F: Breast safety and efficacy of genistein aglycone for postmenopausal bone loss: a follow-up study. J Clin Endocrinol Metab 2008;93:4787–4796.
Dr. Yoshiko Ishimi Project for Bio-index, Nutritional Epidemiology Program, National Institute of Health and Nutrition 1-23-1 Toyama, Shinjuku-ku Tokyo, 162-8636 (Japan) Tel. +81 3 3203 5389, Fax +81 3 3203 7350, E-Mail
[email protected]
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Life-style Related Diseases Yoshikawa T (ed): Food Factors for Health Promotion. Forum Nutr. Basel, Karger, 2009, vol 61, pp 117–128
Probiotics in Primary Prevention of Atopic Dermatitis Geun Eog Ji Department of Food and Nutrition, Seoul National University, Seoul, South Korea
Abstract The incidence of allergic diseases has been increasing in industrialized countries during recent years. Although several environmental factors are thought be involved, lack of moderate level of microbial challenges during the infantile period is known to skew the immune status toward the development of allergic diseases. Various strains of probiotics such as Bifidobacterium, Lactobacillus, and Lactococcus have been assessed for their ability to suppress the occurrence of atopic dermatitis (AD) in animal models and human studies. Although the effect of probiotics on allergic responses is different depending on the strains, doses, and experimental protocols, animal studies generally have shown immunomodulatory activities of probiotics including suppression of specific or nonspecific IgE production, reduction of infiltrated eosinophils and degranulated mast cells, potentiation of regulatory T cell cytokines such as IL-10 and TGF-β relative to IL-4 and IL-5, and potentiation of Th1/ Th2 activity along with reduced symptoms of AD. Several well-designed double-blind placebo-controlled human studies showed that some probiotic strains administered during perinatal period prevented the occurrence of AD but could not consistently show a reduction in specific or nonspecific IgE or a change in specific immunomodulatory cytokines. Taken together, published results suggest that the administration of selected strains of probiotics during the perinatal period may be helpful in Copyright © 2009 S. Karger AG, Basel the prevention of AD.
The gastrointestinal (GI) tract is an immune organ which is continuously exposed to antigens in the form of food, normal bacteria, and pathogens. Despite the numerous antigenic challenges, the mucosal immune system ordinarily maintains GI homeostasis through the orchestrated actions of the various mucosal immune cells. Bifidobacterium and Lactobacillus are major components of the commensal microbes in the GI tract and are frequently used as probiotics. They are known to benefit various physiological responses of the host including immunomodulatory activity. Recently, the potential use of probiotics in the prevention of allergy has drawn considerable attention.
New types of disease are emerging in the modern society. Among those diseases, atopic dermatitis (AD) is one of the most disturbing problems in the developed countries including USA, Europe, and Japan [1]. Prevalence of allergy tends to be lower in the families with a higher number of siblings, and the incidence of allergic diseases is higher in urban areas than rural areas. These observations underline the importance of environmental factors in the occurrence of allergic diseases [1]. In accordance with the phenomenon mentioned above, the so-called ‘hygiene hypothesis’ was put forward [2]. According to the hygiene hypothesis, the use of vaccine and antibiotics and the improved sanitation reduced the incidence of infectious diseases in children. As a result, immune challenges were reduced, which suppressed the potentiation of Th1- relative to Th2-immunity and increased the occurrence of allergy-related disease in children. However, the development of allergy turned out to be more complicated than a simple Th1/Th2 balance theory since an abnormally high level of Th1 or weak activity of regulatory T cells could lead to the occurrence of allergy or allergymediated inflammation. The analysis of epidemiological studies and human clinical studies by Flohr et al. [3] showed that the incidences of hepatitis virus, Helicobacter pylori, tuberculosis, and herpes simplex infections in infants and children were associated with higher frequencies of allergic diseases even though these infections are associated with the increase in Th1 immunity at the infected sites. These contradictory circumstances with the hygiene hypothesis and alleged increase in allergy from either too strong Th1- or Th2-mediated immunity called for a new hypothesis that consider regulatory cells T cells such as Treg as a crucial modulator in the prevention of allergic disorders (fig. 1). Mice raised in a germ-free environment failed to develop oral tolerance and had a persistent Th2-dependent immune response, while reconstitution of intestinal microbes during the neonatal period could reverse this immune deviation [4]. Therefore, establishment of normal intestinal microflora may be crucial in the maintenance of normal gut barrier function and development of tolerogenic immune status [5]. In addition, exposure to farm animals, pets, and day care environment during infantile period are known to be helpful for the introduction of benign challenges including various nonpathogenic microorganisms, which leads to the establishment of protective immunity against allergic disorders in infants. Infants with AD or other allergic diseases show less intestinal colonization of Lactobacillus or Bifidobacterium and more colonization of Clostridium relative to nonallergic infants [6]. In this context, the potential immune regulatory effect of the probiotics in regard to the prevention of allergy has attracted considerable interest among clinical doctors, food microbiologists, and nutrition scientists.
Probiotics in Primary Prevention of Atopic Dermatitis in Human Studies
In the study by Alm et al. [7], the incidence of allergy was lower among the children grown in the family frequently eating traditional lactic bacteria-fermented foods than
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Too clean environment
Th2 dominant
Allergy
Microbial challenge
• Benign challenge • Probiotics
Severe infection Allergy aggravation by Th1–associated inflammation
• Enhanced Treg (Tr1, Th3) activity • Normal Th1/Th2 balance
No allergy
Fig. 1. Relationship between microbial challenge and occurrence of allergy.
those eating mainly sterilized foods. Recently, the administration of probiotic bacteria was reported to help maintain anti-inflammatory and tolerant immunity, which led to the lower prevalence of allergy in the subjects. In our double-blind, randomized placebo-controlled human trial, infants who were perinatally administered a combination of B. bifidum BGN4, B. lactis AD011, and L. acidophilus AD031 showed significantly lower prevalence and cumulative incidence of AD than the placebo group [unpubl. data]. Hattori et al. [8] reported that the children with AD and low number of intestinal Bifidobacterium colonization showed amelioration of allergic symptoms when lyophilized Bifidobacterium was administered orally. In a double-blind, randomized, placebo-controlled trial, prenatal supplementation of Lactobacillus reuteri ATCC 55730 (1 × 108 colony forming units daily) in mothers from gestational week 36 until delivery and subsequent postnatal supplementation in babies from birth until 12 months of age resulted in less IgE-associated eczema at 2-year follow-up, although a preventive effect of probiotics on infant eczema was not confirmed [9]. The oral administration of combined L. rhamnosus and L. reuteri improved the extent of the eczema and decreased serum eosinophil cationic protein levels in children [10]. The effect was more pronounced in patients with a positive skin prick test response and elevated IgE levels. Supplementation of B. lactis Bb-12 or L. rhamnosus GG to the infants with atopic eczema during the weaning period reduced the extent and severity of atopic eczema, which was accompanied by the reduction in serum CD4 and urine eosinophilic protein X. Results from the above two clinical studies suggest that
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probiotics may improve the symptoms of inflammatory responses in allergic diseases beyond the intestinal milieu [11]. An increased traffic of circulating CD34+ hemopoietic precursor cells has been suggested to be a key factor in systemic allergic inflammation [12]. In the 14 6- to 48-year-old allergic patients with clinical symptoms of asthma and/or conjunctivitis, rhinitis, urticaria, AD, food allergy and irritable bowel syndrome, the number of circulating CD34+ hemopoietic precursor cells was decreased when a mixture of L. acidophilus, L. delbrueckii, and Streptococcus thermophilus was administered for 30 days [12]. The possibility of transferring immune regulatory cytokine from mother to infant through mother’s milk was suggested. When 62 pairs of mother and infant were supplemented with probiotics during pregnancy and the breastfeeding period, the level of TGF-β2 was higher in the breast milk from mothers in the probiotics group than from the control group mothers. The incidence of atopic eczema was significantly lower in infants born to the probiotics group mothers even at 2 years after delivery [13]. This result suggested that probiotics administered to mothers during pregnancy or breast feeding period increased the immune protective ability of the mother’s milk and contributed to the protection of infants from suffering atopic eczema. Positive clinical effects of L. rhamnosus GG on the prevention of AD in infants gained attention of the scientific community and generated enthusiasm to use probiotics for the prevention and treatment of various diseases related to the allergy. When L. rhamnosus GG was administered to pregnant mothers and subsequently to infants after delivery, the incidence of AD was reduced by half in comparison with the placebo group [14]. However, a more recent study which employed almost identical study design showed that supplementation of Lactobacillus GG (5 × 109 CFU twice daily during pregnancy and early infancy) did not reduce the incidence or severity of AD in affected children. Instead, it was associated with an increased rate of recurrent episodes of wheezing bronchitis [15]. The effect of different probiotics on children with milk allergy was compared between those fed L. rhamnosus GG and those fed mixed probiotics with 4 strains. Levels of plasma IL-2, IL-4, IL-6, IL-1, TNF-α, TGF-β1, TGF-β2 and C-reactive protein were compared. Among them, levels of C-reactive protein and IL-6 were higher in the L. rhamnosus GG group and the level of IL-10 was higher in the probiotic mixture group. As C-reactive protein and IL-6 are inflammatory cytokine markers and IL-10 is an immune suppressive regulatory factor, the administration of L. rhamnosus GG was thought to increase inflammatory immunity. The potential inflammatory nature of L. rhamnosus was apparent in its association with endocarditis and liver abscess in very rare cases [16, 17]. Therefore, despite the considerable number of reports on the beneficial effect of L. rhamnosus GG on health, caution is warranted for consumption of L. rhamnosus GG by the patient with infectious or inflammatory diseases. More recently, it has been reported that there is an increased risk of mortality in patients with predicted severe acute pancreatitis who use prophylactic probiotics [18]. Another negative result of Lactobacillus use was reported. In a randomized controlled trial, supplementation of L. acidophilus (LAVRI-A1) for the first 6 months of life did not reduce the risk of AD
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in high-risk infants but was associated with increased risk of subsequent cow’s milk sensitization [19]. Recently, two meta-analyses of clinical trials on the prevention and treatment of pediatric allergic diseases by the administration of probiotics have been published. Osborn and Sinn’s [20] meta-analysis of five studies reporting the outcomes of 1,477 infants showed a significant reduction in infant eczema but no significant reduction in atopic eczema confirmed by the skin prick test or specific IgE, though there was significant and substantial heterogeneity between studies. Lee et al. [21] conducted a meta-analysis of 10 double-blind randomized controlled clinical trials which pooled data from 6 prevention studies (n = 1,581) and 4 treatment trials (n = 299) by using fixed effects and random effects models of relative risk ratios and of weighted mean difference, respectively. The results supported the preventive effect of probiotics on pediatric AD but not the treatment effect. As described above, most of the studies on the prevention of allergic diseases have been conducted in infants or children with AD symptoms. A decrease in allergic symptoms and lower levels of total IgE throughout the year were also shown in the elderly people (55–70 years old) consuming yogurt with live culture for 1 year but not in those consuming pasteurized yogurt [22]. However, further studies with welldesigned, placebo-controlled clinical protocols are needed to determine whether any specific probiotic strain may be useful in the management of allergic symptoms in the senior people. Current scientific evidence for the effect of probiotics on primary prevention of allergy is not conclusive but promising. The conflicting results may be due to the differences in study design, host and environmental factors, number and strain of the applied probiotics, which lead to the difficulties in direct comparison between the results from the different studies. Even though the suppression of the occurrence of allergy by probiotics has been documented in human studies, their mechanism of action on the regulation of immune system is not well known. The primary action of probiotics might be through direct contact with GI lymphoid tissue. However, the indirect action of probiotics might also play a partial role in the suppression of allergy. For instance, probiotics might be able to reduce the number of harmful GI bacteria which can cause inflammatory reaction or degrade mucous cell layer and thereby aggravate the allergic reaction or increase permeability of the allergen through the GI epithelial cell layer. Administration of live Bifidobacterium cells was more effective in the suppression of allergy than heat-treated cells or sonicated fragmented cells in the ovalbumin (OVA) allergy animal model [23]. The live cells might have suppressed other harmful intestinal bacteria more effectively or the number of administered bacteria might have increased after colonization in the intestine resulting in the higher dose of active material. The permeability of the intestine was thought to be closely related to the occurrence of allergy. Administration of L. rhamnosus and L. reuteri in children with AD for 6–41 weeks improved GI symptoms and AD and decreased lactulose/mannitol permeability from the lumen to the blood, suggesting that
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administration of probiotics may reduce gut permeability in atopic children [24]. Use of probiotic bacteria was suggested for the management of various diseases associated with increased gut permeability due to impaired gut barrier or intestinal inflammation associated with acute rotavirus diarrhea and various colonic disorders as well as food allergy [25]. If direct interaction of probiotics is the primary mechanism in the prevention of allergic diseases, early infant period would be the most important stage for intervention since the administered probiotics can get access to the yet immature gut immune cells and play a role in their maturation process. Accordingly, most of the allergy preventive effects of the probiotics were shown in infants. In mice, the amount of luminal secretory IgA drastically decreased around the weaning period and presumably transport of the bacterial antigens to the vulnerable immune inductive site was facilitated, thus providing an opportunity for the probiotics to modulate the host immune system via gut immune system and to induce tolerance against allergies [26]. Consequently, the primary administration of L. johnsonii La1 during specific window of weaning period was effective in the prevention of AD manifested at the systemic level.
Effect of Probiotics in Animal Models or in Cell Culture Assays
Gut-associated lymphoid tissue contains Peyer’s patches (PPs) and isolated lymphoid follicles, inductor site of immune responses, and the larmina propria, effector site of immune responses. These immune tissues contain B cells, T cells, dendritic cells (DCs), and macrophages. DCs or/and T-regulatory cells are suggested to play a crucial role in establishing a tolerance in both mucosal and systemic immunity [26–28]. Gut epithelial cells, lymphoid cells, and DCs constantly recognize and interact with bacterial cells or their components such as peptidoglycan, lipoprotein, and lipopolysaccharide using pattern receptor system, including toll-like receptors (TLR), and mediate innate or adaptive immune responses [29]. At the intestinal mucosal layer, probiotic bacteria and their cell components such as peptidoglycan, lipoteichoic acid, intra- and extracellular polysaccharide products, cell-free extracts, and cell walls have been reported to cross-talk with the intestinal epithelial cells, M cells in PPs, and underlying DCs and macrophage cells. The multiple consequences of the cross-talk between the probiotic bacteria and the intestinal mucosa lead to the reinforcement of the intestinal barrier as well as direct modulation of mucosal immune cell functions including cytokine and chemokine release (fig. 2) [30]. Oral intake of the L. pentosus strain induced IFN-γ-producing cells through activation of IL-12 production by CD11c+DCs in a TLR 2- and/or a TLR4-dependent manner. This was confirmed by the observation that the production of IL-12p70 by DCs and IL-12p70 and IFN-γ production by spleen cells significantly decreased in those cells isolated from TLR2–/– or TLR4–/– mice compared with those from wildtype mice [31]. In comparison, the suppressive effect of CpG oligodexoynucleotide
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Intestinal lumen
Maintenance of permeability
Secretory IgA
Contorl of commensal microbiota growth
M cell Intracellular signaling
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Polymeric IgA
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IL-10
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• Local immune homeostasis • Local IgA production • Systemic tolerance
Peyer’s patch and mesenteric lymph node
Fig. 2. Schematic representation of multiple consequences of cross-talk between probiotic bacteria and intestinal mucosa [30].
from L. rhamnosus GG on antigen-specific IgE production in OVA-sensitized mouse model was suggested to be dependent on TLR9 in CD11c+CD8+ DCs [32]. The coapplication of L. plantarum and Der p 1, the major house dust mite allergen of Dermatophagoides pteronyssinus, suppressed specific IgE response and favored the production of INF-γ upon allergen restimulation. This strain was shown to stimulate high IL-12 and moderate IL-10 production in mouse DCs derived from the bone marrow notably through the TLR2-, MyD88-dependent and TLR4-independent pathway [33]. These different results suggest that the interaction of probiotics and DCs are differentially affected by discrete components of the different strains and the types of TLR in DCs. Although the interaction of DCs and probiotics may play a primary and pivotal role, several strains of lactobacilli and bifidobacteria are known to influence immune function through a number of different pathways including effects on enterocytes, antigen-presenting cells including circulating monocytes, DCs, regulatory T cells, and effector T and B cells [34]. DCs that have sampled dietary, self or commensal bacterial antigens may go on to induce tolerogenic T cell responses upon
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migration to the mesenteric lymph nodes [35]. Activation of T cells is regarded as a property of mature DCs that have travelled to draining lymphoid tissue and express high levels of surface MHC class II and costimulatory molecules. Veckman et al. [36] showed that certain probiotics directly enhance the activity of human DC populations to express moderate levels of costimulatory molecules and cytokines thereby facilitating Th1 cell differentiation. Additionally, probiotics induced an increase in regulatory DCs and T cells which then led to immunoregulatory mechanisms mediated in part by release of IL-10 and TGF-β [37]. IL-10 is known to be involved in the activation of Tr1 type regulatory T cells. TGF-β is known to be an important factor to enhance the differentiation of regulatory Th3 cells. Although many in vitro studies in which experimental probiotics were arranged to interact with a specific type of immune cell have provided useful information on their immunomodulatory actions; oftentimes, these in vitro results were contradictory to what have been observed in in vivo studies. Therefore, caution is warranted so that in vitro culture results do not lead to the error-prone interpretation of the role of probiotics in in vivo immune regulation. To compensate the weakness of a simple in vitro experiment, we adopted various transwell coculture systems in which epithelial cells were grown in the insert layer and other cell types such as DCs, spleen cells, or PP cells were grown on the bottom layer [unpubl. data]. In this cocultured experiment with epithelial and DC lines, various probiotic strains either slightly decreased or did not affect the expression of I-Ad, CD86, CD40, and the levels of IL-6 and TNF-α produced. Interestingly, B. lactis increased IL-10 secretion, and L. casei and L. acidophilus increased TGF-β secretion. Further development of a more elaborate in vitro model will help in understanding the role of the probiotics in the regulation of the immune system in relation to their differences in strain, dose, route and timing of exposure. Various in vivo animal experimental models have been employed to characterize and assess how probiotics modulate an allergy-related immune system. Animal models may provide more useful information on the mechanisms of the antiallergic effects of probiotic bacteria than in vitro studies. Depending on the protocols of the animal models, experimental results suggest that the administration of probiotics either potentiate Th1 relative to Th2 or enhance T regulatory cell activity. Orally administered L. casei reduced antigen-specific contact skin sensitivity by controlling the size of the CD8+ effector pool, which was mediated by regulatory CD4+ T cells [38]. Even though several other studies showed that the suppression of antigen-specific IgE by the administration of lactic acid bacteria was associated with the enhanced secretion of Th1-associated cytokines and low levels of Th2-associated cytokines, those studies in which the experimental allergens or probiotic strains were intraperitoneally injected were not included for review in the present study. These experimental models based on intraperitoneal injection do not reflect the real route of entry of antigen or experimental probiotic strain into the human body, and thus make the interpretation of the real mechanism involved difficult. Oral administration of both probiotics and a specific antigen may be more desirable. Several studies
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Naive Sham Live Disrupted Heat killed
a
5
Symptoms on tail
b 4
3
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2 d 1
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b
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Fig. 3. Severity of allergic symptoms on the tails of OVA-sensitized mice treated with various components of B. bifidum BGN4. a Photographs of the tails of the experimental mice. Group 1, naive; group 2, sham; group 3, treated with live BGN4; group 4, treated with disrupted BGN4; group 5, treated with heat-killed BGN4. b Symptom scores of the tails of the experimental mice. Different letters indicate significant differences in Duncan’s multiple range tests (p < 0.05) [23].
assessed the effect of orally administered probiotics in animals which were sensitized to develop allergic symptoms to oral OVA challenge. In this model, oral administration of probiotics suppressed production of the OVA-specific IgE, IgG1 in serum, OVA-specific fecal IgA, and the level of splenic IL-4 production and enhanced the production of splenic INF-γ and IL-10. In addition, the groups treated with probiotics showed ameliorated tail scabs and lower levels of degranulated mast cells in ears and small intestines, and infiltrated eosinophil granules in small intestines [23, 39]. Viable Bifidobacterium was more effective than disrupted or heat-killed cells in suppressing the symptoms of allergy (fig. 3). Antiallergic effects of the probiotics seemed to be manifested at the local and systemic levels and also at the initial and later phases during allergic progression. The observed suppression of IL-4 might be indicative of the potentiation of Th1 cells, and enhanced IL-10 secretion might have partially contributed to the induction of oral tolerance by activating regulatory T cells. Torii et al. [40] showed that oral administration of L. acidophilus L-92 to OVA-induced allergy mice suppressed OVA-specific IgE production and cytokines such as IFN-γ, IL-4 and IL-10. Additionally, antibodies such as total IgE and OVA-specific IgE were produced at a significantly lower level in the splenocytes of L-92-treated mice than those of control mice. In contrast, TGF-β and IgA levels produced by PPs from L-92-treated
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mice were significantly higher than those produced by control mice. In addition, a specific IgE-suppressive effect of B. bifidum G9-1 was suggested to be mediated by Treg cells independent of IFN-γ production [41]. Although the results of the animal experiment were not exactly similar, most of the studies advocated the use of probiotics for the alleviation of allergy. Taken together, oral administration of probiotics has been demonstrated to induce protective immune responses against allergic symptoms at local and systemic levels in animal models and human studies and might provide a rationale to utilize probiotics in the prevention of allergic diseases during the infantile period in humans.
Acknowledgment This work was supported by a grant (A080664) from the Ministry for Health, Welfare and Family Affairs, Republic of Korea.
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26 Inoue R, Nishio A, Fukushima Y, Ushida K: Oral treatment with probiotic Lactobacillus johnsonii NCC533 (La1) for a specific part of the weaning period prevents the development of atopic dermatitis induced after maturation in model mice, NC/ Nga. Br J Dermatol 2007;156:499–509. 27 Williamson E, Westrich GM, Viney JL: Modulating dendritic cells to optimize mucosal immunization protocols. J Immunol 1999;163:3668–3675. 28 Sakaguchi S: Regulatory T cells: key controllers of immunologic self-tolerance. Cell 2000;101:455– 458. 29 Blander JM, Medzhitov R: Toll-dependent selection of microbial antigens for presentation by dendritic cells. Nature 2006;440:808–812. 30 Corthésy B, Gaskins HR, Mercenier A: Cross-talk between probiotic bacteria and the host immune system. J Nutr 2007;137:781S-790S. 31 Koizumi S, Wakita D, Sato T, Mitamura R, Izumo T, Shibata H, Kiso Y, Chamoto K, Togashi Y, Kitamura H, Nishimura T: Essential role of Toll-like receptors for dendritic cell and NK1.1(+) cell-dependent activation of type 1 immunity by Lactobacillus pentosus strain S-PT84. Immunol Lett 2008;120:14–19. 32 Iliev ID, Tohno M, Kurosaki D, Shimosato T, He F, Hosoda M, Saito T, Kitazawa H: Immunostimulatory oligodeoxynucleotide containing TTTCGTTT motif from Lactobacillus rhamnosus GG DNA potentially suppresses OVA-specific IgE production in mice. Scand J Immunol 2008;67:370–376. 33 Hisbergues M, Magi M, Rigaux P, Steuve J, Garcia L, Goudercourt D, Pot B, Pestel J, Jacquet A: In vivo and in vitro immunomodulation of Der p 1 allergen-specific response by Lactobacillus plantarum bacteria. Clin Exp Allergy 2007;37:1286–1295. 34 Prescott SL, Bjorksten B: Probiotics for the prevention or treatment of allergic diseases. J Allergy Clin Immunol 2007;120:255–262. 35 Scheinecker C, McHugh R, Shevach EM, Germain RN: Constitutive presentation of a natural tissue autoantigen exclusively by dendritic cells in the draining lymph node. J Exp Med 2002;196:1079– 1090. 36 Veckman V, Miettinen M, Pirhonen J, Siren J, Matikainen S, Julkunen I: Streptococcus pyogenes and Lactobacillus rhamnosus differentially induce maturation and production of Th1-type cytokines and chemokines in human monocyte-derived dendritic cells. J Leukoc Biol 2004;75:764–771. 37 Rook GAW, Brunet LR: Microbes, immunoregulation, and the gut. Gut 2005;54:317–320. 38 Chapat L, Chemin K, Dubois B, Bourdet-Sicard R, Kaiserlian D: Lactobacillus casei reduces CD8+ T cell-mediated skin inflammation. Eur J Immunol 2004;34:2520–2528.
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39 Kim H, Kwack K, Kim DY, Ji GE: Oral probiotic bacterial administration suppressed allergic responses in an ovalbumin-induced allergy mouse model. FEMS Immunol Med Mic 2005;45:259–267. 40 Torii A, Torii S, Fujiwara S, Tanaka H, Inagaki N, Nagai H: Lactobacillus acidophilus strain L-92 regulates the production of Th1 cytokine as well as Th2 cytokines. Allergol Int 2007;56:293–301.
41 Ohno H, Tsunemine S, Isa Y, Shimakawa M, Yamamura H: Oral administration of Bifidobacterium bifidum G9–1 suppresses total and antigen specific immunoglobulin E production in mice. Biol Pharm Bull 2005;28:1462–1466.
Professor Geun Eog Ji Department of Food and Nutrition, Seoul National University Gwanakro 599 Gwanak-gu Shillimdong (Korea) 151742 Seoul Tel. + 82 2 880 8749, Fax + 82 2 884 0305, E-Mail
[email protected]
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Life-style Related Diseases Yoshikawa T (ed): Food Factors for Health Promotion. Forum Nutr. Basel, Karger, 2009, vol 61, pp 129–135
Astaxanthin Protects Neuronal Cells against Oxidative Damage and Is a Potent Candidate for Brain Food Xuebo Liu ⭈ Toshihiko Osawa Laboratory of Food and Biodynamics, Graduate School of Bioagricultural Science, Nagoya University, Nagoya, Japan
Abstract Astaxanthin (AST) is a powerful antioxidant that occurs naturally in a wide variety of living organisms. Based on the report claiming that AST could cross the brain-blood barrier, the aim of this study was to investigate the neuroprotective effect of AST by using an oxidative stress-induced neuronal cell damage system. The treatment with DHA hydroperoxide (DHA-OOH) or 6-hydroxydopamine (6-OHDA), either of which is a reactive oxygen species (ROS)-inducing neurotoxin, led to a significant decrease in viable dopaminergic SH-SY5Y cells by the MTT assay, whereas a significant protection was shown when the cells were pretreated with AST. Moreover, 100 nM AST pretreatment significantly inhibited intracellular ROS generation that occurred in either DHA-OOH- or 6-OHDA-treated cells. The neuroprotective effect of AST is suggested to be dependent upon its antioxidant potential and mitochondria protection; therefore, it is strongly suggested that treatment with AST may be effective for oxidative stress-associated neurodegeneration and a potential candidate for natural Copyright © 2009 S. Karger AG, Basel brain food.
Astaxanthin (AST; fig. 1), a red-orange carotenoid pigment, occurs naturally in many well-known aquatic animals such as shrimp, crab and salmon. As it belongs to the xanthopyll class of carotenoids, AST is closely related to β-carotene, lutein, and zeaxanthin, sharing with them many of the general metabolic and physiological activities attributed to carotenoids. On the other hand, AST has unique chemical properties based on its molecular structure. The presence of the hydroxyl (OH) and keto (C = O) moieties on each ionone ring explains some of its unique features, i.e. a high antioxidant activity. In recent years, a number of in vitro and in vivo studies on AST have demonstrated its antioxidant effect, for example the quenching effect on singlet oxygen, a strong scavenging effect on superoxide, hydrogen peroxide, and hydroxyl radicals and an inhibitory effect on lipid peroxidation [1–3]. In addition to these, several other biologic activities of AST, including anticancer, anti-inflammatory, antidiabetic, immunomodulatory activities and a neuroprotective effect, have also been reported [4].
O OH
HO
Fig. 1. Chemical structure of AST.
O
Parkinson’s disease (PD) is a neurodegenerative disorder characterized by a preferential loss of the dopaminergic neurons. The mechanism responsible for degeneration of dopaminergic neurons is incompletely understood; however, an increasing body of evidence suggests that oxidative stress, mitochondrial inhibition and impairment of the ubiquitin-proteasome system may be largely involved as major biochemical processes in the degenerative cascade [5]. In our previous study, we demonstrated that reactive oxygen species (ROS)- and mitochondrial dysfunction-mediated apoptotic signaling increased within a few hours after treatment with DHA hydroperoxides (DHA-OOH) and resulted in dopaminergic SH-SY5Y cell death [6]. In addition, the in vitro experimental model using neurotoxic compounds such as 6-hydroxydopamine (6-OHDA) has also revealed that the neuronal cell death was regulated by ROS generation, mitochondrial inhibition and other oxidative stress-related signaling molecules [7–9]. Currently, most efforts to prevent and treat neurodegenerative disorders focus on diet, lifestyle modification and drugs that target the disease processes, and among them, several natural antioxidant food factors have been focused on in recent years, which are also named brain foods. AST is a powerful antioxidant, and Hussein et al. [10] recently reported that AST prevented the ischemia-induced impairment of spatial memory in mice. Although these facts suggested that AST might be a potent candidate for a natural neuroprotective agent, further basic evidence to demonstrate the neuroprotective effect of AST is needed. The human dopaminergic neuronal cell line SH-SY5Y processes many types of substantia nigra neurons [11]. Therefore, in this study we investigated whether AST would prevent DHA-OOH- or 6-OHDA-induced cytotoxicity in SH-SY5Y cells. We examined the effects of AST on cell viability in DHA-OOH- or 6-OHDA-treated cells. The possible mechanisms of AST protection were investigated by measuring intracellular ROS generation and accumulation of AST in the cells.
Methods Materials
AST was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). DHA (purity = 98%) was purchased from the Cayman Chemical Co. (Ann Arbor, Mich., USA). 6-OHDA hydrochloride was obtained from Wako Pure Chemical Industries, Ltd.
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DHA-Hydroperoxide Preparation The DHA-OOH were prepared by the reaction of lipoxidase (from soybeans, Wako Pure Chemical Industries, Ltd.) with docosahexaenoic acid, as previously described [6]. The reaction mixture containing 83.6 mg docosahexaenoic acid, 16 mg lipoxidase and 220 ml borate buffer (200 mm, pH 9.0) was used, and the reaction was carried out in a flask filled with oxygen at room temperature. After incubation for 10 min, to terminate the reaction, HCl was added to the mixture until the solution pH was below 4.0. The formed hydroperoxides were extracted twice with an equal amount of chloroform/methanol (1:1), and the collected chloroform layer was then evaporated. The obtained DHA-OOH was dissolved in ethanol. The identification was performed by high-performance liquid chromatography (HPLC) analysis monitored at A234, and the concentration was quantified using a lipid hydroperoxide kit (Cayman) and compared with a standard curve prepared using authentic 13-HPODE.
Cell Culture and Cell Viability Human dopaminergic neuroblastoma SH-SY5Y cells were grown in Cosmedium-001 (Cosmo-Bio, Tokyo, Japan) containing 5% fetal bovine serum. The cells were seeded on plates coated with polylysine and cultured at 37°C. The cell viability was quantified by a 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, different concentrations of AST were added to the cells for 4 h. Following the removal of excess AST agent, the cells were washed three times with FBS-free DMEM prior to the addition of 10 μm DHA-OOH or 100 μm 6-OHDA for 24 h, followed by further incubation with 0.5% MTT solution (5 mg/ml) for 4 h. The cells were then lysed with 0.04 n HCl in isopropyl alcohol, and the absorbance was read at wavelengths of 550 nm (peak) and 630 nm (bottom).
Analysis of Reactive Oxygen Species Production The intracellular ROS level was detected by flow cytometry using DCHF-DA that is oxidized by hydrogen peroxide or low-molecular-weight peroxides to produce the fluorescent compound 2,7-dichlorofluorescein (DCF). In this study, the SH-SY5Y cells (which had reached approximately 80% confluence) seeded on six-well plates were washed twice with serum-free DMEM and thereafter incubated for 4 h in serum-free DMEM in the presence of 100 nm AST. After washing with serum-free DMEM, the cells were loaded with carboxy-H2DCFDA for 30 min, prior to exposure to 10 μm DHA-OOH or 100 μm 6-OHDA for 30 min. Followed by treatment with 6-OHDA, the cells were washed once with PBS+ and PBS–, respectively, and then collected into vials. The fluorescence of DCF in the supernatant was measured by an EPICS Elite Flow Cytometer (Beckman Coulter, Inc., Fullerton, Calif., USA).
Analysis of Astaxanthin Concentration in Different Fractions of the Cell AST contents in cell membrane, mitochondrial and cytosolic fractionation and media were quantified by HPLC. Five dishes (9-cm cell culture dishes) of confluent SH-SY5Y cells were incubated with 100 nm AST for 4 h. The media were collected. The cells were washed with PBS three times, and then were fractionated into cell membrane, mitochondria and cytosol as described by Pallotti [12]. The media and fractionations were dissolved in 200 ml of acetone and filtered through a
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Fig. 2. Protective effect of AST on DHA-OOH- or 6-OHDAinduced decrease in cell viability measured by MTT assay. a SH-SY5Y cells were incubated with different concentrations of AST for 4 or 24 h, and cell viability was assessed by MTT assay. b, c Cells were incubated with or without 25–1,000 nm AST for 4 or 24 h. Then the media were removed and the cells were washed three times with FBS-free DMEM prior to the addition of 10 μM DHA-OOH or 100 μM 6-OHDA for additional 24 h. Values are percentages of the control (no drugs) of three independent experiments in triplicates and are expressed as mean ± SE (n = 9). ** p < 0.01 and *** p < 0.001 versus control; # p < 0.05 and ## p < 0.01 versus DHA-OOH or 6-OHDA.
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0.45-mm polytetrafluoroethylene membrane filter; then 20 ml of solution was subjected to HPLC-UV using a column of Develosil ODS HG-5 (4.6 × 250 mm, Nomura Kagaku, Japan). Semipreparative HPLC was performed at room temperature using a mobile phase consisting of methanol (95%) and water (5%) with a linear program. The flow rate was set at 0.8 ml/ml, and AST peak was collected by monitoring at 471 nm. AST was quantified relative to calibration with a standard sample. Obtained quantity of AST in media and each cellular fraction was adjusted by added total AST quantity and expressed as percent of added total AST.
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Statistical Analysis All data were analyzed using Bonferroni/Dunn’s multiple comparison procedure.
Results
Effect of Astaxanthin on DHA-OOH- or 6-OHDA-Induced Cell Death SH-SY5Y cells are widely used to study dopaminergic pathogenesis as this cell line expresses some representative dopaminergic markers such as tyrosine hydroxylase and dopamine transporter. Therefore, SH-SY5Y cells can be a suitable model system to study the role of AST against ROS-mediated dopaminergic cell death. In this study, SH-SY5Y cells were pretreated with AST for 4 and 24 h at different concentrations, washed, and then treated with DHA-OOH or 6-OHDA for an additional 24 h. AST itself had no apparent effect on cell viability at a concentration of 25–1,000 nm even for 24 h (fig. 2a). DHA-OOH (10 μm, 24 h) and 6-OHDA (100 μm, 24 h) induced a significant decrease in cell viability by 80 and 70%. The pretreatment of SH-SY5Y cells with AST for 4 h resulted in a dose-dependent protection against DHA-OOH- or 6-OHDA-induced toxicity at a concentration ranging from 25 to 100 nm, and the most significant protection was found at a concentration of 100 nm, with respectively 65 and 84% of the control. The pretreatment with AST at a concentration of 500 and 1,000 nm caused a similar or reduced effect compared to that of 100 nm. In addition, the protective effect of AST was slightly enhanced by a longer pretreatment for 24 h the case of DHA-OOH- and 6-OHDA-treated cells (fig. 2b, c). Effect of Astaxanthin on DHA-OOH- or 6-OHDA-Induced ROS Generation ROS generation has been demonstrated to be a common feature occurring in DHAOOH- or 6-OHDA-treated cells and is also proposed as one of the initial triggers leading to activation of apoptotic signaling. In this study, we examined the effect of AST on ROS generation in SH-SY5Y cells exposed to DHA-OOH or 6-OHDA. Intracellular ROS levels were determined with DCF fluorescence by flow cytometry. As shown in figure 3, exposure of SH-SY5Y cells to DHA-OOH and 6-OHDA led to a 3.5- and 1.8-fold increases, respectively, in DCF signal compared with the control group, whereas AST pretreatment significantly inhibited the increase in DCF fluorescence in the cells treated by both toxins. In addition, we investigated the accumulation of AST in the cells. By HPLC analysis, AST was detected at 0, 9.42, 7.9, and 72.56% of the total administration levels in the cytosolic, mitochondrial, membrane fraction of the cells and the culture medium, respectively (data not shown), suggesting that AST accumulating in the membrane fraction may contribute directly to the protection against ROS-associated cell death by its potent antioxidant property.
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a
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Fig. 3. Protective effects of AST on DHA-OOH- or 6-OHDA-induced ROS generation. DCF fluorescence determination was used to assess intracellular ROS generation in DHA-OOH-treated (a) and 6-OHDA-treated (b) cells. * p < 0.05 and ** p < 0.01 versus control; # p < 0.05 versus DHA-OOH or 6-OHDA.
Conclusion
Advances in understanding the neurodegenerative pathologies are creating new opportunities for the development of neuroprotective therapies. In this work, we have demonstrated that AST, a natural carotenoid and an abundant component in aquatic animals, significantly protected DHA-OOH- or 6-OHDA-induced cellular toxicity in human neuroblastoma dopaminergic SH-SY5Y cells. PD is characterized by a profound loss of dopaminergic neurons in the substantia nigra. Even though the cause of PD remains largely unknown, several lines of evidence strongly suggest the involvement of oxidative stress [13]. Evidence exists that some antioxidants have a neuroprotective effect in the in vitro models of PD. However, the lack of efficacy to penetrate the blood-brain barrier has led to the failure of antioxidants to exhibit the in vivo effect. Tso et al. [14] detected AST in the brain of rats fed with natural AST, suggesting that AST could cross the blood-brain barrier in mammals. The neuroprotective effect of AST in our study is very significant and quite powerful at the nm levels. In addition, Aoi et al. [15] have reported that AST inhibited the formation of linoleic acid-derived hexanoyl lysine adduct in mice skeletal muscle, suggesting that AST might suppress lipid peroxidation occurring in the brain due to the presence of a high concentration of polyunsaturated fatty acids. Together, it is suggested that AST may be used as a brain nutrient to protect the brain content from oxidative stress, neuronal apoptosis, and even brain aging.
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References 1 Miki W: Biological functions and activities of animal carotenoids. Pure Appl Chem 1991;63:141– 146. 2 Palozza P, Krinsky NI: Astaxanthin and canthaxanthin are potent antioxidants in a membrane model. Arch Biochem Biophys 1992;297:291–295. 3 Liu X, Osawa T: Cis astaxanthin and especially 9-cis astaxanthin exhibits a higher antioxidant activity in vitro compared to the all-trans isomer. Biochem Biophys Res Commun 2007;357:187–193. 4 Hussein G, Sankawa N, Goto H, Matsumoto K, Watanabe H: Astaxanthin, a carotenoid with potential in human health and nutrition. J Nat Prod 2006; 69:443–449. 5 Liu X, Yamada N, Maruyama W, Osawa T: Formation of dopamine adducts derived from brain polyunsaturated fatty acid: mechanism for Parkinson’s disease. J Biol Chem 2008;283:34887–34895. 6 Liu XB, Shibata T, Hisaka S, Osawa T: DHA hydroperoxides as a potential inducer of neuronal cell death: a mitochondrial dysfunction-mediated pathway. J Clin Biochem Nutr 2008;43:26–33. 7 Hanrott K, Gudmunsen L, O’Neill MJ, Wonnacott S: 6-hydroxydopamine-induced apoptosis is mediated via extracellular auto-oxidation and caspase 3dependent activation of protein kinase Cdelta. J Biol Chem 2006;281:5373–5382. 8 Chalovich EM, Zhu JH, Caltagarone J, Bowser R, Chu CT: Functional repression of cAMP response element in 6-hydroxydopamine-treated neuronal cells. J Biol Chem 2006;281:17870–17881.
9 Jia Z, Zhu H, Misra HP, Li Y: Potent induction of total cellular GSH and NQO1 as well as mitochondrial GSH by 3H-1,2-dithiole-3-thione in SH-SY5Y neuroblastoma cells and primary human neurons: protection against neurocytotoxicity elicited by dopamine, 6-hydroxydopamine, 4-hydroxy-2-nonenal, or hydrogen peroxide. Brain Res 2008;1197:159–169. 10 Hussein G, Nakamura M, Zhao Q, Iguchi T, Goto H, Sankawa U, Watanabe H: Antihypertensive and neuroprotective effects of astaxanthin in experimental animals. Biol Pham Bull 2005;28:47–52. 11 Takahashi T, Deng Y, Maruyama W, Dostert P, Kawai M, Naoi M: Uptake of a neurotoxin-candidate, (R)-1,2-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline into human dopaminergic neuroblastoma SH-SY5Y cells by dopamine transport system. J Neural Transm Gen Sect 1994;98:107–118. 12 Pallotti F, Baracca A, Hernandez-Rosa E, Walker WF, Solaini G, Lenaz G, Melzi D’Eril GV, Dimauro S, Schon EA, Davidson MM: Biochemical analysis of respiratory function in cybrid cell lines harbouring mitochondrial DNA mutations. Biochem J 2004; 384:287–293. 13 Mariani E, Polidori MC, Cherubini A, Mecocci P: Oxidative stress in brain aging, neurodegenerative and vascular diseases: an overview. J Chromatogr B Analyt Technol Biomed Life Sci 2005;827:65–75. 14 Tso MOM, Lam TT: Method of Retarding and Ameliorating Central Nervous System and Eye Damage. US Patent 1996; 5527533. Board of trustees of the University of Illinois, United States of America. 15 Aoi W, Naito Y, Takanami Y, Ishi T, Kawai Y, Akagiri S, Kato Y, Osawa T, Yoshikawa T: Astaxanthin improves muscle lipid metabolism in exercise via inhibitory effect of oxidative CPT1 modification. Biochem Biophys Res Commun 2008;366:892–897.
Dr. Toshihiko Osawa Laboratory of Food and Biodynamics, Graduate School of Bioagricultural Science, Nagoya University Furo-cho Nagoya 464-8601 (Japan) Tel. +81 52 789 4125, Fax +81 52 789 5296, E-Mail
[email protected]
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Life-style Related Diseases Yoshikawa T (ed): Food Factors for Health Promotion. Forum Nutr. Basel, Karger, 2009, vol 61, pp 136–146
Function of Marine Carotenoids Kazuo Miyashita Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Japan
Abstract Although an effort is made to review marine carotenoids as important bioactive compounds with reference to their presence, and chemical and biofunctional benefits, there has been a relatively little information on the impact of these carotenoids on human health. The potential beneficial effects of marine carotenoids have been studied particularly in astaxanthin and fucoxanthin as they are major marine carotenoids. Both carotenoids show strong antioxidant activity which is attributed to quenching singlet oxygen and scavenging free radicals. The potential role of the carotenoids as dietary antioxidants has been suggested to be one of the main mechanisms for their preventive effects against cancer and inflammatory diseases. However, it would be difficult to explain their biological activities only by their antioxidant activity. We have found the antiobesity and antidiabetic effects as specific and novel bio-functions of fucoxanthin. A nutrigenomic study revealed that fucoxanthin induces uncoupling protein 1 expression in white adipose tissue (WAT) mitochondria to lead to oxidation of fatty acids and heat production in WAT. Fucoxanthin improves insulin resistance and decreases blood glucose level, at least in part, through the downregulation of tumor necrosis factor-α in WAT of animals. Thus, the specific regulation of fucoxanthin on a particular bio-molecule will be responsible for the characteristic chemical structures which differ depending on the length of the polyene, nature of the end group and various substituents they contain. The key structure of carotenoids for the expression of antiobesity effect was suggested to be carotenoid end of the polyene chromophore Copyright © 2009 S. Karger AG, Basel containing an allenic bond and two hydroxyl groups.
It is well known that marine species have characteristic functional properties which cannot be found in land species. Interest in the functionality of marine food resources continues to grow at a faster pace than it was imagined only a few years ago. This is partly due to the fact that the possible prevention of several diseases through dietary marine products has been better understood and recognized by the public. Another factor is the mounting cost of public health care systems. Thus, people keenly want to know of health benefits of food factors, especially marine foods. Exciting developments in nutrigenomics and the human genome project, combined with formulation of food products containing specific bioactives have created new industrial opportunities for food and pharmaceutical companies, but we have only seen the tip of the iceberg for the utilization and research of marine products.
Although there have been many papers published on the health beneficial effects of omega–3 highly unsaturated fatty acids such as eicosapentaenoic acid (20:5n–3) and docosahexaenoic acid (22:6n–3) from marine lipids, the discovery and development of marine nutraceuticals is a relatively new area compared with that of nutraceuticals derived from terrestrial sources. In this paper, I reviewed the published literature with respect to occurrence of marine carotenoids and their physiological benefits, as there has been relatively little information on the physiological effects or beneficial applications of marine carotenoids as compared with the available information on the carotenoids of terrestrial origin.
Marine Carotenoids
In marine environments, carotenoids are widely present in both plants and animals. Palermo et al. [1] reported the presence of β-carotene, zeaxanthin, fucoxanthin and fucoxanthinol (fig. 1) in the red algae. In brown algae, fucoxanthin is the dominant carotenoid [2]. The metabolites of fucoxanthin apo-9⬘-fucoxanthinone and apo13⬘fucoxanthinone were also isolated from brown seaweed [3]. Fucoxanthin is the most abundant one and it contributes more than 10% of the estimated total production of carotenoids in nature [4]. Carotenoids are responsible for the color of many important fish and shellfish products. The distribution of carotenoids in these marine animals varies with species, habitat and their food habits. Commonly found carotenoids from fish are tunaxanthin in yellow fish, astaxanthin (fig. 1) in red fish, zeaxanthin in anchovies, flatfish and shark, tunaxanthin, lutein and zeaxanthin in brackish water fish and lutein and zeaxanthin in fresh water fish [4, 5]. Crustaceans such as shrimp, prawn, lobster, krill and crab contain astaxanthin (fig. 1) as their main pigment present in free forms, esterified forms or as bound to macromolecules such as protein or chitin [4]. Crustaceans absorb the pigments from the diet and deposit them as such or transfer them metabolically to keto or hydroxy derivatives [6]. Astaxanthin and its esters are the main pigment in crustaceans irrespective of the species and the environment from which they are harvested [7]. Astaxanthin was found to be present in both the enantiomeric and meso forms in shrimp Pandalus borealis [8]. Free and esterified carotenoids were also found as the main pigments in deep sea shrimps [9]. Overall, astaxanthin is the main carotenoid pigment found in aquatic animals, while fucoxanthin is specifically abundant in aquatic plants.
Antioxidant Activity of Carotenoids
Reactive oxygen species and oxidative damage to bio-molecules have been widely postulated to cause and aggravate several chronic diseases, including cancer and
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OH
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Fig. 1. Structures of marine carotenoids.
cardiovascular diseases. Carotenoids have been implicated as important dietary nutrients having antioxidant potential. They play a protective role by effectively dissipating excess energy, preventing the formation of reactive oxygen species, and by deactivating singlet oxygen generated during the photosynthetic process. The quenching of singlet oxygen by carotenoids has been attributed mainly to the physical mechanism where the excess energy of singlet oxygen is transferred to carotenoid. The carotenoid with added energy is excited to triplet state and upon losing the energy as heat relaxes to singlet state without change in the structure as follows, 1 3
O2• + 1Car → 3O2 + 3Car• Car• → 1Car + heat
The singlet oxygen quenching rates of carotenoids is characterized by the rate constant kq, where the larger kq values, the faster the quenching reaction. The quenching rate constant (kq = l × mol–1 × s–1) for astaxanthin (2.4 × 1010) was found to be twice that of β-carotene (1.4 × 1010) and 80 times that of the antioxidant tocopherol (0.03 × 1010) [10].
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In addition, dietary carotenoids react with a wide range of free radicals such as CCl3O2•, RSO2•, NO2• and various arylperoxy radicals via electron transfer producing the radical cation of the carotenoid. The free radicals obtain the electron from the other molecule or form an adduct with the other molecule. The electron-rich status of carotenoids makes them more suitable for reaction with the free radicals, thus avoiding the use of cellular components by the free radicals for reactions. Astaxanthin has been found to be more effective than β-carotene in preventing fatty acid peroxidation in chemical solutions [11] and delaying lipid peroxidation in membrane models [12]. Goto et al. [13] demonstrated that the higher antioxidant activity of astaxanthin compared with β-carotene is due to the trapping of radicals at the surface and inside the phospholipid membrane and the unique structure of the terminal ring moiety. We compared the antioxidant effect of polar carotenoids including astaxanthin and astaxanthin-β glucoside on phosphatidylcholine (PC) in liposomes and found that astaxanthin and astaxanthin-β glucosides are highly active antioxidants [14]. This result indicates that the antioxidant effect of carotenoids on PC liposomes depends not only on their ability to scavenge free radicals but also on their location and orientation in the PC liposome system and the extent of their incorporation into PC bilayers.
Importance of Nutrigenomic Study of Carotenoids
The best-known biological function of carotenoids is their established role as provitamin A. Carotenoids such as α- and β-carotene and β-cryptoxanthin can be converted to retinoic acid, an active form of vitamin A. Retinoic acid in its all-trans or 9-cis configuration has highly potent effects on the retinoic acid receptors and the retinoid X receptors. Activation of these nuclear receptors of retinoic acids can influence the transcription of various retinoid response genes [15]. In addition, dietary carotenoids reduce the risk of diseases such as cardiovascular diseases, age-related macular degeneration and cancers. Further, epidemiological studies established a positive correlation between carotenoid consumption and a reduced risk of many kinds of diseases. The antioxidant properties of carotenoids are regarded as the main mechanism by which they afford their beneficial health effects. Much interest has been focused on the potential role of the carotenoids as dietary antioxidants. There is little doubt that, under the right conditions, antioxidant activity of carotenoids can protect cells, tissues and other structures such as lipoproteins. However, the true significance of the antioxidant capacity of carotenoids still remains unclear. Some might argue that the initial focus of the research on the role of carotenoids as antioxidants has set back the progress of research into other biological activities that are independent of the antioxidant properties. The science behind the effects of dietary carotenoids on human health is complex, and simple and straightforward answers to the outstanding questions are unlikely.
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While modulation of the transcriptional activity of carotenoids is known to have an anticancer effect, in most cases, the underlying mechanisms of their other actions still remain uncertain. Certain effects are observed with provitamin A carotenoids that are not elicited by vitamin A itself. Equally, the non-provitamin A carotenoids also are capable of altering patterns of gene and protein expressions and cell function with a specific and important nutritional and biofunctional impact on the body [16]. The nutritional functions of carotenoids depend on their chemical structures, which differ depending on the length of the polyene, nature of the end group and various substituents they contain. The specific regulation of a carotenoid on a particular bio-molecule will be responsible for the carotenoid’s characteristic physiological effect. From this nutrigenomic viewpoint, the physiological effect of allenic and acetylene carotenoids from marine species is very interesting.
Antiobesity and Antidiabetic Effects of Fucoxanthin
Mitochondrial uncoupling proteins (UCPs) are the key molecules for metabolic thermogenesis and are the physiological defense against obesity [17]; their dysfunction leads to the development of obesity [18]. Studies by Serra et al. [19] indicated that carotenoids can positively affect UCP1 expression in brown adipose tissue (BAT). However, as in adult humans the content of BAT is low and most of the fat is stored in white adipose tissue (WAT), we investigated the effect of seaweed lipids on UCP expression and accumulation of fat in WAT of rat and mice [20]. Feeding rats and mice with lipids from the brown seaweed Undaria pinnatifida resulted in reduced abdominal WAT weights and UCP1 expression. As Undaria lipids mainly consists of glycolipids and fucoxanthin, we further examined the antiobesity effect of these two constituents [20]. Feeding with fucoxanthin but not glycolipid resulted in the reduction of WAT weight and clear expression of UCP1 protein. The results indicated the clear antiobesity effect of fucoxanthin by upregulating the expression of UCP1 in WAT resulting in reduced WAT weight (fig. 2). UCP1 expression was found in WAT of 0.2% purified fucoxanthin-fed KK-Ay mice [20], and there was little expression in that of control mice. Expression of UCP1 mRNA was also found in WAT of 0.2% fucoxanthin-fed mice, and little expression in that of control. The finding that fucoxanthin induces both protein and mRNA expression of UCP1 in WAT will give a clue for new dietary antiobesity therapy. An enormous amount of data has been collected on thermogenesis in BAT through UCP1 expression. However, there had been little information on UCP1 expression in WAT induced by a dietary component before the publication of our report. Direct heat production by fat oxidation in WAT, therefore, will reduce the risk of these diseases in humans.
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HO
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Fig. 2. Novel functionalities of fucoxanthin. MCP = Monocyte chemoattractant protein-1.
KK-Ay mice in this study not only developed obesity but also hyperleptinemia and hyperinsulinemia along with insulin resistance. Therefore, glucose levels of mice fed the control diet reached levels higher than 400 mg/dl. On the other hand, mice fed the 0.1 and 0.2% purified fucoxanthin diets had significantly lower blood glucose concentrations of around 220 and 170 mg/dl, respectively [20]. Furthermore, plasma insulin levels decreased in a dose-dependent manner after purified fucoxanthin intake [20].
Relationship between Carotenoid Structure and Antiobesity Activity
When various carotenoids were screened for potential suppression effects on adipocyte differentiation [21, 22], only fucoxanthin, fucoxanthinol, and neoxanthin (fig. 3) showed an encouraging suppressive effect on the differentiation of 3T3L1 adipose cells, while other carotenoids did not show such an effect. The three kinds of carotenoids significantly inhibited intercellular lipid accumulation during
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OCOCH 3
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Fig. 3. Structures of allenic carotenoids.
adipocyte differentiation of 3T3-L1 cells and significantly decreased glycerol-3phosphate, an indicator of adipocyte differentiation, as compared with the control cells. Interestingly, neoxanthin is very similar in structure to fucoxanthinol which has been suggested to be the biologically active form of fucoxanthin (fig. 3). The only structural difference between neoxanthin and fucoxanthinol is the existence of a keto substituent at the end of the polyene chromophore of fucoxanthinol. Thus, it was hypothesized that the specific structure that both carotenoids have is somewhat responsible for the suppressive effect on adipocyte differentiation (fig. 3). In order to test this theory, the effects of neoxanthin and additional 12 kinds of carotenoids (fig. 4) on adipose cell differentiation were analyzed [22]. The result clearly indicated that only neoxanthin showed suppressive effects on lipid accumulation, GPDH activity and aP2 expression in the 3T3-L1 differentiation. However, treatment with (rac)-α-carotene, carotenoids of the keto (citranaxanthin, rhodoxanthin, canthaxanthin) and epoxy (β-carotene 5,6-epoxide) groups did not result in apparent changes in the level of GPDH activity. The same was true for hydroxyl carotenoid (β-cryptxanthin, lutein), epoxy-hydroxy carotenoids (violaxanthin, antheraxanthin, lutein epoxide), and keto-hydroxy cerotenoids (capsorubin). These findings provide further evidence for the theory that the suppressive effects of carotenoids on adipocyte differentiation are related to their structural properties, where the allenic bond
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O
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OH
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Fig. 4. Structures of several selected carotenoids.
is essential for the expression of the activity; carotenoids with an epoxy group, a keto group, or a keto and a hydroxyl group as part of the end group are not active without the allenic bond. When 3T3-L1 cells were treated with fucoxanthin, fucoxanthinol, and neoxanthin, PPARγ, a regulator of adipogenic gene expression, was downregulated by these carotenoids in a dose-dependent manner [21, 22]. These results suggest that fucoxanthin and fucoxanthinol inhibit the adipocyte differentiation of 3T3-L1 cells through downregulation of PPARγ. PPARγ has an important role in the early stages of 3T3-L1 cell differentiation [23, 24], because it is a nuclear transcription factor that regulates adipogenic gene expression [25]. Regulation of PPARγ would be one of the expected mechanisms underlying the antiobesity effect of dietary carotenoids.
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C/EBPβ is a transcriptional activator of PPARγ. C/EBPα and PPARγ promote a level of adipogenesis and sustain each other’s expression during maturation of adipocytes. C/EBPα and PPARγ contribute to adipogenesis by participating in the control of genes involved in lipogenesis, insulin sensitivity, and other pathways. Neoxanthin treatment could have interfered with events occurring downstream of the C/EBPβinduced transcriptional cascade and caused significant inhibition of C/EBPα and PPARγ mRNAs expression in a dose-dependent manner [22].
Anti-Inflammatory Effect of Acetylene Carotenoids
Inflammation is a normal protective response of human body to tissue damage or infection. However, excessive (chronic) inflammation often adversely affects health and gives rise to many diseases. It has been reported that persistence of chronic inflammation exposure increases the risk of obesity, insulin resistance, type 2 diabetes, cardiovascular disease or cancer. The inflammation process is initiated by the synthesis and secretion of proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6. The excessive production of inflammatory cytokines and the subsequent increase in reactive oxygen and nitrogen species are recognized as characteristics of inflammation. These events associated with acute inflammation are usually regulated by the secretion of anti-inflammatory cytokines. The intracellular antioxidants also regulate the development of inflammation. Sea squirts Halocynthia roretzi are a popular seafood in Japan and Korea. They contain acetylenic carotenoids such as halocynthiaxanthin, alloxanthin and diatoxanthin (fig. 1) [26]. We have found that all-trans alloxanthin and all-trans diatoxanthin isolated from H. roretzi suppressed the LPS-induced expression of IL-1β and IL-6 mRNA and protein in RAW264.7 cells [27]. Furthermore, 9-cis isomers of alloxanthin and diatoxanthin also downregulated IL-1β and IL-6 mRNA in RAW264.7 cells. IL-1β has been shown to be an important cytokine in chronic inflammatory diseases [28]. Although LPS stimulation is an acute inflammatory model [29], our results provide useful information regarding the effectiveness of alloxanthin and diatoxanthin with acetylenic structure in preventing both acute-phase and chronic inflammation. Alloxanthin and diatoxanthin also attenuated the expression of COX-2 and iNOS mRNA in RAW264.7 cells stimulated by LPS [27]. Therefore, alloxanthin and diatoxanthin possibly show anti-inflammatory effects through the downregulation of the mRNA for COX-2 and iNOS as well as proinflammatory cytokines in the activated macrophages. Downregulation of IL-1β mRNA by β-carotene was shown to be weaker compared with that of alloxanthin and diatoxanthin. Zeaxanthin did not suppress expression of the proinflammatory cytokine mRNA in RAW264.7 cells treated with LPS in our study. In addition, downregulation of COX-2 and iNOS mRNA by β-carotene and zeaxanthin was also weaker than that of alloxanthin and diatoxanthin.
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Although the mechanism involved has not been elucidated in detail, these results indicate that the structure of the carotenoids, especially with the acetylene bond, is the key to their anti-inflammatory effects.
Conclusion
With the increasing knowledge of biofunctional properties of marine products, utilization of these products has accelerated. Marine oils have been the object of interest of many researchers, as they are a great source of long-chain omega–3 polyunsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid. Marine oils contain little but important bioactive compounds such as carotenoids. Judging from their specific chemical structures, we should pay considerable attention to marine carotenoids because of their expected various functions in human health. From this viewpoint, the antiobesity effect of edible seaweed carotenoid, fucoxanthin, is very interesting, as its molecular mechanism has been made clear and its activity depends on the protein and gene expressions of UCP1 in WAT, although UCP1 is usually only found in BAT. However, little has been done on the nutrigenomic approach to beneficial physiological effects of other carotenoids. Investigations are needed to evaluate the mechanisms with special reference to their regulation of relative gene and protein expression. Unless human intervention studies are conducted, it will be difficult to predict the bigger role of marine carotenoids in disease prevention. Further studies will provide positive results with respect to the application of these carotenoids as nutraceuticals.
Acknowledgment This work was supported by Research and Development Program for New Bio-industry Initiatives of Bio-Oriented Technology Research Advancement Institution, the Grant-in-Aid for Scientific Research and the 21st Century COE Program of the Ministry of Education, Culture, Sports, Science and Technology, Government of Japan.
References 1 Palermo JA, Gros EG, Seldes AM: Carotenoids from three red algae of the Corallinaceae. Phytochemistry 1991;30:2983–2986. 2 Dembitsky VM, Maoka T: Allenic and cumulenic lipids. Prog Lipid Res 2007;46:328–375. 3 Mori K, Ooi T, Hiraoka M, Oka N, Hamada H, Tamura M, Kusumi T: Fucoxanthin and its metabolites in edible brown algae cultivated in deep seawater. Mar Drugs 2004;2:63–72.
Function of Marine Carotenoids
4 Matsuno T: Aquatic animal carotenoids. Fisheries Sci 2001;67:771–783. 5 Goodwin TW: Metabolism, nutrition, and function of carotenoids. Ann Rev Nutr 1986;6:273–297. 6 Davies BH: Carotenoid metabolism in animals: a biochemist’s view. Pure Appl Chem 1985;57:679– 684.
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7 Sachindra NM, Bhaskar N, Mahendrakar NS: Carotenoids in different body components of Indian shrimps. J Sci Food Agri 2005;85:167–172. 8 Renstrøm B, Borch G, Liaaen-Jensen S: Natural occurrence of enantiomeric and meso-astaxanthin 4. Ex Shrimp (Pandalus borealis). Comp Biochem Physiol 1981;69B:621–624. 9 Nègre-Sadargues G, Castillo R, Seginzac M: Carotenoid pigments and tropic behavior of deepsea shrimps (Crustacea, Decapoda, Alvinocarididae) from a hydrothermal area of the Mid-Atlantic Ridge. Comp Biochem Physiol 2000;127A:293–300. 10 Di Mascio P, Murhy ME and Sies H: Antioxidant defense systems: the role of carotenoids, tocopherols, and thiols. Am J Clin Nutr 1991;53:194S–200S. 11 Terao J: Antioxidant activity of β-carotene-related carotenoids in solution. Lipids 1989;24:659–661. 12 Lim BP, Nagao A, Terao J, Tanaka K, Suzuki T, Takama K: Antioxidant activity of xanthophylls on peroxyl radical-mediated phospholipid peroxidation. Biochim Biophys Acta 1992;1126:178–184. 13 Goto S, Kogure K, Abe K, Kimata Y, Kitahama K, Yamashita E, Terada H: Efficient radical trapping at the surface and inside the phospholipid membrane is responsible for highly potent antiperoxidative activity of the carotenoid astaxanthin. Biochim Biophys Acta 2001;1512:251–258. 14 Matsushita Y, Suzuki R, Nara E, Yokoyama A, Miyashita K: Antioxidant activity of polar carotenoids including astaxanthin-β-glucoside from marine bacterium on PC liposomes. Fish Sci 2000; 66:980–985. 15 De Luca LM: Retinoids and their receptors in differentiation, embryogenesis, and neoplasia. FASEB J 1991;5:2924–2933. 16 Chew BP, Park JS: Carotenoid action on the immune response. J Nutr 2004;134:257S–261S. 17 Dulloo AG, Samec S: Uncoupling proteins: their roles in adaptive thermogenesis and substrate metabolism reconsidered. Br J Nutr 2001;86:123– 139. 18 Lowell BB, S-Susullc V, Hamann A, Lawltts, JA, Himms-Hagen J, Boyer BB, Kozak LP, Fller JS: Development of obesity in transgenic mice after genetic ablation of brown adipose tissue. Nature 1993;366:740–742.
19 Serra F, Bonet ML, Puigserver P, Oliver J, Palou A: Stimulation of uncoupling protein 1 expression in brown adipocytes by naturally occurring carotenoids. Intl J Obesity 1999;23:650–655. 20 Maeda H, Hosokawa M, Sashima T, Funayama K, Miyashita K: Fucoxanthin from edible seaweed, Undaria pinnatifida, shows antiobesity effect through UCP1 expression in white adipose tissues. Biochim Biophys Res Commun 2005;332:392–397. 21 Maeda H, Hosokawa M, Sashima T, Takahashi N, Kawada T, Miyashita K: Fucoxanthin and its metabolite, fucoxanthinol, suppress adipocyte differentiation in 3T3-L1 cells. Int J Mole Med 2006;18: 147–152. 22 Okada T, Nakai M, Maeda H, Hosokawa M, Sashima T, Miyashita K: A comparative study on the ability of carotenoids to suppress differentiation of 3T3-L1 cells. J Oleo Sci 2008;57:345–351. 23 Gregoire FM, Smas CM, Sul HS: Understanding adipocyte differentiation. Physiol Rev 1998;78:783– 809. 24 Tontonoz P, Hu E, Spiegelman BM: Stimulation of adipogenesis in fibroblasts by PPARγ2, a lipid-activated transcription factor. Cell 1994;79:1147–1156. 25 Grimaldi PA: The roles of PPARs in adipocyte differentiation. Prog Lipid Res 2001;40:269–281. 26 Matsuno T, Ookubo M, Nishizawa T, Shimizu I: Carotenoids of sea squirts. I. New marine carotenoids, halocynthiaxanthin and mytiloxanthinone from Halocynthia roretzi. Chem Pharm Bull 1984; 32:4309–4315. 27 Konishi I, Hosokawa M, Sashima T, Maoka T, Miyashita K: Suppressive effects of alloxanthin and diatoxanthin from Halocynthia roretzi on the LPSinduced expression of pro-inflammatory genes in RAW264.7 cells. J Oleo Sci 2008;57:181–189. 28 Burger D, Dayer JM, Palmer G, Gabay C: Is IL-1 a good therapeutic target in the treatment of arthritis? Best Pract Res Clin Rheumatol 2006;20:879– 896. 29 Ziegler-Heitbrock HW: Molecular mechanism in tolerance to lipopolysaccharide. J Inflamm 1995;45: 13–26.
Dr. Kazuo Miyashita Faculty of Fisheries Sciences, Hokkaido University 3-1-1 Minato, Hakodate Hokkaido 041-8611 (Japan) Tel. +81 138 548394, Fax +81 138 548394, E-Mail
[email protected]
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Life-style Related Diseases Yoshikawa T (ed): Food Factors for Health Promotion. Forum Nutr. Basel, Karger, 2009, vol 61, pp 147–155
Exercise and Food Factors Wataru Aoi Laboratory of Health Science, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto, Japan
Abstract Habitual exercise is beneficial to health as it improves metabolism, reduces the risk of cardiovascular disease, and maintains the immune system. Appropriate nutrition contributes to acceleration of health promotion due to exercise. Recommended daily allowance is elevated by physical activity and intake of various food factors such carbohydrates, proteins, vitamins, minerals, and other phytochemicals is required to avoid their shortage. Additional dietary food factors are effective not only in supplementation to satisfy the allowance but also in further acceleration of the benefits of fitness. Dietary nutrition is also important to maintain active function in the elderly by preventing aginginduced muscle atrophy and avoiding intense exercise-induced disorders. Recently, several food components have been found to show physiological effects, and some of them are considered to be useful for promoting or alternating the beneficial effects of exercise, maintaining homeostasis, and preventing muscle aging. However, some of these food factors should only be used when there is clear scientific evidence. Also, it is important to understand the physiological changes caused by exercise to use them correctly. This article describes various food factors that have been reported to be effective for improving health promotion, along with the relevant physiological changes that Copyright © 2009 S. Karger AG, Basel occur during exercise.
Appropriate daily exercise prevents common disease and contributes to anti-aging. Exercise increases energy metabolism and cardiovascular function and reduces the risk of arteriosclerosis. Also, moderate exercise activates immunocompetence and is effective for the prevention of inflammatory diseases, infection and cancer. Further, exercise enhances muscle strength, which prevents reduction in active function due to muscle loss in the elderly. Proper nutrition contributes to acceleration of health promotion due to exercise. Previously, a great number of studies have scientifically investigated various food factors that have the potential to exert this function and reported that intake of some nutritional factors is effective for smooth energy metabolism, preventing homeostasis disturbance, recovery from fatigue after exercise, and maintaining muscle mass. The purpose of the food factors can be summarized as follows: (1) acceleration of beneficial effects due to exercise, (2) alternative effect of
Table 1. Exercise and food factors. Physiological functions
Food factors
Replenishment of water
water and electrolytes (sodium) – as isotonic or hypotonic drink
Improvement of energy metabolism
carnitine, astaxanthin, catechin, α-lipoic acid, caffeine, capsaicin, carbohydrates
Building muscle
proteins, BCAAs, β-HMB, glutamine
Prevention of exercise-induced disorder Oxidative damage Muscle damage Immunosuppression
vitamins C and E, carotenoids, polyphenols BCAAs, astaxanthin, vitamin E carbohydrates, vitamins C and E, glutamine
the benefits, and (3) avoiding homeostasis disturbance. When we use the food factors, it is important to consider their intake in terms of appropriate quantity, timing and quality. In addition, we should understand the physiological changes caused by exercise as this would allow effective utilization of the food factors. This article introduces the methods of dietary nutritional management with food factors that have been scientifically demonstrated to have a beneficial influence on health promotion when combined with exercise (table 1).
Replenishment of Water in Exercise
Water is the main constituent of the human body, and it plays an essential role in circulatory function, chemical reactions involved in energy metabolism, elimination of waste products, and maintenance of the body temperature and plasma volume. When the body temperature rises with physical activity or a high ambient temperature, sweating occurs in order to radiate heat, leading to the loss of a large amount of water and electrolytes such as sodium. This loss of body fluid impairs thermoregulation and the circulatory system, leading to a decline of various physiological functions and health problems such as heat exhaustion and heat stroke. Therefore, to maintain homeostasis and avoid disorder, replenishment of water and electrolytes is essential before and during or after exercise [1]. When a large amount of sweating occurred by prolonged exercise, individuals looking to achieve rapid and complete recovery from dehydration should drink 1.5 l of fluid for each kilogram of body weight loss. Generally, it is believed to be useful to drink isotonic beverage that contains electrolytes such sodium, potassium, and chloride at concentrations close to those in body fluids. Also, it has been suggested that intake of hypotonic beverage may exert a similar
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or more rapid effect on replenishment of body water [2] because it is rapidly absorbed from the small intestine along an electrochemical gradient. Furthermore, the sodium concentration and the osmolality of sweat is lower than that of extracellular fluid, so the loss of water with sweating is much greater than the loss of electrolytes, leading to an increase in the plasma osmotic pressure. On the other hand, replenishment of water alone is unlikely to maintain homeostasis of body fluid in prolonged exercise that produces high sweat rates. Taking in only water in prolonged exercise leads to hyponatremia and a decrease in the osmotic pressure of body fluids and inhibits the release of antidiuretic hormone resulting in that water intake is suppressed and the urine output is increased [3]. During prolonged exercise lasting longer than 90 min, fluid drink containing electrolytes and carbohydrate, not water alone, should be considered to sustain carbohydrate oxidation and physical activity [4]. However, it is not clear whether hyperhydration with additional fluids in the extra- and intracellular space before exercise can prevent the disorder and provide physiologic or performance advantage.
Improvement of Energy Metabolism
Energy consumed in muscle during exercise is mainly supplied by carbohydrates and lipids. It is important for health promotion and athletic sports to regulate the metabolism of these two substrates (fig. 1). When energy source during exercise depends on supply from lipids, large energy can be continuously obtained via aerobic metabolism. Thus, the exercise that depends on lipids as energy substrate efficiently accelerates lipolysis of adipose tissue by daily continuation. When energy source is based on carbohydrates, the intramuscular pH will decrease due to increased lactic acid production, which may lead to impairment of muscle contraction. Therefore, elevation of lipid utilization in skeletal muscle is associated with development of obesity and aerobic endurance. In general, the ratio of energy substrates varies with exercise intensity, a habit of exercise, and the level of physical performance. Additionally, the potential effects of several food factors on muscle lipid metabolism in exercise have been investigated, so some of them accelerate the lipid utilization while the efficacy is still controversial. A rate-limiting step of lipid metabolism in myocyte is entry of long-chain fatty acids into mitochondria. Carnitine is essential for the transport of long-chain fatty acids across the outer- and inner-mitochondrial membranes associated with carnitine palmitoyltransferase (CPT). It is a naturally occurring compound that can be synthesized in mammals from the essential amino acids lysine and methionine or ingested through diet. In several human studies, intake of 2–4 g of carnitine before exercise or on a daily basis was reported to increase fat oxidation and reduce the reliance on endogenous carbohydrate stores during exercise [5]. CPT I located on the mitochondrial outer membrane plays an important role in the entry of fatty acids. We
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Glucose
Lipids
Glucose Glycogen
Lipids
Glycolysis
-oxidation
TCA cycle Electron transport ATP
ATP
Energy substrate Carbohydrates
Energy substrate Lipids
Food factors ?
Carbohydrates Lipids
Fig. 1. Energy substrate in muscle during exercise.
found that a novel antioxidant astaxanthin limits the oxidative modification of CPT I by hexanoyl lysine with elevating CPT I activity during exercise, which caused acceleration of body fat reduction by exercise training [6]. Other food compounds which have antioxidative capacity have been also demonstrated. Catechin, one of polyphenols found in Japanese green tea, accelerates utilization of fatty acid as a source of energy production in skeletal muscle during exercise [7]. It was demonstrated that the effect of catechin is related to enhancement of β-oxidation activity and the level of fatty acid translocase/CD36 mRNA in the muscle. The antioxidant α-lipoic acid improves glucose transport activity in skeletal muscle. Intake of α-lipoic acid combined with endurance exercise training further accelerates glucose uptake and insulin signal pathway compared with training alone [8]. Another compound which can affect energy metabolism, caffeine inhibits phosphotidiesterase by promoting catecholamine release and increases hormone-sensitive lipase activity, which leads to an increase in circulating free fatty acids and further improvement of endurance [9]. Capsaicin, obtained from hot red peppers, is likely to enhance fat metabolism by increasing lipolytic hormones and promoting fat oxidation in skeletal muscle [10]. On the other hand, in prolonged exercise such as a marathon, taking carbohydrates immediately before or during exercise is also an effective method of preventing fatigue caused by depletion of energy substrate. Under such conditions, it is desirable
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Protein degradation Apoptosis Undifferentiation
Atrophy
Stumble • Bedridden
Aging
Decrease in metabolic enzyme Switching to IIb fiber
Metabolic dysfunction
Obesity • Arteriosclerosis
Fig. 2. Muscle aging and health problems.
for the athlete to ingest monosaccharides or oligosaccharides, because these are rapidly absorbed and transported to the peripheral tissues.
Building Muscle
Muscle mass decreases due to immobilization, physical inactivity, spaceflight, or aging. Muscle loss leads to a decrease in muscle strength, and especially age-related muscle loss called sarcopenia is involved in various health problems with reduction in quality of life and an increase in healthcare expenditure (fig. 2). Additionally, skeletal muscle is the biggest energy-consuming organ in the body, and therefore the reduction in muscle mass decreases energy metabolic rate, resulting in body fat accumulation, which associates various metabolic diseases. Muscle atrophy occurs due to several factors including apoptosis and a decrease in differentiation capacity of satellite cells as well as protein loss caused by a decrease in protein synthesis and increase in protein degradation. Habitual exercise contributes to prevention of muscle loss by mainly synthesizing proteins. Especially, resistance exercise is aimed at increasing muscle mass because it enhances the secretion and production of growth hormone and various growth factors. It has been shown that the protein intake required for a person performing strength training is higher than that for sedentary individuals [11]. The
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recommended daily protein intake is estimated to be 1.4–1.8 g/kg for persons performing resistance exercise when the intake of calories and carbohydrate is adequate [12]. Not only the amount of protein intake, but also the timing of intake is important for building muscles efficiently. Eating proteins immediately after exercise is more effective for protein synthesis compared with eating proteins several hours later. The cross-sectional area of quadriceps muscle after 12 weeks of resistance training program was shown to be more increased in older men consuming proteins immediately after exercise than 2 h later [13]. Additionally, the synthesis of muscle proteins can be promoted by intake of proteins combined with carbohydrates via the actions of insulin, accelerating the increase in muscle mass and strength [14]. In addition, it has been reported that the intake of amino acids and peptides is beneficial. Free amino acids and small molecules of peptides do not need to be digested, so rapid absorption can be expected. Amino acids are not only utilized for the synthesis of muscle proteins, and some of these molecules also exert a variety of physiological effects. Attention has been focused on the effects of branched-chain amino acids (BCAAs), including valine, leucine, and isoleucine, which are known to be abundant in both muscle proteins and food proteins. BCAAs are metabolized in the muscles and utilized as energy substrates, and their oxidation is enhanced during exercise by activation of branched-chain α-keto acid dehydrogenase [15]. Therefore, when BCAAs are not supplied from diet, muscle proteins are catabolized to obtain them. Furthermore, dietary BCAAs modulate muscle protein metabolism to promote the synthesis and inhibit the degradation of proteins [16], resulting in an anabolic effect on the muscles. Glutamine has also been reported to promote muscle growth by inhibiting protein degradation [17]. It is the most abundant free amino acid in muscle tissue and its intake leads to an increase in myocyte volume, resulting in stimulation of muscle growth. Glutamine is also found at relatively high concentrations in many other human tissues and has an important homeostatic role. Therefore, during catabolic states such as exercise, glutamine is released from skeletal muscle into the plasma to be utilized for maintenance of the glutamine level in other tissues [18]. β-Hydroxy-β-methylbutyrate (β-HMB) is a metabolite of the BCAA leucine, and it increases muscle mass by inhibiting protein degradation via influence on the metabolism of BCAAs. A meta-analysis of studies done between 1967 and 2001 supported the use of β-HMB to augment lean body mass and strength when performing resistance exercise [19]. Several studies have demonstrated that a daily intake of 3.0 g β-HMB for more than 4 weeks led to a greater increase in lean body mass or power compared with the intake of placebo [20].
Prevention of Exercise-Induced Homeostasis Disturbance
Intense physical activity or unaccustomed exercise possibly induces various health disorders such oxidative damage, muscle pain, and immune depression. Exercise
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leads to the production of reactive oxygen species (ROS), mainly via the mitochondrial electron transport chain, xanthin oxidase, and phagocytes. Exerciseinduced ROS oxidize several targets such as proteins, lipids, and DNA in skeletal muscle, heart, and liver, and this is considered a possible cause of health disorders. Previous studies have demonstrated that dietary supplementation of antioxidants such as vitamin E, vitamin C, and carotenoids can decrease oxidative damage induced by intense exercise [21, 22]. Also, ROS generated during exercise could be related to initiation of delayed-onset muscle damage. ROS are generated from mitochondria and endothelium during exercise via elevation of the oxygen uptake of myocytes and ischemia-reperfusion process, which leads to infiltration of phagocytes into the muscles after exercise via redox-sensitive inflammatory cascade. Some antioxidants including vitamin E and astaxanthin could inhibit exercise-induced muscle inflammation [23, 24]. On the other hand, when the muscle damage is induced by a high level of mechanical stress in such resistance exercise or downhill running, the antioxidant factors are likely difficult to prevent inflammation and muscle pain. Although it is known that BCAAs attenuate delayed-onset muscle soreness induced by resistance exercise [25], the exact mechanism is unknown. It is generally believed that moderate exercise enhances immunocompetence and is effective for the prevention of inflammatory diseases, infection, and cancer, while excessive physical activity leads to immunosuppression and an increase in inflammatory and allergic disorders. Susceptibility to infections following excessive physical activity is caused by an increase in the production of immunosuppressive factors such as adrenocortical hormones and anti-inflammatory cytokines, leading to a decrease in the number and activity of circulating natural killer cells and T cells as well as a lower IgA concentration in the saliva [26]. Therefore, persons performing strenuous or unaccustomed exercise are exposed to the risk of impaired immunocompetence. Intake of carbohydrates in prolonged exercise at submaximal intensity attenuates the increase of plasma cortisol and cytokine levels after exercise, which could lead to the inhibition of immunosuppression [26, 27]. Vitamin C and vitamin E have actions that promote immunity, and are essential for T cell differentiation and for maintenance of T cell function [28, 29]. However, there is limited evidence about the effects of vitamin supplementation on immune function in relation to exercise. Glutamine is an important energy source for lymphocytes, macrophages, and neutrophils, and is also an essential amino acid for the differentiation and growth of these cells [18]. Intense exercise decreases plasma glutamine concentration and this may be related to immunosuppression. Castell and Newsholme [30] reported that athletes who ingested glutamine had a lower infection rate after a marathon compared with the placebo group with an increase in the T helper/T suppressor cell ratio. Furthermore, glutamine enhances the activity of intestinal enterobacteria and inhibits the production of cytokines involved in inflammation or immunosuppression.
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Conclusion
Parallel to changes in dietary habits, the growing aging population, and ever-increasing medical costs, people show a growing interest in health and have come to expect dietary food factors. In recent years, various food factors have been evaluated scientifically to determine whether they have any physiological effects such as prevention of diseases. In the literature, even though these effects have been carefully investigated in certain food factors, the efficacy of the latter is still controversial. The effectiveness of the components may differ according to gender and mode of ingestion, so that the optimum method of intake, the quantity and quality of foods to be ingested, and the timing of their intake need to be established in accordance with the purpose of using each food or food component, after understanding the physiological changes induced by exercise. In the future, guidelines for the use and an evaluation system of sports functional foods should be established on the basis of clear scientific evidence related to the individual foods.
References 1 Sawka MN, Burke LM, Eichner ER, Maughan RJ, Montain SJ, Stachenfeld NS: Exercise and fluid replacement. Med Sci Sports Exerc 2007;39:377– 390. 2 Castellani JW, Maresh CM, Armstrong LE, Kenefick RW, Riebe D, Echegaray M, Kavouras S, Castracane VD: Endocrine responses during exercise-heat stress: effects of prior isotonic and hypotonic intravenous rehydration. Eur J Appl Physiol Occup Physiol 1998;77:242–248. 3 Takamata A, Mack GW, Gillen CM, Nadel ER: Sodium appetite, thirst, and body fluid regulation in humans during rehydration without sodium replacement. Am J Physiol 1994;266:R1493–R1502. 4 Latzka WA, Montain SJ: Water and electrolyte requirements for exercise. Clin Sports Med 1999;18:513–524. 5 Brass EP: Supplemental carnitine and exercise. Am J Clin Nutr 2000;72:618S–623S. 6 Aoi W, Naito Y, Takanami Y, Ishii T, Kawai Y, Akagiri S, Kato Y, Osawa T, Yoshikawa T: Astaxanthin improves muscle lipid metabolism in exercise via inhibitory effect of oxidative CPT I modification. Biochem Biophys Res Commun 2008;366:892–897. 7 Murase T, Haramizu S, Shimotoyodome A, Tokimitsu I, Hase T: Green tea extract improves running endurance in mice by stimulating lipid utilization during exercise. Am J Physiol Regul Integr Comp Physiol 2006;290:R1550–R1556.
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8 Henriksen EJ: Exercise training and the antioxidant alpha-lipoic acid in the treatment of insulin resistance and type 2 diabetes. Free Radic Biol Med 2006; 40:3–12. 9 Ryu S, Choi SK, Joung SS, Suh H, Cha YS, Lee S, Lim K: Caffeine as a lipolytic food component increases endurance performance in rats and athletes. J Nutr Sci Vitaminol 2001;47:139–146. 10 Oh TW, Oh TW, Ohta F: Dose-dependent effect of capsaicin on endurance capacity in rats. Br J Nutr 2003;90:515–520. 11 Mccall GE, Byrnes WC, Fleck SJ, Dickinson A, Kraemer WJ: Acute and chronic hormonal responses to resistance training designed to promote muscle hypertrophy. Can J Appl Physiol 1999;24:96–107. 12 Phillips SM: Protein requirements and supplementation in strength sports. Nutrition 2004;20:689– 695. 13 Esmarck B, Andersen JL, Olsen S, Richter EA, Mizuno M, Kjaer M: Timing of postexercise protein intake is important for muscle hypertrophy with resistance training in elderly humans. J Physiol 2001;535:301–311. 14 Borsheim E, Aarsland A, Wolfe RR: Effect of an amino acid, protein, and carbohydrate mixture on net muscle protein balance after resistance exercise. Int J Sport Nutr Exerc Metab 2004;14:255–271. 15 Shimomura Y, Murakami T, Nakai N, Nagasaki M, Harris RA: Exercise promotes BCAA catabolism: effects of BCAA supplementation on skeletal muscle during exercise. J Nutr 2004;134:1583S–1587S.
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16 Phillips GC: Glutamine: the nonessential amino acid for performance enhancement. Curr Sports Med Rep 2007;6:265–268. 17 Antonio J, Street C: Glutamine: a potentially useful supplement for athletes. Can J Appl Physiol 1999;24: 1–14. 18 Castell LM: Glutamine supplementation in vitro and in vivo, in exercise and in immunodepression. Sports Med 2003;33:323–345. 19 Nissen S, Sharp R: Effect of dietary supplements on lean mass and strength gains with resistance exercise: a meta-analysis. J Appl Physiol 2003;94:651– 659. 20 Wilson GJ, Wilson JM, Manninen AH: Effects of beta-hydroxy-beta-methylbutyrate (HMB) on exercise performance and body composition across varying levels of age, sex, and training experience: a review. Nutr Metab (Lond) 2008;5:1. 21 Phillips T, Childs AC, Dreon DM, Phinney S, Leeuwenburgh C: A dietary supplement attenuates IL-6 and CRP after eccentric exercise in untrained males. Med Sci Sports Exerc 2003;35:2032–2037. 22 Kanter MM, Nolte LA, Holloszy JO: Effects of an antioxidant vitamin mixture on lipid peroxidation at rest and postexercise. J Appl Physiol 1993;74:965– 969. 23 Aoi W, Naito Y, Sakuma K, Kuchide M, Tokuda H, Maoka T, Oka S, Toyokuni S, Yoshikawa T: Astaxanthin limits exercise-induced skeletal and cardiac muscle damage in mice. Antiox Redox Sign 2003;5:139–144.
24 Aoi W, Naito Y, Takanami Y, Kawai Y, Sakuma K, Ichikawa H, Yoshida N, Yoshikawa T: Oxidative stress and delayed-onset muscle damage after exercise. Free Radic Biol Med 2004;15:480–487. 25 Shimomura Y, Murakami T, Nakai N, Nagasaki M, Harris RA: Exercise promotes BCAA catabolism: effects of BCAA supplementation on skeletal muscle during exercise. J Nutr 2004;134:1583S–1587S. 26 Gleeson M, Nieman DC, Pederson BK: Exercise, nutrition and immune function. J Sports Sci 2004; 22:115–125. 27 Nehlsen-Cannarella SL, Fagoaga OR, Nieman DC, Henson DA, Butterworth DE, Schmitt RL, Bailey EM, Warren BJ, Utter A, Davis JM: Carbohydrate and the cytokine response to 2.5 h of running. J Appl Physiol 1997;82:1662–1667. 28 Peake JM: Vitamin C: Effects of exercise and requirements with training. Int J Sport Nutr Exerc Metab 2003;13:125–151. 29 Moriguchi S, Muraga M: Vitamin E and immunity. Vitam Horm 2000;59:305–336. 30 Castell LM, Newsholme EA: The effects of oral glutamine supplementation on athletes after prolonged, exhaustive exercise. Nutrition 1997;13:738–742.
Dr. Wataru Aoi Laboratory of Health Science, Graduate School of Life and Environmental Sciences Kyoto Prefectural University Kyoto (Japan) Tel. +81 75 703 5417, Fax +81 75 703 5417, E-Mail
[email protected]
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Chemoprevention and Cancer Yoshikawa T (ed): Food Factors for Health Promotion. Forum Nutr. Basel, Karger, 2009, vol 61, pp 156–169
Molecular Basis for Cancer Chemoprevention by Green Tea Polyphenol EGCG Hirofumi Tachibana Department of Bioscience and Biotechnology, Faculty of Agriculture, and Bio-Architecture Center, Kyushu University, Fukuoka, Japan
Abstract For the past two decades, many researchers have been investigating the potential cancer-preventive and therapeutic effects of green tea. (–)-Epigallocatechin-3-O-gallate (EGCG) has been shown to be the most active and major polyphenolic compound from green tea. The mechanisms of action of EGCG have been extensively investigated. However, the mechanisms for the cancer-preventive activity of EGCG are not completely characterized and many features remain to be elucidated. Recently, we have identified the 67-kDa laminin receptor (67LR) as a cell surface EGCG receptor that confers EGCG responsiveness to many cancer cells at physiological concentrations. This article reviews some of the reported mechanisms and possible targets for the action of EGCG. We especially focus on the current understanding of the signaling pathway for physiologically relevant EGCG through the 67LR for cancer prevention. Our data shed new light on the molecular basis for the cancer-preventive activity of EGCG in vivo and helps in the design of new strategies to prevent cancer. Copyright © 2009 S. Karger AG, Basel
Tea is one of the most widely consumed beverages in the world. It has been demonstrated that tea constituents exhibit various biological and pharmacological properties such anticarcinogenic, antioxidative, antiallergic, antivirus, antihypertensive, antiatherosclerosis, and antihypercholesterolemic activities [1]. Responsible for these activities were shown to be a group of polyphenols, catechins. Major catechins found in tea leaves are (–)-epigallocatechin-3-O-gallate (EGCG), (–)-epigallocatechin (EGC), (–)-epicatechin-3-O-gallate (ECG), (–)-epicatechin (EC). Among the green tea catechins, EGCG is the most abundant, representing ~16.5 wt% of the water-extractable fraction of green tea leaves. In addition, it is the most active catechin in various kinds of physiological activities. Because EGCG is not found in any other plant than tea, EGCG is regarded as a constituent characterizing green tea. Recently, a double-blind, placebo-controlled study on oral administration of green tea catechins (EGCG, 50%) in volunteers with high-grade prostate intraepithelial neoplasia demonstrated that
green tea catechins have potent in vivo chemoprevention activity for human prostate cancer [2]. This impressive evidence has fueled interest in the role of EGCG as chemoprevention of cancer. This article will discuss the effects of green tea polyphenol EGCG on signal transduction pathways that are related to cancer chemoprevention based on the biological importance of the target. In particular, we will focus on the current understanding of EGCG signaling pathway through the 67-kDa laminin receptor (67LR) as the target molecule mediating anticancer effect of EGCG in vivo.
Antioxidants and Pro-Oxidants
Mechanistic investigations of the cancer chemopreventive activity depend on the methodologies available at different time periods. For example, EGCG and other tea polyphenols are well known for their antioxidant activities. Indeed, they have been demonstrated to inhibit carcinogen-induced DNA damage and tumor promoter-induced oxidative stress [3]. These results are consistent with the commonly mentioned idea that tea prevents cancer because tea polyphenols are antioxidants. It is unclear, however, whether this is a general mechanism for cancer prevention, especially in human carcinogenesis when strong carcinogenesis and tumor promoters are not known to be involved. In the 1980s when carcinogen activation and tumor promotion were active areas of research, these events had been proposed as the targets of tea polyphenol action. There are two major problems in extrapolating results observed in cell lines to animal models. (1) The concentration of the test compound used in cell line systems, for example EGCG at 20–100 μm, or higher concentrations, are much higher than those observed in the plasma or tissues in experimental animals or humans after ingestion of tea or related tea preparations [4]. (2) The oxygen partial pressure in a cell culture system (160 mm Hg) is much higher than that in the blood or tissues (<40 mm Hg). Under cell culture conditions, EGCG is not stable, with a half-life of less than 2 h [5]. The half-life can be extended several folds by the addition of superoxide dismutase, suggesting a role for superoxide radical in the oxidation and polymerization of EGCG [6]. Similar to other antioxidants, EGCG and other tea polyphenols may also act as pro-oxidants. They can be oxidized to form phenolic radicals, superoxide radical and hydrogen peroxide. These species may trigger a variety of biochemical reactions and biological responses. In studies of EGCG and other polyphenolic compounds in cell culture, the addition of superoxide dismutase and catalase is recommend to stabilize EGCG and to avoid possible artifacts. Thus, according to this observation, we also added those enzymes to the cell culture systems [7–10]. It is not clear whether prooxidants produced by EGCG-generated reactions occur in low oxygen partial pressure conditions in vivo in cells which generally have strong antioxidative capacity and low oxygen partial pressure. The difference between in vitro and in vivo systems should be considered in studies attempting to elucidate the mechanisms of action of EGCG.
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Bioavailability of EGCG
The bioavailability and biotransformation of tea catechins following tea ingestion has been investigated as well as time to reach maximal concentration in the plasma 1.5–2.5 h after consumption of decaffeinated green tea solids (1.5, 3.0 and 4.5 g) [11]. The catechin levels decreased and were not detectable by 24 h. Although EGCG and ECG were not detected in the urine, 90% of the urinary EC and EGC were excreted by 8 h. Most of the ingested EGCG apparently does not get into the blood, and absolute EGCG is preferentially excreted through the bile to the colon. Glucuronidation, sulfation, methylation, and ring-fission metabolism represent the major metabolic pathways for green tea catechins. Plasma EC and EGC were present mainly in the conjugated form such as glucuronide and sulfate conjugates, whereas 77% of the EGCG was in the free form. EGCG has also been shown to undergo methylation. The maximum plasma concentration of 4⬘,4⬙-di-O-methyl-EGCG is 20% that of EGCG but the cumulative excretion of 4⬘,4⬙-di-O-methyl-EGCG is 10-fold higher than that of EGCG over 24 h [12]. Although most of published studies in cell culture systems used 10–100 μm of EGCG, the blood level of EGCG after consuming the equivalent of 2–3 cups of green tea was 0.1–0.6 μm and for an equivalent of 7–9 cups was still lower than 1 μm [13]. The rather poor bioavailability of tea catechins needs to be considered when we extrapolate results obtained in vitro to situations in vivo.
Possible Direct Targets for the Action of EGCG
Searching for high-affinity proteins that bind to EGCG is the first step to understanding the molecular and biochemical mechanisms of the anticancer effects of tea polyphenols. Several proteins that can directly bind with EGCG have been identified in in vitro models, and these topics are shown in table 1. Some are summarized below.
Proteasome The proteasome is a massive multicatalytic proteinase complex found in all eukaryotic cells and is responsible for degrading most of the cellular proteins. The ubiquitin/ proteasome-dependent degradation pathway plays an essential role in upregulation of cell proliferation, downregulation of cell death, and development of drug resistance in human tumor cells, suggesting that the proteasome is a novel and promising target for cancer chemotherapy. It has been reported that EGCG potently and specifically inhibited the chymotrypsin-like activity of the proteasome both in cell-free systems (IC50 = 86 nm) and tumor cell lines (1–10 μm) [14]. The inhibition of the proteasome by EGCG in several tumor and transformed cell lines results in the accumulation of
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Table 1. Molecular target candidates of EGCG and the effective concentrations Activity
Effective concentration
uPA inhibition H2O2 generation Binding to AhR complex Binding to DNA Cdk2/Cdk4 inhibition -Catenin lysosomal trafficking MMP2 and MMP9 inhibition DNA methyltransferase inhibition ERK1/2 phosphorylation ZAP-70 kinase inhibition Binding to ZAP-70 20s proteasome activity inhibition Fyn kinase inhibition Binding to Fyn Binding to Bcl-2 and Bcl-xL Binding to 67LR Binding to CD4 Vimentin-dependent cell growth inhibition Binding to vimentin
2–10 mM 0.1–1 mM 253 M (Kd) 54 M (Kd) 30 M 25 M 8–13 M (IC50) 7 M (Ki) 5–10 M 3 M (IC50) 0.62 M (Kd) 0.1–0.2 M 5 M (IC50) 0.367 M (Kd) 0.3–0.5 M (Ki) 0.04 M (Kd) 0.01 M (Kd) 15 M 0.003 M (Kd)
two natural proteasome substrates, p27Kip1 and IκB-α, followed by growth arrest in the G1 phase of the cell cycle.
Extracellular Signal-Regulated Protein Kinase 1/2 and Akt MAPKs have received increasing attention as target molecules for cancer prevention and therapy. EGCG inhibited the phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK1/2), and p38 MAPK activity in human fibrosarcoma HT1080 cells. EGCG at 20 μm inhibited the phosphorylation of MAP/ERK kinase 1/2 (MEK1/2), ERK1/2, and ELK-1 in H-ras transformed JB6 mouse epidermal cell line [15]. This study suggested that EGCG decreased the association between RAF-1 and MEK1, and EGCG competitively inhibited the phosphorylation of ELK-1 by ERK1/2 possibly by competing for the binding site on ERK1/2. EGCG inhibited epidermal growth factor-dependent activation of epidermal growth factor receptor (EGFR), and EGFR-dependent activation of ERK1/2 and Akt at the concentration range of 10–50 μm in the immortalized cervical cell line ECE16–1 [16]. Cell-free studies demonstrated that EGCG directly inhibits ERK1/2 and Akt kinase activity at the concentration of more than 5 μm.
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Bcl-2 Family Proteins Bcl-2 family proteins are important regulators of apoptosis. Antiapoptotic members of this family, such as Bcl-2 and Bcl-x, are located on the surface a hydrophobic groove in which they can bind the BH3 domain of the proapoptotic counterparts. This binding is crucial for the regulation of apoptosis in vivo, with pro- and antisurvival proteins neutralizing each other’s function through dimerization. A study using a combination of nuclear magnetic resonance binding assay, fluorescence polarization assay, and computational docking analysis demonstrated the direct binding of EGCG to the BH3 pocket of the antiapoptotic Bcl-2 family proteins and the inhibitory effect of EGCG on the binding of BH3 peptide to Bcl-2 family proteins, Bcl-2 and Bcl-xL with Ki values of 335 and 490 nm, respectively [17]. Whether EGCG can bind directly the intracellular Bcl-2 family proteins in cell culture systems or in vivo still remains unclear.
Vimentin Vimentin, one of the type III intermediate filament (IF) proteins, is a major component of IFs and is expressed during development in a wide range of cells, including mesenchymal cells and in a variety of cultured cell line and tumors. Vimentin is readily phosphorylated by numerous protein kinases such as cyclin-dependent kinases 2 (Cdc2) and cAMP-dependent protein kinase (PKA), thus regulating their functions. Vimentin was recently discovered as a high-affinity EGCG-binding protein with a Kd of 3.3 nm [18]. The protein was isolated from cell lysates of JB6 C141 mouse epidermal cells by an EGCG-sepharose 4B column. Functional studies showed that EGCG inhibited the phosphorylation of vimentin at Ser55 and Ser50 by Cdc2 (IC50 = 17 μm) and PKA (IC50 = 2 μm), respectively.
The 67-kDa Laminin Receptor as a Green Tea Catechin Receptor
It should be noted that most of the effects of EGCG in cell culture systems and cell-free systems have been obtained with higher concentrations than observed in the plasma or tissues of animals or in human plasma after administration of green tea or EGCG. The pharmacokinetic studies in humans indicate that the peak plasma concentration after a single dose of EGCG is <1.0 μm. Furthermore, the intracellular levels of EGCG are much lower than the extracellular levels. Therefore, it is not clear whether the activities observed with high EGCG concentrations in vitro can be observed in vivo and the proposed EGCG-binding molecules as mentioned above are responsible for in vivo physiological activities of EGCG.
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The 67-kDa Laminin Receptor The 67LR is a nonintegrin receptor for laminin, fibronectin and type IV collagen. Expression of the 67LR has been shown to be upregulated in neoplastic cells compared with their normal counterparts and directly correlate with an enhanced invasive and metastatic potential in many malignancies [19]. The receptor has been implicated in laminin-induced tumor cell attachment and migration, as well as in tumor angiogenesis, invasion, and metastasis. Surface expression of the 67LR has also been reported to be a dominant laminin-binding protein expressed in neutrophils, macrophages, and monocytes, which suggests that the receptor may play an important role in the regulation of cell adherence via the basement membrane laminin. Recently, it has become clear that acting as a receptor for laminin is not the only function of the 67LR. This protein also acts as a receptor for pathogenic prion protein, cytotoxic necrotizing factor 1 from Escherichia coli, Sindbis virus, Venezuelan equine encephalitis virus, Dengue virus, and adeno-associated virus.
Discovery of the 67LR as a Green Tea Catechin Receptor To elucidate the exact molecular basis for the action of EGCG, it is necessary to identify the molecular target triggering a specific signaling of EGCG. It is known that the toll-like receptor has the principal role in lipopolysaccharide signaling and that it is necessary to identify the specific receptor as a signal initiator for generating cellular responses to understand the specific cellular signaling of foreign or functional substances. However, the molecular target for physiologically relevant EGCG that can mediate its anticancer effect still remains unknown. Recently, we found that all-transretinoic acid (ATRA) enhanced the binding of EGCG to the cell surface of cancer cells when the binding was monitored on the basis of the increase in response units in a surface plasmon resonance assay [20]. To identify candidates through which EGCG inhibits cell growth, we used a subtraction cloning strategy involving cDNA libraries constructed from cells treated or untreated with ATRA. We isolated a single target that allows EGCG to bind to the cell surface. An analysis of the DNA sequence identified this unknown cell surface candidate as the 67LR. In fact, the expression of this 67LR was enhanced by ATRA treatment. Human lung cancer A549 cells were used to assess how effectively the 67LR elicits EGCG-mediated growth inhibition. Cells transfected with empty vector and treated with EGCG showed no growth inhibition. However, cells transfected with the gene encoding 67LR and treated with EGCG demonstrated considerable inhibition as compared with the cells treated with H2O. We next tested whether the growth inhibitory activity of EGCG correlates with the binding strength of EGCG to the cell surface. We found increased binding of EGCG to the cell surface of cells transfected with the 67LR. EGCG binding to the 67LR-transfected cells was inhibited by treatment
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with an antibody to the 67LR. The predicted Kd value for the binding of EGCG to the 67LR protein is 39.9 nm [20]. Most of the 67LR protein was found to exist in the raft fraction rather than the nonraft fraction [21], and this distribution pattern correlated well with the plasma membrane-associated EGCG level after treating the cells with EGCG, as previously reported [22]. To investigate whether the 67LR can confer a sensitivity to EGCG at physiologically relevant concentrations, we treated the 67LR-transfected A549 cells with two concentrations of EGCG (0.1 and 1.0 μm); these concentrations are similar to the amount of EGCG found in human plasma after drinking more than two or three cups of tea. The growth of the transfected cells was inhibited at both of these concentrations [20]. In addition, this growth-suppressive effect was completely eliminated upon treatment with anti-67LR antibody before the addition of EGCG. Although we have identified the 67LR as a cell surface receptor for EGCG that mediates EGCG-induced cell growth inhibition [20], there is no validation of its implication in EGCG-induced cancer prevention in vivo. We investigated the effect of oral administration of EGCG on subcutaneous tumor growth in C57BL/6N mice challenged with 67LR-ablated B16 cells [7]. We confirmed both silencing of the 67LR by stable RNAi in B16 cells and attenuation of the inhibitory effect of 1 μm EGCG on cell growth in 67LR-ablated B16 cells in vitro. As shown in figure 1, tumor growth was significantly retarded in EGCG-administered mice implanted with the B16 cells harboring a control shRNA, whereas tumor growth was not affected by EGCG in the mice implanted with 67LR-ablated B16 cells, suggesting that the 67LR functions as an EGCG receptor not only in vitro but also in vivo. Together, these observations demonstrate that the cell surface 67LR is the receptor for antitumor action of EGCG at the physiologically relevant concentration.
Inhibition of Cancer Cell Growth by EGCG through the Green Tea Catechin Receptor Phosphorylation of the myosin regulatory light chain (MRLC) at Thr18/Ser19 was shown to increase the actin-activated Mg-ATPase activity of myosin II and the assembly of myosin II filaments, and regulate the association between myosin II and filamentous actin (F-actin). The association of myosin II with F-actin results in the formation of stress fibers in interphase cells and the contractile ring in dividing cells. Control HeLa cells exhibited an organized network of F-actin-containing stress fibers spanning across the cell body. When the cells were incubated with EGCG, the cells retracted and left intercellular gaps. In addition, disappearance of the stress fibers in the central cell body was observed upon treatment with EGCG. Because stress fiber formation has been known to require Thr18/Ser19
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Fig. 1. Suppression of the 67LR in tumor cells results in abrogation of EGCG-induced tumor growth inhibition in murine tumor model. C57BL/6N mice were subcutaneously inoculated with B16 cells stably transfected with control shRNA or 67LR shRNA expression vector. Peroral administration of 0.1% EGCG was started 1 day before the tumor cell inoculation. After 15 days, tumor size was measured. Data are expressed as the mean ± SE of 6 or 7 mice (* p < 0.05 vs. untreated control).
phosphorylation of MRLC, we examined the effect of EGCG on MRLC phosphorylation [9]. We treated HeLa cells for 24 h with three concentrations of EGCG (0.1, 1 or 10 μm). The MRLC phosphorylation was reduced at concentrations between 1 and 10 μm. The phosphorylation of MRLC at Thr18/Ser19 has been shown to be necessary for the formation of the contractile ring in dividing cells. We subjected EGCG-treated cells to FACS analysis using propidium iodide staining to measure the DNA content. EGCG treatment significantly increased the percentage of cells in the G2/M phase. These results suggest that the suppressive effect of EGCG on the MRLC phosphorylation continued at least for 48 h and the increase in G2/M phase cells resulted from the effect of EGCG. EGCG has been known to produce H2O2 when it is added to the media of several cultured cell lines, and H2O2 may exert biological effects on cell growth. We examined whether catalase can change the effect of EGCG on cell growth and MRLC phosphorylation [9]. The addition of catalase could not alter either the EGCG-induced reduction in the MRLC phosphorylation level, the inhibition of the cell growth or the accumulation of the cells in the G2/M phase. To analyze whether the suppressive effect of EGCG on MRLC phosphorylation is mediated by the 67LR, RNAi-mediated gene silencing was utilized to knock down the expression of the 67LR [9]. EGCG significantly reduced the phosphorylation of MRLC in the cells transfected with the empty vector; however, in the cells transfected with shRNA expression vectors for the 67LR, the same concentration
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of EGCG only slightly reduced the phosphorylation. These results indicate that the 67LR does indeed mediate the suppressive effect of EGCG on MRLC phosphorylation. Taken together, these findings suggest that EGCG inhibits the cancer cell growth by reducing the MRLC phosphorylation and this effect is mediated by the 67LR. Epidemiological studies have suggested that the consumption of green tea may decrease colon cancer risk. The Wnt pathway appears to play an important role particularly in colon carcinogenesis. The essential event in Wnt signaling is the stabilization of β-catenin. As the main binding partner of β-catenin at cell-cell junctions, E-cadherin plays a pivotal role in β-catenin stabilization and function. Previous studies have reported that EGCG treatment downregulated the β-catenin protein expression [23] or upregulated E-cadherin protein expression, suggesting that EGCG suppressed Wnt signaling. However, it is still not known how a physiological concentration of EGCG induces cell growth inhibition in colorectal cancer cells. Recently, we found for the first time that a physiologically achievable concentration of EGCG inhibited cell cycle progression of human colon adenocarcinoma Caco-2 cells through the 67LR without affecting Wnt signaling components. Further, we found that EGCG at a physiological concentration decreased the phosphorylation of MRLC at Thr-18/Ser-19 in Caco-2 cells through the 67LR, suggesting that an activation of myosin cytoskeleton is involved in the antiproliferative effect of EGCG at a physiological concentration [8].
Induction of Apoptosis by EGCG through the Green Tea Catechin Receptor Recently, EGCG has been shown to be able to induce growth arrest and subsequent apoptotic cell death in multiple myeloma (MM) cells and primary patient MM cells in vitro, while having no significant effect on the growth of normal cells such as peripheral blood mononuclear cells (PBMCs) and fibroblasts [24]. Treatment with EGCG also led to significant apoptosis in human myeloma cells grown as tumors in SCID mice. The expression of the 67LR was significantly elevated in myeloma cell lines and patient samples compared with normal PBMCs. RNAi-mediated inhibition of the 67LR resulted in abrogation of EGCG-induced apoptosis in myeloma cells, indicating that the 67LR plays an important role in mediating EGCG activity in MM while sparing PBMCs. Evaluation of changes in gene expression profile indicates that EGCG treatment activates distinct pathways of growth arrest and apoptosis in MM cells by inducing the expression of death-associated protein kinase 2, the initiators and mediators of death receptor-dependent apoptosis (Fas ligand, Fas, and caspase 4), p53-like proteins (p73, p63), positive regulators of apoptosis and NF-κB activation (CARD10, CARD14), and cyclin-dependent kinase inhibitors (p16 and p18). These data demonstrate potent and specific antimyeloma activity of EGCG and provide the rationale for its clinical evaluation.
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Green Tea Catechin Receptor Signaling Molecules
Eukaryotic Translation Elongation Factor 1A In an attempt to elucidate the pathways involved in the anticancer action of EGCG, we applied the genetic suppressor element (GSE) methodology. GSEs are short cDNA fragments encoding peptides acting as dominant inhibitors of protein function or antisense RNAs inhibiting gene expression. GSEs behave as dominant selectable markers for the phenotype associated with the repression of the gene from which they derived, thus allowing identification of this gene. For identifying genes mediating cell sensitivity to EGCG, we selected GSEs conferring resistance to EGCG. To search for the mediators of EGCG-induced cell growth inhibition in B16 mouse melanoma cells, we utilized a targeted genetic screen with a GSE complementary DNA library. Among genetic elements protecting cells from EGCG-induced cell growth inhibition, we isolated a GSE that encoded the N terminus of eukaryotic translation elongation factor 1A (eEF1A) [7]. eEF1A is an important component of the eukaryotic translation apparatus and is also known as a multifunctional protein that is involved in a large number of cellular processes [25]. To investigate the role of eEF1A in EGCG-induced cell growth inhibition, we used stable RNAi to silence eEF1A expression in B16 cells. Remarkably, silencing of eEF1A attenuated the inhibitory effect of 1 μm EGCG on cell growth [7]. In contrast, overexpression of eEF1A enhanced the inhibitory effects of 1 μm EGCG on cell growth. This concentration is similar to the amount of EGCG found in human plasma after drinking more than two or three cups of green tea. Given this, we investigated the effect of oral administration of EGCG on subcutaneous tumor growth in C57BL/6N mice challenged with eEF1A-ablated B16 cells [7]. Tumor growth was significantly retarded in EGCG-administered mice implanted with the B16 cells harboring a control shRNA, whereas tumor growth was not affected by EGCG in the mice implanted with eEF1A-ablated B16 cells, indicating that eEF1A is involved in EGCG-induced cancer prevention. These results support our conclusion that eEF1A serves as a mediator for EGCG-induced cancer prevention.
Myosin Phosphatase-Targeting Subunit The phosphorylation of MRLC is regulated by two classes of enzymes: MLC kinases and myosin phosphatase. Previously, we reported that EGCG-induced cell growth inhibition may result from the reduction in the phosphorylation of MRLC at Thr-18/ Ser-19 through the 67LR in HeLa cells [9]. The activity of myosin phosphatase is known to be inhibited by phosphorylation of its targeting subunit MYPT1 at Thr-696 and Thr-853 [26]. We tested the effect of EGCG on the phosphorylation of MYPT1 at Thr-696 and Thr-853 [7]. Intriguingly, although the phosphorylation level at Thr-
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853 was unaffected by EGCG, EGCG induced the dephosphorylation of MYPT1 at Thr-696. Further, this effect correlated with EGCG-induced reduction in the MRLC phosphorylation, suggesting that EGCG activates myosin phosphatase by reducing the MYPT1 phosphorylation level at Thr-696. Next, we investigated whether MYPT1 is involved in anticancer action of EGCG in vivo. In B16 cells, physiological concentrations of EGCG reduced the MYPT1 phosphorylation at Thr-696 and the MRLC phosphorylation. We confirmed both silencing of MYPT1 by stable RNAi in B16 cells and attenuation of the inhibitory effect of 1 μm EGCG on cell growth in MYPT1ablated B16 cells in vitro. We tested the effect of oral administration of EGCG on subcutaneous tumor growth in C57BL/6N mice challenged with MYPT1-ablated B16 cells. Tumor growth was significantly retarded in EGCG-administered mice implanted with the B16 cells harboring a control shRNA, whereas tumor growth was not affected by EGCG in the mice implanted with MYPT-1-ablated B16 cells, suggesting that MYPT1 is indispensable for EGCG-induced cancer prevention.
A Hierarchy of EGCG-Signaling Molecules In both 67LR-ablated HeLa cells and eEF1A-ablated HeLa cells, the inhibitory effect of EGCG on both the phosphorylation of MYPT1 at Thr-696 and the phosphorylation of MRLC was attenuated. In addition, EGCG-induced actin cytoskeleton rearrangement was no longer observed in MYPT1-, eEF1A-, or 67LR-ablated HeLa cells. The involvement of MYPT1 in downstream EGCG-triggered signaling from both the 67LR and eEF1A was further documented by confirming abrogation of 1 μm EGCG-induced reduction in the MYPT1 phosphorylation level at Thr-696 and the MRLC phosphorylation in 67LR- or eEF1A-ablated B16 cells. These results suggest that MYPT1 is involved in downstream EGCG signaling from both the 67LR and eEF1A (fig. 2). It has been reported that MYPT1 binds to eEF1A, and more than half of the total eEF1A (>60%) binds to the actin cytoskeleton. Characterizing the mechanisms by which EGCG induces reduction of the MYPT1 phosphorylation at Thr-696 and reorganization of actin cytoskeleton through eEF1A should help in more precise understanding of cytoskeleton organization, although further experiments are necessary.
Conclusion
Recent studies have highlighted the importance of genetically determined factors in evaluating the role of green tea intake. In a case-control study conducted among Asian-American women, green tea intake appeared to reduce breast cancer risk [27]. Reduction in risk was strongest among persons who had the low-activity catecholO-methyltransferase (COMT) alleles, but not high-activity COMT alleles, suggesting that these individuals were less efficient in eliminating green tea catechins and
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Fig. 2. Model of molecular basis for cancer-preventive activity of EGCG in vivo. EGCG induces myosin phosphatase activation by reducing MYPT1 phosphorylation via both the 67LR and eEF1A. Moreover, we show that the 67LR, eEF1A and MYPT1 are all indispensable for mediating EGCG signaling for cancer prevention in vivo.
may derive the most benefit from these compounds. Yuan’s group reported a low risk of breast cancer among Singapore Chinese women with higher green tea intake and the low-activity genotype of angiotensin-converting enzyme gene. These observations indicate that a further study to elucidate the key molecules determining EGCG responsiveness is indispensable for a better understanding of EGCG activity in vivo.
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Little is known about the mechanism of the chemopreventive action of most phytochemicals, including EGCG. Although previous studies have proposed various different mechanisms for cancer-preventive action of EGCG [28], it remains unclear which EGCG-induced molecular events are relevant in vivo. The essence is the identification of the primary target and the demonstration of specific mechanisms of action in animal models and human tissues. Here, we described that the 67LR, eEF1A, and MYPT1 are all indispensable for EGCG-induced cancer prevention in vivo, and these proteins mediate physiological levels of EGCG-triggered unique signaling for cancer prevention. Our findings suggest that these proteins are ‘master proteins’ that determine the efficacy of cancer-preventive activity of EGCG and have important implications for development and use of EGCG as a cancer-chemopreventive agent. Probably, only a tumor with a high expression level of these ‘master proteins’ is sensitive to physiological concentrations of EGCG, while lower expression of those molecules causes ‘EGCG resistance’. Our results not only illuminate the mechanisms for the cancer-preventive activity of EGCG but should help in the design of new strategies to prevent cancer and underscore the importance of tailoring cancer therapy on the basis of tumor genotype.
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7 Umeda D, Yano S, Yamada K, Tachibana H: Green tea polyphenol epigallocatechin-3-gallate signaling pathway through 67-kDa laminin receptor. J Biol Chem 2008;283:3050–3058. 8 Umeda D, Yano S, Yamada K, Tachibana H: Involvement of 67-kDa laminin receptor-mediated myosin phosphatase activation in antiproliferative effect of epigallocatechin-3-O-gallate at a physiological concentration on Caco-2 colon cancer cells. Biochem Biophys Res Commun 2008;37:172–176. 9 Umeda D, Tachibana H, Yamada K: Epigallocatechin3-O-gallate disrupts stress fibers and the contractile ring by reducing myosin regulatory light chain phosphorylation mediated through the target molecule 67 kDa laminin receptor. Biochem Biophys Res Commun 2005;333:628–635. 10 Fujimura Y, Umeda D, Yano S, Maeda-Yamamoto M, Yamada K, Tachibana H: The 67 kDa laminin receptor as a primary determinant of anti-allergic effects of O-methylated EGCG. Biochem Biophys Res Commun 2007;364:79–85. 11 Yang CS, Chen L, Lee MJ, Balentine D, Kuo MC, Schantz SP: Blood and urine levels of tea catechins after ingestion of different amounts of green tea by human volunteers. Cancer Epidemiol Biomarkers Prev 1998;7:351–354.
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12 Lambert JD, Yang CS: Mechanisms of cancer prevention by tea constituents. J Nutr 2003;133: 3262S–3267S. 13 Lee MJ, Wang ZY, Li H, Chen L, Sun Y, Gobbo S, Balentine DA, Yang CS: Analysis of plasma and urinary tea polyphenols in human subjects. Cancer Epidemiol Biomarkers Prev 1995;4:393–399. 14 Nam S, Smith DM, Dou QP: Ester bond-containing tea polyphenols potently inhibit proteasome activity in vitro and in vivo. J Biol Chem 2001;276:13322– 13330. 15 Chung JY, Park JO, Phyu H, Dong Z, Yang CS: Mechanisms of inhibition of the Ras-MAP kinase signaling pathway in 30.7b Ras 12 cells by tea polyphenols (–)-epigallocatechin-3-gallate and theaflavin3,3⬘-digallate. FASEB J 2001;15:2022–2024. 16 Sah JF, Balasubramanian S, Eckert RL, Rorke EA: Epigallocatechin-3-gallate inhibits epidermal growth factor receptor signaling pathway. Evidence for direct inhibition of ERK1/2 and AKT kinases. J Biol Chem 2004;279:12755–12762. 17 Leone M, Zhai D, Sareth S, Kitada S, Reed JC, Pellecchia M: Cancer prevention by tea polyphenols is linked to their direct inhibition of antiapoptotic Bcl-2-family proteins. Cancer Res 2003;63:8118– 8121. 18 Ermakova S, Choi BY, Choi, HS, Kang BS, Bode AM, Dong Z: The intermediate filament protein vimentin is a new target for epigallocatechin gallate. J Biol Chem 2005;280:16882–16890. 19 Tanaka M, Narumi K, Isemura M, Abe M, Sato Y, Abe T, Saijo Y, Nukiwa T, Satoh K: Expression of the 37-kDa laminin binding protein in murine lung tumor cell correlates with tumor angiogenesis. Cancer Lett 2000;153:161–168. 20 Tachibana H, Koga K, Fujimura Y, Yamada K: A receptor for green tea polyphenol EGCG. Nat Struct Mol Biol 2004;11:380–381.
21 Fujimura Y, Yamada K, Tachibana H: A lipid raftassociated 67 kDa laminin receptor mediates suppressive effect of epigallocatechin-3-O-gallate on FcepsilonRI expression. Biochem Biophys Res Commun 2005;336:674–681. 22 Fujimura Y, Tachibana H, Yamada K: Lipid raftassociated catechin suppresses the FcepsilonRI expression by inhibiting phosphorylation of the extracellular signal-regulated kinase1/2. FEBS Lett 2004;556:204–210. 23 Dashwood WM, Carter O, Al-Fageeh M, Li Q, Dashwood RH: Lysosomal trafficking of betacatenin induced by the tea polyphenol epigallocatechin-3-gallate. Mutat Res 2005;591:161–172. 24 Shammas MA, Neri P, Koley H, Batchu RB, Bertheau RC, Munshi V, Prabhala R, Fulciniti M, Tai YT, Treon SP, Goyal RK, Anderson KC, Munshi NC: Specific killing of multiple myeloma cells by (–)-epigallocatechin-3-gallate extracted from green tea: biologic activity and therapeutic implications. Blood 2006;108:2804–2810. 25 Negrutskii BS, El’skaya AV: Eukaryotic translation elongation factor 1 alpha: structure, expression, functions, and possible role in aminoacyl-tRNA channeling. Prog Nucleic Acid Res Mol Biol 1998; 60:47–78. 26 Hartshorne DJ, Ito M, Erdodi F: Role of protein phosphatase. J Biol Chem 2004;273:5542–5548. 27 Wu AH, Tseng CC, Van Den Berg D, Yu MC: Tea intake, COMT genotype, and breast cancer in Asian-American women. Cancer Res 2003;63:7526– 7529. 28 Khan N., Afaq F, Saleem M, Ahmad N, Mukhtar H: Targeting multiple signaling pathways by green tea polyphenol (–)-epigallocatechin-3-gallate. Cancer Res 2006;66:2500–2505.
Dr. Hirofumi Tachibana Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University 6-10-1 Hakozaki, Higashi-ku Fukuoka 812-8581 (Japan) Tel./Fax +81 92 642 3008, E-Mail
[email protected]
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Chemoprevention and Cancer Yoshikawa T (ed): Food Factors for Health Promotion. Forum Nutr. Basel, Karger, 2009, vol 61, pp 170–181
Chemoprevention by Isothiocyanates: Molecular Basis of Apoptosis Induction Yoshimasa Nakamura Division of Bioscience, Department of Biofunctional Chemistry, Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan
Abstract An important and promising group of compounds that have a cancer-chemopreventive property are organosulfur compounds, such as isothiocyanates (ITCs). Various ITCs are effective chemoprotective agents against chemical carcinogenesis in experimental animals. Several epidemiological studies also indicated that the dietary consumption of ITCs or ITC-containing foods inversely correlates with the risk of developing lung, breast, and colon cancers, providing evidence that they have a potential to prevent cancer in humans. Mechanistically, ITCs are capable of inhibiting both the formation and development of a cancer cell through multiple pathways; i.e. the inhibition of carcinogen-activating cytochrome P450 mono-oxygenases, induction of carcinogen-detoxifying phase 2 enzymes, induction of apoptosis, and inhibition of cell cycle progression. We have clarified the molecular mechanisms underlying the relationship between cell cycle regulation and apoptosis induced by benzyl ITC (BITC), a major ITC compound isolated from papaya (Carica papaya) fruit. We identified phosphorylated Bcl-2 as a key molecule linking p38 MAPK-dependent cell cycle regulation with the c-Jun N-terminal kinase activation by BITC. We also established that BITC exerts the cytotoxic effect more preferentially in the proliferating cells than in the quiescent cells. Furthermore, p53 was found to be a potential negative regulator of apoptosis induction by BITC in normal epithelial cells through inhibition of cell cycle progression at the G0/G1 phase. In contrast, treatment with an excessive concentration of BITC resulted in necrotic cell death in an ATP-dependent manner. This review addresses the biological impact of cell death induction by BITC as well as other ITCs and the Copyright © 2009 S. Karger AG, Basel involved molecules regulating signal pathways.
Isothiocyanates and Chemoprevention
A number of studies support the fact that certain food phytochemicals protect against cancer. An important group of compounds that have this property are organosulfur compounds including isothiocyanates (ITCs; fig. 1a) [1]. ITCs, naturally occurring in abundance in cruciferous vegetables such as broccoli, watercress, brussels sprouts, cabbage, Japanese radish and cauliflower, may play a significant role in affording the
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Fig. 1. Chemistry of ITCs. a Structures and sources of the representative ITCs. b Conversion of GS into ITC by myrosinase. c Metabolism of ITC via the mercapturic acid pathway. GTP = γGlutamyltranspeptidase; CGase = cysteinylglycinase; HAT = histone acetyltransferase.
cancer chemopreventive activity of these vegetables. They are stored as glucosinolates (GSs) in plants and are released when the plant tissue is damaged or ground. Within the plant, GS content can vary between and within members of the cruciferous vegetables depending on cultivation environment and genotype. There are over 120 GSs in various plants, each yielding different aglycone metabolic products including ITCs [1]. For example, sulforaphane (SFN) is one of the best investigated ITCs, which is particularly abundant in broccoli (Brassica oleracea L. var. italica) in the form of its corresponding GS glucoraphanin. β-Phenethyl ITC (PEITC) is also a well-characterized ITC, which is found as its corresponding GS gluconasturtiin in considerable quantities in water cress
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(Nasturtium officinale L.). Benzyl ITC (BITC) is formed from benzyl GS (glucotropaeolin) in papaya (Carica papaya). Although exhibiting a spectrum of biological activities similar to those of SFN and PEITC, BITC is rare in Brassicaceae vegetables. The general structure of GS consists of a β-D-thioglucose group, a sulfonated oxime group, and a variable side chain. The chemopreventive properties of cruciferous vegetables might be attributed to their high content of GSs, which is responsible for their pungent odor and biting taste. The conversion from GSs into ITCs is catalyzed by myrosinase, a thioglucosidase that is physically separated from GSs under normal conditions, but, in the human diet, the myrosinase in cruciferous vegetables is heat inactivated. GSs may also be hydrolyzed in the intestinal tract, because the microflora possesses a myrosinaselike activity [2]. Therefore, most attention has been focused on the cancer-preventive potential of the GSs metabolites, ITCs, as described in this paper. Various ITCs are effective chemoprotective agents against chemical carcinogenesis in experimental animals [for review see 3, 4]. More recently, several epidemiological studies have indicated that the dietary consumption of ITCs or ITC-containing foods inversely correlates with the risk of developing lung, breast, and colon cancers [for review see 5], providing evidence that they have a potential to prevent cancer in humans. The genetic polymorphism of human glutathione S-transferase (GST) isozymes provides another point of view. A more significant association between the urinary ITC level and reduced lung cancer risks among subjects with the deletion of GSTM1 or GSTT1 than with normal genotypes has been reported [5]. The ITC compounds are known as metabolic substrates for GSTM1 and GSTT1 [6], implying that the delayed metabolism and elimination of ITCs in subjects with deletion of GSTM1 or GSTT1 might contribute to maintaining the blood/tissue concentrations of ITCs and thus more sustained effects derived from cruciferous vegetables. Indeed, one study investigating a population in Singapore reported higher ITC excretion among GSTT1-positive individuals in comparison to GSTT1 null [5]. Therefore, these results do not allow us to exclude the possibility that the elevation of the total phase 2 drug metabolizing enzyme activity by ITCs is more significant in these subjects. The impact of polymorphisms on ITC metabolism and bioavailability is one of the most important research areas that will aid in our understanding of response variability to ITCs in human populations. Mechanistically, ITCs are capable of inhibiting both the formation and development of a cancer cell through multiple pathways: inhibition of carcinogen-activating cytochrome P450 mono-oxygenases, induction of carcinogen-detoxifying enzymes, induction of apoptosis, and inhibition of cell cycle progression. ITC can function by blocking initiation via inhibiting phase 1 enzymes through direct interactions with cytochrome P450 enzymes (CYP) or regulating their transcript levels. Phase 1 enzymes usually involve oxidation, reduction, or hydrolysis and generally lead to detoxification, but are also involved in converting procarcinogens to carcinogens. Inhibition of phase 1 enzymes is thus thought to be an important step in blocking chemically-induced carcinogenesis.
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Although CYP inhibition is of interest, the blocking theory of ITCs has focused on phase 2 xenobiotic metabolizing enzyme induction via antioxidant response element (ARE)-driven gene expression on the basis of observations from epidemiological and rodent studies. Several lines of evidence indicate that phase 2 enzymes play a major role in the cellular detoxification of oxidative damaging, genotoxic and carcinogenic chemicals. GSTs are a family of soluble proteins, which conjugate xenobiotics with glutathione (GSH) as well as convert toxic peroxides into the corresponding carbinols. Metabolites after glutathionylation are more hydrophilic and thus biologically inactive. Regulation of the ARE-driven genes is mediated by nuclear factor E2-factor-related factor (Nrf2), a member of the basic leucinezipper NF-E2 family, which binds to ARE sites as a cis-element in the 50-flanking region of the genes for many of these phase 2 enzymes. The biologically active antioxidant effects of ITCs are also via transcriptional activation of various antioxidant proteins, including NAD(P)H:(quinone-acceptor) oxidoreductase 1, GST, glutamate cysteine ligase, heme oxygenase 1, and thioredoxin reductase, regulated by the Nrf2/ARE signaling pathway. Importantly, the induction of these blocking and antioxidant genes after ITC treatment is dependent on Nrf2, as revealed from works done in Nrf2 knockout mice. A succession of reviews has recently addressed the phase 2 enzyme induction by ITCs and their underlying molecular signaling mechanisms [5, 7]. In recent years, cancer-preventive mechanisms dealing with the inhibition of cancer cell development have also attracted considerable attention. In this regard, apoptosis induced by various ITC compounds has been extensively studied mainly in cancer cell lines derived from various tissues, as discussed below.
Chemistry of Isothiocyanates
The metabolism of GSs and ITCs is summarized in figure 1b. The initial reaction involves enzymatic hydrolysis of the GS precursor of ITC found in the plant. This reaction is catalyzed by myrosinase, a β-thioglucosidase, which cleaves the glycone from the GS-forming glucose, hydrogen sulfate and one of many different rearranged aglycones including thiocyanate, ITC, or a nitrile, depending on the GS, reaction pH, and availability of ions [1]. At neutral pH, the major GS hydrolysis products are stable ITCs. The exposure of cells to ITCs leads to rapid and high intracellular accumulation. Intracellular ITC accumulation is quite rapid and its levels may reach 100to 200-fold over the extracellular concentrations. Reduced GSH, which is the most abundant thiol-carrying molecule in a cell (1–10 mM in cells), is known to be primarily responsible for the nucleophilic conjugation of ITCs, which results in the formation of dithiocarbamates (DTCs; R-NH-CS-S-R⬘). The intracellular GSH elevation, resulting from the induction of the GSH biosynthesis, counteracts not only the GSH depletion by ITCs but also the ITC-induced biological responses. Furthermore, it is
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also noteworthy that such a covalent conjugation with GSH is reversible and that the conjugates can undergo either dissociation or replacement reactions to other targets [8]. Several ITCs are effective for inhibiting glyceraldehyde-3-phosphate dehydrogenase, a model protein having active thiol groups, while the nonelectrophilic O-methyl benzylthiocarbamate (Bz-NH-CS-O-CH3) does not [9]. This structureactivity relationship has also been found in some biological activities in cultured cells and animals, including the suppression of the NADPH oxidase-dependent superoxide generation [10], induction of GST [11], and mitochondrial function modification [12]. Therefore, it is very likely that ITCs can modify other intracellular thiol molecules including protein sulfhydryls within the cells. The intracellular potential targets of ITCs have been postulated, e.g. Kelch-like ECH-associated protein 1 and proteasome components in the Nrf2-dependent phase 2 enzyme induction pathway, thioredoxin in the apoptosis signal-regulating kinase 1/c-Jun N-terminal kinase (JNK) signaling pathway, gp91PHOX of NADPH oxidase system, adenine nucleotide translocase for mitochondrial permeability transition control, etc. More recently, tubulin has been identified as one of major in vivo targets for ITC binding, which is the first demonstration showing in vivo adduct formation between an ITC and any intracellular human protein [13]. Covalent binding preference of BITC, PEITC, and SFN to tubulin correlated well with their ability to induce mitosis arrest and apoptosis. This information is essential for increasing our knowledge of the mechanisms underlying ITC-induced apoptosis and will help in designing more efficacious ITC-related compounds for the prevention and therapy of cancer. After absorption, ITCs are predominantly metabolized via the mercapturic acid pathway (fig. 1c). The enzymes that catalyze GSH conjugation to ITCs are a family of GST enzymes, and polymorphisms in these enzymes have a significant impact on overall ITC metabolism, as discussed above. The DTCs formed during ITC digestion are further metabolized into cysteinylglycine, cysteine and N-acetylcysteine (NAC) via the mercapturic acid pathway in the kidney and excreted in the urine. Therefore, the formation of ITC-NAC conjugates, which occur within a day after the consumption of cruciferous vegetables, serves as a metabolic marker of ITCs released from the GSs. The decomposition of thiol conjugates of ITCs was pH dependent [8]. The physiological implication for the influence of pH on thiol-ITC decomposition is that orally administered thiol-ITCs would remain intact when absorbed through the stomach mucosa, but would probably dissociate to ITCs to a much greater extent in the intestinal tract. On the basis of the law of equilibrium process, the plasma and tissue concentrations of GSH could retard the deconjugation of ITC-thiol conjugates in vivo, hence free ITCs would not be readily available from thiol conjugates. Anyway, although the biological activities of DTCs and the ITC-NAC conjugates are not fully understood, they are not ultimately inactive, possibly due to the dissociative conversion into free ITCs [8].
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Isothiocyanates as Apoptosis Inducers
The apoptosis induction by the ITC compounds was first documented by Chen et al. [14] in 1998. Subsequently, these ITCs, as well as other ITCs, including SFN and allyl ITC (AITC), were shown to induce apoptosis in a variety of cultured human and animal cell lines, as well as animal tissues and cancer cell xenografts in vivo [for review, see 15]. Besides the effect of individual ITC compounds, Brassica vegetable juices also showed apoptosis induction in rat colonic mucosal crypts in vivo. The apoptosisinducing potency of ITCs seems to be partly responsible for their chemopreventive efficacy in some animal models. For example, phenylhexyl ITC, which was the most potent preventer of tobacco specific nitrosamine-induced lung cancers in rats was also the most potent apoptosis inducer in HeLa cells. Multiple apoptotic pathways are involved in the treatment of cells with ITCinduced caspase activation. For example, several ITCs simultaneously activate caspase-3 through the mitochondria death pathway (caspase-9), the death receptor pathway (caspase-8), and the endoplasmic reticulum pathway (caspase-12) in HL-60 cells [16]. We demonstrated that BITC induced the mitochondrial cytochrome c release and subsequently activated the mitochondrial death pathway in rat hepatocytes, possibly through a cellular redox status-dependent mechanism [17]. Although the molecular mechanisms underlying the apoptosis induction by ITCs are not fully understood and are cell line dependent, most of the studies focused on the mitochondria death pathway and/or the related molecules including mitogen-activated protein kinases (MAPK) and Bcl2 family proteins. BITC and PEITC induced apoptosis accompanied by an increase in the activity of JNK, and inhibition of JNK with dominant-negative mutants suppressed ITC-induced apoptosis [14]. The activation of JNK may result from oxidative stress induced by ITCs, since JNK is well known to be stress activated, and the pretreatment of the cells with antioxidants, including 2-mercaptoethanol or NAC, blocked the activation [14]. Another report indicated that PEITC inhibits a phosphatase that inactivates JNK [18]. We also provided biological evidence of the involvement of JNK in the Bcl-2 modification, which should be added to the list of the ITC-induced apoptosis signaling pathways [19]. BITC stimulates JNK-dependent Bcl-2 phosphorylation in several human cancer cell lines. Furthermore, we observed that the antiapoptotic Bcl-2 interaction with proapoptotic Bax, which leads to the Bax inactivation, was significantly decreased by the BITC treatment. Therefore, phosphorylated Bcl-2 appears to lose its binding ability with Bax, leading to the relative increment of the active Bax homodimer ratio and thus enhanced susceptibility of the cells to apoptosis. In addition to such potential target proteins in the cytoplasm, it should be noted that ITCs are able to afford a direct damage to mitochondria and induce the release of cytochrome c [12, 17]. The induction of cell cycle arrest by ITCs was first reported by Hasegawa et al. [9] in 1993. AITC, BITC, PEITC, and SFN have been reported to induce G2/M arrest in
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HT29 cells. However, the same ITC may induce cell cycle arrest in different phases such as the G0/G1 and G2/M phases in a cell line-dependent manner. Although the exact molecular mechanisms responsible for cell cycle arrest are still mostly unknown, some potential targets of ITCs have been postulated. For example, the downregulation of cdk1 and cdc25B was also observed in the G2/M cells arrested through AITC, suggesting that cyclin B1, cdk1 and cdc25B may be targeted by AITC [20]. In addition to these molecules, we also reported that the exposure of cells to BITC resulted in the inhibition of G2/M progression and upregulated expression of the G2/M cell cycle arrest-regulating genes including p21 [21], inhibiting cyclin-dependent kinase activity by directly binding to CDK/cyclin complexes including Cdc2/cyclin B1 kinase. It is quite noteworthy that the regulation of the cell cycle progression at the G2/M phase is a prerequisite for apoptosis induced by BITC [19], while, to our knowledge, a direct link between the cell cycle regulation and apoptosis induced by other ITCs including SFN has not yet been documented. BITC is likely to confine the cells in the G2/M phase mainly through the p38 MAPK pathway because only the p38 MAPK inhibitor, at the concentration required to selectively inhibit the p38 MAPK activity, significantly attenuated the accumulation of the inactive phosphorylated Cdc2 protein and the G2/M phase cell numbers. Furthermore, we demonstrated for the first time that the BITC treatment resulted in the G2/M phase-specific phosphorylation of antiapoptotic Bcl-2 protein through a JNK-dependent pathway. Taken together, we concluded that the phosphorylated Bcl-2 is a key molecule linking the p38 MAPKdependent cell cycle regulation with the JNK activation by BITC (fig. 2), providing a novel insight into the understanding of molecular mechanism of the ITC-induced apoptosis. Furthermore, the data concerning the G2/M cell-specific apoptosis induction by BITC led us to the hypothesis that BITC shows a selective cytotoxic effect on the proliferating cells having the higher mitotic division indices. Expectedly, we observed in successful experiments using a presynchronization technique that BITC more preferentially induced the cytotoxic effect in the proliferating cells than the quiescent cells [22]. We also found that BITC induced p53 phosphorylation and accumulation, which was counteracted by caffeine treatment, implying the involvement of an ATM/ ataxia telangiectasia and Rad3-related kinase signaling pathway. Downregulation of p53 by a siRNA resulted in the enhancement of susceptibility to undergo apoptosis by BITC. Depletion of p53 abrogated G0/G1 arrest accompanied by the reduced expression of p21waf1/cip1 and p27kip1 in CCD-18Co cells, suggesting that the cell-cycle progression around the S to G2/M phase might be an important determinant for sensitivity to BITC (fig. 2). In other words, the p53 accumulation maintaining the stay in the G0/G1 phase might play an important role in the resistance to the cytotoxic effect of BITC in the quiescent cells. We thus identified p53 as a novel negative regulator of the apoptosis induction by BITC in the normal epithelial cells through the inhibition of cell-cycle progression at the G0/G1 phase [22]. The p53 tumor suppressor gene plays a key role in the cellular response to genotoxic stress. The mutation or
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Fig. 2. A proposed mechanism for the BITC-induced apoptosis. The BITC-induced p38 activation inhibits cell cycle progression into the G0/G1 phase. The JNK activation in the G2/M phase cells phosphorylates the Bcl-2 protein, which leads to the enhanced susceptibility of the cells to apoptosis. p53 negatively regulates the apoptosis induction by BITC in the normal quiescent cells through inhibition of the cell cycle progression into the S phase.
loss of p53 has been observed in over 50% of all tumors and in almost every tumor type. Although the role of p53 in the ITC-induced apoptosis in cancer cells is controversial [23], these findings implied that BITC has a potential to induce apoptosis in the p53-mutated proliferating precancerous cells in preference to the p53-active normal cells. Consistent with these results, we found that ITCs were preferentially toxic to p53-mutated tumor cells compared with the p53-native tumor cells (unpublished data). The lack of selectivity in the killing of tumor and normal cells is a major obstacle in the apoptosis study and cancer therapy. In principle, chemotherapy drugs and chemopreventive agents possessing a cytotoxic activity poorly discriminate between normal and cancer cells. Blagosklonny et al. [24] envisioned the protection of normal cells that is mainly based on four principles: (1) cancer cells are less dependent on growth factors; (2) it is possible to selectively arrest normal cells in the interphase; (3) inducers of G2/M phase cell cycle arrest are toxic to cycling agents, and (4) cells arrested at an interphase may be insensitive to G2/M arrest inducers. Therefore, we propose that BITC, which induces the cell cycle-specific apoptosis, in combination with reversible and selective inhibitors of the cycle of proliferating epithelial and hematopoietic cells might be able to selectively induce apoptosis in cancer cells.
Isothiocyanates as Stress Inducers
There is abundant evidence that ITCs induce cellular stress themselves. As mentioned above, the exposure of cells to ITCs leads, at least transiently, to a decrease
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in the pool of cellular thiol compounds, especially GSH. The depletion of GSH and other thiol molecules renders cells more susceptible to oxidative stress and stressinduced damage, although the electrophilic reaction of ITCs with the target protein sulfhydryl groups is a prerequisite for the induction of several biological activities. In addition, ITCs also cause rapid increases in the cellular reactive oxygen species (ROS) levels, as evidenced by BITC-treated rat liver epithelial RL34 cells [17]. Although the beneficial effects such as phase II enzyme induction decreased at higher ITC concentrations [25], the increase in the intracellular ROS levels in RL34 cells was dose dependent up to 100 μM BITC [17]. More recently, we observed in experiments using the mitochondrial DNA-deficient (ρ0) cells that the major source of the BITC-stimulated ROS production is the mitochondria electron transfer chain [26]. The treatment of mitochondria isolated from rat liver with BITC resulted in an inhibition of respiration, mitochondrial swelling, and the release of cytochrome c [12, 17], a clear indication that ITCs directly damage mitochondria. Ca2+ also inhibits the mitochondrial respiration-dependent removal of exogenous hydrogen peroxide possibly via respiration inhibition leading to starvation of NADPH, a substrate for GSH reductase. Therefore, the inhibition of mitochondrial respiration by BITC might also be engaged in the ROS accumulation sufficient to induce oxidative damage. The induction of oxidative stress by higher concentrations of ITCs might contribute to a narrow threshold to exhibit apoptosis. The unfavorable effect of higher concentrations of ITCs has also been observed; treatment with an excessive concentration of BITC resulted in severe cytotoxicity with caspase-3 inactivation and no DNA ladder formation [17, 26], leading to necrotic cell death and thus damage to the surrounding cells. Treatment with 2-deoxyglucose, an inhibitor of ATP synthesis, drastically increased the ratio of necrotic dead cells induced by BITC, while it influenced little the ratio of apoptotic cells [26]. Moreover, an analysis using the mitochondrial DNA-deficient HeLa cells demonstrated that the ρ0 cells were more susceptible to the BITC-induced necrosis-like cell death compared to the wild-type (ρ+) cells, whereas the ROS production was significantly inhibited in the ρ0 cells, suggesting that the BITC-induced ROS, derived from mitochondrial respiratory chain, ruled out the contribution to the mechanism of cell death mode switching. In addition, the BITC treatment resulted in a more rapid depletion of ATP in the ρ0 cells than in the ρ+ cells. Furthermore, a caspase inhibitor, Z-VAD-fmk counteracted not only apoptosis, but also necrosis-like cell death induced by BITC, suggesting that increment in this cell death pattern might be due to the interruption of events downstream of a caspase-dependent pathway. Thus, the decline in the intracellular ATP level may play an important role in tuning the mode of cell death by BITC [26]. These results imply that modification of the mitochondrial function by ITCs leading to the proapoptotic cytochrome c release, ROS generation and respiratory alteration might contribute not only to its apoptosis-inducing ability but also to their unfavorable side effects such as necrotic cell death.
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Conclusion and Perspective
Undoubtedly, ITCs possess a potential to exhibit cancer-chemopreventive activity. The transcriptional induction of phase 2 xenobiotic metabolizing enzymes involved in cellular defense against chemical carcinogens and oxidative stress substantially contributes to their preventive activity. Although they are promising and effective anticarcinogen candidates, some in vivo experiments also demonstrated the enhancement of tumorigenesis by ITCs. For instance, both BITC and phenethyl ITC (PEITC) promote urinary bladder carcinogenesis in rats treated with diethylnitrosamine and N-butyl-N-(4-hydroxybutyl)nitrosamine especially dosed during the postinitiation phase, which is possible through necrotic cytotoxicity [25]. Our results support the idea that the intake of excess amount of ITCs, which induces necrotic cell death, would provoke an inflammation reaction which could contribute to promoting the carcinogenesis process. Since the employed dose was higher than that required for chemoprevention [27], the promotional effect of ITCs could be dependent on their concentration. Moreover, we also obtained the puzzling result that the treatment with the typical phenolic antioxidant unexpectedly enhanced oxidative stress and thus tumor promotion even when the application dose was increased [28]. Thus ITCs experimentally possess both carcinogenic and anticarcinogenic activity in a dosedependent manner. Ye et al. [29] demonstrated that the peak plasma concentrations were 0.94–2.27 μM ITC equivalent, most of which might exist as DTCs, at 1 h after dosing the broccoli sprout extract containing 200 μmol total ITCs, consistent with the earlier observation. Our study of necrosis induction by BITC examined the concentration of BITC up to 100 μM as a suprapharmacological but locally achievable dose, since the recent preclinical evaluation revealed that ITCs concentration in the gastric lumina was temporally achieved to approximately 600–2,000 μM after the consumption of the broccoli extract [30]. Therefore, more attention should be paid to the dose administered as a supplement of condensed extracts, and thus carefully designed pharmacokinetic studies are needed before clinical testing of ITCs can begin. Further studies not only on the involvement and/or disturbance of intracellular oxidative stress in cell cycle arrest and apoptosis induction by ITCs but also on the optimization of the ITC dose or dose scheme for human study are necessary. In addition, strategies that use multiple agents with different modes of action rather than individual single agents should produce results with higher efficacy and lower toxicity. Therefore, there is a need for investigation concerning the combinatory use of certain food chemicals with ITCs to gain synergistic interaction for an anticancer effect and lowered harmful effects.
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References 1 Fahey JW, Zalcmann AT, Talalay P: The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochemistry 2001; 56:5–51. 2 Getahun SM, Chung FL: Conversion of glucosinolates to isothiocyanates in humans after ingestion of cooked watercress. Cancer Epidemiol Biomark Prev 1999;8:447–451. 3 Hecht SS: Inhibition of carcinogenesis by isothiocyanates. Drug Metab Rev 2000;32:395–411. 4 Conaway CC, Yang Y, Chung FL: Isothiocyanates as cancer chemopreventive agents: their biological activities and metabolism in rodents and humans. Curr Drug Metab 2002;3:233–255. 5 Zhang Y: Cancer-preventive isothiocyanates: measurement of human exposure and mechanism of action. Mutat Res 2004;555:173–190. 6 Kolm RH, Danielson UH, Zhang Y, Talalay P, Mannervik B: Isothiocyanates as substrates for human glutathione transferases: structure-activity studies. Biochem J 1995;311:453–459. 7 Zhang Y, Gordon GB: A strategy for cancer prevention: stimulation of the Nrf2-ARE signaling pathway. Mol Cancer Ther 2004;3:885–893. 8 Conaway CC, Krzeminski J, Amin S, Chung FL: Decomposition rates of isothiocyanate conjugates determine their activity as inhibitors of cytochrome p450 enzymes. Chem Res Toxicol 2001;14:1170– 1176. 9 Hasegawa T, Nishino H, Iwashima A: Isothiocyanates inhibit cell cycle progression of HeLa cells at G2/M phase. Anticancer Drugs 1993;4:273–279. 10 Miyoshi N, Takabayashi S, Osawa T, Nakamura Y: Benzyl isothiocyanate inhibits excessive superoxide generation in inflammatory leukocytes: implication for prevention against inflammation-related carcinogenesis. Carcinogenesis 2004;25:567–575. 11 Nakamura Y, Morimitsu Y, Uzu T, Ohigashi H, Murakami A, Naito Y, Nakagawa Y, Osawa T, Uchida K: A glutathione S-transferase inducer from papaya: rapid screening, identification and structure-activity relationship of isothiocyanates. Cancer Lett 2000; 157:193–200. 12 Kawakami M, Harada N, Hiratsuka M, Kawai K, Nakamura Y: Dietary isothiocyanates modify mitochondrial functions through their electrophilic reaction. Biosci Biotechnol Biochem 2005;69:2439– 2444. 13 Mi L, Xiao Z, Hood BL, Dakshanamurthy S, Wang X, Govind S, Conrads TP, Veenstra TD, Chung FL: Covalent binding to tubulin by isothiocyanates. A mechanism of cell growth arrest and apoptosis. J Biol Chem 2008;283:22136–22146.
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14 Chen YR, Wang W, Kong AN, Tan TH: Molecular mechanisms of c-Jun N-terminal kinase-mediated apoptosis induced by anticarcinogenic isothiocyanates. J Biol Chem 1998;273:1769–1775. 15 Keum YS, Jeong WS, Kong AN: Chemoprevention by isothiocyanates and their underlying molecular signaling mechanisms. Mutat Res 2004;555:191– 202. 16 Zhang Y, Tang L, Gonzalez V: Selected isothiocyanates rapidly induce growth inhibition of cancer cells. Mol Cancer Ther 2003;2:1045–1052. 17 Nakamura Y, Kawakami M, Yoshihiro A, Miyoshi N, Ohigashi H, Kawai K, Osawa T, Uchida K: Involvement of the mitochondrial death pathway in chemopreventive benzyl isothiocyanate-induced apoptosis. J Biol Chem 2002;277:8492–8499. 18 Chen YR, Han J, Kori R, Kong AN, Tan TH: Phenylethyl isothiocyanate induces apoptotic signaling via suppressing phosphatase activity against c-Jun N-terminal kinase. J Biol Chem 2002;277: 39334–39342. 19 Miyoshi N, Uchida K, Osawa T, Nakamura Y: A link between benzyl isothiocyanate-induced cell cycle arrest and apoptosis: involvement of mitogen-activated protein kinases in the Bcl-2 phosphorylation. Cancer Res 2004;64:2134–2142. 20 Xiao D, Srivastava SK, Lew KL, Zeng Y, Hershberger P, Johnson CS, Trump DL, Singh SV: Allyl isothiocyanate, a constituent of cruciferous vegetables, inhibits proliferation of human prostate cancer cells by causing G2/M arrest and inducing apoptosis. Carcinogenesis 2003;24:891–897. 21 Miyoshi N, Uchida K, Osawa T, Nakamura Y: Benzyl isothiocyanate modifies expression of the G2/M arrest-related genes. Biofactors 2004;21:23–26. 22 Miyoshi N, Uchida K, Osawa T, Nakamura Y: Selective cytotoxicity of benzyl isothiocyanate in the proliferating fibroblastoid cells. Int J Cancer 2007;120:484–492. 23 Wu X, Kassie F, Mersch-Sundermann V: Induction of apoptosis in tumor cells by naturally occurring sulfur-containing compounds. Mutat Res 2005;589: 81–102. 24 Blagosklonny MV, Bishop PC, Robey R, Fojo T, Bates SE: Loss of cell cycle control allows selective microtubule-active drug-induced Bcl-2 phosphorylation and cytotoxicity in autonomous cancer cells. Cancer Res 2000;60:3425–3428. 25 Zhang Y, Li J, Tang L: Cancer-preventive isothiocyanates: dichotomous modulators of oxidative stress. Free Radic Biol Med 2005;38:70–77.
Nakamura
26 Miyoshi N, Watanabe E, Osawa T, Okuhira M, Murata Y, Ohshima H, Nakamura Y: ATP depletion alters the mode of cell death induced by benzyl isothiocyanate. Biochim Biophys Acta 2008;1782:566– 573. 27 Hirose M, Yamaguchi T, Kimoto N, Ogawa K, Futakuchi M, Sano M, Shirai T: Strong promoting activity of phenylethyl isothiocyanate and benzyl isothiocyanate on urinary bladder carcinogenesis in F344 male rats. Int J Cancer 1998;77:773–777. 28 Nakamura Y, Torikai K, Ohto Y, Murakami A, Tanaka T, Ohigashi H: A simple phenolic antioxidant protocatechuic acid enhances tumor promotion and oxidative stress in female ICR mouse skin: dose-and timing-dependent enhancement and involvement of bioactivation by tyrosinase. Carcinogenesis 2000;21:1899–1907.
29 Ye L, Dinkova-Kostova AT, Wade KL, Zhang Y, Shapiro TA, Talalay P: Quantitative determination of dithiocarbamates in human plasma, serum, erythrocytes and urine: pharmacokinetics of broccoli sprout isothiocyanates in humans. Clin Chim Acta 2002;316:43–53. 30 Gasper AV, Traka M, Bacon JR, Smith JA, Taylor MA, Hawkey CJ, Barrett DA, Mithen RF: Consuming broccoli does not induce genes associated with xenobiotic metabolism and cell cycle control in human gastric mucosa. J Nutr 2007;137:1718–1724.
Dr. Yoshimasa Nakamura Graduate School of Natural Science and Technology Faculty of Agriculture, Okayama University Okayama 700-8530 (Japan) Tel. +81 86 251 8300, Fax +81 86 251 8388, E-Mail
[email protected]
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Chemoprevention and Cancer Yoshikawa T (ed): Food Factors for Health Promotion. Forum Nutr. Basel, Karger, 2009, vol 61, pp 182–192
Ginger-Derived Phenolic Substances with Cancer Preventive and Therapeutic Potential Joydeb Kumar Kundu ⭈ Hye-Kyung Na ⭈ Young-Joon Surh National Research Laboratory of Molecular Carcinogenesis and Chemoprevention, College of Pharmacy, Seoul National University, Seoul, South Korea
Abstract Ginger, the rhizomes of Zingiber officinale Roscoe (Zingiberaceae), has widely been used as a spice and condiment in different societies. Besides its food-additive functions, ginger has a long history of medicinal use for the treatment of a variety of human ailments including common colds, fever, rheumatic disorders, gastrointestinal complications, motion sickness, diabetes, cancer, etc. Ginger contains several nonvolatile pungent principles viz. gingerols, shogaols, paradols and zingerone, which account for many of its health beneficial effects. Studies conducted in cultured cells as well as in experimental animals revealed that these pungent phenolics possess anticarcinogenic properties. This chapter summarizes updated information on chemopreventive and chemotherapeutic effects of ginger-derived phenolic substances and their underlying mechanisms. Copyright © 2009 S. Karger AG, Basel
Cancer is one of the most debilitating diseases taking out millions of lives each year throughout the world. Till now, all efforts in developing anticancer therapies have only been reflected in the limited success in prolonging survival against a growing challenge of chemotherapy resistance, high-cost medication and poor quality of life issues. Based on the advances in identifying cancer causes and unraveling the molecular basis of cancer, it is now estimated that approximately 30–40% of cancers are preventible [1]. One of the most practical and feasible ways to prevent cancer is chemoprevention, which refers to the use of nontoxic chemical substances to interfere with the multistage carcinogenesis. The concept of chemoprevention appears as a timely and optimistic approach to reduce the global burden of cancer. Numerous dietary phytochemicals including those present in common spices, such as ginger, turmeric, garlic, red chili peppers, etc. have been identified as potential chemopreventive agents [2]. Many of these spice-derived chemopreventive phytochemicals also exert cancer chemotherapeutic effects by selectively inducing apoptosis, arresting the growth of
cancer cells, and sensitizing chemoresistant tumors to conventional chemotherapy. This chapter highlights the cancer chemopreventive and chemotherapeutic potential of some pungent phenolic substances present in the rhizomes of ginger (Zingiber officinale Roscoe, Zingiberaceae).
Pungent Phenolic Substances Derived from Ginger
Ginger (Zingiber officinale Roscoe, Zingiberaceae), is one of the most widely used spices and a common condiment in various foods and beverages. Besides its extensive use as a spice, the rhizome of ginger has long been used in the oriental herbal medicine for the management of various human ailments including colds, fever, rheumatic disorders, gastrointestinal discomforts, motion sickness, and leprosy. As a traditional medicine, ginger has antiemetic, diuretic, anti-inflammatory, analgesic, carminative, stimulant, antioxidative, and antipyretic effects [3, 4]. The pungent principles of ginger include gingerols, shogaols, paradols, and zingerone [4] (structures shown in figure 1). These pungent phenolics of ginger possess antioxidant, anti-inflammatory and anticarcinogenic activities, which will be discussed in the following sections. Figure 2 illustrates the biochemical basis of cancer chemopreventive and therapeutic potential of ginger-derived pungent phenolic substances.
Antioxidative Effects
Oxidative stress and inflammation are the key pathophysiologic features implicated in multistage carcinogenesis [5]. The antitumor promoting effects of ginger have been ascribed to the antioxidative and anti-inflammatory properties of its pungent phenolics. [6]-Gingerol suppressed the generation of superoxides in differentiated human promyelocytic leukemia (HL-60) cells stimulated with a prototype tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) [4]. Like [6]-gingerol, [6]-paradol inhibited TPA-induced H2O2 production and myeloperoxidase activity in mouse skin and superoxide generation in HL-60 cells [6]. [6]-Paradol also attenuated the formation of 8-hydroxy-deoxyguanosine, a marker of DNA damage, in calf thymus DNA exposed to H2O2 followed by UV irradiation [6]. Oxidative stress induced by reactive nitrogen species (RNS), such as nitric oxide (NO) and peroxynitrite, can also cause DNA damage and contribute to carcinogenesis [5]. Zingerone efficiently scavenged peroxynitrite in rat prostatic endothelial cells stimulated with 3-morpholinosydnonimine hydrochloride (SIN-1) and reduced the formation of nitrotyrosine in vitro, suggesting the potential of this ginger-derived pungent phenolic against RNSinduced tissue damage [7]. Dehydrozingerone isolated from Zingiber officinale and its synthetic analogs showed free radical-scavenging activity in the 1,1-diphenyl-2-
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O
O
OH
H3OC
(CH2)n
H3CO
(CH2)n
HO
HO
Shogaols
Gingerols
[6]-Shogaol: n = 4 [10]-Shogaol: n = 8
[4]-Gingerol: n = 2 [6]-Gingerol: n = 4 [8]-Gingerol: n = 6 [10]-Gingerol: n = 8 [12]-Gingerol: n = 10 O H3OC
O H3OC HO
HO Zingerone
(CH2)n Paradols [6]-Paradol: n = 4 [8]-Paradol: n = 6
Fig. 1. Chemical structures of some representative pungent phenolics derived from ginger.
picrylhydrazyl (DPPH) radical quencher assay [8]. The structure-activity relationship analysis revealed that the number and the position of hydroxyl groups on the aromatic ring and a double bond between C-3 and C-4 played a critical role in exerting the antioxidant activity [8]. [6]-Gingerol also suppressed peroxynitrite-induced oxidative single-strand DNA breaks and protein nitrosylation [9]. Masuda et al. [10] examined the structure-activity relationship of gingerol and related compounds. According to their study, alkyl substitution at –10, 12 and 14 carbon contributed to free radical-scavenging activity and protection against autoxidation of oils [10]. A novel [6]-gingerdiol glucosides, 5-O-beta-d-glucopyranosyl-3-hydroxy-1-(4hydroxy-3-methoxyphenyl)decane exerted as strong antioxidative activity as that of the aglycon [6]-gingerdiol [11]. Besides the inhibitory effects on superoxide generation and free radical scavenging ability, some ginger phenolics protect against oxidative stress through induction of antioxidant enzymes. One of the key transcription factors that regulate the expression of a battery of genes encoding antioxidant enzymes is nuclear factor-E2-related factor 2 (Nrf2). In resting cells, Nrf2 remains sequestered in cytoplasm by its inhibitory counter part Kelch-like ECH-associated protein-1 (Keap1). Oxidative or covalent modification of critical cysteine residues of Keap1 results in the dissociation of Nrf2 from Keap1 and subsequently facilitates the nuclear translocation and DNA binding of Nrf2 [2]. [10]-Shogaol has been reported to alkylate the cysteine 151 residue of recombinant human Keap1 [12]. Therefore, it is speculated that [10]-shogaol may enhance nuclear accumulation of Nrf2 and upregulate antioxidant/detoxifying enzyme expression.
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Blockade of proinflammatory signaling
Inhibition of tumor cell proliferation
Chemoprevention
Protection against oxidative and nitrosative stress
Ginger-derived phenolics
Inhibition of angiogenesis Suppression of tumor metastasis
Chemotherapy
Induction of apoptosis in cancer cells
Sensitization of chemoresistant cells
Fig. 2. Biochemical mechanisms underlying anticarcinogenic effects of ginger-derived phenolic substances.
Anti-Inflammatory Effects
Inappropriate functioning of a distinct set of proinflammatory mediators, such as cytokines, chemokines, cyclooxygenase-2 (COX-2), prostaglandins, inducible nitric oxide synthase (iNOS), and NO, has been implicated in the carcinogenic process [5]. [6]-Gingerol inhibited the liberation of proinflammatory cytokines, such as interleukin (IL)-12, tumor necrosis factor-α (TNF-α) and IL-1β from lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages [13]. Topical application of [6]-gingerol [6] or [6]-paradol [14] significantly attenuated TPAinduced mouse ear edema and epidermal vascular permeability. Moreover, [6]-gingerol ameliorated TPA-induced inflammation in mouse skin by suppressing the expression of COX-2 through inactivation of a ubiquitous eukaryotic transcription factor nuclear factor-κB (NF-κB) [15]. Likewise, topical application of [6]-gingerol diminished ultraviolet B-induced expression of COX-2 protein and its mRNA transcript as well as activation of NF-κB in mouse skin in vivo and in HaCaT keratinocytes in culture [16]. A standardized fraction of the ginger extract containing gingerols suppressed LPS-induced COX-2 expression in differentiated human leukemia U937 cells, while a shogaol-rich fraction failed to inhibit COX-2 expression [17]. Moreover, partially purified fractions of fresh [18] or commercially available
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processed dry ginger [19] containing gingerols and/or gingerol derivatives significantly reduced LPS-induced PGE2 production in human leukemia cells. Evaluation of the effects of pungent oleoresin principles of ginger on COX-2 enzyme activity showed that [8]-paradol and [8]-shogaol inhibited the COX-2 activity in IL-1βstimulated human airways epithelial (A549) cells [20]. In this study, analysis of the structure-activity relationship of ginger constituents revealed that the relative COX-2 inhibitory effects of pungent principles of ginger depends on the lipophilicity of the alkyl side chain, a substitution pattern of hydroxy and carbonyl groups on the side chain, and a substitution pattern of hydroxy and methoxy groups on the aromatic moiety [20]. In another study, [6]-gingerol significantly inhibited the expression of iNOS and the production of NO in LPS-stimulated J774.1 murine macrophages [21].
Antitumor-Promoting Effects
[6]-Gingerol and [6]-paradol inhibited epidermal growth factor (EGF)-induced transformation of cultured mouse epidermal JB6 cells following apparently different mechanisms [22]. While [6]-paradol inhibited transformation of JB6 cells stimulated with EGF through induction of apoptosis, [6]-gingerol inhibited cell transformation by blocking EGF-induced transactivation of activator protein-1 (AP-1), a ubiquitous transcription factor involved in tumor promotion [22]. Transforming growth factor-β1 (TGF-β1) promotes tumor progression in the late stages of carcinogenesis through induction of epithelial-mesenchymal transition. [6]-Gingerol blocked TGFβ1-induced epithelial-mesenchymal transition in c-Ha-ras-transfected immortalized human keratinocyte (HaCaT) cells [23]. Both [6]-gingerol and [6]-paradol attenuated chemically induced mouse skin tumor promotion [6, 14]. Topical application of these ginger phenolics inhibited TPA-induced ornithine decarboxylase activity, a biochemical hallmark of tumor promotion, in mouse skin [6, 14]. Dietary administration of gingerol (0.02%) significantly decreased the number of intestinal tumors in male F344 rats treated with the carcinogen azoxymethane [24]. One of the etiological factors for the development of gastric and colon cancer is the infection with Helicobacter pylori. A fractionated methanol extract of dried ginger rhizomes containing gingerols inhibited the growth of 19 strains of H. pylori, including 5 CagA+ strains in culture [25]. In another study, [6]-gingerol significantly reduced the formation of HCl-induced gastric lesions [26], a predisposing factor for gastric carcinogenesis. The gastroprotective effect of [6]-gingerol was blocked by treatment with a vanilloid receptor-1 (VR1) antagonist, ruthenium red, suggesting that the compound may exert chemopreventive effects on gastric acid-induced carcinogenesis through interaction with VR1 [26].
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Inhibition of the Growth and Proliferation of Tumor Cells
Ginger phenolics have been reported to suppress the growth of tumor xenografts. While [6]-shogaol suppressed the growth of human gastric cancer xenografts in athymic nude mice, [6]-gingerol alone merely displayed a growth inhibitory effect. However, when cotreated with TNF-related apoptosis-inducing ligand (TRAIL), [6]-gingerol significantly potentiated the growth-inhibitory effect of TRAIL [27]. The compound arrested the growth of two human pancreatic cancer cell lines, one expressing mutant p53 and the other expressing wild-type p53, in the G1 phase of the cell cycle [28]. This study also demonstrated that [6]-gingerol decreased expression of cell cycle-regulatory proteins, such as cyclin A and cyclin-dependent kinase, thereby reducing the phosphorylation of retinoblastoma protein and subsequent blockade of S phase entry [28]. [6]-Shogaol exerted antiproliferative effects in two transgenic mouse ovarian cancer cell lines, C1 (genotype: p53–/–, c-myc, K-ras) and C2 (genotype: p53+/+, c-myc, Akt) [29]. In this study, [4]-, [6]-, [8]- and [10]-gingerols also showed moderate antiproliferative effects [29].
Induction of Apoptosis in Cancerous or Transformed Cells
[6]-Gingerol [30] and [6]-shogaol [30, 31] induced apoptosis in human promyelocytic leukemia (HL-60) cells. [6]-Gingerol-induced apoptosis in HL-60 cells was prevented by catalase, suggesting that the compound induced cell death through generation of reactive oxygen species (ROS) [31]. In another study, [6]-shogaol, but not [6]-gingerol, induced cell death in human colorectal carcinoma COLO 205 cells through mitochondrial ROS generation, cytochrome c release, caspase activation, and DNA fragmentation [32]. Moreover, the induction of apoptosis by [6]-shogaol in these cells was associated with increased expression of proapoptotic Bax and inhibition of antiapoptotic Bcl-2 and Bcl-xL. The expression of growth arrest and DNA damage-inducible transcription factor-153 and its mRNA transcript was also induced by [6]-shogaol. In addition, [6]-shogaol enhanced the expression of Fas and FasL in COLO 205 cells [32]. The most potent cytotoxic effect of [6]-shogaol was observed against human nonsmall cell lung adenocarcinoma (A549), ovarian cancer (SK-OV-3), melanoma (SK-MEL-2) and colon cancer (HCT15) cells [29]. Treatment of oral squamous carcinoma KB cells with [6]-paradol or other structurally related derivatives, including [10]-paradol, [3]-dehydroparadol, [6]-dehydroparadol, and [10]-dehydroparadol, induced apoptosis through activation of caspase-3 [33]. While [6]-gingerol reduced the viability of gastric cancer cells by inhibiting cIAP1 through the blockade of NF-κB activation, the decrease in viability of these cells by [6]-shogaol resulted from the damage of microtubules and induction of mitotic arrest [27]. The induction of apoptosis by [6]-shogaol in Mahlavu cells, a poorly differentiated and p53-mutated human hepatoma cell line with high expression of multidrug
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resistance gene-1 and Bcl-2, was mediated via caspase activation [34]. [6]-Shogaol treatment increased ROS generation and glutathione (GSH) depletion in these cells. Cotreatment with N-acetylcysteine, an antioxidant and a precursor of GSH biosynthesis, almost completely abrogated the apoptotic effects of [6]-shogaol, suggesting that ROS generation and GSH depletion are the major contributing factors in [6]-shogaol-induced apoptosis of Mahlavu cells [34]. Treatment of mutant p53-expressing human pancreatic cancer cells with [6]-gingerol revealed that the compound increased the phosphorylation of Akt/protein kinase B and induced apoptosis. In contrast, no signs of early apoptosis were detected in wild-type p53-expressing pancreatic cancer cells treated with [6]-gingerol [28]. These results indicate that [6]-gingerol can circumvent the resistance of mutant p53expressing cells towards chemotherapy by inducing apoptotic cell death, while it exerts cytostatic effects in wild-type p53-expressing pancreatic cancer cells by inducing growth arrest [28]. [6]-Gingerol restored testosterone-depleted levels of p53 and increased the expression of Bax and activation of caspase-9 and caspase-3 in androgen-sensitive human prostate cancer (LNCaP) cells and in murine prostate. In addition, the compound downregulated testosterone-induced expression of antiapoptotic proteins Bcl-2 and survivin in both LNCaP cells and mouse prostate in vivo [35]. [6]-Gingerol induced apoptosis in human colorectal cancer cells by accumulating cells at the G1 phase of cell cycle arrest [36]. In this study, the induction of apoptosis by [6]-gingerol was associated with a decrease in cyclin D1 resulting from reduced nuclear translocation of β-catenin and proteolysis of cyclin D1, and an increase in the expression of nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1), which is a cytokine with proapoptotic and antitumorigenic properties [36].
Antiangiogenic and Antimetastatic Effects
Angiogenesis is a process of forming new vasculature inside benign tumors to nourish the rapidly proliferating preneoplastic cells with adequate oxygen and nutrient supply. Major proangiogenic factors involved in tumor angiogenesis include vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), IL-8, iNOS, angiopoietins, etc. Targeted inhibition of these proangiogenic factors leads to the reduced growth and survival of tumor cells. The eukaryotic transcription factor NF-κB is constitutively activated in epithelial ovarian cancer cells and contributes to increased transcription of the aforementioned angiogenic factors. Treatment of cultured epithelial ovarian cancer cells with ginger extracts induced profound growth inhibition in A2780, SKOV3, and ES-2 cells [37]. Ginger extracts suppressed the activation of NF-κB and diminished the secretion of VEGF and IL-8 in ES-2 cells [38]. This study also reported that [6]-shogaol, but not gingerols, significantly inhibited the growth of A2780 cells in culture. Investigation of individual pungent ingredients of ginger on the expression or secretion of angiogenic factors is still limited.
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Kim et al. demonstrated that [6]-gingerol blocked VEGF-induced capillary-like tube formation by endothelial cells, and strongly inhibited sprouting of these cells in the rat aorta and the formation of new blood vessels in the mouse cornea. The compound also inhibited VEGF- or bFGF-induced proliferation of human endothelial cells [38]. Almost 90% of cancer mortality is associated with tumor metastasis. [6]-Gingerol was found to be the most active component of Keishi-ka-kei-to, a traditional Chinese herbal medicine composed of crude extracts from five medicinal plants (Cinnamomi cortex, Paeoniae radix, Zizyphi fructus, Zingiberis rhizoma and Glycyrrhizae radix), which inhibited experimental pulmonary metastasis in mice implanted with B16F10 melanoma cells [39]. Likewise, intraperitoneal administration of [6]-gingerol to mice receiving i.v. injection of B16F10 melanoma cells reduced the number of lung metastasis [38]. Treatment of human breast cancer (MDA-MB-231) cells with [6]-gingerol led to a concentration-dependent decrease in cell migration and motility, and reduced mRNA expression and activities of matrix metalloproteinase (MMP)-2 or MMP-9, which are well-known markers of tumor metastasis [40].
Chemosensitizing Effects
One of the reasons for the growing trend of chemotherapy failure is the emergence of chemoresistance. P-Glycoprotein (P-gp), a product of multi-drug resistance gene-1, is considered as a key player in developing chemoresistance. P-gp is frequently overexpressed in tumor cells resistant to multiple anticancer agents. By actively refluxing drugs from cells, P-gp reduces intratumoral concentrations of chemotherapeutic drugs and hence lowers their efficacy. [6]-Gingerol inhibited the efflux of P-gp substrates in human multidrug-resistant carcinoma KB-C2 cells, suggesting that the compound may facilitate intratumoral accumulation of anticancer drugs [41].
Conclusion
Besides their value as a common spice, the rhizomes of ginger have been used since antiquity as a component of oriental medicine. Mounting evidence from in vitro and in vivo studies suggest that the organic pungent vanilloid compounds of ginger possess chemopreventive and chemotherapeutic potential. Ginger phenolics have been shown to target critical intracellular signaling molecules involved in regulating cellular redox status, inflammation, tumor cell proliferation, cancer cell apoptosis, tumor angiogenesis and metastasis (fig. 3). Some of these pungent phenolics of ginger have also been reported to inhibit chemotherapy-induced adverse reactions, thereby improving the quality of life of cancer patients. Detailed pharmacokinetic studies to
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Ginger phenolics
ROS/RNS
DNA damage and genetic alterations
Upstream kinases (e.g. Akt, MAP Kinases, etc.)
Transcription factors (e.g. NF-B, AP-1, etc.)
Proinflammatory mediators Initiated cells
(e.g. COX-2, iNOS, IL, TNF-␣, etc.)
Cyclins, Bcl-2, Bcl-xL,VEGF, MMPs
Preneoplastic cells
Neoplasia
Fig. 3. Potential molecular targets for anticarcinogenic effects of ginger and its pungent ingredients.
obtain a safe dosage regimen and a well-designed human chemoprevention trial to verify the anticarcinogenic activity of individual vanilloids are needed before ginger phenolics can be used clinically.
Acknowledgements This study was supported by the research grants from the BioGreen 21 Program (No. 2007301-034027), Rural Development Administration, and from the Biofoods Research Program, Ministry of Health and Welfare, Republic of Korea. Joydeb Kumar Kundu is a recipient of Brain Korea (BK21) Assistant Professorship at the Applied Pharmaceutical Life Science Research Division of Seoul National University.
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14 Park KK, Chun KS, Lee JM, Lee SS, Surh Y-J: Inhibitory effects of [6]-gingerol, a major pungent principle of ginger, on phorbol ester-induced inflammation, epidermal ornithine decarboxylase activity and skin tumor promotion in ICR mice. Cancer Lett 1998;129:139–144. 15 Kim SO, Kundu JK, Shin YK, Park JH, Cho MH, Kim TY, Surh Y-J: [6]-Gingerol inhibits COX-2 expression by blocking the activation of p38 MAP kinase and NF-κB in phorbol ester-stimulated mouse skin. Oncogene 2005;24:2558–2567. 16 Kim JK, Kim Y, Na KM, Surh Y-J, Kim TY: [6]-Gingerol prevents UVB-induced ROS production and COX-2 expression in vitro and in vivo. Free Radic Res 2007;41:603–614. 17 Lantz RC, Chen GJ, Sarihan M, Solyom AM, Jolad SD, Timmermann BN: The effect of extracts from ginger rhizome on inflammatory mediator production. Phytomedicine 2007;14:123–128. 18 Jolad SD, Lantz RC, Solyom AM, Chen GJ, Bates RB, Timmermann BN: Fresh organically grown ginger (Zingiber officinale): composition and effects on LPS-induced PGE2 production. Phytochemistry 2004;65:1937–1954. 19 Jolad SD, Lantz RC, Chen GJ, Bates RB, Timmermann BN: Commercially processed dry ginger (Zingiber officinale): composition and effects on LPS-stimulated PGE2 production. Phytochemistry 2005;66:1614– 1635. 20 Tjendraputra E, Tran VH, Liu-Brennan D, Roufogalis BD, Duke CC: Effect of ginger constituents and synthetic analogues on cyclooxygenase-2 enzyme in intact cells. Bioorg Chem 2001;29:156– 163. 21 Ippoushi K, Azuma K, Ito H, Horie H, Higashio H: [6]-Gingerol inhibits nitric oxide synthesis in activated J774.1 mouse macrophages and prevents peroxynitrite-induced oxidation and nitration reactions. Life Sci 2003;73:3427–3437. 22 Bode AM, Ma WY, Surh Y-J, Dong Z: Inhibition of epidermal growth factor-induced cell transformation and activator protein 1 activation by [6]-gingerol. Cancer Res 2001;61:850–853. 23 Davies M, Robinson M, Smith E, Huntley S, Prime S, Paterson I: Induction of an epithelial to mesenchymal transition in human immortal and malignant keratinocytes by TGF-beta1 involves MAPK, Smad and AP-1 signalling pathways. J Cell Biochem 2005;95:918–931. 24 Yoshimi N, Wang A, Morishita Y, Tanaka T, Sugie S, Kawai K, Yamahara J, Mori H: Modifying effects of fungal and herb metabolites on azoxymethaneinduced intestinal carcinogenesis in rats. Jpn J Cancer Res 1992;83:1273–1278.
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25 Mahady GB, Pendland SL, Yun GS, Lu ZZ, Stoia A: Ginger (Zingiber officinale Roscoe) and the gingerols inhibit the growth of Cag A+ strains of Helicobacter pylori. Anticancer Res 2003;23:3699– 3702. 26 Horie S, Yamamoto H, Michael GJ, Uchida M, Belai A, Watanabe K, Priestley JV, Murayama T: Protective role of vanilloid receptor type 1 in HCl-induced gastric mucosal lesions in rats. Scand J Gastroenterol 2004;39:303–312. 27 Ishiguro K, Ando T, Maeda O, Ohmiya N, Niwa Y, Kadomatsu K, Goto H: Ginger ingredients reduce viability of gastric cancer cells via distinct mechanisms. Biochem Biophys Res Commun 2007;362: 218–223. 28 Park YJ, Wen J, Bang S, Park SW, Song SY: [6]-Gingerol induces cell cycle arrest and cell death of mutant p53-expressing pancreatic cancer cells. Yonsei Med J 2006;47:688–697. 29 Kim JS, Lee SI, Park HW, Yang JH, Shin TY, Kim YC, Baek NI, Kim SH, Choi SU, Kwon BM, Leem KH, Jung MY, Kim DK: Cytotoxic components from the dried rhizomes of Zingiber officinale Roscoe. Arch Pharm Res 2008;31:415–418. 30 Lee E, Surh Y-J: Induction of apoptosis in HL-60 cells by pungent vanilloids, [6]-gingerol and [6]-paradol. Cancer Lett 1998;134:163–168. 31 Wei QY, Ma JP, Cai YJ, Yang L, Liu Zl: Cytotoxic and apoptotic activities of diarylheptanoids and gingerol-related compounds from the rhizome of Chinese ginger. J Ethnopharmacol 2005;102:177– 184. 32 Pan MH, Hsieh MC, Kuo JM, Lai CS, Wu H, Sang S, Ho CT: 6-Shogaol induces apoptosis in human colorectal carcinoma cells via ROS production, caspase activation, and GADD 153 expression. Mol Nutr Food Res 2008;52:527–537. 33 Keum YS, Kim J, Lee KH, Park KK, Surh Y-J, Lee JM, Lee SS, Yoon JH, Joo SY, Cha IH, Yook JI: Induction of apoptosis and caspase-3 activation by chemopreventive [6]-paradol and structurally related compounds in KB cells. Cancer Lett 2002; 177:41–47.
34 Chen CY, Liu TZ, Liu YW, Tseng WC, Liu RH, Lu FJ, Lin YS, Kuo SH, Chen CH: 6-shogaol (alkanone from ginger) induces apoptotic cell death of human hepatoma p53 mutant Mahlavu subline via an oxidative stress-mediated caspase-dependent mechanism. J Agric Food Chem 2007;55:948–954. 35 Shukla Y, Prasad S, Tripathi C, Singh M, George J, Kalra N: In vitro and in vivo modulation of testosterone mediated alterations in apoptosis related proteins by [6]-gingerol. Mol Nutr Food Res 2007; 51:1492–1502. 36 Lee SH, Cekanova M, Baek SJ: Multiple mechanisms are involved in 6-gingerol-induced cell growth arrest and apoptosis in human colorectal cancer cells. Mol Carcinog 2008;47:197–208. 37 Rhode J, Fogoros S, Zick S, Wahl H, Griffith KA, Huang J, Liu JR: Ginger inhibits cell growth and modulates angiogenic factors in ovarian cancer cells. BMC Complement Altern Med 2007;7:44. 38 Kim EC, Min JK, Kim TY, Lee SJ, Yang HO, Han S, Kim YM, Kwon YG: [6]-Gingerol, a pungent ingredient of ginger, inhibits angiogenesis in vitro and in vivo. Biochem Biophys Res Commun 2005;335:300– 308. 39 Suzuki F, Kobayashi M, Komatsu Y, Kato A, Pollard RB: Keishi-ka-kei-to, a traditional Chinese herbal medicine, inhibits pulmonary metastasis of B16 melanoma. Anticancer Res 1997;17:873–878. 40 Lee HS, Seo EY, Kang NE, Kim WK: [6]-Gingerol inhibits metastasis of MDA-MB-231 human breast cancer cells. J Nutr Biochem 2008;19:313–319. 41 Nabekura T, Kamiyama S, Kitagawa S: Effects of dietary chemopreventive phytochemicals on P- glycoprotein function. Biochem Biophys Res Commun 2005;327:866–870.
Professor Young-Joon Surh College of Pharmacy, Seoul National University Shillim-dong, Kwanak-gu Seoul 151-742 (South Korea) Tel. +82 2 880 7845, Fax +82 2 874 9775, E-Mail
[email protected]
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Chemoprevention and Cancer Yoshikawa T (ed): Food Factors for Health Promotion. Forum Nutr. Basel, Karger, 2009, vol 61, pp 193–203
Chemoprevention with Phytochemicals Targeting Inducible Nitric Oxide Synthase Akira Murakami Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
Abstract A regulated low level of nitric oxide (NO) production in the body is essential for maintaining homeostasis (neuroprotection, vasorelaxation, etc.), though certain pathophysiological conditions associated with inflammation involve de novo synthesis of inducible NO synthase (iNOS) in immune cells, including macrophages. A large body of evidence indicates that many inflammatory diseases, such as colitis and gastritis, as well as many types of cancer, occur through sustained and elevated activation of this particular enzyme. The biochemical process of iNOS protein expression is tightly regulated and complex, in which the endotoxin lipopolysaccharide selectively binds to toll-like receptor 4 and thereby activates its adaptor protein MyD88, which in turn targets downstream proteins such as IRAK and TRAF6. This leads to functional activation of key protein kinases, including IkB kinases and mitogen-activated protein kinases (MAPKs), such as p38 MAPK, JNK1/2, and ERK1/2, all of which are involved in activating key transcription factors, including nuclear factor-κB and activator protein-1. In addition, the production of proinflammatory cytokines such as interferon-γ and interleukin-12 potentiates iNOS induction in autocrine fashions. Meanwhile, an LPS-stimulated p38 MAPK pathway plays a pivotal role in the stabilization of iNOS mRNA, which has the AU-rich element in its 3⬘-untranslated region, for rapid NO production. Thus, suppression and/or inhibition of the abovementioned signaling molecules may have a great potential for the prevention and treatment of inflammation-associated carcinogenesis. In fact, there have been numerous reports of phytochemicals found capable of targeting NO production by unique mechanisms, including polyphenols, terpenoids, and others. This review article briefly highlights the molecular mechanisms underlying endotoxin-induced iNOS expression in macrophages, and also focuses on promising natural agents that may be useful for anti-inflammation and anticarcinogenesis strategies. Copyright © 2009 S. Karger AG, Basel
Roles of Nitric Oxide in Inflammation-Associated Cancer
Nitric oxide (NO), a gaseous free radical, is produced by NO synthase (NOS), which catalyzes the conversion of L-arginine into L-citrulline, with NADPH, tetrahydrobiopterine, FAD, FMN, molecular oxygen (3O2), and protoporphyrin known to be its
cofactors. NOS is classified into subfamilies with constitutive or inducible characteristics, according to the location of expression in the body and manner of expression. Constitutive NOS is detected in neuronal tissues (nNOS) and vascular endothelial cells (eNOS), whereas inducible NOS (iNOS) is expressed in many cell types, including macrophages, microglial cells, keratinocytes, hepatocytes, astrocytes, and vascular endothelial and epithelial cells, under both normal and pathological conditions. Constitutive NOS subtypes have essential roles in the maintenance of homeostasis, e.g. regulating blood vessel tone (eNOS), and serving as a neurotransmitter and neuromodulator (nNOS) in nonadrenergic and noncholinergic nerve endings. NO generation from constitutive NOS enzymes is constant and within the nanomolar range. In contrast, in response to infectious and other proinflammatory stimuli, iNOS protein is synthesized de novo to produce a micromolar range of NO. Numerous reports have implicated that sustained and/or excess NO generation occurs in pathogenic conditions, while it is mediated mostly by iNOS in inflammatory cells. In particular, iNOS has drawn considerable attention for its critical functions in inflammationrelated diseases such as cancer. It is essential to point out that NO is coupled with superoxide anion radical (O2–) to form peroxynitrite (ONOO–) in a diffusion-dependent manner. ONOO– is extremely reactive, as compared with NO or O2– alone, though NO has been shown to have a chemical potential to cause DNA damage by nitration, nitrosation, and oxidation [1]. Recently, Sawa et al. [2] described a significant finding that treatment of macrophages with endotoxin led to the formation of a nitrated derivative of cGMP, 8-nitroguanosine 3⬘,5⬘-cyclic monophosphate (8-nitro-cGMP), which underwent S-guanylation of the highly nucleophilic cysteine sulfhydryls of Keap1, suggesting its role in cancer cell survival and antiapoptosis. In addition, the same research group documented that 8-nitroguanosine, another DNA base modified by ONOO–, induced a G to T transversion in the gpt gene and also significantly increased levels of abasic sites in DNA [3]. In addition to those mutagenic properties, excess NO generation may increase the metastatic potential of tumor cells by inducing vascular endothelial cell growth factor, matrix metalloproteinases (MMPs), and adhesion molecules. Some studies using iNOS gene-deficient mice have reported the essential roles of NO in tumor development. For example, Ahn and Ohshima [4] demonstrated that iNOS gene knockout Apc(Min/+) mice developed significantly fewer adenomas in both the small and large intestines as compared to Apc(Min/+) iNOS(+/+) mice. Also, Kohno et al. [5] reported that ONO-1714, a selective iNOS inhibitor, suppressed colitis-related colon cancer development in Apc(Min/+) mice, which was consistent with a previous study of chemically-induced colon carcinogenesis in rats [6]. Infection with Helicobacter pylori, a definitive mutagenic bacterium, has been shown to increase the risk of gastritis and resultant gastric carcinogenesis by chronic inflammation, which is evoked by immune cells activated by upregulated iNOS protein. Nam et al. [7] also observed that the incidence of gastric adenocarcinomas in iNOS-deficient mice exposed to combined N-methyl-N-nitrosourea administration
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with Helicobacter pylori infection was significantly lower than that of wild-type mice, indicating the importance of iNOS expression in the onset of gastric carcinogenesis. The above-mentioned background information led us to speculate that iNOS inhibition and/or suppression is a promising and attractive avenue for regulating chronic inflammation-associated gastrointestinal carcinogenesis.
Signal Transduction Pathways for iNOS Expression
The molecular mechanisms underlying iNOS expression are highly complex, and occasionally differ among different types of cells and stimuli. Thus, a brief overview is given here, mostly in reference to iNOS expression in LPS-challenged macrophages. iNOS mRNA expression is regulated during several distinguishable stages in a complex manner, and one of the earliest induction mechanisms is posttranscriptional regulation (fig. 1). Most, if not all, proinflammatory genes contain the AU-rich element (ARE) in the 3⬘-untranslated region (UTR), which has some critical roles in regard to the stability of mRNA [8]. LPS specifically binds to toll-like receptor 4 (TLR4) and thereby activates numerous protein kinases, including p38 mitogen-activated protein kinase (MAPK). Several independent reports of different cell types have shown that activation of the p38 MAPK pathway plays a central role in the stabilization of a number of pro-inflammatory mRNAs. Further, MAPK-activated protein kinase 2, a substrate for p38 MAPK, induces the phosphorylation of certain proteins, including heterogeneous nuclear ribonucleoprotein A0 [9] and Hu antigen R, which bind to AREs, thereby contributing to a rapid synthesis of iNOS protein via a posttranscriptional mechanism [10]. In contrast, other ARE-binding proteins, including TTP and TIA-1, promote decay of ARE-containing mRNA independently from regulating deadenylation, while the role of AUF1 remains controversial [11]. As noted above, LPS binding to TLR4 activates its adaptor protein MyD88, which in turn targets downstream proteins such as IRAK and TRAF6 (fig. 2). This leads to the multiple activation of protein kinases such as IκB kinase (IKK) and MAPKs (p38 MAPK, JNK1/2, ERK1/2, and others), all of which are involved in the activation of central iNOS gene transcription factors, i.e. nuclear factor κB (NFκB) and activator protein (AP)-1. Lee et al. [12] presented an important finding that both NFκB and AP-1 binding sites are located on the iNOS promoter, and necessary for iNOS induction. Meanwhile, both LPS-produced interleukin (IL)-12 and interferon (IFN)-γ are known to amplify iNOS induction signaling via autocrine loops, and function in an interdependent manner. IL-12, a 70-kDa heterodimeric protein, is critical to the initiation and progression of Th-1-type responses, and one of the initial cytokines released in response to microbial infection. IL-12p70 is composed of two disulfide-linked subunits of IL-12p35 and IL-12p40. The latter is constitutively expressed by macrophages and monocytes, while synthesis of the 35-kDa subunit is a rate-limiting activity that is crucial for bioactive IL-12. This cytokine is activated by posttranslational processing
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Fig. 1. Possible molecular mechanisms underlying LPS-induced stabilization of iNOS mRNA, which carries ARE in 3⬘-UTR, in macrophages. LPS = Lipopolysaccharide; LBP = LPS-binding protein; MyD88 = myeloid differentiation factor 88; IRAK = IL-1 receptor-associated kinase; TRAF6 = TNF receptorassociated factor 6; MKK = MAPK kinase; MK-2 = MAPK-activated protein kinase 2; hnRNP = heterogeneous nuclear ribonucleoprotein; HuR = human antigen R; TIA-1 = T-cell intracellular antigen-1; TTP = tristetraprolin.
with a complex mechanism [13]. The promoter region of the IL-12p40 gene contains LPS-responsive elements, including C/EBPβ, AP-1, and NFκB [14]. The binding of IL-12 to its receptors, IL-12RB1 and IL-12RB2, leads to phosphorylation of Janus kinase (JAK)2 and tyrosine kinase (TYK)2 for activating the signal transducer and activator of transcription (STAT)4, which partially regulates IFN-γ induction. In parallel, LPS-upregulated interferon regulatory factor (IRF)-1 mediates IFN-γ production via transcriptional regulation, whereas IRF-2 is mediated via posttranscriptional regulation [15]. In contrast, IFN-γ mRNA is regulated by a posttranscriptional mechanism [16]. In addition, LPS-produced IFN-γ has been demonstrated to act on the JAK2/ STAT1 system for IL-12p35 induction [17] and thereby contribute to iNOS activation.
Promising Phytochemicals Suppress iNOS Expression
Resveratrol, a phytoalexin found in grapes, is a stilbene-type polyphenol reported to exhibit a marked potential for cancer prevention (fig. 3). An early work by
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Fig. 2. Complex molecular mechanisms of LPS-induced iNOS expression in macrophages. TIRAP = TIR-domain-containing adaptor protein; TRIF = Toll-IL-1R domain-containing adaptor inducing IFNβ; IRF1 = interferon response factor 1; TYK2 = tyrosine kinase 2.
Tsai et al. [18] showed that resveratrol suppressed LPS-induced iNOS mRNA expression through the suppression of IκB degradation and, thus, NFκB blockade in macrophages, which was confirmed by several other investigations, including ours [19]. While modulation of MAPK activities has been reported to be generally dependent on concentration in the medium as well as the presence or absence of stimuli [20], the upstream target(s) of this agent have not been fully elucidated. Brouet and Ohshima were the first to report that curcumin attenuates LPS- and IFNγ-induced iNOS expression, and NO2– production in macrophages [21]. This compound is found in the rhizomes of Curcurma longa L. (Zingiberaceae), widely used as a yellow pigment in curry. A typical crude extract of the rhizomes of C. longa contain 70–76% curcumin, 16% demethoxycurcumin, and 8% bisdemethoxycurcumin. Chan et al. [22] also reported the efficacy of curcumin in vivo, which attenuated LPS-induced iNOS expression in rat livers, while Pan et al. [23] indicated that this suppression was related to IKK inhibition. Although the direct target of curcumin in macrophages is yet to be fully elucidated, studies with other experimental systems have suggested that this agent acts on the MAPK, PKC, and JAK/STAT pathways, which are located upstream of iNOS transcription and posttranscription [20].
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Fig. 3. Chemical structures of phytochemicals that suppress iNOS expression. Curcumin, ACA, and zerumbone occur in the rhizomes of C. longa, Alpinia galangal, and Z. zerumbet, respectively. EGCG is abundant in green tea. Resveratrol is present in grapes and wine, while both auraptene and nobiletin are the major constituents of citrus peel.
In particular, it is well established that c-Jun and c-Fos form a heterodimer, the AP-1 transcription factor, which is responsible for iNOS gene induction. Interestingly, curcumin decreased the amount of c-Fos protein by stimulating the formation of unstable hyperphosphorylated protein, thereby reducing the formation of a functionally active c-Jun/c-Fos complex [24], though this finding was obtained with mouse fibroblasts, not macrophages. Similarly, this yellow pigment attenuated JNK activation in various Jurkat T cells stimulated with various stimuli, while suppression may be due to interfering with MEKK1, an upstream kinase of JNK [24]. Lin and Lin [25] published a pioneering work showing that certain food phytochemicals have a pronounced ability to suppress iNOS expression, one of which is (–)-epigallocatechin-3-gallate (EGCG). Thereafter, a number of reports supported their findings, including in vivo and ex vivo efficacy results [26]. Dong et al. [27] proposed a hypothesis stating that EGCG-mediated suppression of AP-1-dependent transcriptional activity and DNA binding activity occurred through inhibition of JNK but not ERK1/2 in JB6 mouse epidermal cells. In contrast, our recent data indicate that EGCG generates superoxide anion to stimulate the JNK1/2-c-Fos/c-Jun
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Fig. 4. ACA, nobiletin, zerumbone, and auraptene attenuated iNOS expression at different stages. ACA suppressed MAPK activation; nobiletin did not affect either AP-1 or NF-κB, but it suppressed iNOS mRNA expression. Zerumbone accelerated the degradation of iNOS mRNA, which contains ARE at the 3⬘-UTR (c.f., fig. 1), whereas auraptene did not attenuate mRNA expression, but inhibited its protein synthesis, suggesting that it targets the translational step [31].
ACA MAPKs AP-1/NFB Nobiletin iNOS gene transcription Zerumbone iNOS mRNA stabilization Auraptene iNOS mRNA translation
pathway for activating AP-1 in HT-29 human colorectal cancer cells, thereby inducing MMP-7, a proteinase responsible for tumor cell development and invasion [28]. This discrepancy can be attributable, at least in part, to differences in the cell types. Meanwhile, a recent report noted induction of endothelium-dependent vasodilatation as an intriguing property of EGCG, which was implied to be primarily based on rapid activation of eNOS by a phosphatidylinositol 3-kinase-, PKA-, and an Aktdependent increase in eNOS activity [29]. Punathil et al. [30] also showed that EGCG inhibits mammary cancer cell migration through the inhibition of NO/NOS and guanylate cyclase. We are constantly searching for functionally novel phytochemicals that are able to attenuate iNOS induction in macrophages. Activity-guided separation of extracts from edible plants indigenous to Southeast Asian countries has identified 1⬘-acetoxychavicol acetate (ACA, from Alpinia galanga, Zingiberaceae) and zerumbone (from Zingiber zerumbet, Zingiberaceae), as well as auraptene and nobiletin (from citrus fruits), which are readily available from natural sources and/or chemically synthesized products [31]. Although their action mechanisms have not been fully elucidated, the anti-inflammatory activities of these compounds based on suppression of inflammatory cell activation may play major roles in their chemopreventive effects. Previously, we evaluated their iNOS suppressive activities and underlying molecular mechanisms with RAW264.7 macrophages [32]. Activation of p38 MAPK was not attenuated by zerumbone, ACA, or nobiletin (fig. 4). However, ACA substantially blocked both JNK1/2 and ERK1/2 activation, whereas both zerumbone and nobiletin allowed LPS-induced
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activation of those protein kinases. Furthermore, the phosphorylation of Akt at Ser473 was detectable in nontreated cells and enhanced by LPS in a time-dependent manner. None of those 3 compounds demonstrated any inhibition. In addition, ACA did not allow LPS-induced IκB degradation, whereas both zerumbone and nobiletin were virtually inactive, except that the nuclear protein level of NFκBp65 in nobiletin-treated cells was comparable to that in nontreated cells. LPS treatment markedly elevated the activities of the transcription factors of NFκB and AP-1, and it is notable that ACA and nobiletin significantly suppressed those transcription factors, whereas zerumbone had no effect. Taken together, ACA targets both JNK1/2 and ERK1/2, and nobiletin may interfere with coactivators, such as CREB-binding protein/p300, which suppress the transactivation of NF-κB and AP-1. Zerumbone allowed LPS-induced MAPK/Akt activation and transcriptional activation of those transcription factors, whereas it abrogated iNOS mRNA induction. Thus, zerumbone may target MK-2 or downstream molecules for destabilizing mRNA. In addition, auraptene might disturb the translation process of iNOS mRNA, since we previously found that it targets the translational step of proMMP-7 by inactivating ERK1/2 in HT-29 human adenocarcinoma cells, whose action mechanism differs from that of rapamycin, an inhibitor of mTOR [33]. Because auraptene did not affect the level of iNOS mRNA expression but suppressed its protein induction, a similar mechanism might be involved. Those results indicate that these 4 dietary factors have different molecular mechanisms for iNOS protein suppression, while their use in combinations may lead to synergistic results with higher efficacy and lower toxicity. The above-mentioned natural compounds have also been reported to show cancerpreventive activities in many types of rodent models. However, it is unclear whether their putative iNOS suppression abilities in vivo have a causal link to their cancer preventive effects, though some reports have indicated iNOS suppression. For example, topical application of resveratrol and its derivative, pterostilbene, suppressed phorbol ester-induced iNOS expression in mouse epidermis [34]. When injected into the peritoneum of ovalbumin-sensitized mice, curcumin suppressed iNOS expression in lung tissue [35], while it also inhibited iNOS activity in SD rats that had hepatic warm ischemia/reperfusion injuries [36]. Of interest, NCB 02, a standardized curcumin preparation, suppressed 2,4-dinitrochlorobenzene-induced ulcerative colitis and iNOS expression in rats [37], with a similar finding reported for curcumin itself [38]. Finally, oral administration of curcumin reduced iNOS mRNA expression levels in the livers of LPS-injected mice [39].
Conclusions
Involvement of chronic iNOS expression in the onset of inflammation-associated carcinogenesis has been reported; however, the molecular mechanisms related to
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phytochemical attenuation of iNOS induction remain to be fully elucidated. Although there is a large body of evidence indicating phytochemical-affected signal transduction pathways, knowledge of their direct binding or associated molecular targets is limited. Nevertheless, ongoing identification of the binding proteins of tea catechin [40] and other flavonoids [41, 42] is an encouraging progress, and undoubtedly an important future direction for this research field.
Acknowledgement The author thanks long-time collaborators Prof. Hajime Ohigashi of Fukui Prefectural University and Prof. Takuji Tanaka of Kanazawa Medical University, Japan, as well as Mr. Tomohiro Shigemori for his technical assistance.
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13 Murphy FJ, Hayes MP, Burd PR: Disparate intracellular processing of human IL-12 preprotein subunits: atypical processing of the P35 signal peptide. J Immunol 2000;164:839–847. 14 Trinchieri G: Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev Immunol 2003;3:133–146. 15 Salkowski CA, Barber SA, Detore GR, Vogel SN: Differential dysregulation of nitric oxide production in macrophages with targeted disruptions in IFN regulatory factor-1 and -2 genes. J Immunol 1996;156:3107–3110. 16 Wang JG, Collinge M, Ramgolam V, Ayalon O, Fan XC, Pardi R, Bender JR: LFA-1-dependent HuR nuclear export and cytokine mRNA stabilization in T cell activation. J Immunol 2006;176:2105–2113. 17 Negishi H, Fujita Y, Yanai H, Sakaguchi S, Ouyang X, Shinohara M, Takayanagi H, Ohba Y, Taniguchi T, Honda K. Evidence for licensing of IFN-gammainduced IFN regulatory factor 1 transcription factor by MyD88 in Toll-like receptor-dependent gene induction program. Proc Natl Acad Sci USA 2006; 103:15136–15141. 18 Tsai SH, Lin-Shiau SY, Lin JK: Suppression of nitric oxide synthase and the down-regulation of the activation of NFkappaB in macrophages by resveratrol. Br J Pharmacol 1999;126:673–680. 19 Murakami A, Matsumoto K, Koshimizu K, Ohigashi H: Effects of selected food factors with chemopreventive properties on combined lipopolysaccharide- and interferon-gamma-induced IkappaB degradation in RAW264.7 macrophages. Cancer Lett 2003;195:17– 25. 20 Rahman I, Biswas SK, Kirkham PA: Regulation of inflammation and redox signaling by dietary polyphenols. Biochem Pharmacol 2006;72:1439–1452. 21 Brouet I, Ohshima H: Curcumin, an anti-tumour promoter and anti-inflammatory agent, inhibits induction of nitric oxide synthase in activated macrophages. Biochem Biophys Res Commun 1995; 206:533–540. 22 Chan MM, Huang HI, Fenton MR, Fong D: In vivo inhibition of nitric oxide synthase gene expression by curcumin, a cancer preventive natural product with anti-inflammatory properties. Biochem Pharmacol 1998;55:1955–1962. 23 Pan MH, Lin-Shiau SY, Lin JK: Comparative studies on the suppression of nitric oxide synthase by curcumin and its hydrogenated metabolites through down-regulation of IkappaB kinase and NFkappaB activation in macrophages. Biochem Pharmacol 2000;60:1665–1676.
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24 Surh YJ, Chun KS, Cha HH, Han SS, Keum YS, Park KK, Lee SS: Molecular mechanisms underlying chemopreventive activities of anti-inflammatory phytochemicals: down-regulation of COX-2 and iNOS through suppression of NF-kappa B activation. Mutat Res 2001;480–481:243–268. 25 Lin YL, Lin JK: (–)-Epigallocatechin-3-gallate blocks the induction of nitric oxide synthase by down- regulating lipopolysaccharide-induced activity of transcription factor nuclear factor-kappaB. Mol Pharmacol 1997;52:465–472. 26 Song EK, Hur H, Han MK: Epigallocatechin gallate prevents autoimmune diabetes induced by multiple low doses of streptozotocin in mice. Arch Pharm Res 2003;26:559–563. 27 Dong Z, Ma W, Huang C, Yang CS: Inhibition of tumor promoter-induced activator protein 1 activation and cell transformation by tea polyphenols, (–)-epigallocatechin gallate, and theaflavins. Cancer Res 1997;57:4414–4419. 28 Kim M, Murakami A, Kawabata K, Ohigashi H: (–)-Epigallocatechin-3-gallate promotes pro-matrix metalloproteinase-7 production via activation of the JNK1/2 pathway in HT-29 human colorectal cancer cells. Carcinogenesis 2005;26:1553–1562. 29 Lorenz M, Wessler S, Follmann E, Michaelis W, Dusterhoft T, Baumann G, Stangl K, Stangl V: A constituent of green tea, epigallocatechin-3-gallate, activates endothelial nitric oxide synthase by a phosphatidylinositol-3-OH-kinase-, cAMP-dependent protein kinase-, and Akt-dependent pathway and leads to endothelial-dependent vasorelaxation. J Biol Chem 2004;279:6190–6195. 30 Punathil T, Tollefsbol TO, Katiyar SK: EGCG inhibits mammary cancer cell migration through inhibition of nitric oxide synthase and guanylate cyclase. Biochem Biophys Res Commun 2008;375:162–167. 31 Murakami A, Ohigashi H: Targeting NOX, INOS and COX-2 in inflammatory cells: chemoprevention using food phytochemicals. Int J Cancer 2007; 121:2357–2363. 32 Murakami A, Shigemori T, Ohigashi H: Zingiberaceous and citrus constituents, 1⬘-acetoxychavicol acetate, zerumbone, auraptene, and nobiletin, suppress lipopolysaccharide-induced cyclooxygenase-2 expression in RAW264.7 murine macrophages through different modes of action. J Nutr 2005;135: 2987S–2992S. 33 Kawabata K, Murakami A, Ohigashi H: Citrus auraptene targets translation of MMP-7 (matrilysin) via ERK1/2-dependent and mTOR-independent mechanism. FEBS Lett 2006;580:5288–5294.
Murakami
34 Cichocki M, Paluszczak J, Szaefer H, Piechowiak A, Rimando AM, Baer-Dubowska W: Pterostilbene is equally potent as resveratrol in inhibiting 12-O- tetradecanoylphorbol-13-acetate activated NFkappaB, AP-1, COX-2, and iNOS in mouse epidermis. Mol Nutr Food Res 2008;52:S62–S70. 35 Moon DO, Kim MO, Lee HJ, Choi YH, Park YM, Heo MS, Kim GY: Curcumin attenuates ovalbumininduced airway inflammation by regulating nitric oxide. Biochem Biophys Res Commun 2008;375: 275–279. 36 Shen SQ, Zhang Y, Xiang JJ, Xiong CL: Protective effect of curcumin against liver warm ischemia/reperfusion injury in rat model is associated with regulation of heat shock protein and antioxidant enzymes. World J Gastroenterol 2007;13:1953– 1961. 37 Venkataranganna MV, Rafiq M, Gopumadhavan S, Peer G, Babu UV, Mitra SK: NCB-02 (standardized curcumin preparation) protects dinitrochlorobenzene- induced colitis through down-regulation of NFkappa-B and iNOS. World J Gastroenterol 2007; 13:1103–1107.
38 Camacho-Barquero L, Villegas I, Sanchez-Calvo JM, Talero E, Sanchez-Fidalgo S, Motilva V, Alarcon de la Lastra C: Curcumin, a Curcuma longa constituent, acts on MAPK p38 pathway modulating COX-2 and iNOS expression in chronic experimental colitis. Int Immunopharmacol 2007;7:333–342. 39 Chan MM, Huang HI, Fenton MR, Fong D: In vivo inhibition of nitric oxide synthase gene expression by curcumin, a cancer preventive natural product with anti-inflammatory properties. Biochem Pharmacol 1998;55:1955–1962. 40 Tachibana H, Koga K, Fujimura Y, Yamada K: A receptor for green tea polyphenol EGCG. Nat Struct Mol Biol 2004;11:380–381. 41 Lee KW, Kang NJ, Heo YS, Rogozin EA, Pugliese A, Hwang MK, Bowden GT, Bode AM, Lee HJ, Dong Z: Raf and MEK protein kinases are direct molecular targets for the chemopreventive effect of quercetin, a major flavonol in red wine. Cancer Res 2008; 68:946–955. 42 Lee KW, Kang NJ, Rogozin EA, Kim HG, Cho YY, Bode AM, Lee HJ, Surh YJ, Bowden GT, Dong Z: Myricetin is a novel natural inhibitor of neoplastic cell transformation and MEK1. Carcinogenesis 2007; 28:1918–1927.
Dr. Akira Murakami Division of Food Science and Biotechnology Graduate School of Agriculture, Kyoto University Kyoto 606-8502 (Japan) Tel. +81 75 753 6282, Fax +81 75 753 6284, E-Mail
[email protected]
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Chemoprevention and Cancer Yoshikawa T (ed): Food Factors for Health Promotion. Forum Nutr. Basel, Karger, 2009, vol 61, pp 204–216
Chemoprevention of Tocotrienols: The Mechanism of Antiproliferative Effects Sayori Wada Laboratory of Health Science, Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto, Japan
Abstract Tocotrienols have been reported as antitumor agents and widely commercialized as an antioxidant dietary supplement. Tocotrienols have more significant biological activity than tocopherols, although serum level of tocotrienols is much lower than that of tocopherols. This may be because intracellular concentration of tocotrienols was revealed to be significantly higher compared with tocopherols, and tocotrienol accumulation is observed in tumor. Previous reports have suggested antiproliferative effect, induction of apoptosis, modulation of cell cycle, antioxidant activity, inhibition of angiogenesis, and suppression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity as anticarcinogenesis mechanisms of tocotrienols both in vivo and in vitro. Extension of the duration of host survival was observed in tumor-implanted mice treated with tocotrienol. Tocotrienols induce apoptosis mainly via mitochondria-mediated pathway. Cell cycle arrest is due to suppression of cyclin D by tocotrienols. Tocotrienols also inhibit vascularization-reducing proliferation, migration and tube formation. Malignant proliferation demands elevation of HMG CoA reductase activity, and tocotrienols suppress its activity. Tocotrienol treatment decreases oncogene expression and increases the level of tumor suppressors. Only a few clinical trials to determine the effects of tocotrienol on cancer prevention or treatment have been carried out. There is no convincing or probable evidence of the role of tocotrienols in cancer prevention, while α-tocopherol has been suggested to have a limited anti-prostate cancer potential. Neither beneficial activity nor adverse effect of tocotrienol has sufficiently been explored so far. The above-mentioned mechanisms of tocotrienols seem to be promising for cancer prevention; however, further clinical studies are warranted to assess the Copyright © 2009 S. Karger AG, Basel efficacy and safety of tocotrienol.
Natural vitamin E, a liposoluble vitamin, is a mixture of two classes of compounds, tocopherols and tocotrienols, each consisting of 4 isoforms: α-, β-, γ- and δ-tocopherol, and α-, β-, γ- and δ-tocotrienol. The structural difference between tocopherols and tocotrienols is that tocopherols have a saturated phytyl chain, and tocotrienols have an unsaturated phytyl chain; therefore, tocotrienols are isoprenoids while tocopherols are not. Barley, rice oat bran and olive oils have been reported to be rich in
α-tocotrienol, and barley, rice oat bran and palm oils in γ- and δ-tocotrienol [1]. The content of α-, β-, γ-, δ-tocopherol, and α-, β-, γ- and δ-tocotrienol are 256, –, 316, 70, –, 143, 32, 286 ppm in palm oil, and 324, 18, 53, –, 236, –, 349, 0 ppm in rice bran, respectively. Soy bean and corn oil contain plenty of α-, β-, γ- and δ-tocopherol: 101, –, 593, 264 ppm in soy bean and 112, 50, 602, 18 ppm in corn oil, but not tocotrienols [2]. As it is an antioxidant, tocotrienol is also provided as a dietary supplement all over the world. The preventive effects of various dietary food factors against cancer have been studied, and vitamin E, especially tocotrienol, has also been shown to participate in such antitumor effects. Although both tocopherol and tocotrienol have been reported as antiproliferative agents or tumor promotion inhibitors, tocotrienol exerts more significant effects than does tocopherol. Tocopherols have been studied especially for their neuroprotective and cardioprotective effects. The accumulation of tocotrienols in the cells is much greater than that of tocopherols. This might be one of the reasons why tocotrienols have a more significant physical effect than tocopherols. Antioxidant effects, suppression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity, proapoptotic effects, regulation of mitogenesis, and antiangiogenic potential have been proposed as mechanisms of the antiproliferative effects of these compounds. Several previous studies have demonstrated that γ-tocotrienol and δ-tocotrienol exert more potent antiproliferative activity among tocotrienol isoforms. In this article, we focus on the possible chemopreventive potential of tocotrienol.
Bioavailability of Tocotrienols
The concentration of tocotrienols in plasma and tissues, except adipose tissue, is lower than that of tocopherols in human and animals after oral administration [3–5]. Tocopherols and tocotrienols were found to be degraded via the same metabolic pathway, but tocotrienols were degraded to a larger extent [6 and its references]. The mean daily intakes of α-, β-, γ-, δ-tocopherol, and α-, β-, γ- and δ-tocotrienol in human were 8.76, 0.69, 7.58, 1.56, 1.44, 2.51, 0.20 and 0.06 mg, respectively [7]. Plasma levels of tocotrienol were found to be much lower than those of tocopherol, i.e. approximately 0.3 μM of tocotrienol in humans and 1–10 μM in various animals [3–5 and references within]. In rats, the level of tocotrienol was the highest at 8 h after intragastric administration in the liver, lung, serum, mesenteric lymph nodes and spleen. On the other hand, in some adipose tissues, tocotrienol levels were found to be the highest at 24 h after administration. In contrast, tocotrienol was undetectable in blood clots, brain, thymus, testes and muscle. The respective concentrations of α- and γ-tocotrienol were reported to be 32.1 and 12.1 μg/g in the liver and 9.7 and 9.0 μg/g in the lungs, respectively [3].
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Antiproliferative Effect of Tocotrienols
Tocotrienols were also found to inhibit the proliferation in several cancer cell lines in a dose- and time-dependent manner, and the mechanisms of their antiproliferative effect are thought to be induction of apoptosis and also, to some extent, cell cycle arrest. The IC50 of tocotrienol has been reported in various cells; the IC50 of α-, γ- and δ-tocotrienol was found to be approximately 5–10, 4–6 and 3–5 μM in mammary malignant epithelial cells [8 and its references]. In murine melanoma cells, the IC50 of α-, γ- and δ-tocotrienol was 110, 20 and 10 μM [1]. The IC50 of γ-tocotrienol in human leukemia cells was 4 μM [9 and its references]. Our group showed that the IC50 of γand δ-tocotrienol was 27.4 and 9.6 μM in human hepatocellular carcinoma HepG2 cells. Intracellular concentration of δ-tocotrienol in HepG2 cells was increased in a dose- and time-dependent manner; when cells were incubated with 0, 10, 20, 40 and 100 μM of δ-tocotrienol for 8 h the concentrations were (mean ± SE) 0.015 ± 0.0048, 7.9 ± 1.8, 18 ± 0.64, 42 ± 2.0 and 190 ± 31 nmol/mg protein, and when incubated for 24 h they were 0.0096 ± 0.0041, 7.5 ± 0.93, 17 ± 1.7, 69 ± 7.2 and 250 ± 42 nmol/ mg protein, respectively. In the study, the relative antiproliferative potential of tocotrienol isoforms were characterized as δ-tocotrienol > β-tocotrienol > α-tocotrienol = γ-tocotrienol in human hepatocellular carcinoma cells [10]. Importantly, vitamin E exerts significant antiproliferative effects in malignant cells, but not in normal cells. Tocotrienol also induced apoptosis in human breast cancer cells, but not in normal human mammary epithelial cells [11]. Highly malignant mouse mammary epithelial cells were more sensitive to the antiproliferative effects of tocotrienol than were preneoplastic or neoplastic cells [8].
Antitumor Activity of Tocotrienols in vivo
Some in vivo studies of the antitumor effects of vitamin E have been reported (table 1). Intraperitoneal injection of γ-tocotrienol, but not α-tocopherol, has shown a slight life-prolonging effect in sarcoma transplanted mice [12]. In the study of C57BL female mice implanted with melanomas, a tocotrienolenriched diet delayed tumor growth and prolonged survival rate [1]. Mice were fed with 928 μmol/kg of γ-tocotrienol for 10 days before implantation. Tumor weight in the tocotrienol-treated group was significantly decreased at 28 days after implantation. 2 μmol/kg of γ-tocotrienol diet increased the duration of host survival, mean survival was 13.83 days in controls and 18.67 in the tocotrienol diet group [1]. Oral administration of tocotrienols also resulted in significant suppression of liver and lung carcinogenesis in male C3H/He mice, and also in the glycerol-induced lung tumor promotion in 4NQO-initiated male ddY mice. In the liver carcinogenesis experiment, the control group developed 7.6 ± 6.9 tumors/mouse, whereas the 0.05% tocotrienol
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Table 1. Anticarcinogenetic effects of tocotrienol in animal studies Effect
Source dosing period
Dose administration route
Animal
Type of tumor
Reference
Prolongs life
γ-T3
i.p.
mice
fibrosarcoma, inoculated i.p.
[12]
Retards onset; reduces tumor growth and size
TRF 20 weeks
1 mg/day p.o.
mice
human breast cancer, injected
[14]
Retards tumor growth
γ-T3 10 days
mice
melanoma, implanted into the flanks
[1]
Increases host survival
γ-T3 1 month
2.2 mmol/kg p.o.
Reduces number of tumor nodules
T3 mixture 40 weeks
2.25 mg/day p.o.
mice
HCC, spontaneously developed
[10]
T3 mixture 25 weeks
2.25 mg/day p.o.
mice
lung adenocarcinoma, two-stage induced
[10]
Decreases ALP and GST in mammary glands
TRF 6 months
10 mg/kg gastric intubation
rat
mammary carcinoma, DMBA induced
[16]
Decreases ALP, GST, lipid peroxidation, LDL oxidation
TRF 6 months
10 mg/kg gastric intubation
rat
hepatocarcinoma, DEN/ [17] AAF-induced
Suppresses tumor cell-induced vessel formation
T3 extract 5 days
10 mg/day p.o.
mice
colorectal adenocarcinoma, implanted
928 μmol/kg p.o.
[15]
T3 = Tocotrienol; HCC = hepatocellular carcinoma; ALP = alkaline phosphatase; GST = glutathione S-transferase; DMBA = 12-dimethylbenz[a]anthracene; DEN = diethylnitrosamine; AAF = 2-acetylaminofluorene.
mixture group exhibited 1.4 ± 1.0 tumors/mouse. Histologically, tumors were identified as well-differentiated hepatocellular carcinoma. In the lung carcinogenesis experiment, oral administration of tocotrienol mixture resulted in a significantly lower mean number of tumors (adenocarcinoma) per mouse, i.e. approximately 22% of the number in the control group. The mean tocotrienol mixture intake was 2.25 mg/day [10]. Specific accumulation of γ- and δ-tocotrienol in tumors was observed in C3H/ HeN mice that had been implanted with murine hepatoma cells and fed with 0.1%
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γ- and δ-tocotrienol [13]. This accumulation might be one of the critical phenomena of antitumor activity of tocotrienols. Oral intake of tocotrienol-rich fraction (TRF), 1 mg/day for 20 weeks, retarded onset and growth of tumor in human breast cancer MCF cells that had been injected in athymic nude mice. cDNA array of tumor tissues showed that TRF treatment modulates immune response [14]. An in vivo angiogenesis study has also been reported. Oral administration of tocotrienol-rich oil suppresses tumor cell-induced vessel formation in mice implanted with human colorectal adenocarcinoma (DLD-1) cells. Embryonic angiogenesis was suppressed by δ-tocotrienol treatment in an in vivo study [15]. Long-term intake of TRF reduced the activity of alkaline phosphatase, the marker of neoplastic transformation, in rats [16, 17].
Regulation of Mitogenesis by Tocotrienols
Tocotrienol also affects the cell cycle (fig. 1a). Several studies demonstrate that α-tocopherol and RRR-α-tocopheryl succinate (α-TOS), which is a semisynthetic analogue of vitamin E, induce cell cycle block. In osteosarcoma cell line MG63, α-TOS induced cell cycle arrest rather than apoptosis [18 and its references]. Despite the existence of only a few studies, γ-tocotrienol induces G1 phase arrest. In murine melanoma cells, treatment with 20 μM of γ-tocotrienol for 3 h induced G1 phase arrest. The ratio of the G1 phase in control cells and tocotrienol-treated cells is 56.4 and 74.0%, respectively. γ-Tocotrienol treatment also induces apoptosis that ranges from 0.7% in the control group to 7.4% in the treated group [9]. G0/G1 phase arrest is induced by γ-tocotrienol at the concentration of 60 μM (24-, 48-hour incubation) in human gastric adenocarcinoma SGC-7901 cells. γ-Tocotrienol increases the ratio of the G0/G1 phase from 53.06% in the control group to 65.14% in the γ-tocotrienol group at 48 h. The ratio of the G2/M phase is decreased from 13.25% in the control group to 4.16% in treated group, and the S phase values are not changed. The expression of cyclin D1 was decreased in the 60-μM γ-tocotrienol group, after 24- and 48-hour incubation periods in a dose-dependent manner, compared with the negative control [19]. Cyclin D1 is a key regulator of the G1 to S phase progression and plays an important role in tumor development and progression. TRF after a 24-hour incubation period caused G0/G1 phase arrest and sub-G1 accumulation in three different cell lines of human prostate cancer cells (LNCaP, androgen responsive; DU145, androgen refractory, and PC-3, androgen refractory). In LNCaP cells, the ratios of the G0/G1 phase are 68.2, 74.1, 77.2 and 75.3% in 0, 10, 20 and 40 μg/ml TRF treated group, respectively. Incubation with TRF concentrations of 0, 10, 20 and 40 μg/ml results in the ratios of 54.8, 56.9, 61.4 and 68.2% of the G0/ G1 phase in DU 145 cells and 47.2, 51.7, 59.4 and 62.5% in PC-3 cells, respectively [20].
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␥-T3, T3E
HMG CoA HMG CoA reductase
Cyclin D ␥-T3 TRF
␥-T3 Mevalonate
G1
S
Farnesyl-PP
G0
G2
Exogenous T3
M
Non-sterol
a
Sterol
b T3
P PDK
P AKt
P eNOS
NO Angiogenesis
c Fig. 1. The mechanism of cancer prevention by tocotrienols. a Modulation of cell cycle; γ-tocotrienol (γ-T3) and TRF induce G0/G1 phase arrest. γ-Tocotrienol and T3E decrease cyclin D, which promotes progression through the G1-S phase of the cell cycle and plays pivotal roles in tumor progression. δ-Tocotrienol tends to induce S phase arrest but not significantly. b Suppression of HMG CoA reductase activity; HMG CoA reductase activity is increased in tumor cells. HMG CoA reductase activity is suppressed by exogenous isoprenoid such as tocotrienol, but tumor cells resist its cholesterolmediated suppression. c Inhibition of angiogenesis; δ-tocotrienol inhibits phosphorylation of PDK, Akt and endothelial nitric oxide synthase (eNOS).
6-O-carboxypropyl-α-tocotrienol (T3E) is a redox-silent analogue of α-tocotrienol, which is synthesized to overcome the disadvantage of tocotrienol and reinforce the anticancer activity; 24- and 48-hour incubation with T3E induces G1 phase arrest in the cell cycle in human lung adenocarcinoma A549 cells. The sub-G1 population was increased by T3E treatment at 48 h but not at 24 h. T3E also decreases the protein expression of cyclin D, which is required for G1/S progression in the cell cycle [21]. In human hepatocellular carcinoma HepG2 cells, a tendency toward S phase arrest in a δ-tocotrienol-treated group was observed in a dose-dependent manner, but the result was not statistically significant. An 8-hour treatment with δ-tocotrienol concentrations of 0, 10, 20, 40 and 100 μM results in 33 ± 5.7, 35 ± 4.6, 36 ± 3.2, 42 ± 4.6 and 42 ± 3.8% of the S-phase, respectively [10]. In contrast, tocotrienols do not affect the cell cycle in normal cells. For example, in rat pancreatic stellate cells, at the concentration of 20 μM, 24-hour incubation of TRF
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does not affect cell cycle [22]. In virally transformed normal human prostate epithelial cell (PZ-HPV-7), TRF does not affect the cell cycle, although G0/G1 phase arrest was detected in three prostate cancer cell lines [20]. Some studies concluded that tocotrienols do not affect the cell cycle; a TRF showed no effect on the cell cycle in human colon carcinoma cells [23].
Proapoptotic Effect of Tocotrienols and Its Pathway
Concerning apoptosis, previous reports conclude that γ-tocotrienol and δ-tocotrienol have more apoptosis-inducing potency than α-tocotrienol. δ-Tocotrienol exerts more significant proapoptosis effects than α- and γ-tocotrienol in both estrogen-responsive MCF-7 and nonresponsive MDA-MB-231 breast cancer cells [18 and its references]. δ-Tocotrienol suppresses cell proliferation more effectively than α-, β- or γ-tocotrienol in hepatocellular carcinoma HepG2 cells [10]. We characterized the dose- and time-dependent proapoptotic activity of tocotrienol in hepatocellular carcinoma cells. At a concentration of 100 μM, δ-tocotrienoltreated cells caused apoptosis at 4 h, which was much earlier than when the cells were treated with 40 μM tocotrienol; in these cells, apoptosis at 40 μM was significant from 16 to 48 h, compared with the control cells. [10]. Similarly, a significant increase in apoptosis has been observed after 18-hour incubation with 120 μM TRF in human colon carcinoma cell line RKO by flow cytometric analysis [23]. Certain pathways of tocotrienol-induced apoptosis have also been reported in the literature (fig. 2). For example, δ-tocotrienol was shown to induce apoptosis via the TGF-β, Fas and JNK signaling pathways in human breast cancer cell line MDA-MB-435 [11]. Tocotrienol induced caspase-9 after activating p53 and increasing the Bax/Bcl-2 ratio in human colon carcinoma cell line RKO [23]. In murine mammary cancer cell line +SA, tocotrienol induced the expression of caspase-8 but not caspase-9 [24]. γ-Tocotrienol induced a mitochondrial disruption pathway without affecting Bax/Bcl-2 expression in human breast cancer cell line MDA-MB-231. 24-, 48- and 72-hour incubation with γ-tocotrienol (8 μM) suppresses ErbB3 receptor tyrosine phosphorylation, subsequent reduction in PI3K/ PDK-1/Akt mitogenic signaling in mouse neoplastic mammary epithelial cell line +SA. 18- and 24-hour incubation with γ-tocotrienol (25, 50 μM) induced poly (ADP ribose)-polymerase (PARP) cleavage, caspase-3, -8, -9 activity and upregulated Bax, Bid in human hepatoma cell line Hep3B [18 and its references]. 72-hour incubation with γ-tocotrienol (4 μM) reduces EGF-dependent PI3K/PDK-1/Akt, and NF-κB mitogenic signaling in human neoplastic mammary epithelial cell line +SA. 0- to 24-hour incubation with γ-tocotrienol (20 μM) induces caspase-8 activation, suppresses PI3K/PDK-1/Akt mitogenic signaling pathway and subsequent reduction in intracellular FLIP level in mouse neoplastic mammary epithelial cell +SA cells [18, 25 and its references]. 12-, 24-, 36-, 48- and 60-hour incubation with
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Pathway of apoptosis ␣-TEA or ␦-T3
TNF
␥-T3
TRF
TNFR TRADD TRAF2
P PDK-1 p53
␥-T3
IKKK
P Akt
Bcl-2
Bax
TGF- R-II
IKK
Daxx
Cytochrome c
Bcl-xL
Bad
NF-B
TRF or ␥-T3 FLIP
JNK 1
Apaf-1 Caspase-9
XIAP
P Caspase-8
P c-Jun
Caspase-3
Cleaved PRAP
Apoptosis
Fig. 2. Possible apoptosis-inducing pathway by tocotrienols.
γ-tocotrienol (60 μM) activates caspase-3 and PARP (via mitochondrial pathway) in adenocarcinoma cell line SGC-7901 [19]. γ-Tocotrienol (25 μM) inhibits NF-κB signaling pathway through inhibition of receptor-interacting protein and TAK1 in several cells, myeloid (KBM-5), multiple myeloma (U266), lung adenocarcinoma (H1299), embryonic kidney (A293) and breast carcinoma (MCF-7) cells. At the downstream of NF-κB, there are proteins associated with apoptosis (IAP1, IAP2, Bcl-xl, Bcl-2, cFLIP, XIAP, Bfl-1, TRAF1 and survivin), proliferation (cyclin D1, COX2 and c-Myc), invasion (matrix metalloproteinase 9 and intercellular adhesion molecule 1) and angiogenesis (vascular endothelial growth factor). δ-Tocotrienol also downregulates these carcinogenesis-related proteins and promotes apoptosis [26]. Twenty-four-hour incubation with T3E decreases the levels of Bcl-xL and inhibits RhoA geranyl geranylation, which induce apoptosis in human lung adenocarcinoma A549 cells [21].
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Antiangiogenic Effects of Tocotrienols
Induction of angiogenesis is an important process of tumor progression. Tocotrienol, but not tocopherol inhibits the proliferation of bovine aortic endothelial cells in a dose-dependent manner. All tocotrienol isomers inhibit cell proliferation in BAEC cells. The median inhibition concentration for α-, β-, γ- and δ-tocotrienol was calculated as 38.0, 6.3, 11.2 and 5.1 μM, respectively. Tocotrienols inhibit tube formation, but do not have any effect on migration, except high-dose δ-tocotrienol, which significantly suppresses migration [27]. Oral administration of tocomin 50 (tocotrienol extract from palm oil; 10 mg/day containing 1.4 mg α-tocotrienol, 2.4 mg γ-tocotrienol, 0.6 mg δ-tocotrienol and 1.4 mg α-tocopherol) suppresses tumor cell-induced vessel formation in human colorectal adenocarcinoma (DLD-1) cells implanted in mice. Treatment with 1,000 μg of δ-tocotrienol causes suppression of embryonic angiogenesis but not in α-tocopherol treatment in vivo. In human umbilical vein endothelial (HUVEC) cells, all tocotrienol isomers suppress fibroblast growth factor-induced proliferation, migration and tube formation. Suppression of PDK and Akt, promotion of the apoptosis signal-regulating kinase and p38 phosphorylation are detected by δ-tocotrienol treatment in HUVEC cells [15] (fig. 1c).
Effects of Tocotrienols on Gene Expression
cDNA array analysis of cancer-related gene expression is performed in estrogendependent (MCF-7) and estrogen-independent (MDA-MB231) human breast cancer cell lines. 72-hour incubation with tocotrienol-rich fraction (TRF) at the concentration of 8 μg/ml increases gene expression of the 23-kDa highly basic protein and c-myc-binding protein MM-1, and decreases one of interferon-inducible proteins 9-27 in both MCF-7 and MDA-MB231 cells. The 23-kDa highly basic protein is related to the cell cycle, and the c-myc-binding protein MM-1 and interferon-inducible protein 9-27 are related to oncogenes and tumor suppressors [28]. Another cDNA array study demonstrated that TRF oral administration upregulated interferon-inducible transmembrane protein 1 gene and CD59 glycoprotein precursor gene in nude mice inoculated with human breast cancer cells. The c-myc gene was downregulated by TRF treatment [14]. In human hepatocellular carcinoma HepG2 cells, toxicology-related gene expression array was carried out after treatment with or without 20 μM of δ-tocotrienol for 8, 16, 24, 48 and 72 h. Only the expression of cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) was significantly higher in δ-tocotrienol-treated cells than in the control cells for all time periods. After a 48- and 72-hour incubation period, the expression of nitric oxide synthase 3 is elevated in treated cells. δ-Tocotrienol increases the expression of cystationase
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for 16 and 24 h, and that of intercellular adhesion molecule 1 and G protein-coupled receptor 3 for 8 and 16 h. The gene expression of cadherin 1, type 1, E-cadherin is decreased by δ-tocotrienol for 24, 48 and 72 h [10].
Regulation of Tumor HMG CoA Reductase
Tocotrienols have also been previously reported to decrease hepatic cholesterol production by suppression of HMG CoA reductase activity. HMG CoA reductase activity is subject to complex feedback regulation at the transcriptional, translational and posttranslational levels by both the sterol end product, cholesterol and a nonsterol product derived from the mevalonate pathway. Sterol-mediated transcriptional feedback is the primary regulatory mechanism in normal cells. In tumor cells, MHG CoA reductase activity is elevated and dysregulated. Maintaining the pools of essential mevalonate-diverted mevalonate-derived intermediates is required for the malignant proliferation. γ-Tocotrienol lowers HMG CoA reductase mass and increases reductase degradation in cultured cells. This suppression of tumor HMG CoA reductase might be one of the antitumor mechanisms of tocotrienols [29 and its references] (fig. 1b).
Clinical Trials
Only a few clinical trials of tocotrienol in cancer prevention have been reported. In Finland, 29,133 male smokers aged 50–69 were registered in the α-Tocopherol, β-Carotene Cancer Prevention Study. After up to 19 years of follow-up, 1,732 were diagnosed with incident prostate cancer. α-Tocopherol supplementation (50 mg daily for 5–8 years) significantly reduced prostate cancer risk. There was, however, no association between prostate cancer and the individual dietary tocopherols and tocotrienols, which were estimated based on a 276-item food frequency questionnaire and food chemical analyses [30]. In this trial, there was no tocotrienol supplement-treated group. It is well known that DNA damage, which is induced by e.g. free radicals to change DNA structure, causes carcinogenesis. Since tocotrienols are antioxidants, a randomized, double-blind placebo-controlled study was undertaken to assess the effect of Tri E tocotrienol on DNA damage. 64 subjects aged 37–78 years were treated with 160 mg of Tri E tocotrienol (daily) for 6 months. Blood samples were analyzed for DNA damage using comet assay, frequency of sister chromatid exchange and chromosome 4 aberrations. Tri E tocotrienol supplementation was found to reduce DNA damage, as shown by a comet assay and reduction of sister chromatid exchange frequency, urinary 8-hydroxy-2⬘-deoxyguanosine [31]. This suppressive effect of DNA damage might be one of the mechanisms of cancer prevention of tocotrienols, even though it is not a direct antitumor activity of tocotrienols.
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Inflammation is also a risk factor of cancer. C-reactive protein (CRP) is an acutephase protein produced by the liver in response to inflammation. 203 healthy adult participants who consumed <4 daily servings of fruits and vegetable and were either active or passive smokers were randomized to receive a placebo or vitamin C (515 mg/day) or antioxidant mixture (515 mg vitamin C, 371 mg α-tocopherol, 171 mg γ-tocopherol, 252 mg mixed tocotrienols and 95 mg α-lipoic acid per day). Vitamin C supplementation reduced plasma CRP level, whereas antioxidant mixture and placebo did not change it significantly [32]. Another clinical trial also concluded that a combination of vitamin C and vitamin E (α-tocopherol) over 3 years in healthy men failed to reduce plasma CRP, TNF-α, or interleukin-6 [32].
Possible Adverse Effects of Tocotrienols
Even though tocotrienol seems to elicit hypervitaminosis, as it is a lipid soluble vitamin, only few adverse effects have been reported. Oral administration of tocotrienol for 13 weeks causes a decrease in mean corpuscular volume, increase in the albumin/ globulin ratio, elevation of alkaline phosphatase and adrenal weight gain in Fischer 344 rats [33]. A 52-week study of exposure to tocotrienol was performed by the same group. Wistar Hannover rats fed with tocotrienol developed nodular hepatocellular hyperplasia [34]. As noted previously in a clinical trial, the antioxidant mixture including 252 mg of mixed tocotrienols negates the antioxidant effect of vitamin C. The enzyme CYP1A1, which is induced by δ-tocotrienol, activates carcinogenic polycyclic aromatic hydrocarbons. Thus, tocotrienol might have the potential to enhance carcinogenesis in some persons, such as tobacco smokers, and this might be one of the reasons why the antioxidant mixture that included tocotrienol could not be effective. The 48- and 72-hour incubation with δ-tocotrienol elevated the mRNA level of endothelial nitric oxide synthetase in human hepatocellular carcinoma cells; endothelial nitric oxide synthetase is a risk factor of angiogenesis, as mentioned previously [10]. In any case, it is important to assess the balance between beneficial and adverse effects, and to consider means of maintaining safe administration.
Conclusion
Tocotrienol is a popular dietary supplement for beautiful skin since it has antioxidant properties. The focus on tocotrienols is not only due to their cancer-preventing effect but also because tocotrienols can be used for cosmetic purposes. In this article, tocotrienols have been shown to have biological anticancer activities, proapoptotic effects and antiangiogenic potential, suppress carcinogenesis and
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HMG CoA reductase activity and regulate mitogenesis. In in vitro studies, the tumor suppressive effects of tocotrienols have almost come to conclusion, and its mechanisms have been proposed. Tocotrienol’s apoptosis-inducing pathway is mainly caspase dependent, and partially caspase independent. G0/G1 phase arrest is caused by δ-tocotrienol through cyclin D reduction. Tocotrienol improves hyperlipidemia by suppressing HMG CoA reductase activity. Inactivation of HMG CoA reductase also suppresses tumor progression since intermediates derived by mevalonate are required for progression. Phosphorylation of PDK/Akt pathway is halted by tocotrienol; this is the one of the mechanisms of suppression of angiogenesis. In animal studies, tocotrienols reduced the size and number of tumors in carcinogenesis-induced models, increased the duration of host survival and suppressed neoangiogenesis in tumor-implanted mice. It is an interesting finding that tocotrienols induce immune response, e.g. though suppression of CD74/Li gene expression. The risk of long-term administration of tocotrienol is still unclear, although few examinations demonstrated adverse effects. The mechanisms of antitumor activity of tocotrienols have been revealed, whereas the clinical evidence is not sufficient. There has been no clear evidence that tocotrienol treatment reduces cancer risk. Additional trials and examinations of tocotrienol will be required for its effective and safe usage.
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24 Shah S, Gapor A, Sylvester PW: Role of caspase-8 activation in mediating vitamin E-induced apoptosis in murine mammary cancer cells. Nutr Cancer 2003;45:236–246. 25 Shah SJ, Sylvester PW: Gamma-tocotrienol inhibits neoplastic mammary epithelial cell proliferation by decreasing Akt and nuclear factor kappaB activity. Exp Biol Med (Maywood) 2005;230:235–241. 26 Ahn KS, Sethi G, Krishnan K, Aggarwal BB: Gamma-tocotrienol inhibits nuclear factor-kappaB signaling pathway through inhibition of receptorinteracting protein and TAK1 leading to suppression of antiapoptotic gene products and potentiation of apoptosis. J Biol Chem 2007;282:809–820. 27 Miyazawa T, Inokuchi H, Hirokane H, Tsuzuki T, Nakagawa K, Igarashi M: Anti-angiogenic potential of tocotrienol in vitro. Biochemistry (Mosc) 2004; 69:67–69. 28 Nesaretnam K, Ambra R, Selvaduray KR, Radhakrishnan A, Canali R, Virgili F: Tocotrienolrich fraction from palm oil and gene expression in human breast cancer cells. Ann N Y Acad Sci 2004; 1031:143–157. 29 Mo H, Elson CE: Studies of the isoprenoid-mediated inhibition of mevalonate synthesis applied to cancer chemotherapy and chemoprevention. Exp Biol Med (Maywood) 2004;229:567–585. 30 Weinstein SJ, Wright ME, Lawson KA, et al: Serum and dietary vitamin E in relation to prostate cancer risk. Cancer Epidemiol Biomarkers Prev 2007;16: 1253–1259. 31 Chin SF, Hamid NA, Latiff AA, et al: Reduction of DNA damage in older healthy adults by Tri E Tocotrienol supplementation. Nutrition 2008;24:1– 10. 32 Block G, Jensen C, Dietrich M, Norkus EP, Hudes M, Packer L: Plasma C-reactive protein concentrations in active and passive smokers: influence of antioxidant supplementation. J Am Coll Nutr 2004; 23:141–147. 33 Nakamura H, Furukawa F, Nishikawa A, et al: Oral toxicity of a tocotrienol preparation in rats. Food Chem Toxicol 2001;39:799–805. 34 Tasaki M, Umemura T, Inoue T, et al: Induction of characteristic hepatocyte proliferative lesion with dietary exposure of Wistar Hannover rats to tocotrienol for 1 year. Toxicology 2008;250:143–150.
Dr. Sayori Wada Laboratory of Health Science, Division of Applied Life Sciences Graduate School of Life and Environmental Sciences, Kyoto Prefectural University 1-5 Hangi-cho Shimogamo Sakyo-ku Kyoto 606-8522 (Japan) Tel. +81 75 703 5484, Fax +81 75 703 5416, E-Mail
[email protected]
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Author Index
Abe, K. 1 Aoi, W. 147 Crifò, C. 75
Na, H.-K. 182 Nagao, A. 55 Naito, Y. 39 Nakamura, Y. 170 Nakashim, Y. 10
Goto, T. 25 Osawa, T. 129 Hirai, S. 25 Ho, C.-T. 64 Ide, T. 10 Iida, H. 10 Ishimi, Y. 104 Ji, G.E. 117 Kang, J. -H. 95 Katsuta, M. 10 Kawada, T. 25 Kim, C.-S. 95 Kundu, J.K. 182
Salerno, C. 75 Siems, W. 75 Sommerburg, O. 75 Surh, Y.-J. 182 Tachibana, H. 156 Takahashi, N. 25 Terao, J. 87 Uemura, T. 25 Wada, S. 204 Wang, Y. 64 Wiswedel, I. 75
Liu, X 129 Miyashita, K. 136 Murakami, A. 193
Yasumoto, S. 10 Yoshikawa, T. 39 Yu, R. 95
217
Subject Index
ACC, see Acyl-CoA carboxylase 1’-Acetoxychavicol acetate, inducible nitric oxide synthase suppression in cancer chemoprevention 199, 200 Acetylene carotenoids, anti-inflammatory activity 144, 145 ACO, see Acyl-CoA oxidase Acyl-CoA carboxylase (ACC), nuclear receptor regulation of expression and food effects 28, 29 Acyl-CoA oxidase (ACO), nuclear receptor regulation of expression and food effects 31 AD, see Atopic dermatitis Adipocyte-specific triglyceride lipase (ATGL), nuclear receptor regulation of expression and food effects 30 Adiponectin, obesity-induced inflammation 99 Akt epigallocatechin gallate receptor signaling in cancer chemoprevention 159 tocotrienol effects in cancer chemoprevention 212 Allyl isothiocyanate, obesity-induced inflammation modulation 101 Angiogenesis ginger phenol effects in cancer chemoprevention 188, 189 tocotrienol inhibition in cancer chemoprevention 212 Apoptosis, cancer chemoprevention ginger phenols 187, 188 isothiocyanate induction 175–177 laminin 67LR receptor in induction 164 tocotrienol induction 210–212 Astaxanthin beneficial biological effects 129, 130
218
neuroprotection studies cell culture and viability assays 131 concentration determination in cell fractions 131 DHA hydroperoxide and 6-hydroxydopamine neurotoxicity effects cell death 133 reactive oxygen species generation 133 materials 130, 131 reactive oxygen species assay 131 structure 129, 130, 138 ATGL, see Adipocyte-specific triglyceride lipase Atopic dermatitis (AD), probiotics and prevention animal model studies 122–126 cell culture studies 122, 123 overview 117, 118 primary prevention studies in humans 118–122 BCAAs, see Branched-chain amino acids Bcl-2 epigallocatechin gallate receptor signaling in cancer chemoprevention 160 ginger phenol effects in cancer chemoprevention 187 isothiocyanate actions in cancer chemoprevention 175, 176 Benzyl isothiocyanate, see Isothiocyanates Bifidobacterium, see Probiotics Branched-chain amino acids (BCAAs) muscle metabolism 152 prevention of exercise-induced homeostasis disturbance 153
Cancer chemoprevention, see Epigallocatechin gallate; Ginger; Isothiocyanates; Nitric oxide synthase; Vitamin E Capsaicin, obesity-induced inflammation modulation 99–101 Carnitine, sesame seed lignan effects on liver levels 12, 18–20 Carnitine palmitoyl transferase-1 (CPT1) food-exercise interactions 149, 150 nuclear receptor regulation of expression and food effects 30, 31 β-Carotene breakdown product analysis adenine nucleotide translocator targeting 80 formation conditions 81, 82 identification 79 mitochondria membrane potential measurements 78 preparation 77 respiration measurements 78, 79 toxicity prevention with antioxidants 83, 84 neutrophil studies degradation products 78, 79 incubation 77 overview of effects 76, 77, 82–84 preparation and formation 77 sulfhydryl analysis 78, 80, 81 supplementation studies 75, 76 Carotenoids, see also specific carotenoids bioavailability and absorption 56–58 breakdown products, see β-Carotene functions 55, 56, 60–62 marine carotenoids acetylene carotenoids and antiinflammatory activity 144, 145 antiobesity activity and structure-activity relationships 141–144 antioxidant activity 137–139 fucoxanthin antiobesity and antidiabetic effects 140, 141 nutrigenomics 139, 140 types and structures 137, 138 metabolism 59, 60 types and structures 55, 56 Catechins, see Epigallocatechin gallate; Flavonoids C/EBPβ, marine carotenoid effects on activity 144
Subject Index
Cocoa polyphenols, functional food genomics and antiobesity effects 5 COX-2, see Cyclooxygenase-2 CPT1, see Carnitine palmitoyl transferase-1 β-Cryptoxanthine, peroxisome proliferatoractivated receptor expression regulation 35, 36 Curcumin inducible nitric oxide synthase suppression in cancer chemoprevention 197, 198 obesity-induced inflammation modulation 101 Cyclooxygenase-2 (COX-2), ginger phenol effects in cancer chemoprevention 185, 186 Diosgenin, antagonism of LXRs 36 DNA microarray, sesame seed lignan regulation of liver fatty acid metabolism gene expression 12–18, 22, 23 Epidermal growth factor (EGF), ginger phenol effects in cancer chemoprevention 186 EGCG, see Epigallocatechin gallate EGF, see Epidermal growth factor Epigallocatechin gallate (EGCG) bioavailability 158 cancer chemoprevention cell line and animal models 157 gene polymorphisms and risk reduction 166, 167 mechanisms of action Akt 159 Bcl-2 160 extracellular signal regulated kinase 1/2 159 prospects for study 168 proteasome 158, 159 vimentin 160 overview 156, 157 inducible nitric oxide synthase suppression in cancer chemoprevention 198, 199 laminin 67LR receptor apoptosis induction role 164 cancer cell growth inhibition modulation 162–164 discovery as green tea catechin receptor 161, 162 functional overview 161 signaling elongation factor 1A 165 hierarchy of signaling molecules 166
219
Epigallocatechin gallate (EGCG) (continued) myosin phosphatase targeting subunit 165, 166 metabolism 65, 66, 69–72 sources 156 Equol, see Soybean isoflavones ERK1/2, see Extracellular signal regulated kinase 1/2 Exercise bone metabolism effects with soybean isoflavones 109 food factor interactions energy metabolism improvement 149–151 hydration 148, 149 muscle mass 151, 152 overview 147, 148 prevention of exercise-induced homeostasis disturbance 152–154 Extracellular signal regulated kinase 1/2 (ERK1/2), epigallocatechin gallate receptor signaling in cancer chemoprevention 159 FAS, see Fatty acid synthase FATPs, see Fatty acid transport proteins Fatty acid metabolism, see Lipid metabolism Fatty acid synthase (FAS), nuclear receptor regulation of expression and food effects 28, 29 Fatty acid transport proteins (FATPs), nuclear receptor regulation of expression and food effects 31, 32 Flavonoids, see also specific flavonoids antioxidant activity quercetin metabolites nerve model 90, 91 vascular system 91, 92 structure-activity relationships 88–90 beneficial effects 65, 66 catechins 65 food sources 64, 65 metabolism biological activities of metabolites 71 colon 70, 71 conjugation 68–70 epigallocatechin gallate 65, 66, 69–72 overview 66, 67 Phase I biotransformation 67, 68 polymethoxyflavones 64 pro-oxidants 92, 93 synthesis 87
220
Food genomics, see also Nutrigenomics food comparison with drugs 2, 3 food safety genomics low-allergen wheat 6 neoculin sweetener 6–8 functional food genomics cocoa polyphenol and antiobesity effects 5 royal jelly and osteogenesis facilitation 5, 6 sesamin regulation of β-oxidation and aldehyde dehydrogenase induction 5 soy protein and lipid metabolism 4, 5 nutrigenomics-based evaluation of functional food 3, 4 Fucoxanthin, antiobesity and antidiabetic effects 140, 141 Functional food genomics, see Food genomics Genomics, see Food genomics; Nutrigenomics Ginger anti-inflammatory effects 185, 186 antioxidant effects 183, 184 cancer chemoprevention by phenols angiogenesis inhibition 188, 189 apoptosis 187, 188 cell growth and proliferation inhibition 187 chemosensitization 189 metastasis inhibition 188, 189 prospects for study 189, 190 tumor promotion inhibition 186 pungent phenols 183, 184 uses 183 Glyceraldehyde-3-phosphate dehydrogenase, isothiocyanate inhibition 174 Green tea, polyphenols 156, 157 HEL, see N-(Hexanonyl)lysine N-(Hexanonyl)lysine (HEL), oxidative stressinduced posttranslational modification 44–46 HMG CoA reductase, tocotrienol regulation in tumors 213 HNE, see 4-Hydroxy-2-nonenal Hormone-sensitive lipase (HSL), nuclear receptor regulation of expression and food effects 30 HSL, see Hormone-sensitive lipase 4-Hydroxy-2-nonenal (HNE), oxidative stressinduced posttranslational modification 43, 44
Subject Index
IL-6, see Interleukin-6 Interleukin-6 (IL-6), obesity-induced inflammation 98 Isoflavones, see Soybean isoflavones Isoprenols, peroxisome proliferator-activated receptor expression regulation 33 Isothiocyanates cancer chemoprevention apoptosis induction 175–177 cellular stress induction 177, 178 mechanisms 172, 173 prospects for study 179 food sources 170, 171 metabolism 171–174 types and structures 171 Keap1, ginger phenol effects in cancer chemoprevention 184 Lactobacillus, see Probiotics Laminin 67LR receptor, see Epigallocatechin gallate Lipid metabolism, see also Obesity-induced inflammation nuclear receptor genomics and foods diosgenin antagonism of LXRs 36 gene expression regulation acyl-CoA carboxylase 28, 29 acyl-CoA oxidase 31 adipocyte-specific triglyceride lipase 30 carnitine palmitoyl transferase-1 30, 31 fatty acid synthase 28, 29 fatty acid transport proteins 31, 32 hormone-sensitive lipase 30 lipoprotein lipase 31 overview 26, 27 stearoyl-CoA desaturase-1 28, 29 sterol response element-binding protein-1 28, 29 peroxisome proliferator-activated receptor food agonists and antagonists β-cryptoxanthine 35, 36 isoprenols 33 phytic acid 34 phytol 34 receptors structures 26, 27 types 25, 26
Subject Index
sesame seed lignan nutrigenomic studies animals and diets 11 carnitine and lignan level evaluation in liver and serum 18–20 DNA microarray analysis of liver fatty acid metabolism gene expression 12–18, 22, 23 gene regulation mechanisms 21 lignan preparations 10, 11 sesamin regulation of β-oxidation and aldehyde dehydrogenase induction 5 Lipoprotein lipase (LPL), nuclear receptor regulation of expression and food effects 31 LPL, see Lipoprotein lipase Macrophage, obesity-induced inflammation 96, 97 MAPK, see Mitogen-activated protein kinase Marine carotenoids, see Carotenoids MCP-1, see Monocyte chemoattractant protein-1 Metabolic syndrome, see Lipid metabolism; Obesity-induced inflammation Metastasis, ginger phenol effects in cancer chemoprevention 189 Mitochondria, carotenoid breakdown product studies membrane potential measurements 78 preparation 77 respiration measurements 78, 79 toxicity prevention with antioxidants 83, 84 Mitogen-activated protein kinase (MAPK), inducible nitric oxide synthase expression regulation 195 Monocyte chemoattractant protein-1 (MCP-1), obesity-induced inflammation 98, 99 NAFLD, see Nonalcoholic fatty liver disease Neoculin, sweetener use and food safety genomics 6–8 Neutrophil carotenoid breakdown product studies degradation products 78, 79 incubation 77 oxidative stress-induced posttranslational modification 46, 47 NF-B, see Nuclear factor-B Nitric oxide synthase (NOS) inducible enzyme expression regulation 195, 196
221
Nitric oxide synthase (NOS) (continued) phytochemical suppression in cancer chemoprevention 196–200 nitric oxide in inflammation-associated cancer 193–195 Nitrotyrosine, oxidative stress-induced posttranslational modification 47–51 Nonalcoholic fatty liver disease (NAFLD), nitrosative stress and protein modification 49 NOS, see Nitric oxide synthase Nuclear factor-B (NF-B), tocotrienol effects in cancer chemoprevention 211 Nutrigenomics, see also Lipid metabolism functional food evaluation 3, 4 marine carotenoids 139 sesame seed lignan studies animals and diets 11 carnitine and lignan level evaluation in liver and serum 18–20 DNA microarray analysis of liver fatty acid metabolism gene expression 12–18, 22, 23 gene regulation mechanisms 21 lignan preparations 10, 11 sesamin regulation of β-oxidation and aldehyde dehydrogenase induction 5 Obesity-induced inflammation macrophages in adipose tissue 96, 97 mediators adiponectin 99 interleukin-6 98 modulation by functional food factors phytochemicals 99–101 monocyte chemoattractant protein-1 98, 99 tumor necrosis factor-␣ 97, 98 overview 95 OPTM, see Oxidative stress-induced posttranslational modification Osteoporosis, soybean isoflavones and bone metabolism studies animal studies 107 equol food factors affecting production 111, 112 intestinal bacteria synthesis and isolation 110, 111 status and importance in bone health 110
222
exercise combination studies 109 observational and intervention studies 108, 109 osteoporosis prevention 105, 114 safety evaluation in Japan 112, 113 Oxidative stress-induced posttranslational modification (OPTM) cysteine modification 40, 41 elimination of proteins 42 N-(hexanonyl)lysine 44–46 4-hydroxy-2-nonenal 43, 44 mechanisms 41, 42 neutrophil-dependent oxidative stress 46, 47 nitrotyrosine 47–51 types 39, 40 p53, isothiocyanate actions in cancer chemoprevention 176, 177 Parkinson’s disease (PD) astaxanthin antioxidant activity, see Astaxanthin oxidative stress 130, 134 PARP, see Poly(ADP ribose)-polymerase PD, see Parkinson’s disease Peroxisome proliferator-activated receptors (PPARs) food agonists and antagonists β-cryptoxanthine 35, 36 isoprenols 33 phytic acid 34 phytol 34 gene expression regulation acyl-CoA carboxylase 28, 29 acyl-CoA oxidase 31 adipocyte-specific triglyceride lipase 30 carnitine palmitoyl transferase-1 30, 31 fatty acid synthase 28, 29 fatty acid transport proteins 31, 32 hormone-sensitive lipase 30 lipoprotein lipase 31 overview 26, 27 stearoyl-CoA desaturase-1 28, 29 sterol response element-binding protein-1 28, 29 marine carotenoid effects on expression 143, 144 P-glycoprotein, ginger phenol effects 189 Phenethyl isothiocyanate, see Isothiocyanates Physical activity, see Exercise
Subject Index
Phytic acid, peroxisome proliferator-activated receptor expression regulation 34 Phytol, peroxisome proliferator-activated receptor expression regulation 34 Poly(ADP ribose)-polymerase (PARP), tocotrienol effects in cancer chemoprevention 210 PPARs, see Peroxisome proliferator-activated receptors Probiotics, atopic dermatitis prevention animal model studies 122–126 cell culture studies 122, 123 overview 117, 118 primary prevention studies in humans 118–122 Proteasome, epigallocatechin gallate receptor signaling in cancer chemoprevention 158, 159 Proteomics, see Oxidative stress-induced posttranslational modification Quercetin, antioxidant activity of metabolites nerve model 90, 91 vascular system 91, 92 Reactive oxygen species, see Astaxanthin; Oxidative stress-induced posttranslational modification Royal jelly, functional food genomics and osteogenesis facilitation 5, 6 SCD1, see Stearoyl-CoA desaturase-1 Sesame seed lignans nutrigenomics studies animals and diets 11 carnitine and lignan level evaluation in liver and serum 12, 18–20 DNA microarray analysis of liver fatty acid metabolism gene expression 12–18, 22, 23 gene regulation mechanisms 21 lignan preparations 10, 11 sesamin regulation of β-oxidation and aldehyde dehydrogenase induction 5 types 10–11 Soy protein, functional food genomics and lipid metabolism 4, 5 Soybean isoflavones bone metabolism studies animal studies 107 equol
Subject Index
food factors affecting production 111, 112 intestinal bacteria synthesis and isolation 110, 111 status and importance in bone health 110 exercise combination studies 109 observational and intervention studies 108, 109 osteoporosis prevention 105, 114 overview of benefits 104, 105 safety evaluation in Japan 112, 113 types and metabolites 105, 106 SREBP1, see Sterol response element-binding protein-1 Stearoyl-CoA desaturase-1 (SCD1), nuclear receptor regulation of expression and food effects 28, 29 Sterol response element-binding protein-1 (SREBP1), nuclear receptor regulation of expression and food effects 28, 29 Tangeritin, metabolism 68, 69 TNF-␣, see Tumor necrosis factor-␣ Tocotrienols, see Vitamin E TRAIL, ginger phenol effects in cancer chemoprevention 187 Tumor necrosis factor-␣ (TNF-␣), obesityinduced inflammation 97, 98 UCP1, see Uncoupling protein-1 Uncoupling protein-1 (UCP1), marine carotenoid effects on expression 140, 145 Vimentin, epigallocatechin gallate receptor signaling in cancer chemoprevention 160 Vitamin Cm prevention of exercise-induced homeostasis disturbance 153 Vitamin E prevention of exercise-induced homeostasis disturbance 153 tocotrienols in cancer chemoprevention adverse effects 214 angiogenesis inhibition 212 antitumor activity in vivo 206–208 apoptosis induction 210–212 bioavailability 205 cell proliferation inhibition 206 clinical trials 213, 214 gene expression regulation 212, 213 mitogenesis regulation 208–210
223
Vitamin E (Continued) prospects for study 214, 215 tumor HMG CoA reductase regulation 213 types 204, 205
224
Wheat, food safety genomics of low-allergen wheat 6 Zingerone, obesity-induced inflammation modulation 101
Subject Index